Rectified Hydrocarbons by-products from synthetic process of Turpentine and acid, alcohols fraction

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Key study: Test method OECD 422. GLP study. The NOAEL of the test substance for parental and reproductive toxicity in rats by oral route was determined to be 600 mg/kg bw/day since no adverse effects were observed at the highest dose tested.

Supporting studies. Experimental results from reproductive studies performed with the component terpineol and the analogue alpha terpinyl acetate are available:

Terpineol: Test method according to OECD Guideline 422 with GLP. The NOEL for parental reproduction was found 300 mg/kg bw/day in males and 100 mg/kg bw/day in females, and the NOEL for development toxicity was established in 1000 mg/kg bw/day, the highest dose tested.

Alpha terpinyl acetate. Repeated dose toxicity study with test method similar to OECD Guideline 408. No effects were seen on male and female gonads after 20-weeks of administration of the test substance.

Extended one-generation reproductive toxicity: Data waiving (study scientifically not necessary): According to the column 1 of REACH Annex IX, the extended one generation reproductive toxicity study does not need to be conducted because the available repeated dose toxicity studies do not indicate adverse effects on reproductive organs or tissues nor reveal any other concern in relation with reproductive toxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 January 2019 – 21 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals.
- Females (if applicable) nulliparous and non-pregnant: yes
- Weight at study initiation: 377.18-381.03 g (range of average values of each group of males at first day of dosing); 271.24-272.21 g (range of average values of each group of females at first day of dosing)
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with a stainless-steel mesh top grill having facilities for holding pelleted food and drinking water in a water bottle fitted with a stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
Acclimatization: maximum of 3 animals of same sex per cage.
Pre mating: 2 animals of the same sex and group per cage.
Mating: 2 animals (one male and one female) of the same group per cage.
Post mating: After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
Recovery animals: 3 animals of same sex per cage.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the experimental period. Deep bore-well water passed through a Reverse Osmosis Unit was provided in plastic water bottles with stainless-steel sipper tubes.
- Acclimation period: at least 5 days. Females were screened for normal oestrous cycles (4 to 5 days) in a two weeks pre-treatment period before initiation of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2-23.1ºC
- Humidity (%): 47-67 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item was weighed into a clean beaker and a little volume of vehicle was added into the beaker and mixed well with glass rod. The formulation was transferred into a measuring cylinder, the beaker was rinsed with some more amount of vehicle also transferred into the measuring cylinder. This procedure was repeated until the entire quantity of the test item formulation was transferred into the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get the desired concentration for the different dose levels.
Test item formulations were prepared daily before administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is not miscible with distilled water and clearly miscible with corn oil at the concentration of 100 mg/mL as per the in-house miscibility test. Hence, corn oil was selected as vehicle for test item formulation preparations. Corn oil is universally accepted and routinely used vehicle in oral toxicity studies.
- Concentration in vehicle: 10, 30 and 60 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): A1712001

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test item in dose formulations was established in a preliminary study. The test item formulations were stable at room temperature for 6 hours at the concentrations of 0.5 mg/mL and 200 mg/mL in corn oil. However, freshly prepared test item formulations were administered to the animals.
Homogeneity and dose formulation analysis for dose concentration verification was done by a validated analytical method UV-Vis spectrophotometer (study nº BIO-ANM 1305). Sampling and analysis of formulations were performed during week 1 (13-02-2019) and week 5 (08-03-2019) of the treatment. Approximately 10 mL of samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control.
Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was ≤10%.

Duration of treatment / exposure:

Main group males: two weeks pre-mating, during mating and up to the day before sacrifice during the post-mating period (total of 36 days of treatment).
Main group females main group: two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.
Recovery animals: same period as for the main group females until the first scheduled sacrifice of dams and kept without treatment for a further 14 days observation.

Frequency of treatment:

Once a day

Details on study schedule:

F1 animals were not mated.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

G1 - Vehicle control + G1R - Vehicle Control Recovery Group

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

G2 - Low dose

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

G3 - Mid dose

Dose / conc.:

600 mg/kg bw/day (actual dose received)

Remarks:

G4 - High dose + G4R - High dose Recovery Group

No. of animals per sex per dose:

Main Group: 12
Recovery Group: 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Doses were decided based on the results of a dose range finding study in which the No Observed Effect Level (NOEL) was found to be 100 mg/kg bw/day and Low Observed Effect Level (LOEL) was found to be 300 mg/kg bw/day, as the dose of 100 mg/kg bw/day did not reveal any treatment related effects in either sex, the dose of 300 mg/kg bw/day revealed treatment related clinical signs of toxicity and reduction in body weight and feed consumption and the dose of 1000 mg/kg bw/day revealed treatment related clinical signs of toxicity, mortality, reduction in body weight and feed consumption.

Positive control:

None

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the treatment on day 1 and weekly thereafter during treatment for all the animals. Signs noted were included, but not be limited to, changes in skin, fur, eyes and mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.

BODY WEIGHT: Yes
- Time schedule for examinations: The main group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The main group females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period. The recovery group animals (both males and females) were weighed at receipt, on the first day of dosing, weekly thereafter and at termination.

FOOD CONSUMPTION: yes
-feed consumption was measured for main group animals (both males and females) once a week during premating and weekly once for main group males during the post mating period. Feed consumption was not measured during the mating period for both males and females. Thereafter, feed consumption for main group females was recorded during gestation days 0 to 7, 7 to 14 and 14 to 20 and on lactation days 1 to 4, 4 to 7 and 7 to 13. Feed consumption was measured for recovery group animals (both males and females) once a week throughout the experimental period. Average feed intake per rat (g/rat/day) was calculated using the amount of feed given and left over in each cage and the number of rats surviving in each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once before treatment for all animals, during end of the dosing period for main group males (on day 34) and during lactation period for main group females (on lactation day 13) of vehicle control and high dose main group animals and during last week (day 64) for all recovery group animals (both males and females).
- Dose groups that were examined: vehicle control and high dose main group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for males after completion of 36 days of treatment and for females on lactation day 14).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 males and 5 females randomly selected from each main group and all recovery group animals.
- Parameters checked: Haemoglobin concentration (HGB), Haematocrit (HCT), Erythrocyte count (RBC), Total leukocyte count (WBC), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLT), Mean platelet volume (MPV), Reticulocyte count (Retic), Absolute reticulocyte count, Differential leucocytes count (DLC), Absolute differential leucocytes count (DLC). Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated by an "OptiClot-4 coagulation analyzer".

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for males after completion of 36 days of treatment and for females on lactation day 14).
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 males and 5 females randomly selected from each main group and all recovery group animals.
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Cholinesterase, Total Protein, Albumin, Total bilirubin, Glucose, Total Cholesterol, Creatinine, Urea, Blood urea nitrogen (BUN), Triglycerides, Phosphorous, Calcium, Globulin. Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using Prolyte Na/K/Cl analyzer (Diamond Diagnostics).

URINALYSIS: Yes
- Time schedule for collection of urine: on the day of scheduled terminal sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 randomly selected males of each main group and all recovery animals.
- Parameters checked: Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Microalbumin, Leucocytes. In addition pH, nitrite and specific gravity were also analyzed. After analysis of the above parameters, the urine was subjected for centrifugation at 1500 rpm for 3 minutes. Then the urine was subjected for microscopic examination for urine sediments.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Towards the end of the dosing period for males (on day 36) and during lactation period for females (on lactation day 13). Towards the end of the recovery period (shortly prior to scheduled sacrifice on day 66) for the recovery group.
- Dose groups that were examined: all main and recovery groups.
- Battery of functions tested: Home Cage Observations (convulsions, tremors and palpebral closure), Handling Observations, Open Field Observations, Sensory Observations, Neuromuscular Observations, Physiological Observation (Rectal temperature), Grip strength assessment and Motor activity assessment.

OTHER:

Thyroxine Hormone (T4) Level estimation: Blood samples were collected for measurement of serum T4 levels on the following schedule:
a. Two pups per litter on lactation day 4 based on the following conditions:
- Two female pups in order to retain more male pups for nipple retention on PND 13.
- No pups were eliminated when litter size dropped below 10 pups/litter.
- Only one pup was eliminated and used for blood collection in case of only one pup was available above ten.
b. All main group females (dams) at termination (lactation day 14).
c. Two pups per litter (same sex) at termination (lactation day 13).
d. All adult main group males, at termination (after completion of 36 days of treatment).

Oestrous cyclicity (parental animals):

Estrous cycles were monitored during the acclimatization to evaluate its normal oestrous cyclicity (4 to 5 days). Only females with normal oestrous cyclicity were selected for the treatment. For the main group females, the vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating. Oestrous cyclicity was also monitored on the day of sacrifice for main group females.

Sperm parameters (parental animals):

Parameters examined in all parental male in the control and high dose groups: testis and epididymis weight. Also, a detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen or any cell or stage specificity of testicular findings.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
The number of pups born (dead and live) in a litter, sex and external observations were recorded at birth. Individual body weight of live pups on lactation day 1 (within 24 hours of parturition), 4, 7 and 13 was recorded. The anogenital distance of each pup was measured on postnatal day 4 (lactation day 4). The number of nipples/areolae in male pups was counted on postnatal day 13 (lactation day 13). Fertility index for dams and sires, pup survival index and sex ratio at birth were calculated.
Also, blood samples were collected for measurement of serum T4 levels on the following schedule:
1. Two pups per litter on lactation day 4 based on the following conditions:
- Two female pups in order to retain more male pups for nipple retention on PND 13.
- No pups were eliminated when litter size dropped below 10 pups/litter.
- Only one pup was eliminated and used for blood collection in case of only one pup was available above ten.
2. Two pups per litter (same sex) at termination (lactation day 13).

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after completion of 36 days of treatment.
- Maternal animals: All surviving animals on lactation day 14
- Recovery animals: All surviving animals after completion of 14 days observation from the first scheduled sacrifice of dams.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed on lactation day 4 (those pups selected for blood collection) and on lactation day 13 (the rest of pups)
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

Statistics:

After verification, the data was subjected to statistical analysis using SPSS software, version 22. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05). The statistical analysis was followed to the parameters as mentioned in the table 3 below.

Reproductive indices:

See table 2 below.

Offspring viability indices:

See table 2 below.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs of toxicity observed at groups G1/G1R and G2 animals of either sex throughout the experimental period.
The groups G3 and G4/G4R animals of either sex did not reveal any clinical signs of toxicity for the first three days of the treatment period. Thereafter, slight wet perineum was noted during several occasions in animals of either sex of these dose groups and found recovered later during the treatment period.
The detailed clinical examination revealed treatment related wet perineum and perinasal staining at groups G3 and G4/G4R during treatment period and found recovered later in the study.
These observed clinical signs at both the dose group animals of G3 and G4/G4R are considered as treatment related due to reduced mean body weight, percent change in mean body weight in either sex during these periods and also the observations are dose dependant when compared with control group.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no mortality/morbidity observed at any dose group during the experimental period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment related changes noted in mean body weight and percent change in body weight with respect to day 1 at group G2 when compared with vehicle control group animals of either sex during the experimental period. However, statistically significant reduction in percent change in mean body weight with respect to day 1 at group G2 animals of either sex was noted. This change is considered as incidental due to lack of consistency and no clinical signs were noted and no effects were noted in feed consumption during this period when compared with control group.
In groups G3 and G4 animals, statistically significant reduction in percent change in body weight with respect to day 1 on days 7, 14, 21, 28 and 35 in males and days 7 and 14 in females was noted.
In group G4R animals, statistically significant reduction in percent change in body weight with respect to day 1 on days 7, 14, 28 and 35 in males, and reduction in mean body weight on day 28 and 35 and percent change in body weight with respect to day 1 during the entire experimental period in females, was noted.
The observed changes at these dose levels [300 mg/kg and 600 mg/kg] are considered as treatment related due to occurrence of treatment related clinical signs of toxicity and also the observed changes are dose dependant and consistent during the experimental period.
There were no changes observed in the gestation body weight and percent change in body weight during gestation period at G2 and G3 dose group animals. A slight reduction in mean gestation body weight was noted on GD 0, 7, 14 and 20 at group G4 animals when compared with other dose groups and vehicle control group. This observation can be considered as treatment related due to significant reduction in mean body weight at this dose group during pre-mating period and also due to occurrence of clinical signs of toxicity during treatment period.
There were no changes observed in the lactation body weight and percent change in body weight during lactation period at groups G2 and G3 when compared with control group animals. A slight reduction in mean lactation body weight was noted throughout the lactation period and the reduction is statistically significant during lactation day 1 to 4 at group G4 animals when compared with other dose groups and vehicle control group. This observation can be considered as treatment related due to significant reduction in mean body weight at this dose group during pre-mating period, gestation period and also due to occurrence of clinical signs of toxicity during treatment period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Statistically significant reduction in mean feed consumption was noted during pre-mating period (week 1) of main group animals in either sex at all the tested dose groups when compared with vehicle control group. This occurrence is considered as incidental at group G2 and G3 of either sex as the mean feed consumption was comparable with vehicle control group during week 2. However, the reduction at group G4 can be considered as treatment related as the reduction was continued during week 2 also and this effect is correlated with reduction in percent change in body weight at this dose level.

There were no treatment related changes observed in the mean gestation feed consumption at group G2 and G3 when compared with vehicle control group. A slight reduction in mean gestation feed consumption was noted during GD 0 to 7, 7 to 14 and 14 to 20 at group G4 when compared with other dose groups and vehicle control group. This observation can be considered as treatment related due to consistency in reduction of feed consumption at this dose group during pre-mating period and also due to occurrence of clinical signs of toxicity during treatment period.

There were no treatment related changes observed in the feed consumption during lactation period at all the tested dose groups when compared with control group animals.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ocular changes observed in G1 and G4 group animals of either sex during the ophthalmological examination conducted towards end of the dosing period and no ocular changes observed in G1R and G4R group animals of either sex during the ophthalmological examination conducted at the end of recovery period.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant changes were noted in haematology parameters when compared with their concurrent control groups: increase in activated prothrombin time at group G4 males; decrease in haemoglobin at group G2 females; decrease in total leucocyte count, absolute lymphocytes and activated prothrombin time at group G4R males and increase in mean platelet volume at group G4R males. These changes are considered as incidental and not treatment related, due to lack of dose dependency and similar changes were not observed in other sex.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant decrease in total bilirubin at group G3 males and statistically significant decrease in triglycerides at group G4R males were noted when compared with their concurrent control groups. These changes are considered as incidental and not treatment related, due to lack of dose dependency and similar changes were not observed in other sex.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no changes observed in urinalysis parameters at any of the tested dose main group males and tested dose group recovery group animals of either sex conducted at termination.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes noted in neurological/functional examinations like home cage, handling, open field, sensory, neuromuscular, physiological observations and assessment of grip strength and motor activity at all the tested dose group animals of either sex from both main and recovery group animals when compared with concurrent vehicle control group animals.
However, statistically significant increase in mean number of rearing during open field examination was noted at groups G2 and G4 females when compared with vehicle control group females. This change is considered as incidental but not treatment related due to lack of dose dependency and no effects were noted in other functional observation battery parameters at these dose levels.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related histopathological findings were noticed in this study.
Lesions considered to be spontaneous and incidental were observed in treated group and control group animals. These lesions consisted of interstitial mononuclear cell infiltrate and concretions in prostate gland, and the presence of calculi in kidney.
1 female from high dose group G4 revealed mononuclear cells infiltration at sub-urothelium of kidney. This lesion was considered spontaneous because of lack of consistency.
3 males from G4 group revealed presence of concretions in prostate gland. Inflammation and concretions of the prostate gland is common background finding in rats and mice, which increases with age (Dianne C., et.al, 2012).
Females from vehicle control group and high dose group revealed presence of placental scar characterized by brown-pigmented nodules at the uterine-mesometrial boundary. The presence of discrete pigmented nodules along the uterine-mesometrial boundary was reported. Each nodule overlies a site of placental detachment at parturition, or the site of the resorption of a feto-placental unit or decidualized implantation site. These nodules were often called placental scars and may persist for many months postpartum, perhaps even the lifespan of a rodent (Janet M., 1990).
Presences of ultimobranchial cyst or ectopic thymus in thyroid were congenital lesions and it does not have toxicological significance.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Serum Thyroxine (T4) Levels: There were no treatment related changes in serum Thyroxine (T4) hormone levels noted at all the tested dose group males. However, statistically significant decrease in T4 levels of males at group G4 was noted when compared with vehicle control group. This change is considered as incidental and not treatment related as the observations do not occur in dose dependant manner and also the values obtained are within historical control range. As no treatment related changes were noted in males the assessment of T4 in blood samples from the dams was not performed.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no changes (irregularities) observed in the oestrus cyclicity of females at any of the tested dose group females during pre-mating treatment, mating treatment and on lactation day 14 of dams.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No effects were detected during examination of spermatogenesis stages in the male gonads and histopathology of interstitial testicular cell structure.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

There were no changes observed in the copulatory interval and number of conceiving days at groups G2 and G3 when compared with vehicle control group. However, an increased copulatory interval was noted at group G4 when compared with vehicle control group. This change is considered as treatment related due to occurrence of treatment related clinical signs and reduced mean body weights at this dose group during the treatment period.

There were no changes observed in the gestation length, litter delivered, number of pups delivered, sex ratio, live birth index at groups G2 and G3 when compared with vehicle control group. However, a treatment related statistically significant reduction in mean gestation length at group G4 was noted when compared with vehicle control group. This observation is considered as treatment related due to observed clinical signs during the treatment period at this dose level.

Key result

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: parental general (systemic) toxicity

Key result

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: Increased copulatory interval and gestation length observed at high dose tested (600 mg/kg bw/day).

Key result

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: parental general toxicity and sexual function and fertility

Remarks on result:

other: No adverse effects were found at any dose tested.

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no changes noted in daily observation of pups at all the tested and vehicle control groups.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no mortalities of pups during the 13-day lactation period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes observed in mean pup weight of either sex during lactation period at all the tested dose groups when compared with vehicle control group. However, statistically significant increase in mean male and female pup weight on lactation day 4 at groups G2 and G4; statistically significant increase in mean female pup weight on LD 7 at group G4 were noted when compared with vehicle control. These changes are considered incidental and not treatment related as the obtained weights are within historical control data and also the changes are not dose dependant.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes noted in any of the pups sacrificed.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Serum Thyroxine (T4) Levels: There were no effects found in serum Thyroxine (T4) hormone levels noted at lactation day 13 pups. As no effects were noted in lactation day 13 pups the assessment of T4 in blood samples from the day 4 pups was not performed.
Anogenital Distance of Pups on Lactation Day 4: There were no treatment related changes observed in mean pup ano-genital distance ratio of either sex during measured on LD 4 at all the tested dose groups when compared with vehicle control group. However, statistically significant decrease in mean male and female mean pup ano-genital distance ratio at groups G3 and G4 was noted when compared with vehicle control. These changes cannot be considered as treatment related effects as the obtained values are within historical control range and also no treatment related effects noted in mean pup weight on the day of measurement of ano-genital distance.
Nipple Retention in Male Pups on Lactation Day 13: There were no occurrences of nipples in male pups at any of the tested doses on lactation day 13.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Development of the offspring

Remarks on result:

other: No treatment related effects were observed at the highest dose tested (600 mg/kg bw/day)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Development of the offspring

Remarks on result:

other: No adverse effects were observed at any dose level tested.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 4. Summary of vaginal smear examination for determination of oestrus cyclicity

Group & Dose (mg/kg body weight/day)	Total No. of Females	No. of Females with Regular Oestrus Cyclicity during Pre-mating and Mating	No. of Females confirmed with pregnancy	No. of Females with Regular Oestrus Cyclicity on Lactation Day 14	No. of Females with Irregular Oestrus Cyclicity during Pre-mating, Mating and on Lactation day 14
G1 & 0	12	12	10	10	0
G2 & 100	12	12	11	11	0
G3 & 300	12	12	10	10	0
G4 & 600	12	12	10	10	0

Table 5. Summary of cohabitation record

Group & Dose (mg/kg body weight)		Copulatory Interval (Days)		Conceiving Days (1 to 5)	Conceiving Days (6 to 14)
G1 & 0	Mean	8.17	n	5	7
	±SD	6.37
	n	12	%	41.7	58.3
G2 & 100	Mean	8.50	n	4	8
	±SD	5.96
	n	12	%	33.3	66.7
G3 & 300	Mean	8.58	n	5	7
	±SD	6.14
	n	12	%	41.7	58.3
G4 & 600	Mean	10.42	n	3	9
	±SD	5.81
	n	12	%	25.0	75.0

Table 6. Summary of gestation length and delivery data record

Group & Dose (mg/kg body weight)		Gestation Length (Days)	Litter Size (No.)	Live Pups (No.)			Dead Pups (No.)			Sex Ratio (M/F) at Birth	Live Birth Index (%)
				Total (No.)	Male (No.)	Female (No.)	Total (No.)	Male (No.)	Female (No.)
G1 & 0	Mean	22.20	11.60	11.60	4.50	7.10	0.00	0.00	0.00	0.83	100.00
	±SD	0.42	3.31	3.31	2.76	2.64	0.00	0.00	0.00	0.91	0.00
	n	10	10	10	10	10	10	10	10	10	10
G2 & 100	Mean	22.73	10.82	10.82	6.09	4.73	0.00	0.00	0.00	1.55	100.00
	±SD	0.65	2.52	2.52	3.18	1.68	0.00	0.00	0.00	1.10	0.00
	n	11	11	11	11	11	11	11	11	11	11
G3 & 300	Mean	22.40	12.00	12.00	6.10	5.90	0.00	0.00	0.00	1.33	100.00
	±SD	0.52	2.31	2.31	2.47	2.33	0.00	0.00	0.00	1.06	0.00
	n	10	10	10	10	10	10	10	10	10	10
G4 & 600	Mean	23.40*	10.10	10.10	5.70	4.40	0.00	0.00	0.00	1.47	100.00
	±SD	0.70	2.02	2.02	1.77	1.35	0.00	0.00	0.00	0.83	0.00
	n	10	10	10	10	10	10	10	10	10	10

n: Number of dams

* Statistically significant (P<0.05) change than the vehicle control group.

Table 7.1. Summary of litter observation record during lactation period. LD1 to 4

Group & Dose (mg/kg body weight)		No. of Live Pups At Birth	During LD 1 to 4							Sex Ratio (M/F) at LD 4	No. of Survived Pups during LD 1 to 4	Pup Survival Index (%) LD1 to 4
			Live Pups (No.)				Dead Pups (No.)
			Total (No.)	Male (No.)	Female (No.)		Total (No.)	Male (No.)	Female (No.)
G1 & 0	Mean	11.60	11.60	4.50	7.10		0.00	0.00	0.00	0.83	11.60	100.00
	±SD	3.31	3.31	2.76	2.64		0.00	0.00	0.00	0.91	3.31	0.00
	n	10	10	10	10		10	10	10	10	10	10
G2 & 100	Mean	10.82	10.82	6.09	4.73		0.00	0.00	0.00	1.55	10.82	100.00
	±SD	2.52	2.52	3.18	1.68		0.00	0.00	0.00	1.10	2.52	0.00
	n	11	11	11	11		11	11	11	11	11	11
G3 & 300	Mean	12.00	12.00	6.10	5.90		0.00	0.00	0.00	1.33	12.00	100.00
	±SD	2.31	2.31	2.47	2.33		0.00	0.00	0.00	1.06	2.31	0.00
	n	10	10	10	10		10	10	10	10	10	10
G4 & 600	Mean	10.10	10.10	5.70	4.40		0.00	0.00	0.00	1.47	10.10	100.00
	±SD	2.02	2.02	1.77	1.35		0.00	0.00	0.00	0.83	2.02	0.00
	n	10	10	10	10		10	10	10	10	10	10

Table 7.2. Summary of litter observation record during lactation period. LD4 to 7

Group & Dose (mg/kg body weight/day)		Live Pups (No.) on LD 4				Pups Sacrificed for Blood Collection on LD 4 (No.)				Live Pups (No.) on LD 4 after Sacrificed for Blood Collection			During LD 4 to 7						Sex Ratio (M/F) at LD 7	No. of Survived Pups during LD 4 to 7	Pup Survival Index (%) during LD 4 to 7
													Live Pups (No.)			Dead Pups (No.)
		Male (No.)	Female (No.)	Total (No.)		Male (No.)	Female (No.)	Total (No.)		Male (No.)	Female (No.)	Total (No.)	Male (No.)	Female (No.)	Total (No.)	Male (No.)	Female (No.)	Total (No.)
G1 & 0	Mean	4.50	7.10	11.60		0.00	1.30	1.30		4.50	5.80	10.30	4.50	5.80	10.30	0.00	0.00	0.00	1.57	10.30	100.00
	±SD	2.76	2.64	3.31		0.00	0.95	0.95		2.76	2.39	2.54	2.76	2.39	2.54	0.00	0.00	0.00	2.70	2.54	0.00
	n	10	10	10		10	10	10		10	10	10	10	10	10	10	10	10	10	10	10
G2 & 100	Mean	6.09	4.73	10.82		0.00	1.18	1.18		6.09	3.55	9.64	6.09	3.55	9.64	0.00	0.00	0.00	3.31	9.64	100.00
	±SD	3.18	1.68	2.52		0.00	0.98	0.98		3.18	2.16	1.69	3.18	2.16	1.69	0.00	0.00	0.00	3.65	1.69	0.00
	n	11	11	11		11	11	11		11	11	11	11	11	11	11	11	11	11	11	11
G3 & 300	Mean	6.10	5.90	12.00		0.00	1.50	1.50		6.10	4.40	10.50	6.10	4.40	10.50	0.00	0.00	0.00	2.47	10.50	100.00
	±SD	2.47	2.33	2.31		0.00	0.71	0.71		2.47	2.22	1.84	2.47	2.22	1.84	0.00	0.00	0.00	3.19	1.84	0.00
	n	10	10	10		10	10	10		10	10	10	10	10	10	10	10	10	10	10	10
G4 & 600	Mean	5.70	4.40	10.10		0.00	0.80	0.80		5.70	3.60	9.30	5.70	3.60	9.30	0.00	0.00	0.00	1.79	9.30	100.00
	±SD	1.77	1.35	2.02		0.00	0.92	0.92		1.77	1.07	1.25	1.77	1.07	1.25	0.00	0.00	0.00	0.88	1.25	0.00
	n	10	10	10		10	10	10		10	10	10	10	10	10	10	10	10	10	10	10

Table 7.3. Summary of litter observation record during lactation period. LD7 to 13

Group & Dose (mg/kg body weight)		Live Pups (No.) on LD 7				During LD 7 to 13							Sex Ratio (M/F) at LD 7	No. of Survived Pups during LD 7 to 13	Pup Survival Index (%) during LD 7 to 13
						Live Pups (No.)				Dead Pups (No.)
		Male (No.)	Female (No.)	Total (No.)		Male (No.)	Female (No.)	Total (No.)		Male (No.)	Female (No.)	Total (No.)
G1 & 0	Mean	4.50	5.80	10.30		4.50	5.80	10.30		0.00	0.00	0.00	1.57	10.30	100.00
	±SD	2.76	2.39	2.54		2.76	2.39	2.54		0.00	0.00	0.00	2.70	2.54	0.00
	n	10	10	10		10	10	10		10	10	10	10	10	10
G2 & 100	Mean	6.09	3.55	9.64		6.09	3.55	9.64		0.00	0.00	0.00	3.31	9.64	100.00
	±SD	3.18	2.16	1.69		3.18	2.16	1.69		0.00	0.00	0.00	3.65	1.69	0.00
	n	11	11	11		11	11	11		11	11	11	11	11	11
G3 & 300	Mean	6.10	4.40	10.50		6.10	4.40	10.50		0.00	0.00	0.00	2.47	10.50	100.00
	±SD	2.47	2.22	1.84		2.47	2.22	1.84		0.00	0.00	0.00	3.19	1.84	0.00
	n	10	10	10		10	10	10		10	10	10	10	10	10
G4 & 600	Mean	5.70	3.60	9.30		5.70	3.60	9.30		0.00	0.00	0.00	1.79	9.30	100.00
	±SD	1.77	1.07	1.25		1.77	1.07	1.25		0.00	0.00	0.00	0.88	1.25	0.00
	n	10	10	10		10	10	10		10	10	10	10	10	9

Table 8.1. Summary of pup observation record during lactation period. At birth

Parameter ↓	Group & Dose (mg/kg body weight)
	G1 & 0	G2 & 100	G3 & 300	G4 & 600
No. of Females with Live Pups at Birth	10	11	10	10
No. of Females Confirmed as Non-Pregnant	2	1	2	2
Litter Size (No.)	116	119	120	101
No. of Dead Pups	0	0	0	0
No. of Cannibalized Pups	0	0	0	0
No. of Live Pups at Birth	116	119	120	101
No. of Live Pups with No Abnormality Detected (No.)	116	119	120	101

Table 8.2. Summary of pup observation record during lactation period. LD1 to 4

Parameter ↓	Group & Dose (mg/kg body weight)
	G1 & 0	G2 & 100	G3 & 300	G4 & 600
No. of Females with Live Pups on LD 4	10	11	10	10
No. of Dead Pups during LD 1 to 4	0	0	0	0
No. of Cannibalised Pups during LD 1 to 4	0	0	0	0
No. of Live Pups during LD 1 to 4 (No.)	116	119	120	101
No. of Live Pups with No Abnormality Detected during LD 1 to 4	116	119	120	101
No. of Pups Sacrificed on LD 4 for blood collection	13	13	15	8

Table 8.3. Summary of pup observation record during lactation period. LD4 to 7

Parameter ↓	Group & Dose (mg/kg body weight)
	G1 & 0	G2 & 100	G3 & 300	G4 & 600
No. of Females with Live Pups on LD 7	10	11	10	10
No. of Dead Pups during LD 4 to 7	0	0	0	0
No. of Cannibalised Pups during LD 4 to 7	0	0	0	0
No. of Live Pups during LD 4 to 7	103	106	105	93
No. of Live Pups with No Abnormality Detected during LD 4 to 7	103	106	105	93

Table 8.4. Summary of pup observation record during lactation period. LD7 to 13

Parameter ↓	Group & Dose (mg/kg body weight)
	G1 & 0	G2 & 100	G3 & 300	G4 & 600
No. of Females with Live Pups on LD 13	10	11	10	10
No. of Dead Pups during LD 7 to 13	0	0	0	0
No. of Cannibalised Pups during LD 7 to 13	0	0	0	0
No. of Live Pups during LD 7 to 13	103	106	105	93
No. of Live Pups with No Abnormality Detected during LD 7 to 13	103	106	105	93

Table 9. Summary record of mean pup weight (g) during lactation period

Group & Dose (mg/kg body weight)		Mean Pup Weight (g) on LD1			Mean Pup Weight (g) on LD 4			Mean Pup Weight (g) on LD 7			Mean Pup Weight (g) on LD 13
		Male	Female		Male	Female		Male	Female		Male	Female
G1 & 0	Mean	6.93	6.51		11.16	10.96		15.16	14.68		25.22	24.57
	±SD	0.35	0.50		0.31	0.41		0.68	0.87		0.38	0.44
	n	9	10		9	10		9	10		9	10
G2 & 100	Mean	6.98	6.64		11.74*	11.57*		15.44	14.89		25.11	24.92
	±SD	0.35	0.42		0.40	0.48		0.45	0.61		0.24	0.63
	n	11	11		11	11		11	11		11	11
G3 & 300	Mean	6.87	6.46		11.64	11.21		15.42	14.92		25.30	24.87
	±SD	0.23	0.26		0.35	0.41		0.35	0.41		0.27	0.44
	n	10	10		10	10		10	10		10	10
G4 & 600	Mean	6.71	6.37		11.82*	11.49*		15.53	15.43*		25.13	24.71
	±SD	0.19	0.26		0.63	0.48		0.62	0.54		0.48	0.92
	n	10	10		10	10		10	10		10	10

n: Number of dams

* Statistically significant (P<0.05) change than the vehicle control group.

Table 10. Summary of mean pup anogenital distance ratio record

Group & Dose (mg/kg body weight)	AGD Ratio
		Mean Male AGD Ratio	Mean Female AGD Ratio
G1 & 0	Mean	2.10	1.18
	±SD	0.09	0.06
	n	9	10
G2 & 100	Mean	2.04	1.12
	±SD	0.07	0.05
	n	11	11
G3 & 300	Mean	1.98*	1.10*
	±SD	0.05	0.04
	n	10	10
G4 & 600	Mean	1.95*	1.10*
	±SD	0.04	0.06
	n	10	10

n: Number of dams

* Statistically significant (P<0.05) change than the vehicle control group.

Table 11. Summary of male pup nipple/areolae retention (no.) record

Group & Dose (mg/kg body weight)		No. of Nipples/Areolae on Lactation Day 13
G1 & 0	Mean	0.00
	±SD	0.00
	n	10
G2 & 100	Mean	0.00
	±SD	0.00
	n	11
G3 & 300	Mean	0.00
	±SD	0.00
	n	10
G4 & 600	Mean	0.00
	±SD	0.00
	n	10

n: Number of dams

Table 12. Summary of uteri observation record

Group & Dose (mg/kg body weight)		No. of Corpora lutea	No. of Implantations	Implantation Index (%)	Pre-Implantation Loss (%)	Post-Implantation Loss (%)	Pre-natal Loss (No.)	Post-natal Loss (At birth to LD 13) (%)	Post-natal Loss (At birth to LD 13) (No.)	No. of Early Resorptions	No. of Late Resorptions
G1 & 0	Mean	11.60	11.60	100.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	±SD	3.31	3.31	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	n	10	10	10	10	10	10	10	10	10	10
G2 & 100	Mean	10.82	10.82	100.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	±SD	2.52	2.52	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	n	11	11	11	11	11	11	11	11	11	11
G3 & 300	Mean	12.00	12.00	100.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	±SD	2.31	2.31	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	n	10	10	10	10	10	10	10	10	10	10
G4 & 600	Mean	10.10	10.10	100.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	±SD	2.02	2.02	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	n	10	10	10	10	10	10	10	10	10	10

Conclusions:

The NOAEL of the test substance for parental and reproductive toxicity in rats by oral route was determined to be 600 mg/kg bw/day since no adverse effects were observed at the highest dose tested.

Executive summary:

A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of the test substance Pine oil 50% by oral administration in rats was conducted according to OECD guideline 422, in accordance with GLP principles. A total of 116 (58 males + 58 females) Sprague Dawley rats were distributed to four main groups and two recovery groups to detect delayed occurrence and recovery from toxic effects. Each main group (G1, G2, G3 and G4) consisted of 12 males and 12 females and each recovery group (G1R and G4R) consisted of 5 males and 5 females. The animals in G1/G1R group were administered with vehicle (Corn oil), and the animals in G2, G3 and G4/G4R groups were administered with test item at the dose levels of 100, 300 and 600 mg/kg body weight based on the results of a dose range finding study performed with the same species at doses of 100, 300 and 1000 mg/kg bw for a period of 14 days. The test item was administered to the main group males for a period of 36 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period), to the main group females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 and to the recovery group animals for a period of 50 days with a 14 days recovery. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight. The stability of test item formulations in corn oil was established before initiation of the treatment. Dose formulation analysis for homogeneity and concentration verification was performed during weeks 1 and 5 of the treatment period and the results were within acceptable limits.

All the main group and recovery group animals were observed for clinical signs, mortality and morbidity, detailed clinical examination, body weight, feed consumption and ophthalmological and neurological/functional examinations. The clinical pathological (haematology, clinical chemistry and urinalysis) examinations were conducted for 5 randomly selected animals from each group per sex for main group and from all the animals for recovery group at termination. The gross pathology and organ weighing were performed on the day of termination for all the main and recovery group animals. All the main group females were observed for maternal body weight and feed consumption during gestation and lactation. The main group females were evaluated for oestrus cyclicity. Each dam was observed for mating performance, fertility performance, gestation length, litter size, number and percentage of live/dead pups, live birth index, sex ratio and observation of litter throughout the lactation period. Histopathological examination was conducted on all the tissues collected from the main group vehicle control and high dose group animals sacrificed at termination. The pups were observed once daily for clinical signs and external examinations, weighed individually on postnatal day (PND) 1, 4, 7 and 13, measured for anogenital distance on PND 4, observed for retention of any nipples/areolae in male pups on PND 13 and observed for gross pathological observations at termination. The analysis of thyroxine hormone (T4) levels in serum collected at termination was performed for main group males and PND 13 pups.

There were no clinical signs, no mortality/morbidity, no effects of test item on mean body weight, feed consumption, no changes in ophthalmological examination and neurological/functional examination, clinical pathology, organ weights and gross pathology noted at group G2 of either sex. The group G3 and G4/G4R animals of either sex revealed treatment related wet perineum and recovered later in the study. A treatment related reduction in body weight was noted at both G3 and G4/G4R groups of either sex. No treatment related changes were noted in clinical pathology and organ weights and no gross pathological changes were noted at group G3 and G4/G4R. There were no treatment related changes in oestrus cyclicity, maternal and lactation body weight and feed consumption, mating performance, fertility performance, gestation length, litter size, number and percentage of live/dead pups, live birth index, sex ratio and observation of litter at group G2 and G3. A treatment related reduction in mean body weight and feed consumption during gestation and lactation periods and increased gestation length was noted at group G4. There were no treatment related changes occurred in histopathological examination conducted for group G4 animals in either sex. There were no clinical signs and no external abnormalities noted in pups from all the tested dose groups. No treatment related changes in mean pup weight and anogenital distance noted at all the tested dose groups. The gross pathology conducted for the pups at all the tested dose groups revealed no changes. No occurrences of nipples/areolae in male pups from all the dose groups observed on PND 13.There were no treatment related changes noted in serum thyroxine hormone (T4) levels.

The No Observed Effect Level (NOEL) for parental general toxicity was determined to be 100 mg/kg bw/day based on treatment related clinical signs of toxicity and reduction in body weight found at the dose of 300 mg/kg bw/day. The No Observed Effect Level (NOEL) for parental reproduction was established at 300 mg/kg bw/day based on the increased copulatory interval and gestation length observed at the high dose tested (600 mg/kg bw/day). Finally, the No Observed Adverse Effect Level (NOAEL) for parental and reproductive toxicity was determined to be 600 mg/kg bw/day as no adverse effects were found on general toxicity, fertility or development of offspring at any dose level tested.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

18 September 2012 – 28 March 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Test method according to OECD guideline 422 and GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

(mating females were only administered test chemical up to 4 days of lactation instead of 13 days)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River. Atsugi Breeding Center.
- Age at study initiation: 10 weeks
- Weight at study initiation: 350-420 g (378 g average in males); 215-257 g (231 g average in females)
- Housing: bracket-type metal net cage (W 254 × D 350 × H 170 mm). During mating (1 male: 1 female) and from 17th day of pregnancy to 4th day of lactation a plastic cage (W340 × D400 × H185 mm) was used.
- Diet (e.g. ad libitum): NMF solid form (radiation sterilization, Oriental Yeast Co., Ltd.), ad libitum.
- Water (e.g. ad libitum): Tap water (metal cage) and from bottle (during time of lactation in plastic cage), ad libitum.
- Acclimation period: 20 days (including 3 days of quarantine)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24ºC
- Humidity (%): 39-65 %
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Preparation of the test solutions was conducted by taking a required amount of the test substance with a glass syringe for each concentration and accurately weighing it in a beaker. The weighed test substance was transferred to a measuring cylinder. The beaker was washed several times with a small amount of solvent, and the liquid was also added to the measuring cylinder. The mixture was shaken by hand and mixed, and it was visually confirmed that it was completely dissolved. Next, the solvent was added to the measuring cylinder to the required amount of test material.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1 male/1 female
- Length of cohabitation: up to 14 days
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A sample of 10 mL of each formulation prepared for administration in the first and last week of the dosing period was analysed for achieved concentration of the test substance. The mean concentrations of terpineol in test formulations analysed for the study were within applied limits (99.5 to 103.0% of nominal concentrations), confirming accurate formulation.
The determination of the concentrations was performed by HPLC.

Duration of treatment / exposure:

Males: From 14th day before mating up to 30 days after mating (total 44 days).
Females: From 14th day before mating up to 4 days of lactation (total 41-51 days).
Non-mating female group: 44 days

Frequency of treatment:

Once a day, 7 days a week

Details on study schedule:

Study period: 9 October 2012 (administration start day) – 16 December 2012 (animal test end day)
Note: after administration period some animals from the control and high dose groups were kept for further 14 days without treatment to detect delayed occurrence and recovery from toxic effects.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Mating male group: 12 per sex per dose (including 5 in the control and high dose group for 14-day observation after treatment)
Mating female group: 12 per sex per dose
Non-mating female group: 10 (control group, including 5 for 14-day observation after treatment) and 10 (high dose group, including 3 for 14-day observation after treatment)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
A preliminary range finding study was conducted with doses of 250, 500 and 1000 mg/kg bw/ day. One male in the 1000 mg/kg bw/day dose group showed a decrease in body weight and food intake, a decrease in feces volume, a decrease in locomotor activity and a decrease in respiratory rate, and died on the 12 th day of administration. A high value of liver and kidney weight and a high value of urea nitrogen and total protein were observed in autopsy of surviving males of high dose group. In each female of 500 and 1000 mg/kg bw/day group, a significant reduction in walking and muscle tone and a licking gait were observed only on 1 day of administration. Therefore, in this study, 1000 mg/kg bw/day at which obvious signs of toxicity is expected was set as high dose, and 300 and 100 mg/kg bw/day were set as medium and low dose, respectively, divided by the common ratio of about 3.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily during the quarantine period, three times daily for the administration period (before administration, immediately after administration and 1 to 3 hours after administration), twice daily during the recovery period (morning, afternoon)
- Cage side observations: The presence or absence of abnormalities in general conditions such as life and death, appearance of the body, posture, behavior and emissions (urine, feces) were observed. Regular observation accompanied by handling of animals after 2 weeks of administration was also carried out as observation of detailed general condition.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed general condition observation was performed once for all individuals, before administration. Also, males and non-mating females were observed once a week during the administration period and during the recovery period. Females in the mating group were observed once a week during the pre-mating period, during the mating period, during the pregnancy period and during the lactation period.

BODY WEIGHT: Yes
- Time schedule for examinations: For all individuals, body weight was measured on acclimated days 1, 3, 8, 13 and 20 and on the day of administration 1, 8, 15, 22, 29, 36, 42, 44 and on the day of necropsy. For the recovered animals further recovered the body weight was measured on days 1, 8 and 14 and on the day of necropsy. Body weights of female mating group were measured on day of administration 1, 8, 15, 22, on day of gestation 0, 7, 14, and on day 0 and 4 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes.
-Food consumption was determined daily by weighing the residue. Measurements were not made during the mating period.

OTHER:
-URINE EXAMINATION:
During the last week of the administration period and during the recovery period a urinalysis was performed in 5 rats in each group. These animals were housed in a cage prepared with urine collector, and under fasting and free water ingestion urine was collected for 4 and 20 hours. For the first 4 hours urine collected pH and urine volume were taken. Osmotic pressure, electrolytes (sodium, potassium and chlorine) and urine volume were measured for the 20 hours urine obtained.

- HAEMATOLOGY:
The day before necropsy after administration period and after 14-day observation period, blood samples were obtained from 5 animals per sex and per group after overnight withdrawal of food, and under anaesthesia induced by isoflurane. The following were measured using a Siemens Advia 120 haematology analyser: Erythrocyte count (RBC), Haemoglobin concentration (HGB), Haematocrit (HCT), Mean cell volume (MCV), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Reticulocyte count (Retic), Platelet count (PLT) and White blood cell count (WBC). Differential leucocyte count (Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M) and Large unstained cells (LUC)) were also measured. Prothrombin time (PT), Activated partial thromboplastin time (APTT) and Fibrinogen (FIB) were measured by automatic blood coagulation analyzer ACL Elite Pro.

- BLOOD CHEMISTRY:
At the same time and using the same animals as for haematology, further blood samples were collected and examined using a Toshiba analyzer TBA-120 FR. Serum was analysed for: Alkaline phosphatase (ALP), Total bile acid (TBA), Total cholesterol (T-CHO), Triglyceride (TG), Phospholipids (PL), Total bilirubin (T-BIL), Glucose (GLU), Urea nitrogen (BUN), Creatinine (CRNN), Sodium (Na), Potassium (K), Chlorine (Cl), Canoesium (Ca), Inorganic phosphorus (P), Total protein (TP) and Anolebumin (ALB). Heparinized blood was examined for Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Lactate dehydrogenase (LDH) and Gamma-glutamyl transpeptidase (GTP).

Oestrous cyclicity (parental animals):

Estrous cycle was measured during pre-mating period: count of estrous, mean duration of cycle, number and index of animals with abnormal estrous cycle.

Sperm parameters (parental animals):

Parameters examined in male parental:
testis: weight, sperm count, vacuolation/atrophy of seminiferous tubular, multinucleated giant cells, etc.
epididymis: weight, sperm count, cell debris (lumen), granuloma

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in offspring: The number of live births and stillbirths were counted on the 0th day of delivery. The sex and weight were measured for each individual. The stillborn pups were fixed with Bouin's solution and stored. Living pups were checked for death once daily until 4th day of lactation. On the 4th day after birth, the sex of all surviving pups was judged, the body weight was measured for each individual, and the presence or absence of abnormalities were observed visually. Then, they were immersed in Bouin's solution under deep anesthesia of isoflurane, fixed and stored.

GROSS EXAMINATION OF DEAD PUPS: yes. The dead pups were observed visually for presence of abnormalities and then fixed with Bouin's solution and stored.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- All animals were subject to a detailed necropsy, which included a visual examination of the external tissues and organs of the head, thorax and abdomen. For mating females, the number of uterine implantation sites and number of corpora lutea were also recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
- ORGAN WEIGHTS:
The absolute weight and relative weight of the following organs were measured: Brain, pituitary, thyroid (including parathyroid gland), adrenal gland, spleen, thymus, tongue, heart, liver, Kidney, testis, epididymis, prostate, seminal vesicle (including coagulated gland), ovary and uterus.
-HISTOPATHOLOGY:
For all individuals, the following organs / tissues were fixed for histopathology examination: cerebrum, cerebellum, sciatic nerve, spinal cord (chest), the eyeball, optic nerve, pituitary, thyroid, parathyroid, adrenal gland, spleen, thymus, submandibular lymph node, mesenteric lymph node, heart, aortic aorta, trachea, lung (including bronchus), tongue, larynx, oesophagus, stomach, duodenum, jejunum, ileum, cecum, colon, kidney, testis, ovary, epididymis, uterus, prostate, seminal vesicle (coagulating glands mate), skin (inguinal region), mammary gland (inguinal region), sternum, femur (including bone marrow) and femoral skeletal muscle. Samples of any abnormal tissues were also retained and processed for examination. Testis and epididymis were fixed with Bouin's fixative.

Postmortem examinations (offspring):

GROSS NECROPSY
- Fracture head, facial nostrils and upper and lower jaws, distortion or fracture of the lips, size and shape of the eyes and ears, left and right balance, distortion and size of the chest body, distortion and size of the internal organs were observed with attention to the length, shape, number, loss of the tail, length and shape of limbs, number of fingers, defects and abnormal cases different from normal.

Reproductive indices:

Sex cycle abnormality rate (%) = (number of individuals showing sexual cycle abnormality / number of observed female individuals) × 100
Mating rate (%) = (number of animals mating / number of living animals) × 100
Fertilization rate (%) = (number of males who pregnant female / number of male copulated) × 100
Conception rate (%) = (number of pregnant females / number of mated females) × 100

Offspring viability indices:

Birth rate (%) = (number of births of babies born / number of pregnant females) × 100
Delivery rate (%) = (number of births / number of implantation sites) × 100
4-day survival rate (%) = (number of surviving offspring on 4th day / number of births) × 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

CAGE SIDE OBSERVATIONS: There was no abnormality in any animal.
SIGNS AND ARENA OBSERVATIONS: Wiggling was observed on the first day of pregnancy in one female in the mating group of 1000 mg/kg/day administration group.
In addition, two animals were observed gait abnormality and jumping respectively but they were temporary findings and it was judged that there was no relation with the administration of the test substance because it was not related to the dose. Furthermore, facial cleansing behavior was observed in another animal, which is a normal behavior observed when the animal calms down, and is not related to administration of the test substance.

General condition of surviving animals: In the 1000 mg/kg/day dose group, 4 males had nursing behaviour and 1 male a decrease in feces volume, and in females, a decrease in feces volume was observed. In 3 females of the non - mating group, decrease in fecal volume, scaling, decreased motor activity, wiggling gait or decrease in respiration rate were observed.
These symptoms were not seen during the recovery period, thus it was judged as no treatment related.

Gripping strength: There was no significant difference between the control group and each test substance administration group.

Motor activity: There was no significant difference between the control group and each test substance administration group. Meanwhile, in the females of the non-mating group, a significant low value was observed in the measurement values of 0 - 10 and 10 - 20 intervals in the 1000 mg/kg/day dose group compared with the control group, but there was no significant difference in the total measurement value (0-60 interval) and it was equivalent to the measurement value of the control group at the end of the recovery period week. Thus it was judged to be a change within the variation range.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

6 females in the 1000 mg/kg/day dose group were found dead or euthanized. These animals showed worsening status before moribund and death, suppression and reduction in body weight gain, low food consumption, decreased fecal volume, scraping, wiggling, decreased motor activity, decreased respiratory rate, abdominal drainage and decrease in body temperature. In addition, it was mainly characterized by malnutrition, reduction of spleen and thymus, downsizing of kidney (renal tubular dilation, vacuolization of proximal renal tubule, vacuolation of distal tubule / collecting duct, papillary necrosis, single cell necrosis of teat canal, regeneration of the papillary collecting duct, papillary cell infiltration), single cell necrosis of the transitional epithelial cell, hypertrophy / hyperplasia of the transitional epithelial cell, adrenal gland cell vacuolation, eosinophilic droplets and vacuolation of hepatocytes in the liver. Other secondary changes accompanying state deterioration include atrophy of various lymphoid tissues, atrophy of colonic mucosa, land mucosa and uterine atrophy or reduction of zymogen granules of pancreas, mesenterium lymph nodes, submandibular lymph nodes and thymus.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In males, suppression of body weight gain was observed in the 1000 mg/kg/day dose group, and a significant lower value was observed in body weight gain during the administration period. In the group administered with 300 mg/kg/day or less, the body weight transition was similar to that of the control group, and no significant difference was observed.
In the mating group females, body weight was lower than that of the control group through premating, pregnancy and lactation period in the 1000 mg/kg/day dose group, but no significant difference was observed. A significant low value was observed on the 0th day of lactation compared to the control group in the 300 mg/kg/day administration group, but it was a temporary change, and the weight gain increase in the subsequent lactation period was found to be a significant high value. It was judged that it was not related to administration of the test substance. In the 100 mg/kg/day administration group, no significant difference was observed between the control group and the control group.
In the non-mating group females, mean body weight decreased due to the moribund / death cases on day 4 of administration in the 1000 mg/kg/day dose group, and a significant lower value was observed than in the control group. However, it is a transient fluctuation due to the moribund / death cases, not an influence on the body weight by administration of the test substance.
In the 1000 mg/kg/day dose group, both sexes showed weight gain above the control group during the recovery period, but no significant difference was found between the treated group and the control group.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In males, a significant high value was observed on the 30th and 44th days of administration in the 1000 mg/kg/day dose group, but it was judged as a change within the variation range because it was temporary. In the group administered with 300 mg/kg/day or less, the food consumption was equivalent to that of the control group, and no significant difference was observed.
In the mating group females, there was no significant difference between the control groups and the test substance administration group in the pre-mating, pregnancy and lactation periods.
In the non-mating group females, a significant low value was observed on the 8th day of administration compared with the control group in the 1000 mg/kg/day dose group, but it was judged that it was a slight temporary change and was not related to the administration of the test substance.
In the 1000 mg/kg/day dose group, males showed food intake over the control group during the recovery period, and a significant difference was observed on administration 1 and 14 days. In females there was no significant difference between the control group and the 1000 mg/kg/day dose group.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

During administration and recovery period, a significant high value of water intake was observed in the 1000 mg/kg/day dose group of males and non-mating group females as compared with the control group.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males, a significant lower value of hemoglobin was observed in the 1000 mg/kg/day dose group compared to the control group. This difference was not seen during the recovery period, thus it was judged as no treatment related.
In the mating group females, no change was observed by administration of the test substance. In addition, compared with the control group, the leukocyte count, the basophil count and the large unstained cell count were significantly lower in the 100 mg/kg/day administration group. Significantly high levels of white blood cell count, eosinophil count and monocyte count were observed in the 300 mg/kg/day administration group, but there was no significant difference in the leukocyte percentage and there was no relation with the dose and the females of the non-mating group. It is considered that it is temporary fluctuation accompanying increase / decrease of mild leukocyte counts at the beginning of lactation after childbirth because there is no such change, and it was judged to be within the physiological variation range.
Moreover, a significant shortening of the prothrombin time was observed in the group administered with 300 mg/kg/day, but it was judged within the physiological variation range because it was slight shortening and there was no toxicological significance.
Significantly low levels of hemoglobin and hematocrit and significant high values of mean red blood cell hemoglobin concentration and significant high levels of fibrinogen were observed in non-mating group females as compared with the control group. In addition, although a significant low value of eosinophil ratio was observed in the 1000 mg/kg/day dose group compared with the control group, it was a slight change and there was no significant difference in the eosinophil count, so it was judged as an accidental change.
During recovery period, significantly low platelet count was observed in the 1000 mg/kg/day dose group compared with the control group, and the lymphocyte ratio was significantly lower in the 1000 mg/kg/day dose group in the non-mating group female, but since it was not recognized during administration period and it was a slight change in the background data, it was judged that it was not related to the administration of the test substance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In male, a significant high value was observed in GTP in the 1000 mg/kg/day dose group compared to the control group. This difference was not seen during the recovery period, thus it was judged as no treatment related.
In the mating group females, a significant high value was found in GTP in the group administered with 300 mg/kg/day compared to the control group, but it was a slight change and it was not related to the dose, so it was judged within the physiological variation range.
In the non-mating group females, significant high values of calcium and inorganic phosphorus were found in the background data of the test facility, but this change was equivalent to the control group of the mating group female, thus it was judged that there was no relation with the administration of the test substance.
At the end of recovery period, significant low value for total bilirubin and significant high value for calcium in the male 1000 mg/kg/day dose group, and significant high value of total cholesterol in the 1000 mg/kg/day dose group of non-mating group females were observed. These were minor changes compared with background data and results at the end of the administration period, thus it was judged that there was no relation with the administration of the test substance.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

During administration period: Regarding qualitative items, neither sex was abnormal in any animals. Regarding quantitative items, a significant high value of urine volume accompanied by a significant high value of water intake and a significant low value of urinary osmotic pressure were observed in the 1000 mg/kg/day dose group of males and non-mating group females as compared with the control group.
During the recovery period: Regarding the qualitative items, neither sex was abnormal in any animals. For quantitative items, high or significant urine volume with a significant high value of water intake and significant low value of urinary osmotic pressure were observed in the 1000 mg/kg/day dose group of males and non-mating females compared with the control group. Sodium, potassium and chlorine emissions were high or significantly high.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

During dosing period, neither animal had any abnormality in the auditory reaction, approach reaction, contact reaction, pain sense reaction, pupillary reflex, and airborne forward reflection.
In the landing leg width, there was no effect of administration of the test substance. In the non-mating group females, a significant high value was observed at 6 weeks of administration in the 1000 mg/kg/day administration group compared with the control group, but since it was equivalent to the measurement value of the control group at the end of the recovery period, it was judged as no treatment related.

During recovery period, neither animal had any abnormality in the auditory reaction, approach reaction, contact reaction, pain sense reaction, pupillary reflex, and airborne forward reflection.
There was no significant difference between the control group and the 1000 mg/kg/day administration group in the landing leg open width.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Adrenal gland: The vacuolation (minor or mild) of cortical cells was found in the group administered 300 mg/kg/day or more in the mating group female and in the 1000 mg/kg/day dose group of the non-mating group female.

Testis: Atrophy (moderate) of seminiferous tubules, multinucleated giant cells (minor to moderate) and vacuolization of seminiferous tubules (minor, mild) were found in the1000 mg/kg/day dose group. The testicular disorder caused by the administration of the test substance was considered to be the cause of female infertility.
At the end of the recovery period, atrophy of seminiferous tubules (mild to moderate), multinucleated giant cells (minor) and vacuolization of seminiferous tubules (minor, mild) were found in the1000 mg/kg/day dose group. Mature spermatozoa were not observed, but the length of seminal epithelium was higher than the examination at the end of administration and recovery was noted.

Epididymis: Reduction of sperm (mild to severe) and cell debris content of the lumen (mild, moderate) were observed in the 1000 mg/kg/day dose group, reflecting the influence on the testes by administration of the test substance.
At the end of the recovery period, reduction of sperm (moderate) and cell debris content of the lumen (mild, moderate) were seen in the 1000 mg/kg/day dose group. Although it was thought that it was an image reflecting the change of the testes, it was thought that it will disappear with the recovery of the testes in the future.

Kidney: The vacuolization (minor) of the proximal tubule was observed in the 300 mg/kg/day dose group in the mating group female, the vacuolization (minor, mild) in the distal tubule / collecting duct was in the male, the mating group female and the non-mating group female in the 1000 mg/kg/day dose group, the regeneration of the cortical renal tubule (minor, mild) was in the group administered with 300 mg/kg/day or more in males and in the 1000 mg/kg/day dose group of non-mating females, renal tubule dilation (minor, mild), single cell necrosis of papillary duct (minor) and cortical cell infiltration (minor) were observed in the 1000 mg/kg/day dose group of males and non-mating group females, regeneration (minor) of papillary collecting duct was in the 1000 mg/kg/day male group. Papillary necrosis (minor, mild) and papillary cell infiltration (minor) were found in the 1000 mg/kg/day dose group of mating female and non-mating females. In addition, an increase in the frequency of eosinophilic corpuscles in renal tubules was observed in the male group receiving 300 mg/kg/ day or more. Eosinophilic bodies of renal tubular epithelial cells were positive for α2μ globulin immunohistochemistry and were negative for PAS staining and were considered to be derived from α2μ globulin.
At the end of the recovery period, papillary necrosis (minor) was observed in the 1000 mg/kg/day dose group of male and non-mating females, regeneration (mild) of cortical renal tubules was observed 1000 mg/kg/day dose group of males. Although renal papillary necrosis was not seen at the end of male administration, similar changes were observed in mating and non-mating females, and even in male, regeneration of papillary collecting ducts and single-cell necrosis of papilla were seen, thus it was thought that the change at the end of the administration remained, but the degree of finding was minor. In females, animals with strong papillary changes were deceased or euthanized, and it seemed that they recovered for those with minor changes. Regeneration of cortical renal tubules decreased frequency and showed recovery.

Liver: centrilobular, hepatocytic hypertrophy (minor) was found in the 1000 mg/kg/day dose group of male; hepatocyte vacuolation (minor, mild) was found in the 1000 mg/kg/day dose group of mating female and the non-mating female.

Urinary bladder: Atrophy of umbrella cells (minor, mild) were observed in the 1000 mg/kg/day dose group of males, mating females and non-mating females. Vacuolation of umbrella cells (minor to moderate) were observed in the 300 mg/kg/day dose group of males and mating females. Hypertrophy / hyperplasia of epithelial cells (minor, mild) was observed in the male, mating group female and non - mating group females administered with 1000 mg/kg/day.

Pancreas: An increase in the frequency of decreased zymogen granules (minor to moderate) was observed in the 300 mg/kg/day dose group of mating females and in the 1000 mg/kg/day dose group of non-mating females.

In two female animals all littermate died on 2nd day of lactation in the 300 and 1000 mg/kg/day dose group, respectively. In both cases the death of all littermate was considered to be a possibility of deterioration of the condition of the mother animal.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No sexual cycle abnormalities were found, and no significant difference was observed between the control group and each test substance administration group in the sexual cycle abnormality rate, development of oestrus stages and the average cycle duration.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Testis: Atrophy (moderate) of seminiferous tubules, multinucleated giant cells (minor to moderate) and vacuolization of seminiferous tubules (minor, mild) were found in the1000 mg/kg/day dose group. The testicular disorder caused by the administration of the test substance was considered to be the cause of female infertility.
At the end of the recovery period, atrophy of seminiferous tubules (mild to moderate), multinucleated giant cells (minor) and vacuolization of seminiferous tubules (minor, mild) were found in the1000 mg/kg/day dose group. Mature spermatozoa were not observed, but the length of seminal epithelium was higher than the examination at the end of administration and recovery was noted.

Epididymis: Reduction of sperm (mild to severe) and cell debris content of the lumen (mild, moderate) were observed in the 1000 mg/kg/day dose group, reflecting the influence on the testes by administration of the test substance.
At the end of the recovery period, reduction of sperm (moderate) and cell debris content of the lumen (mild, moderate) were seen in the 1000 mg/kg/day dose group. Although it was thought that it was an image reflecting the change of the testes, it was thought that it will disappear with the recovery of the testes in the future.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

The copulation ratio was 100% for control group and each test substance administration group. There was no significant difference in the number of days required for copulation between the control group and each test substance administration group.

Insemination index (males) and fertility index (females) were 100% for control group and for administration doses of 300 mg/kg/day or less. However, in the 1000 mg/kg/day dose group Insemination index (males) was 2/11 (18.2%) and fertility index was 2/10 (20%), with significant difference (p<0.01) from control group.

No significant differences were found between the control group and each test substance-administered group in terms of gestation ratio, pregnancy period, number of corpora lutea, implantation number, implantation ratio, delivery ratio, number of stillborn babies and number of live-borns.

All pregnant animals were delivered on pregnancy day 21 to 23, and the placenta and amniotic membrane were processed normally.

In two female animals all littermate died on 2nd day of lactation in the 300 and 1000 mg/kg/day dose group, respectively. Since these conditions are deteriorated in the histopathologic examination, it is suggested that it was influenced by the administration of the test substance.

In addition, decrease in nursing behaviour was observed from day 2 to 4 of lactation period in 1 animal in the 100 mg/kg/day administration group. This effect is not dose-dependent and thus it was judged as not related with administration of test substance.

Key result

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive performance

Remarks on result:

other: Effects observed at the highest dose tested (1000 mg/kg bw/day)

Key result

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Remarks on result:

other: Effects observed at the dose tested of 300 mg/kg bw/day

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no significant difference in the birth rate and 4-day survival rate between the control group and each test substance-administered group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no significant difference between the control group and each test substance administration group.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

None of the dead pups examined had macroscopic abnormality in control and any treated group.
There was no macroscopic abnormality in any pup examined on postnatal day 4.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There was no significant difference in the sex ratio of liveborns and live pups on day 4 between the control group and each test substance administration group.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Remarks on result:

other: No effects observed at the highest dose tested (1000 mg/kg bw/day)

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Based on the findings of this study, the NOEL for parental reproduction was 300 mg/kg bw/day in males and 100 mg/kg bw/day in females, and the NOEL for development toxicity was 1000 mg/kg bw/day, the highest dose tested.

Executive summary:

A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of terpineol (CAS 8000-41-7) by oral administration in rats was conducted according to OECD guideline 422, in accordance with GLP principles. Terpineol was diluted with corn oil and added to Sprague-Dawley SPF rats (12 per sex per dose) at doses of 0, 100, 300 and 1000 mg/kg bw/day, from day 14 before mating up to 30 days after mating for males (total 44 days) and up to 4 days of lactation for females (total 41-51 days). A non-mating female group (10) was exposed at doses of 0 and 1000 mg/kg bw/day for 44 days. Also, after administration period some animals from the control and high dose groups (5 males of mating group, 5 females of control and 3 females of high dose of non-mating group) were kept for further 14 days without treatment to detect delayed occurrence and recovery from toxic effects.

During the study, data was recorded on clinical condition, performance under detailed physical and arena examination, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, haematology, blood chemistry, urinalysis, oestrous cycles, mating performance and fertility and gestation length. Organ weight, macroscopic and microscopic pathology investigations were undertaken in the adults. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed, and macroscopic pathology investigations were undertaken.

In the 1000 mg/kg/day dose group of the mating group female, 9/12 cases were infertile, 2/12 cases were pregnant, and 1/12 cases died on day 3 pregnancy. Female infertility was considered to be caused by the effects found in histopathology of testis for the same dose group: Atrophy (moderate) of seminiferous tubules, multinucleated giant cells (minor to moderate) and vacuolization of seminiferous tubules (minor, mild). For epididymis, reduction of sperm (mild to severe) and cell debris content of the lumen (mild, moderate) were observed in the 1000 mg/kg/day dose group, reflecting the influence on the testes by administration of the test substance. No oestrous cycle abnormalities were found, and no significant difference was observed between the control group and each test substance administration group in the sexual cycle abnormality rate, development of oestrous stages and the average cycle duration. The copulation ratio was 100% for control group and each test substance administration group. There was no significant difference in the number of days required for copulation between the control group and each test substance administration group. Insemination index (males) and fertility index (females) were 100% for control group and for administration doses of 300 mg/kg/day or less. However, in the 1000 mg/kg/day dose group, insemination index (males) was 2/11 (18.2%) and fertility index was 2/10 (20%), with significant difference (p<0.01) from control group. No significant differences were found between the control group and each test substance-administered group in terms of gestation ratio, pregnancy period, number of corpora lutea, implantation number, implantation ratio, delivery ratio, number of stillborn babies and number of live-borns. In two female animals all littermate died on 2nd day of lactation in the 300 and 1000 mg/kg/day dose group, respectively. Since these conditions were supported by the histopathological examination, it is suggested that it was influenced by the administration of the test substance.

Based on these findings, the NOEL for parental reproduction was established at 300 mg/kg bw/day for males and 100 mg/kg bw/day for females, and the NOEL for development toxicity was determined as the highest dose tested, i.e. 1000 mg/kg bw/day.

Reference 3

Endpoint:

reproductive toxicity, other

Remarks:

(20 week repeated dose toxicity study)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD TG 408

Deviations:

yes

Remarks:

(no data on details of test animals and environmental conditions)

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Osborne-Mendel

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Housing: The animals were housed individually in wire cages
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): ad libitum

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

- DIET PREPARATION
- Rate of preparation of diet (frequency): weekly

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

7% loss of test compound from lab animal diet during a 7-day period.

Duration of treatment / exposure:

20 weeks

Frequency of treatment:

Fresh diets were made and distributed weekly.

Dose / conc.:

10 000 ppm

Remarks:

Equivalent to 400 mg/kg bw/ day (based on food consumption of 40 g/kg bw/day included in the
Guidance on information requirements and chemical safety assessment R8, table R 8 -17)

Dose / conc.:

2 500 ppm

Remarks:

Equivalent to 100 mg/kg bw/ day (based on food consumption of 40 g/kg bw/day included in the
Guidance on information requirements and chemical safety assessment R8, table R 8 -17)

Dose / conc.:

1 000 ppm

Remarks:

Equivalent to 40 mg/kg bw/ day (based on food consumption of 40 g/kg bw/day included in the
Guidance on information requirements and chemical safety assessment R8, table R 8 -17)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes, weekly

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes; At the termination of the experiments the rats were sacrificed and exsanguinated. The tissues of all the rats were examined macroscopically at the time of sacrifice. The viscera were removed and the liver, kidneys, spleen, heart, and testes were weighed. Tissues from rats dying during the experiment were examined for gross changes and were preserved if autolysis was not advanced. Organs were not weighed but abnormalities and the suspected reason for death were noted.

HISTOPATHOLOGY: Yes; Organs weighed at termination, the remaining abdominal and thoracic viscera, and one hind leg, for bone, bone marrow, and muscle, were preserved in 10% buffered formalin-saline solution for histopathological examination. For routine histopathology, sections were embedded in paraffin wax and stained with haematoxylin and eosin. Detailed microscopic examinations were done on 6 or 8 rats, evenly divided by sex, from the high dose group and the control group. If changes attributable to the test compound were found in the high dose group, additional animals on lower dosage levels were examined as indicated.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No microscopic change in the tissues in the 10000 ppm exposure group.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

> 10 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

haematology

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: Equivalent to NOAEL > 400 mg/kg bw.

Key result

Critical effects observed:

no

Dose descriptor:

NOAEL

Remarks on result:

not measured/tested

Key result

Reproductive effects observed:

no

Conclusions:

No effects were seen on male and female gonads after 20-weeks of administration of the test substance.

Executive summary:

In a 20 -weeks oral exposure study, Osborne-Mendel rats (10/dose/sex) were administered alpha-Terpinyl Acetate via diet intake at concentrations of 0 (control), 10000, 2500 and 1000 ppm. Animals were then observed for mortality, weight, food intake and general condition. Haematological examinations were made at termination. At the termination of the experiments the rats were sacrificed and exsanguinated. The tissues of all the rats were examined macroscopically at the time of sacrifice. The viscera were removed and the liver, kidneys, spleen, heart, and testes were weighed. Detailed microscopic examinations was done on the animals from the high dose group and the control group. No effect on growth or haematology, and no macroscopic or microscopic change in the tissues in the 10000 ppm exposure group were observed. No effect on growth or haematology, and no macroscopic change in the tissues in the 2500 and 1000 ppm exposure groups. No microscopic examination was performed on rats exposed to 2500 and 1000 ppm. According to the Guidance on information requirements and chemical safety assessment R8 table R 8 -17, rats eat 40 to 50 g/kg bw/day. 10000 ppm is equivalent to 1% in the diet. Consequently, rats exposed to 10000 ppm in the diet consumed between 400 and 500 mg Terpinyl-Acetate-Alpha per kg bodyweight per day. Therefore, the NOAEL was calculated to be higher than 400 mg/kg bw.

Reference 4

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
According to the column 1 of REACH Annex IX, the extended one generation reproductive toxicity study does not need to be conducted because the available repeated dose toxicity studies do not indicate adverse effects on reproductive organs or tissues nor reveal any other concern in relation with reproductive toxicity.

Reason / purpose for cross-reference:

data waiving: supporting information

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

A key study is available with a Klimisch score of 1.
Also some available supporting studies on components/analogue substances with Klimisch score of 2 have been included in the dossier.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Key study: The test substance Pine oil 50% was tested in a combined repeated dose toxicity study with the reproduction /developmental toxicity screening test according to OECD guideline 422, in accordance with GLP principles. Sprague Dawley rats were administered with test item at the dose levels of 100, 300 and 600 mg/kg body weight previously selected according to the results of a dose range finding study performed with the same species at doses of 100, 300 and 1000 mg/kg bw for a period of 14 days. The treatment of male and female rats with test substance at dose levels up to 600 mg/kg-bw/day by repeated oral (gavage) administration revealed no adverse effects on the reproductive performance or fertility of rats.The No Observed Effect Level (NOEL) for parental general toxicity was determined to be 100 mg/kg bw/day based on treatment related clinical signs of toxicity and reduction in body weight found at the dose of 300 mg/kg bw/day. The No Observed Effect Level (NOEL) for parental reproduction was established at 300 mg/kg bw/day based on the increased copulatory interval and gestation length observed at the high dose tested (600 mg/kg bw/day). However, these effects were found along with other systemic effects (clinical and weight changes) and thus, it is reasonable to consider these reproductive effects as non- specific secondary effects to those systemic effects.

Finally, the No Observed Adverse Effect Level (NOAEL) for parental and reproductive toxicity was determined to be 600 mg/kg bw/day since the treatment related clinical sings of toxicity observed at doses of 300 and 600 mg/kg bw/day were found recovered later during the treatment period in all cases. Furthermore, although increased copulatory interval and reduced gestation length were observed at 600 mg/kg bw/day, no other fertility or reproduction subsequent effects in fertility index, parturition, litter size or live birth index were found and also no treatment related histopathological changes were noted in testes, epididymides, seminal vesicle with coagulating gland, prostate or ovaries at this dose level.

Supporting studies with individual main components/analogues:

A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of terpineol (CAS 8000-41-7) by oral administration in rats was conducted according to OECD guideline 422, in accordance with GLP principles. Terpineol was added to Sprague-Dawley SPF rats (12 per sex per dose) at doses of 0, 100, 300 and 1000 mg/kg bw/day, from day 14 before mating up to 30 days after mating for males (total 44 days) and up to 4 days of lactation for females (total 41-51 days). The NOEL for parental reproduction was established at 300 mg/kg bw/day for males and 100 mg/kg bw/day for females, and the NOEL for development toxicity was determined as the highest dose tested, i.e. 1000 mg/kg bw/day.

In a 20 -weeks oral exposure study, Osborne-Mendel rats (10/dose/sex) were administered alpha-Terpinyl Acetate via diet intake at concentrations of 0 (control), 10000, 2500 and 1000 ppm. The NOAEL was calculated to be higher than 400 mg/kg bw.

Effects on developmental toxicity

Description of key information

Key study: Test method OECD 422. GLP study. The NOAEL of the test substance for parental and developmental toxicity in rats by oral route was determined to be 600 mg/kg bw/day since no adverse effects were observed at the highest dose tested.

Key study: Test method OECD 414. GLP study. The NOAEL for systemic, developmental maternal toxicity and fetal (pre-natal developmental) toxicity is estimated as 900 mg/kg bw/day as no adverse observed effects were found up to the highest dose tested.

Supporting studies: Experimental results from pre-natal developmental studies performed with the components cineole, d-limonene and alpha terpinene are available:

 

Cineole in rats: In a reproductive toxicity study performed by oral route (gavage) with 1,8-cineole using Wistar rats, the results obtained provide evidence that possibly the test substance presents maternal and fetal toxicity, but it is concluded that further studies need to be conducted. The NOAEL was not established since effects in maternal body weight were found at the lowest dose tested (250 mg/kg bw/day).

D-limonene in rats: The NOAEL for maternal toxicity was considered to be 591 mg/kg bw/day based on the deaths and decreased bodyweight gain. The NOAEL for fetal toxicity was considered to be 591 mg/kg bw/day based on the delayed skeletal formation and decreased bodyweight gain.

D-limonene in mice: The NOAEL for maternal and fetal toxicity was considered to be 591 mg/kg bw/day based on the decreased bodyweight gain in dams and increased incidences of abnormal bone formation in fetuses.

D-limonene in rabbits: D-limonene was not teratogenic in rabbit fetuses and the NOAEL for fetal toxicity was considered to be greater than 1000 mg/kg bw/day. The NOAEL for maternal toxicity was considered to be 250 mg/kg bw/day based on the decreased bodyweight gain.

Alpha terpinene in rats: The NOAEL of alpha terpinene for embryofoetal toxicity was determined to be 30 mg/kg body weight by the oral route based on reported signs of delayed ossification and a higher incidence of minor skeletal malformations observed at doses of 60 mg/kg bw or more.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 January 2019 – 21 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals.
- Females (if applicable) nulliparous and non-pregnant: yes
- Weight at study initiation: 377.18-381.03 g (range of average values of each group of males at first day of dosing); 271.24-272.21 g (range of average values of each group of females at first day of dosing)
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with a stainless-steel mesh top grill having facilities for holding pelleted food and drinking water in a water bottle fitted with a stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
Acclimatization: maximum of 3 animals of same sex per cage.
Pre mating: 2 animals of the same sex and group per cage.
Mating: 2 animals (one male and one female) of the same group per cage.
Post mating: After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
Recovery animals: 3 animals of same sex per cage.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the experimental period. Deep bore-well water passed through a Reverse Osmosis Unit was provided in plastic water bottles with stainless-steel sipper tubes.
- Acclimation period: at least 5 days. Females were screened for normal oestrous cycles (4 to 5 days) in a two weeks pre-treatment period before initiation of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2-23.1ºC
- Humidity (%): 47-67 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item was weighed into a clean beaker and a little volume of vehicle was added into the beaker and mixed well with glass rod. The formulation was transferred into a measuring cylinder, the beaker was rinsed with some more amount of vehicle also transferred into the measuring cylinder. This procedure was repeated until the entire quantity of the test item formulation was transferred into the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get the desired concentration for the different dose levels.
Test item formulations were prepared daily before administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is not miscible with distilled water and clearly miscible with corn oil at the concentration of 100 mg/mL as per the in-house miscibility test. Hence, corn oil was selected as vehicle for test item formulation preparations. Corn oil is universally accepted and routinely used vehicle in oral toxicity studies.
- Concentration in vehicle: 10, 30 and 60 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): A1712001

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test item in dose formulations was established in a preliminary study. The test item formulations were stable at room temperature for 6 hours at the concentrations of 0.5 mg/mL and 200 mg/mL in corn oil. However, freshly prepared test item formulations were administered to the animals.
Homogeneity and dose formulation analysis for dose concentration verification was done by a validated analytical method UV-Vis spectrophotometer (study nº BIO-ANM 1305). Sampling and analysis of formulations were performed during week 1 (13-02-2019) and week 5 (08-03-2019) of the treatment. Approximately 10 mL of samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control.
Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was ≤10%.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: No
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- Any other deviations from standard protocol: No

Duration of treatment / exposure:

Main group males: two weeks pre-mating, during mating and up to the day before sacrifice during the post-mating period (total of 36 days of treatment).
Main group females main group: two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.
Recovery animals: same period as for the main group females until the first scheduled sacrifice of dams and kept without treatment for a further 14 days observation.

Frequency of treatment:

Once a day

Duration of test:

Main group Males: 13 February - 21 march 2019
Main group Females: 13 February - 18 April 2019
Recovery Animals: 13 February - 03 April 2019

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

G1 - Vehicle control + G1R - Vehicle Control Recovery Group

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

G2 - Low dose

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

G3 - Mid dose

Dose / conc.:

600 mg/kg bw/day (actual dose received)

Remarks:

G4 - High dose + G4R - High dose Recovery Group

No. of animals per sex per dose:

Main Group: 12
Recovery Group: 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Doses were decided based on the results of a dose range finding study in which the No Observed Effect Level (NOEL) was found to be 100 mg/kg bw/day and Low Observed Effect Level (LOEL) was found to be 300 mg/kg bw/day, as the dose of 100 mg/kg bw/day did not reveal any treatment related effects in either sex, the dose of 300 mg/kg bw/day revealed treatment related clinical signs of toxicity and reduction in body weight and feed consumption and the dose of 1000 mg/kg bw/day revealed treatment related clinical signs of toxicity, mortality, reduction in body weight and feed consumption.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the treatment on day 1 and weekly thereafter during treatment for all the animals. Signs noted were included, but not be limited to, changes in skin, fur, eyes and mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.

BODY WEIGHT: Yes
- Time schedule for examinations: The main group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The main group females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period. The recovery group animals (both males and females) were weighed at receipt, on the first day of dosing, weekly thereafter and at termination.

FOOD CONSUMPTION: yes
-feed consumption was measured for main group animals (both males and females) once a week during premating and weekly once for main group males during the post mating period. Feed consumption was not measured during the mating period for both males and females. Thereafter, feed consumption for main group females was recorded during gestation days 0 to 7, 7 to 14 and 14 to 20 and on lactation days 1 to 4, 4 to 7 and 7 to 13. Feed consumption was measured for recovery group animals (both males and females) once a week throughout the experimental period. Average feed intake per rat (g/rat/day) was calculated using the amount of feed given and left over in each cage and the number of rats surviving in each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once before treatment for all animals, during end of the dosing period for main group males (on day 34) and during lactation period for main group females (on lactation day 13) of vehicle control and high dose main group animals and during last week (day 64) for all recovery group animals (both males and females).
- Dose groups that were examined: vehicle control and high dose main group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for males after completion of 36 days of treatment and for females on lactation day 14).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 males and 5 females randomly selected from each main group and all recovery group animals.
- Parameters checked: Haemoglobin concentration (HGB), Haematocrit (HCT), Erythrocyte count (RBC), Total leukocyte count (WBC), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLT), Mean platelet volume (MPV), Reticulocyte count (Retic), Absolute reticulocyte count, Differential leucocytes count (DLC), Absolute differential leucocytes count (DLC). Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated by an "OptiClot-4 coagulation analyzer".

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for males after completion of 36 days of treatment and for females on lactation day 14).
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 males and 5 females randomly selected from each main group and all recovery group animals.
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Cholinesterase, Total Protein, Albumin, Total bilirubin, Glucose, Total Cholesterol, Creatinine, Urea, Blood urea nitrogen (BUN), Triglycerides, Phosphorous, Calcium, Globulin. Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using Prolyte Na/K/Cl analyzer (Diamond Diagnostics).

URINALYSIS: Yes
- Time schedule for collection of urine: on the day of scheduled terminal sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 randomly selected males of each main group and all recovery animals.
- Parameters checked: Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Microalbumin, Leucocytes. In addition pH, nitrite and specific gravity were also analyzed. After analysis of the above parameters, the urine was subjected for centrifugation at 1500 rpm for 3 minutes. Then the urine was subjected for microscopic examination for urine sediments.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Towards the end of the dosing period for males (on day 36) and during lactation period for females (on lactation day 13). Towards the end of the recovery period (shortly prior to scheduled sacrifice on day 66) for the recovery group.
- Dose groups that were examined: all main and recovery groups.
- Battery of functions tested: Home Cage Observations (convulsions, tremors and palpebral closure), Handling Observations, Open Field Observations, Sensory Observations, Neuromuscular Observations, Physiological Observation (Rectal temperature), Grip strength assessment and Motor activity assessment.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on lactation day 14 (dams)
- Organs examined: see table 1

OTHER:
Thyroxine Hormone (T4) Level estimation: Blood samples were collected for measurement of serum T4 levels on the following schedule:
a. Two pups per litter on lactation day 4 based on the following conditions:
- Two female pups in order to retain more male pups for nipple retention on PND 13.
- No pups were eliminated when litter size dropped below 10 pups/litter.
- Only one pup was eliminated and used for blood collection in case of only one pup was available above ten.
b. All main group females (dams) at termination (lactation day 14).
c. Two pups per litter (same sex) at termination (lactation day 13).
d. All adult main group males, at termination (after completion of 36 days of treatment).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes.
- Number of implantations: Yes.
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
Ovaries and uterus were examined for histopathology.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: No data
- Head examinations: No data

Statistics:

After verification, the data was subjected to statistical analysis using SPSS software, version 22. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05). The statistical analysis was followed to the parameters as mentioned in the table 3 below.

Indices:

See table 2 below.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs of toxicity observed at groups G1/G1R and G2 animals of either sex throughout the experimental period.
The groups G3 and G4/G4R animals of either sex did not reveal any clinical signs of toxicity for the first three days of the treatment period. Thereafter, slight wet perineum was noted during several occasions in animals of either sex of these dose groups and found recovered later during the treatment period.
The detailed clinical examination revealed treatment related wet perineum and perinasal staining at groups G3 and G4/G4R during treatment period and found recovered later in the study.
These observed clinical signs at both the dose group animals of G3 and G4/G4R are considered as treatment related due to reduced mean body weight, percent change in mean body weight in either sex during these periods and also the observations are dose dependant when compared with control group.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no mortality/morbidity observed at any dose group during the experimental period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment related changes noted in mean body weight and percent change in body weight with respect to day 1 at group G2 when compared with vehicle control group animals of either sex during the experimental period. However, statistically significant reduction in percent change in mean body weight with respect to day 1 at group G2 animals of either sex was noted. This change is considered as incidental due to lack of consistency and no clinical signs were noted and no effects were noted in feed consumption during this period when compared with control group.
In groups G3 and G4 animals, statistically significant reduction in percent change in body weight with respect to day 1 on days 7, 14, 21, 28 and 35 in males and days 7 and 14 in females was noted.
In group G4R animals, statistically significant reduction in percent change in body weight with respect to day 1 on days 7, 14, 28 and 35 in males, and reduction in mean body weight on day 28 and 35 and percent change in body weight with respect to day 1 during the entire experimental period in females, was noted.
The observed changes at these dose levels [300 mg/kg and 600 mg/kg] are considered as treatment related due to occurrence of treatment related clinical signs of toxicity and also the observed changes are dose dependant and consistent during the experimental period.
There were no changes observed in the gestation body weight and percent change in body weight during gestation period at G2 and G3 dose group animals. A slight reduction in mean gestation body weight was noted on GD 0, 7, 14 and 20 at group G4 animals when compared with other dose groups and vehicle control group. This observation can be considered as treatment related due to significant reduction in mean body weight at this dose group during pre-mating period and also due to occurrence of clinical signs of toxicity during treatment period.
There were no changes observed in the lactation body weight and percent change in body weight during lactation period at groups G2 and G3 when compared with control group animals. A slight reduction in mean lactation body weight was noted throughout the lactation period and the reduction is statistically significant during lactation day 1 to 4 at group G4 animals when compared with other dose groups and vehicle control group. This observation can be considered as treatment related due to significant reduction in mean body weight at this dose group during pre-mating period, gestation period and also due to occurrence of clinical signs of toxicity during treatment period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Statistically significant reduction in mean feed consumption was noted during pre-mating period (week 1) of main group animals in either sex at all the tested dose groups when compared with vehicle control group. This occurrence is considered as incidental at group G2 and G3 of either sex as the mean feed consumption was comparable with vehicle control group during week 2. However, the reduction at group G4 can be considered as treatment related as the reduction was continued during week 2 also and this effect is correlated with reduction in percent change in body weight at this dose level.

There were no treatment related changes observed in the mean gestation feed consumption at group G2 and G3 when compared with vehicle control group. A slight reduction in mean gestation feed consumption was noted during GD 0 to 7, 7 to 14 and 14 to 20 at group G4 when compared with other dose groups and vehicle control group. This observation can be considered as treatment related due to consistency in reduction of feed consumption at this dose group during pre-mating period and also due to occurrence of clinical signs of toxicity during treatment period.

There were no treatment related changes observed in the feed consumption during lactation period at all the tested dose groups when compared with control group animals.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ocular changes observed in G1 and G4 group animals of either sex during the ophthalmological examination conducted towards end of the dosing period and no ocular changes observed in G1R and G4R group animals of either sex during the ophthalmological examination conducted at the end of recovery period.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant changes were noted in haematology parameters when compared with their concurrent control groups: increase in activated prothrombin time at group G4 males; decrease in haemoglobin at group G2 females; decrease in total leucocyte count, absolute lymphocytes and activated prothrombin time at group G4R males and increase in mean platelet volume at group G4R males. These changes are considered as incidental and not treatment related, due to lack of dose dependency and similar changes were not observed in other sex.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant decrease in total bilirubin at group G3 males and statistically significant decrease in triglycerides at group G4R males were noted when compared with their concurrent control groups. These changes are considered as incidental and not treatment related, due to lack of dose dependency and similar changes were not observed in other sex.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no changes observed in urinalysis parameters at any of the tested dose main group males and tested dose group recovery group animals of either sex conducted at termination.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes noted in neurological/functional examinations like home cage, handling, open field, sensory, neuromuscular, physiological observations and assessment of grip strength and motor activity at all the tested dose group animals of either sex from both main and recovery group animals when compared with concurrent vehicle control group animals.
However, statistically significant increase in mean number of rearing during open field examination was noted at groups G2 and G4 females when compared with vehicle control group females. This change is considered as incidental but not treatment related due to lack of dose dependency and no effects were noted in other functional observation battery parameters at these dose levels.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes observed in the absolute and relative organ weights at any of the tested dose group animals of either sex in both main and recovery group animals.
However, statistically significant increase in absolute and relative liver weight at group G4, statistically significant decrease in absolute seminal vesicles with coagulating glands at group G3 males; statistically significant decrease in absolute heart weight at group G4R females were noted when compared with concurrent vehicle control group. These changes are considered as incidental and not treatment related as the observations do not occur in dose dependant manner, are not found in both sexes and also no gross or histopathological changes were noted in these organs at this dose level.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes observed during necropsy at any of the main and recovery group animals of either sex.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related histopathological findings were noticed in this study.
Lesions considered to be spontaneous and incidental were observed in treated group and control group animals. These lesions consisted of interstitial mononuclear cell infiltrate and concretions in prostate gland, and the presence of calculi in kidney.
1 female from high dose group G4 revealed mononuclear cells infiltration at sub-urothelium of kidney. This lesion was considered spontaneous because of lack of consistency.
3 males from G4 group revealed presence of concretions in prostate gland. Inflammation and concretions of the prostate gland is common background finding in rats and mice, which increases with age (Dianne C., et.al, 2012).
Females from vehicle control group and high dose group revealed presence of placental scar characterized by brown-pigmented nodules at the uterine-mesometrial boundary. The presence of discrete pigmented nodules along the uterine-mesometrial boundary was reported. Each nodule overlies a site of placental detachment at parturition, or the site of the resorption of a feto-placental unit or decidualized implantation site. These nodules were often called placental scars and may persist for many months postpartum, perhaps even the lifespan of a rodent (Janet M., 1990).
Presences of ultimobranchial cyst or ectopic thymus in thyroid were congenital lesions and it does not have toxicological significance.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Serum Thyroxine (T4) Levels: There were no treatment related changes in serum Thyroxine (T4) hormone levels noted at all the tested dose group males. However, statistically significant decrease in T4 levels of males at group G4 was noted when compared with vehicle control group. This change is considered as incidental and not treatment related as the observations do not occur in dose dependant manner and also the values obtained are within historical control range. As no treatment related changes were noted in males the assessment of T4 in blood samples from the dams was not performed.

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions ocurred in any dose group.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No pre or post-implantation losses were observed during the study.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No resorptions were detected at any dose group during the study.

Early or late resorptions:

no effects observed

Description (incidence and severity):

No resorptions were detected at any dose group during the study.

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead pups were counted at any dose group during the study.

Changes in pregnancy duration:

effects observed, treatment-related

Description (incidence and severity):

There were no changes observed in the gestation length at groups G2 and G3 when compared with vehicle control group. However, a treatment related statistically significant reduction in mean gestation length at group G4 was noted when compared with vehicle control group. This observation is considered as treatment related due to observed clinical signs during the treatment period at this dose level.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There were no statistically significant differences observed in the number of dams between the treated groups and the vehicle control group.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

There were no changes observed in the copulatory interval and number of conceiving days at groups G2 and G3 when compared with vehicle control group. However, an increased copulatory interval was noted at group G4 when compared with vehicle control group. This change is considered as treatment related due to occurrence of treatment related clinical signs and reduced mean body weights at this dose group during the treatment period.

There were no changes (irregularities) observed in the oestrus cyclicity of females at any of the tested dose group females during pre-mating treatment, mating treatment and on lactation day 14 of dams.

Key result

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: parental general (systemic) toxicity

Key result

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

changes in pregnancy duration

other: Changes in the copulatory interval and number of conceiving days

Remarks on result:

other: Increased copulatory interval and gestation length observed at high dose tested (600 mg/kg bw/day).

Key result

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal general and developmental toxicity

Remarks on result:

other: No adverse effects were found at any dose tested.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes observed in mean pup weight of either sex during lactation period at all the tested dose groups when compared with vehicle control group. However, statistically significant increase in mean male and female pup weight on lactation day 4 at groups G2 and G4; statistically significant increase in mean female pup weight on LD 7 at group G4 were noted when compared with vehicle control. These changes are considered incidental and not treatment related as the obtained weights are within historical control data and also the changes are not dose dependant.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no statistically significant differences observed in the live birth index (Number of pups born alive / Total number of pups born X 100) between the treated groups and the vehicle control group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no statistically significant differences observed in the sex ratio of pups between the treated groups and the vehicle control group.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no statistically significant differences observed in the litter size (mean number of pups delivered per dam) between the treated groups and the vehicle control group.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

There were no statistically significant differences observed in pup survival from lactation day 1 to 13 between the treated groups and the vehicle control group.

External malformations:

no effects observed

Description (incidence and severity):

No external abnormalities were detected in any of the pups sacrificed.

Skeletal malformations:

not specified

Visceral malformations:

no effects observed

Description (incidence and severity):

No visceral abnormalities were detected in any of the pups sacrificed.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Serum Thyroxine (T4) Levels: There were no effects found in serum Thyroxine (T4) hormone levels noted at lactation day 13 pups. As no effects were noted in lactation day 13 pups the assessment of T4 in blood samples from the day 4 pups was not performed.
Anogenital Distance of Pups on Lactation Day 4: There were no treatment related changes observed in mean pup ano-genital distance ratio of either sex during measured on LD 4 at all the tested dose groups when compared with vehicle control group. However, statistically significant decrease in mean male and female mean pup ano-genital distance ratio at groups G3 and G4 was noted when compared with vehicle control. These changes cannot be considered as treatment related effects as the obtained values are within historical control range and also no treatment related effects noted in mean pup weight on the day of measurement of ano-genital distance.
Nipple Retention in Male Pups on Lactation Day 13: There were no occurrences of nipples in male pups at any of the tested doses on lactation day 13.

Key result

Dose descriptor:

NOEL

Effect level:

600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Remarks on result:

other: No treatment related effects were observed at the highest dose tested (600 mg/kg bw/day)

Key result

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Remarks on result:

other: No adverse effects were observed at any dose level tested.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 4. Summary of vaginal smear examination for determination of oestrus cyclicity

Group & Dose (mg/kg body weight/day)	Total No. of Females	No. of Females with Regular Oestrus Cyclicity during Pre-mating and Mating	No. of Females confirmed with pregnancy	No. of Females with Regular Oestrus Cyclicity on Lactation Day 14	No. of Females with Irregular Oestrus Cyclicity during Pre-mating, Mating and on Lactation day 14
G1 & 0	12	12	10	10	0
G2 & 100	12	12	11	11	0
G3 & 300	12	12	10	10	0
G4 & 600	12	12	10	10	0

Table 5. Summary of cohabitation record

Group & Dose (mg/kg body weight)		Copulatory Interval (Days)		Conceiving Days (1 to 5)	Conceiving Days (6 to 14)
G1 & 0	Mean	8.17	n	5	7
	±SD	6.37
	n	12	%	41.7	58.3
G2 & 100	Mean	8.50	n	4	8
	±SD	5.96
	n	12	%	33.3	66.7
G3 & 300	Mean	8.58	n	5	7
	±SD	6.14
	n	12	%	41.7	58.3
G4 & 600	Mean	10.42	n	3	9
	±SD	5.81
	n	12	%	25.0	75.0

Table 6. Summary of gestation length and delivery data record

Group & Dose (mg/kg body weight)		Gestation Length (Days)	Litter Size (No.)	Live Pups (No.)			Dead Pups (No.)			Sex Ratio (M/F) at Birth	Live Birth Index (%)
				Total (No.)	Male (No.)	Female (No.)	Total (No.)	Male (No.)	Female (No.)
G1 & 0	Mean	22.20	11.60	11.60	4.50	7.10	0.00	0.00	0.00	0.83	100.00
	±SD	0.42	3.31	3.31	2.76	2.64	0.00	0.00	0.00	0.91	0.00
	n	10	10	10	10	10	10	10	10	10	10
G2 & 100	Mean	22.73	10.82	10.82	6.09	4.73	0.00	0.00	0.00	1.55	100.00
	±SD	0.65	2.52	2.52	3.18	1.68	0.00	0.00	0.00	1.10	0.00
	n	11	11	11	11	11	11	11	11	11	11
G3 & 300	Mean	22.40	12.00	12.00	6.10	5.90	0.00	0.00	0.00	1.33	100.00
	±SD	0.52	2.31	2.31	2.47	2.33	0.00	0.00	0.00	1.06	0.00
	n	10	10	10	10	10	10	10	10	10	10
G4 & 600	Mean	23.40*	10.10	10.10	5.70	4.40	0.00	0.00	0.00	1.47	100.00
	±SD	0.70	2.02	2.02	1.77	1.35	0.00	0.00	0.00	0.83	0.00
	n	10	10	10	10	10	10	10	10	10	10

n: Number of dams

* Statistically significant (P<0.05) change than the vehicle control group.

Table 7.1. Summary of litter observation record during lactation period. LD1 to 4

Group & Dose (mg/kg body weight)		No. of Live Pups At Birth	During LD 1 to 4							Sex Ratio (M/F) at LD 4	No. of Survived Pups during LD 1 to 4	Pup Survival Index (%) LD1 to 4
			Live Pups (No.)				Dead Pups (No.)
			Total (No.)	Male (No.)	Female (No.)		Total (No.)	Male (No.)	Female (No.)
G1 & 0	Mean	11.60	11.60	4.50	7.10		0.00	0.00	0.00	0.83	11.60	100.00
	±SD	3.31	3.31	2.76	2.64		0.00	0.00	0.00	0.91	3.31	0.00
	n	10	10	10	10		10	10	10	10	10	10
G2 & 100	Mean	10.82	10.82	6.09	4.73		0.00	0.00	0.00	1.55	10.82	100.00
	±SD	2.52	2.52	3.18	1.68		0.00	0.00	0.00	1.10	2.52	0.00
	n	11	11	11	11		11	11	11	11	11	11
G3 & 300	Mean	12.00	12.00	6.10	5.90		0.00	0.00	0.00	1.33	12.00	100.00
	±SD	2.31	2.31	2.47	2.33		0.00	0.00	0.00	1.06	2.31	0.00
	n	10	10	10	10		10	10	10	10	10	10
G4 & 600	Mean	10.10	10.10	5.70	4.40		0.00	0.00	0.00	1.47	10.10	100.00
	±SD	2.02	2.02	1.77	1.35		0.00	0.00	0.00	0.83	2.02	0.00
	n	10	10	10	10		10	10	10	10	10	10

Table 7.2. Summary of litter observation record during lactation period. LD4 to 7

Group & Dose (mg/kg body weight/day)		Live Pups (No.) on LD 4				Pups Sacrificed for Blood Collection on LD 4 (No.)				Live Pups (No.) on LD 4 after Sacrificed for Blood Collection			During LD 4 to 7						Sex Ratio (M/F) at LD 7	No. of Survived Pups during LD 4 to 7	Pup Survival Index (%) during LD 4 to 7
													Live Pups (No.)			Dead Pups (No.)
		Male (No.)	Female (No.)	Total (No.)		Male (No.)	Female (No.)	Total (No.)		Male (No.)	Female (No.)	Total (No.)	Male (No.)	Female (No.)	Total (No.)	Male (No.)	Female (No.)	Total (No.)
G1 & 0	Mean	4.50	7.10	11.60		0.00	1.30	1.30		4.50	5.80	10.30	4.50	5.80	10.30	0.00	0.00	0.00	1.57	10.30	100.00
	±SD	2.76	2.64	3.31		0.00	0.95	0.95		2.76	2.39	2.54	2.76	2.39	2.54	0.00	0.00	0.00	2.70	2.54	0.00
	n	10	10	10		10	10	10		10	10	10	10	10	10	10	10	10	10	10	10
G2 & 100	Mean	6.09	4.73	10.82		0.00	1.18	1.18		6.09	3.55	9.64	6.09	3.55	9.64	0.00	0.00	0.00	3.31	9.64	100.00
	±SD	3.18	1.68	2.52		0.00	0.98	0.98		3.18	2.16	1.69	3.18	2.16	1.69	0.00	0.00	0.00	3.65	1.69	0.00
	n	11	11	11		11	11	11		11	11	11	11	11	11	11	11	11	11	11	11
G3 & 300	Mean	6.10	5.90	12.00		0.00	1.50	1.50		6.10	4.40	10.50	6.10	4.40	10.50	0.00	0.00	0.00	2.47	10.50	100.00
	±SD	2.47	2.33	2.31		0.00	0.71	0.71		2.47	2.22	1.84	2.47	2.22	1.84	0.00	0.00	0.00	3.19	1.84	0.00
	n	10	10	10		10	10	10		10	10	10	10	10	10	10	10	10	10	10	10
G4 & 600	Mean	5.70	4.40	10.10		0.00	0.80	0.80		5.70	3.60	9.30	5.70	3.60	9.30	0.00	0.00	0.00	1.79	9.30	100.00
	±SD	1.77	1.35	2.02		0.00	0.92	0.92		1.77	1.07	1.25	1.77	1.07	1.25	0.00	0.00	0.00	0.88	1.25	0.00
	n	10	10	10		10	10	10		10	10	10	10	10	10	10	10	10	10	10	10

Table 7.3. Summary of litter observation record during lactation period. LD7 to 13

Group & Dose (mg/kg body weight)		Live Pups (No.) on LD 7				During LD 7 to 13							Sex Ratio (M/F) at LD 7	No. of Survived Pups during LD 7 to 13	Pup Survival Index (%) during LD 7 to 13
						Live Pups (No.)				Dead Pups (No.)
		Male (No.)	Female (No.)	Total (No.)		Male (No.)	Female (No.)	Total (No.)		Male (No.)	Female (No.)	Total (No.)
G1 & 0	Mean	4.50	5.80	10.30		4.50	5.80	10.30		0.00	0.00	0.00	1.57	10.30	100.00
	±SD	2.76	2.39	2.54		2.76	2.39	2.54		0.00	0.00	0.00	2.70	2.54	0.00
	n	10	10	10		10	10	10		10	10	10	10	10	10
G2 & 100	Mean	6.09	3.55	9.64		6.09	3.55	9.64		0.00	0.00	0.00	3.31	9.64	100.00
	±SD	3.18	2.16	1.69		3.18	2.16	1.69		0.00	0.00	0.00	3.65	1.69	0.00
	n	11	11	11		11	11	11		11	11	11	11	11	11
G3 & 300	Mean	6.10	4.40	10.50		6.10	4.40	10.50		0.00	0.00	0.00	2.47	10.50	100.00
	±SD	2.47	2.22	1.84		2.47	2.22	1.84		0.00	0.00	0.00	3.19	1.84	0.00
	n	10	10	10		10	10	10		10	10	10	10	10	10
G4 & 600	Mean	5.70	3.60	9.30		5.70	3.60	9.30		0.00	0.00	0.00	1.79	9.30	100.00
	±SD	1.77	1.07	1.25		1.77	1.07	1.25		0.00	0.00	0.00	0.88	1.25	0.00
	n	10	10	10		10	10	10		10	10	10	10	10	9

Table 8.1. Summary of pup observation record during lactation period. At birth

Parameter ↓	Group & Dose (mg/kg body weight)
	G1 & 0	G2 & 100	G3 & 300	G4 & 600
No. of Females with Live Pups at Birth	10	11	10	10
No. of Females Confirmed as Non-Pregnant	2	1	2	2
Litter Size (No.)	116	119	120	101
No. of Dead Pups	0	0	0	0
No. of Cannibalized Pups	0	0	0	0
No. of Live Pups at Birth	116	119	120	101
No. of Live Pups with No Abnormality Detected (No.)	116	119	120	101

Table 8.2. Summary of pup observation record during lactation period. LD1 to 4

Parameter ↓	Group & Dose (mg/kg body weight)
	G1 & 0	G2 & 100	G3 & 300	G4 & 600
No. of Females with Live Pups on LD 4	10	11	10	10
No. of Dead Pups during LD 1 to 4	0	0	0	0
No. of Cannibalised Pups during LD 1 to 4	0	0	0	0
No. of Live Pups during LD 1 to 4 (No.)	116	119	120	101
No. of Live Pups with No Abnormality Detected during LD 1 to 4	116	119	120	101
No. of Pups Sacrificed on LD 4 for blood collection	13	13	15	8

Table 8.3. Summary of pup observation record during lactation period. LD4 to 7

Parameter ↓	Group & Dose (mg/kg body weight)
	G1 & 0	G2 & 100	G3 & 300	G4 & 600
No. of Females with Live Pups on LD 7	10	11	10	10
No. of Dead Pups during LD 4 to 7	0	0	0	0
No. of Cannibalised Pups during LD 4 to 7	0	0	0	0
No. of Live Pups during LD 4 to 7	103	106	105	93
No. of Live Pups with No Abnormality Detected during LD 4 to 7	103	106	105	93

Table 8.4. Summary of pup observation record during lactation period. LD7 to 13

Parameter ↓	Group & Dose (mg/kg body weight)
	G1 & 0	G2 & 100	G3 & 300	G4 & 600
No. of Females with Live Pups on LD 13	10	11	10	10
No. of Dead Pups during LD 7 to 13	0	0	0	0
No. of Cannibalised Pups during LD 7 to 13	0	0	0	0
No. of Live Pups during LD 7 to 13	103	106	105	93
No. of Live Pups with No Abnormality Detected during LD 7 to 13	103	106	105	93

Table 9. Summary record of mean pup weight (g) during lactation period

Group & Dose (mg/kg body weight)		Mean Pup Weight (g) on LD1			Mean Pup Weight (g) on LD 4			Mean Pup Weight (g) on LD 7			Mean Pup Weight (g) on LD 13
		Male	Female		Male	Female		Male	Female		Male	Female
G1 & 0	Mean	6.93	6.51		11.16	10.96		15.16	14.68		25.22	24.57
	±SD	0.35	0.50		0.31	0.41		0.68	0.87		0.38	0.44
	n	9	10		9	10		9	10		9	10
G2 & 100	Mean	6.98	6.64		11.74*	11.57*		15.44	14.89		25.11	24.92
	±SD	0.35	0.42		0.40	0.48		0.45	0.61		0.24	0.63
	n	11	11		11	11		11	11		11	11
G3 & 300	Mean	6.87	6.46		11.64	11.21		15.42	14.92		25.30	24.87
	±SD	0.23	0.26		0.35	0.41		0.35	0.41		0.27	0.44
	n	10	10		10	10		10	10		10	10
G4 & 600	Mean	6.71	6.37		11.82*	11.49*		15.53	15.43*		25.13	24.71
	±SD	0.19	0.26		0.63	0.48		0.62	0.54		0.48	0.92
	n	10	10		10	10		10	10		10	10

n: Number of dams

* Statistically significant (P<0.05) change than the vehicle control group.

Table 10. Summary of mean pup anogenital distance ratio record

Group & Dose (mg/kg body weight)	AGD Ratio
		Mean Male AGD Ratio	Mean Female AGD Ratio
G1 & 0	Mean	2.10	1.18
	±SD	0.09	0.06
	n	9	10
G2 & 100	Mean	2.04	1.12
	±SD	0.07	0.05
	n	11	11
G3 & 300	Mean	1.98*	1.10*
	±SD	0.05	0.04
	n	10	10
G4 & 600	Mean	1.95*	1.10*
	±SD	0.04	0.06
	n	10	10

n: Number of dams

* Statistically significant (P<0.05) change than the vehicle control group.

Table 11. Summary of male pup nipple/areolae retention (no.) record

Group & Dose (mg/kg body weight)		No. of Nipples/Areolae on Lactation Day 13
G1 & 0	Mean	0.00
	±SD	0.00
	n	10
G2 & 100	Mean	0.00
	±SD	0.00
	n	11
G3 & 300	Mean	0.00
	±SD	0.00
	n	10
G4 & 600	Mean	0.00
	±SD	0.00
	n	10

n: Number of dams

Table 12. Summary of uteri observation record

Group & Dose (mg/kg body weight)		No. of Corpora lutea	No. of Implantations	Implantation Index (%)	Pre-Implantation Loss (%)	Post-Implantation Loss (%)	Pre-natal Loss (No.)	Post-natal Loss (At birth to LD 13) (%)	Post-natal Loss (At birth to LD 13) (No.)	No. of Early Resorptions	No. of Late Resorptions
G1 & 0	Mean	11.60	11.60	100.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	±SD	3.31	3.31	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	n	10	10	10	10	10	10	10	10	10	10
G2 & 100	Mean	10.82	10.82	100.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	±SD	2.52	2.52	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	n	11	11	11	11	11	11	11	11	11	11
G3 & 300	Mean	12.00	12.00	100.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	±SD	2.31	2.31	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	n	10	10	10	10	10	10	10	10	10	10
G4 & 600	Mean	10.10	10.10	100.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	±SD	2.02	2.02	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	n	10	10	10	10	10	10	10	10	10	10

Conclusions:

The NOAEL of the test substance for parental and developmental toxicity in rats by oral route was determined to be 600 mg/kg bw/day since no adverse effects were observed at the highest dose tested.

Executive summary:

A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of the test substance Pine oil 50% by oral administration in rats was conducted according to OECD guideline 422, in accordance with GLP principles. A total of 116 (58 males + 58 females) Sprague Dawley rats were distributed to four main groups and two recovery groups to detect delayed occurrence and recovery from toxic effects. Each main group (G1, G2, G3 and G4) consisted of 12 males and 12 females and each recovery group (G1R and G4R) consisted of 5 males and 5 females. The animals in G1/G1R group were administered with vehicle (Corn oil), and the animals in G2, G3 and G4/G4R groups were administered with test item at the dose levels of 100, 300 and 600 mg/kg body weight based on the results of a dose range finding study performed with the same species at doses of 100, 300 and 1000 mg/kg bw for a period of 14 days. The test item was administered to the main group males for a period of 36 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period), to the main group females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 and to the recovery group animals for a period of 50 days with a 14 days recovery. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight. The stability of test item formulations in corn oil was established before initiation of the treatment. Dose formulation analysis for homogeneity and concentration verification was performed during weeks 1 and 5 of the treatment period and the results were within acceptable limits.

All the main group and recovery group animals were observed for clinical signs, mortality and morbidity, detailed clinical examination, body weight, feed consumption and ophthalmological and neurological/functional examinations. The clinical pathological (haematology, clinical chemistry and urinalysis) examinations were conducted for 5 randomly selected animals from each group per sex for main group and from all the animals for recovery group at termination. The gross pathology and organ weighing were performed on the day of termination for all the main and recovery group animals. All the main group females were observed for maternal body weight and feed consumption during gestation and lactation. The main group females were evaluated for oestrus cyclicity. Each dam was observed for mating performance, fertility performance, gestation length, litter size, number and percentage of live/dead pups, live birth index, sex ratio and observation of litter throughout the lactation period. Histopathological examination was conducted on all the tissues collected from the main group vehicle control and high dose group animals sacrificed at termination. The pups were observed once daily for clinical signs and external examinations, weighed individually on postnatal day (PND) 1, 4, 7 and 13, measured for anogenital distance on PND 4, observed for retention of any nipples/areolae in male pups on PND 13 and observed for gross pathological observations at termination. The analysis of thyroxine hormone (T4) levels in serum collected at termination was performed for main group males and PND 13 pups.

There were no clinical signs, no mortality/morbidity, no effects of test item on mean body weight, feed consumption, no changes in ophthalmological examination and neurological/functional examination, clinical pathology, organ weights and gross pathology noted at group G2 of either sex. The group G3 and G4/G4R animals of either sex revealed treatment related wet perineum and recovered later in the study. A treatment related reduction in body weight was noted at both G3 and G4/G4R groups of either sex. No treatment related changes were noted in clinical pathology and organ weights and no gross pathological changes were noted at group G3 and G4/G4R. There were no treatment related changes in oestrus cyclicity, maternal and lactation body weight and feed consumption, mating performance, fertility performance, gestation length, litter size, number and percentage of live/dead pups, live birth index, sex ratio and observation of litter at group G2 and G3. A treatment related reduction in mean body weight and feed consumption during gestation and lactation periods and increased gestation length was noted at group G4. There were no treatment related changes occurred in histopathological examination conducted for group G4 animals in either sex. There were no clinical signs and no external abnormalities noted in pups from all the tested dose groups. No treatment related changes in mean pup weight and anogenital distance noted at all the tested dose groups. The gross pathology conducted for the pups at all the tested dose groups revealed no changes. No occurrences of nipples/areolae in male pups from all the dose groups observed on PND 13.There were no treatment related changes noted in serum thyroxine hormone (T4) levels.

The No Observed Effect Level (NOEL) for parental general toxicity was determined to be 100 mg/kg bw/day based on treatment related clinical signs of toxicity and reduction in body weight found at the dose of 300 mg/kg bw/day. The No Observed Effect Level (NOEL) for maternal developmental toxicity was established at 300 mg/kg bw/day based on the increased copulatory interval and gestation length observed at the high dose tested (600 mg/kg bw/day). The No Observed Effect Level (NOEL) for developmental toxicity was established at 600 mg/kg bw/day based no effects found in offspring at any dose tested.

Finally, the No Observed Adverse Effect Level (NOAEL) for parental and developmental toxicity was determined to be 600 mg/kg bw/day as no adverse effects were found on maternal or developmental toxicity at any dose level tested.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Test method equivalent to OECD guideline 414

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

(2 treated groups with fewer than 16 pregnant dams)

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Oswaldo Cruz Central Animal House breeding stock.
- Age at study initiation: no data
- Weight at study initiation: 227 ± 22 to 240 ± 23 g
- Housing: animals were housed in standard plastic cages with stainless-steel cover lids and wood shavings as bedding.
- Diet (e.g. ad libitum): pelleted diet (Nuvital ®, Nuvilab Ltd, Curitiba, PR, Brazil), ad libitum.
- Water (e.g. ad libitum): Tap water, ad libitum.
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1ºC
- Humidity (%): 70 %
- Photoperiod: dark/light cycle (lights on from 10.00 hr to 22.00 hr).

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

(Mazola ®)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
No info.

VEHICLE
- Concentration in vehicle: no info.
- Amount of vehicle (if gavage): 3.75 g/kg bw

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: Mating was performed by transferring two females to the cage of one male for 2 hr (08.00-10.00 hr).
- M/F ratio per cage: 1 male/2 female
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

From the 6th to 15th day of pregnancy

Frequency of treatment:

Daily

Duration of test:

21 days (from day 0 to day 21 of pregnancy)

Dose / conc.:

30 mg/kg bw/day

Dose / conc.:

60 mg/kg bw/day

Dose / conc.:

125 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

No. of animals per sex per dose:

Pregnant female rats per group: 24 (control), 14 (30 mg/kg bw/d), 18 (60 mg/kg bw/d), 25 (125 mg/kg bw/d) and 15 (250 mg(kg bw/d)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Not specified.

Maternal examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed on days 0, 6 up to 15 and 21 of pregnancy.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: On day 21 of pregnancy the female rats were anaesthetized with ethyl ether inhalation and killed by decapitation. The gravid uterus was weighed with its contents.

Ovaries and uterine content:

The uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes.
- Number of corpora lutea: Yes.
- Number of implantations: Yes. The number of implantation sites was determined by the method of Salewski (1964).
- Number of resorptions: Yes.
- Other: Sex ratio, weight and viability of fetuses were determined.

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [1/3 per litter]. By a microsectioning technique adapted from Sterz (1977). Heart, lungs, thymus, spleen, liver and kidneys of foetuses, which were microdissected, were also weighed.
- Skeletal examinations: Yes: [2/3 per litter]. According to the method of Dawson.

Statistics:

Data were evaluated by one-way analysis of variance or, alternatively, by the Kruskal-Wallis test whenever the data did not fit a normal distribution. Differences between groups were tested by the two-sided Student's t-test or Mann-Whitney U-test. Proportions were analysed by the chi-square test or Fischer's exact test. Statistical evaluation was performed using a MINITAB program (MTB, University of Pennsylvania, 1984), and a difference was considered significant at P < 0.05.

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality:

not specified

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was no statistically significant difference in pregnancy weight gain between the control and the groups treated with 30 and 60 mg TER/kg body weight but marked reductions in weight gain during the treatment period (days 6-15) were observed in rats exposed to the two highest doses tested (125 and 250mg/kg body weight).
Furthermore, a statistically significant reduction in total pregnancy weight gain minus gravid uterus weight was found at these two highest doses tested (125 and 250mg/kg body weight).

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

Treatment did not influence drinking-water consumption.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

At caesarian section no gross pathological alteration was found in the maternal organs of any group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Neither the number of corpora lutea graviditatis/dam nor the number of visible implantation sites/litter were altered by TER over the dose range tested.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

In any of the TER-treated groups the number of resorptions/litter and the ratio of resorptions/implantation sites were not increased above that of the controls.

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Description (incidence and severity):

No alteration in the number of live foetuses/litter was noted in TER treated dams.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

effects observed, treatment-related

Description (incidence and severity):

The ratio of pregnant (i.e. rats with implantation sites detected by the method of Salewski at term)/sperm-positive treated dam did not differ significantly from that of the control group in rats treated with doses up to 125 mg TER/kg body weight but it was reduced at 250 mg TER/kg body weight.

Other effects:

no effects observed

Key result

Dose descriptor:

LOEL

Effect level:

125 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

LOEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

changes in number of pregnant

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

A significant reduction in foetal body weight was noted at the highest dose (250mg/kg body weight). No statistically significant reduction in foetal body weight was observed at doses lower than 250 mg/kg body weight.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no substance-related and/or statistically significant differences between the treated groups and the control group in the number of viable fetuses.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in the treated groups was comparable with the control fetuses. The differences observed in comparison to the control are without any biological relevance.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Except for a higher frequency of kinky tail in the group exposed to 30 mg TER/kg body weight and a higher proportion of foetuses with haematoma at the highest dose (250mg/kg body weight), no salient finding was revealed by external examination.
The increased frequency of tail abnormalities (bent tip and kinky tail) observed in foetuses exposed to TER was not related to dose and, in addition, the spontaneous frequency of kinky tail is relatively high in this rat strain (1%).

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Signs of delayed ossification (poorly ossified and not ossified bones as well as irregular spongy bones) were noted at doses higher than 30mg TER/kg body weight.
Also, a dose-related increase in the number of foetuses showing one or more abnormalities was found at doses higher than 30mg TER/kg body weight. These skeletal anomalies were manly higher incidences of os squamosum irregularly shaped, os supraoccipitale incompletely ossified, shorter ribs, extra ribs (cervical), sternum dislocated and os processus deltoid irregularly shaped.

Visceral malformations:

no effects observed

Description (incidence and severity):

No increase in visceral malformations was observed in TER-treated groups.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The reduction in foetal weight noted at the highest dose was accompanied by a decrease in the absolute weights of heart, liver, lungs and thymus. The reduction in thymus weight was particularly pronounced and the relative weight [thymus weight (mg)/foetal weight (g)] of this organ was also decreased. In contrast with the effects of TER on the weight of other foetal organs, the kidneys were heavier in the groups treated with 125 and 250mg TER/kg body weight.

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: skull

skeletal: sternum

skeletal: rib

Description (incidence and severity):

The overall increase in the occurrence of skeletal anomalies seems to result, to a large extent, from higher incidences of os squamosum irregularly shaped, os supraoccipitale incompletely ossified, shorter ribs, extra ribs (cervical), sternum dislocated and os processus deltoid irregularly shaped.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

60 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1: Maternal weight gain of rats treated orally with alpha-terpinene on days 6-15 of pregnancy (a)

	alpha-terpinene (mg/kg bw/day)
Treatment	0	30	60	125	250
Treated females	28	15	20b	26	27
Pregnant females	24	14	18	25	15
Pregnant/sperm positive females (%)	86	93	90	96	56*
Maternal weight (g)
Day 0	227±20	230±22	229±11	227±18	240±23
Day 21	348±29	347±39	357±23	341±28	324±29*
Gravid uterus weight (g)	71.8±18.1	72.8±23.1	77.0±19.9	76.3±11.0	63.0±18.7
Maternal weight gain (g)
Days 0-6	27.5±8.2	30.8±8.4	31.1±9.0	29.1±7.8	27.3±7.6
Days 6-11	13.6±5.7	16.9±5.7	11.8±5.8	6.3±7.1*	-17.8±12.9*
Days 6-15	30.7±21.9	35.7±9.4	29.2±6.8	21.0±9.1*	1.4±9.7*
Days 15-21	63.0±11.4	64.5±15.2	67.9±12.0	63.9±17.6	55.1±19.3
Days 0-21	121.2±21.9	131.1±23.2	128.3±17.4	114.1±22.1	83.7±27.1*
Days 0-21 (minus uterus weight)	49.4±15.6	58.3±11.5	51.2±14.4	37.7±19.0*	20.7±13.7*

a One pregnant female delivered on day 20.

b Percentage of pregnant females was analysed by the chi-square test. All other parameters were analysed by one-way analysis of variance and Student’s t-test. Values are mean ± SD.

*p < 0.05 v. controls.

Table 2: Parameters assessed at caesarean section of rats treated orally with alpha-terpinene on days 6-15 of pregnancy (a)

	alpha-terpinene (mg/kg bw/day)
	0	30	60	125	250
Corpa lutea	12.5±3.0	12.9±2.1	12.6±2.5	12.9±1.8	12.1±2.9
Implantation sites
Total	306	179	232	327	189
Per litter	12.6±3.2	12.8±3.8	12.9±3.1	13.2±1.9	12.6±2.4
Resorptions
Total	37	27	15	27	24
Resorptions/implantations (%)	12.1	15.1	6.5	8.2	12.7
Live foetuses
Total	275	158	218	299	165
Foetuses/implantations (%)	88	85	94	92	87
Per litter	11.5±3.1	11.2±3.9	12.1±3.1	11.9±1.9	11.0±3.3
Foetal weight (g)
Individual	4.7±0.3	4.8±0.4*	4.8±0.4*	4.7±0.4	4.1±0.5*
Litter	4.7±0.2	4.9±0.3	4.8±0.3	4.7±0.4	4.0±0.4*
Sex ratio (M/F)	139/130	70/82	115/102	160/140	85/80

a Proportions were analysed by the chi-square test. All other parameters were analysed by one-way analysis of variance and Student’s t-test. Values are mean ± SD.

*p < 0.05 v. controls.

Table 3: Signs of delayed ossification in foetuses of rats treated with alpha-terpinene on days 6-15 of pregnancy (a)

	alpha-terpinene (mg/kg bw/day)
	0	30	60	125	250
Foetuses examined	189	109	151	207	114
Foetuses with signs of delayed ossification (%)	11.1	14.7	53.0*	73.4*	88.6*
Foetuses (%) with retarded ossification in
Skull bones	0.5	4.6*	2.6	2.9*	16.6*
Vertebral column	1.6	0.9	22.5*	34.8*	21.0*
Sternum	11.6	5.5*	45.0*	70.0*	87.7*
Ribs	0	0	6.0*	13.5*	6.1*
Forelimbs	1.6	1.8	13.2*	9.2*	9.6*
Hindlimbs	4.8	9.2	37.7*	37.2*	47.4*

Signs of delayed ossification: not ossified (whole bone not stained); poorly ossified (whole bone is poorly ossified); and irregular spongy bones.

a Data were analysed by the chi-square test.

*p < 0.05 v. controls.

Table 4: Externally visible and visceral anomalies in foetuses of rats treated orally with alpha-terpinene on days 6-15 of pregnancy (a)

	alpha-terpinene (mg/kg bw/day)
Treatment	0	30	60	125	250
External examination (no. of foetuses)	275	158	218	299	165
Foetuses with anomalies (%)
Haematoma	10 (3.6)	7 (4.4)	7 (3.2)	16 (5.3)	13 (7.9)
Tail
Bent end	1 (0.4)	0	2 (0.9)	6 (2.0)	4 (2.4)
Kinky	3 (1.1)	10 (6.3)*	3 (1.4)	6 (2.0)	5 (3.0)
Pale	0	0	1 (0.4)	0	0
Oedema	1 (0.4)	0	0	0	0
Irregular positioning of forepaws	0	1 (0.6)	0	4 (1.3)	0
Irregular positioning of hindpaws	2 (0.7)	2 (1.3)	1 (0.4)	3 (1.0)	2 (1.2)
Visceral examination (no. of foetuses)	86	49	67	92	51
Foetuses with anomalies (%)
Spleen (ectopic)	1 (1.2)	0	0	0	0
Heart (smaller)	0	1 (2.0)	0	0	0
Liver (smaller)	1 (1.2)	0	0	0	0
Adrenal gland (smaller)	0	0	1 (1.5)	0	0
Testes (ectopic)	3 (3.5)	0	1 (1.5)	1 (1.1)	0
Ureter (thicker)	0	0	0	0	1 (2.0)

a Proportions were analysed by the chi-square test or, alternatively, by Fischer’s exact test.

*p < 0.05 v. controls

Table 5: Foetal organ weight in rats treated orally with alpha-terpinene on days 6-15 of pregnancy (a)

	alpha-terpinene (mg/kg bw/day)
Treatment	0	30	60	125	250
Foetuses examined	86	49	67	92	51
Foetal body weight (g)	4.9±0.5	5.3±0.5	5.3±0.4	5.2±0.5	4.2±0.5*
Foetal organ weights (mg)
Spleen	4.9±1.5	4.0±1.8	4.8±1.6	4.7±1.8	4.7±1.4
Heart	29.1±5.0	29.9±5.0	28.7±4.0	29.2±5.0	26.5±5.0*
Liver	370.0±66.0	372.0±48.0	375.0±39.0	362±80.0	335.0±64.0
Kidneys
Right	10.8±2.0	11.1±1.7	12.2±1.7*	12.1±2.4*	11.8±2.0*
Left	10.4±2.2	10.4±1.6	11.4±1.7*	11.1±2.0*	12.0±2.0*
Lung	143.0±18.0	139.0±12.0	142.0±14.0	138.0±15.0*	131.0±24.0*
Thymus	7.6±1.1	7.4±1.5	8.0±1.4	7.5±1.6	5.3±1.7*

a Data were analysed by one-way analysis of variance and Student’s t-test. Values are mean ± SD.

*p < 0.05 v. controls.

Table 6: Skeletal anomalies in foetuses of rats treated orally with alpha-terpinene on days 6-15 of pregnancy (a)

	alpha-terpinene (mg/kg bw/day)
	0	30	60	125	250
Foetuses examined	189	109	151	207	114
Foetuses with skeletal anomalies (%)	19.6	27.5	33.1	61.3	89.5
Foetuses (%) showing anomalies in:
Skull	5.3	8.2	16.5*	34.8*	63.1*
Os basisphenoid Bifurcated	0	0.9	0.7	0	0
Os basoccipitale Irregular shape	0	0	0	0	1.7
Os squamosum Irregular shape	4.8	6.4	13.2*	24.6*	35.1*
Os frontale Distance too large	0	0.9	0	0	0.9
Os interparietale Bone hole	0	0	0	0	0.9
Os palatinum Bone hole	0	0	1.3	1.4	0.9
Os parietale Distance too large	0	0.9	0	1.0	0.9
Os suproccipitale
Discontinuous	0	0	0	0	0.9
Gap	0	0	0	0.5	0.9
Incomplete ossification	0.5	0.9	2.6	12.6*	36.0*
Os tympanicum Discontinuous	0	0	0	0	0.9
Vertebral column	0	0.9	0	0	2.6
Atlas
Thicker	0	0	0	0	1.7
Cervical vertebra
Irregular shape	0	0	0	0	0.9
Fused	0	0	0	0	0.9
Thoracic vertebra
Fused with rib	0	0	0	0	0.9
Two ossification centra	0	0.9	0	0	0.9
Ribs	6.9	10.1	8.6	20.3*	53.5*
Shorter	5.8	6.4	6.0	19.8*	50.0*
Extra
Cervical	0.5	0.9	1.3	1.0	7.0*
Lumbar	0.5	2.7	1.3	1.0	0.9
Sternum	5.8	5.5	3.3	8.2	11.4*
Dislocated	5.8	5.5	3.3	8.2	11.4*
Forelimbs	2.6	5.5	6.6	17.9*	6.1
Irregular position	0.5	0.9	0	2.9	0
Os processus deltoid
Bone hole	1.6	0	1.3	1.9	3.5
Irregular shape	0.5	4.6	6.6	14.5	2.6

a Data were analysed by the chi-square test.

*p < 0.05 v. controls.

Conclusions:

Under the test conditions, alpha terpinene can adversely affect embryofoetal development in the rat at oral doses higher than 60 mg/kg body weight and is toxic to the mother at oral doses higher than 125 mg/kg body weight. Thus, the NOAEL of alpha terpinene for embryofoetal toxicity was determined to be 30 mg/kg body weight.

Executive summary:

A pre-natal developmental toxicity test was performed with alpha terpinene following a method equivalent to OECD Guideline 414. Alpha terpinene dissolved in corn oil at doses of 30, 60, 125 and 250 mg/kg body weight was given by gavage to female Wistar rats from day 6 to 15 of pregnancy. Caesarean sections were performed on day 21 of pregnancy. The number of implantation sites, living and dead foetuses, resorptions and corpora lutea were recorded. All foetuses were weighed and examined for externally visible malformations. One-third of the foetuses of each litter were evaluated for visceral anomalies. The remaining foetuses were examined for skeletal malformations. A reduction in body weight minus uterine weight at term indicated that the two highest doses tested (125 and 250mg/kg bw) were maternally toxic. No increase in the ratio of resorptions/implantations was observed over the dose range tested. The highest dose (250 mg/kg bw) reduced the ratio of pregnant/treated female. A decrease in foetal body weight and an increase in foetal kidney weights were noted at 250 mg /kg bw. Signs of delayed ossification (poorly ossified and not ossified bones as well as irregular spongy bones) and a higher incidence of minor skeletal malformations were observed at doses of 60 mg/kg bw or more. These findings indicate that the NOAEL for alpha terpinene-induced embryofoetotoxicity can be set at 30 mg/kg bw by the oral route.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

(The study does not provide specified information on abnormalities findings)

GLP compliance:

not specified

Remarks:

No information reported

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Department of Physiology and Pharmacology of Federal University of Pernambuco (UFPE,
Pernambuco, Brazil)
- Females (if applicable) nulliparous and non-pregnant: yes
- Diet (e.g. ad libitum): industrialized dry food (Presence®, Purina, Brazil), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 ºC
- Humidity (%): 55-65 %
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: gavage

Vehicle:

other: Tween-80 aqueous solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
1,8-cineole (CIN) was emulsified in a 1% Tween-80 aqueous solution before administration to the
animals.
Volume administered: 10 mL/kg bw

VEHICLE
Tween-80 (CAS No: 9005-65-6) was obtained from Sigma-Aldrich® (St. Louis, MO, USA).

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 2 female/1 male
- Length of cohabitation: nulliparous female rats were distributed in polyethylene cages, into contact with a male rat during the dark phase of the environmental cycle. The next day, male rats were removed from the cages and the female vaginal lavage was carried out with 0.9% physiological saline solution.
- Proof of pregnancy: The spermatozoids viewing associated with the diagnosis of the estrous phase of the estral cycle was considered indicative of pregnancy (Cooper et al., 1993). The observation of the presence of sperm in the vaginal smear has defined the 1st day of pregnancy.

Duration of treatment / exposure:

Preimplantation period: From 1st to 6th day of pregnancy.
Organogenic period: From 7th to 14th day of pregnancy.

Frequency of treatment:

Daily

Duration of test:

From 1st to 21st day of pregnancy

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control (Group I)

Dose / conc.:

250 mg/kg bw/day

Remarks:

Group II

Dose / conc.:

500 mg/kg bw/day

Remarks:

Group III

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Group IV

No. of animals per sex per dose:

Preimplantation period: 9-10
Organogenic period: 7-9

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- During pregnancy, the rats were evaluated for survival, altered appearance and any clinical signs of toxicity, such as changes in food and water intake, piloerection, diarrhea, changes in locomotor activity and vaginal bleeding.

BODY WEIGHT: Yes
- Time schedule for examinations: daily.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: uterine horns, ovaries, fetuses and placentae

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: The fetuses and placentae were weighed (absolute mass).
- Number of corpora lutea: Yes.
- Number of implantations: Yes.
- Number of resorptions: Yes.
- Other:
Ovaries (right and left) were weighed

Fetal examinations:

Live and dead fetuses were recorded.
The fetuses were observed macroscopically for any visible abnormalities.

Statistics:

Values were expressed as mean ± standard error of mean (S.E.M.) and the differences are analyzed by variance analysis (ANOVA) followed by Dunnett’s test or Student’s T test for unpaired samples. The implantation and resorption indexes, as well as pre-implantation and post-implantation loss rates, were analyzed using the Kruskal–Wallis test followed by the Dunn test, when necessary.The level of significance for rejection of the null hypothesis was set at 5% (p < 0.05). Statistical analyses were performed using GraphPad Prism 5.0®.

Indices:

Implantation index: total number of implantation sites/total number of corpora lutea × 100)
Resorption index: total number of resorption sites/total number of implantation sites × 100
Preimplantation loss rate: (number of corpora lutea − number of viable implantations)/number of corpora lutea × 100
Post-implantation loss rate: (number of implantations − number of live fetuses)/number of implantations × 100

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs of toxicity, such as salivation, piloerection, diarrhea, changes in locomotor activity or changes in behavior at any of the doses administered.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

Treatment at any dose produced no deaths in pregnant rats treated during pre-implantation or organogenesis.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significant decrease was observed in maternal weight gain during pre-implantation (1st to 6th day) and organogenesis (7th to 14th day) in females treated with all doses, and during pregnancy (1st to 20th day) in females treated with 1000 mg/kg during the preimplantation period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No changes in the intake of food were observed during pregnancy in either period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

No changes in the intake of water were observed during pregnancy in either period.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

During the pre-implantation or organogenesis periods, the ovary and placental masses were similar in all treated experimental groups.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

During the pre-implantation or organogenesis periods, the implantation index and the loss rate of pre-and post-implantation were similar in all treated experimental groups.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

During the pre-implantation or organogenesis periods, the resorption index was similar in all treated experimental groups.

Early or late resorptions:

no effects observed

Description (incidence and severity):

During the pre-implantation or organogenesis periods, the resorption index was similar in all treated experimental groups.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

Dead fetuses were observed during the pre-implantation and organogenesis periods at doses of 500 and 1000 mg/kg, and also in the control group. Since there was no reduction in the mass of fetuses in animals treated with 1000 mg/kg during the organogenesis period, it can be inferred that there is a relation between reduced maternal body mass gain and a reduction in the mass of rat foetuses treated with this dose during the pre-implantation period, but that needs to be better investigate.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

During the organogenesis period, a single rat at a dose of 1000 mg/kg presented vaginal bleeding on the 13th day of gestation and laparotomy did not reveal either live or dead fetuses.
There was a reduction in the number of corpora lutea in females treated with 250 mg/kg during the organogenesis, when compared to the control group.

Key result

Dose descriptor:

LOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduction in the mass of fetuses were observed in females treated with 1000 mg/kg in the preimplantation period.
Since there was no reduction in the mass of fetuses in animals treated with 1000 mg/kg during the organogenesis period, it can be inferred that there is a relation between reduced maternal body mass gain and a reduction in the mass of rat foetuses treated
with this dose during the pre-implantation period, but that needs to be better investigate.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

not specified

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks on result:

other: Due to the reduction of maternal body mass induced at the dose of 250 mg/kg in the pre-implantation and organogenesis periods, it was not possible to determine the NOAEL.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1: Reproductive parameters of female Wistar rats treated with 1,8-cineole (CIN) from 1st to the 6th day of pregnancy (preimplantation period).

Reproductive parameters	control	250 mg/kg	500 mg/kg	1000 mg/kg
Pregnant rats	9	10	10	10
Mass gain in the pre-implantation period (g)a	15.29±1.51	-1.98±2.93*	0.66±3.53*	-6.12±3.73*
Mass gain in the pregnancy period (g)a	73.82±7.53	73.04±6.53	71.14±3.64	50.12±7.57*
Number of live fetuses	96	116	117	84
Number of dead fetuses	1	0	0	4
Offspring/dam relationshipa	10.67±0.88	11.60±1.05	11.60±0.52	8.80±1.45
Fetuses mass (g)a	2.35±0.06	2.32±0.10	2.29±0.071.60	1.60±0.28*
Placentae mass (g)a	0.51±0.02	0.47±0.01	0.45±0.01	0.42±0.05
Ovary mass (mg/100g)a	22.87±0.87	18.70±1.25	22.16±1.83	20.96±1.63
Number of implantation sites	107	123	121	94
Number of resorption sites	10	7	4	6
Number of corpora luteaa	12.89±0.54	12.70±1.11	12.10±0.43	11.80±0.99
Implantation index (%)b	100	100	100	89.96
Resorption index (%)b	0	0	0	6.67
Pre-implantation loss (%)b	0	0	0	10.05
Post-implantation loss (%)b	0	0	0	3.33

Implantation index (total number of implantation sites/total number of corpora lutea × 100), resorption index (total number of resorption sites/total number of implantation sites × 100, pre-implantation loss rate (number of corpora lutea − number of viable implantations/number of corpora lutea × 100) and post-implantation loss rate (number of implantations − number of live fetuses/number of implantations × 100). The values are expressed as mean ± S.E.M.(a) or median (b). *Statistically different from control group (ANOVA followed by Dunnett's test, p < 0.05).

Table 2: Reproductive parameters of female Wistar rats treated with 1,8-cineole (CIN) from 7th to the 14th day of pregnancy (organogenic period).

Reproductive parameters	control	250 mg/kg	500 mg/kg	1000 mg/kg
Pregnant rats	8	9	8	7
Mass gain in the organogenic period (g)a	20.05±1.60	-3.00±3.22*	-1.60±4.20*	0.37±8.89*
Mass gain in the pregnancy period (g)a	74.79±5.13	56.67±4.72	54.91±9.38	57.17±10.34
Number of live fetuses	85	90	81	62
Number of dead fetuses	1	0	1	0
Offspring/dam relationshipa	10.63±0.84	10.00±0.60	10.13±1.10	8.85±1.33
Fetuses mass (g)a	2.33±0.01	2.42±0.12	2.34±0.18	2.34±0.23
Placentae mass (g)a	0.47±0.030.47	±0.010.49	±0.020.50	0.50±0.03
Ovary mass (mg/100g)a	24.69±1.87	23.45±1.47	24.77±2.36	25.99±2.38
Number of implantation sites	91	90	80	64
Number of resorption sites	7	10	3	12
Number of corpora luteaa	13.75±0.83	11.11±0.48*	11.88±0.74	12.14±0.96
Implantation index (%)b	78.3	100	88.31	78.57
Resorption index (%)b	6.25	8.33	0	0
Pre-implantation loss (%)b	21.27	0	11.69	21.43
Post-implantation loss (%)b	0	8.33	0	0

Implantation index (total number of implantation sites/total number of corpora lutea × 100), resorption index (total number of resorption sites/total number of implantation sites × 100, pre-implantation loss rate (number of corpora lutea − number of viable implantations/number of corpora lutea × 100) and post-implantation loss rate (number of implantations − number of live fetuses/number of implantations × 100). The values are expressed as mean ± S.E.M.(a) or median (b). *Statistically different from control group (ANOVA followed by Dunnett's test, p < 0.05).

Conclusions:

In a reproductive toxicity study performed by oral route (gavage) with 1,8-cineole using Wistar rats, the results obtained provide evidence that possibly the test substance presents maternal and fetal toxicity, but it is concluded that further studies need to be conducted. The NOAEL was not established since effects in maternal body weight were found at the lowest dose tested (250 mg/kg bw/day).

Executive summary:

A reproductive toxicity study was performed by oral route (gavage) with 1,8-cineole using female Wistar rats. The pregnant rats were randomly distributed into 8 groups (n = 7-10 per group). 4 groups were assigned to treatment during the pre-implantation period (1st to 6th day of pregnancy) and 4 groups to treatment during organogenesis (7th to 14th day of pregnancy). Test substance was administered to all animals of both pregnancy periods with doses of 0 (control, consisted only in 1% Tween-80 aqueous solution), 250, 500 and 1000 mg / kg bw/day.

The test substance produced no deaths or signs of toxicity in pregnant rats treated during the pre-implantation or organogenesis periods. No alterations were observed in maternal food and water intake during the course of pregnancy in both periods, although, during the pre-implantation period, a reduction in body mass gain was observed in females at all doses. After discontinuation of this treatment, the situation was reversed, as evidenced by the lack of difference in body mass gain during pregnancy (1st to 20th day). A similar response was observed during organogenesis. With regard to reproductive parameters, the data showed no significant differences in the number of fetuses, placentae and ovary masses, implantation or resorption index, or pre-implantation and post-implantation losses.

Dead fetuses were observed during the pre-implantation and organogenesis periods at doses of 500 and 1000 mg/kg, and also in the control group. Since there was no reduction in the mass of fetuses in animals treated with 1000 mg/kg during the organogenesis period, it can be inferred that there is a relation between reduced maternal body mass gain and a reduction in the mass of rat foetuses treated with this dose during the pre-implantation period, but that needs to be better investigate.

These results provide evidence that possibly the test substance presents maternal and fetal toxicity, but it is concluded that further studies need to be conducted. The NOAEL was not established since effects in maternal body weight were found at the lowest dose tested (250 mg/kg bw/day).

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Prenatal developmental toxicity study: Groups of pregnant ICR mice (20/dose: 15 for teratogenicity study, 5 for postnatal development) were administered orally with d-limonene at dose levels of 0, 591 and 2363 mg/kg bw/day for 6 days from Day 7 to 12 of gestation and evaluated for developmental and postnatal development toxicity.

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

ICR

Details on test animals or test system and environmental conditions:

No data

Route of administration:

oral: unspecified

Vehicle:

not specified

Details on exposure:

No data

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Details on mating procedure:

no data

Duration of treatment / exposure:

6 days (gestation Day 7-12)

Frequency of treatment:

Once daily

Duration of test:

Gestatation Day 0 to postnatal week 7

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

591 mg/kg diet

Dose / conc.:

2 363 mg/kg bw/day

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

No data

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data
General behaviour observed, but no data regarding the frequency of observation

BODY WEIGHT: Yes
- Time schedule for examinations: daily

POST-MORTEM EXAMINATIONS: No data

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: No data
Examinations included: Number of implantations, number of resorptions and foetus bodyweight and placental weight

Fetal examinations:

- External examinations: Yes
- Visceral examinations: Yes
- Skeletal examinations: Yes

Statistics:

statistical significance difference of effects from controls were calculated at 5% and 1% levels.

Indices:

No data

Historical control data:

No data

Clinical signs:

no effects observed

Description (incidence and severity):

No anomalies were observed in the general behavior of dams during the period of gestation

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significant decrease of bodyweight gain in pregnant mice was observed at 2363 mg/kg bw/day

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Description (incidence and severity):

No anomalies were observed in the general behavior of dams during the period of gestation

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

591 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

A significant decrease of bodyweight gain was observed in male offsprings born to dams given drug orally at 2363 mg/kg bw/day, but there were not differences in weaning rate, sensory function, organ
weight and histological findings of the testis and ovary comparing with those of control.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

An incidence of lumber rib and fused rib in the fetuses increased significantly at 2363 mg/kg bw/day comparing with those of control.
In the observation of skeletal development in fetuses, retarded ossification of proximal phalanx of fore limb, metatarsal bone and proximal phalanx of hind limb were observed. However, these retarded
ossifications were restored to normal during postnatal development.

Visceral malformations:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

A significant decrease of bodyweight gain was observed in male offsprings born to dams given drug orally at 2363 mg/kg bw/day, but there were not differences in weaning rate, sensory function, organ
weight and histological findings of the testis and ovary comparing with those of control.

Key result

Dose descriptor:

NOAEL

Effect level:

591 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: rib

Description (incidence and severity):

Increase in incidence of lumber rib and fused rib compared to control.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

2 363 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

no

Relevant for humans:

not specified

Table 1: Effects of d-limonene on development of mouse fetuses.

	Control	591	2363
No. of mothers	15	15	15
No. of implantations	162	179	154
(mean ± S.E.)	(10.80 ± 0.20)	(11.93 ± 0.13)	(10.27 ± 0.21)
No. of dead fetuses	9	1.1	6
(mean ± S.E.)	(0.60 ± 0.05)	(0.73 ± 0.10)	(0.40 ± 0.05)
No. of resorbed fetuses	18	20	20
(mean ± S.E.)	(1.20 ± 0.05)	(1.33 ± 0.10)	(1.33 ± 0.10)
No. of live fetuses	135	148	125
Sex ratio (Male/Female)	1.33	0.83	1.13
Fetuses Body weight (g) Male (mean ± S.E.)	1.34 ± 0.02	1.24 ± 0.01	1.28 ± 0.02
Female (mean ± S.E.)	1.28 ± 0.02	1.22 ± 0.01	1.19 ± 0.02
Placental weight (mg) Male (mean ± S.E.)	94 (2 ± 2.0)	86 ( 0 ± 1.7)	89 (3 ± 1.7)
Female (mean ± S.E.)	87 (8 ± 2.6)	81 (5 ± 1.9)	83 (5 ± 2.2)
External observation No. of fetuses examined	135	148	128
No. of fetuses malformed	0	4	0
Cleft palate	0	4	0
Incidence (%)	0	2.7	0
Visceral observation No. of fetuses examined	71	76	68
No. of fetuses malformed	4	4	3
Enlargement of foramen ovale	4	4	3
Incidence (%)	5.6	5.3	4.4

Table 2: Effects of d-limonene on skeletal development of mouse fetuses

Dose (mg/kg bw)	Control	591	2363
No. of fetuses examined	64	72	61
Variation Lumbar rib (%)	17 (26.6)	12 (16.7)	28 (46.7) *
Cervical rib (%)	1 (1.6)	(5.6)	1 (1.7)
Fused rib (%)	0 (0)	0 (1)	5 (8.3) *
Crooked rib (%)	2 (3.1)	0 (1)	0 (0)
Asymmetry of sternebrae (%)	2 (1.7)	7 (9.7)	7 (11.7)
Fused sternebrae	0 (0)	1 (1.4)	1 (1.7)
No. of ossification Sternebrae	5.99 ± 0.01	5.97 ± 0.02	5.94 ± 0.03
Fore limb Metacarpal bone	8.00 ± 0	7.97 ± 0.03	8.00 ± 0
Proximal phalanx	7.50 ± 0.13	7.67 ± 0.16	6.87 ± 0.30 *
Middle phalanx	1.16 ± 0.23	2.14 ± 0.29 **	2.18 ± 0.29 **
Distal phalanx	9.08 ± 0.32	9.72 ± 0.20	9.13 ± 0.36
Hind limb Metatarsal bone	10.00 ± 0	9.92 ± 0.05	9.80 ± 0.09 *
Proximal phalanx	8.12 ± 0.23	8.03 ± 0.23	7.23 ± 0.39 *
Middle phalanx	0.09 ± 0.09	0.33 ± 0.18	0.20 ± 0.11
Distal phalanx	9.47 ± 0.24	9.72 ± 0.20	9.30 ± 0.31
Caudal vertebrae	7.12 ± 0.95	6.50 ± 0.21	7.00 ± 0.25

* Significantly different from the control at 5% level.

** Significantly different from the control at 1% level.

Table 3: Effects of d-limonene on postnatal development of mouse offsprings

		d-Limonene (mg/kg bw)
	Control	591	2363
No. of mothers	5	5	5
No. of implantations	51	52	50
(mean ± S.E.)	(10.80 ± 0.33)	(10.41 ± 0.96)	(10.03 ± 0.63)
No. of offsprings	50	46	39
No. of dead offsprings at birth	0	0	0
Sensory function	Normal	Normal	Normal
No. of live offsprings At birth	50	46	39
1st week	50	46	39
2nd week	50	46	39
3rd week	50	46	39
4th week	50	46	39
5th week	50	46	39
6th week	50	46	39
7th week	50	46	39
Weanling rate (%)	100	100	100

Table 4: Effects of d-limonene on gross differentiation of mouse offsprings

		d-Limonene (mg/kg bw)
Gross differentiation	Control	591	2363
Opening of the ear-shell	3.5 ± 0.07	3.6 ± 0.08	4.1 ± 0.08
Coating with the hair	5.0 ± 0.00	4.11 ± 0.08	5.2 ± 0.06
Odontiasis	9.8 ± 0.07	9.3 ± 0.07	9. 1 ± 0.04
Opening of the eyelid	13.3 ± 0.08	12.9 ± 0.06	13.6 ± 0.09
Descending of the testis	23.0 ± 0.20	23.3 ± 0.11	25.0 ± 0.27
Opening of the vaginal orifice	29.5 ± 0.26	30.5 ± 0.16	30.6 ± 0.15

Table 5: Absolute organ weights of postnatal mouse offsprings born to mothers given d-limonene

Sex

Dose (mg/kg bw)

No. of offsprings

Final BW (g)

Thyroids (mg)

Thymus (g)

Lungs (g)

Heart (g)

Spleen (g)

Kidneys (g)

Liver (g)

Adrenals (g)

Testes (g) or Ovaries (mg)

Female

Control

27

34.2 ± 0.50

5.43 ± 0.18

75.72 ± 4.14

199.26 ± 4.70

158.22 ± 2.13

131.54 ± 9.96

0.63 ± 0.02

2.06 ± 0.09

8.39 ± 0.49

217.52 ± 1.65

591

27

34.9 ± 0.44

6.04 ± 0.68

65.26 ± 4.26

203.76 ± 2.95

166.66 ± 7.58

121.66 ± 4.74

0.63 ± 0.02

2.08 ± 0.07

8.29 ± 0.67

209.76 ± 9.23

2363

23

32.2 ± 0.77

5.41 ± 0.62

64.08 ± 3.64

189.60 ± 6.69

158.96 ± 3.12

125.52 ± 3.04

0.58 ± 0.02

2.14 ± 0.04

8.53 ± 0.86

200.20 ± 0.39

Male

Control

23

27.3 ± 0.50

4.80 ± 0.22

81.47 ± 4.38

172.80 ± 7.20

118.98 ± 3.91

121.76 ± 6.48

0.37 ± 0.01

1.37 ± 0.02

11.71 ± 0.40

13.38 ± 0.68

591

19

28.8 ± 0.45

4.29 ± 0.20

69.44 ± 5.52

174.26 ± 5.66

129.96 ± 3.62

112.68 ± 2.79

0.38 ± 0.01

1.37 ± 0.04

11.36 ± 0.43

16.98 ± 1.71

2363

16

28.1 ± 0.34

3.53 ± 0.41 *

63.95 ± 9.72

171.75 ± 5.70

135.15 ± 6.89

116.15 ± 5.78

0.38 ± 0.01

1.46 ± 0.03 *

11.61 ± 0.30 *

17.93 ± 1.30 *

* Significantly different from the control at 5% level.

Table 6: Relative organ weights per 100 g body weights of postnatal mouse offsprings born to mothers given d-limonene

Sex

Dose (mg/kg bw)

No. of offsprings

Final BW (g)

Thyroids(mg/100 g)

Thymus(mg/100 g)

Lungs(mg/100 g)

Heart(mg/100 g)

Spleen(mg/100 g)

Kidneys(g/100 g)

Liver(g/100 g)

Adrenals(mg/100 g)

Testes or Ovaries (mg/100 g)

Female

Control

27

34.2 ± 0.50

15.80 ± 0.44

220.36 ± 10.67

580.50 ± 13.59

461.18 ± 8.65

386.55 ± 39.35

1.83 ± 0.07

6.01 ± 0.26

24.48 ± 1.55

634.84 ± 19.06

591

27

34.9 ± 0.44

17.20 ± 1.93

185.29 ± 8.48 *

581.21 ± 9.42

474.60 ± 717.10

346.85 ± 12.29

1.80 ± 0.05

5.93 ± 0.14

23.55 ± 1.48

596.69 ± 14.95

2363

23

32.2 ± 0.77

16.55 ± 2.64

191.59 ± 6.25

568.43 ± 13.67

477.39 ± 11.90

376.91 ± 10.23

1.73 ± 0.03

6.44 ± 0.29

25.74 ± 2.82

602.30 ± 34.26

Male

Control

23

27.3 ± 0.50

17.13 ± 18.47

291.98 ± 34.66

619.50 ± 8.54

425.00 ± 32.37

437.67

1.31 ± 0.04

4.89 ± 0.08

41.89 ± 1.41

47.85 ± 2.42

591

19

28.8 ± 0.45

15.06 ± 0.61 *

242.35 ± 13.57

611.98 ± 17.12

456.02 ± 4.93 *

397.44 ± 20.40

1.33 ± 0.02

4.81 ± 0.11

39.96 ± 1.73

59.59 ± 5.78

2363

16

28.1 ± 0.34

12.60 ± 1.44 *

229.38 ± 35.46

612.74 ± 20.32

482.14 ± 24.21 *

414.30 ± 17.34

1.37 ± 0.04

5.23 ± 0.09 *

41.45 ± 1.27

64.33 ± 5.38 *

* Significantly different from the control at 5% level.

Table 7: Summaried data on postnatal development of mouse offsprings

		d-limonene (mg/kg bw)
	Control	591	2363
External observation
No. of offsprings examined	50	46	39
No. of offsprings malformed	0	0	0
Visceral observation
No. of offsprings examined	50	46	39
No. of offsprings malformed	0	0	0
Skeletal observation
No. of offsprings examined	50	46	39
No. of offsprings malformed	0	0	0
Skeletal variation
Lumbar rib (%)	17 (34.0)	23 (50.0)	20 (51.3)
Fusion of 13th and lumbar rib (%)	0	0	1 (2.6)
Fusion of lumbar vertebra (%)	1 (2.0)	1 (2.2)	1 (2.6)
Crooked tail (%)	0	0	1 (2.6)

Conclusions:

Under the test conditions, the NOAEL for maternal and fetal toxicity was considered to be 591 mg/kg bw/day based on the decreased bodyweight gain in dams and increased incidences of abnormal bone formation in fetuses.

Executive summary:

In a prenatal developmental toxicity study, d-limonene was administered orally to groups of pregnant ICR mice (20/dose: 15 for teratogenicity study, 5 for postnatal development) at dose levels of 0, 591 and 2363 mg/kg bw/day for 6 days from Day 7 to 12 of gestation. Bodyweights of pregnant mice were recorded during organogenesis. Caesarean sections were performed and the number of dead, live or resorbed fetuses, sex ratio and number of implantation sites were recorded. Fetuses were weighed and examined for external, visceral and skeletal malformations. Number of live offsprings, sensory functions, gross differentiation and organ weights of offsprings were recorded until postnatal week 7.

 

A significant decrease of bodyweight gain in pregnant mice was observed at 2363 mg/kg bw/day. However, no anomalies were observed in the general behavior of dams during the period of gestation. An incidence of lumber rib and fused rib in the fetuses increased significantly at 2363 mg/kg bw/day comparing with those of control. In the observation of skeletal development in fetuses, retarded ossification of proximal phalanx of fore limb, metatarsal bone and proximal phalanx of hind limb were observed. However, these retarded ossifications were restored to normal during postnatal development. A significant decrease of bodyweight gain was observed in male offsprings born to dams given drug orally at 2363 mg/kg bw/day, but there were not differences in weaning rate, sensory function, organ weight and histological findings of the testis and ovary comparing with those of control.

 

Under the test conditions, the NOAEL for maternal and fetal toxicity was considered to be 591 mg/kg bw/day based on the decreased bodyweight gain in dams and increased incidences of abnormal skeletal formation in fetuses at 2363 mg/kg bw/day.

Reference 5

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Prenatal developmental toxicity study: Groups of pregnant Japanese white rabbits were administered orally with d-limonene at dose levels of 250, 500 and 1000 mg/kg bw/day for 13 days from Day 6 to 18 of gestation and evaluated for teratogenicity.

GLP compliance:

not specified

Limit test:

no

Species:

rabbit

Strain:

other: Japanese white

Details on test animals or test system and environmental conditions:

no data

Route of administration:

oral: unspecified

Vehicle:

not specified

Details on exposure:

no data

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

no data

Details on mating procedure:

No data

Duration of treatment / exposure:

13 days (gestation Day 6-18)

Frequency of treatment:

Once daily

Duration of test:

Gestatation Day 0 to postnatal Day 49

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

10 (in 0-500 mg/kg bw/day groups) or 18 (in 1000 mg/kg bw/day group) pregnant females

Control animals:

yes

Details on study design:

no data

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

General behaviour observed, but no data regarding the frequency of observation

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
mean daily food consumption by treatment group is reported

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data


POST-MORTEM EXAMINATIONS: No data

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: No data
Examinations included: Number of implantations, number of resorptions and foetus bodyweight and placental weight

Fetal examinations:

- External examinations: Yes: about 90% per litter
- Visceral examinations: Yes: about 90% per litter
- Skeletal examinations: Yes: about 90% per litter

Statistics:

statistical significance difference of effects from controls were calculated at 5% and 1% levels.

Indices:

no data

Historical control data:

no data

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No anomalies were observed in the general behaviour of dams given 250 and 500 mg/kg of d-limonene during the gestation.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Treatment with the highest dose level (1000 mg/kg) of d-limonene resulted in death of dams with less than 40% mortality.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The significant decrease of body- weight gain was temporarily observed in dams given 500 and 1000 mg/kg of d-limonene

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The significant decrease of food consumption was temporarily observed in dams given 500 and 1000 mg/kg of d-limonene

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Visceral and skeletal examinations revealed some anomalies such as incomplete lobulation of the lungs, enlargement of the foramen ovale and retarded ossification of the middle phalanx of fore limbs in addition to the 5th sternebrae. These did not appear to be dose-dependent and restored to normal during the postnatal development.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Visceral and skeletal examinations revealed some anomalies such as incomplete lobulation of the lungs, enlargement of the foramen ovale and retarded ossification of the middle phalanx of fore limbs in addition to the 5th sternebrae. These did not appear to be dose-dependent and restored to normal during the postnatal development.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Other non specific anomalies involved the lumber ribs in fetuses and offsprings, formation of the accessory ossification center of the 5th sternebrae in offsprings and the atrial septal defect detected in only 2 fetuses of a litter from dams treated with 250 mg/kg bw/day of d-limonene.

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1: Effect of d-limonene on prenatal development of rabbit fetuses

Dose (mg/kg bw)	Control	250	500	1000
No. of pregnant animals	10	10	10	18
No. of dead clams	0	0	0	6
(%)				33
No. of examined clams	10	10	10	10
No. of implantations	96	94	85	91
(mean ± S.E.)	9.50 ± 0.25	9.40 ± 0.21	8.50 ± 0.33	9.10 ± 0.25
No. of resorbed fetuses	5	4	4	8
No. of dead fetuses	3	5	0	3
No. of live fetuses	88	85	81	80
Sex ratio (Male/Female)	0.73 (37/51)	1.13 (45/50)	0.62 (31/50)	1.11 (38/42)
Fetus body weight (g)
Male (mean ± S.E.)	44.39 ± 1.33	48.09 ± 1.07 *	44.76 ± 1.51	43.22 ± 0.96
Female (mean ± S.E.)	45.64 ± 1.00	47.45 ± 1.08	46.14 ± 1.21	45.13 ± 1.10
Placental weight (g)
Male (mean ± S.E.)	5.76 ± 0.17	5.84 ± 0.17	5.95 ± 0.29	5.77 ± 0.19
Female (mean ± S.E.)	5.87 ± 0.19	5.70 ± 0.15	6.16 ± 0.18	5.87 ± 0.23

* Significantly different from the control at 5% level

Table 2: Prenatal examinations of rabbit fetuses

Dose (mg/kg bw)	Control	250	500	1000
External examination
No. of examined fetuses	91	90	81	83
No. of malforrned fetuses	0	0	0	0
Visceral examination
No. of examined fetuses	88	85	81	80
No. of malformed fetuses
Atrial septal defect (%)	0	2 (2.4)	0	0
No. of minor abnormality
Incomplete lobulation of lungs (%)	11 (12.5)	16 (18.8)	19 (23.5)	19 (23.8)
Enlargement of foramen ovale (%)	2 (2.3)	2 (2.4)	5 (6.2)	4 (5.0)
Skeletal examination
No. of examined fetuses	86	87	81	80
No. of malformed fetuses	0	0	0	0
No. of variation
Left lumbar rib (%)	18 (20.9)	26 (29.9)	14 (17.3)	25 (31.3)
Right lumbar rib (%)	16 (18.6)	22 (25.3)	14 (17.3)	22( 27.5)
Ossification pattern
Retarded ossification of 5th sternebrae (%)	11 (12.8)	14 (16.1)	8 (9.9)	18 (22.5)
Retarded ossification of middle phalanx of fore limbs (%)	2 (2.3)	3 (3.4)	0	6 (7.4)

Table 3: Absolute organ weights of rabbit offsprings

	Male				Female
	Control	250	500	1000	Control	250	500	1000
No. of offsprings	13	12	8	13	10	13	16	9
Final body weight (g)	893.0 ± 45.3	1021.2 ± 45.4 **	931.9 ± 55.5	957.3 ± 52.4	1005.5 ± 53.7	1093.1 ± 46.6	860.0 ± 31.6 *	1071.7 ± 58.1
Liver (g)	36.49 ± 2.52	45.08 ± 1.52 *	38.67 ± 3.10	34.91 ± 2.76	41.79 ± 3.39	13.95 ± 3.18	34.68 ± 1.90	48.30 ± 6.14
Lungs (g)	5.53 ± 0.35	6.25 ± 0.26	5.98 ± 0.36	5.46 ± 0.18	5.91 ± 0.25	5.94 ± 0.28	5.72 ± 0.18	5.93 ± 0.45
Heart (g)	2.63 ± 0.17	3.50 ± 0.16 **	2.86 ± 0.17	3.04 ± 0.19	3.19 ± 0.17	3.38 ± 0.21	2.72 ± 0.12 *	3.34 ± 0.20
Spleen (g)	0.71 ± 0.05	0.77 ± 0.04	0.71 ± 0.08	0.81 ± 0.04	0.64 ± 0.06	0.74 ± 0.05	0.74 ± 0.04	0.78 ± 0.07
Thymus (g)	2.31 ± 0.20	2.51 ± 0.23	2.11 ± 0.38	1.96 ± 0.11	2.40 ± 0.30	2.77 ± 0.19	1.671.14 *	2.36 ± 0.31
Kidneys (g)	7.67 ± 0.42	9.80 ± 0.46 **	8.29 ± 0.29	8.58 ± 0.52	8.82 ± 0.46	8.40 ± 0.30	7.86 ± 0.37	9.56 ± 0.55
Thyroids (mg)	75.89 ± 8.35	102.68 ± 4.18*	86.74 ± 10.97	80.93 ± 7.41	82.78 ± 8.00	89.90 ± 4.11	75.75 ± 5.43	96.30 ± 9.67
Adrenals (mg)	69.65 ± 6.02	9.1.93 ± 1.06 **	78.79 ± 5.89	71.36 ± 6.00	82.23 ± 4.37	94.24 ± 5.07	87.64 ± 4.18	105.39 ± 15.11
Testes or Ovaries (mg)	180.42 ± 17.15	272.86 ± 16.46 **	185.62 ± 23.78	162.84 ± 20.59	46.31 ± 7.90	46.10 ± 2.80	43.53 ± 2.69	47.77 ± 3.59

* Significantly different from the control at 5% level

** Significantly different from the control at 1% level

Table 4: Relative organ weights per 100 g of rabbit offsprings

	Male				Female
	Control	250	500	1000	Control	250	500	1000
No. of offsprings	13	12	8	13	10	13	16	9
Final body weight (g)	893.0 ± 45.3	1021.2 ± 45.4 **	931.9 ± 55.5	957.3 ± 52.4	1005.5 ± 53.7	1093.1 ± 46.6	860.0 ± 31.6 *	1071.7 ± 58.1
Liver (g/100 g)	4.08 ± 0.25	3.99 ± 0.22 *	4.16 ± 0.26	3.52 ± 0.10	4.25 ± 0.17	4.03 ± 0.21	4.02 ± 0.16	4.04 ± 0.32
Lungs (g/100 g)	0.61 ± 0.03	0.55 ± 0.03	0.65 ± 0.04	0.58 ± 0.03	0.62 ± 0.02	0.55 ± 0.02 *	0.67 ± 0.03	0.51 ± 0.02 **
Heart (g/100 g)	0.29 ± 0.01	0.31 ± 0.01	0.31 ± 0.01	0.31 ± 0.02	0.33 ± 0.01	0.31 ± 0.01	0.32 ± 0.01	0.29 ± 0.01 *
Spleen (g/100 g)	0.08 ± 0.01	0.07 ± 0.01	0.08 ± 0.01	0.08 ± 0	0.07 ± 0.01	0.07 ± 0.01	0.09 ± 0	0.07 ± 0.01
Thymus (g/100 g)	0.25 ± 0.02	0.22 ± 0.02	0.25 ± 0.03	0.20 ± 0.01 *	0.24 ± 0.03	0.25 ± 0.01	0.195 ± 0.01	0.20 ± 0.02
Kidneys (g/100 g)	0.85 ± 0.04	0.86 ± 0.04	0.90 ± 0.03	0.88 ± 0.02	0.91 ± 0.02	0.80 ± 0.01	0.91 ± 0.01	0.83 ± 0.03 *
Thyroids (mg/100 g)	8.21 ± 0.69	9.10 ± 0.51	9.21 ± 0.87	8.18 ± 0.42	8.40 ± 0.56	8.41 ± 0.45	8.81 ± 0.57	8.13 ± 0.41
Adrenals (mg/100 g)	7.82 ± 0.60	8.37 ± 0.40	8.58 ± 0.77	7.18 ± 0.28	8.61 ± 0.51	8.79 ± 0.57	9.15 ± 0.42	9.04 ± 0.14
Testes or Ovaries (mg/100 g)	23.68 ± 1.31	19.68 ± 0.94 *	19.48 ± 1.69	16.35 ± 1.56	5.15 ± 1.19	4.33 ± 0.32	5.19 ± 0.43	3.84 ± 0.32

* Significantly different from the control at 5% level

** Significantly different from the control at 1% level

Table 5: Effects of d-limonene on gross differentiations of rabbit offsprings

	Control	250	500	1000
No. of examined offsprings	23	25	24	22
Days of gross differentiation after birth Opening of the ear-shell
6th day (%)	0	0	1 (4.2)	0
7th day (%)	23 (100)	25 (100)	23 (95.8)	22 (100)
Coating with the hair
2nd day (%)	7 (30.4)	0	0	0
3rd day (%)	16 (69.6)	25 (100)	24 (100)	22 (100)
Odontiasis
At birth (%)	23 (100)	25 (100)	24 (100)	22 (100)
Opening of the eyelids
9th day (%)	0	0	0	3 (13.6)
10th day (%)	11 (47.8)	4 (16.0)	13 (54.2)	5 (22.7)
11th day (%)	4 (17.4)	15 (60.0)	10 (41.7)	12 (54.5)
12th day (%)	3 (13.0)	5 (20.0)	1 (4.2)	2 (9.1)
13th day (%)	5 (21.7)	1 (4.0)	0	0

Table 6: Effects of d-limonene on postnatal development of rabbit offsprings

	Control	250	500	1000
No of dams	3	3	3	3
No. of still-birth (Male/Female)	1 (1/0)	0	0	1 (0/1)
No. of offsprings (Male/Female) At birth	28 (15/13)	27 (14/13)	26 (8/18)	27 (15/12)
1st week	28 (15/13)	27 (14/13)	25 (8/17)	26 (14/12)
2nd week	26 (14/12)	27 (14/13)	25 (8/17)	26 (14/12)
3rd week	24 (14/10)	27 (14/13)	25 (8/17)	25 (14/11)
4th week	23 (13/10)	26 (13/13)	25 (8/17)	22 (13/ 9)
5th week	23 (13/10)	25 (12/13)	25 (8/17)	22 (13/ 9)
6th week	23 (13/10)	25 (12/13)	25 (8/17)	22 (13/ 9)
7th week	23 (13/10)	25 (12/13)	24 (8/16)	22 (13/ 9)
Weanling rate (%)	79.3 (81.2/76.9)	92.6 (85.7/100)	92.3 (100/88.9)	78.6 (86.7/69.2)

Table 7: Postnatal examinations of rabbit offsprings

	Control	250	500	1000
No. of dams	3	3	3	3
No. of examined offsprings	23	25	24	22
Sensory function	Normal	Normal	Normal	Normal
External examination No. of malformed offsprings	0	0	0	0
Visceral examination No. of malformed offsprings	0	0	0	0
No. of minor abnormality Incomplete lobulation of lungs (%)	2 (8.7)	1 (4.0)	0	0
Accessory spleen (%)	2 (8.7)	0	0	0
Protrusion of gall bladder (%)	1 (4.3)	1 (4.0)	0	0
Skeletal examination No. of malformed offsprings	0	0	0	0
No. of variation Left lumbar rib (%)	4 (17.4)	4 (16.0)	4 (16.7)	4 (18.2)
Right lumbar rib (%)	2 (8.7)	6 (24.0)	6 (25.0)	4 (18.2)
Translocation of caudal vertebrae (%)	1 (4.3)	0	1 (4.2)	0
Ossification pattern Retarded ossification of 5th sternebrae (%)	0	2 (8.0)	0	1 (4.5)
Accessory ossification center of 5th sternebrae (%)	1 (4.3)	2 (8.0)	0	3 (13.6)

Conclusions:

Under the test conditions, d-limonene was not teratogenic in rabbit fetuses and the NOAEL for fetal toxicity was considered to be greater than 1000 mg/kg bw/day. The NOAEL for maternal toxicity was considered to be 250 mg/kg bw/day based on the decreased bodyweight gain.

Executive summary:

In a prenatal developmental toxicity study, d-limonene was administered orally to groups of pregnant Japanese white rabbits at dose levels of 250, 500 and 1000 mg/kg bw/day for 13 days from Day 6 to 18 of gestation. Food consumption and bodyweights of pregnant rabbits were recorded during organogenesis. Caesarean sections were performed and the number of dead, live or resorbed fetuses, sex ratio and number of implantation sites were recorded. Fetuses were weighed and examined for external, visceral and skeletal malformations. Treatment with the highest dose level (1000 mg/kg bw/day) of d-limonene resulted in death of 6/18 dams (33% mortality). The significant decrease of bodyweight gain and food consumption were temporarily observed in dams given 500 and 1000 mg/kg bw/day of d-limonene, but no anomalies were observed in the general behavior of dams given 250 and 500 mg/kg bw/day of d-limonene during the gestation. External examination of fetuses showed no anomalies. Visceral and skeletal examinations revealed some anomalies such as incomplete lobulation of the lungs, enlargement of the foramen ovale and retarded ossification of the middle phalanx of fore limbs in addition to the 5th sternebrae. These did not appear to be dose-dependent and restored to normal during the postnatal development. Other non specific anomalies involved the lumber ribs in fetuses and offsprings, formation of the accessory ossification center of the 5th sternebrae in offsprings and the atrial septal defect detected in only 2 fetuses of a litter from dams treated with 250 mg/kg bw/day of d-limonene. Under the test conditions, d-limonene was not teratogenic in rabbit fetuses and the NOAEL for fetal toxicity was considered to be greater than 1000 mg/kg bw/day. The NOAEL for maternal toxicity was considered to be 250 mg/kg bw/day based on the decreased bodyweight gain.

Reference 6

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Prenatal developmental toxicity study: Groups of pregnant Wistar rats (20/dose: 15 for teratogenicity study, 5 for postnatal development) were administered orally with d-limonene at dose levels of 0, 591 and 2869 mg/kg bw/day suspended with 1% gum-arabic solution for 7 days from Day 9 to 15 of gestation and evaluated for developmental toxicity.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

No data

Route of administration:

oral: unspecified

Vehicle:

other: 1% gum-arabic solution

Details on exposure:

Volume administered: 5 mL/kg bw for all doses

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

None

Details on mating procedure:

no data

Duration of treatment / exposure:

7 days (gestation Day 9-15)

Frequency of treatment:

Once daily

Duration of test:

Gestation Day 0 to postnatal week 7

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

591 mg/kg bw/day

Dose / conc.:

2 869 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20 pregnant rats

Control animals:

yes, concurrent vehicle

Details on study design:

No data

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data
General behaviour observed, but no data regarding the frequency of observation

BODY WEIGHT: Yes
- Time schedule for examinations: daily

POST-MORTEM EXAMINATIONS: No data

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: No data
Examinations included: Number of implantations, number of resorptions and foetus bodyweight and placental weight

Fetal examinations:

- External examinations: Yes
- Visceral examinations: Yes
- Skeletal examinations: Yes

Statistics:

statistical significance difference of effects from controls were calculated at 5% level.

Indices:

No data

Historical control data:

No data

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Deaths (40%) and decreased bodyweight gain at 2869 mg/kg bw/day

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Deaths (40%) and decreased bodyweight gain at 2869 mg/kg bw/day

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

591 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Delayed ossification of fetuses metacarpal bone and proximal phalanx at 2869 mg/kg bw/day was caused significantly, compared with the control group, but this was restored to normal within several weeks after birth.

Visceral malformations:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

A tendency of decreased bodyweight was noted in postnatal male offsprings born to mothers treated at 2869 mg/kg bw/day, compared with the control group.
Thymus, spleen and ovaries weights decreased in offsprings born to mothers treated at 2869 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Effect level:

591 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

other: Decreased bodyweight gain (male offsprings) and organ weights at 2869 mg/kg bw/day

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: skeletal: metacarpals and proximal phalanges

Description (incidence and severity):

Prolongation of the ossification of metacarpals and proximal phalanges

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

2 869 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

no

Relevant for humans:

not specified

Table 1: Body weight changes in pregnant rats treated orally with d-limonene

Dose (mg/kg bw)	Gestational days					Gain
	0	9	12	16	20
Control	214.80 ± 32.55	248.70 ± 28.92	265.60 ± 30.53	289.85 ± 35.01	325.30 ± 44.01	105.50 ± 29.67
591	221.25 ± 39.58	254.65 ± 41.16	264.80 ± 39.94	290.10 ± 38.37	325.60 ± 52.75	103.95 ± 19.38
2869	214.75 ± 4.57	258.75 ± 32.76	248.92 ± 26.27	263.17 ± 22.96 *	305.00 ± 27.07	90.25 ± 22.38

* Significantly different from the control, P <0.05

Table 2: Effects of d-limonene on rat fetuses

Dose (mg/kg bw)	Control	591	2869
No. of mothers	15	15	15
Mortality of mothers (%)	0	0	40
No. of total implants	12.73 ± 2.96	12.18 ± 3.65	10.44 ± 3.71
No. of dead fetuses	0	0	0
No. of resorbed fetuses	1.00 ± 1.10	1.47 ± 2.42	0.89 ± 0.73
No. of live fetuses	176	162	87
Sex ratio (Male/Female)	0.69	1.22	0.85
Fetuses body weight (g) Male	3.71 ± 0.45	3.53 ± 0.35	3.73 ± 0.52
Female	3.46 ± 0.44	3.38 ± 0.45	3.63 ± 0.40
Placental weight(g) Male	0.49 ± 0.07	0.49 ± 0.10	0.48 ± 0.06
Female	0.47 ± 0.07	0.46 ± 0.06	0.44 ± 0.05
Malformation External	0	0	0
Visceral	1	0	0

Table 3: Effects of d-limonene on skeletal development of rat fetuses

Dose (mg/kg bw)	Control	591	2869
No. of examined fetuses	83	84	42
Variation Shortness of 13th rid	1	0	0
Lumbar rid	0	1	2
Asymmetry of sternebrae	0	0	1
Ossification Delayed ossification of parietal bone	2	0	0
Non-ossification of occipital bone	0	4	1
Non-ossification of parietal bone	0	3	0
No. of ossified metacarpal bone	7.69 ± 0.72	7.49 ± 0.84	6.97 ± 0.96 *
No. of ossified proximal phalanx (Forelimb)	2.48 ± 1.71	2.25 ± 1.81	0.55 ± 1.28 *
No. of ossified metatarsal bone	7.98 ± 0.56	8.01 ± 011	8.00 ± 0
No. of ossified sternebraea	5.47 ± 0.98	5.60 ± 0.71	5.52 ± 0.73
No. of ossified caudal vertebrae	3.76 ± 0.64	3.80 ± 0.57	3.95 ± 0.68

* Significantly different from the control, P <0.05

Table 4: Body weight changes of postnatal rat offsprings born to mothers treated orally with d-limonene

Postnatal weeks	Males			Females
	Dose (mg/kg bw)			Dose (mg/kg bw)
	Control	591	2869	Control	591	2869
0	5.19 ± 0.55	5.46 ± 0.52	4.79 ± 0.46	4.93 ± 0.62	5.09 ± 0.68	4.89 ± 0.66
1	13.06 ± 1.50	12.49 ± 0.99	10.62 ± 1.54 *	12.82 ± 1.59	12.22 ± 1.07	10.66 ± 1.84
2	26.06 ± 3.12	24.86 ± 2.74	22.67 ± 5.05 *	25.88 ± 3.35	24.31 ± 2.42	22.72 ±.3.92
3	41.91 ± 5.89	39.55 ± 5.14	40.77 ± 5.16	41.08 ± 5.59	38.56 ± 4.37	38.39 ± 4.96
4	73.12 ± 9.89	71.33 ± 8.87	67.67 ± 8.02	69.66 ± 9.43	67.38 ± 6.71	66.01 ± 9.29
5	122.32 ± 12.25	116.47 ± 12.78	112.77 ± 12.65 *	109.57 ± 10.61	107.58 ± 7.95	107.10 ± 13.34
6	176.04 ± 15.80	164.68 ± 16.69	163.11 ± 17 .85 *	141.39 ± 10.54	140.11 ± 9.40	139.45 ± 14.22
7	235.52 ± 17.72	222.43 ± 18.57	213.64 ± 20.10 *	173.06 ± 8.89	169.65 ± 11.13	167.84 ± 15.86

* Significantly different from the control, P <0.05

Table 5: Effects of d-limonene on postnatal development of the rats

Dose (mg/ kg bw)	Days of postnatal development
	Opening of the ear-shell	Coating with the hair	Odontiasis	Opening of the eyelid	Descending of the testis	Opening of the vaginal orifice
Control	2.55 ± 0.76	5.51 ± 0.91	10.1 ± 0.96	14.83 ± 0.55	22.5 ± 1.30	35.6 ± 2.50
591	2.09 ± 0.82	6.00 ± 0	10.4 ± 0.71	15.00 ± 0.76	21.6 ± 1.39	35.5 ± 1.75
2869	2.41 ± 0.49	8.50 ±0.50	10.4 ± 1.85	15.14 ± 0.75	21.27 ± 0.57	35.93 ± 2.20

Table 6: Effects of d-limonene on development of rat offsprings

Dose (mg/kg)	Control	591	2869
No. of mothers	5	5	5
Mortality of mothers	0	0	40
No. of offspring from birth to the 7th week 0	61	65	33
1	53	63	30
2	53	63	30
3	53	63	28
4	53	63	28
5	53	63	28
6	53	63	28
7	53	63	28
External abnormality	0	0	0
No. of total implants	13.6 ± 3.1	14.8 ± 1.7	13.7 ± 1.7
No. of dead fetuses at birth	4	4	5
Parturient rate	95	92	93
Weaning rate	89	97	85

Table 7: Absolute organ weights of postnatal rat offsprings born to mothers treated orally with d-limonene

Sex

Dose (mg/kg bw)

No. of offsprings.

Final BW (g)

Pituitary (mg)

Thyroids (mg)

Thymus (g)

Lungs (g)

Heart (g)

Spleen (g)

Kidneys (g)

Liver (g)

Adrenals (g)

Testes (g) or Ovaries (mg)

Male

Control

27

235.5 ± 17.7

10.04 ± 1.95

14.06 ± 2.52

0.77 ± 0.08

1.15 ± 0.10

0.82 ± 0.08

0.75 ± 0.11

2.18 ± 0.36

11.55 ± 1.14

38.85 ± 7.37

2.15 ± 0.17

591

29

222.4 ± 18.6

9.58 ± 4.84

13.12 ± 2.77

0.72 ± 0.09

1.10 ± 0.11

0.81 ± 0.08

0.69 ± 0.08

2.10 ± 0.20

11.24 ± 1 .49

36.77 ± 6.93

2.13 ± 0.28

2869

11

213.6 ± 20.1

9.69 ± 0.65

14.41 ± 3.60

0.66 ± 0.08 *

1.19 ± 0.17

0.80 ± 0.07

0.63 ± 0.07 *

2.23 ± 0.38

11.64 ± 1.64

43.23 ± 10.91

2.18 ± 0.14

Female

Control

25

173.1 ± 8.9

11.18 ± 3.15

12.26 ± 1.32

0.59 ± 0.08

0.97 ± 0.11

0.66 ± 0.07

0.52 ± 0.06

1.72 ± 0.22

8.88 ± 0.85

45.51 ± 8.01

77.24 ± 22.01

591

34

169.7 ± 11.7

10.05 ± 3.34

11.57 ± 1.62

0.54 ± 0.07

0.96 ± 0.07

0.67 ± 0.05

0.50 ± 0.06

1.60 ± 0.15 *

8.16 ± 0.85

46.56 ± 8.45

85.84 ± 42.52 *

2869

17

167.8 ± 15.9

9.95 ± 1.87

12.31 ± 1.80

0.51 ± 0.07 *

0.94 ± 0.10

0.62 ± 0.06

0.44 ± 0.04 *

1.62 ± 0.17 *

8.50 ± 0.58

47.15 ± 6.63

63.15 ± 7.99

* Significantly different from the control, P <0.05

Table 8: Relative organ weights per 100 g body weights of postnatal rat offsprings born to mothers treated orally with d-limonene

Sex

Dose (mg/kg bw)

No. of offsprings.

Final BW (g)

Pituitary (mg/100 g)

Thyroids (mg/100 g)

Thymus (mg/100 g)

Lungs (mg/100 g)

Heart (mg/100 g)

Spleen (mg/100 g)

Kidneys (g/100 g)

Liver (g/100 g)

Adrenals (mg/100 g)

Testes or Ovaries (mg/100 g)

Male

Control

27

235.5 ± 17.7

4.31 ± 0.78

5.98 ± 0.99

0.33 ± 0.04

0.48 ± 0.04

0.36 ± 0.03

0.31 ± 0.05

0.93 ± 0.08

4.89 ± 0.36

16.49 ± 2.71

0.90 ± 0.04

591

29

222.4 ± 18.6

4.02 ± 0.50

5.93 ± 0.86

0.33 ± 0.04

0.49 ± 0.04

0.37 ± 0.04

0.33 ± 0.09

0.95 ± 0.07

5.10 ± 0.37

16.39 ± 2.53

0.95 ± 0.08 *

2869

11

213.6 ± 20.1

4.23 ± 0.43

5.93 ± 0.66

0.28 ± 0.03 *

0.51 ± 0.05

0.35 ± 0.02

0.27 ± 0.02 *

0.96 ± 0.06

5.03 ± 0.27

17.23 ± 2.26

0.95 ± 0.09

Female

Control

25

173.1 ± 8.9

6.09 ± 1.08

7.08 ± 0.85

0.34 ± 0.05

0.54 ± 0.04

0.38 ± 0.04

0.30 ± 0.04

0.99 ± 0.12

5.14 ± 0.42

26.38 ± 4.71

47.94 ± 9.78

591

34

169.7 ± 11.7

5.82 ± 0.81

6.85 ± 0.96

0.32 ± 0.04

0.54 ± 0.12

0.40 ± 0.03

0.30 ± 0.03

0.95 ± 0.07

4.83 ± 0.37

27.61 ± 3.76

46.74 ± 10.76

2869

17

167.8 ± 15.9

6.27 ± 1.90

7.31 ± 0.90

0.31 ± 0.03 *

0.57 ± 0.08

0.37 ± 0.03

0.27 ± 0.04 *

0.94 ± 0.06

5.14 ± 0.33

26.75 ± 2.93

36.89 ± 4.25 *

* Significantly different from the control, P <0.05

Conclusions:

Under the test conditions, the NOAEL for maternal toxicity was considered to be 591 mg/kg bw/day based on the deaths and decreased bodyweight gain. The NOAEL for fetal toxicity was considered to be 591 mg/kg bw/day based on the delayed skeletal formation and decreased bodyweight gain.

Executive summary:

In a developmental toxicity study, d-limonene was administered orally to groups of pregnant Wistar rats (20/dose: 15 for teratogenicity study, 5 for postnatal development) at dose levels of 0, 591 and 2869 mg/kg bw/day suspended with 1% gum-arabic solution for 7 days from Day 9 to 15 of gestation. Bodyweight of pregnant rats were recorded on Days 0, 9, 12, 16 and 20 during organogenesis. Caesarean sections were performed and the number of dead, live or resorbed fetuses, sex ratio and number of implantation sites were recorded. Fetuses were weighed and examined for external, visceral and skeletal malformations. Number of live offsprings, gross differentiation and organ weights of offsprings were recorded until postnatal week 7.

 

At 2869 mg/kg bw/day, maternal bodyweight decreased and several mothers (40%) died for a period of the treatment, but at 591 mg/kg bw/day, no changes were observed. Delayed ossification of fetuses metacarpal bone and proximal phalanx at 2869 mg/kg bw/day was caused significantly, compared with the control group, but this was restored to normal within several weeks after birth. A decreased tendency of bodyweight was noted in postnatal male offsprings born to mothers treated at 2869 mg/kg bw/day, compared with the control group. Thymus, spleen and ovaries weights decreased in offsprings born to mothers treated at 2869 mg/kg.

 

Under the test conditions, the NOAEL for maternal toxicity was considered to be 591 mg/kg bw/day based on the deaths and decreased bodyweight gain. The NOAEL for fetal toxicity was considered to be 591 mg/kg bw/day based on the delayed skeletal formation and decreased bodyweight gain.

Reference 7

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 11-05-2022 to 03-11-2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Biotechnology India Pvt. Ltd, Charles River Technology Licensee, CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) Registration No.: 1808/PO/RcBt/S/15/CPCSEA.
- Age at study initiation: 10-11 weeks (at receipt)
- Weight at study initiation: 258-272 g (range of averages across groups at gestation day 0)
- Housing: During all the phases of experiment (pre-mating/acclimatization/mating/post-mating), animals were housed in sterilized standard polycarbonate cage (size: L 43 x B 28 x H 21 cm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube.
Processed corncob granules were provided as bedding material and were changed along with the cage twice a week.
- Diet (e.g. ad libitum): ad libitum. Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG)
- Water (e.g. ad libitum): ad libitum. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimatization period: The healthy animals were acclimatized for a period of five (first batch of animals left for cohabitation) to nineteen days (last batch of animals left for cohabitation) to experimental room conditions before cohabitation and were observed for clinical signs once daily.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 to 22.9ºC
- Humidity (%): 47 to 64%
- Air changes (per hr): 12 to 15
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle.

IN-LIFE DATES: From: To: 11 May 2022 to 23 June 2022

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared and administered to the animals within the established stability periods. The required quantity of test item was weighed in a clean glass beaker. The test item was mixed with a small quantity of vehicle using glass rod and transferred into measuring cylinder. Again, a small quantity of vehicle was added to rinse the beaker and transferred into the measuring cylinder. The rinsing procedure of beaker was repeated many times to ensure the transfer of entire contents to the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get the desired concentrations of test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was clearly miscible with corn oil (non-aqueous solution) at the concentration of 225 mg/mL considering the dose volume of 4 mL/kg body weight as per the in-house miscibility test. Corn oil is the universally accepted vehicle for oral toxicity studies.
- Concentration in vehicle: 25, 75 and 225 mg/mL.
- Amount of vehicle (if gavage): 4 mL/kg bw.
- Lot/batch no. (if required): N12009002

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and dose formulation analysis for dose concentration verification was done by a validated analytical method UV-Vis spectrophotometer (study nº BIO-ANM 1956). The stability of the test item in dose formulations was established in this preliminary study. The test item formulations were stable at room temperature for 48 hours at the concentrations of 0.5 mg/mL and 250 mg/mL in corn oil. Dose formulations were prepared and administered to the animals within the established stability period.

Sampling and analysis of all dose formulations were performed during during first and last week (week 5) of the treatment. Approximately 5 mL of samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control. Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was ≤10%.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2 ratio (one male and two females)
- Length of cohabitation: until evidence of copulation was observed or for two weeks.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: Not described.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

Gestation days 5 to 20.

Frequency of treatment:

Once daily

Duration of test:

Acclimatization: 12 May 2022 to 30 May 2022
Cohabitation: 17 May 2022 to 02 June 2022
Treatment: 23 May 2022 to 22 June 2022
Necropsy: 08 June 2022 to 23 June 2022

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1 - Vehicle control

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

G2 - Low dose

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

G3 - Mid dose

Dose / conc.:

900 mg/kg bw/day (nominal)

Remarks:

G4 - High dose

No. of animals per sex per dose:

25 presumed pregnant females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses of 100, 300 and 900 mg/kg/day were selected based on the results of a OECD 422 study (Study No. BIO-DTX 002) and a 14-Day Dose range finding study (BIO-DTX 001). In the 14-Day DRF study, 100, 300 and 1000 mg/kg/day were tested wherein one mortality was observed at 1000 mg/kg/day. Also as per BIO-DTX 002 study, although not considered adverse, the dose of 300 mg/kg/day [mid dose] revealed treatment related clinical signs of toxicity and reduction in body weight, the dose of 600 mg/kg/day [high dose] revealed treatment related effects in feed consumption, increased copulatory interval and reduced gestation length.
- Fasting period before blood sampling for (rat) dam thyroid hormones: rats not fasted.
- Time of day for (rat) dam blood sampling: within a 2-hour time period on the morning of the day of caesarean section/necropsy.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on gestation days (GD) 0, 3, 5, 8, 11, 14, 17, 20 and 21 (day of caesarean section for the study animals).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes / No / No data
- Individual animal feed intake of mated females was recorded during gestation day
(GD) 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17, 17 to 20 and 20 to 21.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: Ovaries, Uterus with cervix, thyroid and parathyroid glands. Histopathological examination was conducted for thyroid along with the parathyroid from all the dose group animals sacrificed at termination.

OTHER: Blood samples were collected before caesarean section, on GD 21, from all dams (including non-pregnant females) for assessment of thyroid hormones (T4, T3 and TSH).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

- Plasma: No
- Serum: Yes
- Volume collected: From each animal 3 blood samples of approximately 1 mL each

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: individual fetus was measured for its anogenital distance on the day of caesarean section.

Statistics:

After verification, the data was subjected to various statistical analysis using SPSS software version 27. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05). The statistical analysis was followed to the parameters as mentioned in the table below.

Indices:

- Corrected Body weight (g) = (Gestation day 20 body weight - Gestation day 5 body weight) Gravid uterus weigh
- Percent of Live Fetuses per dam = (Number of Live Fetuses / Litter Size) x 100
- Percent of Dead Fetuses per dam = (Number of Dead Fetuses/ Litter Size) x 100
- Percent of Early Resorptions (%) per dam = (Number of Early Resorptions/Number of Implantation sites) x 100
- Percent of Late Resorptions (%) per dam = (Number of Late Resorptions / Number of Implantation sites)x 100
- Pre-implantation Loss (%) per dam = [(Number of Corpora lutea - Number of Implantation sites)/Number of Corpora lutea]x 100
- Post-implantation Loss (%) per Dam = [(Number of Implantation sites - Number of Viable fetuses )/ Number of Implantation sites]x 100
- Sex Ratio (m/f) = Number of live male fetuses / Number of live female fetuses
- Male/Female Fetuses (%) = (Number of live male/female fetuses/ Total number of live fetuses)x 100
- Ano-genital Distance Ratio = Ano-genital distance (mm) / Cube root of Fetal weight (g)
- Fetal incidence (%) = (Number of Fetuses with particular observation per group/Total number of Fetuses examined per group)x 100
- Litter incidence (%) = (Number of Litters/dams with particular observation per group/Total number of Litters per group) x 100

Historical control data:

Historical control data of the same species and strain was used for comparisons but information not provided in the report.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity noted in any of the animals from all the tested dose groups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no morbidity/mortality noted at all the tested dose groups and vehicle control group animals during the experimental period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Gestation/maternal body weight gain:
In group G2, there were no changes noted in mean gestational (maternal) body weight and percent change in mean gestational (maternal) body weight gain during pregnancy throughout the treatment period when compared with the vehicle control group.
In group G3, a slight reduction in percent change in mean gestational (maternal) body weight gain was noted during mid-pregnancy. This reduction is statistically significant during GD 8 to 11 when compared with the vehicle control group. However, during late pregnancy the animals shown normal increase in body weight.
In group G4, a slight reduction in percent change in mean gestational (maternal) body weight gain was noted during early and mid-pregnancy. These reductions are statistically significant during GD 5 to 8 and 8 to 11 when compared with the vehicle control group. However, during late pregnancy the animals shown normal increase in body weight.

Gravid uterus weight and Corrected body weight for maternal gain:
In groups G2 and G3, there were no changes noted in mean gravid uterus weight and mean maternal body weight change corrected for gravid uterus weight when compared with the vehicle control group.
In group G4, a statistically significant reduction in mean maternal body weight change corrected for gravid uterus weight (both absolute and percent) when compared with the vehicle control group was observed. These changes are considered as a secondary effect caused due to stress induced by the test item exposure, but not adverse.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In groups G2 and G3, there were no changes noted in mean gestational (maternal) feed consumption when compared with the vehicle control group.
In group G4, a minimal and statistically significant reduction in mean gestational (maternal) feed consumption during GD 20 to 21 was noted when compared with the vehicle control group. This change is considered as a secondary effect caused due to stress induced by the test item exposure, but not adverse.
The noted statistically significant increase in mean feed consumption during GD 3 to 5 in group G4 is considered incidental and toxicologically irrelevant.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes noted for mean serum T3 levels in any of the tested dose groups when compared to vehicle control group. The noted statistically significant decrease in mean serum T3 levels in group G4 is considered as incidental and un-related to treatment.
There were no changes noted for mean serum T4 levels in any of the tested dose groups when compared to vehicle control group.
There were no test item-related changes noted for mean serum TSH levels in any of the tested dose groups when compared to vehicle control group. The noted statistically significant decrease in mean serum TSH levels in group G4 is considered as incidental and unrelated to treatment as the obtained mean values are well within historical control range of same species and strain and also due to lack of dose dependency.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes noted in any of the animals from all the tested dose groups and vehicle control group during conduct of the necropsy.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related histopathological findings in thyroid and parathyroid glands of all treated animals.
However, ultimobranchial cysts and ectopic thymus in thyroid gland were noted in this study. These changes were considered incidental findings as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats (Elizabeth McInnes, 2012).

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions observed in any of the animals from all the tested dose groups.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

The mean number of corpora lutea per litter was 11.81, 10.86, 11.38 and 11.68 for groups G1, G2, G3 and G4 respectively. There were no changes or no statistically significant differences noted in all the tested dose groups when compared with vehicle control group for mean number of corpora lutea.

The mean number of implantations per litter was 11.19, 9.52, 10.57 and 11.05 for groups G1, G2, G3 and G4 respectively. There were no test item related changes or no statistically significant differences noted in all the tested dose groups when compared with vehicle control group for mean number of implantations.

The mean percentage of pre-implantation loss per litter was 7.50, 14.93, 10.13 and 11.01 for groups G1, G2, G3 and G4 respectively. There were no changes or no statistically significant differences noted for percentage of pre-implantation loss per litter in all the tested dose groups.

The mean percentage of post-implantation loss per litter was 14.04, 15.93, 22.35 and 21.01 for groups G1, G2, G3 and G4 respectively. There were no changes noted for mean percentage of post-implantation loss per litter in group G2 when compared to the vehicle control group. However, the occurrences were little high in both mid and high dose groups when compared to the vehicle control group. This increase in post-implantation loss per litter can be considered as treatment related due to noted increase in no. of resorptions at these dose levels.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The following incidences of early resorptions were noted from the dams with total implantation loss:
- In group G1, 1 (out of 25) presumed pregnant females was evidenced with only
2 implantation sites and both were early resorptions.
- In group G3, 2 (out of 25) presumed pregnant females was evidenced with implantation sites, one dam noted with 8 early resorptions and the other with single early resorption.
The following incidences of late resorptions were noted from the dams with total implantation loss:
- In group G2, 1 (out of 25) presumed pregnant females was evidenced with single implantation site consisted of late resorption.
- In group G4, 3 (out of 25) presumed pregnant females was evidenced with implantation sites, two dams were noted with a single late resorption and another dam noted with two late resorptions.

The interpretation of results was made by excluding the data of dams with total implantation loss.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

There were no early resorptions noted in any of the dams with live fetuses (G1: 21 dams; G2: 21 dams; G3: 21 dams and G4: 19 dams) during caesarean sectioning from all the tested and vehicle control groups.

The mean number of late resorptions per litter (dam with live fetuses) was 1.10, 1.10, 1.90 and 1.68 and the percentage of late resorptions was 14.04, 15.93, 22.35 and 18.91 for groups G1, G2, G3 and G4 respectively. There were no statistically significant differences noted for mean number and percentage of late resorptions per dam across dose groups when compared to the vehicle control group. However, a slight increase in occurrences of mean number of late resorptions per litter was noted in groups G3 and G4 when compared with vehicle control group. These noted changes are not considered as adverse but can be considered as secondary effects caused due to test item exposure.

Dead fetuses:

no effects observed

Description (incidence and severity):

The mean number of dead fetuses per litter was 0.00, 0.00, 0.00 and 0.11 with a percentage of 0.00, 0.00, 0.00 and 2.63. There were no statistically significant differences noted for mean number of dead fetuses in all the tested dose groups when compared to the vehicle control group.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

A total of 21 (out of 25), 21 (out of 25), 21 (out of 25) and 19 (out of 25) females from group G1, G2, G3 and G4 were found with live fetuses yielding to pregnancy with pregnancy rates of 84%, 84%, 84% and 76% respectively.

Other effects:

no effects observed

Description (incidence and severity):

A total of 212 (21), 177 (21), 182 (21) and 176 (19) fetuses (litters) were available for gross maternal-fetal examinations from G1, G2, G3 and G4 respectively. There were no test item-related maternal-fetal alterations such as changes in amniotic fluid, placental and umbilical cord noted in any of the fetuses examined from all the tested dose group litters and also no statistically significant differences occurred.
The noted single occurrence of discoloration of amniotic fluid from group G3 is considered incidental.

Key result

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

early or late resorptions

pre and post implantation loss

Remarks on result:

other: Related treatment non-adverse effects at mid dose (G3, 300 mg/kg bw/d)

Key result

Dose descriptor:

NOAEL

Effect level:

900 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: Highest concentration tested, no adverse effects observed

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no changes noted in mean fetal weight of either sex across the dose groups when compared with the vehicle control group.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The mean number of live fetuses per litter was 10.10, 8.43, 8.67 and 9.26 with a percentage of 100.00, 100.00, 100.00 and 97.37 for both sexes combined; 5.57, 4.24, 5.00 and 4.47 for male fetuses per litter with a percentage of 59.72, 47.83, 56.50 and 50.30; and 4.52, 4.19, 3.67 and 4.79 for female fetuses per litter with a percentage of 40.28, 52.17, 43.50 and 49.70 in groups G1, G2, G3 and G4 respectively. There were no statistically significant differences noted for mean number of live fetuses (combined and or sex wise) in all the tested dose groups when compared to the vehicle control group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex ratio per litter calculated as the mean of male/female fetuses per litter was
1.48, 1.01, 1.73 and 1.07 for groups G1, G2, G3 and G4 respectively. There were no test item related changes noted and no statistically significant differences noted for mean male/female sex ratio across the dose groups when compared to the control group.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean litter sizes, assessed as the total number of fetuses in uteri (live plus dead) per dam, was 10.10, 8.43, 8.67 and 9.37 for groups G1, G2, G3 and G4, respectively.
There were no changes or no statistically significant differences noted in all the tested dose groups when compared with vehicle control group for mean litter size.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

There were no changes noted in mean fetal anogenital distance and mean fetal anogenital distance ratio per litter of either sex across the dose groups when compared with the vehicle control group.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related fetal structural and developmental external malformations or variations noted for any of the fetuses examined from all the tested dose group litters and also no statistically significant differences occurred.
Also, some sporadic fetal external alterations (see table below) were noted across the dose groups and vehicle control group. These developmental and structural alterations are considered incidental as these observations are comparable with the vehicle control group and/or also these developmental alterations are common for this species and strain.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related fetal structural malformations or variations noted for any of the fetuses examined from all the tested dose group litters during skeletal examination. The noted statistically significant increase in number and percent of fetal incidences of unossified sternebra no. 1 in group G3, decrease in number and percent of fetal incidences of dumbbell shaped thoracic vertebra no. 13 in all the tested dose group when compared with vehicle control group is considered as incidental and toxicologically insignificant.

Also, some sporadic fetal skeletal alterations (see table below) were noted across the dose groups and vehicle control group. These structural and skeletal alterations are considered incidental as these observations are comparable with the vehicle control group and also these alterations are common for this species and strain. Also, the noted alterations in ossifications, i.e. delayed ossification, incomplete ossification, dumbbell shaped ossification from various regions from all the tested dose groups and control group are considered as transient findings and are reported to resolve shortly with the development of fetus (Reference: Review article: Bone development in laboratory mammals used in developmental toxicity studies, John M. DeSesso and Anthony R. Scialli).

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related fetal structural and developmental visceral/soft tissue malformations or variations noted for any of the fetuses examined from all the tested dose group litters and also no statistically significant differences occurred.
Also, some sporadic fetal visceral/soft tissue and cranial alterations (see table below) were noted across the dose groups and vehicle control group. These developmental soft tissue alterations are considered incidental as these observations are comparable with the vehicle control group and also these developmental alterations are common for this species and strain.

Other effects:

no effects observed

Description (incidence and severity):

There were no changes noted in mean crown rump length per litter of either sex across the dose groups when compared with the vehicle control group.

Key result

Dose descriptor:

NOAEL

Effect level:

900 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest dose tested, no related treatment effects observed

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

TABLE 1. SUMMARY OF CLINICAL SIGNS OF TOXICITY AND MORTALITY RECORD

Group & Dose (mg/kg body weight/day)	Clinical Signs and Mortality Record
	Clinical Signs of Toxicity (No. of Animals revealed)	No. of Mortalities / Total No. of animals
G1 & 0	N (25)	0/25
G2 & 100	N (25)	0/25
G3 & 300	N (25)	0/25
G4 & 900	N (25)	0/25

N: Normal.

 

TABLE 2. SUMMARY OF GESTATION BODY WEIGHT (g) RECORD 

Group & Dose (mg/kg body weight/day)		Body Weight (g) on Gestation Day
		0	3	5	8	11	14	17	19	20	21
G1 & 0	Mean	262.42	274.00	283.44	293.51	307.44	321.51	343.98	377.97	393.02	262.42
	±SD	15.39	15.42	17.01	17.14	16.50	21.48	23.00	32.44	34.72	15.39
	n	21	21	21	21	21	21	21	21	21	21
G2 & 100	Mean	258.24	270.27	278.76	289.62	302.59	318.08	335.77	367.16	377.72	258.24
	±SD	20.09	20.25	20.25	22.50	22.50	22.41	25.08	35.06	39.83	20.09
	n	21	21	21	21	21	21	21	21	21	21
G3 & 300	Mean	268.38	280.61	288.87	298.65	308.74	318.60	338.69	371.24	384.74	268.38
	±SD	20.28	21.27	22.64	23.43	24.80	26.23	28.34	34.03	37.06	20.28
	n	21	21	21	21	21	21	21	21	21	21
G4 & 900	Mean	271.79	285.87	294.50	298.95	307.45	320.69	340.01	368.15	378.36	271.79
	±SD	19.77	20.29	20.36	20.01	22.76	20.94	22.07	35.04	42.53	19.77
	n	19	19	19	19	19	19	19	19	19	19

SD: Standard Deviation; n: Number of Dams.            

Note: The data of 3, 3, 2 & 3 non-pregnant animals from groups G1, G2, G3 and G4 respectively and the data of 1, 1, 2 & 3 dams with total implantation loss from groups G1, G2, G3 and G4 respectively, are excluded for mean calculations and statistical analysis, but presented in individual animal data.  

 

TABLE 3. SUMMARY OF PERCENT CHANGE IN GESTATION BODY WEIGHT (%) RECORD 

Group & Dose (mg/kg body weight/day)		Percent Change in Body Weight Gain (%) during Gestation Day
		0 to 3	3 to 5	5 to 8	8 to 11	11 to 14	14 to 17	17 to 19	19 to 20	20 to 21
G1 & 0	Mean	4.44	3.43	3.57	4.79	4.54	7.03	9.78	3.97	4.44
	±SD	1.54	1.15	1.80	1.93	2.98	3.06	3.74	1.81	1.54
	n	21	21	21	21	21	21	21	21	21
G2 & 100	Mean	4.69	3.17	3.87	4.51	5.17	5.57	9.28	2.81	4.69
	±SD	1.50	1.42	1.90	1.45	2.04	2.71	4.96	1.81	1.50
	n	21	21	21	21	21	21	21	21	21
G3 & 300	Mean	4.57	2.94	3.40	3.38*	3.21	6.35	9.61	3.61	4.57
	±SD	1.54	1.70	2.06	1.88	2.46	3.41	4.13	2.08	1.54
	n	21	21	21	21	21	21	21	21	21
G4 & 900	Mean	5.20	3.04	1.55*	2.81*	4.39	6.07	8.16	2.62	5.20
	±SD	1.34	1.37	2.13	2.00	2.37	3.36	5.71	2.25	1.34
	n	19	19	19	19	19	19	19	19	19

SD: Standard Deviation; n: Number of Dams.            

Note: The data of 3, 3, 2 & 3 non-pregnant animals from groups G1, G2, G3 and G4 respectively and the data of 1, 1, 2 & 3 dams with total implantation loss from groups G1, G2, G3 and G4 respectively, are excluded for mean calculations and statistical analysis, but presented in individual animal data. 

*: Statistically significant (P<0.05) change than the control group.

 

TABLE 4. SUMMARY OF GRAVID UTERUS WEIGHT (g) AND MATERNAL BODY WEIGHT CHANGE CORRECTED FOR GRAVID UTERINE WEIGHT (g) 

Group & Dose (mg/kg body weight/day)		Body Weight Change (g) from GD 5 to GD 20	Gravid Uterus Weight (g)	Corrected Body weight
				g	%
G1 & 0	Mean	109.58	77.82	31.76	11.28
	±SD	29.91	28.04	14.32	5.12
	n	21	21	21	21
G2 & 100	Mean	98.96	64.14	34.82	12.49
	±SD	27.89	32.47	18.25	6.52
	n	21	21	21	21
G3 & 300	Mean	95.86	62.97	32.90	11.42
	±SD	30.47	31.28	12.16	4.15
	n	21	21	21	21
G4 & 900	Mean	83.86	65.16	18.70*	6.28*
	±SD	38.68	37.48	22.56	7.91
	n	19	19	19	19

*: Statistically significant (P<0.05) change than the control group. 

 

TABLE 5. SUMMARY OF AVERAGE GESTATION FEED CONSUMPTION (g/animal/day) RECORD 

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g): GD 0 to 3	Feed Consumption (g): GD 3 to 5	Feed Consumption (g): GD 5 to 8	Feed Consumption (g): GD 8 to 11	Feed Consumption (g): GD 11 to 14	Feed Consumption (g): GD 14 to 17	Feed Consumption (g): GD 17 to 20	Feed Consumption (g): GD 20 to 21
G1, F & 0	Mean	18.22	21.66	20.23	22.09	24.03	25.51	27.87	27.91
	SD	2.44	3.71	2.55	2.87	3.14	3.52	3.36	2.30
	n	21	21	21	21	21	21	21	21
G2, F & 100	Mean	18.64	21.25	19.73	21.94	25.04	25.44	27.81	26.10
	SD	2.46	2.77	2.91	2.83	2.28	2.92	3.69	3.20
	n	21	21	21	21	21	21	21	21
G3, F & 300	Mean	18.85	22.30	19.83	21.83	22.88	24.92	28.52	27.00
	SD	2.91	4.02	4.46	4.08	3.02	3.36	2.25	3.32
	n	21	21	21	21	21	21	21	21
G4, F & 900	Mean	19.28	24.99*	18.51	20.94	23.08	25.20	27.84	24.63*
	SD	2.11	4.31	3.50	3.06	3.60	3.45	3.46	4.41
	n	19	19	19	19	19	19	19	19

*: Statistically significant (P<0.05) change than the control group. 

 

TABLE 6. SUMMARY OF UTERI OBSERVATIONS PER LITTER RECORD 

 

Excluding the data of dams with total implantation loss:

Group & Dose (mg/kg body weight/day)		Corporaluetea (No.)	Implantation sites (No.)	Litter Size (No.)	Live Fetuses (No.)			Dead Fetuses (No.)	Early Resorptions (No.)	Late Resorptions (No.)
					Total	Male	Female
G1 & 0	Mean	11.81	11.19	10.10	10.10	5.57	4.52	0.00	0.00	1.10
	±SD	3.28	3.74	4.21	4.21	2.52	2.44	0.00	0.00	1.26
	n	21	21	21	21	21	21	21	21	21
G2 & 100	Mean	10.86	9.52	8.43	8.43	4.24	4.19	0.00	0.00	1.10
	±SD	3.95	4.62	5.13	5.13	3.18	2.91	0.00	0.00	1.34
	n	21	21	21	21	21	21	21	21	21
G3 & 300	Mean	11.38	10.57	8.67	8.67	5.00	3.67	0.00	0.00	1.90
	±SD	4.24	4.62	4.67	4.67	3.19	2.42	0.00	0.00	1.37
	n	21	21	21	21	21	21	21	21	21
G4 & 900	Mean	11.68	11.05	9.37	9.26	4.47	4.79	0.11	0.00	1.68
	±SD	5.18	5.53	5.88	6.00	3.41	3.44	0.46	0.00	2.43
	n	19	19	19	19	19	19	19	19	19

 

Including the data of pregnant animals and dams with total implantation loss:

Group & Dose (mg/kg body weight/day)		Corporaluetea (No.)	Implantation sites (No.)	Litter Size (No.)	Live Fetuses (No.)			Dead Fetuses (No.)	Early Resorptions (No.)	Late Resorptions (No.)
					Total	Male	Female
G1 & 0	Mean	11.45	10.77	9.64	9.64	5.32	4.32	0.00	0.09	1.05
	±SD	3.61	4.14	4.63	4.63	2.73	2.57	0.00	0.43	1.25
	n	22	22	22	22	22	22	22	22	22
G2 & 100	Mean	10.50	9.14	8.05	8.05	4.05	4.00	0.00	0.00	1.09
	±SD	4.21	4.86	5.32	5.32	3.23	2.98	0.00	0.00	1.31
	n	22	22	22	22	22	22	22	22	22
G3 & 300	Mean	10.87	10.04	7.91	7.91	4.57	3.35	0.00	0.39	1.74
	±SD	4.44	4.86	5.11	5.11	3.37	2.53	0.00	1.67	1.42
	n	23	23	23	23	23	23	23	23	23
G4 & 900	Mean	10.55	9.73	8.09	8.00	3.86	4.14	0.09	0.00	1.64
	±SD	5.62	6.16	6.36	6.44	3.52	3.60	0.43	0.00	2.26
	n	22	22	22	22	22	22	22	22	22

SD: Standard Deviation; n: Number of Dams.

 

TABLE 7. SUMMARY OF MATERNAL DATA PER LITTER RECORD 

 

Excluding the data of dams with total implantation loss:

Group & Dose (mg/kg body weight/day)		Pre-implantation loss (%)	Post-Implantation Loss (%)	Live Fetuses (%)	Dead Fetuses (%)	Early Resorptions (%)	Late Resorptions (%)	Male/Female Sex ratio	Male Fetuses (%)	Female Fetuses (%)
G1 & 0	Mean	7.50	14.04	100.00	0.00	0.00	14.04	1.48	59.72	40.28
	±SD	13.17	20.74	0.00	0.00	0.00	20.74	0.87	18.81	18.81
	n	21	21	21	21	21	21	19	21	21
G2 & 111	Mean	14.93	15.93	100.00	0.00	0.00	15.93	1.01	47.83	52.17
	±SD	16.85	21.53	0.00	0.00	0.00	21.53	0.76	25.31	25.31
	n	21	21	21	21	21	21	18	21	21
G3 & 333	Mean	10.13	22.35	100.00	0.00	0.00	22.35	1.72	56.50	43.50
	±SD	9.42	21.26	0.00	0.00	0.00	21.26	1.93	23.90	23.90
	n	21	21	21	21	21	21	18	21	21
G4 & 1000	Mean	11.01	21.01	97.37	2.63	0.00	18.91	1.07	50.30	49.70
	±SD	18.53	29.81	11.47	11.47	0.00	28.27	0.78	29.39	29.39
	n	19	19	19	19	19	19	14	19	19

 

Including the data of pregnant animals and dams with total implantation loss:

Group & Dose (mg/kg body weight/day)		Pre-implantation loss (%)	Post-Implantation Loss (%)	Live Fetuses (%)	Dead Fetuses (%)	Early Resorptions (%)	Late Resorptions (%)	Male/Female Sex ratio	Male Fetuses (%)	Female Fetuses (%)
G1 & 0	Mean	9.44	13.40	95.45	0.00	4.55	13.40	1.40	52.71	42.29
	±SD	15.72	20.46	21.32	0.00	21.32	20.46	0.91	18.37	16.81
	n	22	22	22	22	22	22	20^@	20^@	20^@
G2 & 111	Mean	17.28	15.21	95.45	0.00	0.00	19.76	0.96	42.33	52.40
	±SD	19.80	21.28	21.32	0.00	0.00	27.62	0.77	19.42	20.81
	n	22	22	22	22	22	22	19^@	19^@	19^@
G3 & 333	Mean	12.15	20.41	91.30	0.00	8.70	20.41	1.55	49.32	40.68
	±SD	15.04	21.27	28.81	0.00	28.81	21.27	1.91	22.72	20.62
	n	23	23	23	23	23	23	20^@	20^@	20^@
G4 & 1000	Mean	17.84	18.15	84.09	2.27	0.00	29.96	0.88	38.57	43.78
	±SD	24.76	28.57	35.81	10.66	0.00	38.69	0.82	22.82	24.87
	n	22	22	22	22	22	22	17^@	17^@	17^@

SD: Standard Deviation; n: Number of Dams

@: The data of 2, 3, 3 & 5 dams noted with single sex (male or female) from groups G1, G2, G3 and G4 respectively is excluded for mean calculations.

 

TABLE 8 SUMMARY OF ABSOLUTE ORGAN WEIGHT (g), TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT RELATIVE TO TERMINAL BODY WEIGHT (%) RECORD

Group & Dose (mg/kg body weight/day)		Absolute Thyroid along with parathyroid weight (g)	Terminal Body Weight (g)	Relative Thyroid along with parathyroid weight (%)
G1 & 0	Mean	0.0211	378.83	0.0056
	±SD	0.0032	46.28	0.0010
	n	25	25	25
G2 & 100	Mean	0.0249*	369.34	0.0068*
	±SD	0.0052	44.15	0.0015
	n	25	25	25
G3 & 300	Mean	0.0246*	374.42	0.0066
	±SD	0.0050	43.35	0.0013
	n	25	25	25
G4 & 900	Mean	0.0238	362.40	0.0067*
	±SD	0.0053	49.15	0.0017
	n	25	25	25

*: Statistically significant (P<0.05) change than the control group.

 

TABLE 9. SUMMARY OF SERUM TRIIODOTHYRONINE (T3) LEVELS (ng/mL) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Serum T3 Levels (ng/mL)
G1 & 0	Mean	2.208
	±SD	0.177
	n	21
G2 & 100	Mean	2.282
	±SD	0.240
	n	21
G3 & 300	Mean	2.138
	±SD	0.172
	n	21
G4 & 900	Mean	2.043*
	±SD	0.138
	n	19

SD: Standard Deviation; n: Number of Dams; ng/mL: Nano gram per milliliter.

*: Statistically significant (P<0.05) change than the control group 

 

TABLE 10 SUMMARY OF SERUM THYROXINE (T4) LEVELS (ng/mL) RECORD 

Group, Sex & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
G1 & 0	Mean	61.357
	±SD	12.401
	n	21
G2 & 100	Mean	60.950
	±SD	11.226
	n	21
G3 & 300	Mean	67.155
	±SD	11.976
	n	21
G4 & 900	Mean	61.267
	±SD	15.443
	n	19

SD: Standard Deviation; n: Number of Dams; ng/mL: Nano gram per milliliter. 

 

TABLE 11 SUMMARY OF SERUM THYROID STIMULATING HORMONE (TSH) LEVELS (μIU/mL) RECORD 

Group, Sex & Dose (mg/kg body weight/day)		Serum TSH Levels (µIU/mL)
G1 & 0	Mean	1.050
	±SD	0.220
	n	21
G2 & 100	Mean	1.033
	±SD	0.264
	n	21
G3 & 300	Mean	1.129
	±SD	0.317
	n	21
G4 & 900	Mean	0.766*
	±SD	0.275
	n	19

SD: Standard Deviation; n: Number of Dams.

*: Statistically significant (P<0.05) change than the control group. 

 

TABLE 12 SUMMARY OF MEAN FETAL WEIGHT, MEAN FETAL CROWN RUMP LENGTH, MEAN FETAL ANOGENITAL DISTANCE AND RATIO RECORD 

Group & Dose (mg/kg body weight/day)		Fetal weight (g)			Crown rump Length (mm)			Anogenital distance measurement (mm)			Cube root of fetal weight			Anogenital distance ratio
		Male	Female		Male	Female		Male	Female		Male	Female		Male	Female
G1 & 0	Mean	5.64	5.32		41.46	40.06		4.66	2.52		1.78	1.74		2.60	1.45
	±SD	0.48	0.38		1.86	1.81		0.17	0.16		0.05	0.04		0.19	0.09
	n	21	19		21	19		21	19		21	19		21	19
G2 & 100	Mean	5.66	5.38		41.20	40.11		4.59	2.54		1.78	1.75		2.59	1.46
	±SD	0.87	0.71		1.86	1.81		0.21	0.11		0.09	0.08		0.16	0.07
	n	20	19		20	19		20	19		20	19		20	19
G3 & 300	Mean	5.35	4.90		40.16	39.45		4.61	2.51		1.74	1.69		2.65	1.49
	±SD	0.81	0.75		1.98	2.25		0.22	0.18		0.09	0.09		0.15	0.12
	n	20	19		20	19		20	19		20	19		20	19
G4 & 900	Mean	5.44	5.16		40.84	39.17		4.59	2.60		1.75	1.72		2.62	1.51
	±SD	0.84	0.88		2.63	1.79		0.27	0.12		0.09	0.10		0.14	0.12
	n	17	16		17	16		17	16		17	16		17	16

SD: Standard Deviation; n: Number of Dams.

Note: The presented mean values are group mean of all litters from each group which was calculated from mean of individual fetal values obtained from each litter sex wise.  

 

TABLE 13 SUMMARY RECORD OF FETAL EXTERNAL EXAMINATION PER LITTER 

Variations

Group & Dose (mg/kg body weight/day)			G1 & 0		G2 & 100		G3 & 300		G4 & 900
Litters Evaluated for External Examination		No.	21		21		21		19
Fetuses Evaluated for External Examination		No.	211		177		182		176
Region/ Tissue↓	Alteration↓
General	Subcutaneous haemorrhagic spot
	Litter Incidences	No. (%)	7	33.3	4	19.0	4	19.0	6	31.6
	Fetal Incidences	No. (%)	12	5.7	6	3.4	7	3.8	7	4.0

No.: Number; %: Percentage. 

 

TABLE 14 SUMMARY OF FETAL VISCERAL EXAMINATION PER LITTER RECORD 

Variations                                                                                                                                                                                                                                             

Group & Dose (mg/kg body weight/day)			G1 & 0		G2 & 100		G3 & 300		G4 & 900
Litters Evaluated for Visceral Examination		No.	21		21		21		19
Fetuses Evaluated for Visceral Examination		No.	103		85		89		87
Region/Tissue↓	Alteration↓
Ureters	Dilation (Bilateral)
	Litter Incidences	No. (%)	0	0.0	1	4.8	1	4.8	0	0.0
	Fetal Incidences	No. (%)	0	0.0	1	1.2	1	1.1	0	0.0
Kidneys	Renal pelvis dilation (Bilateral)
	Litter Incidences	No. (%)	5	23.8	0	0.0	2	9.5	1	5.3
	Fetal Incidences	No. (%)	6	5.8	0	0.0	2	2.2	1	1.1
Spleen	Discoloured
	Litter Incidences	No. (%)	4	19.0	1	4.8	1	4.8	0	0.0
	Fetal Incidences	No. (%)	4	3.9	1	1.2	1	1.1	0	0.0
Lungs	Discoloured
	Litter Incidences	No. (%)	1	4.8	0	0.0	0	0.0	0	0.0
	Fetal Incidences	No. (%)	1	1.0	0	0.0	0	0.0	0	0.0

 No.: Number; %: Percentage; * Statistically significant (P<0.05) change than the control group. 

Malformation                                                                                                                                                                                  

Group & Dose (mg/kg body weight/day)			G1 & 0		G2 & 100		G3 & 300		G4 & 900
Litters Evaluated for Visceral Examination		No.	21		21		21		19
Fetuses Evaluated for Visceral Examination		No.	103		85		89		87
Region/Tissue↓	Alteration↓
Brain	Dilated lateral ventricles
	Litter Incidences	No. (%)	1	4.8	0	0.0	1	4.8	1	5.3
	Fetal Incidences	No. (%)	1	1.0	0	0.0	1	1.1	1	1.1
Eye	Retina fold
	Litter Incidences	No. (%)	1	4.8	0	0.0	0	0.0	0	0.0
	Fetal Incidences	No. (%)	1	1.0	0	0.0	0	0.0	0	0.0
Eye	Altered texture
	Litter Incidences	No. (%)	0	0.0	0	0.0	1	4.8	0	0.0
	Fetal Incidences	No. (%)	0	0.0	0	0.0	1	1.1	0	0.0
Naso-pharyngeal tract	Nasal conchae enlarge
	Litter Incidences	No. (%)	1	4.8	1	4.8	0	0.0	0	0.0
	Fetal Incidences	No. (%)	1	1.0	1	1.2	0	0.0	0	0.0
Naso-pharyngeal tract	Nasal cavity large
	Litter Incidences	No. (%)	0	0.0	1	4.8	0	0.0	1	5.3
	Fetal Incidences	No. (%)	0	0.0	1	1.2	0	0.0	1	1.1
Ureters	Dilation (Bilateral)
	Litter Incidences	No. (%)	1	4.8	0	0.0	1	4.8	1	5.3
	Fetal Incidences	No. (%)	1	1.0	0	0.0	1	1.1	1	1.1

 

TABLE 15 SUMMARY OF SKELETAL EXAMINATION OF FETUSES PER LITTER RECORD 

Variations                                                                                                                                   

Group & Dose (mg/kg body weight/day)			G1 & 0		G2 & 100		G3 & 300		G4 & 900
Litters Evaluated for Skeletal Examination		No.	21		21		21		19
Fetuses Evaluated for Skeletal Examination		No.	111		92		94		93
Region↓	Alteration↓
Skull	Interparietal - Incomplete ossification
	Litter Incidences	No. (%)	3	14.3	2	9.5	1	4.8	3	15.8
	Fetal Incidences	No. (%)	3	2.7	2	2.2	1	1.1	3	3.2
Skull	Interparietal - Bipartite ossification
	Litter Incidences	No. (%)	1	4.8	0	0.0	0	0.0	0	0.0
	Fetal Incidences	No. (%)	1	0.9	0	0.0	0	0.0	0	0.0
Skull	Interparietal - Poor ossification
	Litter Incidences	No. (%)	0	0.0	0	0.0	1	4.8	0	0.0
	Fetal Incidences	No. (%)	0	0.0	0	0.0	1	1.1	0	0.0
Skull	Parietal - Bipartite ossification
	Litter Incidences	No. (%)	0	0.0	0	0.0	1	4.8	0	0.0
	Fetal Incidences	No. (%)	0	0.0	0	0.0	2	2.1	0	0.0
Skull	Parietal - Incomplete ossification
	Litter Incidences	No. (%)	0	0.0	0	0.0	0	0.0	1	5.3
	Fetal Incidences	No. (%)	0	0.0	0	0.0	0	0.0	1	1.1
Skull	Supra occipital - Unossified
	Litter Incidences	No. (%)	0	0.0	0	0.0	1	4.8	0	0.0
	Fetal Incidences	No. (%)	0	0.0	0	0.0	1	1.1	0	0.0
Skull	Supra occipital - Incomplete ossification
	Litter Incidences	No. (%)	0	0.0	0	0.0	1	4.8	1	5.3
	Fetal Incidences	No. (%)	0	0.0	0	0.0	2	2.1	1	1.1
Skull	Zygomatic arch - Incomplete ossification (Unilateral)
	Litter Incidences	No. (%)	1	4.8	0	0.0	1	4.8	1	5.3
	Fetal Incidences	No. (%)	1	0.9	0	0.0	1	1.1	1	1.1
Skull	Zygomatic arch - Incomplete ossification (Bilateral)
	Litter Incidences	No. (%)	1	4.8	1	4.8	1	4.8	1	5.3
	Fetal Incidences	No. (%)	1	0.9	1	1.1	2	2.1	1	1.1
Skull	Zygomatic arch - Unossified (Bilateral)
	Litter Incidences	No. (%)	0	0.0	0	0.0	0	0.0	1	5.3
	Fetal Incidences	No. (%)	0	0.0	0	0.0	0	0.0	1	1.1
Sternum	Sternebra No. 1 - Incomplete ossification
	Litter Incidences	No. (%)	1	4.8	0	0.0	0	0.0	1	5.3
	Fetal Incidences	No. (%)	1	0.9	0	0.0	0	0.0	1	1.1
Sternum	Sternebra No. 1 - Unossified
	Litter Incidences	No. (%)	0	0.0	0	0.0	3	14.3	0	0.0
	Fetal Incidences	No. (%)	0	0.0	0	0.0	3*	3.2*	0	0.0
Sternum	Sternebra No. 2 - Incomplete ossification
	Litter Incidences	No. (%)	0	0.0	1	4.8	0	0.0	0	0.0
	Fetal Incidences	No. (%)	0	0.0	1	1.1	0	0.0	0	0.0

No.: Number; %: Percentage.

Variations                                                                                                                                      

Group & Dose (mg/kg body weight/day)			G1 & 0		G2 & 100		G3 & 300		G4 & 900
Litters Evaluated for Skeletal Examination		No.	21		21		21		19
Fetuses Evaluated for Skeletal Examination		No.	111		92		94		93
Region↓	Alteration↓
Sternum	Sternebra No. 5 - Incomplete ossification
	Litter Incidences	No. (%)	3	14.3	3	14.3	4	19.0	2	10.5
	Fetal Incidences	No. (%)	4	3.6	3	3.3	4	4.3	4	4.3
Sternum	Sternebra No. 5 - Unossified
	Litter Incidences	No. (%)	3	14.3	1	4.8	0	0.0	2	10.5
	Fetal Incidences	No. (%)	4	3.6	1	1.1	0	0.0	4	4.3
Sternum	Sternebra No. 5 - Bipartite ossification
	Litter Incidences	No. (%)	0	0.0	1	4.8	0	0.0	0	0.0
	Fetal Incidences	No. (%)	0	0.0	2	2.2	0	0.0	0	0.0
Sternum	Sternum No. 6 - Incomplete ossification
	Litter Incidences	No. (%)	2	9.5	1	4.8	1	4.8	1	5.3
	Fetal Incidences	No. (%)	2	1.8	1	1.1	2	2.1	1	1.1
Sternum	Sternum No. 6 - Unossified
	Litter Incidences	No. (%)	1	4.8	0	0.0	3	14.3	2	10.5
	Fetal Incidences	No. (%)	1	0.9	0	0.0	3	3.2	3	3.2
Ribs	Rib No. 14 - Ossification site (Unilateral)
	Litter Incidences	No. (%)	2	9.5	3	14.3	3	14.3	2	10.5
	Fetal Incidences	No. (%)	2	1.8	6	6.5	3	3.2	2	2.2
Ribs	Rib No. 14 - Ossification site (Bilateral)
	Litter Incidences	No. (%)	2	9.5	3	14.3	2	9.5	1	5.3
	Fetal Incidences	No. (%)	2	1.8	3	3.3	3	3.2	1	1.1
Ribs	Rib No. 14 - Rudimentary (Unilateral)
	Litter Incidences	No. (%)	0	0.0	1	4.8	1	4.8	0	0.0
	Fetal Incidences	No. (%)	0	0.0	1	1.1	1	1.1	0	0.0
Ribs	Rib No. 11 - Wavy
	Litter Incidences	No. (%)	0	0.0	0	0.0	0	0.0	1	5.3
	Fetal Incidences	No. (%)	0	0.0	0	0.0	0	0.0	1	1.1
Ribs	Rib No. 12 - Wavy
	Litter Incidences	No. (%)	0	0.0	0	0.0	0	0.0	1	5.3
	Fetal Incidences	No. (%)	0	0.0	0	0.0	0	0.0	1	1.1
Ribs	Rib No. 13 – Wavy (Bilateral
	Litter Incidences	No. (%)	0	0.0	0	0.0	0	0.0	1	5.3
	Fetal Incidences	No. (%)	0	0.0	0	0.0	0	0.0	1	1.1
Thoracic Vertebra	Centrum No. 5 - Dumbbell
	Litter Incidences	No. (%)	1	4.8	0	0.0	0	0.0	0	0.0
	Fetal Incidences	No. (%)	1	0.9	0	0.0	0	0.0	0	0.0

No.: Number; %: Percentage 

Variations                                                                                                                                      

Group & Dose (mg/kg body weight/day)			G1 & 0		G2 & 100		G3 & 300		G4 & 900
Litters Evaluated for Skeletal Examination		No.	21		21		21		19
Fetuses Evaluated for Skeletal Examination		No.	111		92		94		93
Region/Tissue↓	Alteration↓
Thoracic Vertebra	Centrum No. 10 - Dumbbell
	Litter Incidences	No. (%)	3	14.3	2	9.5	5	23.8	4	21.1
	Fetal Incidences	No. (%)	5	4.5	2	2.2	5	5.3	4	4.3
Thoracic Vertebra	Centrum No. 11 - Dumbbell
	Litter Incidences	No. (%)	5	23.8	6	28.6	5	23.8	6	31.6
	Fetal Incidences	No. (%)	6	5.4	6	6.5	5	5.3	7	7.5
Thoracic Vertebra	Centrum No. 12 - Dumbbell
	Litter Incidences	No. (%)	6	28.6	6	28.6	6	28.6	5	26.3
	Fetal Incidences	No. (%)	9	8.1	8	8.7	6	6.4	7	7.5
Thoracic Vertebra	Centrum No. 13 - Dumbbell
	Litter Incidences	No. (%)	6	28.6	1	4.8	1	4.8	1	5.3
	Fetal Incidences	No. (%)	6	5.4	1*	1.1*	1*	1.1*	1	1.1*
Thoracic Vertebra	Centrum No. 8 - Bipartite ossification
	Litter Incidences	No. (%)	0	0.0	0	0.0	0	0.0	1	5.3
	Fetal Incidences	No. (%)	0	0.0	0	0.0	0	0.0	1	1.1
Thoracic Vertebra	Centrum No. 10 - Bipartite ossification
	Litter Incidences	No. (%)	2	9.5	0	0.0	1	4.8	1	5.3
	Fetal Incidences	No. (%)	2	1.8	0	0.0	1	1.1	1	1.1
Thoracic Vertebra	Centrum No. 11 - Bipartite ossification
	Litter Incidences	No. (%)	1	4.8	3	14.3	2	9.5	2	10.5
	Fetal Incidences	No. (%)	1	0.9	3	3.3	2	2.1	2	2.2
Thoracic Vertebra	Centrum No. 12 - Bipartite ossification
	Litter Incidences	No. (%)	1	4.8	2	9.5	1	4.8	1	5.3
	Fetal Incidences	No. (%)	1	0.9	3	3.3	2	2.1	1	1.1
Thoracic Vertebra	Centrum No. 5 - Asymmetrical ossification
	Litter Incidences	No. (%)	0	0.0	0	0.0	0	0.0	1	5.3
	Fetal Incidences	No. (%)	0	0.0	0	0.0	0	0.0	1	1.1
Thoracic Vertebra	Centrum No. 10 - Asymmetrical ossification
	Litter Incidences	No. (%)	0	0.0	0	0.0	1	4.8	0	0.0
	Fetal Incidences	No. (%)	0	0.0	0	0.0	1	1.1	0	0.0
Thoracic Vertebra	Centrum No. 11 - Asymmetrical ossification
	Litter Incidences	No. (%)	1	4.8	3	14.3	2	9.5	1	5.3
	Fetal Incidences	No. (%)	1	0.9	3	3.3	2	2.1	1	1.1
Thoracic Vertebra	Centrum No. 12 - Asymmetrical ossification
	Litter Incidences	No. (%)	2	9.5	0	0.0	2	9.5	3	15.8
	Fetal Incidences	No. (%)	2	1.8	0	0.0	2	2.1	3	3.2

No.: Number; %: Percentage. 

Variations                                                                                                                                      

Group & Dose (mg/kg body weight/day)			G1 & 0		G2 & 100		G3 & 300		G4 & 900
Litters Evaluated for Skeletal Examination		No.	21		21		21		19
Fetuses Evaluated for Skeletal Examination		No.	111		92		94		93
Region↓	Alteration↓
Thoracic Vertebra	Centrum No. 10 - Incomplete ossification
	Litter Incidences	No. (%)	1	4.8	0	0.0	1	4.8	0	0.0
	Fetal Incidences	No. (%)	1	0.9	0	0.0	1	1.1	0	0.0
Thoracic Vertebra	Centrum No. 11 - Incomplete ossification
	Litter Incidences	No. (%)	1	4.8	0	0.0	1	4.8	0	0.0
	Fetal Incidences	No. (%)	1	0.9	0	0.0	1	1.1	0	0.0
Thoracic Vertebra	Centrum No. 12 - Incomplete ossification
	Litter Incidences	No. (%)	1	4.8	0	0.0	0	0.0	0	0.0
	Fetal Incidences	No. (%)	1	0.9	0	0.0	0	0.0	0	0.0
Lumbar Vertebra	Centrum No. 1 - Dumbbell
	Litter Incidences	No. (%)	1	4.8	0	0.0	0	0.0	0	0.0
	Fetal Incidences	No. (%)	1	0.9	0	0.0	0	0.0	0	0.0
Lumbar Vertebra	Centrum No. 2 - Dumbbell
	Litter Incidences	No. (%)	0	0.0	0	0.0	1	4.8	0	0.0
	Fetal Incidences	No. (%)	0	0.0	0	0.0	1	1.1	0	0.0
Lumbar Vertebra	Centrum No. 1 - Asymmetrical ossification
	Litter Incidences	No. (%)	1	4.8	0	0.0	0	0.0	1	5.3
	Fetal Incidences	No. (%)	1	0.9	0	0.0	0	0.0	1	1.1
Sacral Vertebra	Arch No. 4 - Incomplete ossification (Unilateral)
	Litter Incidences	No. (%)	1	4.8	1	4.8	1	4.8	1	5.3
	Fetal Incidences	No. (%)	1	0.9	1	1.1	1	1.1	1	1.1
Sacral Vertebra	Arch No. 4 - Incomplete ossification (Bilateral)
	Litter Incidences	No. (%)	2	9.5	1	4.8	2	9.5	2	10.5
	Fetal Incidences	No. (%)	3	2.7	1	1.1	2	2.1	3	3.2
Caudal Vertebra	Arch No. 1 - Incomplete ossification (Unilateral)
	Litter Incidences	No. (%)	0	0.0	0	0.0	1	4.8	1	5.3
	Fetal Incidences	No. (%)	0	0.0	0	0.0	1	1.1	1	1.1
Caudal Vertebra	Arch No. 1 - Incomplete ossification (Bilateral)
	Litter Incidences	No. (%)	2	9.5	1	4.8	2	9.5	1	5.3
	Fetal Incidences	No. (%)	2	1.8	1	1.1	2	2.1	1	1.1

No.: Number; %: Percentage. 

Variations                                                                                                                                      

Group & Dose (mg/kg body weight/day)			G1 & 0		G2 & 100		G3 & 300		G4 & 900
Litters Evaluated for Skeletal Examination		No.	21		21		21		19
Fetuses Evaluated for Skeletal Examination		No.	111		92		94		93
Region↓	Alteration↓
Pelvic Girdle	Pubis - Incomplete ossification (Bilateral)
	Litter Incidences	No. (%)	1	4.8	0	0.0	0	0.0	0	0.0
	Fetal Incidences	No. (%)	1	0.9	0	0.0	0	0.0	0	0.0
Pelvic Girdle	Ileum - Unossified (Bilateral)
	Litter Incidences	No. (%)	0	0.0	0	0.0	1	4.8	0	0.0
	Fetal Incidences	No. (%)	0	0.0	0	0.0	1	1.1	0	0.0
Pelvic Girdle	Ischium - Unossified (Bilateral)
	Litter Incidences	No. (%)	0	0.0	0	0.0	2	9.5	0	0.0
	Fetal Incidences	No. (%)	0	0.0	0	0.0	2	2.1	0	0.0
Pelvic Girdle	Pubis - Unossified (Bilateral)
	Litter Incidences	No. (%)	0	0.0	0	0.0	1	4.8	0	0.0
	Fetal Incidences	No. (%)	0	0.0	0	0.0	1	1.1	0	0.0
Fore - Limb	Proximal phalanges No. 3 and 4 - Unossified (Bilateral)
	Litter Incidences	No. (%)	5	23.8	3	14.3	2	9.5	4	21.1
	Fetal Incidences	No. (%)	5	4.5	3	3.3	3	3.2	4	4.3
Fore - Limb	Metacarpal No. 5 - unossified (Bilateral)
	Litter Incidences	No. (%)	3	14.3	2	9.5	1	4.8	2	10.5
	Fetal Incidences	No. (%)	3	2.7	2	2.2	4	4.3	2	2.2

Malformation                                                                                                                              

Group & Dose (mg/kg body weight/day)			G1 & 0		G2 & 100		G3 & 300		G4 & 900
Litters Evaluated for Skeletal Examination		No.	21		21		21		19
Fetuses Evaluated for Skeletal Examination		No.	111		92		94		93
Region↓	Alteration↓
Skull	Zygomatic arch - Misshapen (Bilateral)
	Litter Incidences	No. (%)	0	0.0	0	0.0	1	4.8	0	0.0
	Fetal Incidences	No. (%)	0	0.0	0	0.0	1	1.1	0	0.0
Ribs	Rib No. 7 - Nodulated (Unilateral)
	Litter Incidences	No. (%)	0	0.0	0	0.0	0	0.0	1	5.3
	Fetal Incidences	No. (%)	0	0.0	0	0.0	0	0.0	1	1.1
Ribs	Rib No. 8 - Nodulated (Unilateral)
	Litter Incidences	No. (%)	1	4.8	0	0.0	0	0.0	1	5.3
	Fetal Incidences	No. (%)	1	0.9	0	0.0	0	0.0	1	1.1
Ribs	Rib No. 9 - Nodulated (Unilateral)
	Litter Incidences	No. (%)	1	4.8	0	0.0	0	0.0	1	5.3
	Fetal Incidences	No. (%)	1	0.9	0	0.0	0	0.0	1	1.1
Ribs	Rib No. 10 - Nodulated (Unilateral)
	Litter Incidences	No. (%)	1	4.8	0	0.0	0	0.0	0	0.0
	Fetal Incidences	No. (%)	1	0.9	0	0.0	0	0.0	0	0.0
Ribs	Rib No. 11 - Short (Unilateral)
	Litter Incidences	No. (%)	0	0.0	0	0.0	0	0.0	1	5.3
	Fetal Incidences	No. (%)	0	0.0	0	0.0	0	0.0	1	1.1
Ribs	Rib No. 12 - Short (Unilateral)
	Litter Incidences	No. (%)	0	0.0	0	0.0	0	0.0	2	10.5
	Fetal Incidences	No. (%)	0	0.0	0	0.0	0	0.0	2	2.2
Ribs	Rib No. 13 - Short (Unilateral)
	Litter Incidences	No. (%)	1	4.8	1	4.8	0	0.0	1	5.3
	Fetal Incidences	No. (%)	1	0.9	1	1.1	0	0.0	1	1.1
Thoracic	Centrum No. 8 - Split
	Litter Incidences	No. (%)	0	0.0	0	0.0	1	4.8	0	0.0
	Fetal Incidences	No. (%)	0	0.0	0	0.0	1	1.1	0	0.0
Thoracic	Centrum No. 10 - Split
	Litter Incidences	No. (%)	1	4.8	1	4.8	1	4.8	1	5.3
	Fetal Incidences	No. (%)	1	0.9	1	1.1	1	1.1	1	1.1
Thoracic	Centrum No. 11 - Split
	Litter Incidences	No. (%)	1	4.8	1	4.8	1	4.8	0	0.0
	Fetal Incidences	No. (%)	1	0.9	1	1.1	1	1.1	0	0.0
Thoracic	Centrum No. 12 - Split
	Litter Incidences	No. (%)	0	0.0	0	0.0	0	0.0	1	5.3
	Fetal Incidences	No. (%)	0	0.0	0	0.0	0	0.0	1	1.1

No.: Number; %: Percentage 

 

Conclusions:

In a pre-natal developmental toxicity study conducted in Sprague Dawley Rats, the NOAEL of the test substance for both maternal and fetal toxicity was determined to be 900 mg/kg body weight/day under the experimental conditions employed.

Executive summary:

The developmental toxicity of the test item was determined using OECD Guideline 414 under GLP conditions. The study used Sprague Dawley rats to test the daily oral administration via gavage of the test item from day 5 until day 20 of gestation. A total of 100 mated female Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3 and G4) consisted of 25 presumed pregnant females. The animals in group G1 were administered with vehicle (corn oil), the animals in groups G2, G3 and G4 were administered with test item at the dose levels of 100, 300 and 900 mg/kg body weight/day for low, mid, and high dose groups respectively, based on a previous study (OECD 422). On day GD21 all animals were euthanized, and tissue, organs and fetuses extracted and examined. During the experiment, any clinical signs, mortality, body weight development, feed consumption were recorded. On GD21 the animals were euthanized and were subject to gross necropsy and relevant organs like ovaries, uterus with cervix and thyroid and parathyroid glands extracted. Histopathology of thyroid along with the parathyroid was performed. Each dam was observed for number of live fetuses, litter size, sex ratio, number of implantation sites and number of resorptions. The assessment of thyroid hormones T4, T3 and TSH was conducted. All the fetuses collected from each litter were weighed and measured for its crown rump length and anogenital distance. All the fetuses were subjected to external examination. All the even numbered fetuses were subjected to fresh visceral (soft tissue) examination and fixed head sections examination. All the odd numbered fetuses were subjected to skeletal examination.

For maternal toxicity assessments, there were no clinical signs of toxicity and no mortality/morbidity noted in any of the dams in all tested dose groups. The mean absolute and relative thyroid along with the parathyroid weight did not reveal any changes and no test item-related changes were noted in mean thyroid hormonal levels (T3, T4 and TSH) in all the tested dose groups. There were no gross pathological changes noted in any of the dams during caesarean section and no test item-related microscopic changes were noted in thyroid along with the parathyroid of all dams during histopathological examination.

In group G2, there were no differences noted in mean maternal body weight, percent change in maternal body weight gain, body weight corrected for maternal increase and maternal feed consumption. There were no effects noted in mean number of live fetuses per litter, litter size, sex ratio, number of corpora lutea, number of implantation sites, number of incidences of resorptions, pre-and post-implantation losses per dam.

In group G3, a slight reduction in percent change in mean gestational (maternal) body weight gain was noted during mid-pregnancy. However, during late pregnancy the animals shown normal increase in body weight. There were no effects noted in mean number of live fetuses per litter, litter size, sex ratio, number of corpora lutea, number of implantation sites and number of incidences of resorptions. A slight increase in occurrence of mean number of late resorptions per litter and mean percentage of post-implantation loss was noted at this dose level.

In group G4, a slight reduction in percent change in mean gestational (maternal) body weight gain was noted during early and mid-pregnancy. However, during late pregnancy the animals shown normal increase in body weight. A reduction in mean maternal body weight change corrected for gravid uterus weight (both absolute and percent) was noted. These changes are considered as a secondary effect caused due to stress induced by the test item exposure. There were no effects noted in mean number of live fetuses per litter, litter size, sex ratio, number of corpora lutea, number of implantation sites and number of incidences of resorptions. A slight increase in occurrence of mean number of late resorptions per litter and mean percentage of post-implantation loss was noted at this dose level.

For fetal (pre-natal developmental) toxicity assessment, there were no test item-related changes noted in mean fetal weight, mean fetal crown rump length and mean fetal anogenital distance measurement / ratio per litter in either sex in all the tested dose groups. There was no test item-related fetal developmental and or structural alterations noted from all the tested dose group litters when subjected to fetal external, visceral and skeletal examinations. The observed fetal external/visceral/skeletal developmental variations and or malformations are considered as incidental and unrelated to treatment as these findings occurred infrequently or at a frequency similar to the vehicle control group and did not occur in a dose-dependent manner. Also, the observed mean litter/fetal proportions were within the lab historical control range of this species and strain.

 

Based on the obtained results from the study, the NOEL and LOEL for both systemic and developmental maternal toxicity could be established as 100 and 300 mg/kg bw/day respectively, based on the slight decrease in percent change in mean body weight during mid pregnancy and minimal increase in occurrence of mean percent of late resorptions and mean percentage of post-implantation loss at the latter dose level. These effects are not considered as adverse but could be secondary effects caused due to test item exposure. Also, the occurred changes are minimal and not accompanied with any other relevant systemic and reproductive toxicity end points.

 

The NOAEL for systemic, developmental maternal toxicity and fetal (pre-natal developmental) toxicity is estimated as 900 mg/kg bw/day as no adverse observed effects were found up to the highest dose tested.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Two key studies (OECD 422 and OECD 414) are available with a Klimisch score of 1.
Also some available supporting studies on components/analogue substances with Klimisch score of 2 have been included in the dossier.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Key study : Test method OECD 422. GLP study. The test substance Pine oil 50% was tested in a combined repeated dose toxicity study with the reproduction /developmental toxicity screening test according to OECD guideline 422, in accordance with GLP principles. Sprague Dawley rats were administered with test item at the dose levels of 100, 300 and 600 mg/kg body weight previously selected according to the results of a dose range finding study performed with the same species at doses of 100, 300 and 1000 mg/kg bw for a period of 14 days. The treatment of male and female rats with test substance at dose levels up to 600 mg/kg-bw/day by repeated oral (gavage) administration revealed revealed no maternal toxicity nor adverse effects on the reproductive performance or fertility. There were no indications of developmental effects in the offspring. The No Observed Effect Level (NOEL) for parental general toxicity was determined to be 100 mg/kg bw/day based on treatment related clinical signs of toxicity and reduction in body weight found at the dose of 300 mg/kg bw/day. The No Observed Effect Level (NOEL) for maternal developmental toxicity was established at 300 mg/kg bw/day based on the increased copulatory interval and gestation length observed at the high dose tested (600 mg/kg bw/day). However, these effects were found along with other systemic effects (clinical and weight changes) and thus, it is reasonable to consider these reproductive effects as non- specific secondary effects to those systemic effects. The No Observed Effect Level (NOEL) for developmental toxicity was established at 600 mg/kg bw/day based no effects found in offspring at any dose tested.

Finally, the No Observed Adverse Effect Level (NOAEL) for parental and developmental toxicity was determined to be 600 mg/kg bw/day since the treatment related clinical sings of toxicity observed at doses of 300 and 600 mg/kg bw/day were found recovered later during the treatment period in all cases. Furthermore, although increased copulatory interval and reduced gestation length were observed at 600 mg/kg bw/day, no other fertility or reproduction subsequent effects in fertility index, parturition, litter size or live birth index were found and also no treatment related histopathological changes were noted in testes, epididymides, seminal vesicle with coagulating gland, prostate or ovaries at this dose level.

 

Key study : Test method OECD 414. GLP study. The developmental toxicity of the test item was determined using OECD Guideline 414 under GLP conditions. The study used Sprague Dawley rats to test the daily oral administration via gavage of the test item from day 5 until day 20 of gestation. A total of 100 mated female Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3 and G4) consisted of 25 presumed pregnant females. The animals in group G1 were administered with vehicle (corn oil), the animals in groups G2, G3 and G4 were administered with test item at the dose levels of 100, 300 and 900 mg/kg body weight/day for low, mid, and high dose groups respectively, based on a previous study (OECD 422).

Based on the obtained results from the study, the NOEL and LOEL for both systemic and developmental maternal toxicity could be established as 100 and 300 mg/kg bw/day respectively, based on the slight decrease in percent change in mean body weight during mid pregnancy and minimal increase in occurrence of mean percent of late resorptions and mean percentage of post-implantation loss at the latter dose level. These effects are not considered as adverse but could be secondary effects caused due to test item exposure. Also, the occurred changes are minimal and not accompanied with any other relevant systemic and reproductive toxicity end points.

The NOAEL for systemic, developmental maternal toxicity and fetal (pre-natal developmental) toxicity is estimated as 900 mg/kg bw/day as no adverse observed effects were found up to the highest dose tested.

 

Supporting studies with individual main components:

 

A reproductive toxicity study was performed by oral route (gavage) with 1,8-cineole using female Wistar rats. The pregnant rats were randomly distributed into 8 groups (n = 7-10 per group). 4 groups were assigned to treatment during the pre-implantation period (1st to 6th day of pregnancy) and 4 groups to treatment during organogenesis (7th to 14th day of pregnancy). The test substance was administered to all animals of both pregnancy periods with doses of 0 (control, consisted only in 1% Tween-80 aqueous solution), 250, 500 and 1000 mg / kg bw/day. The NOAEL was not established since effects in maternal body weight were found at the lowest dose tested (250 mg/kg bw/day).

 

In a prenatal developmental toxicity study, d-limonene was administered orally to groups of pregnant Japanese white rabbits at dose levels of 250, 500 and 1000 mg/kg bw/day for 13 days from Day 6 to 18 of gestation. The NOAEL for fetal toxicity was considered to be greater than 1000 mg/kg bw/day.

 

In another prenatal developmental toxicity study, d-limonene was administered orally to groups of pregnant ICR mice (20/dose: 15 for teratogenicity study, 5 for postnatal development) at dose levels of 0, 591 and 2363 mg/kg bw/day for 6 days from Day 7 to 12 of gestation. The NOAEL for maternal and fetal toxicity was considered to be 591 mg/kg bw/day.

 

In a third developmental toxicity study, d-limonene was administered orally to groups of pregnant Wistar rats (20/dose: 15 for teratogenicity study, 5 for postnatal development) at dose levels of 0, 591 and 2869 mg/kg bw/day for 7 days from Day 9 to 15 of gestation. The NOAEL for maternal and fetal toxicity was considered to be 591 mg/kg bw/day.

 

A pre-natal developmental toxicity test was performed with alpha terpinene following a method equivalent to OECD Guideline 414. Alpha terpinene dissolved in corn oil at doses of 30, 60, 125 and 250 mg/kg body weight was given by gavage to female Wistar rats from day 6 to 15 of pregnancy. Caesarean sections were performed on day 21 of pregnancy. Signs of delayed ossification (poorly ossified and not ossified bones as well as irregular spongy bones) and a higher incidence of minor skeletal malformations were observed at doses of 60 mg/kg bw or more. These findings indicate that the NOAEL for alpha terpinene-induced embryofoetotoxicity can be set at 30 mg/kg bw by the oral route. Nevertheless, the number of ossification centres affected in relation to the number examined was very small, even at the highest dose level tested indicating that the effect of treatment on the developing foetus was minimal. Also, it cannot be excluded that the observed maternal reproductive effects are secondary to general maternal toxicity. Thus, additional data would be needed to confirm the (absence of) observed effects.

Justification for classification or non-classification

Based on the available information, the substance is not classified for toxicity to reproduction in accordance with CLP Regulation (EC) no 1272/2008.