thiacloprid (ISO);(Z)-3-(6-chloro-3-pyridylmethyl)-1,3-thiazolidin-2-ylidenecyanamide;{(2Z)-3-[(6-chloropyridin-3-yl)methyl]-1,3-thiazolidin-2-ylidene}cyanamide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

2-generation (rat, OECD 416):

NOAEL P0 (general and reproductive toxicity): 50 ppm (nominal concentration, corresponding to actual average daily dose of ca. 2.7 mg/kg bw/day for males and ca. 3.3 mg/kg bw/day for females)

NOAEL P1 (general toxicity): 50 ppm (nominal concentration, corresponding to actual average daily dose of ca. 2.6 mg/kg bw/day for males and ca. 3.5 mg/kg bw/day for females)

NOAEL P1 (reproductive toxicity): 300 ppm (nominal concentration, corresponding to actual average daily dose of ca. 16.4 mg/kg bw/day for males and ca. 22.0 mg/kg bw/day for females)

NOAEL F1 (pup developmental toxicity): 50 ppm (nominal concentration, corresponding to actual average daily dose of ca. 2.7 mg/kg bw/day for males and ca. 3.3 mg/kg bw/day for females in P0 adults)

NOAEL F2 (pup developmental toxicity): 50 ppm (nominal concentration, corresponding to actual average daily dose of ca. 2.6 mg/kg bw/day for males and ca. 3.5 mg/kg bw/day for females in P1 adults)

 

LOAEL P0 (general and reproductive toxicity): 300 ppm (nominal concentration, corresponding to actual average daily dose of ca. 16.4 mg/kg bw/day for males and ca. 20.4 mg/kg bw/day for females)

LOAEL P1 (general toxicity): 300 ppm (nominal concentration, corresponding to actual average daily dose of ca. 16.4 mg/kg bw/day for males and ca. 22.0 mg/kg bw/day for females)

LOAEL P1 (reproductive toxicity): 600 ppm (nominal concentration, corresponding to actual average daily dose of ca. 32.1 mg/kg bw/day for males and ca. 44.0 mg/kg bw/day for females)

LOAEL F1 (pup developmental toxicity): 300 ppm (nominal concentration, corresponding to actual average daily dose of ca. 16.4 mg/kg bw/day for males and ca. 20.4 mg/kg bw/day for females in P0 adults)

LOAEL F2 (pup developmental toxicity): 300 ppm (nominal concentration, corresponding to actual average daily dose of ca. 16.4 mg/kg bw/day for males and ca. 22.0 mg/kg bw/day for females in P1 adults)

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Nov 1995 - 19 August 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

adopted 1983

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

adopted 2001

Deviations:

yes

Remarks:

housing temperature slightly different compared to guideline, sperm parameters not examined, weights of uterus, prostate, epididymis, brain, seminal vesicles, kidney, spleen, pituitary and adrenal gland not noted, organs from pups not weighted

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SASCO Inc., Saint Louis, Missouri, USA
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 7 weeks; (F1) at weaning
- Weight at study initiation: (P) Males: 217.4 - 222.6 g; Females: 148.3 - 151.4 g; (F1) Males: 177.9 - 216.6 g; Females: 145.1 - 160.0 g
- Fasting period before study: not specified
- Housing: Animals were individually housed (except during the mating period) in stainless steel cages suspended over bedding of deotized animal cage board. During the gestation and lactation phases, females were housed individually in polycarbonate cages with BedO-Cobs bedding.
- Use of restrainers for preventing ingestion: no
- Diet: Purina Rodent Laboratory Chow 5001-4 Etts form, ad libitum
- Water: municipal water, ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8 - 25.6
- Humidity (%): 40 - 70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 Nov 1995 To: 19 Aug 1996

Route of administration:

oral: feed

Vehicle:

other:

Remarks:

acetone and corn oil, both of which were added at 1% of the diet for all dose groups, respectively

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): basal diet
- Storage temperature of food: in a freezer at -23 °C

VEHICLE
- Justification for use and choice of vehicle: not provided

The appropriate amount of test substance was dissolved/suspended in acetone and corn oil for each dose level. Then, the corn oil mixture was added to the diet, whereas acetone was again used during diet preparation to rinse the equipment. The corn oil was added at 1% of the diet for all dose groups. The same quantity of acetone was added to the feed of all dose groups.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: up to 21 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually in plastic nesting cage

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration:
The concentration of the test substance in the diet was determined by liquid chromatographic analysis. The concentration of the test substance in the rodent ration was verified for dietary mixtures of Weeks 1, 10, 18, 27, and 36. The mean, standard deviation (SD), and coefficient of cariation (CV) of the chemical concentration were determined for each test level. The mean concentration for the 50 ppm test level was 45.9 ppm (91.7%, CV = 8%), for the 300 ppm level mean was 279 ppm (93.2%, CV = 7%) and for the 600 ppm level mean was 542 ppm (90.3%, CV = 7%).

Homogeneity:
The distribution of the test substance in the diet was determined at 20 and 2000 ppm. The rodent ration was mixed with the test item. Three samples were taken for analysis from each of three areas (top, middle, and bottom) for a total of nine samples. The mean concentration, SD, and CV were determined for the nine samples from each concentration. The results obtained, revealed that the test substance mixed in rodent ration at test concentrations of 20 and 2000 ppm was
homogeneously distributed.

Stability:
The stability of the test substance mixed in rodent ration at 20 and 2000 ppm was assessed under freezer conditions (-23 °C) for 28 days and at room temperature (22 °C) for 14 days. Therefore, a sample from each ration batch was taken immediately after the mixing was complete. After seven days in the freezer one portion was removed for room temperature testing.
The portion stored at room temperature was sampled on days 0, 1, 3, 7, 10, and 14 post freezer storage. The portion remaining in the freezer was sampled for analysis on days7, 14, 21, and 28.
The results of the stability analysis revealed that the test substance mixed with rodent ration at 20 and 2000 ppm was stable at freezer temperature for 28 days, and at room temperature for 7 days at 20 ppm and for 14 days at 2000 ppm.

Duration of treatment / exposure:

(P) Males: 10 weeks before mating
(P) Females: 10 weeks before mating, 3 weeks during mating, 3 weeks during resulting pregnancies, 3 weeks through weaning of their F1 offspring.
(F1) Males: 12 weeks at weaning, during growth into adulthood, mating and production of an F2 generation, until weaning of the F2 generation.
(F1) Females: 10 weeks at weaning, during growth into adulthood, mating and production of the F2 generation, until weaning of the F2 generation.

Frequency of treatment:

Continuously via the diet.

Details on study schedule:

- F1 parental animals (P1) were not mated until 10 weeks after selection from the F1 litters.
- Selection of parents from F1 generation: at weaning
- Age at mating of the mated animals in the study: approximately 17 weeks

Dose / conc.:

50 ppm

Remarks:

corresponding to actual dose ingested:
2.7 mg/kg bw/day for P generation males (P0)
3.3 mg/kg bw/day for P generation females (P0)
2.6 mg/kg bw/day for F1 parental males (P1)
3.5 mg/kg/day for F1 parental females (P1)

Dose / conc.:

300 ppm

Remarks:

corresponding to actual dose ingested:
16.4 mg/kg bw/day for P generation males (P0)
20.4 mg/kg bw/day for P generation females (P0)
16.4 mg/kg bw/day for F1 parental males (P1)
22.0 mg/kg/day for F1 parental females (P1)

Dose / conc.:

600 ppm

Remarks:

corresponding to actual dose ingested:
32.3 mg/kg bw/day for P generation males (P0)
41.0 mg/kg bw/day for P generation females (P0)
32.1 mg/kg bw/day for F1 parental males (P1)
44.0 mg/kg/day for F1 parental females (P1)

No. of animals per sex per dose:

30 (P generation (P0))
30 (F1 parental animals (P1))

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The doses for this study were selected based on the results from a pilot dietary reproduction study in rats, a 13-week dietary subchronic study in rats, a 2-week subacute dietary study in rats and preliminary body weight data from the chronic/carcinogenicity study in rats.

Positive control:

not included

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: moribundity, mortality, and clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week

BODY WEIGHT: Yes
- Time schedule for examinations:
Female: during mating once a week, during gestation on Days 0, 6, 13, and 20 and during lactation on Days 0, 4, 7, 14, and 21
Males: once a week

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: No data

Oestrous cyclicity (parental animals):

Vaginal smears were taken for three weeks from ten P and F1 females/dose level prior to mating. The vaginal smears were analyzed microscopically and classified as diestrus, proestrus, or estrus based on the cytology observed.

Sperm parameters (parental animals):

Parameters examined in [P/F1] male parental generations: testis weight, epididymis weight
No other sperm parameters were examined.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1/F2] offspring:
The number of live and stillborn pups was recorded for each litter. Litter counts and clinical observations were performed daily from Days 0 to 21. Individual pup weights were recorded as soon as possible after completion of delivery (day 0), and on Days 4, 7, 14, and 21.

GROSS EXAMINATION OF DEAD PUPS: no

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
- Method: Animals were sacrificed by carbon dioxide asphyxiation.
- Male animals: All surviving animals after completion of the mating phase.
- Maternal animals: All surviving animals after the last litter of each generation was weaned (when Day 24 of gestation was reached or 24 days after the last day of co-housing).

GROSS NECROPSY
The necropsy consisted of a systematic gross examination of each animal's general physical condition, body orifices, external and internal organs and tissues. The uteri from all dams were examined for implantation sites, and the number of implantation sites was recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Microscopic examination of tissues and organs was performed as follows:
Coagulating gland, cervix, epididymides (caput, corpus, and cauda), liver, thyroid, pituitary gland, prostate gland, seminal vesicles, uterus, vagina, gross lesions, and physical identifier were fixed in buffered 10% formalin. Ovaries and testes were fixed in Bouin's Fluid. Tissues were stained with hematoxylin and eosin (H&E). Verhoeff-Van Gieson and periodic acid-Schiff stains were applied to recuts of the cervix, vagina, and uterus of all P females with dystocia and
several control and 600 ppm P females undergoing normal parturition and sacrificed at scheduled termination. With the exception of the physical identifier (tail tattoo) and skulls containing maloccluded teeth without any other accompanying morphologic lesions, all tissues were examined histologically.
The following organs were weighted: gonad, liver, and thyroid
Relative organ weights were also calculated (organ weight/terminal body weight X 100).

Postmortem examinations (offspring):

SACRIFICE
- Method: Weanlings were sacrificed by carbon dioxide asphyxiation. Pups culled on Day 4 and pups born to dams which died or were sacrificed while delivering were sacrificed by intracranial injection of Fatal-Plus (Vortech Pharmaceuticals, Michigan, USA).
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at approximately 21 days of age.

The F1 offspring not selected as parental animals and all F2 offspring animals (below referred to as offspring) were subjected to postmortem examinations (macroscopic and microscopic examination) as follows:

GROSS NECROPSY
The necropsy consisted of a systematic gross examination of each animal's general physical condition, body orifices, external and internal organs and tissues. All offspring lesions were recorded, but none were collected for microscopic examination. For pups found dead on day 0 postpartum, the ability of the lungs to float in water was used to determine if the pups were stillborn.

HISTOPATHOLOGY / ORGAN WEIGTHS
Offspring were not examined microscopically. Organ weights of offspring were not noted.

Statistics:

Statistical significance was determined at p ≤ 0.05 for all tests with the exception of Bartlett's test, in which a probability value of p ≤ 0.001 was used. All tests were two-tailed, except for adult pathology evaluations. Body weight and food consumption data, were analyzed by analyses of variance (ANOVA) and if significant differences were shown, Dunnett's test was used to identify significant differences from the control group. Number of estrous cycles and estrous cycle length were analyzed by the Kruskal-Wallis test. If significant differences were shown, the Mann-Whitney U-test was used to identify statistical significance between groups. Insemination length, gestation length, litter size, viability index, birth index, live birth index, number of stillborn pups per litter, percent of male pups, and number of implantation sites were analyzed by the Kruskal-Wallis test. If significant differences were shown, Dunn's test was used to identify significant differences from the control group. Clinical signs, number of dams with cannibalized pups, mating index, fertility index, and gestation index, were analyzed using the Chi-square test. If significant differences were shown, Fisher's exact test was used to identify significant differences from the control group. A Bonferroni adjustment of the p value was used with the Fisher's exact test. Term body weights and organ weights were evaluated by Bartlett's test for homogeneity. If the data were homogeneous, an ANOVA was performed followed by Dunnett's t-test on parameters showing a significant effect by ANOVA. If the data were nonhomogeneous, a Kruskal-Wallis ANOVA was performed followed by the Mann-Whitney U-test to identify statistical significance between groups. The pup necropsy and adult micropathology lesion frequencies were analyzed using the Chi-square test. If significant differences were shown, Fisher's exact test was used to identify significant differences from the control group.

Reproductive indices:

The following indices were calculated for each dose group:
Mating index (%) = (number of inseminated females / number of females co-housed) * 100
Fertility index (%) = (number of pregnant females / number of inseminated females) * 100
Gestation index (%) = (number of females with live pups / number of pregnant females) * 100
Birth index (%) = (total number of pups born/litter / total number of implantation sites/dam) * 100
Live birth index (%) = (number of pups born per litter / total number of pups per litter) * 100

Offspring viability indices:

Viability index (%) = (number of live pups per litter on day 4 (pre-culling) / number of live pups born per litter) * 100
Lactation index (%) = (number of live pups per litter on day 21 / number of live pups per litter on day 4 (post-culling)) * 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no compound-related clinical signs for adults.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

mortality observed, treatment-related

Description (incidence):

Control: One male P adult was early sacrificed (lying on site).
50 ppm: One female P adult treated was found dead due to teeth caught in feeder.
300 ppm: Three females P adults died due to dystocia. Furthermore, one female was early sacrificed due to dystocia.
600 ppm: Three females P adults were early sacrificed due to dystocia.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Premating:
50 and 300 ppm: No statistically significant differences in body weight were observed.
600 ppm: Treatment-related statistically significant lower body weights were observed. The lower body weights were 4-5% below the control animals starting on Day 56 of the premating phase. No statistically significant differences in body weight were observed for P males.
Gestation:
50 and 300 ppm: During gestation, P dams showed no compound-related effect on body weight gain.
600 ppm: Statistically significant and compound-related lower body weights were observed in the high-dose group when compared to the control group (5% and 6% on gestation days 13 and 20, respectively). The lower body weights during gestation were a continuation of the low body weights seen in the high-dose group females during the premating phases.

Lactation:
50 and 300 ppm: During lactation, P dams showed no compound-related effect on body weight gain.
600 ppm: Statistically significant and compound-related lower body weights were observed in the P high-dose group. The body weights for the P high-dose group animals were between 7 and 8% lower than the control group. As during the gestation phase, these lower body weights reflected the lower body weight observed during the premating phases. For details, please refer to the attached background material (attachment 1).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Premating:
There was no compound-related effect on food consumption for the P males and females. There were statistically significant alterations in food consumption on one or more occasions in females given 50 or 300 ppm and males given 600 ppm, however, these were considered as incidental findings.

Gestation:
There was no compound-related effect on food consumption during the gestation phase.

Lactation:
There was no compound-related effect on food consumption during the lactation phase.

For details, please refer to the attached background material (attachment 2).

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not specified

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

300 and 600 ppm: Micropathology evaluation revealed treatment-related findings in the liver and thyroid, which corresponded to treatment-related weight changes in these organs.

Liver
In the liver, hepatocytomegaly was present in P adults of both sexes given 300 or 600 ppm. Even though this alteration occurred at a high incidence, it was generally only minimal to slight in severity. Hepatocytomegaly was further characterized by an increase in nuclear and cytoplasmic volume in centrilobular hepatocytes. The increased cytoplasm in the enlarged hepatocytes was eosinophilic and generally homogeneous, whereas the nuclear morphology was unremarkable except for the noted enlargement. Hepatocytomegaly in mid- and high-dose group adults might explain the increased liver weights recorded at necropsy. In addition to hepatocytomegaly, minimal to moderate hepatocellular necrosis occurred in each of the mid- and high-dose group P females which died or were sacrificed early due to dystocia. The necrosis was distributed in the parenchyma and appeared to be an acute response with minimal inflammatory infiltrate of neutrophils. Cytoplasmic vacuolization was frequent in centrilobular hepatocytes in P males, but was an incidental finding since it occurred equally across all dose groups including controls.

Thyroid gland
In the thyroid gland, follicular cell hypertrophy was present in the high-dose group P males and in the mid-and high-dose group P females. This alteration was fairly subtle and was characterized by increased follicular cell size combined with some tendency to decreased overall follicle size. Increased follicular cell cytoplasm indicated by a change from low to high cuboidal cell shape was the basis for diagnosis of hypertrophy. Follicular cell hypertrophy in adults given 300 or 600 ppm was believed to be the reason for increased thyroid weights recorded at necropsy.

All remaining microscopic findings were considered incidental findings unrelated to treatment with the test compound. Furthermore, there were no microscopic changes in the reproductive tracts of the P females which would explain the dystocia observed at 300 and 600 ppm. Special stains were additionally performed to evaluate connective tissue in the cervix of these animals, but again there were no significant microscopic changes.

For details, please refer to the attached background material (attachment 4).

In an expert statement (M-282359-01-1) the effects observed in liver (minimal to moderate hepatocellular hypertrophy) were assessed as indicative of an adaptive response of the liver to the enhanced need for metabolization of the test substance. The same applies to the effects seen in the thyroid gland (slight hypertrophy of thyroid epithelium) which were interpreted as an adaptation to the increased need of the organism for thyroidal hormones due to their faster metabolization caused by liver enzyme induction. Therefore, neither of these effects are regarded as adverse. However, the minimal to moderate hepatocellular necrosis in the liver observed in the mid- and high-dose females needs to be regarded as an adverse effect.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no compound-related effect on estrous cycling (i.e., the number of cycles in a three-week period and the cycle length).

Reproductive function: sperm measures:

not examined

Description (incidence and severity):

not applicable

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Dystocia at 300 and 600 ppm: There was an increase in the number of P animals in the mid- and high-dose groups, which had difficulty delivering (0, 0, 4 and 3 animals in the control, low-, mid- and high-dose groups, respectively). As dystocia is historically not a common finding, it appears that the dystocia observed may have been compound-related. However, in the F1 generation no dystocia was observed. Therefore, it cannot be concluded from this study whether or not the dystocia observed in the P dams was due to compound administration.

A compound-related effect on the insemination length, mating, fertility, implantation sites, gestation indices, birth index, litter size, pup gender or lactation index was not detected.

For details, please refer to the attached background material (attachment 5).

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: corresponding to 2.7 mg/kg bw/day (males) and 3.3 mg/kg bw/day (females)

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: corresponding to 2.7 mg/kg bw/day (males) and 3.3 mg/kg bw/day (females)

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: corresponding to 16.4 mg/kg bw/day (males) and 20.4 mg/kg bw/day (females)

Key result

Dose descriptor:

LOAEL

Remarks:

reproductive toxicity

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: corresponding to 16.4 mg/kg bw/day (males) and 20.4 mg/kg bw/day (females)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 ppm

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):

There were no compound-related clinical signs for adults.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

mortality observed, non-treatment-related

Description (incidence):

50 ppm: There were no deaths or early sacrifices in the low-dose group.
300 and 600 ppm: For the F1 adults there were two deaths (one control female and one high-dose group male) and one early sacrifice due to hard palate fracture (mid-dose group male).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Premating:
50 and 300 ppm: During premating, no compound-related effect on body weight gain was observed.
600 ppm: Treatment-related statistically significantly lower body weights were observed for F1 males and females given 600 ppm of the test substance via diet. For F1 males and females, statistically significantly lower body weights ranging from 14-19% and 8-9%, respectively, below the control group were observed beginning on day 0 of the premating phase. For both the F1 males and females, the lower body weights were due to the low body weight of the F1 adults as pups.

Gestation:
50 and 300 ppm: During gestation, F1 dams showed no compound-related effect on body weight gain.
600 ppm: Statistically significantly and compound-related lower body weights were observed in the high-dose group of F1 dams when compared to the control group. The lower body weights during gestation were a continuation of the low body weights seen in the high-dose group females during the F1 premating phases.

Lactation:
50 and 300 ppm: During lactation, F1 dams showed no compound-related effect on body weight gain.
600 ppm: Statistically significantly and compound-related lower body weights were observed in the F1 high-dose group. The body weights for the F1 high-dose group animals were 5 and 10% lower than the body weights in the control group. As during the gestation phase, these lower body weights reflected the lower body weight observed during the F1 premating phases.
For details, please refer to the attached background material (attachment 1).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Premating:
50 ppm: There were no statistically significant alterations in food consumption noted at this dose level.
300 ppm: There were statistically significant alterations in food consumption on one or more occasions in males given 300 ppm, however, these were considered as incidental findings.
600 ppm: For the F1 males and females there was a compound related increase in food consumption in the high-dose group. For the F1 males the food consumption ranged from 3-9% above the food consumption of the control group, with statistically significant values being observed for all weeks except weeks 1 and 11. For the F1 females the food consumption ranged from 0-8% above the control group, with statistically significantly higher food consumption being observed for five of the ten weeks during the premating phase.

Gestation:
There was no compound-related effect on food consumption during the gestation phase.

Lactation:
There was no compound-related effect on food consumption during the lactation phase.

For details, please refer to the attached background material (attachment 2).

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not specified

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

300 and 600 ppm: Treatment-related alterations in organ weights were observed for liver and thyroid as follows:
- Liver weights (absolute and/or relative) were statistically significantly increased in the high-dose F1 group (males and females) and in the mid-dose group (females).
- Thyroid weights were also statistically significantly increased in F1 adults at 300 ppm (males) and 600 ppm (both sexes).

Statistical increases in relative weights for testes in F1 males and ovaries in F1 females were considered incidental and unrelated to treatment. The absolute organ weights in these dose groups were within the range of normal biological variability compared to controls.
For details, please refer to the attached background material (attachment 3 and 8).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Various other gross observations were noted in F1 adults of both sexes, but all of these observations were considered incidental findings unrelated to treatment with the test compound.

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

300 and 600 ppm: Micropathology evaluation revealed treatment-related findings in the liver and thyroid, which corresponded to treatment-related weight changes in these organs.

Liver
In the liver, hepatocytomegaly was present in F1 adults given 300 or 600 ppm. Even though this alteration occurred at a high incidence, it was generally only minimal to slight in severity. Hepatocytomegaly was further characterized by an increase in nuclear and cytoplasmic volume in centrilobular hepatocytes. The increased cytoplasm in the enlarged hepatocytes was eosinophilic and generally homogeneous, whereas the nuclear morphology was unremarkable except for the noted enlargement. Hepatocytomegaly in mid- and high-dose group adults might explain the increased liver weights recorded at necropsy. Cytoplasmic vacuolization was frequent in centrilobular hepatocytes in F1 males, but was an incidental finding since it occurred equally across all dose groups including controls.

Thyroid gland
In the thyroid gland, follicular cell hypertrophy was present in the mid-and high-dose group F1 animals (both sexes). This alteration was fairly subtle and was characterized by increased follicular cell size combined with some tendency to decreased overall follicle size. Increased follicular cell cytoplasm indicated by a change from low to high cuboidal cell shape was the basis for diagnosis of hypertrophy. Follicular cell hypertrophy in adults given 300 or 600 ppm was believed to be the reason for increased thyroid weights recorded at necropsy.

As explained for the P0 generation, the effects observed in liver (minimal to moderate hepatocellular hypertrophy) of the P1 generation were assessed in an expert statement (M-282359-01-1) as indicative of an adaptive response of the liver to the enhanced need for metabolization of the test substance. The same applies to the effects seen in the thyroid gland (slight hypertrophy of thyroid epithelium) which were interpreted as an adaptation to the increased need of the organism for thyroidal hormones due to their faster metabolization caused by liver enzyme induction. Therefore, neither of these effects are regarded as adverse.

All remaining microscopic findings were considered incidental findings unrelated to treatment with the test compound.

For details, please refer to the attached background material (attachment 4).

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no compound-related effect on estrous cycling.

Reproductive function: sperm measures:

not examined

Description (incidence and severity):

not applicable

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

There was no compound-related effect on the insemination length, mating, fertility, implantation sites, gestation indices, birth index, litter size, pup gender or lactation index.
Although dystocia was observed among the P animals in the mid- and high-dose groups, no dystocia was observed in the F1 generation.

For details, please refer to the attached background material (attachment 5).

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: corresponding to 2.6 mg/kg bw/day (males) and 3.5 mg/kg bw/day (females)

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: corresponding to 16.4 mg/kg bw/day (males) and 22.0 mg/kg bw/day (females)

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: corresponding to 16.4 mg/kg bw/day (males) and 22.0 mg/kg bw/day (females)

Key result

Dose descriptor:

LOAEL

Remarks:

reproductive toxicity

Effect level:

600 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: corresponding to 32.1 mg/kg bw/day (males) and 44.0 mg/kg bw/day (females)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 ppm

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):

There were no compound-related clinical signs.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Viability index and cannibalization:
There was no compound-related effect on the viability index. However, there was a non-statistical, but markedly lower pup viability index in the P0 high-dose group (97 and 83 for the control and high-dose groups, respectively). The low viability index in the high-dose group was due to the cannibalization of pups. Excluding the cannibalized pups, the viability index for the control and high-dose groups are 99 and 91, respectively. The latter is not considered compound-related, since the viability index for the F2 generation control group was 94 and the historical control range for the viability indices is 91-100 (please refer to attachment 7). Although the cause of the cannibalization is not known, based on clinical signs and weight, the pups which were cannibalized were normal. Cannibalization is defined as pups, which were missing as well as pups for which remnants were observed. In the high-dose group (17 pups), as compared to the control group (5 pups), during F1 breedings there was a markedly greater number of pups cannibalized. However, there was no statistically significant difference between the control and treated groups for the number of dams with cannibalized pups.

Live birth index (stillbirths):
There was a non-statistically significant, but possible compound-related reduction in the live birth index in the high-dose group. This was manifested by a reduction in the live birth index of the F1 generation (live birth index for the control, low-, mid-, and high-dose groups in the F1 generation was 99, 96, 95, 91, respectively). However, the live birth index for the high-dose group (of both generations) of the current study was 6-7% below the historical control range. The live birth indices for the F1 generation low- and mid-dose groups were not considered to be significantly lower than the control group for the following reasons:
1. Similar findings were not observed in the F2 generation.
2. The control value for the F2 breeding was comparable to the values for the low- and mid-dose groups.
3. There was no dose response.
4. Similar values have been observed for the live birth index in the historical control data base (please refer to attachment 7).

For details, please refer to the attached background material (attachment 5).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Birth weight:
There was no compound-related effect on birth weight.

Pup weights:
300 ppm: There was a compound-related decrease in pup weights for the F1 mid-dose group, which occurred on Days 14 and 21. The body weights were 8% below the control group for the F1 pups.
600 ppm: There was a statistically significant and compound-related decrease in pup weights for the F1 high-dose group. This decrease occurred on days 7 to 21. The body weights ranged from 8-15% below the control group for the F1 pups.

For details, please refer to the attached background material (attachment 6).

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

not applicable

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Sexual maturation:

not examined

Description (incidence and severity):

not applicable

Anogenital distance (AGD):

not examined

Description (incidence and severity):

not applicable

Nipple retention in male pups:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

not applicable

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A low incidence of various gross observations was noted in F1 and F2 pups; all of these observations were considered incidental findings unrelated to treatment with the test compound.

Histopathological findings:

not examined

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Developmental immunotoxicity:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: corresponding to 2.7 mg/kg bw/day (males) and 3.3 mg/kg/day (females) in P0 adults.

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

other: live birth index

Remarks on result:

other: corresponding to 16.4 mg/kg bw/day (males) and 20.4 mg/kg/day (females) in P0 adults.

Key result

Critical effects observed:

no

Clinical signs:

not examined

Description (incidence and severity):

not applicable

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Viability index and cannibalization:
There was no compound-related effect on the viability index. In the high-dose group (14 pups), as compared to the control group (3 pups), during F2 breedings there was a markedly greater number of pups cannibalized. However, there was no statistically significant difference between the control and treated groups for the number of dams with cannibalized pups. As the pups were healthy prior to cannibalization (based on clinical signs and body weight), the cannibalization was not the consequence of unhealthy pups.

Live birth index (stillbirths):
There was a non-statistically significant, but possible compound-related reduction in the live birth index in the high-dose group. This was manifested by a reduction in the live birth index of F2 generations (live birth index for the control, low-, mid-, and high-dose groups in the F2 generation was 96, 96, 97, 90, respectively). In addition, the live birth index for the high-dose group was 6-7% below the historical control range.

For details, please refer to the attached background material (attachment 5).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

300 ppm: There was a statistically significant compound-related decrease in pup weights for the F2 mid-dose group, which occurred on days 14 and 21. The body weights were 9% below the control group for the F2 pups.
600 ppm: There was a statistically significant and compound-related decrease in pup weights for the F2 high-dose group. This decrease occurred on days 7 to 21. The body weights ranged from 11-20% below the control group for the F2 pups.

For details, please refer to the attached background material (attachment 6).

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

not applicable

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Sexual maturation:

not examined

Description (incidence and severity):

not applicable

Anogenital distance (AGD):

not examined

Description (incidence and severity):

not applicable

Nipple retention in male pups:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

not applicable

Gross pathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings:

not examined

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Developmental immunotoxicity:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: corresponding to 2.6 mg/kg bw/day (males) and 3.5 mg/kg bw/day (females) in the P1 generation

Key result

Dose descriptor:

LOAEL

Generation:

F2

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: corresponding to 16.4 mg/kg bw/day (males) and 22.0 mg/kg bw/day (females) in the P1 generation

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

300 ppm

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Average daily doses:

Actual doses were 0, 45.9 (91.7%, CV = 8%), 279 (93.2%, CV = 7%), and 542 ppm (90.3%, CV = 7%). These doses corresponded to average daily doses as follows:

P male: 0, 2.7, 16.4 and 32.3 mg/kg bw/day

P female: 0, 3.3, 20.4 and 41.0 mg/kg bw/day

F1 male: 0, 2.6, 16.4 and 32.1 mg/kg bw/day

F1 female: 0, 3.5, 22.0 and 44.0 mg/kg bw/day

Conclusions:

In total, the study was performed under GLP conditions and is in accordance with OECD TG 416 (adopted 1983). Deviations to the current guideline (adopted 2001) were minor and concerned the housing temperature and parameters that were not examined such as sperm parameters and weights of specific pups and/or parental organs. The study is therefore considered reliable and valid. Based on the findings of this study, a NOAEL of 50 ppm (corresponding to 3.3 mg/kg bw/day for P females and 3.5 mg/kg bw/day for F1 females) was derived for general toxicity based on decreased body weights during premating, gestation, and lactation and due to alterations in liver and thyroid weights and micropathology findings (hepatocellular necrosis). The neonatal NOAEL was 50 ppm, based on a possible compound-induced decrease in the live birth index and decreased pup body weights. The reproductive toxicity NOAEL for the test substance was 50 ppm, based on the compound-induced dystocia.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

2.7 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The toxicity to reproduction was assessed in a 2-generation study conducted with male and female rats according to OECD TG 416 (1983), under GLP conditions. The study is considered of reliable quality and validity (RL1), fulfills the criteria of a key study and thus, is suitable for assessment of the present endpoint.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

To assess the effect of the test item on reproduction, a 2-generation key study conducted with rat including a range-finding study is available.

2-generation reproduction range-finding study was conducted with the test substance in Sprague-Dawley rats to determine dose levels for a 2-generation study in rats (M-000911-01-2). The test substance was admixed with basal rodent diet at concentrations of 0, 100, 400, and 1600 ppm and offered to male and female animals from the P (comprised of 28 male and 28 female rats) and F1 generations. P male and female animals received test or control diet for a minimum of 28 days prior to mating and during mating interval. All males were euthanized on day 42 after completion of the mating period, while females were continued on diet during gestation and throughout a 21-day post-delivery lactation period. No clinical signs were observed in adults or progeny. The highest dose caused a reduced feed intake and decreased body weight gains. Histopathologically, hepatocellular hypertrophy in the liver, and elongated follicular cells in the thyroids were detected at 1600 ppm. Also at 400 ppm, liver effects were observed in one female and thyroid effects in one male. Neonatal mortality was increased and body weight gain of viable pups was reduced at 1600 ppm. Therefore, 100 ppm was a NOAEL in this study based on liver (hepatocellular hypertrophy) and thyroid (elongated follicular cells) effects at 400 ppm.

 

A GLP-compliant 2-generation reproductive toxicity study in rats conducted according to OECD TG 416 was considered as key study (M-001304-01-1). During this study, rats were treated with nominal dose levels of 0, 50, 300 or 600 ppm of the test substance via basal rodent diet, corresponding to actual doses of 0, 45.9, 279, and 542 ppm. The P and F1 adults were comprised of 30 rats/sex/group. P male and female rats received test or control diet throughout the study, beginning at 7 weeks of age for the P adults and at weaning for the F1 adults. Prior to breeding, the animals received treated diet for a 10-week period. The average daily doses were 0, 2.7, 16.4 and 32.3 mg/kg bw/day in P males, 0, 3.3, 20.4 and 41.0 mg/kg bw/day in P females, 0, 2.6, 16.4 and 32.1 mg/kg bw/day in F1 males, and 0, 3.5, 22.0 and 44.0 mg/kg bw/day in F1 females. P adults were mated to produce F1 litters and F1 were mated to produce F2 litters. The following effects of the test substance on P adults were evaluated: body weight, food consumption, clinical signs, estrous cycling, mating, fertility, gestation length, and litter size. The offspring were evaluated with regards to body weight, food consumption, clinical signs, estrous cycling, mating, fertility, gestation length, and litter size. Gross necropsy evaluations were performed on all adults and pups, and histopathological evaluation of reproductive organs, the pituitary, liver, thyroid and gross lesions was performed on all P and F1 adults.

Parental animals (P0 and F1):

In parental animals, no compound-related clinical signs were observed. There were no deaths or early sacrifices in the low-dose group, but four female P0 adults were found dead. Three out of those four deaths were attributed to dystocia and were noted in animals given 300 ppm of the test substance. One female treated with 50 ppm was found dead due to teeth caught in the feeder. Five P0 adults were early sacrificed, one male from the control group, one female given 300 ppm and three females given 600 ppm. Four female rats were early sacrificed due to dystocia. There were no deaths or early sacrifices in the low-dose group for the F1 adults. However, two deaths (one control and one high-dose group animal) and one early sacrifice due to hard palate fracture (mid-dose group) occurred. In parental animals body weight gains were decreased at 600 ppm for P0 and F1 females during the premating phase and for F1 males. During the gestation phase lower body weights with normal weight gains were measured in the P0 and F1 high-dose group. Also during the lactation phase lower body weights were observed in the P0 and F1 high-dose group. Terminal body weights were decreased in high-dose F1 males and female adults. There was no compound-related effect on food consumption for the P0 males and females during premating, gestation and lactation phases. After the highest dose, food consumption was increased in F1 males in pre- and post-mating and in F1 females during premating. There was no compound-related effect on food consumption for F1 females during the gestation or lactation phases. Treatment-related alteration in organ weights of the P0 and F1 adults were observed for liver and thyroid as follows: Liver weights were statistically increased in the P0 and F1 adults given 300 and 600 ppm of the test substance. Thyroid weights were also statistically increased in P0 and F1 adults at 300 ppm and 600 ppm. Three high-dose group P0 females and four mid-dose group P0 females died or were sacrificed early due to abnormal parturition (dystocia). All of these pregnancies were near expected termination. Gross observations in these animals generally included pallor, wet/stained perineal areas (ventrum), red vaginal discharge, and partial or complete failure to deliver with dead pups in utero. Pinpoint red foci in the liver were also noted in one of the 600 ppm P0 females. Treatment-related gross pathological findings were not observed in F1 adults. Micropathology evaluation revealed treatment-related findings in the liver and thyroid, which corresponded to treatment-related weight changes in these organs. In the mid- and high-dose group P0 and F1 adults, generally in both sexes, hepatocytomegaly in the liver and thyroid follicular cell hypertrophy were seen. In dams, which died or were sacrificed due to dystocia red foci in the liver and hepatocellular necrosis were detected. There was no compound-related effect on the estrous cycle, insemination length, mating, fertility, implantation sites, gestation indices, birth index, litter size, pup gender or lactation index. There was an increase in the number of P0 animals in the mid- and high-dose groups, which had difficulty delivering. As dystocia is historically not a common finding, it appears that the dystocia may have been compound-related. However, in the F1 generation no dystocia was observed. Therefore, it cannot be concluded from this study whether or not the dystocia observed in the P0 dams was due to compound administration. Regarding dystocia two expert statements are available (M-282359-01-1 and M-293264-01-1). In the 2-generation study, dystocia and failed delivery were recorded in the parental generation in 4/30 dams at dietary dose levels of 300 ppm (corresponding to approximately 20.4 mg/kg b/day) and at 3/30 dams after 600 ppm (corresponding to approximately 41.0 mg/kg bw/day). All females with dystocia had evidence of hepatocellular necrosis. Whereas the other effects observed in the liver (minimal to moderate hepatocellular hypertrophy) can be assessed as indicative of an adaptive response of the liver to the enhanced need for metabolization of the test substance, the minimal to moderate hepatocellular necrosis in the liver observed in the mid- and high-dose females needs to be regarded as an adverse effect. However, this effect did exclusively occur in pregnant dams, not in males or non-pregnant females. As cited in M-282359-01-1, dystocia was also observed in a supplemental 1-generation study in 2-4 of 30 dams at a dietary dose of 1000 ppm (corresponding to approximately 75 mg/kg bw/day), but not at 300 ppm (corresponding to approximately 23 mg/kg bw/day). Furthermore, dystocia was not observed after short-term exposure to the test substance. According to the expert statement, this supports the observation that a high exposure with the test substance for a long time is necessary to cause severe liver damage via metabolic overload of the liver, which can lead to dystocia in pregnant dams. Furthermore, it has been noted that prolongation of gestation and dystocia are known to be easily induced in rats with progesterone or progestogens as well as with non-steroidal anti-inflammatory drugs, which block prostaglandin-synthesis. In further studies a key effect of the test substance was shown to be a slight increase in progesterone levels due to the induction of enzymes of the steroid biosynthesis pathway. Increased progesterone levels can in turn cause dystocia, since a marked decrease in progesterone levels is mandatory in rats to initiate labor and delivery. However, dystocia was considered to be a rat-specific effect with no relevance for human risk assessment due to the following reasons:

1. In humans, progesterone levels remain high during parturition and therefore slightly enhanced progesterone levels in pregnant women will not be able to provoke dystocia.

2. A clear NOAEL exists for liver enzyme induction (please refer to IUCLID section 7.5).

3. A 250 fold margin of safety exists between the NOAEL for liver enzyme induction and the ADI (Acceptable Daily Intake).

Beside dystocia, there was a possible compound-related reduction in the live birth index in the high-dose group. This was manifested by a reduction in the live birth index of the F1 generation, which did not reach statistical significance. However, the live birth index for the high-dose group (of both generations) of the current study was 6-7 % below the historical control range. The live birth indices for the F1 generation low- and mid-dose groups were not considered to be significantly lower than the control group for the following reasons:

1. Similar findings were not observed in the F2 generation.

2. The control value for the F2 breeding was comparable to the values for the low- and mid-dose groups.

3. There was no dose response.

4. Similar values have been observed for the live birth index in the historical control data base.

F1 and F2 pups:

There was no compound-related effect on pup weights at birth. However, the pup weights were decreased in the F1 and F2 mid- and high-dose groups on days 14 and 21, and days 7-21, respectively.

In total, the study was performed under GLP conditions and is in accordance with OECD TG 416 (adopted 1983).Deviations to the current guideline (adopted 2001) concerned the housing temperature and parameters that were not examined such as sperm parameters and weights of specific pups and/or parental organs. The study is therefore considered reliable and valid. Based on the findings of this study, a NOAEL of 50 ppm for maternal toxicity based on decreased body weights during premating, gestation, and lactation and due to alterations in liver and thyroid weights and micropathology findings (hepatocellular necrosis). The neonatal NOAEL was 50 ppm, based on a possible compound-induced decrease in the live birth index and decreased pup body weights. The reproductive toxicity NOAEL for the test substance was 50 ppm, based on the possible compound-induced dystocia.

Effects on developmental toxicity

Description of key information

Developmental toxicity (rat, OECD 414):

NOAEL (maternal): 10 mg/kg bw/day
LOAEL (maternal): 50 mg/kg bw/day
NOAEL (developmental): 10 mg/kg bw/day
LOAEL (developmental): 50 mg/kg bw/day

Developmental toxicity (rabbit, OECD 414):

NOAEL (maternal): 2 mg/kg bw/day
LOAEL (maternal): 10 mg/kg bw/day
NOAEL (developmental): 2 mg/kg bw/day
LOAEL (developmental): 10 mg/kg bw/day

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Jul 1995 - 09 May 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 1981

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 2018

Deviations:

yes

Remarks:

thyroid weight, thyroid histopathology and thyroid hormones (T4, T3 and TSH) not investigated, anogential distance not measured, justification for choice of vehicle not provided

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Age at study initiation: not specified
- Weight at study initiation: more than 300 g for males and 182 - 239 g for females
- Housing: During adaptation, females were kept in groups in Type III Makrolon® cages. Starting from gestation day 0 they were individually accommodated in Type II Makrolon® cages on low-dust wood shavings. The males were kept individually in Type III Makrolon® cages.
- Diet standard rat diet (Altromin® 1324, produced by the Altromin company in Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.0 ± 3.0
- Humidity (%): approximately 50
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 11 Jul 1995 To: 09 May 1996

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% aqueous carboxymethyl cellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was formulated in 0.5% aqueous carboxymethyl cellulose suspension. Animals received a uniform volume of 10 mL/kg bw per oral gavage.

OTHER:
The animals of the control group received vehicle only (0.5% carboxymethyl cellulose) at the same volume. After insemination was ascertained, 28 females each were allocated to four experimental groups according to a computer-generated randomization plan. Due to inadequate skeletal and cartilage staining for unknown reasons in single fetuses from several litters of the different dose groups 7 additional inseminated females were allocated non-randomized to the study at each dose level in order to obtain a minimum of 20 litters for complete skeletal and cartilage evaluation in this study. The animals were treated daily from day 6 to 19 post coitum (p.c.).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability of the test article/vehicle mixtures concentration were determined by HPLC. The analytical data verified that the test material content in the toxicology test mixture agreed with the target concentration within defined limits. The concentration were 101.0 - 108.0% (for 0.2 mg/mL), 81.1 - 111.0% (for 1.0 mg/mL) and 104.0 - 112.0% (for 5.0 mg/mL). Furthermore, the analytical data verified that the test material was homogeneously distributed and chemically stable within the concentration range of 0.02 mg/mL to 20.0 mg/mL. Under current sample preparation and handling conditions stability and homogeneity was assured at room temperature for a period of at least 8 days.

Details on mating procedure:

The animals were mated by placing two females overnight into a Type III cage together with one male rat. If sperm was detected in the vaginal smear taken on the morning following mating, this day was regarded as day 0 of gestation.

Duration of treatment / exposure:

The animals were treated daily from day 6 to 19 p.c.

Frequency of treatment:

daily

Duration of test:

14 days

Dose / conc.:

2 mg/kg bw/day

Dose / conc.:

10 mg/kg bw/day

Dose / conc.:

50 mg/kg bw/day

No. of animals per sex per dose:

35

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels used were selected according to a preceding developmental toxicity dose-range finding study. In this study, dose level of 2, 10 and 50 mg/kg bw were tested. Effects on maternal animals were only observed at 50 mg/kg bw/day shown as reduced food consumption and body weight gain. The same dose level caused decreased fetal weights and increased number of fetuses with skeletal retardations. The external, visceral and skeletal examination of the fetuses revealed no treatment-related malformations. Thus, the test substance revealed embryotoxicity (retardation) in a maternally toxic dose range, therefore, the same dose levels were proposed for the main developmental toxicity study.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: From day 0 to 20 p.c. all animals were inspected twice daily.
- Cage side observations checked: general condition of the rats (appearance, behavior), any alterations concerning their excretory products

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: The body weights of the animals were determined on day 0 p.c. and daily from day 6 to 20 p.c.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, food consumption was calculated as g per animal per day
The feed consumption of the animals on gestation days 0 - 6, 6 - 11, 11 - 16 and 16 - 20 was determined based on the differences in weight of feed provided and feed which remained unconsumed.

WATER CONSUMPTION: Yes
Water consumption was determined by estimation of the remaining quantities in the water bottles.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 p.c.
- Organs examined: the uterus, uterine contents, number of corpora lutea

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Individual weight and appearance of the placentas, number of live fetuses, sex of live fetuses, individual weight of fetuses

Blood sampling:

- Plasma: No data
- Serum: No data

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter], however, during skeletal and cartilage evaluation it turned out that inadequate staining had occurred in fetuses from several litters. Therefore, 7 additional inseminated females per group were assigned to the study.
- Head examinations: No data
- Anogenital distance of all live rodent pups: no
- Methods:
For visceral malformations and other findings deviating from normal, evaluation of about half of the fetuses were done by razor blade sectioning according to the modified WILSON technique. For findings in abdominal, pelvic, thoracic organs and skeletal and cartilage, DAWSON technique modified by the addition of cartilage staining (method described by INOUYE, modified) was used. Furthermore, the skin was viscerated and removed, the cartilage was stained with alcian blue GX, the fetuses were cleared with dilute potassium hydroxide solution, the skeletal system was stained with alizarin red S, and the cartilage and skeletal system were assessed.

Statistics:

Animals without implantation sites were excluded from statistical evaluation. Animals with total resorption were not taken into account for calculation of group mean values of body weights, body weight gains and feed intakes. Differences between the control and treated groups were considered significant when p<0.05. Statistical significance was tested using the following methods:
- Analysis of Variance (ANOVA) and in case of significant results Dunnett's t-Test as posthoc test for: feed intakes, body weights and body weight gains, uterus weights, corrected body weight gains, number of corpora lutea per dam, number of implantations per dam, number of live fetuses per dam and as percentage of implantations per dam, placental weights, fetal weights
- CHI2 test (correction according to Yates) for: fertility rate, gestation rate, number of fetuses or litters with malformations
- 2 by N CHI2 test; in case of significant differences Fisher's exact test with Bonferroni correction for: number of implantations per group, number of preimplantation losses per group, number of postimplantation losses, early resorptions, late resorptions or dead fetuses per group, number of live fetuses per group in percent of implantations, number of male or female fetuses or fetuses with undeterminable sex per group, number of fetuses or litters with skeletal findings
- Kruskall-Wallis test and in case of significant differences Dunn's test for: number of preimplantation losses per dam, number of postimplantation losses, early resorptions, late resorptions or dead fetuses per dam, number of male or female fetuses or fetuses with undeterminable sex per dam

Indices:

No indices were calculated.

Historical control data:

Yes, available in the study report and in an expert statement. For details, please refer to the attached background material (attachment 8).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No relevant treatment-related clinical observations were made in this study up to and including 50 mg/kg bw/day except for urinary and fecal excreta as follows:
2 and 10 mg/kg bw/day: No treatment-related changes were noted after treatment up to and including 10 mg/kg bw/day.
50 mg/kg bw/day: Urinary and also fecal excreta were affected by the treatment at 50 mg/kg bw/day. Towards and during the second half of gestation animals displayed increased urine excretion, which can be explained by the increased water consumption occurring at the same time. Decreased amounts of feces were observed mainly during the first few days after initiation of treatment which is highly correlated with the decreased feed intake detected during the same time period at this dose level. Six dams of the high dose group also displayed light discoloration of the feces, a finding which could be due to the elimination of test material which is a pale-yellow powder.
For details, please refer to the attached background material (attachment 1).

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

no mortality observed

Description (incidence):

No mortality occurred in this study up to and including 50 mg/kg bw/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Severe transient body weight loss was observed during days 6 to 9 p.c. at 50 mg/kg bw/day resulting in significantly decreased absolute body weight during the entire treatment period. Furthermore, mean maternal body weight gain was significantly decreased during days 6 to 9, 14 to 15 and 17 to 18 p.c. at 50 mg/kg bw/day. No effect on body weight development of the females was observed at 2 and 10 mg/kg bw/day.
For details, please refer to the attached background material (attachment 2).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

2 and 10 mg/kg bw/day: No treatment-related changes in food consumption were noted after treatment up to and including 10 mg/kg bw/day.
50 mg/kg bw/day: Feed consumption was decreased throughout the entire treatment period at the high dose level (50 mg/kg bw/day) with pronounced effect during day 6 to 11 p.c. whereas during day 11 to 20 p.c. the observed effect on feed consumption was less severe.
For details, please refer to the attached background material (attachment 3).

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

effects observed, treatment-related

Description (incidence and severity):

2 and 10 mg/kg bw/day: No treatment-related changes in water consumption were noted after treatment up to and including 10 mg/kg bw/day.
50 mg/kg bw/day: Water consumption was affected by the treatment at 50 mg/kg bw/day. During the first days after initiation of treatment an increased incidence of animals with decreased water consumption was observed at the high dose level whereas more animals displayed increased water consumption towards and during the second half of gestation at this dose level. For details, please refer to the attached background material (attachment 1).

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

not applicable

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

2, 10 and 50 mg/kg bw/day: No treatment related gross pathological findings were evident at necropsy up to and including the dose of 50 mg/kg bw/day.

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Number of abortions:

not specified

Description (incidence and severity):

not applicable

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

2 and 10 mg/kg bw/day: No alterations in the rate of post-implantation loss was noted at dose levels of up to and including 10 mg/kg bw/day.
50 mg/kg bw/day: The elevated incidence of late resorptions in animals with viable fetuses resulted in a significantly increased rate of post-implantation loss in the high dose group (50 mg/kg bw/day).
For details, please refer to the attached background material (attachment 4).

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

2 and 10 mg/kg bw/day: The resorption rate was unaffected at dose levels of up to and including 10 mg/kg bw/day.
50 mg/kg bw/day: Total resorption of the conceptus was observed in one female at the 50 mg/kg bw/day dose level. However, in the rat strain used single total resorptions occur spontaneously as demonstrated by historical data collected during 1991 to 1994 in the laboratory performing this study. Data from those studies indicate that single total resorptions can occur by chance, apparently independent of treatment. Since marked maternal toxicity was visible at the high dose level, it can not be excluded that the single total resorption observed at this dose level is related to the treatment with the test compound.
For details, please refer to the attached background material (attachment 4).

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

2 and 10 mg/kg bw/day: The resorption rate was unaffected at dose levels of up to and including 10 mg/kg bw/day.
50 mg/kg bw/day: Incidence of late resorptions (total number per group) in animals with viable fetuses was elevated in the high dose group (50 mg/kg bw/day). Since the incidence of late resorptions at this dose level was above the range of historical control data from this rat strain, it is considered to be related to the treatment with the test compound.
For details, please refer to the attached background material (attachment 4).

Dead fetuses:

no effects observed

Description (incidence and severity):

The number of dead fetuses was not affected by the treatment.

Changes in pregnancy duration:

not specified

Description (incidence and severity):

not applicable

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The number of pregnant animals were not affected by the treatment.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Weight and appearance of placentas:
The weight of the placentas was unaffected by the treatment at doses of up to and including 50 mg/kg bw/day. Necrotic placental borders were observed in all dose groups including the control animals without dose-dependency. The incidence of necrotic placental borders appeared to be increased at dose levels of 2 mg/kg bw/day and above when compared to control animals. However, this is a common finding, displayed no dose-dependency and was at all dose levels within the incidence range of untreated control animals of former studies, therefore, this observation is considered incidental and not related to the treatment with the test compound. It is therefore concluded that the weight and appearance of the placentas was not affected up to and including 50 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed at this dose level

Key result

Dose descriptor:

NOAEL

Remarks:

maternal developmental toxicity

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed at this dose level

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Key result

Dose descriptor:

LOAEL

Remarks:

maternal developmental toxicity

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

pre and post implantation loss

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

2 and 10 mg/kg bw/day: The mean fetal weight was unaffected by the treatment at dose levels of up to and including 10 mg/kg bw/day.
50 mg/kg bw/day: The mean fetal weight recorded at cesarean section was statistically significantly decreased.
For details, please refer to the attached background material (attachment 4).

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The number of live fetuses was not affected by the treatment.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No changes in sex ratio were observed in any treatment group when compared to the control group.

Changes in litter size and weights:

not examined

Description (incidence and severity):

not applicable

Anogenital distance of all rodent fetuses:

not examined

Description (incidence and severity):

not applicable

Changes in postnatal survival:

not examined

Description (incidence and severity):

not applicable

External malformations:

effects observed, treatment-related

Description (incidence and severity):

50 mg/kg bw/day: At cesarean section forelimb deviations were observed in 2 fetuses at 50 mg/kg bw/day. Although these limb anomalies were observed in a single litter, this finding is considered to be related to treatment with the test compound since these findings were confirmed by skeletal evaluation (limb bone dysplasia) and since similar findings were also observed during skeletal evaluation of other fetuses in several litters at the 50 mg/kg bw dose level. All other observations made during external examination are not considered to be compound-related findings because of lack of dose-dependency or correspondingly frequent incidences in historical control animals of other studies with similar study design. At the high dose level one fetus with multiple malformations (gastrochisis, shortened mandible, cleft palate, limb deviations, etc.) was observed. Historical control data for malformations demonstrated that single fetuses with multiple malformations including those observed in this fetus may also occur spontaneously in untreated control animals of this rat strain. But, considering the increased incidence of late resorptions and the fetal skeletal findings (see below) together with the distinct maternal toxicity at the high dose level (50 mg/kg bw/day) it can not be excluded that the single multiple malformation observed in this dose group might be related to treatment with the test compound.
For details, please refer to the attached background material (attachment 5 and 9).

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

It has to be noted, that during skeletal and cartilage evaluation, an inadequate staining had occurred in fetuses from several litters. Therefore, 7 additional inseminated females per group were assigned to the study and with regards to skeletal and cartilage examination, the data generated were evaluated and reported separately for completely evaluated litters and for litters where complete evaluation was not possible due to insufficient staining.

2 and 10 mg/kg bw/day: Some statistically significant deviations were noticed at the 2 and 10 mg/kg bw/day dose levels, which should not be regarded to be of toxicological relevance because of the lack of dose-dependency or comparable rate of occurrence in control groups of previously conducted studies with similar study design in the same rat strain.
50 mg/kg bw/day: Several statistically significant observations were recorded with respect to ossification and skeletal variations. In all these cases statistically significant findings were made due to cases of impaired ossification (retardations) in different skeletal localizations. These observations were apparent in an individual and on a per litter basis. The findings considered toxicologically relevant were cases of retardations (impaired ossification) or variations (wavy ribs, asymmetrical sternebrae) at the high dose level. Further, an increased total incidence of malformations observed was especially due to an increased incidence of extremital bone dysplasia (tubular bones, clavicula and scapula). These limb bone malformations are prevalent spontaneous malformations in the rat strain used. The incidence at which they were observed in this study was however elevated at the high dose level.
For details, please refer to the attached background material (attachment 6 and 7).

Additional information on dysplasia of limb bones were given in an expert statement (M-031344-01-1) and the observed effects in the present rat developmental toxicity study were further assessed in a position paper (M-278407-01-1). With regards to the increased incidence of dysplastic limb bones in the fetuses of the highest dose group, it was noted, that these incidences were within the historical control range. Further it was stated, that if a treatment relationship would be assumed, then it would most likely be related to the observed maternal toxicity. In addition, a clear dose-response relationship was not visible: The next lower level (10 mg/kg bw/day) did not cause maternal toxicity and hence dysplastic limb bones were not observed. At the low dose of 2 mg/kg bw/day this finding was seen at an incidence within the historical control range. In total, the incidence showed a high variability so that a treatment relationship is questionable, especially since this finding occurs spontaneously in the rat strain used at a rather high and variable rate. This was supported by the results of further toxicity studies: In a study, which was conducted at the same time period, 3.7% of the fetuses and 23.1% of the litters in the control group were affected. In another study, the highest incidence of this finding was detected in the control animals, with a fetal incidence of 2.7% and a litter incidence of 14.3%. This demonstrates that even the effects in the highest dose group with a fetal incidence of 3.0% and a litter incidence of 20.7% were covered by, or were comparable to control incidences. Furthermore, if litters from dams mated with certain males (for which an association to an increased malformation rate could be suspected), are excluded, the incidence of findings would be smaller. In the years 1994 up to 1999, the historical control incidence was 0-3.45% in the fetuses and from 0-23.07% in the litters, which shows that the incidences observed in all groups, even in the 50 mg/kg bw group, did not exceed historical control ranges. For details, please refer to the attached background material (attachment 8) and to the respective endpoint summary.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

10 and 50 mg/kg bw/day: Slightly increased incidence of renal pelvis dilatation were noted at the 10 and 50 mg/kg bw dose levels. However, renal pelvis is not considered to be a compound-related finding because of the lack of dose-dependency or correspondingly frequent incidences in historical control animals of other studies with similar study design.
For details, please refer to the attached background material (attachment 5 and 9).

Other effects:

not examined

Description (incidence and severity):

not applicable

Details on embryotoxic / teratogenic effects:

not reported

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed at this dose level

Key result

Dose descriptor:

LOAEL

Remarks:

developmental toxicity

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

external malformations

skeletal malformations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: skeletal: limb, ribs, sternebrae

Description (incidence and severity):

Incidence of extremital bone dysplasia together with multiple malformations (one fetus) as well as an increased incidence of skeletal retardations (impaired ossification) and variations (wavy ribs, asymmetrical sternebrae) at the highest dose tested.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The study was performed under GLP conditions and according to OECD TG 414 (adopted 1981). Notable deviations from the OECD guideline mainly concerned thyroid parameters, which were not examined. However, the study is considered reliable and valid. Under the conditions of this study, no clear and indisputable evidence for a specific developmental toxicity potential of the test substance was determined since all developmental effects observed were manifest only at dose levels which also induced severe maternal toxicity and/or are common spontaneous malformations in the rat strain used. The NOAEL was 10 mg/kg bw/day for maternal toxicity and developmental toxicity based on maternal and fetal body weight, maternal food consumption and embryotoxic effects at 50 mg/kg bw/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Jun 1995 - 15 Sep 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 1981

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 2018

Deviations:

yes

Remarks:

housing temperature slightly different compared to guideline, thyroid weight, histopathology not investigated, justification for choice of vehicle not provided

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. Karl Thomae GmbH, Biberach, Germany
- Age at study initiation: not specified
- Weight at study initiation: 2185 to 3130 g
- Housing: Animals were kept individually in Macrolon® cages with perforated cage sheets.
- Diet: standard diet (Ssniff® rabbit diet K - Z), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3
- Humidity (%): approximately 60
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 21 Jun To: 15 Sep 1995

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% aqueous carboxymethyl cellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Administration formulations were prepared daily using a suspension of 0.5% carboxymethyl cellulose in demineralized water, which has no effect on the investigated parameters. Animals received a uniform volume of 5 mL/kg bw per oral gavage.

OTHER:
The animals of the control group received vehicle only (0.5% carboxymethyl cellulose) at the same volume. The animals were treated daily from day 6 to 28 post coitum (p.c.) between 8 and 12 o'clock a.m.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Investigations on the homogeneity and stability after 8 days storage of the active ingredient in samples of low (0.02 mg/mL), medium (0.2 mg/mL) and high concentrations (20 mg/mL) covering the concentrations used in this study had been performed before the start of study. Homogeneity and stability were confirmed in these investigations by HPLC. The analytical data verified that the test material content in the toxicology test mixture agreed with the target concentration within defined limits. The concentrations were 93.8 - 94.3% (for 0.4 mg/mL), 96.5 - 97.0% (for 2.0 mg/mL) and 107.7 - 109.8% (for 9.0 mg/mL). Furthermore, the analytical data verified that the test material was homogeneously distributed and chemically stable within the concentration range of 0.02 mg/mL to 20.0 mg/mL. Under current sample preparation and handling conditions stability and homogeneity was assured at room temperature for a period of at least 8 days.

Details on mating procedure:

One male rabbit was mated with one female rabbit under observation. About one hour after the first mating had occurred the same animals were mated again. It was recorded which female was mated with which male. The day on which the copulation was observed was considered as day 0 of gestation.

Duration of treatment / exposure:

The animals were treated daily from day 6 to 28 p.c.

Frequency of treatment:

daily

Duration of test:

22 days

Dose / conc.:

2 mg/kg bw/day

Dose / conc.:

10 mg/kg bw/day

Dose / conc.:

45 mg/kg bw/day

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels used were selected according to a range finding developmental toxicity study with the test substance in rabbits. In this study, dose level of 3, 10, 30, 40 or 50 mg/kg bw/day were tested. Effects on maternal animals were observed at 30 (slight), 40 (moderate) and 50 mg/kg bw/day (severe) shown as reduced food consumption and/or body weight gain. Developmental toxicity (retardation) could not be excluded at the 40 mg/kg bw/day level and was evident at the 50 mg/kg bw/day level (retardation, embryolethality, increase in variations and in common malformations). Therefore, dose levels of 0, 2, 10 or 45 mg/kg bw/day were proposed as dose levels for the main developmental toxicity study with the test substance in rabbits.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: general condition (appearance, behavior), and alterations concerning the excretory products

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: The body weights of the animals were determined on day 0 p.c. and daily from day 6 to day 29 p.c.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, food consumption was calculated as g per animal per day
The feed intake of the animals was determined from the difference in weight between the feed offered and the feed not consumed for the following days of gestation: day 0 - 6 , day 6 - 11, day 11 - 16, day 16 - 21, day 21 - 24 and day 24 - 29 p.c.

WATER CONSUMPTION: Yes
Water intake was assessed by visual estimation of the quantities left over.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29 p.c. (by intravenous injection of 2 mL T61 ad us. vet., Hoechst AG). The animals which aborted were sacrificed after abortion had occurred.
- Organs examined: uterus weight, number of corpora lutea and implantations, individual weights and appearance of placentas

Ovaries and uterine content:

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

Blood sampling:

- Plasma: No data
- Serum: No data

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]
- Methods:
The fetuses were eviscerated according to the modified STAPLES technique including a transverse section through the brain. The cartilage was stained with Alcian blue (method described by INOUYE, modified). Subsequently, the fetuses were cleaned with dilute potassium hydroxide solution and stained with Alizarin red S according to the modified DAWSON technique. After evaluation of the fetuses, the viscera were discarded.

Statistics:

Differences between the control and treated groups were considered significant when p<0.05. Statistical significance was tested using the following methods:
- Analysis of Variance (ANOVA); in case of significances Dunnett's test for feed intakes, body weights and body weight gains, uterus weights, corrected body weight gains, number of corpora lutea per dam, number of implantations per dam, number of live fetuses per dam and as percentage of implantantions per dam, placental weights and fetal weights
- CHI2 test (correction according to Yates) for fertility rate, gestation rate and number of fetuses or litters with malformations
- 2 by N CHI2 test; in case of significant differences Fisher's exact test with Bonferroni correction for number of implantations per group, number of preimplantation losses per group, number of postimplantation losses, early resorptions, late resorptions or dead fetuses per group, number of live fetuses per group in percent of implantations, number of male or female fetuses or fetuses with undeterminable sex per group and number of fetuses or litters with skeletal findings
- Kruskall-Wallis test; in case of significant differences Dunn's test for number of preimplantation losses per dam, number of postimplantation losses, early resorptions, late resorptions or dead fetuses per dam and number of male or female fetuses or fetuses with undeterminable sex per dam

Indices:

No indices were calculated.

Historical control data:

Yes, available in the study report. For details, please refer to the attached background material (attachment 6).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The following treatment-related effects were observed for urinary and fetal excretion:
Urine
2 mg/kg bw/day: Reddish excretion for more than one day was evident in one female, which revealed an incidental resorption of all implants.
10 mg/kg bw/day: The number of females with decreased urination was slightly increased. The finding of light yellow urine in only 2 females was considered incidental as light yellow discoloration may occur spontaneously in individual rabbits of the strain used.
45 mg/kg bw/day: The number of females with decreased urination was slightly increased. Several females showed a light yellow discoloration of urine. Reddish excretion for more than one day was evident in two females which also revealed total resorption at cesarean section.

Feces
2 mg/kg bw/day: No effects on feces were observed.
10 and 45 mg/kg bw/day: The incidence of females with reduced feces was increased at the 10 mg/kg bw/day and 45 mg/kg bw/day levels including one female without feces on single days in the 45 mg/kg bw/day group. This correlates with the decreased feed intake at dose levels of 10 mg/kg bw/day and above.

No further treatment-related clinical signs were observed. Findings with respect to appearance and behavior did not reveal a dose dependency and were therefore considered incidental.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

no mortality observed

Description (incidence):

No mortality occurred at any dose level.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

2 mg/kg bw/day: The body weight gains during treatment and gestation as well as the corrected body weight gain were unaffected.
10 mg/kg bw/day: The females lost weight after start of treatment until day 11 p.c. which resulted in a statistically significantly decreased weight gain from day 6 to 11 p.c. and correlated with the statistically significantly decreased feed intakes during the same period. From day 11 p.c. on the females of this group gained weight at a rate comparable to the control group. However, the overall weight gain during the treatment and gestation period as well as corrected body weight gain were slightly lower than in the control group.
45 mg/kg bw/day: The females showed distinct weight loss from day 6 to 13 p.c. correlating to the severely decreased feed intakes during this time period. Body weight gains during the treatment and whole gestation period were distinctly decreased and corrected body weight gain was slightly decreased.
For details, please refer to the attached background material (attachment 2).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

2 mg/kg bw/day: The feed intakes were unaffected.
10 mg/kg bw/day: Feed intakes were decreased from day 6 to 16 p.c. which reached statistical significance on Day 6 to 11 p.c.
45 mg/kg bw/day: Feed intakes were decreased during most of the treatment period which reached statistical significance on Day 6 to 21 and Day 24 to 29 p.c. Feed intake was severely affected from Day 6 to 16 p.c.
For details, please refer to the attached background material (attachment 1).

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

effects observed, treatment-related

Description (incidence and severity):

2 and 10 mg/kg bw/day: water intakes of the females were unaffected by treatment at levels up to and including 10 mg/kg body weight/day.
45 mg/kg bw/day: The number of females showing decreased water intakes was increased.

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

not applicable

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

2 and 10 mg/lg bw/day: No dose related gross pathological findings were ascertained in the females at levels up to and including 10 mg/kg bw/day.
45 mg/kg bw/day: Effects on the gastrointestinal tract (hardened contents in the stomach, marked vascular pattern of the intestines) as part of the systemic effects observed at this dose level were mainly seen in animals which aborted or resorbed all their implants. One female with total resorption showed a cystocele with hardening and reddish discoloration of urinary bladder and partial necrosis of liver. A treatment related effect for this isolated finding is not assumed.

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

2 mg/kg bw/day: One female aborted on day 24 p.c. This single abortion was considered incidental as no abortion occurred at the 10 mg/kg bw/day level and as isolated cases of abortions may occur spontaneously in the strain of rabbits used.
10 mg/kg bw/day: No abortion occurred at this dose level.
45 mg/kg bw/day: Two females aborted on day 23 p.c. or on day 28 p.c. A treatment related effect was assumed due to distinct impairment of feed intakes and body weight gains observed in the animals.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Preimplantation loss
No effects were noted with regards to preimplantation loss after oral application of the test substance up to and including 45 mg/kg bw/day.

Postimplantation loss
2 and 10 mg/kg bw/day: The postimplantation loss was unaffected by treatment at levels up to and including 10 mg/kg bw/day.
45 mg/kg bw/day: The postimplantation loss was increased (statistically significantly when calculated per group only) due to the total resorptions in three females of this group. When postimplantation loss was calculated excluding these females with total resorption it was unaffected by treatment at levels up to and including 45 mg/kg bw/day and correspondingly was the number of fetuses.

For details, please refer to the attached background material (attachment 3).

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

2 mg/kg bw/day: The single total resorption was considered incidental as isolated cases of total resorptions may occur spontaneously.
10 mg/kg bw/day: No total resorptions were noted for this dose level.
45 mg/kg bw/day: Three total resorptions were noted. The total resorption of one of the three affected females was possibly caused by the cystocele evident at necropsy while a treatment-related effect has to be assumed for the total resorption of the other two females.
For details, please refer to the attached background material (attachment 3).

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

Early resorption
2 and 10 mg/kg bw/day: No effects on early resorption were noted.
45 mg/kg bw/day: The number of dams with implantations having early absorption was statistically increased, whereas no effects were observed in dams with viable fetuses.

Late resorption
2, 10 and 45 mg/kg bw/day: No effects on late resorption were noted.

For details, please refer to the attached background material (attachment 3).

Dead fetuses:

no effects observed

Description (incidence and severity):

The number of dead fetuses was not affected by the treatment.

Changes in pregnancy duration:

not specified

Description (incidence and severity):

not applicable

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The number of pregnant animals were not affected by the treatment.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Weight and appearance of placentas:
2 and 10 mg/kg bw: The weight of placentas was unaffected by treatment.
45 mg/kg bw/day: The placental weight was decreased. This decrease was considered to be marginal as statistical significance was only achieved when calculation was performed on an individual basis.

There were no treatment related gross pathological findings in the placentas. Incidental findings (partial necrosis or missing placental lobe and coarse-grained placentas) observed in the dose groups did not reveal a dose dependency or were comparable to spontaneous placental findings in the strain of rabbits used or only appeared in a single female.

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

2 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed at this dose level

Key result

Dose descriptor:

NOAEL

Remarks:

maternal developmental toxicity

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed at this dose level

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

Key result

Dose descriptor:

LOAEL

Remarks:

maternal developmental toxicity

Effect level:

45 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

number of abortions

pre and post implantation loss

total litter losses by resorption

other: decreased placental weights

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

2 mg/kg bw/day: The fetal body weight was unaffected.
10 mg/kg bw/day: The fetal weight was decreased, however, this only achieved statistical significance when calculated on an individual basis and not when calculated on a litter basis. Therefore, the effect on fetal weight in this group was only considered marginal.
45 mg/kg bw/day: The fetal weight was statistically significantly decreased when calculated on a litter and on an individual basis.
For details, please refer to the attached background material (attachment 3).

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the number of live fetuses between the treatment and control groups.

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

2 and 10 mg/kg bw/day: The percentage of male or female fetuses did not differ from the control figures to any marked extent at levels up to and including 10 mg/kg bw/day.
45 mg/kg bw/day: A statistically significantly lower percentage of males were noted when compared to the control group. However, postimplantation loss of the females evaluated for sex distribution of their fetuses was unaffected at levels up to and including 45 mg/kg bw/day and external and visceral evaluation of the fetuses did not result in any indication of feminization of male fetuses. In addition, data from different groups of studies performed in the same laboratory (1988 - 1994) show that the sex ratio has spontaneously varied within a large scattering range in the strain of rabbits used (please also refer to attachment 7). Therefore, the lower percentage of male fetuses at this dose level was considered incidental.

Changes in litter size and weights:

not examined

Description (incidence and severity):

not applicable

Anogenital distance of all rodent fetuses:

not examined

Description (incidence and severity):

not applicable

Changes in postnatal survival:

not examined

Description (incidence and severity):

not applicable

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

2 and 10 mg/kg bw/day: The incidence of malformed fetuses was comparable to the control group.
45 mg/kg bw/day: The increased incidence of malformed fetuses in this group is mainly due to arthrogryposis which is the most frequent spontaneous malformation in the strain of rabbits used. The incidence of fetuses with arthrogryposis in this group (4.4%) lay within the upper range of historical controls (5.6 %).
For details, please refer to the attached background material (attachment 5).
The increased incidence of forelimb arthrogryposis in the present developmental toxicity study were further assessed in a position paper (M-278407-01-1). As mentioned above, this finding is the most common spontaneous malformation in the strain of rabbits used and it is sometimes regarded as variation only. It is most likely the consequence of restriction of fetal movements in the uterus, which results in a caudal flexure of the forelimb in the region of the wrist (carpal joint). In this study fetal incidences of this finding (called ventral flexure of the region of the wrist), do not suggest a treatment relationship. Furthermore, all incidences, including the incidence of the highest dose group were within the historical control range.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

2 and 10 mg/kg bw/day: Examination for skeletal retardations and variations did not reveal treatment related findings at levels up to and including 10 mg/kg bw/day.
45 mg/kg bw/day: Retarded ossification of different localisations (phalanges, metacarpals, calcanei, cervical and caudal vertebrae, hyoid bone) evident by statistically significant differences were noted when calculated on a fetal basis and for the phalanges when calculated on a litter basis, also. Further, the incidence of fetuses with supernumerary 13th ribs without supernumerary lumbar vertebra (variation) was marginally increased. The difference did not achieve statistical significance. Nevertheless, a relationship to treatment cannot be totally excluded for this finding as two additional fetuses of the 45 mg/kg bw/day group revealed supernumerary 13th ribs combined with a supernumerary lumbar vertebra.
All dose groups: A higher stage of ossification of the 5th sternebra (statistically significantly when calculated on an individual basis) was observed which showed no dose dependency and the percentage of affected fetuses was within the range of historical controls. Therefore, this finding was considered incidental.
For details, please refer to the attached background material (attachment 4).

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no visceral findings in the fetuses which were attributed to treatment as the findings were different in type and only occurred in the fetuses of one female in each dose group.
For details, please refer to the attached background material (attachment 5).

Other effects:

not examined

Description (incidence and severity):

not applicable

Details on embryotoxic / teratogenic effects:

not reported

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

2 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed at this dose level

Key result

Dose descriptor:

LOAEL

Remarks:

developmental toxicity

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

other: retarded ossification

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: supernumerary rib

Description (incidence and severity):

A treatment related increase in fetuses with supernumerary 13th ribs with supernumerary lumbar vertebra (common malformation) and with supernumerary 13th ribs only (variation) could not be totally excluded at the highest dose tested. However, a specific teratogenic potential of the test substance is excluded as all malformations observed in the dose groups were of a common type and correlated with systemic maternal toxicity.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

10 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The study was performed under GLP conditions and according to OECD TG 414 (adopted 1981). Notable deviations from the OECD guideline were minor. Thus, the study is considered reliable and valid. Under the conditions of this study, no clear and indisputable evidence for a specific developmental toxicity potential of the test substance was determined since all developmental effects observed were manifest only at dose levels which also induced maternal toxicity and/or are common spontaneous malformations in the rabbit strain used. The NOAEL was 2 mg/kg bw/day for maternal and developmental toxicity based on decreased feed consumption (maternal), body weights and excretion rates, effects on the gastrointestinal tract and intrauterine development (abortions and total resorptions), marginally decreased fetal weights, retarded ossification, a marginal increase in common malformations and a possible increase in variations at 10 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

2 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

The developmental toxicity was assessed in rodent (rat) and non-rodent (rabbit), according to OECD TG 414 (1981), under GLP conditions. Both studies are considered of reliable quality and validity (RL1 or RL2), fulfilling the criteria of a key study. Thus, both are suitable for assessment of the present endpoint.

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

To assess developmental toxicity, the effects of the test material were investigated in rats and rabbits.

The first developmental study considered as key study was conducted in accordance with GLP and OECD 414 adopted in 1981 (M-000832-01-1). In this study, groups of 35 inseminated Wistar rats each were treated daily, by gavage, with the test substance formulated in 0.5% aqueous carboxymethyl cellulose suspension from day 6 to day 19 post coitum (p.c.) with doses of 0, 2, 10, and 50 mg/kg bw/day, respectively. The fetuses were delivered by cesarean section on day 20 of gestation. The doses were based on the results of a pilot developmental toxicity study in rats in which doses of 0, 2, 10, and 50 mg/kg bw/day were tested. Effects on maternal animals were only observed at 50 mg/kg bw/day shown as reduced food consumption and body weight gain. The same dose level caused decreased fetal weights and increased number of fetuses with skeletal retardations. The external, visceral and skeletal examination of the fetuses revealed no treatment-related malformations. Thus, the test substance revealed embryotoxicity (retardation) in a maternally toxic dose range, therefore, the same dose levels were proposed for the main developmental toxicity study.

Maternal toxicity:

In the main study, no clinical signs or increased mortalities were detected at any dose, appearance, behavior of the dams were unaffected by the treatment up to and including 50 mg/kg bw/day. Feed consumption of the dams was distinctly decreased especially during the first few days after initiation of treatment at 50 mg/kg bw/day leading to body weight losses during the first few days after initiation of treatment and to a reduction of total body weight gain so that cumulative and corrected body weight gain during gestation were significantly decreased at 50 mg/kg bw/day. Water consumption and the amount of excreted feces were decreased during the first few days after initiation of treatment at 50 mg/kg bw/day whereas increased water consumption and urine excretion were observed towards and during the second half of gestation at the high dose level (50 mg/kg bw/day). At gross necropsy no test compound-related gross pathological findings were evident in any of the experimental groups.

Developmental toxicity:

At cesarean section an increased incidence of late resorptions and one total resorption were observed at 50 mg/kg bw/day resulting in a slightly decreased number of viable fetuses at the high dose level. Weight and appearance of the placentas were not affected at any dose tested whereas fetal weight was decreased at the highest dose level. Visceral examination of the fetuses did not reveal treatment-related findings up to and including 50 mg/kg bw/day. At external and skeletal evaluation, an increased incidence of extremital bone dysplasia together with one fetus with multiple malformations as well as an increased incidence of skeletal retardations (impaired ossification) and variations (wavy ribs, asymmetrical sternebrae) at the highest dose were observed. Additional information on dysplasia of limb bones were given in an expert statement (M-031344-01-1) and the observed effects in the present rat developmental toxicity study were further assessed in a position paper (M-278407-01-1). With regards to the increased incidence of dysplastic limb bones in the fetuses of the highest dose group, it was noted, that these incidences were within the historical control range. Further it was stated, that there was a lack of a clear dose-response relationship. Since the incidence showed a high variability, a treatment relationship is questionable, especially because this finding occurs spontaneously in the rat strain used at a rather high and variable rate. However, the data from the main study are indicative of impaired embryonic or fetal development at a maternal toxic dose of 50 mg/kg bw/day with an increased incidence of postimplantation loss, retarded development and an increase in common malformations and variations. Nevertheless, since these effects occurred only at doses, which were toxic to the dams, and as all effects are common in the rat strain used, they are not considered to be indicative of a specific developmental toxic potential.

In conclusion, under the conditions of this study, no clear and indisputable evidence for a specific developmental toxicity potential of the test substance was determined since all developmental effects observed were manifest only at dose levels, which also induced severe maternal toxicity and/or are common spontaneous malformations in the rat strain used. The NOAEL was 10 mg/kg bw/day for maternal toxicity and developmental toxicity based on maternal and fetal body weight, maternal food consumption and embryotoxic effects at 50 mg/kg bw/day.

In the second key study, the developmental toxicity after oral administration was investigated in rabbits in accordance with GLP and the OECD guideline 414 adopted in 1981 (M-000780-01-1). 24 female rabbits each were daily treated orally by gavage with the test substance formulated in a suspension of 0.5 % carboxymethyl cellulose in demineralized water, from day 6 to 28 p.c. with doses of 0, 2, 10 or 45 mg/kg bw/day. On the 29th day of gestation the fetuses were delivered by cesarean section. The doses were based on the results of a pilot study in which doses of 0, 3, 10, 30, 40, and 50 mg/kg bw/day were tested. In this pilot study, effects on maternal animals were observed at 30 (slight), 40 (moderate) and 50 mg/kg bw/day (severe) shown as reduced food consumption and/or body weight gain. Developmental toxicity (retardation) could not be excluded at the 40 mg/kg bw/day level and was evident at the 50 mg/kg bw/day level (retardation, embryolethality, increase in variations and in common malformations). Therefore, dose levels of 0, 2, 10 or 45 mg/kg bw/day were proposed as dose levels for the main developmental toxicity study with the test substance in rabbits.

Maternal toxicity:

In the main study, no clinical signs were observed. There were 2 abortions at 45 mg/kg bw/day. Food consumption was reduced at 10 mg/kg bw/day and above. Water uptake was reduced at the highest dose. The females lost weight during the first week of treatment at levels of ≥ 10 mg/kg bw/day with distinct losses at 45 mg/kg bw/day. This resulted in decreased weight gains during treatment and gestation. The number of females with decreased urination was slightly increased at 10 and 45 mg/kg bw/day. The finding of light yellow urine in only 2 females was considered incidental as light yellow discoloration may occur spontaneously in individual rabbits of the strain used. Gross necropsy of the females revealed findings in the gastro-intestinal tract (hardened contents in the stomach, marked vascular pattern of the intestines) at the 45 mg/kg bw/day level mainly in animals, which aborted or resorbed all their implants.

Developmental toxicity:

Due to 2 abortions and 3 resorptions at 45 mg/kg bw/day, the gestation rate was reduced at this dose level. The postimplantation loss of females with viable fetuses (excluding females with total resorption) and correspondingly the number of fetuses as well as fetal sex were unaffected at levels up to and including 45 mg/kg bw/day. Placental weights were marginally decreased at 45 mg/kg bw/day. Fetal weights were marginally reduced at 10 mg/kg bw/day and above. The skeletal ossification of the fetuses was retarded at different localizations (phalanges, metacarpals, calcanei, cervical and caudal vertebrae, hyoid bone) at the 45 mg/kg bw/day dose level. The incidence of common malformations, mainly of arthrogryposis, was marginally increased at 45 mg/kg bw/day. The increased incidence of forelimb arthrogryposis in the present developmental toxicity study was further assessed in a position paper (M-278407-01-1). This finding is the most common spontaneous malformation in the strain of rabbits used and it is sometimes regarded as variation only. It is most likely the consequence of restriction of fetal movements in the uterus, which results in a caudal flexure of the forelimb in the region of the wrist (carpal joint). In this study fetal incidences of this finding (called ventral flexure of the region of the wrist), do not suggest a treatment relationship. Furthermore, all incidences, including the incidence observed in the highest dose group were within the historical control range. Beside arthrogryposis, a treatment-related marginal increase in fetuses with supernumerary 13th ribs combined with supernumerary lumbar vertebra (common malformation) or with supernumerary 13th ribs only (variation) could not be completely excluded at the 45 mg/kg bw/day dose level.

In summary, under the conditions of this study, no clear and indisputable evidence for a specific developmental toxicity potential of the test substance was determined since all developmental effects observed were manifest only at dose levels, which also induced maternal toxicity and/or are common spontaneous malformations in the rabbit strain used.The NOAEL was 2 mg/kg bw/day for maternal and developmental toxicity based on decreased feed consumption (maternal), body weights and excretion rates, effects on the gastrointestinal tract and intrauterine development (abortions and total resorptions), marginally decreased fetal weights, retarded ossification, a marginal increase in common malformations and a possible increase in variations at 10 mg/kg bw/day.

Mode of Action Analysis / Human Relevance Framework

Please refer to the information provided in the endpoint summary.

Justification for classification or non-classification

The available data on reproductive toxicity meet the criteria for classification according to Regulation (EC) 1272/2008 and are therefore sufficient for classification of the test substance for reprotoxicity. The substance is listed in Annex VI of Regulation (EC) 1272/2008 (Index No. 613-325-00-3) as a reproductive toxicant category 1B (Repr. 1B; H360FD).