2-(2-heptadec-8-enyl-2-imidazolin-1-yl)ethanol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No effects on fertility has been observed up to a top dose of 100/60 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

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 same study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3650

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Amine O
- Analytical purity: 96.6% imidazolines
- Isomers composition: The test substance is a uvcb substance consisting of 96.6% imidazolines
- Lot/batch No.:05774HP8
- Substance type: Organic
- Physical state: Liquid
- Stability under test conditions: Stable

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-12 weeks (males/females)
- Weight at study initiation: ca. 350 g males, 192 g females
- Housing: individually in Makrolon type M III cages
- Diet: Ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: Bottles, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Humidity: 30 - 70%
- Air changes: 10 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a solution. To prepare the solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then the vehicle (drinking water) was filled up to the desired volume, buffered to pH 7.5 with HCl and subsequently mixed using a magnetic stirrer. Each preparation was prepared once, divided in daily portions and stored deep frozen until the daily use.

VEHICLE
- Amount of vehicle: 10 mL/kg bw

Details on mating procedure:

P0 animals were mated 13 days after the beginning of treatment. Each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group. Mating was accomplished by placing the female in the cage of the male mating partner from about 16.00 h until 07.00-09.00 h of the following morning. Deviations from these specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm was detected was denoted "gestation day (GD) 0" and the following day "GD 1".

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses of the test-substance preparations were carried out at the Analytical Chemistry Laboratory of the Experimental Toxicology and Ecology of BASF SE. The stability of the test substance in drinking water (neutrally buffered) at room temperature for a period of 10 days was proven before the start of the administration period (Project No.: 1Y0472/098050). Homogeneity was given because the test substance was completely miscible with water and solutions were considered to be homogenous without further analysis. Concentration control analyses of the test-substance preparations were performed in samples of all concentrations at the start and at the end of the administration period.

Duration of treatment / exposure:

Males: 31 days
Females 51 days

Frequency of treatment:

daily

Details on study schedule:

The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females.
P0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all P0 females.
A detailed clinical observation (DCO) was performed in all P0 animals before test substance administration and thereafter at weekly intervals.
Food consumption of the P0 parents was determined regularly during premating and after the mating period and during the gestation and lactation periods in dams.
In general, body weights of P0 animals were determined once a week. During gestation and lactation period, P0 females were weighed on gestation days (GD) 0, 7, 14 and 20, on the parturition day (postnatal day [PND] 0) and on PND 4.
Pups were sexed on PND 0 and weighed one day after birth and on PND 4. Their viability was recorded twice daily on each working day or only in the morning on Saturday and Sunday.
Clinicochemical and hematological examinations as well as urinalyses were performed in 5 randomly selected P0 parental animals of either sex towards the end of the administration period.
At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 randomly selected P0 parental males and females per test group. All surviving P0 parental animals were sacrificed assessed by gross pathology. Organ weights were recorded and a histopathological examination was performed

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Remarks:

Highest dose: First 7 (females) and 8 days (males) of the study

Dose / conc.:

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

Remarks:

Highest dose: Reduced dose level from study day 8 (females) / 9 (males) onwards, toxic effects were seen at 100 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a previously performed 14-days test study the test article was administered by gavage to groups of 3 male and 3 female Wistar rats at dose levels of 0, 50, 100, 200, and 600 mg/kg body weight/day (mg/kg bw/d). Drinking water containing 1% Carboxylmethylcellulose and Cremophor EL® (5 mg/100 mL) served as vehicle. In this study, the oral administration of the test substance caused severe adverse effects in both sexes, i.e. premature deaths and poor general state, at dose levels of 600 and 200 mg/kg bw/d. Only slight effects on food and water consumption and body weight data were observed at 100 mg/kg bw/d.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied. A cageside examination was conducted before and after treatment for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented for each animal. The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g., animal could not litter, umbilical cord not cut) were documented on an individual dam basis. On weekdays (except public holidays) the littering behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of littering was considered to be a 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 x 37.5 cm with side borders of 25 cm high). The following parameters were examined: abnormal behavior during “handling”, fur, skin, salivation, exophthalmos, lacrimation, pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, feces (appearance/consistency), urine, other findings.

BODY WEIGHT: Yes
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning).
The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals:
- During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
- Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
- Females without a litter were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION
Food consumption was determined once a week (in a period of 7 days) for male and female parental animals, with the following exceptions:
- Food consumption was not determined during the mating period (male and female P0 animals).
- Food consumption of the P0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
- Food consumption of P0 females, which gave birth to a litter, was determined on PND 0 and 4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).

OTHER:
The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. On study day 30, a functional observational battery and motor activity measurement were carried out in the first five randomly male animals per test group. The females were allowed to litter and rear their pups until day 4 after parturition. On PND 4, all pups were sacrificed and examined. On study day 50, a functional observational battery and motor activity measurement was carried out in 5 randomly selected P0 parental female animals (with litter) per test group. From the first 5 male animals and the selected 5 female animals urinalysis were carried out on study days 29 (males) and 46 (females). Clinicochemical and hematological examinations were carried out on study days 31 (males) and 51 (females). At the end of the study (males: study day 31, females: study day 51), the animals were sacrificed after a fasting period (withdrawal of food) for at least 16-20 hours.

Oestrous cyclicity (parental animals):

not evaluated

Sperm parameters (parental animals):

not evaluated

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, pup weight, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead. All surviving pups (sacrificed on PND 4), all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Adrenal glands, Brain, Cauda epididymis, Epididymides, Heart, Kidneys, Liver, Ovaries, Pituitary gland, Prostate, Seminal vesicles with coagulation glands, Spleen, Testes, Thymus, Thyroid glands, Uterus with cervix

HISTOPATHOLOGY: Yes
The following organs or tissues were fixed in 4% formaldehyde solution or in modified Davidson’s solution: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Coagulation glands, Colon, Duodenum, Epididymides (modified Davidson’s solution), Esophagus, Eyes with optic nerve, Female mammary gland, Femur with knee joint, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (mesenteric and axillary lymph nodes), Nose (nasal cavity), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Pharynx, Pituitary gland, Prostate, Rectum, Salivary glands (mandibular and sublingual glands), Seminal vesicle with coagulation glands, Sciatic nerve, Skeletal muscle, Skin, Spinal cord (cervical, thoracic and lumbar cords), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testes (modified Davidson’s solution), Thymus, Thyroid glands/parathyroid glands, Trachea, Urinary bladder, Uterus, Vagina. Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table presented in 'Any other information on materials and mthods incl. tables'.

Postmortem examinations (offspring):

All surviving pups (sacrificed on PND 4), all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.

Statistics:

DUNNETT-test (two-sided): Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of pups delivered per litter, implantation sites, post implantation loss.
FISHER'S EXACT test: Male and female mating index, male and female fertility index, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters with affected pups at necropsy.
WILCOXON-test (one-sided): Proportions of affected pups per litter with necropsy observations.
KRUSKAL-WALLIS test (two-sided): Feces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON test (two-sided).

Reproductive indices:

Male mating index, Male fertility index, Female mating index, Female fertility index, Gestation index, Live birth index, Post implantation loss

Offspring viability indices:

All pups delivered from the P0 parents were examined as soon as possible on the day of birth to determine the total number of pups and the number of liveborn and stillborn pups in each litter. Pups, which died before the first determination of their status on the day of birth, were defined as stillborn pups. At the same time, the pups were examined for macroscopically evident changes. Viability index and sex ratio were calculated.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Because of severe clinical findings seen in animals of test group 3 (100 mg/kg bw/d), i.e. respiration sounds, reduced body weights and the death of one female animal on study day 3, the dose level in test group 3 was reduced to 60 mg/kg w/d for the females on study day 8 as well as for the males on study day 9.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

In test group 3 (100 and 60 mg/kg bw/d) one female animal was found dead within the first week of the study. In addition, one male animal and one female animal of test group 3 (100 and 60 mg/kg bw/d) were sacrificed in a moribund state in study week 2.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In test group 3 (100 and 60 mg/kg bw/d) male animals’ body weight was significantly lower in week 2. In the same test group body weight loss was observed in male and female (not significant) animals between study weeks 0-1 and, in summary, body weight change values of male animals were significantly lower between study weeks 0-4. No effects on body weight data were observed in male and female animals of test groups 1 and 2 (5 and 20 mg/kg bw/d) during the entire study period. The same was true for female animal of test group 3 (100 and 60 mg/kg bw/d) during gestation and lactation periods.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Significantly decreased food consumption in male animals of test group 3 (100 and 60 mg/kg bw/d) was only observed during the first two study weeks. Significantly decreased food consumption in female animals of test group 3 (100 and 60 mg/kg bw/d) was observed in the first study week. Both findings were assessed as being related to treatment. Impaired food consumption was not observed in male and female animals of test groups 1 and 2 (5 and 20 mg/kg bw/d). The same was true for female animals of test group 3 (100 and 60 mg/kg bw/d) during gestation and lactation periods.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related, adverse changes among haematological parameters were observed.
In female rats of test groups 1 (5 mg/kg bw/d) and 3 (100 and 60 mg/kg bw/d) the platelet counts were lower compared to controls. These values were within the historical control ranges (platelet counts: 765-1009 Giga/L). Therefore, the alterations were regarded as incidental rather than treatment-related. In females of test group 2 (20 mg/kg bw/d) the relative lymphocyte counts, and in females of test group 3 (100 and 60 mg/kg bw/d) the relative large unstained cell (LUC) counts were lower compared to controls. These changes in the differential blood cell counts were not accompanied by a change of the total white blood cell counts, and the lymphocyte and LUC cell counts were within the historical control ranges (relative lymphocyte counts: 66.7-83.9%, relative LUC counts: 0.2-0.5%). The alterations were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related, adverse changes among clinical chemistry parameters were measured. In males of test group 3 (100 and 60 mg/kg bw/d) the creatinine values were decreased. This was the only alteration among the clinical pathology parameters in rats of this sex in this test group. Therefore, it was regarded as a maybe treatment-related but non-adverse effect (ECETOC, Technical Report No. 85, 2002).

Urinalysis findings:

no effects observed

Description (incidence and severity):

No treatment-related adverse changes among urinalysis parameters were measured.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No test substance-related or spontaneous findings in male and female animals of all test groups during the home cage observation were observed.
The open field observations did not reveal any test substance-related findings in male and female animals of all test groups. During sensimotor tests, there were no test substance-related findings in male and female animals of all test groups. Any deviations from "zero values" were equally distributed between test substance-treated groups and controls or occurred in single animals only. Therefore, these observations were considered as being incidental. There were no significant deviations concerning single intervals and the overall motor activity (summation of all intervals) in male and female animals of all test groups in comparison to the concurrent control group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

MALES
For all F0 parental males, which were placed with F0 females to generate F1 pups, mating was confirmed. Thus, the male mating index was 100% in test groups 0 to 2 and 89% in test group 3. Fertility was proven for all of the F0 parental males of test groups 0 (control) and 1 (5 mg/kg bw/d) within the scheduled mating interval for the F1 litter. One male of test group 2 and one male of test group 3 did not generate F1 pups. Thus, the male fertility index ranged between 78% and 100% and did not show statistically significant differences. These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.


FEMALES
The female mating index calculated after the mating period for F1 litter was 100% for the test groups 0, 1 to 2 (0, 5 and 20 mg/kg bw/d, respectively) and 89% to the test group 3 (100 and 60 mg/kg bw/d). The mean duration until sperm was detected (GD 0) amounted to 2.7, 1.7, 2.4, and 2.8 days (0, 5, 20 and 100 and 60 mg/kg bw/d, respectively). Consequently, the differences between the test groups were assessed as being spontaneous in nature and without biological relevance. All sperm-positive animals of test groups 0 (control) and 1 (5 mg/kg bw/d) delivered pups or had implantations in utero. One female of test group 2 (20 mg/kg bw/d) and one female of test group 3 (100 and 60 mg/kg bw/d) did not become pregnant. The fertility index varied between 88% and 100% and did not show statistically significant differences. The mean duration of gestation values were comparable between all test groups and varied between 21.9 and 22.1 days. The gestation index was 100% in all test groups. Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (13.8, 13.1, 12.2, and 12.1 implants per dam in test groups 0- 3). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any significant differences between the test groups, and the mean number of F1 pups delivered per dam remained unaffected (12.8, 12.4, 11.9, and 11.0 pups per dam at 0, 5, 20 and 100 and 60 mg/kg bw/d, respectively). The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100% (test group 0), 98% (test groups 1 and 2) and 99% (test group 3). Moreover, the number of stillborn pups was comparable between the test groups and a dose-response relationship was not observed.

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

reproductive performance

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

gross pathology

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

gross pathology

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

jejunum

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

immune system

Organ:

thymus

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Clinical signs:

no effects observed

Description (incidence and severity):

The F1 pups did not show adverse clinical signs up to scheduled sacrifice on PND 4.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The mean number of delivered pups per dam and the rate of liveborn and stillborn pups were comparable between the test substance-treated groups and the control group. The respective values reflect the normal range of biological variation inherent in the strain used in this study. One pup in test group 3 (100 and 60 mg/kg bw/d) died on PND 1. One pup in test group 2 (20 mg/kg bw/d) was cannibilized on PND 1. The viability index as indicator for pup mortality between PND 0-4 was 100% for test groups 0 (control) and 1 (5 mg/kg bw/d) and 99% in test group 2 (20 mg/kg bw/d) and 3 (100 and 60 mg/kg bw/d).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the concurrent control values.

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:

effects observed, non-treatment-related

Description (incidence and severity):

All stillborn pups of test groups 1 (5 mg/kg bw/d), 2 (20 mg/kg bw/d) and 3 (100 and 60 mg/kg bw/d) did not show any abnormal findings. On day 4 all pups were sacrificed scheduled and only incidental findings were obtained. In test group 0 (0 mg/kg bw/d) one pup showed dextrocardia, in test group 2 (20 mg/kg bw/d) one pup showed hemorrhagic testis, hydronephrosis, hydroureter and distended urinary bladder. In one pup of test group 2 (20 mg/kg bw/d) infarct of liver was observed. All findings were assessed as being incidental and can be found in the historical control data. A relation to dosing was excluded.

Histopathological findings:

not examined

Other effects:

no effects observed

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 60 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

60 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a GLP-compliant OECD 422 study, the test article was administered orally by gavage to groups of 10 male and 10 female Wistar rats (P0 parental animals) at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; vehicle control; test group 0), 5 mg/kg bw/d (test group 1), 20 mg/kg bw/d (test group 2) and 100 mg/kg bw/d (test group 3). Because of severe clinical findings seen in animals of test group 3 (100 mg/kg bw/d), i.e. respiration sounds, reduced body weights and the death of one female animal on study day 3, the dose level in test group 3 was reduced to 60 mg/kg bw/d for the females on study day 8 as well as for the males on study day 9. Drinking water (neutrally buffered for test substance preparations) served as vehicle. P0 animals were mated 13 days after the beginning of treatment to produce litter (F1 generation pups). Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear. P0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all P0 females. A detailed clinical observation (DCO) was performed in all P0 animals before test substance administration and thereafter at weekly intervals. Food consumption of the P0 parents was determined regularly during premating and after the mating period and during the gestation and lactation periods in dams. In general, body weights of P0 animals were determined once a week. During gestation and lactation period, P0 females were weighed on gestation days (GD) 0, 7, 14 and 20, on the parturition day (postnatal day [PND] 0) and on PND 4. Pups were sexed on PND 0 and weighed one day after birth and on PND 4. Their viability was recorded twice daily on each working day or only in the morning on Saturday and Sunday. Clinicochemical and haematological examinations as well as urinalyses were performed in 5 randomly selected P0 parental animals of either sex towards the end of the administration period. At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 randomly selected P0 parental males and females per test group. All surviving P0 parental animals were sacrificed assessed by gross pathology. Organ weights were recorded and a histopathological examination was performed.

 

In test group 3 (100 mg/kg bw/d between study days 0-7 [females] and 0-8 [males], 60 mg/kg bw/d onwards from study day 8 [females] and 9 [males]), one female animal was found dead in study week 1. One male and one female animal were sacrificed moribund state in study week 2. Poor general state was observed in one female animal in study week 0, in two male and two female animals in study weeks 1 and 2, in one male animal in study weeks 3 and 4 and in one female animal in study weeks 6 and 7 as well as in one female animal from GD 0 and 1, and from GD 7 onwards. Reduced nutritional state was recorded in one male animal in study week 2, one female animal in study week 0 and two female animals in study weeks 1 and 2 as well as in one female animal from GD 0 to 5. Respiratory sounds were reported in several male and female P0 animals during the entire study period. Furthermore, piloerection was seen in one female animal in study week 0 and one female female animal in study week 2 had diarrhea. Significantly decreased food consumption was reported in males during the first two study weeks and in females in the first study week. In addition, significantly decreased male animals’ body weight in week 2 and significantly decreased body weight change in week 0-1 and in summary between weeks 0-4 were seen. The male animal which was sacrificed moribund showed severe dilation of the gastro-intestinal tract (stomach, jejunum, colon and cecum) with severe organ size reduction of the thymus. The female animal which was sacrificed moribund showed severe dilation of the gastro-intestinal tract (stomach, jejunum and cecum) with severe organ size reduction of the thymus. No test-substance related, relevant findings were observed regarding fertility and clinical pathology. No findings in test groups 1 (5 mg/kg bw) and 2 (20 mg/kg bw), F1 animals were free of effects in all groups. Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats the no observed adverse effect level (NOAEL) for reproductive performance and fertility in male and female Wistar rats was 60 mg/kg bw/d. The NOAEL for general, systemic toxicity of the test substance was 20 mg/kg bw/d for male and female P0 parental animals based on clinical and pathological findings. The NOAEL for developmental toxicity was 60 mg/kg bw/d.

Effects on developmental toxicity

Description of key information

No effects on development up to PND4 has been observed up to a top dose of 100/60 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010

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 same study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3650

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Amine O
- Analytical purity: 96.6% imidazolines
- Isomers composition: The test substance is a uvcb substance consisting of 96.6% imidazolines
- Lot/batch No.:05774HP8
- Substance type: Organic
- Physical state: Liquid
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10 - 12 weeks (males/females)
- Weight at study initiation: ca. 350g males, 192 g females
- Housing: Individually in Makrolon type M III cages
- Diet: Ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: Bottles, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Humidity: 30 - 70%
- Air changes: 10 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a solution. To prepare the solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then the vehicle (drinking water) was filled up to the desired volume, buffered to pH 7.5 with HCl and subsequently mixed using a magnetic stirrer. Each preparation was prepared once, divided in daily portions and stored deep frozen until the daily use.

VEHICLE
- Amount of vehicle: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses of the test-substance preparations were carried out at the Analytical Chemistry Laboratory of the Experimental Toxicology and Ecology of BASF SE. The stability of the test substance in drinking water (neutrally buffered) at room temperature for a period of 10 days was proven before the start of the administration period (Project No.: 1Y0472/098050). Homogeneity was given because the test substance was completely miscible with water and solutions were considered to be homogenous without further analysis. Concentration control analyses of the test-substance preparations were performed in samples of all concentrations at the start and at the end of the administration period.

Details on mating procedure:

P0 animals were mated 13 days after the beginning of treatment. Each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group. Mating was accomplished by placing the female in the cage of the male mating partner from about 16.00 h until 07.00-09.00 h of the following morning. Deviations from these specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm was detected was denoted "gestation day (GD) 0" and the following day "GD 1".

Duration of treatment / exposure:

Males: 31 days
Females 51 days

Frequency of treatment:

daily

Duration of test:

The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females. P0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all P0 females. A detailed clinical observation (DCO) was performed in all P0 animals before test substance administration and thereafter at weekly intervals. Food consumption of the P0 parents was determined regularly during premating and after the mating period and during the gestation and lactation periods in dams. In general, body weights of P0 animals were determined once a week. During gestation and lactation period, P0 females were weighed on gestation days (GD) 0, 7, 14 and 20, on the parturition day (postnatal day [PND] 0) and on PND 4. Pups were sexed on PND 0 and weighed one day after birth and on PND 4. Their viability was recorded twice daily on each working day or only in the morning on Saturday and Sunday. Clinicochemical and haematological examinations as well as urinalyses were performed in 5 randomly selected P0 parental animals of either sex towards the end of the administration period. At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 randomly selected P0 parental males and females per test group. All surviving P0 parental animals were sacrificed assessed by gross pathology. Organ weights were recorded and a histopathological examination was performed

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Remarks:

Highest dose: First 7 (females) and 8 days (males) of the study

Dose / conc.:

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

Remarks:

Highest dose: Reduced dose level from study day 8 (females) / 9 (males) onwards, toxic effects were seen at 100 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a previously performed 14-days test study the test article was administered by gavage to groups of 3 male and 3 female Wistar rats at dose levels of 0, 50, 100, 200, and 600 mg/kg body weight/day (mg/kg bw/d). Drinking water containing 1% Carboxylmethylcellulose and Cremophor EL® (5 mg/100 mL) served as vehicle. In this study, the oral administration of the test substance caused severe adverse effects in both sexes, i.e. premature deaths and poor general state, at dose levels of 600 and 200 mg/kg bw/d. Only slight effects on food and water consumption and body weight data were observed at 100 mg/kg bw/d.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied. A cageside examination was conducted before and after treatment for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented for each animal. The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g., animal could not litter, umbilical cord not cut) were documented on an individual dam basis. On weekdays (except public holidays) the littering behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of littering was considered to be a 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 x 37.5 cm with side borders of 25 cm high). The following parameters were examined: abnormal behavior during “handling”, fur, skin, salivation, exophthalmos, lacrimation, pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, feces (appearance/consistency), urine, other findings.

BODY WEIGHT: Yes
In general, the body weight of the female parental animals was determined once a week at the same time of the day (in the morning).
The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals:
- During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
- Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
- Females without a litter were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION
Food consumption was determined once a week (in a period of 7 days) for female parental animals, with the following exceptions:
- Food consumption was not determined during the mating period (female P0 animals).
- Food consumption of the P0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
- Food consumption of P0 females, which gave birth to a litter, was determined on PND 0 and 4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).

OTHER:
The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. The females were allowed to litter and rear their pups until day 4 after parturition. On PND 4, all pups were sacrificed and examined. On study day 50, a functional observational battery and motor activity measurement was carried out in 5 randomly selected P0 parental female animals (with litter) per test group. From the selected 5 female animals urinalysis was carried out on study day 46. Clinicochemical and haematological examinations were carried out on study day 51 females. At the end of the study (females: study day 51), the animals were sacrificed after a fasting period (withdrawal of food) for at least 16-20 hours.

Ovaries and uterine content:

Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: After sacrifice of the female animals the uterus and ovaries were removed and the implantation sites were counted. To determine the number of implantation sites in apparently non-pregnant animals, the uteri from those females were stained in 10% ammonium sulfide solution for about 5 minutes. Then, the respective uteri were rinsed carefully with fresh tap water. The implantation sites were recorded for the calculation of the postimplantation loss.

Fetal examinations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, pup weight, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead. All surviving pups (sacrificed on PND 4), all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.
- External examinations: Yes: all per litter

Statistics:

DUNNETT-test (two-sided): Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of pups delivered per litter, implantation sites, post implantation loss.
FISHER'S EXACT test: Female mating index, female fertility index, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters with affected pups at necropsy.
WILCOXON-test (one-sided): Proportions of affected pups per litter with necropsy observations.
KRUSKAL-WALLIS test (two-sided): Feces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON test (two-sided).

Indices:

Female mating index, Female fertility index, Gestation index, Live birth index, Post implantation loss, Viability index and sex ratio.

Historical control data:

All findings have been assessed with respect to historical control data.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs of toxicity were only seen in test group 3 (100 and 60 mg/kg bw/d). Poor general state was observed in study weeks 1 and 2 in two female animals and in one female animal in study weeks 6 and 7. Reduced nutritional state was also observed in study week 2 in two female animals in study weeks 1 and 2 and one female animal in week 0. In females respiratory sounds were observed in nine animals in study week 0, in eight animals in study week 1, in three animals in study week 2 and also in two animals in study week 6 and in study week 7 one animal. Piloerection was observed in study week 0 in one female animal. Diarrhea was observed in study week 2 in one female animal. No treatment-related findings were seen in any animal of test groups 1 and 2 (5 and 20 mg/kg bw/d).

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

In test group 3 (100 and 60 mg/kg bw/d) one female animal was found dead within the first week of the study. In addition, one female animal of test group 3 (100 and 60 mg/kg bw/d) was sacrificed in a moribund state in study week 2.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In test group 3 (100 and 60 mg/kg bw/d) body weight loss was observed in female animals between study weeks 0-1 (not significant). No effects on body weight data were observed in female animals of test groups 1 and 2 (5 and 20 mg/kg bw/d) during the entire study period. The same was true for female animal of test group 3 (100 and 60 mg/kg bw/d) during gestation and lactation periods.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Significantly decreased food consumption in female animals of test group 3 (100 and 60 mg/kg bw/d) was observed in the first study week. This finding was assessed as being related to treatment. Impaired food consumption was not observed female animals of test groups 1 and 2 (5 and 20 mg/kg bw/d). The same was true for female animals of test group 3 (100 and 60 mg/kg bw/d) during gestation and lactation periods.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related, adverse changes among haematological parameters were observed.
In female rats of test groups 1 (5 mg/kg bw/d) and 3 (100 and 60 mg/kg bw/d) the platelet counts were lower compared to controls. These values were within the historical control ranges (platelet counts: 765-1009 Giga/L). Therefore, the alterations were regarded as incidental rather than treatment-related. In females of test group 2 (20 mg/kg bw/d) the relative lymphocyte counts, and in females of test group 3 (100 and 60 mg/kg bw/d) the relative large unstained cell (LUC) counts were lower compared to controls. These changes in the differential blood cell counts were not accompanied by a change of the total white blood cell counts, and the lymphocyte and LUC cell counts were within the historical control ranges (relative lymphocyte counts: 66.7-83.9%, relative LUC counts: 0.2-0.5%). The alterations were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related, adverse changes among clinical chemistry parameters were measured.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No treatment-related adverse changes among urinalysis parameters were measured.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No test substance-related or spontaneous findings in female animals of all test groups during the home cage observation were observed.
The open field observations did not reveal any test substance-related findings in female animals of all test groups. During sensimotor tests, there were no test substance-related findings in female animals of all test groups. Any deviations from "zero values" were equally distributed between test substance-treated groups and controls or occurred in single animals only. Therefore, these observations were considered as being incidental. There were no significant deviations concerning single intervals and the overall motor activity (summation of all intervals) in female animals of all test groups was in comparison to the concurrent control group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Both absolute- and relative organ weight changes did not reveal any relation to treatment for planned sacrificed animals.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Regarding pathology, animals, which died intercurrently or were sacrificed moribund showed some macroscopic and/or microscopic lesions in the gastro-intestinal tract. Dilation of stomach, jejunum, cecum or colon, mucosal/villous atrophy, acute inflammation and erosion(s) and intra-epithelial abscesses in forestomach were observed in one animal. In addition, lesions in some lymphoid organs indicating secondary stress related changes were observed such as severe organ size reduction of thymus (atrophy), lymphoid depletion in spleen and axillary lymph node and lymphocytolysis in axillary and mesenteric lymph nodes. In one animal of test group 3 (100 and 60 mg/kg bw/d) that died on study day 3, also bilateral cortical hypertrophy of the adrenal cortex was observed indicating prolonged stress response. These changes mentioned for animals at moribund sacrifice and spontaneous death were considered to be treatment-related.

No treatment-related macroscopic and/or microscopic changes were observed in animals autopsied at planned sacrifice. For these planned sacrificed animals, all macroscopic and/or microscopic and organ weight changes observed were considered to be normal background changes, bearing no relation to treatment.

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):

Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (13.8, 13.1, 12.2, and 12.1 implants per dam in test groups 0- 3). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any significant differences between the test groups, and the mean number of F1 pups delivered per dam remained unaffected (12.8, 12.4, 11.9, and 11.0 pups per dam at 0, 5, 20 and 100 and 60 mg/kg bw/d, respectively).

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 100% (test group 0), 98% (test groups 1 and 2) and 99% (test group 3). Moreover, the number of stillborn pups was comparable between the test groups and a dose-response relationship was not observed.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

The mean duration of gestation values were comparable between all test groups and varied between 21.9 and 22.1 days.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): The mean duration of gestation values were comparable between all test groups and varied between 21.9 and 22.1 days.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All sperm-positive animals of test groups 0 (control) and 1 (5 mg/kg bw/d) delivered pups or had implantations in utero. One female of test group 2 (20 mg/kg bw/d) and 1 female of test group 3 (100 and 60 mg/kg bw/d) did not become pregnant. The fertility index varied between 88% and 100% and did not show statistically significant differences.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Basis for effect level:

clinical signs

gross pathology

mortality

Dose descriptor:

LOAEL

Effect level:

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

Based on:

act. ingr.

Basis for effect level:

clinical signs

gross pathology

mortality

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the concurrent control values.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The mean number of delivered pups per dam and the rate of liveborn and stillborn pups were comparable between the test substance-treated groups and the control group. The respective values reflect the normal range of biological variation inherent in the strain used in this study.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 4 did not show biologically relevant differences.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

One pup in test group 3 (100 and 60 mg/kg bw/d) died on PND 1. One pup in test group 2 (20 mg/kg bw/d) was cannibalized on PND 1. The viability index as indicator for pup mortality between PND 0-4 was 100% for test groups 0 (control) and 1 (5 mg/kg bw/d) and 99% in test group 2 (20 mg/kg bw/d) and 3 (100 and 60 mg/kg bw/d).

External malformations:

not examined

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Description (incidence and severity):

GROSS PATHOLOGICAL FINDINGS
All stillborn pups of test groups 1 (5 mg/kg bw/d), 2 (20 mg/kg bw/d) and 3 (100 and 60 mg/kg bw/d) did not show any abnormal findings. On day 4 all pups were sacrificed scheduled and only incidental findings were obtained. In test group 0 (0 mg/kg bw/d) one pup showed dextrocardia, in test group 2 (20 mg/kg bw/d) one pup showed hemorrhagic testis, hydronephrosis, hydroureter and distended urinary bladder. In one pup of test group 2 (20 mg/kg bw/d) infarct of liver was observed. All findings were assessed as being incidental and can be found in the historical control data. A relation to dosing was excluded.

Key result

Dose descriptor:

NOAEL

Effect level:

> 60 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

not examined

Key result

Developmental effects observed:

no

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

60 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

In a GLP-compliant OECD 422 study, the test article was administered orally by gavage to groups 10 female Wistar rats (P0 parental animals) at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; vehicle control; test group 0), 5 mg/kg bw/d (test group 1), 20 mg/kg bw/d (test group 2) and 100 mg/kg bw/d (test group 3). Because of severe clinical findings seen in animals of test group 3 (100 mg/kg bw/d), i.e. respiration sounds, reduced body weights and the death of one female animal on study day 3, the dose level in test group 3 was reduced to 60 mg/kg bw/d for the females on study day 8. Drinking water (neutrally buffered for test substance preparations) served as vehicle. P0 animals were mated 13 days after the beginning of treatment to produce litter (F1 generation pups). Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear. P0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all P0 females. A detailed clinical observation (DCO) was performed in all P0 animals before test substance administration and thereafter at weekly intervals. Food consumption of the P0 parents was determined regularly during premating and after the mating period and during the gestation and lactation periods in dams. In general, body weights of P0 animals were determined once a week. During gestation and lactation period, P0 females were weighed on gestation days (GD) 0, 7, 14 and 20, on the parturition day (postnatal day [PND] 0) and on PND 4. Pups were sexed on PND 0 and weighed one day after birth and on PND 4. Their viability was recorded twice daily on each working day or only in the morning on Saturday and Sunday. Clinicochemical and hematological examinations as well as urinalyses were performed in 5 randomly selected P0 parental animals of either sex towards the end of the administration period. At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 randomly selected P0 parental females per test group. All surviving P0 parental animals were sacrificed assessed by gross pathology. Organ weights were recorded and a histopathological examination was performed.

 

In test group 3 (100 mg/kg bw/d between study days 0-7 [females], 60 mg/kg bw/d onwards from study day 8 [females]), one female animal was found dead in study week 1. One female animal was sacrificed moribund state in study week 2. Poor general state was observed in one female animal in study week 0 and in one female animal in study weeks 6 and 7 as well as in one female animal from GD 0 and 1, and from GD 7 onwards. Reduced nutritional state was recorded in one female animal in study week 0 and two female animals in study weeks 1 and 2 as well as in one female animal from GD 0 to 5. Respiratory sounds were reported in several female P0 animals during the entire study period. Furthermore, piloerection was seen in one female animal in study week 0 and one female female animal in study week 2 had diarrhea. Significantly decreased food consumption was reported in females in the first study week. The female animal which was sacrificed moribund showed severe dilation of the gastro-intestinal tract (stomach, jejunum and cecum) with severe organ size reduction of the thymus. No test-substance related, relevant findings were observed regarding fertility and clinical pathology. No findings in test groups 1(5 mg/kg bw) and 2 (20 mg/kg bw), F1 animals were free of effects in all groups. Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats the no observed adverse effect level (NOAEL) for developmental toxicity was 60 mg/kg bw/d.

Justification for classification or non-classification

Based on the available data, classification for reproductive/developmental toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.