7,7,9(or 7,9,9)-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, oral (OECD 422), rat:

reproductive toxicity: NOAEL >= 1000 mg/kg bw/day for males and females of the parental generation

systemic toxicity: NOAEL = 100 mg/kg bw/day for males and 300 mg/kg bw/day for females of the parental generation

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Apr - 20 Jun 2017

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:

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

Version / remarks:

adopted in 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

adopted in 2000

GLP compliance:

yes (incl. QA statement)

Remarks:

Országos Gyógyszerészeti és Élelmezés-egészségügyi Intézet, Budapest, Hungary

Limit test:

no

Species:

rat

Strain:

other: Hsd.Han: of Wistar origin

Details on species / strain selection:

The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt.Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 83 - 85 days (males), 83 - 85 days (females)
- Weight at study initiation: 330 - 384 g (males), 208 - 246 g (females); The weight variation did not exceed ± 20 per cent of the mean weight.
- Housing: 2 animals of the same sex per cage (before mating), 1 male and 1 female per cage (mating), individually (pregnant females), 2 animals per cage (males after mating), 2 or 3 animals of the same sex per cage (recovery animals) in Type III polypropylene/polycarbonate (Size: 22 x 32 x 19 cm (width x length x height)); certified laboratory wood bedding (Lignocel® Hygienic Animal Bedding, J. Rettenmaier & Söhne GmbH+Co.KG, Rosenberg, Germany) suitable as nesting material
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice – breeding and maintenance (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 20 days

DETAILS OF FOOD AND WATER QUALITY: The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used. Water quality control analysis and microbiological assessment are performed once in every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service (Budapest,Hungary).


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

polyethylene glycol 400 (PEG 400)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was formulated in the vehicle at concentrations of 20, 60 and 200 mg/mL in the formulation laboratory of the Test Facility beforehand not longer than for three days before the administration and was stored in a refrigerator until use.

VEHICLE
- Justification for use and choice of vehicle: The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front.
- Concentration in vehicle: 20, 60 and 20 mg/mL
- Amount of vehicle: 5 mL/kg bw
- Lot/batch no.: 15I040501 (batch number 1), 16I284004 (batch number 2)

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: until copulation occurred
- Proof of pregnancy: vaginal plug / 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.
- After successful mating each pregnant female was caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing formulations (control of concentration) was performed twice during the study. Five aliquots of 1 mL of each formulation (20, 60 and 200 mg/mL) and five aliquots of 1 mL control substance (vehicle) were taken and analyzed. The samples were stored at 5 ± 3 °C before the analysis. Concentration of the test item in the dosing formulations varied in the range of 101% to 105% in comparison to the nominal values. The formulation samples were homogenous at both analytical occasions. Recovery of the test material from formulations in the vehicle was 102 and 99% at ~1 and ~300 mg/mL concentrations, respectively. A sufficient stability and homogeneity in the chosen vehicle was verified over the range of relevant concentrations at the appropriate frequency of preparation in a separate analytical report. The test material proved to be stable at room temperature for 1 day (recovery was 106% of starting concentrations at 1 mg/mL and 105% at 300 mg/mL) and at 5 ± 3°C for 3 days (recovery was 102% of starting concentrations at 1 mg/mL and 105% at 300 mg/mL).

Duration of treatment / exposure:

56 days (males, control and test groups)
56, 57 or 64 days (females, control and test groups, depending on the effectiveness of mating)
55 days and 14 day post-exposure observation period (recovery groups)

Frequency of treatment:

once daily at similar time (± 2 h), 7 days/week

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:

Treatment period: 12/sex (control and test groups)
Treatment and recovery period: 5/sex (control group and 1000 mg/kg bw/day)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen on the basis of the results of a preliminary toxicity screening test with the tets material in rats. The high and mid-high doses were chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.
- Post-exposure recovery period in satellite groups: 14 days

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily and once daily
- Cage side observations included: morbidity and mortality (twice daily), general clinical observations (once daily)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at the times of weekly weighing, prior to and during the mating until necropsy; weekly during the recovery period

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of dosing and weekly thereafter and on the day of necropsy (parental males); on the first day of dosing and weekly thereafter and on gestation days 0, 7, 14 and 21 and on days 0 (within 24 h after parturition), 4 and 13 post-partum (parental females); on day of necropsy (females subjected to organ weighing); weekly during treatment and post-treatment observation period (recovery groups)

FOOD CONSUMPTION:
The food consumption was determined weekly by reweighing the non-consumed diet during the treatment period except mating phase (pre-mating days 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13). Food consumption of male animals was also determined by weekly interval during post-mating period. The food consumption of animals assigned to the recovery groups were weighed by weekly interval during the treatment and post-treatment observation period.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of necropsy (control and test groups), at the end of the 14 days post-treatment period (recovery groups)
- Anaesthetic used for blood collection: Yes (Isofluran)
- Animals fasted: Yes
- How many animals: 5 males and 5 females randomly selected from each group (control and test groups), all animals (recovery groups)
- Parameters examined: white blood cell (leukocyte) count, red blood cell (erythrocyte) count, hemoglobin concentration, hematocrit (relative volume of erythrocytes), mean corpuscular (erythrocyte) volume, mean corpuscular (erythrocyte) hemoglobin, mean corpuscular (erythrocyte) hemoglobin concentration, platelet (thrombocyte) count, reticulocytes, differential white blood cell count (neutrophil, monocyte, lymphocyte, basophil, eosinophil), blood coagulation (activated partial thromboplastin time, prothrombin time)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of necropsy (control and test groups), at the end of the 14 days post-treatment period (recovery groups)
- Animals fasted: Yes
- How many animals: 5 males and 5 females randomly selected from each group (control and test groups), all animals (recovery groups)
- Parameters examined: alanine aminotransferase activity, aspartate aminotransferase activity, total bilirubin concentration, creatinine concentration, urea concentration, glucose concentration, cholesterol concentration, bile acids, sodium concentration, potassium concentration, albumin concentration, total protein concentration

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last exposure week (Day 56) but before the blood sampling
- Dose groups that were examined: 5 male and 5 female animals randomly selected from each group
- Battery of functions tested: sensory activity / grip strength / motor activity / other: modified Irwin test

IMMUNOLOGY: No

OTHER: Determination of serum levels of thyroid hormones (T4) from all parental male animals at termination on Day 54.

Oestrous cyclicity (parental animals):

Estrous cycle was monitored by examining vaginal smears each day before the treatment started from each animal being considered for study for two weeks. Estrous cycle was evaluated and considered at randomization. Vaginal smears were also prepared and estrous cycle was monitored daily from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating. Vaginal smears were also prepared on the day of the necropsy. Vaginal smears were stained with 1% aqueous methylene blue solution and were examined with a light microscope.

Sperm parameters (parental animals):

Parameters examined in all parental males of the control and high dose group:
testis weight, epididymis weight, other: quantity and morphology of spermatogenic cells (spermatogonia, spermatocytes, spermatids and spermatozoa) representing different stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 4 pups/sex/litter; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, other: runts (pups that are significantly smaller than normal pups)

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead; on the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn) from pups died after the birth (dead pups)

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after the optionally extended post-mating period on Day 56.
- Maternal animals: All surviving animals not selected for toxicology examinations on post-partum days 14 - 22 or shortly thereafter (Days 57 or 64). All surviving animals selected for toxicology examinations shortly after post-partum days 15 - 21 (Day 56).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs of male adults were weighed: brain, testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole. In addition, for 5 males and females randomly selected from each group, and for recovery animals, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed. Detailed histological examination was performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals of control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure and on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma. Additionally, these organs were processed and examined histologically in not mated male animals (1/17 at 100 mg/kg bw/day and 1/17 at 300 mg/kg bw/day), in non-pregnant female and its male pair at 100 mg/kg bw/day and in one dam at 300 mg/kg bw/day based on macroscopic observation. Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose including recovery groups. Histological examinations were performed on the liver in all animals and on organs with macroscopic findings (thymus, uterus) in the low and mid dose groups.

Postmortem examinations (offspring):

SACRIFICE
- All offspring were sacrificed on postnatal day 13 or shortly thereafter.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Dead pups and pups euthanized at day 13 post-partum, or shortly thereafter, were carefully examined for gross abnormalities.

GROSS NECROPSY
- Gross necropsy consisted of examinations for gross abnormalities.

OTHER: The serum level of T4 (thyroid hormone) was determined in pups sacrificed on Day 13.

Statistics:

The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible. For evaluation of data obtained during the recovery period, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the Two-Sample t-test was performed. Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.

Reproductive indices:

Copulatory index males [%] = Number of males with confirmed mating/Total number of males cohabited x 100
Copulatory index females [%] = Number of sperm positive females/Total number of females cohabited x 100
Fertility index males [%] = Number of males impregnating a female/Total number of males with confirmed mating x 100
Fertility index females [%] = Number of pregnant females/Number of sperm positive females x 100
Gestation index [%] = Number of females with live born pups/Number of pregnant females x 100

Offspring viability indices:

Post-implantation mortality (intrauterine mortality) [%] = (Number of implantations - Number of liveborns)/Number of implantations x 100
Post-natal mortality [%] = (Number of liveborns - Number of live pups on postnatal day 13)/Number of liveborns x 100
Survival index [%] = Number of live pups on postnatal day 13/Number of pups born x 100
Sex ratio [%] = (Number of pups examined - Number of males (females))/Number of pups examined x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

MALES
Control, 100, 300 and 1000 mg/kg bw/day: soft stool caused by the vehicle from Day 3 up to the termination of treatment

FEMALES
Control, 100, 300 and 1000 mg/kg bw/day: soft stool caused by the vehicle from Day 3 up to the termination of treatment
Control: alopecia on the forelimbs and on the abdomen in 1/12 animal from gestation Day 9 and gestation Day 21, respectively, up to the termination of the treatment
300 mg/kg bw/day: alopecia on the forelimbs in 1/12 animal between lactation days 4 and 14
1000 mg/kg bw/day: alopecia on the abdomen in 1/11 dams between lactation day 0 and 17

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

MALES
1000 mg/kg bw/day: statistically significant slightly increased mean body weight gain comparing to their control during the first week of the recovery period; slightly increased summarized body weight gain during the post-treatment observation period (not statistically significant)

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

MALES
1000 mg/kg bw/day: statistically significant slightly decreased mean daily food consumption during the second week of the premating period

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):

MALES
100 mg/kg bw/day: statistically significant slightly decreased mean percentage of basophil granulocytes, mean concentration of hemoglobin and mean hematocrit value; statistically significant shorter mean activated thromboplastin time
300 mg/kg bw/day: statistically significant slightly decreased mean percentage of basophil granulocytes, mean concentration of hemoglobin and mean hematocrit value
1000 mg/kg bw/day: statistically significant slightly decreased mean percentage of basophil granulocytes; statistically significant slightly decreased mean percentage of lymphocytes at the end of the recovery period

FEMALES
1000 mg/kg bw/day: statistically significant decreased mean percentage of neutrophil granulocytes, statistically significant increased percentage of lymphocytes and mean platelet count

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

MALES
100 mg/kg bw/day: statistically significant slightly decreased mean concentration of albumin
300 mg/kg bw/day: statistically significant slightly decreased mean concentration of albumin
1000 mg/kg bw/day: statistically significant slightly decreased mean activity of alanine aminotransferase; statistically significant slightly decreased mean concentration of potassium in the recovery period

FEMALES
300 mg/kg bw/day: statistically significant slightly increased mean urea concentration
1000 mg/kg bw/day: statistically significant slightly increased mean urea and creatinine concentration

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

MALES
Control: acute thymic hemorrhage in 1/5 animal; hyperplasia of bronchus associated lymphoid tissue in 1/5 animal; alveolar emphysema of minimal degree in 1/5 animal at the end of the recovery period; hyperplasia of bronchus associated lymphoid tissue in 1/5 animal at the end of the recovery period; renal cyst in 1/5 animal
300 mg/kg bw/day: centrilobular microvesicular vacuolation in hepatocytes/ hepatic lipidosis in 3/12 animals; acute thymic hemorrhage in 1/5 animal (non-treatment-related)
1000 mg/kg bw/day: centrilobular microvesicular vacuolation in hepatocytes/ hepatic lipidosis in 7/12 animals; alveolar emphysema in 1/5 animal (non-treatment-related); acute thymic hemorrhage in 1/5 animal (non-treatment-related); alveolar emphysema of minimal degree in 1/5 animal at the end of the recovery period (non-treatment-related)

FEMALES
Control: dilatation of uterine horns in 2/12 animals; alveolar emphysema in 1/5 animal; acute thymic hemorrhage in 1/5 animal; hyperplasia of bronchus associated lymphoid tissue in 1/5 animal at the end of the recovery period
300 mg/kg bw/day: dilatation of uterine horns in 1/12 animal (non-treatment-related)
1000 mg/kg bw/day: dilatation of uterine horns in 1/12 animal at the end of the treatment period and in 2/5 animals at the end of the recovery period (non-treatment-related); centrilobular microvesicular vacuolation in hepatocytes/ hepatic lipidosis in 3/12 animals; alveolar emphysema in 1/5 animal (non-treatment-related); acute thymic hemorrhage in 1/5 animal (non-treatment-related); hyperplasia of bronchus associated lymphoid tissue in 1/5 animal (non-treatment-related)

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Description (incidence and severity):

There were no statistically significant differences in the thyroid hormone (free T4) level in parental male animals compared to the control.

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

MALES
100 mg/kg bw/day: statistically significant slightly decreased copulatory index
300 mg/kg bw/day: statistically significant slightly decreased copulatory index

FEMALES
100 mg/kg bw/day: statistically significant slightly decreased fertility index
300 mg/kg bw/day: statistically significant increased mean number of pre-coital days and mean number of conceiving days
1000 mg/kg bw/day: statistically significant slightly decreased fertility index

CLINICAL SIGNS
Soft stool was observed in all animals at all dose groups and was attributed to the vehicle. Alopecia is a common spontaneous finding in this strain of experimental rats and is seen also in untreated rats. The effect occurred in single animals of control, mid and high dose groups in this study. Therefore, it was considered to be an incidental finding and not related to the treatment with the test item.

BODY WEIGHT AND BODY WEIGHT GAIN
Statistical significant slightly increased mean body weight gain was observed in males during the first week of the recovery period at the highest dose group. This resulted in a slightly higher summarized body weight gain during the post-treatment observation period and caused no significant difference with respect to the control in the mean body weight. This difference was not considered treatment-related.

FOOD CONSUMPTION
The slightly lower mean daily food consumption in males of the high dose group (1000 mg/kg bw/day) compared to controls was with minor degree and was transient and therefore was judged to be toxicologically not relevant.

HAEMATOLOGICAL FINDINGS
The differences in several haemotological parameters with respect to control were judged to be indicative of biological variation and toxicologically not relevant. Changes in basophil granulocytes, hemoglobin, hematocrit and activated thromboplastin time in treated male animals were of minor degree and not related to the doses at the end of the treatment period. Moreover, all values were well within the historical ranges and statistical significances were mainly originated from the relative high value of the control group (hemoglobin, hematocrit and activated thromboplastin time). Slight changes in the neutrophil granulocytes, lymphocytes and platelet count were only observed in female animals at the end of the post-treatment observation period but not at the end of the treatment period.

CLINICAL BIOCHEMISTRY FINDINGS
Higher concentrations of urea and creatinine were indicative of altered renal function in in female animals at 300 or 1000 mg/kg bw/day. Changes in concentrations of creatinine and urea in serum were considered to be signs of adaptation of the organ to the altered demands. Without supporting histopathological changes, these effects were judged to be toxicologically not relevant. The slight, but statistically significant differences compared to the controls with respect to albumin and potassium concentration, were considered to have little or no toxicological relevance. Decrease in enzyme activity of alanine aminotransferase has no toxicological relevance without tissue damage. Albumin concentrations changed only in the lower dose groups but not at the high dose.

GROSS PATHOLOGICAL FINDINGS
Hemorrhage in the thymus was due to circulatory disturbance developed during the exsanguination procedure. Hydrometra, related to the female sexual cycle, is a frequent observation in experimental rats. Cecum dilatation was an individual change probably due to an increased water accumulation as there were no accompanying histological findings. Alopecia on the skin is a common observation in this strain of experimental rats, which occurred with similar incidence in the control and test item treated female animals (300 and 1000 mg/kg bw/day) in this study. Therefore, these macroscopic changes were considered to be independent from the treatment with the test item.

ORGAN WEIGHTS
The slight increases in liver weights in females at 300 mg/kg bw/day were not considered to be toxicologically relevant due to the small degree and in the lack of associated histopathological alterations as well as in the absence of similar changes in the high dose group.

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC
The dilatation of uterine horns observed in females is not associated with inflammatory or other pathological lesions and is a slight neuro-hormonal phenomenon and was in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.
The treatment related observed hepatic lipidosis was of minimal or mild degree and and is considered to be a slight reversible liver injury and it might be in connection with a disturbance of energy metabolism of affected hepatocytes.
The acute changes in the lungs (alveolar emphysema) and thymus (hemorrhage) in control or high dose treated animals were considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguinations.
Hyperplasia of bronchus associated lymphoid tissue as observed in males and females of the control as well as of the highest dose group is a physiological immune-morphological phenomenon, without toxicological significance.

REPRODUCTIVE PERFORMANCE
The slight, but statistically significant differences in the copulatory index observed in males at 100 and 300 mg/kg bw/day as well as in the fertility index at 100 and 1000 mg/kg bw/day and in the mean number of pre-coital and conceiving days at 300 mg/kg bw/day in females were considered to be indicative of biological variation due to the lack of a clear and consistent dose-response relationsship. All values were within or marginal to historical control mean.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects at highest dose tested

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

100 mg/kg bw/day: swollen abdomen, smaller than normal size and dyspnea
300 mg/kg bw/day: swollen abdomen, smaller than normal size and dyspnea
1000 mg/kg bw/day: decreased percentage of of not suckled pups

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Control: one dead offspring on postnatal day 13
100 mg/kg bw/day: one stillborn offspring, two dead pups on postnatal day 0, two dead pups on postnatal day 13
300 mg/kg bw/day: two stillborn pups, two dead pups
1000 mg/kg bw/day: one stillborn pup, one dead offspring

Body weight and weight changes:

no effects observed

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:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the thyroid hormone (free T4) level in Day 13 pups compared to the control.

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):

Control: no milk in the stomach of 1/1 dead offspring
100 mg/kg bw/day: hydrops fetalis in 1/1 stillborn; hole on the cranium, paleness and black colored content in the intestines in 1/2 dead pup on postnatal day 0; swollen abdomen and stomach as well as gas filled intestines in 1/2 dead pup on postnatal day 13
300 mg/kg bw/day: gas filled stomach and intestines in 1/2 dead pup; autolyzed visceral organs in 2/2 pups
1000 mg/kg bw/day: misshapen head (lack of eyes, mouth and nostrils) in 1/1 stillborn pup

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

SEX DISTRIBUTION
1000 mg/kg bw/day: statistically significant slightly decreased mean number of male pups per litter

ANOGENITAL DISTANCE
100 and 300 mg/kg bw/day: statistically significant slightly shorter anogenital distance in male pups (non-treatment-related)
1000 mg/kg bw/day: statistically significant slightly shorter anogenital distance in female pups (non-treatment-related)

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

GROSS PATHOLOGICAL FINDINGS
The observed alterations (hydrops fetalis, hole on the cranium, misshapen head) are spontaneous variation or malformation in experimental rats of this strain.

SEX DISTRIBUTION
The difference in the mean number of male pups per litter compared to the control was with minor degree and was due to the relative high value of control (6.4 ± 1.7 vs. historical control mean: 5.4 ± 2). Therefore, it was considered to be toxicologically not relevant.

ANOGENITAL DISTANCE
Due to the minor degree, the slight differences regarding the anogenital distance in males and females compared to the control were not considered to be toxicologically relevant.

Dose descriptor:

NOAEL

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: no effects at highest dose tested

Critical effects observed:

no

Reproductive effects observed:

no

Table 1: Summary of clinical signs in males (pre-mating, mating and post-mating periods)

Observations	Control		100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
	Treatment period*	Recovery period			Treatment period*	Recovery period
Normal	17/17	5/5	12/12	12/12	17/17	5/5
Soft stool	17/17	5/5	12/12	12/12	17/17	5/5

Frequency of observations: number of animals (cage) with observation/number of animals (cage) examined

* Including animals of recovery group

 

Table 2: Summary of clinical signs in females

Observations	Control	100 mg/kg bw/day	300 mg/kg bw/day	600 mg/kg bw/day
Pre-mating and mating periods
Normal	12/12	12/12	12/12	12/12
Soft stool	12/12	12/12	12/12	12/12
Post-mating periods
Normal	-	1/1	-	1/1
Soft stool	-	1/1	-	1/1
Gestation period
Normal	11/12	11/11	12/12	11/11
Soft stool	12/12	11/11	12/12	11/11
Skin: Alopecia	1/12	0/11	0/12	0/11
Lactation period
Normal	11/12	11/11	11/12	10/11
Soft stool	12/12	11/11	12/12	11/11
Skin: Alopecia	1/12	0/11	1/12	1/11
Recovery animals
Normal	5/5	5/5	5/5	5/5
Soft stool	5/5	5/5	5/5	5/5

 

Table 3: Summary of body weight gain in males

Group		Body weight gain (g) between
		Pre-mating days			Mating and post-mating days						Total
		0 – 7	7 – 13	0 – 13	13 – 20	20 – 27	27 – 34	34 – 41	41 – 48	48 - 55	0 – 55
Control	Mean	17.9	17.2	35.1	14.5	11.3	10.2	7.9	10.9	11.5	101.4
	SD	4.5	4.1	6.1	4.7	4.2	6.7	5.1	3.5	4.5	17.2
	n	17	17	17	17	17	17	17	17	17	17
100 mg/kg bw/day	Mean	18.3	15.6	33.9	15.7	10.8	11.0	8.4	10.9	12.1	102.8
	SD	7.7	5.6	10.1	4.6	5.2	4.0	4.8	8.4	7.1	18.9
	n	12	12	12	12	12	12	12	12	12	12
300 mg/kg bw/day	Mean	15.8	16.6	32.4	10.8	12.7	10.2	10.0	13.6	10.8	100.4
	SD	6.2	4.1	9.1	8.5	6.8	4.3	4.0	4.7	3.7	22.2
	n	12	12	12	12	12	12	12	12	12	12
1000 mg/kg bw/day	Mean	16.3	15.0	31.3	12.2	11.5	13.1	7.4	11.2	8.0	94.6
	SD	5.6	4.3	8.1	7.5	4.6	5.4	3.4	4.9	4.3	20.0
	n	17	17	17	17	17	17	17	17	17	17
		NS	NS	NS	NS	NS	NS	NS	NS	NS	NS

NS = Not Significant

 

Table 4: Summary of body weight gain in males of the recovery group

Group		Body weight gain (g) between
		Recovery days
		0 – 6	6 – 13	0 – 13
Control	Mean	4.8	0.8	5.6
	SD	3.8	3.1	6.5
	n	5	5	5
1000 mg/kg bw/day	Mean	11.0*	3.6	14.6
	SD	4.4	4.6	7.8
	n	5	5	5
		*	NS	NS

* p < 0.05

 

Table 5: Summary of body weight gain in females

Group		Body weight gain (g) between
		Pre-mating days			Treatment days					Total
		0 – 7	7 – 13	0 – 13	13 – 20	20 – 27	27 – 34	34 – 41	41 – 48	0 – 55
Control	Mean	8.2	7.2	15.4	6.0	2.6	7.0	4.0	1.8	39.6
	SD	6.4	7.1	6.8	4.0	7.6	4.5	2.6	4.9	10.0
	n	17	17	17	5	5	5	5	5	5
100 mg/kg bw/day	Mean	11.3	6.4	17.8	-	-	-	-	-	-
	SD	9.1	7.7	4.5	-	-	-	-	-	-
	n	12	12	12	-	-	-	-	-	-
300 mg/kg bw/day	Mean	13.3	5.1	18.3	-	-	-	-	-	-
	SD	7.1	5.0	9.0	-	-	-	-	-	-
	n	12	12	12	-	-	-	-	-	-
1000 mg/kg bw/day	Mean	10.1	8.4	18.5	8.8	-2.2	9.6	0.6	5.0	39.4
	SD	9.6	6.4	9.5	2.3	6.9	4.2	4.5	1.2	9.4
	n	17	17	17	5	5	5	5	5	5
		NS	NS	NS	NS	NS	NS	NS	NS	NS

NS = Not Significant

 

Table 6: Summary of body weight gain in females of the recovery group

Group		Body weight gain (g) between
		Recovery days
		0 – 6	6 – 13	0 – 13
Control	Mean	0.8	0.0	0.8
	SD	7.7	1.9	7.3
	n	5	5	5
1000 mg/kg bw/day	Mean	6.6	-1.0	5.6
	SD	6.66	3.9	3.0
	n	5	5	5
		NS	NS	NS

NS = Not Significant

 

 

Table 7: Summary of food consumption in males

Group		Daily mean food consumption (g/animal/day)
		Pre-mating days		Post-mating days					Recovery period
		0 – 7	7 – 13	20 – 27	27 – 34	34 – 41	41 – 48	48 – 55	Day 0 – 6	Day 6 – 13
Control	Mean	25.9	27.4	24.9	24.9	24.5	25.0	25.1	24.4	24.6
	SD	1.93	1.46	1.59	1.86	1.25	1.26	1.54	0.55	0.35
	n	8.00	8.00	8.00	8.00	8.00	8.00	8.00	2.00	2.00
100 mg/kg bw/day	Mean	26.0	27.1	24.8	24.9	24.9	25.1	24.9	-	-
	SD	1.52	2.03	1.09	0.85	0.73	1.66	1.66	-	-
	n	6	6	5	5	6	6	6	-	-
	±%	0	-1	0	0	1	0	-1	-	-
300 mg/kg bw/day	Mean	25.8	26.2	24.4	24.6	24.6	25.1	25.1	-	-
	SD	2.22	1.31	1.38	1.16	0.94	1.27	1.16	-	-
	n	6	6	6	6	6	6	6	-	-
	±%	-1	-4	-2	-1	0	1	0	-	-
1000 mg/kg bw/day	Mean	24.7	25.2*	24.0	24.2	24.0	24.3	23.6	25.1	25.6
	SD	2.30	1.80	2.16	1.91	1.65	1.54	1.3	1.73	2.54
	n	8	8	8	8	8	8	8	2	2
	±%	-5	-8	-4	-3	-2	-3	-6	3	4
		NS	DN	NS	NS	NS	NS	NS	NS	NS

* p < 0.05

NS = Not Significant

DN = Duncan´s multiple range test

 

Table 8: Summary of food consumption in females

Group			Daily mean food consumption (g/animal/day)
		Pre-mating days		Post-mating days						Recovery period
		0 – 7	7 – 13	13 – 20	20 – 27	27 – 34	34 – 41	41 – 48	48 – 55	Day 0 – 6	Day 6 – 13
Control	Mean	19.7	19.4	17.7	19.3	18.3	17.7	17.3	17.0	16.9	17.3
	SD	2.36	1.06	1.94	0.12	0.99	0.62	0.84	0.00	1.77	0.61
	n	8	8	2	2	2	2	2	2	2	2
100 mg/kg bw/day	Mean	19.8	19.0	-	-	-	-	-	-	-	-
	SD	1.53	1.26	-	-	-	-	-	-	-	-
	n	6	6	-	-	-	-	-	-	-	-
	±%	0	-2	-	-	-	-	-	-	-	-
300 mg/kg bw/day	Mean	18.9	18.8	-	-	-	-	-	-	-	-
	SD	1.18	1.69
	n	6	6	-	-	-	-	-	-	-	-
	±%	-4	-3	-	-	-	-	-	-	-	-
1000 mg/kg bw/day	Mean	18.6	19.0	17.4	16.5	17.8	16.6	17.5	16.8	18.6	18.9
	SD	0.96	2.00	0.62	1.92	1.03	1.16	0.45	0.13	0.12	0.02
	n	8	8	2	2	2	2	2	2	2	2
	±%	-6	-2	-2	-14	-3	-6	1	-1	10	10
		NS	NS	NS	NS	NS	NS	NS	NS	NS	NS

NS = Not Significant

 

 

Table 9: Summary of hematology

Group		Basophil [%]	Hemoglobin [g/L]	Hematocrit [L/L]	Neutrophil [%]	Lymphocyte [%]	Mean platelet count [×10E9/L]	Activated partial thromboplastin time [sec]
MALES – TREATMENT PERIOD
Control	Mean	0.16	175.40	0.47	23.80	72.40	891.60	23.60
	SD	0.05	9.34	0.03	5.06	5.74	118.00	2.60
	n	5	5	5	5	5	5	5
100 mg/kg bw/day	Mean	0.04*	161.60**	0.43**	25.08	70.62	901.40	20.48*
	SD	0.09	4.72	0.01	5.03	5.26	88.05	0.96
	n	5	5	5	5	5	5	5
	±%	-75	-8	-9	5	-2	1	-13
300 mg/kg bw/day	Mean	0.02**	163.80**	0.44*	20.96	74.20	897.40	21.72
	SD	0.04	2.95	0.00	3.74	4.21	137.05	1.91
	n	5	5	5	5	5	5	5
	±%	-88	-7	-6	-12	2	1	-8
1000 mg/kg bw/day	Mean	0.04**	169.00	0.45	17.64	77.98	796.60	22.22
	SD	0.05	3.67	0.01	6.23	7.44	54.62	1.43
	n	5	5	5	5	5	5	5
	±%	-75	-4	-5	-26	8	-11	-6
		DN	DN	U	NS	NS	NS	DN
MALES – RECOVERY PERIOD
Control	Mean	0.08	176.20	0.47	16.44	78.94	823.80	19.48
	SD	0.04	3.11	0.01	3.44	3.44	83.96	3.49
	n	5	5	5	5	5	5	5
1000 mg/kg bw/day	Mean	0.02	107.40	0.46	22.28	72.40*	833.40	20.24
	SD	0.04	5.81	0.01	5.06	4.94	42.55	1.85
	n	5	5	5	5	5	5	5
	±%	-75	-3	-2	36	-8	1	4
		NS	NS	NS	NS	*	NS	NS
FEMALES – TREATMENT PERIOD
Control	Mean	0.10	159.20	0.43	25.68	69.78	1016.20	23.44
	SD	0.22	8.32	0.01	7.76	7.54	151.26	1.11
	n	5	5	5	5	5	5	5
100 mg/kg bw/day	Mean	0.04	169.00	0.45	16.70	79.02	1079.80	23.94
	SD	0.09	9.67	0.02	3.57	2.94	181.70	2.09
	n	5	5	5	5	5	5	5
	±%	-60	6	4	-35	13	6	2
300 mg/kg bw/day	Mean	0.10	152.00	0.41	21.18	75.50	1064.20	22.12
	SD	0.10	15.02	0.03	9.78	10.17	195.41	1.62
	n	5	5	5	5	5	5	5
	±%	0	-5	-4	-18	8	5	-6
1000 mg/kg bw/day	Mean	0.10	162.80	0.43	21.90	73.34	996.20	21.80
	SD	0.10	5.67	0.01	4.39	5.74	79.97	0.70
	n	5	5	5	5	5	5	4
	±%	0	2	1	-15	5	-2	-7
		NS	NS	NS	NS	NS	NS	NS
FEMALES – RECOVERY PERIOD
Control	Mean	0.14	160.20	0.44	18.94	74.84	744.40	21.76
	SD	0.13	9.86	0.02	2.23	3.40	75.68	1.33
	n	5	5	5	5	5	5	5
1000 mg/kg bw/day	Mean	0.04	164.20	0.45	15.18*	80.86*	852.00*	20.30
	SD	0.09	5.45	0.01	1.92	2.28	67.62	1.18
	n	5	5	5	5	5	5	5
	±%	-71	2	3	-20	8	14	-7
		NS	NS	NS	*	*	*	NS

* p < 0.05, **p < 0.01

NS = Not Significant

U = Mann-Whitney U-test vs. Control

DN = Duncan´s multiple range test

 

Table 10: Summary of clinical biochemistry findings

Group		Albumin [g/L]	Alanine aminotransferase activity [U/L]	Urea [mmol/L]	Creatinine [µmol/L]	Potassium [mmol/L]
MALES – TREATMENT PERIOD
Control	Mean	37.16	70.98	8.10	28.04	4.71
	SD	1.08	18.19	0.99	3.22	0.54
	n	5	5	5	5	5
100 mg/kg bw/day	Mean	35.04**	56.78	8.22	31.24	4.67
	SD	1.33	13.38	0.90	2.22	0.15
	n	5	5	5	5	5
	±%	-6	-20	1	11	-1
300 mg/kg bw/day	Mean	35.74*	56.76	8.20	29.90	4.74
	SD	0.69	8.28	1.12	3.21	0.22
	n	5	5	5	5	5
	±%	-4	-20	1	7	1
1000 mg/kg bw/day	Mean	36.70	49.66*	8.68	30.50	4.57
	SD	0.76	6.45	1.23	1.47	0.45
	n	5	5	5	5	5
	±%	-1	-30	7	9	-3
		DN	DN	NS	NS	NS
MALES – RECOVERY PERIOD
Control	Mean	36.36	60.22	8.04	27.28	4.79
	SD	0.92	7.23	0.63	1.19	0.19
	n	5	5	5	5	5
1000 mg/kg bw/day	Mean	35.60	69.60	1.72	26.40	4.42*
	SD	0.69	11.83	0.31	3.50	0.19
	n	5	5	5	5	5
	±%	-2	16	11	-3	-8
		NS	NS	NS	NS	*
FEMALES – TREATMENT PERIOD
Control	Mean	34.24	53.30	7.87	27.56	4.39
	SD	0.46	12.42	1.32	1.44	0.47
	n	5	5	5	5	5
100 mg/kg bw/day	Mean	35.02	50.46	8.90	30.34	4.13
	SD	1.04	4.34	0.90	2.34	0.18
	n	5	5	5	5	5
	±%	2	-5	13	10	-6
300 mg/kg bw/day	Mean	34.26	64.36	10.07*	30.42	4.65
	SD	1.23	5.54	1.12	2.01	0.38
	n	5	5	5	5	5
	±%	0	21	28	10	6
1000 mg/kg bw/day	Mean	33.48	54.94	10.35*	32.30*	4.39
	SD	0.37	19.24	1.97	3.78	0.20
	n	5	5	5	5	5
	±%	-2	3	32	17	0
		NS	NS	DN	DN	NS
FEMALES – RECOVERY PERIOD
Control	Mean	39.22	53.32	7.66	31.86	4.19
	SD	1.70	14.32	0.81	4.40	0.31
	n	5	5	5	5	5
1000 mg/kg bw/day	Mean	39.04	62.18	8.24	32.98	3.95
	SD	1.69	20.47	0.66	4.23	0.15
	n	5	5	5	5	5
	±%	0	17	8	4	-6
		NS	NS	NS	NS	NS

* p < 0.05, **p < 0.01

NS = Not Significant

DN = Duncan´s multiple range test

 

Table 11: Summary of necropsy findings

Organs	Observations	Frequency of observations per group
		Control		100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
		Main group	Recovery group			Main group	Recovery group
MALES
	No macroscopic finding	11/12	4/5	12/12	10/12	8/12	5/5
Thymus	Hemorrhages	1/12	0/5	0/12	1/12	1/12	0/5
Kidney	3 mm cavity at the renal medulla	0/12	1/5	0/12	0/12	0/12	0/5
Liver	Pale	0/12	0/5	0/12	1/12	4/12	0/5
FEMALES - DAMS
	No macroscopic finding	8/12	5/5	11/11	9/12	6/11	3/5
Thymus	Hemorrhages	1/12	0/5	0/11	0/12	1/11	0/5
Uterus	Hydrometra	2/12	0/5	0/11	1/12	1/11	2/5
Cecum	Dilatation	0/12	0/5	0/11	0/12	3/11	0/5
Liver	Pale	0/12	0/5	0/11	0/12	3/11	0/5
Skin	Alopecia	1/12	0/5	0/11	1/12	1/11	0/5
FEMALES – NON-PREGNANT
	No macroscopic finding	NA	NA	1/1	NA	0/1	NA
Uterus	Hydrometra	NA	NA	0/1	NA	1/1	NA

Frequency of observation =number of animals with observations / number of animals examined

NA = not applicable

 

Table 12: Summary of organ weights

		Body weight [g]	Organ weight [g]		Organ weight relative to body weight [%]		Organ weight relative to brain weight [%]
Group			Liver	Kidney	Liver	Kidney	Liver	Kidney
MALES – TREATMENT PERIOD
Control	Mean	448.4	11.31	2.70	2.604	0.622	518.31	123.81
	SD	24.45	1.05	0.17	0.179	0.037	40.16	7.90
	n	12	5	5	5	5	5	5
100 mg/kg bw/day	Mean	443.5	11.65	2.62	2.632	0.595	533.25	120.14
	SD	29.02	1.86	0.22	0.235	0.039	70.18	7.46
	n	12	5	5	5	5	5	5
	±%	-1	3	-3	1	-4	3	-3
300 mg/kg bw/day	Mean	444.8	12.43	2.77	2.740	0.610	578.38	129.37
	SD	34.75	0.84	0.32	0.201	0.057	43.08	19.33
	n	12	5	5	5	5	5	5
	±%	-1	10	3	5	-2	12	4
1000 mg/kg bw/day	Mean	430.7	12.07	12.82	2.768	0.647	555.54	129.84
	SD	34.57	1.25	0.30	0.263	0.082	43.19	14.08
	n	12	5	5	5	5	5	5
	±%	-4	7	4	6	4	7	5
		NS	NS	NS	NS	NS	NS	NS
MALES – RECOVERY PERIOD
Control	Mean	448.8	11.68	2.54	2.599	0.566	436.09	116.69
	SD	23.21	1.19	0.19	0.193	0.018	45.94	7.51
	n	5	5	5	5	5	5	5
1000 mg/kg bw/day	Mean	458.2	11.93	2.82*	2.607	0.618	544.92	129.01
	SD	27.07	0.94	0.13	0.187	0.049	41.69	9.65
	n	5	5	5	5	5	5	5
	±%	2	2	11	0	9	2	11
		NS	NS	*	NS	NS	NS	NS
FEMALES – TREATMENT PERIOD
Control	Mean	248.8	7.92	1.78	3.183	0.716	392.68	88.44
	SD	7.29	0.45	0.12	0.137	0.052	15.47	7.80
	n	5	5	5	5	5	5	5
100 mg/kg bw/day	Mean	263.6	8.29	1.83	3.142	0.695	406.12	89.58
	SD	8.88	0.63	0.09	0.152	0.026	40.55	4.22
	n	5	5	5	5	5	5	5
	±%	6	5	3	-1	-3	3	1
300 mg/kg bw/day	Mean	270.8*	9.45*	1.88	3.481	0.695	469.07*	93.36
	SD	14.20	1.23	0.11	0.295	0.049	61.20	6.89
	n	5	5	5	5	5	5	5
	±%	9	19	6	9	-3	19	6
1000 mg/kg bw/day	Mean	264.0	9.00	1.94	3.404	0.737	447.20	96.67
	SD	13.11	1.05	0.13	0.288	0.063	40.25	5.75
	n	5	5	5	5	5	5	5
	±%	6	14	9	7	3	14	9
		DN	DN	NS	NS	NS	DN	NS
FEMALES – RECOVERY PERIOD
Control	Mean	255.6	6.99	1.56	2.738	0.614	350.45	78.40
	SD	18.20	0.86	0.12	0.309	0.057	47.29	6.87
	n	5	5	5	5	5	5	5
1000 mg/kg bw/day	Mean	264.6	7.17	1.67	2.705	0.630	371.57	86.39
	SD	18.70	0.81	0.12	0.118	0.021	33.63	4.98
	n	5	5	5	5	5	5	5
	±%	4	3	7	-1	3	6	10
		NS	NS	NS	NS	NS	NS	NS

* p < 0.05, **p < 0.01

NS = Not Significant

DN = Duncan´s multiple range test

 

Table 13: Summary of histopathological findings

Organs	Observations	Incidence of observations per group
		Control		100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
		Main group	Recovery group			Main group	Recovery group
MALES
Kidneys	Cyst	0/5	1/5	-	-	0/5	0/5
Liver	Centrilobular vacuolation in hepatocytes	0/12	0/5	0/12	3/12	7/12	0/5
Lungs	Alveolar emphysema	0/5	1/5	-	-	1/5	1/5
	Hyperplasia of BALT	1/5	1/5	-	-	0/5	0/5
Thymus	Acute hemorrhage	1/5	0/5	-	1/1	1/5	0/5
FEMALES
Kidneys	Cyst	0/5	0/5	-	-	0/5	0/5
Liver	Centrilobular vacuolation in hepatocytes	0/12	0/5	0/11	0/11	3/12	0/5
Lungs	Alveolar emphysema	1/5	0/5	-	-	1/5	0/5
	Hyperplasia of BALT	0/5	1/5	-	-	1/5	0/5
Thymus	Acute hemorrhage	1/5	0/5	-	-	1/5	0/5
Uterus with cervix	Dilatation	2/12	0/5	0/1	1/1	1/12	2/5

Incidence of observation =number of animals with observations / number of animals examined

BALT = bronchus associated lymphoid tissue

 

Table 14: Summary of clinical observation and fate of offspring

Observations/Fate of pup		Group [mg/kg bw/day]
		Control	100	300	1000
Number of pups examined (liveborns)		139	120	144	116
No signs	N	97	107	101	82
	%	70	89	70	71
Cold	N	38	10	29	33
	%	27	8	20	28
No milk in the stomach	N	42	14	27	21
	%	30	12	19	18
Swollen abdomen	N	0	1	1	0
	%	0	1	1	0
Smaller than others	N	0	1	1	0
	%	0	1	1	0
Cianotic	N	0	0	1	1
	%	0	0	1	1
Dispnoe	N	0	0	1	0
	%	0	0	1	0
Found dead	N	1	2	2	1
	%	1	2	1	1
Missing	N	1	2	3	1
	%	1	2	2	1
Interim euthanazia	N	42	33	44	37
	%	30	28	31	23
Terminal euthanazia	N	95	83	95	87
	%	68	69	66	75

 

Table 15: Summary data of reproductive ability

Values		Group [mg/kg bw/day]
		Control	100	300	1000
MALES
No. of males paired		12	12	12	12
Copulatory index	%	100	92**	92**	100
FEMALES
Pre-coital interval (days)	Mean	1.2	0.6	2.2*	2.1	DN
	SD	1.1	0.7	1.5	1.0
	N	12	12	12	12
Conceiving days	Mean	2.2	1.6	3.2*	3.1	DN
	SD	1.1	0.7	1.5	1.0
	N	12	12	12	12
Fertility index	%	100	92**	100	92**

* p < 0.05 CH², ** p < 0.01 CH²

NS = Not Significant

DN = Duncan´s multiple range test

 

Table 16: Summary of extra uterine mortality and sex distribution of offspring – litter data

Values			Control	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Number of litters		N	12	11	12	11
Number of viable pups
on post-natal day 0	Total	N	11.6	10.8	11.7	10.5	NS
		SD	1.7	2.9	2.5	2.5
	Male	N	6.4	5.5	6.4	4.5	* DN
		SD	1.7	1.9	2.0	1.8
	Female	N	5.2	5.4	5.3	5.9	NS
		SD	1.5	1.8	1.2	2.3
on post-natal day 4	Total	N	11.5	10.6	11.6	10.4	NS
		SD	1.7	2.9	2.4	2.6
	Male	N	6.4	5.4	6.3	4.5	NS
		SD	1.7	1.9	2.0	1.8
	Female	N	5.1	5.3	5.3	5.8
		SD	1.5	1.9	1.2	2.5
on post-natal day 13	Total	N	7.9	7.5	7.9	7.9	NS
		SD	0.3	1.2	0.3	0.8
	Male	N	4.1	3.9	3.9	3.8
		SD	0.5	0.8	0.7	1.3
	Female	N	3.8	3.6	4.0	4.1
		SD	0.6	0.7	0.4	1.3

* p < 0.05

NS = Not Significant

DN = Duncan´s multiple range test

 

Table 17: Summary of anogenital distance of offspring

Group		Anogenital distance (AGD) on post-natal day 4
		Absolute (mm)	Normalized (AGD/^3√bw)
MALES
Control	Mean	6.27	2.85
	SD	0.55	0.22
	n	77	77
	N	12	12
100 mg/kg bw/day	Mean	6.19	2.77*
	SD	0.57	0.25
	n	59	59
	N	11	11
300 mg/kg bw/day	Mean	6.12	2.76*
	SD	0.46	0.19
	n	76	76
	N	12	12
1000 mg/kg bw/day	Mean	6.24	2.79
	SD	0.56	0.21
	n	50	50
	N	11	11
		NS	DN
FEMALES
Control	Mean	3.31	1.52
	SD	0.47	0.18
	n	61	61
	N	12	12
100 mg/kg bw/day	Mean	3.31	1.51
	SD	0.47	0.19
	n	58	58
	N	11	11
300 mg/kg bw/day	Mean	3.29	1.49
	SD	0.46	0.20
	n	63	63
	N	12	12
1000 mg/kg bw/day	Mean	3.16	1.44*
	SD	0.37	0.15
	n	64	64
	N	11	11
		NS	DN

* p < 0.05

NS = Not Significant

DN = Duncan´s multiple range test

n = number of offspring

N = Number of litters

Conclusions:

The test material had no effect on reproductive performance.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.7, of Regulation (EC) No 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A reliable sub-acute study regarding reproductive/developmental toxicity is available for the test substance.

The potential reproductive or developmental toxicity of the test substance was assessed in a sub-acute combined repeated dose toxicity study with the reproductive/developmental toxicity screening test in Hsd.Han: Wistar rats performed according to OECD Guideline 422 and in compliance with GLP. Three groups of 12 male and 12 female rats received the test substance in polyethylene glycol as vehicle at doses of 100, 300 or 1000 mg/kg bw/day orally via gavage. A control group of 12 animals/sex received the vehicle only. In addition, 5 animals/sex were added to the control and high dose group to assess the reversibility of any effects observed at the high dose level (recovery group). All animals of the parental generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before necropsy (altogether for 56 days). Females were additionally exposed through the gestation period and up to lactation days 13 - 21, i.e. up to the day before necropsy (altogether for 56, 57 or 64 days). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, lactation as well as development of offspring. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating. The dams were allowed to litter, and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on post-natal day 13 or shortly thereafter. Blood samples were collected for determination of serum levels of thyroid hormones (T4) from all parental male animals at termination and from all pups on post-natal day 13. Gross pathology of the parental males included body weight, brain weight and weight of the testes, epididymides, prostate and seminal vesicles with coagulating gland (as a whole) determination. Histopathological examinations was performed in all parental animals of the control and high dose group on ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control or high dose group. These organs were processed and examined histologically in not mated male animals (at 100 mg/kg bw/day and 300 mg/kg bw/day) and in non-pregnant female and its male partner at 100 mg/kg bw/day and in one dam at 300 mg/kg bw/day based on macroscopic observation. Five dams and the male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, blood analysis (hematology and clinical chemistry), gross necropsy, organ weighing and histopathology. Animals allocated to the recovery group were observed for additional 14 days after termination of treatment and subjected to clinical pathology and gross pathology examinations, organ weighing and full histological examinations.

At the lowest dose (100 mg/kg bw/day) no treatment-related changes in any of the parameters were observed.

An oral dose of 300 mg/kg bw/day induced pale liver in one male animal. Histopathological examination revealed centrilobular microvesicular vacuolation in the hepatocytes (hepatic lipidosis) in male animals at the termination of treatment.

At the highest dose of 1000 mg/kg bw/day pale liver with high incidence at necropsy as well as hepatic lipidosis at histopathological examination was observed in male and female animals. These test item related hepatic and hepatocellular changes were considered reversible as these were not seen at the end of the 14-day post treatment observation period.

There were no test item related changes in body weight and body weight gain, food consumption, hematology, organ weights and clinical chemistry parameters. The T4 serum levels were similar between control and treatment groups. A test item influence on the estrous cycle as well as on the reproductive performance was not detected at any dose level. Intrauterine mortality was not affected. Thus, under the conditions of this study, the NOAEL of the test substance for systemic toxicity of the parental generation following oral administration via gavage for 56 days is 100 mg/kg bw/day in male Wistar rats. The corresponding NOAEL in female Wistar rats following oral administration via gavage for 56, 57 or 64 days is 300 mg/kg bw/day. In terms of reproductive effects, no treatment-related findings were observed at any dose level tested; therefore, the high dose of 1000 mg/kg bw/day is considered to be the NOAEL for reproductive toxicity.

Effects on developmental toxicity

Description of key information

Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, oral (OECD 422), rat:

systemic toxicity: NOAEL = 1000 mg/kg bw/day for offspring, NOAEL = 100 mg/kg bw/day for males and 300 mg/kg bw/day for females of the parental generation

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.7, of Regulation (EC) No 1907/2006.

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

A reliable sub-acute study regarding reproductive/developmental toxicity is available for the test substance.

The potential reproductive or developmental toxicity of the test substance was assessed in a sub-acute combined repeated dose toxicity study with the reproductive/developmental toxicity screening test in Hsd.Han: Wistar rats performed according to OECD Guideline 422 and in compliance with GLP. Three groups of 12 male and 12 female rats received the test substance in polyethylene glycol as vehicle at doses of 100, 300 or 600 mg/kg bw/day orally via gavage at concentrations of 0, 25, 75 and 150 mg/mL corresponding to a 4 mL/kg bw dosing volume. A control group of 12 animals/sex received the vehicle only. In addition, 5 animals/sex were added to the control and high dose group to assess the reversibility of any effects observed at the high dose level (recovery group). All animals of the parental generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before necropsy (altogether for 56 days). Females were additionally exposed through the gestation period and up to lactation days 13 - 21, i.e. up to the day before necropsy (altogether for 56, 57 or 64 days). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, lactation as well as development of offspring. The dams were allowed to litter, and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on post-natal day 13 or shortly thereafter. Blood samples were collected for determination of serum levels of thyroid hormones (T4) from all pups per litter at termination on post-natal day 13.

No adverse effect on mortality, clinical signs, body weight or necropsy findings were detected in the offspring terminated as scheduled. Thyroid homone levels (T4) in pups on post-natal day 13 were not affected. The anogenital distance (male and female) or nipple retention (male) was not affected due to treatment with the test substance. For the parental animals pale livers and histopathological changes in the liver (hepatic lipidosis) were observed at 300 mg/kg bw/day for males and 1000 mg/kg bw/day for females. Thus, under the conditions of this study, the NOAEL of the test substance for systemic toxicity of the parental generation following oral administration via gavage for 56 days is 100 mg/kg bw/day in male Wistar rats. The corresponding NOAEL in female Wistar rats following oral administration via gavage for 56, 57 or 64 days is 300 mg/kg bw/day. The corresponding NOAEL for the offspring is 1000 mg/kg bw/day.

Justification for classification or non-classification

The available data on toxicity to reproduction do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.