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Diss Factsheets

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(2-chlor-5-pyridyl-methyl)-cyanimino-1,3-thiazolidin
EC Number:
601-147-9
Cas Number:
111988-49-9
Molecular formula:
C10H9ClN4S
IUPAC Name:
3-(2-chlor-5-pyridyl-methyl)-cyanimino-1,3-thiazolidin

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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).

Effect levels (P0)

open allclose all
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)

Target system / organ toxicity (P0)

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

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

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).

Effect levels (P1)

open allclose all
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)

Target system / organ toxicity (P1)

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

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined
Description (incidence and severity):
not applicable

Effect levels (F1)

open allclose all
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.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

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

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined
Description (incidence and severity):
not applicable

Effect levels (F2)

open allclose all
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

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.