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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

2-generation reproductive toxicity (rat, OECD 416):

NOAEL (general parental toxicity): 150 ppm (corresponding to 10.2 mg/kg bw/day (males) and 11.8 mg/kg bw/day (females))

LOAEL (general parental toxicity): 500 ppm (corresponding to 32.7 mg/kg bw/day (males) and 37.9 mg/kg bw/day (females))

NOAEL (pup development): 150 ppm (corresponding to 10.2 mg/kg bw/day (males) and 11.8 mg/kg bw/day (females))

LOAEL (pup development): 500 ppm (corresponding to 32.7 mg/kg bw/day (males) and 37.9 mg/kg bw/day (females))

NOAEL (reproduction): 500 ppm (corresponding to 32.7 mg/kg bw/day (males) and 37.9 mg/kg bw/day (females))

LOAEL (reproduction): 2500 ppm (corresponding to 179.6 mg/kg bw/day (males) and 212.9 mg/kg bw/day (females))

Link to relevant study records

Referenceopen allclose all

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Jun 1998 - 6 Apr 1999
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 2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
adopted 1983
Deviations:
no
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 7 - 8 wks; (F1) 3 wks
- Weight at study initiation: (P) Males: 261.0 - 272.3 g; Females: 185.1 - 188.6 g
- Housing: individually (except during mating phase) in suspended stainless steel cages
- Diet: Purina Mills Rodent Lab Chow 5001-4, ad libitum
- Water: municipal tap water, ad libitum
- Acclimation period: at least six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 26 Jun 1988 To: 6 Apr 1999
Route of administration:
oral: feed
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with: Purina Mills Rodent Lab Chow 5001-4 and corn oil as carrier for the compound and to control dust formation
- Storage temperature of food: under refrigerator conditions
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 consecutive days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear (daily examination); referred to as Day 0 of gestation
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity and stability of the test substance in rodent feed was verified prior to initiation of the study. Feed samples were taken from each dietary level on study weeks 1, 2, 3 and at monthly intervals thereafter. Samples were maintained at refrigerator conditions prior to analysis. The test item was found to be homogenously distributed since the coefficient of variation (CV) of the samples is less than 10%. The test item was also found to be stable in feed for up to 14 days (room temperature). The mean analytical determined dietary concentrations of test item found in the diet were 91, 88, and 89% of nominal concentrations for the 150, 500, and 2500 ppm dose groups, respectively.
Frequency of treatment:
continously via the diet
Details on study schedule:
Selection of parents from F1 generation when pups were 21 days of age.
Dose / conc.:
150 ppm
Remarks:
Corresponding to the actual dose ingested during the premating phases of both generations combined: 10.2 mg/kg bw/day (males), 11.8 mg/kg bw/day (females)
Dose / conc.:
500 ppm
Remarks:
Corresponding to the actual dose ingested during the premating phases of both generations combined: 32.7 mg/kg bw/day (males), 37.9 mg/kg bw/day (females)
Dose / conc.:
2 500 ppm
Remarks:
Corresponding to the actual dose ingested during the premating phases of both generations combined: 179.6 mg/kg bw/day (males), 212.9 mg/kg bw/day (females)
No. of animals per sex per dose:
(P0): 30
(P1): 30
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose selection was performed on basis of results obtained in previous studies (chronic toxicity study and pilot reproduction toxicity study). The selected dose range was expected to produce evidence of toxicity at the highest dietary concentration and no parental or reproductive effects at the lowest dietary concentration.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once a day during weekends and holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week and if an animal showed clinical signs during daily cage side observations

BODY WEIGHT: Yes
- Time schedule for examinations: once a week during premating (males + females), once per week during mating period and until sacrifice (males), during gestation body weight of dams was measured on Days 0, 6, 13 and 20, during lactation dam body weight was measured on Days 0, 4, 7, 14 and 21.

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
Oestrous cyclicity (parental animals):
Oestrous cycle was characterized for all females by examining daily vaginal smears over a three-week period prior to mating and just prior to termination.
Sperm parameters (parental animals):
For all P0 and P1 male parental animals, sperm was collected from one testes and one epididymis for enumeration of homogenization-resistant spermatids and cauda epididymal sperm reserves, respectively. Additionally, sperm motility and sperm morphology were evaluated from the distal portion of the vas deferens.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If 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 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD) (only F2 on lactation Day 0).

Further, in F1 offspring grown to adults also the following parameters were examined:
clinical signs, body weights, vaginal opening, preputial separation and food consumption.

GROSS EXAMINATION OF DEAD PUPS:
yes, gross necropsy was performed born or found dead
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals following the mating phase
- Maternal animals: All surviving animals following the weaning of their respective litters
-Female animals that were sperm positive and/or had an internal vaginal plug but did not deliver were sacrificed after GD 24. Females which were never observed as being inseminated and/or with an internal vaginal plug were sacrificed at least 24 days after the completion of the mating phase, if they did not deliver. In these animals in addition to gross necropsy an evaluation of patency of the cervical/uterine os was performed.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examination. The uterus was excised and the implantation sites were counted.

HISTOPATHOLOGY / ORGAN WEIGHTS
Ovaries, uterus, brain, pituitary, thymus, liver, kidneys, adrenal glands, spleen, testes, epididymides, seminal vesicles and prostate were removed and weighed. Microscopical examination: cervix, epididymis, gross lesions, adrenals, liver, ovaries, pituitary, prostate, testicles, seminal vesicles/coagulating gland, uterus, and vagina. Histopathological evaluations were conducted on control and top dose group, except for reproductive organs which were analysed for all animals demonstrating affected fertility (failed to mate, conceive, sire, or deliver healthy offspring).

Furthermore, a quantitative evaluation of the primordial ovarian follicles was conducted in F1 dams if any of the following were ascribed to treatment: 1) a reduction in ovarian weight and abnormal ovarian histopathology findings, 2) abnormal oestrous cyclicity and female infertility, and/or 3) depletion is testicular spermatid counts in the F1 males and evidence of germ cell depletion during testicular histopathology evaluations.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of macroscopical examination for any structural abnormalities or pathological changes, particularly as they may relate to the organs of the reproductive system
-Pups found dead were examined for possible defects and/or cause of death

HISTOPATHOLOGY / ORGAN WEIGTHS
Additionally the brain, spleen, and thymus were weighed from one randomly selected pup/sex/litter in those pups examined macroscopically
Statistics:
Parametric data (including body weight gain and food consumption) were analyzed using a univariate Analysis of Variance (ANOVA), and if significant differences were observed a Dunnett's Test was performed. Nonparametric data (e.g., number of oestrous cycles, litter site, and number of implantation sites) were first analyzed by the Kruskal-Wallis test and then subjected to Dunn's Test if significant differences were identified. Nonparametric dichotomous data (e.g. fertility and gestation indices) were initially analyzed by the Chi-Square Test and if significance was observed between groups then by the Fisher's Exact Test with the Bonferroni adjustment. To the extent possible, the frequency of gross lesions were first examined visually, then, in the event of questionable distribution, by statistical analysis using the Chi-Square and Fisher's exact tests. Comparisons were made at both the 0.05 and 0.01 levels of significance. Sperm morphology and count were analyzed in the following way: The homogeneity of variances was tested with the folded form of the F statistic at the 0.01 significance level. If variances were not significantly different, the high dose group was compared with the control with the Cochran and Cox t-test. Animals with less than 100 available sperm were not included in the analysis of sperm morphology. For sperm motility, a trend test was conducted in SAS PROC MULTTEST using the ordinal dose scale and the permutation adjusted p-value. If the trend test with 4 groups was significant (p <= 0.05), the high dose group was deemed significantly different from control and the trend test was repeated with the 3 remaining groups (control, low and mid dose). The analysis process continued until the trend test was not statistically significant.
Reproductive indices:
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 per litter / total number of implantation sites per litter) * 100

Live birth index (%) = (number of pups born per litter / total number of pups per litter) * 100

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

-inseminated females include pregnant females not observed sperm positive or to have had an internal vaginal plug
-pregnant females include females which did not deliver, but had implantation sites
Offspring viability indices:
Viability index = (number of live pups/litter on Day 4 (pre-culling) / number of live pups born/litter) * 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs such as lacrimation of eyes, hair thinning or chromorhinorrhea of the nose were only observed in individual cases and without any dose-dependency. They are considered to be of no toxicological relevance.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female rat each of the control and 2500 ppm group died before sacrifice. Both deaths were considered to be incidental and not compound-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decreased body weights were observed in male and female rats of the 2500 ppm dose group from Day 7 onwards. Male body weights were decreased by 10 % compared to control on Day 126. Female body weights were decreased by 13% compared to control on Day 70. During gestation phase, also a decreased body weight was noted for females of the top dose group (-12 to -14% compared to control). During lactation period, body weights of dams were decreased at all time points (2500 ppm) or on lactation Day 14 (500 ppm), respectively. No effect on body weight or weight changes during any study period was noted in the 150 ppm group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was affected in male and female rats during the pre-mating phase in the 2500 ppm dose group. In the males, the food consumption was decreased during the first week and then increased afterwards for all subsequent time points, relative to control. In the females, food consumption was also decreased during the first week, then similar to control during weeks 2 and 3 and then increased for the remainder of pre-mating period. In the 500 ppm group a significantly lowered food consumption was observed during the first two weeks of pre-mating phase. The decrease in food consumption noted during the first weeks of a dietary study are not unusual and typically related to feed palatability, and as such are not considered to be toxicologically meaningful. During gestation phase, food consumption of females dosed with 2500 ppm test item was increased (+11 % compared to control) from days 6 to 13 of this phase. No effect on food consumption was noted during lactation phase.
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
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, non-treatment-related
Description (incidence and severity):
All histopathological lesions were considered to be incidental and not compund-related.
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):
No significant effects on either oestrous cycle length or number of cycles were observed. The term cycle stage data was not statistically analyzed for significant findings as there were no histological ovarian findings.
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Sperm morphology and total sperm count were not affected in the top dose group when compared to control and thus not analyzed in the 150 and 500 ppm groups. A statistically significant decrease in sperm motility (percent progressively motile, but not percent motile) was observed in the 2500 ppm group. This effect is considered to be secondary to body weight changes also observed at this dose level. However, according to the RAC opinion, classification of the test substance for fertility and sexual function as Repr. 2 (H361f) is warranted mainly based on delayed male sexual maturation supported by alterations in sperm motility. No effects on sperm motility were noted in the 150 and 500 ppm groups. For details please refer to attachment 07.
Reproductive performance:
no effects observed
Description (incidence and severity):
The mating index was 90, 100, 100, and 100%, the fertility index was 92, 97, 93, and 97%, and the gestation index was 100, 100, 100, and 100% for the control, 150, 500 and 2500 ppm groups, respectively. There were no significant effects on days to insemination, gestation length, or the median number of implants.
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
other: no adverse effect observed
Remarks on result:
other: Corresponding to 32.7 mg/kg bw/d (males) and 37.9 mg/kg bw/d (females)
Key result
Dose descriptor:
LOAEL
Remarks:
reproductive toxicity
Effect level:
2 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: Corresponding to 179.6 mg/kg bw/d (males) and 212.9 mg/kg bw/d (females)
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
150 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: Corresponding to 10.2 mg/kg bw/d (males) and 11.8 mg/kg bw/d (females)
Key result
Dose descriptor:
LOAEL
Remarks:
general toxicity
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: Corresponding to 32.7 mg/kg bw/d (males) and 37.9 mg/kg bw/d (females)
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
2 500 ppm
System:
male reproductive system
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs such as lacrimation of eyes, hair thinning or chromorhinorrhea of the nose were only observed in individual cases and without any dose-dependency. They are considered to be of no toxicological relevance.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female rat of the 150 ppm group was sacrificed unscheduled and exhibited a severe lung abscess with inhalation pneumonia. It was considered to be incidental and not compound-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decreased body weights were observed in male and female rats of the 2500 ppm dose group throughout pre-mating. Male body weights were decreased by 21 % compared to control on Day 0 and an 18% decrease on Day 119. Female body weights were decreased constantly by 16 - 17% compared to control throughout the pre-mating phase. During gestation phase, also a decreased body weight was noted for females of the top dose group (-13 to -16% compared to control). During lactation period, body weights of dams were decreased at all time points (2500 ppm). No effect on body weight or weight changes during any study period was noted in the 150 and 500 ppm groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was significantly increased in male and female rats during the pre-mating phase in the 2500 ppm dose group. In the pre-mating phase this effect ranged from 10 - 27 % (males) and 10- 30 % (females) as compared to control. During gestation phase, food consumption of females dosed with 2500 ppm test item was increased (+11 % compared to control) from days 6 to 13 of this phase. Food consumption was significantly increased during the last two weeks of lactation phase in the top dose females. No effects on food consumption during any phase was observed for the 150 and 500 ppm groups.
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
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):
Significantly decreased organ weights in both males and females of the 2500 ppm group were found for the thymus, but considered an indirect effect related to decreased body weight noted for this dietary level. All other observations related to organ weight changes are considered incidental since no dose-response relationships were established. See attachment 06 for details.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All gross necropsy observations were considered to be incidental and not compound-related.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All histopathological lesions were considered to be incidental and not compund-related.
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):
No significant effects on either oestrous cycle length or number of cycles were observed. The term cycle stage data was not statistically analyzed for significant findings as there were no histological ovarian findings.
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Sperm morphology and total sperm count were not affected in the top dose group when compared to control and thus not analyzed in the 150 and 500 ppm groups. A statistically significant decrease in sperm motility (percent progressively motile and percent motile) was observed in the 2500 ppm group. No effects on sperm motility were noted in the 150 and 500 ppm groups.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
The mating index was 93, 97, 93, and 93%, the fertility index was 82, 89, 75, and 100%, and the gestation index was 100, 100, 95, and 100% for the control, 150, 500 and 2500 ppm groups, respectively. There were no significant effects on days to insemination, gestation length, or the median number of implants.
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
500 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other:
Remarks on result:
other: Corresponding to 32.7 mg/kg bw/d
Key result
Dose descriptor:
LOAEL
Remarks:
reproductive toxicity
Effect level:
2 500 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive function (sperm measures)
Remarks on result:
other: Corresponding to 179.6 mg/kg bw/d
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: Corresponding to 32.7 mg/kg bw/d (males) and 37.9 mg/kg bw/d (females)
Key result
Dose descriptor:
LOAEL
Remarks:
general toxicity
Effect level:
2 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: Corresponding to 179.6 mg/kg bw/d (males) and 212.9 mg/kg bw/d (females)
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
2 500 ppm
System:
male reproductive system
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Only in individual litters pups with clinical signs such as weakness or bruising were observed.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
An increased incidence (statistically not significant) of stillborn pups was noted in the 2500 ppm dose group (2.3 % overall incicdence). However, the live-birth index, which describes the relative number of live pups born versus the total number of pups born per litter was not affected. In reproductive toxicity studies conducted in the testing laboratory on the Sprague-Dawley rat over the past three years, the incidence of pup stillbirths in the control groups has typically ranged from 0 to 3.9%. Therefore, this effect is considered to be within normal variations.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 2500 ppm group (both sexes), mean pup body weights were significantly decreased relative to control troughtout lactation, as well as pup body weight change. In the 500 ppm group, pup body weight change was significantly affected from lactation Day 7 to 14. These effects are considered secondary to general maternal toxicity. No changes in body weight or body weight change were observed in the 150 ppm group.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
not applicable (food consumption for F1 generation was measured beginning with 10-week premating phase; see P1 section above)
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:
effects observed, treatment-related
Description (incidence and severity):
Preputial separation was observed on days significantly different from control in the 500 and 2500 ppm groups, while no effect in the 150 ppm group was noted. Time to vaginal opening was affected in the 2500 ppm group compared to control, but not in either 150 or 500 ppm groups. The effects observed are considered a consequence of the reduced pup growth demonstrated by retarded body weights and body weight changes in these groups.
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:
effects observed, treatment-related
Description (incidence and severity):
In the 2500 ppm group organ weights in male and female pups were decreased for the brain, thymus and spleen (absolute weights). For the same animals the relative brain weight was increased, while relative thymus and spleen weights were decreased. At the 500 ppm dose level absolute thymic weights were significantly decreased in male pups. See attachment 06 for details.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Gross necropsy observations made for pups sacrificed after 21 days and found dead pups were considered to be incidental and not compound-related. However, it should be noted, that lungs that did not float were observed in the stillborn pups described above.
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:
LOAEL
Generation:
F1
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
body weight and weight gain
Remarks on result:
other: Corresponding to 32.7 mg/kg bw/d (males) and 37.9 mg/kg bw/d (females)
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
150 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: Corresponding to 10.2 mg/kg bw/d (males) and 11.8 mg/kg bw/d (females)
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
500 ppm
System:
male reproductive system
Organ:
not specified
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Only in individual litters pups with clinical signs such as weakness or bruising were observed.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
An increased incidence (statistically not significant) of stillborn pups was noted in the 2500 ppm dose group (3.7 % overall incidence). However, the live-birth index, which describes the relative number of live pups born versus the total number of pups born per litter was not affected. In reproductive toxicity studies conducted in the testing laboratory on the Sprague-Dawley rat over the past three years, the incidence of pup stillbirths in the control groups has typically ranged from 0 to 3.9%. Therefore, this effect is considered to be within normal variations.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 2500 ppm group (both sexes), mean pup body weights were significantly decreased relative to control from lactation Day 7 through weaning, as well as pup body weight change. In the 150 and 500 ppm groups, pup body weights and body weight changes were not affected. The noted effects are considered secondary to general maternal toxicity.
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):
no effects observed
Description (incidence and severity):
There was no statistically significant effect on anogenital distance found.
Nipple retention in male pups:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In the 2500 ppm group organ weights in male and female pups were decreased for the brain, thymus and spleen (absolute weights). For the same animals the relative brain weight was increased, while relative thymus and spleen weights were decreased. Absolute and relative changes in brain weights were consistent with decreases in pup terminal body weights and thus not considered to be directly compound related.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Gross necropsy observations made for pups sacrificed after 21 days and found dead pups were considered to be incidental and not compound-related. However, it should be noted, that lungs that did not float were observed in the stillborn pups described above.
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:
LOAEL
Generation:
F2
Effect level:
2 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Remarks on result:
other: Corresponding to 179.6 mg/kg bw/d (males) and 212.9 mg/kg bw/d (females)
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: Corresponding to 32.7 mg/kg bw/d (males) and 37.9 mg/kg bw/d (females)
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
2 500 ppm
Organ:
not specified
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
2 500 ppm
Treatment related:
not specified
Relation to other toxic effects:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified
Conclusions:
The study was performed under GLP conditions and is in accordance with OECD TG 416 (adopted 1983). No apparent deviations to the current version of the guideline (adopted 2001) were reported. The study is therefore considered reliable and valid. Based on the findings of this study, a NOAEL for parental toxicity of 150 ppm (10 mg/kg bw/day) based on decreased body weight of dams during lactation was established. The NOAEL for effects on neonatal parameters was 150 ppm (10 mg/kg bw/day) based on decreased pup body weight and the subsequent effect on preputial separation. The NOAEL for reproductive effects was 500 ppm (32.7 mg/kg bw/day) based on the increased incidence of stillborn pups, decreased sperm motility and morphology effects at 2500 ppm.
Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
13 May - 31 Aug 1997
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 2001
Deviations:
yes
Remarks:
The study was planned as a dose range finding study and therefore terminated after succession of the first generation. This guideline deviation was intended and has no impact on the validity of the results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
adopted 1983
Deviations:
yes
Remarks:
The study was planned as a dose range finding study and therefore terminated after succession of the first generation. This guideline deviation was intended and has no impact on the validity of the results.
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Rats were used as the test system as they are one of the US EPA, EU, OECD, and Japanese MAFF suggested species for reproductive toxicity studies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Sasco Inc., Kingston, NY
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: (P) 8 - 10 wks
- Weight at study initiation: (P) Males: 248.0 - 330.5 g; Females: 187.1 - 229.5 g
- Housing: individually (except for mating period) in suspended stainless steel cages
- Diet: Purina Mills Rodent Lab Chow 5001-4, ad libitum
- Water: municipal tap water, ad libitum
- Acclimation period: at least six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8 - 25.6
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 13 May To: 31 Aug 1997
Route of administration:
oral: feed
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 7 consecutive days
- Proof of pregnancy: vaginal smears were taken each morning and analyzed for vaginal plug and/or sperm. Day of insemination is referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged: individually in plastic nesting cages
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity and stability of the test substance in rodent feed was verified concurrent to study conduct. Feed samples were taken from each dietary level on study weeks 1, 2, 3, 4, 8, 11, and 14. Samples were maintained at refrigerator conditions prior to analysis. The test item was found to be homogenously distributed since the coefficient of variance (CV) of the samples is less than 10%. The test item was also found to be stable in feed for up to 14 days (room temperature). The mean analytical determined dietary concentrations of test item found in the diet were 86.5, 86.3, 88.3 and 88.8% of nominal concentrations for the 50, 150, 500, and 1000 ppm dose groups, respectively.
Duration of treatment / exposure:
Dosing was performed for eight weeks prior to mating, and then throughout mating phase (up to 7 consecutive days) as well as gestation and lactation period.
Frequency of treatment:
continously via the diet
Dose / conc.:
50 ppm
Remarks:
Corresponding to the actual dose ingested:
premating: 2.6 - 3.5 mg/kg bw/d (males), 3.1 - 3.6 mg/kg bw/d (females)
gestation: 3.2 - 3.5 mg/kg bw/d
lactation: 5.1 - 9.0 mg/kg bw/d
Dose / conc.:
100 ppm
Remarks:
Corresponding to the actual dose ingested:
premating: 5.2 - 7.0 mg/kg bw/d (males), 6.1 - 7.3 mg/kg bw/d (females)
gestation: 5.9 - 6.8 mg/kg bw/d
lactation: 12.0 - 18.6 mg/kg bw/d
Dose / conc.:
500 ppm
Remarks:
Corresponding to the actual dose ingested:
premating: 26.0 - 33.9 mg/kg bw/d (males), 31.9 - 36.6 mg/kg bw/d (females)
gestation: 31.7 - 36.4 mg/kg bw/d
lactation: 62.9 - 94.5 mg/kg bw/d
Dose / conc.:
1 000 ppm
Remarks:
Corresponding to the actual dose ingested:
premating: 49.4 - 68.7 mg/kg bw/d (males), 62.8 - 74.3 mg/kg bw/d (females)
gestation: 66.6 - 70.8 mg/kg bw/d
lactation: 120.3 - 191.8 mg/kg bw/d
No. of animals per sex per dose:
20
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: range finding study
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once on weekends and holidays)
- Cage side observations checked: mortality, moribundity, behavioural changes and overt toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week and in the event a possible clinical sign was observed during the cageside observation

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during premating phase. During mating phase only males were weighed once a week. During gestation, dam body weight was measured on days 0, 6, 13, and 20. During lactation, dam body weight was measured on days 0, 4, 7, 14, and 21.

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 from the consumption and body weight gain data: Yes
Oestrous cyclicity (parental animals):
Oestrous cycle was characterized for all females by examining daily vaginal smears over a three-week period prior to mating and just prior to termination.
Sperm parameters (parental animals):
not examined
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If 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 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities. Pup body weights were recorded as soon as possible following parturition and on lactation days 4, 7, 14 and 21.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed following mating phase
- Female animals: All surviving animals after weaning of their respective litters (lactation day 21). Dams that lost their entire litter prior to lactation day 21 were sacrificed. Females which were never observed as being inseminated and/or with an internal vaginal plug were sacrificed and necropsied at least 24 days after the completion of the mating phase if they did not deliver.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the thoracic, and abdominal viscera. The uterus was excised and the implantation sites, if present, counted and the uterus was weighed.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were collected and fixed: physical identifier, cervix, uterus, vagina, ovaries, gross lesions, epididymis, prostate, seminal vesicles, and testicles. The following organs were weighed: liver, kidney, spleen, testes, and ovaries.
Postmortem examinations (offspring):
SACRIFICE
- The size of each litter was adjusted on lactation day 4 to yield as close as possible, four males and four females per litter. F1 offspring not culled on lactation day 4 were maintained with the dam until weaning and sacrificed at the latest at 21 days of age.
- These animals were subjected to postmortem examinations as follows: gross external and internal necropsy

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
No tissues were weighed or collected from the weanlings
Statistics:
Parametric data (including body weight gain and food consumption) was analyzed using a univariate Analysis of Variance (ANOVA), and if significant differences were observed, a Dunnett's Test was performed. Nonparametric data (e.g., number of oestrous cycles, litter size, and number of implantation sites) were first analyzed by the Kruskal-Wallis test and then subjected to Dunn's Test if significant differences were identified. Nonparametric dichotomous data (e.g. fertility and gestation indices) were initially analyzed by the Chi-Square Test and if significant differences were observed between groups then by the Fisher's Exact Test with the Bonferroni adjustment. To the extent possible, the frequency of gross lesions was first examined visually, then, in the event of questionable distribution, by statistical analysis using the Chi-square and Fisher's exact tests. Comparisons were made at both the 0.05 and 0.01 levels of significance.
Reproductive indices:
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 per litter / total number of implantation sites per litter) * 100

Live birth index (%) = (number of pups born per litter / total number of pups per litter) * 100

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

-inseminated females include pregnant females not observed sperm positive or to have had an internal vaginal plug
-pregnant females include females which did not deliver, but had implantation sites
Offspring viability indices:
Viability index = (number of live pups/litter on Day 4 (pre-culling) / number of live pups born/litter) * 100
Clinical signs:
no effects observed
Description (incidence and severity):
not applicable
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
no mortality observed
Description (incidence):
not applicable
Body weight and weight changes:
no effects observed
Description (incidence and severity):
not applicable
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption was statistically significant decreased in the 500 ppm male and 1000 ppm female rats during the first week of premating phase. These effects are considered incidental to treatment and are within normal variation for this endpoint and rodent strain.
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
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:
no effects observed
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
Histopathological examinations were not performed, since they were not deemed necessary due to lack of effect on organ weights and gross findings.
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):
Oestrous cycles were determined prior to termination, however, due to absence of any effects on uterine or ovary weights these results are not reported.
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
not applicable
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
The mating index was 90, 100, 80, 100 and 90%, the fertility index was 83.3, 100, 87.5, 75, and 83.3%, the gestation index was 100, 95, 100, 93.3, and 100%, for the control, 50, 100, 500 and 1000 ppm groups, respectively.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: Corresponding to the actual dose ingested: premating: 49.4 - 68.7 mg/kg bw/d (males), 62.8 - 74.3 mg/kg bw/d (females) gestation: 66.6 - 70.8 mg/kg bw/d lactation: 120.3 - 191.8 mg/kg bw/d
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs were only observed in individual cases (e.g. wounds/cuts) or in the control group (general weakness (15/178), live pups have no milk in the stomach (11/178)) and are thus not considered treatment-related.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
No treatment-related effect on the number of stillborn pups or pups found dead was observed. These events were only noted in individual cases.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
not applicable
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:
no effects observed
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:
F1
Effect level:
>= 1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: Corresponding to the actual dose ingested: premating: 49.4 - 68.7 mg/kg bw/d (males), 62.8 - 74.3 mg/kg bw/d (females) gestation: 66.6 - 70.8 mg/kg bw/d lactation: 120.3 - 191.8 mg/kg bw/d
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
The study was performed under GLP conditions and in accordance with OECD TG 416 (adopted 1983), but based on the study design of an one-generation reproductive toxicity study with the F1 generation sacrificed on Day 21 of lactation. The study was performed for dose finding purposes for a full 2 generation reproductive toxicity study. The study is therefore considered reliable and valid. Based on the findings in this study, a NOAEL of 1000 ppm is assessed due to the absence of any toxic effect on the parents or offspring. Thus, future reproductive toxicity studies will have to include a dietary concentration greater than 1000 ppm of the test item.
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
32.7 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
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, 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 on reproduction of the test item, a 2-generation key study conducted with rat is available.

 

Reproductive toxicity

 

In the 2-generation reproductivity study (M-031280-03-1), male and female Sprague-Dawley rats were fed with the test substance over two generations; all parental animals (P0 and P1) received the test substance via the diet throughout the study period. The doses were selected based on a dose-range finding experiment (M-027255-01-1), which was conducted under GLP conditions and in accordance with OECD TG 416 (2-generation reproduction toxicity study), but was terminated after the conclusion of the first generation (weaning of F1 pups). In this dose-range finding study, 20 male and 20 female Sprague-Dawley rats wer dosed with 0, 50, 100, 500 or 1000 ppm of the test substance continously via the diet. Dosing was performed for eight weeks prior to mating, and then throughout the mating phase (up to 7 consecutive days) as well as through the gestation and lactation phases. It was shown, that the top-dose (1000 ppm) did neither induce adverse effects in parental animals nor in F1 pups. Based on the findings in this study, a NOAEL of 1000 ppm was derived due to the absence of any toxic effect on the parents or offspring.

 

The key study (M-031280-03-1), was also performed under GLP conditions and in accordance with OECD TG 416. In this study, 30 male and 30 female Sprague-Dawley rats per group were continuously dosed with the test item via the diet throughout the entire study period at 0, 150, 500 or 2500 ppm (corresponding to 10.2, 32.7 and 179.6 mg/kg bw/day (males) and 11.8, 37.9 and 212.9 mg/kg bw/day (females), respectively). Dosing startet at 7 to 8 weeks of age (first day of premating, P0 generation) and 3 weeks of age (weaning, F1 generation) and continued through mating, gestation and lactation phases. Animals were observed for clinical signs and monitored for changes in body weight and food consumption. In addition, all adult animals were evaluated for treatment-related effects on oestrous cycling, mating, fertility and gestation length. Litters were evaluated for effects on pup body weight, litter size, sex ratio, pup viability, onset of preputial separation and vaginal patency (F1 pups selected to become P1) and anogenital distance (F2 pups). P1 animals were evaluated as described for P0. Further examinations included a gross necropsy for all males, all dams following weaning and Day 21 pups not selected to become F1 generation parents. Adult evaluations included weighing, microscopic examinations of uterus, brain, pituitary, thymus, kidneys, adrenal glands, spleen, liver, testes, ovaries, epididymides, seminal vesicles and prostate. Males were also evaluated for sperm motility, morphology and count. Pup analysis included weighing spleen, brain and thymus of one male and female per litter on Day 21. No remarkable clinical signs were observed during any phase of either generation. Statistically significant decreases in body weight and affected food consumption in males and females of the 2500 ppm group during the pre-mating, mating, gestation and lactation phases of both generations as well as statistically significantly decreased body weight in the 500 ppm group during the lactation phase of the first generation were observed. The 150 ppm group was free of effects on body weight or food consumption. Statistically significantly reduced thymus and spleen weights compared to control were observed primarily in the 2500 ppm group, however, based on the concurrent decrease in body weight observed in these animals, these effects were considered secondary to general toxicity. There were no remarkable gross or microscopic findings observed in the males or females in either generation. No effects on any reproductive parameters were noted in either generation. No effects were observed on the ooestrus cycle or ovarian follicular development. In the males, sperm motility was decreased, relative to control in the 2500 ppm group. However, sperm count and morphology were unaffected and there were no remarkable histological findings involving the male reproductive tract. There were no effects on sperm measures noted in any other group. Litter effects were limited to decreased pup weight (primarily in the 2500 ppm group, both generations; to a lesser extent in the 500 ppm group, first generation). This reduction in pup body weight might have resulted in increased time to which developmental landmarks (preputial separation and vaginal opening) were observed in the 2500 ppm group and to a lesser extent in the 500 ppm group. However, as mentioned by RAC, the significant delay in preputial separation was present despite normal post-weaning growth in F1 pups of the 500 ppm group and thus, it is considered treatment-related by RAC. Sexual maturation of females was affected only at highest dose level. No other effects on developmental landmarks were observed. A slight and statistically not significant increased incidence of stillbirths was noted for the 2500 ppm group, but live-birth index was not affected. Effects on pup organ weights (primarily in the 2500 ppm group) were considered secondary to the general toxicity, as demonstrated by decreased body weight as discussed for the adults.

In conclusion, the available study is reliable and valid and can be used as a key study. The NOAEL for general toxicity in the parental (P0) generation was 150 ppm (corresponding to 10.2 mg/kg bw/day (males) and 11.8 mg/kg bw/day (females)) based on decreased body weight of dams during lactation. The NOAEL for effects on developmental parameters (F1 generation) was 150 ppm (10.2 mg/kg bw/day (males) and 11.8 mg/kg bw/day (females)) based on decreased pup body weight and delayed preputial separation. The NOAEL for reproductive effects was 500 ppm (32.7 mg/kg bw/day) based the increased incidence of stillborn pups and decreased sperm motility at 2500 ppm.

Effects on developmental toxicity

Description of key information

Developmental toxicity (rat, OECD 414):

NOAEL (maternal toxicity): 10 mg/kg bw/day

LOAEL (maternal toxicity): 40 mg/kg bw/day

NOAEL (developmental toxicity): ≥ 125 mg/kg bw/day

 

Developmental toxicity (rabbit, OECD 414):

NOAEL (maternal toxicity): 10 mg/kg bw/day

LOAEL (maternal toxicity): 25 mg/kg bw/day

NOAEL (developmental toxicity): 25 mg/kg bw/day

LOAEL (developmental toxicity): 75 mg/kg bw/day

 

2-generation (rat, OECD 416):

NOAEL (parental toxicity): 150 ppm (corresponding to 10.2 mg/kg bw/day (males) and 11.8 mg/kg bw/day (females))

LOAEL (parental toxicity): 500 ppm (corresponding to 32.7 mg/kg bw/day (males) and 37.9 mg/kg bw/day (females))

NOAEL (pup development): 150 ppm (corresponding to 10.2 mg/kg bw/day (males) and 11.8 mg/kg bw/day (females))

LOAEL (pup development): 500 ppm (corresponding to 32.7 mg/kg bw/day (males) and 37.9 mg/kg bw/day (females))

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 May - 19 Jun 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Deviations from current guideline version (e.g. ano-genital distance was not determined). For details please refer to materials and methods section.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 2018
Deviations:
yes
Remarks:
No blood sampling for analysis of T3, T4 and TSH, weight of thyroid gland not determined, no histopathology of thyroid gland. Anogenital distance of foetuses not determined. Temperature slightly out of range (18 to 26°C instead of 19 to 25°C).
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 1981
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD R BR VAF/PlusR
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan
- Age at study initiation: approx. 10 weeks
- Weight at study initiation: 201 - 231 g
- Housing: individually (except during cohabitation period)
- Diet: Certified Rodent Diet #5002 (PMI Feeds, St. Louis, Missouri), ad libitum
- Water: reverse osmosis water with chlorine added as a bacteriostat, ad libitum
- Acclimation period: six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): min. 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 26 May To: 19 Jun 1997
Route of administration:
oral: gavage
Vehicle:
other: aqueous 0.5% methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Suspensions of test substance were prepared weekly at the given concentrations. The test substance was considered 100% pure for the purpose of dosage calculations.

VEHICLE
- Concentration in vehicle: 1, 4, or 12.5 mg/mL
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no.: 56H0439
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken at the beginning and end of the dosing period for concentration analyses. No test item contamination of the vehicle was observed. All samples for the 1, 4, and 12.5 mg/mL concentrations (10, 40 and 125 mg/kg bw/day dosages, respectively) were within 10% range of target concentration, except for the 1 mg/mL concentration from the first preparation, which was 15% above target (range: 98.4 - 115 %). Homogeneity of the test item in the vehicle was demonstrated in samples from the top, middle and bottom of a sample formulation. The relative standard deviation was not greater than 3.3% and the test item therefore considered homogenously distributed in the samples. Stability of the test item in the vehicle was established for up to 14 days (-3% deviation from Day 0).
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: 4 days
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy (DG 0)
Duration of treatment / exposure:
From gestation day (DG) 6 until DG 19.
Frequency of treatment:
Daily
Duration of test:
From DG 0 until DG 20
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
40 mg/kg bw/day
Dose / conc.:
125 mg/kg bw/day
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dosages were selected on the basis of a dosage-range study
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Before and one hour after dosage (DGs 6-19) and on DG 20. Rats were also observed for viability at least twice daily.
- Cage side observations checked: clinical observation of effects of the test item, abortions, premature deliveries, and deaths

BODY WEIGHT: Yes
- Time schedule for examinations: on DG 0, daily during dosage period (DG 6-19) and on the day of Caesarean-section (DG 20).

FOOD CONSUMPTION: Yes
- Food consumption was determined on DGs 0, 6, 9, 12, 15, 18, and 20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Gross necropsy of the thoracic, abdominal and pelvic viscera was performed. To confirm pregnancy status, uteri from rats that appeared nonpregnant were stained with 10% ammonium sulfide. Tissues with gross lesions were preserved in neutral buffered 10% formalin for possible future evaluation. All other maternal tissues were discarded.
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: distribution of implantation sites, live and dead foetuses
Blood sampling:
- Plasma: No
- Serum: No
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter (those used for soft tissue examination)
Statistics:
Clinical observation and other proportion data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution.
Continuous data (e.g., maternal body weights, body weight changes, feed consumption values and litter averages for percent male foetuses, percent resorbed conceptuses, foetal body weights, foetal anomaly data and foetal ossification site data) were analyzed using Bartlett's Test of Homogeneity of Variances and the Analysis of Variance, when appropriate [i.e., Bartlett's Test was not significant (p>=0.05)]|. If the Analysis of Variance was significant (p<=0.05), Dunnett's Test was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate [i.e., Bartlett's Test was significant (p<=0.05)], the Kruskal-Wallis Test was used, when less than or equal to 75% ties were present. In cases where the Kruskal-Wallis Test was statistically significant (p<=0.05), Dunn's Method of Multiple Comparisons was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher's Exact Test was used to analyze the data.
Count data obtained at Caesarean-sectioning of the dams were evaluated using the procedures described above for the Kruskal-Wallis Test.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical observations included localized alopecia on the limbs, underside, back and/or neck, chromorhinorrhea and missing/broken incisors. All observations were considered non-treatment related, since the incidences were not dosage-dependent or the observations occurred in only 1 or 2 rats and/or are common events in the laboratory environment.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
Not applicable
Mortality:
no mortality observed
Description (incidence):
Not applicable
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No effect on body weight or body weight changes was observed at the 10 mg/kg bw/day dosage level. In the 40 mg/kg bw/day group, body weight gain was significantly decreased (p<=0.01) compared to control. In the 125 mg/kg bw/day group a significant weight loss (p<= 0.01) was observed on DGs 6 to 9 and a significantly reduced weight gain (p<=0.01) in the entire dosage period (DGs 6 to 20) as well as through the entire gestation period (DGs 0 to 20). Therefore, average body weights were significantly reduced (p<=0.05 or p<=0.01) on DGs 7 through 20. Please refer to attachment 01.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Feed consumption (absolute+relative) was lower (p<=0.05 or p<=0.01) in 40 mg/kg bw/day dosed rats compared to control on DGs 6 to 9. In the 125 mg/kg bw/day group, feed consumption was decreased (p<=0.05 or p<=0.01) throughout the complete exposure period (calculated as DGs 6 to 20) and entire gestation period (calculated as DGs 0 to 20). No effect on food consumption was noted in the 10 mg/kg bw/day group. Please refer to attachment 02.
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
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, non-treatment-related
Description (incidence and severity):
Gravid uterine weights did not significantly differ among the four groups.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Necropsy findings consisted of a portion of the intermediate lobe of the liver adhered to the diaphragm, which was thin at the point of adhesion, for one 10 mg/kg/day dosage group dam (16230); slight dilation of the pelvis of each kidney for one 40 mg/kg/day dosage group dam (16261) and a dark red area on the dorsal surface of the left apical lobe of the lung for one 125 mg/kg/day dosage group darn (16292). All necropsy findings were considered unrelated to the test substance because each occurred in only one rat. The pregnancies of these dams were unaffected by the presence of these gross lesions.
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
Details on results:
Not applicable
Number of abortions:
no effects observed
Description (incidence and severity):
No abortions occurred during the study.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Not applicable
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Not applicable
Early or late resorptions:
no effects observed
Description (incidence and severity):
Not applicable
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead fetuses were observed.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Not applicable
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
23/25, 22/25, 24/25, and 25/25 rats (92%, 88%, 96% and 100%, respectively) were pregnant in the control, 10, 40, and 125 mg/kg bw/day dose groups, respectively.
Other effects:
not examined
Description (incidence and severity):
Not applicable
Key result
Dose descriptor:
LOAEL
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
other: food consumption
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no adverse effect observed
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
not examined
Description (incidence and severity):
not applicable
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
not applicable
Changes in sex ratio:
no effects observed
Description (incidence and severity):
not applicable
Changes in litter size and weights:
no effects observed
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):
Umbilical hernia occurred in one control group fetus. This foetus had no other soft tissue or skeletal alterations.
One control group foetus had an edematous body (anasarca). Skeletal examination of this foetus revealed a short fibula and tibia of each hindlimb and variations in thoracic vertebral ossification (bifid centra of the 9th, 11th and 12th thoracic vertebrae).
A depressed eye bulge occurred in one 10 mg/kg bw/day foetus and in one 40 mg/kg bw/day dosage group fetus. Skeletal examination of the 10 mg/kg bw/day dosage group fetus revealed a small right eye socket as the only alteration. Microphthalmia of the right eye was first identified at soft tissue examination of the 40 mg/kg bw/day dosage group foetus.
No other gross external fetal malformations or variations occurred in this study.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In individual cases fused, waved or additional ribs, short fibula and tibia in the hindlimbs, bifid centrum in thoracic vertebra, and affected ossifications of sternum or pelvis were noted. These events were considered to be not treatment-related, since no dose-dependency was observed and the findings were within normal variations.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In individual cases umbilical artery that descended to the left of the bladder, absent innominate artery or aortic arch dorsal to the trachea and esophagus were found. No dose-dependency was observed for these events and hence they are not considered treatment-related.
Other effects:
not examined
Description (incidence and severity):
not applicable
Details on embryotoxic / teratogenic effects:
Combination of malformations and variations resulted in 10 (43.5%), 10 (45.4%), 10 (41.7%) and 11 (44.0%) litters in the four respective dosage groups with fetuses with any alterations observed. In these same respective dosage groups, totals of 14 (4.7%), 10 (3.4%), 13 (3.9%) and 17 (5.0%) fetuses had any alterations observed, and averages of 4.7%, 3.4%, 3.9% and 4.9% of the fetuses per litter had an alteration.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 125 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of any developmental toxicity effect
Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
external: eye
skeletal: sternum
skeletal: rib
skeletal: hindlimb
visceral/soft tissue: cardiovascular
Key result
Developmental effects observed:
no
Conclusions:
The current study was performed under GLP conditions and conformed the US EPA OTS 798.4900 guideline. The study is also in accordance with OECD TG 414 (adopted 2018) with some restrictions, such as no examination of anogenital distance of fetuses, and no blood sampling for analysis of thyroid hormones T4, T3 and TSH. Nevertheless, the study is reliable and valid. The developmental toxicity NOAEL in Sprague-Dawley rats was determined to be greater than 125 mg/kg bw/day due to the absence of any developmental toxicity effect in this study. A NOAEL for maternal toxicity was determined to be 10 mg/kg bw/day due to decreased body weight gain and food consumption in the 40 mg/kg bw/day group.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 May - 20 Jun 1997
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 2018
Deviations:
yes
Remarks:
weight of the thyroid gland not determined and no histopathology of the thyroid gland
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 1981
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Remarks:
[Hra:(NZW)SPF]
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Covance Research Products, Inc., Denver, Pennsylvania
- Age at study initiation: 6 - 8 months
- Weight at study initiation: 3.3 - 4.6 kg
- Housing: individually in stainless-steel cages
- Diet (e.g. ad libitum): Certified Rabbit Chow #5322 (PMI Feeds, Inc., St. Louis, Missouri), 150 g per day from arrival until DG 6 and from DG 6 onwards (first day of dosage) 180 g per day.
- Water: reverse osmosis water with added chlorine as a bacteriostat, ad libitum
- Acclimation period: 1 day

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 - 22
- Humidity (%): 30 - 70
- Air changes (per hr): min. 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 18 May To: 20 Jun 1997
Route of administration:
oral: gavage
Vehicle:
other: aqueous 0.5% methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Suspensions of test substance were prepared weekly at the given concentrations. The test substance was considered 100% pure for the purpose of dosage calculations.

VEHICLE
- Concentration in vehicle: 1, 2.5, 7.5 or 10 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required): 56H0439
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken at the beginning and end of the dosing period for concentration analyses. No test item contamination of the vehicle was observed. All samples for the 1, 2.5, 7.5, and 10 mg/mL concentrations (10, 25, 75 and 100 mg/kg bw/day, respectively) were within 10% range of target concentration (95.6 - 104.0% of target concentration). Homogeneity of the test item in the vehicle was demonstrated in samples from the top, middle and bottom of a sample formulation. The relative standard deviation was not greater than 3.3% and the test item therefore considered homogenously distributed in the samples. Stability of the test item in the vehicle was established for up to 14 days (-3% deviation from Day 0).
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
From gestation day (DG) 6 until DG 28.
Frequency of treatment:
Daily
Duration of test:
From DG 0 until DG 29.
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
25 mg/kg bw/day
Dose / conc.:
75 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
No. of animals per sex per dose:
23
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were selected based on the results of a dose-range finding study
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Before and one hour after dosage (DGs 6 - 28) and on DG 29. Animals were also observed for viability at least twice daily and once daily for general appearance before the dosage period.
- Cage side observations checked: clinical observation of effects of the test item, abortions, premature deliveries, and deaths

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: on DG 0, during the dosage period (DGs 6 - 28) and on the day of caesarean-section (DG 29)

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was determined daily

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Gross necropsy of the thoracic, abdominal and pelvic viscera was performed. To confirm pregnancy status, uteri from rabbits that appeared nonpregnant were stained with 10% ammonium sulfide. Tissues with gross lesions were preserved in neutral buffered 10% formalin for possible future evaluation.
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: distribution of implantation sites, live and dead foetuses
Blood sampling:
- Plasma: No
- Serum: No
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Cavitated organs were examined in all foetuses by dissection
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter; other half was used for brain examination in situ
Statistics:
Clinical observation and other proportion data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution.
Continuous data (e.g., maternal body weights, body weight changes, feed consumption values and litter averages for percent male foetuses, percent resorbed conceptuses, foetal body weights, foetal anomaly data and foetal ossification site data) were analyzed using Bartlett's Test of Homogeneity of Variances and the Analysis of Variance, when appropriate [i.e., Bartlett's Test was not significant (p>0.05)]. If the Analysis of Variance was significant (p<=0.05), Dunnett's Test was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate [i.e., Bartlett's Test was significant (p<=0.05)], the Kruskal-Wallis Test was used, when less than or equal to 75% ties were present. In cases where the Kruskal-Wallis Test was statistically significant (p<=0.05), Dunn's Method of Multiple Comparisons was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher's Exact Test was used to analyze the data.
Count data obtained at Caesarean-sectioning of the does were evaluated using the procedures described above for the Kruskal-Wallis Test.
Three does had litters of three or fewer foetuses; two does from the 25 mg/kg bw/day group and one from the 100 mg/kg bw/day group. As such events can skew data distributions, statistical analyses were performed with and without these data. These data were excluded from body weight, food consumption, Caesarean-sectioning, litter observations and foetal ossification site summary tables.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Increased incidences of scant faeces were noted in all dose groups, but were considered not treatment-related at the 10 mg/kg bw/day level (3/23, two of those only on one day). At doses of 25 mg/kg bw/day and higher scant faeces lasted for more than two days. No faeces in the cage pan was also observed for one of these 75 mg/kg bw/day does and for 11 100 mg/kg bw/day dosage group does. A dosage-dependent increase in orange urine was also noted from dose level 25 mg/kg bw/day and above. One doe of the high-dose group that was then sacrificed in moribund state, showed decreased motor activity and lost righting reflex. All other observed signs were considered unrelated to the test substance because they occurred in only one high dosage group doe or no dosage-dependency was observed (e.g. localized alopecia). Please refer to attachment 02.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
mortality observed, treatment-related
Description (incidence):
In the 75 mg/kg bw/d group, 2/23 animals died/were sacrificed moribund on gestation days 25 or 27, respectively. In the 100 mg/kg bw/d group 3/23 animals died or were sacrificed in moribund condition (DGs 17, 19, 20). In the very same animals, weight loss, reduced feed consumption and reduced faecal output were observed, too. Please refer to attachment 02.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The 75 and 100 mg/kg/day dosage groups had reduced weight gains for the entire dosage period and the entire gestation period. These values were statistically significant for the 100 mg/kg/day dosage group (p<=0.01). Reflecting the mortality and abortion incidences, these effects of the test substance were most severe in the 75 mg/kg/day dosage group on DGs 12 through 21, and in the 100 mg/kg/day dosage group on DGs 6 though 15. Maternal body weights were significantly reduced in the 100 mg/kg/day dosage group on DGs 14 to 29. Although the differences were not statistically significant, the 75 and 100 mg/kg/day dosage groups had biologically important reductions in gravid uterine weights. Corrected maternal body weight gains and body weights were reduced only in the 100 mg/kg/day dosage group. Please refer to attachment 01.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Absolute and relative food consumption was significantly decreased in the 75 and 100 mg/kg bw/day dosage groups (p<=0.05 or p<=0.01) for the entire dosage period (calculated as DGs 6 to 29). These parameters were reduced in both the 75 and 100 mg/kg/day dosage groups throughout the entire dosage period, with statistically significant reductions (p<=0.05 or p<=0.01) in both parameters on DGs 21 to 24 in the 75 mg/kg/day dosage group and on DGs 6 to 24 (absolute) or DGs 6 to 21 (relative) in the 100 mg/kg/day dosage group. Dose levels of 25 mg/kg bw/day and lower did not affect food consumption compared to controls. Please refer to attachment 03.
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
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):
Although the differences were not statistically significant, the 75 and 100 mg/kg bw/day dosage groups had biologically significant lower gravid uterine weights (89.1 and 81.1% of control animals). Please refer to attachment 01.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In individual cases a gastric trichobezoar or parovarian cysts were found. These observations were considered to be not treatment-related, because the incidences were not dosage-dependent.
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
Details on results:
N/A
Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
Abortions occurred in 3 (14.3%), 1 (4.5%) and 6 (27.3%) does of the control, 75 and 100 mg/kg bw/day groups, respectively. The abortions in the 100 mg/kg bw/day group are considered treatment-related. Please refer to attachment 04.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
The increase in total, early and late resorptions and percent resorbed conceptuses per litter at Caesarean-delivery were minimal and occurred at incidences within the historical control ranges of the testing facility. Increased incidence of post-implantation loss was observed in the 100 mg/kg bw/day group, but the observed postimplantation loss was not sufficient to result in a significant reduction in live litter size of the highest dose group. Please refer to attachment 04 and 07 for details.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
Only one litter was lost due to resorption on DG 29 (100 mg/kg bw/day groups).
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
Increased incidence of post-implantation loss (resorption) was observed in the 100 mg/kg bw/day group. The litters of the two high-dose does that were found dead were resorbed. Please refer to attachment 04.
Dead fetuses:
no effects observed
Description (incidence and severity):
not applicable
Changes in pregnancy duration:
effects observed, treatment-related
Description (incidence and severity):
Two does in each the 75 and 100 mg/kg bw/day groups prematurely delivered a litter on DG 29. Please refer to attachment 04.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Pregnancy occurred in 2:1(91.3%), 23 (100%), 22 (95.6%), 22 (95.6%) and 23 (100%) of the 23 presumed pregnant does assigned to the 0 (Vehicle), 10, 25, 75 and 100 mg/kg bw/day dosage groups, respectively.
Other effects:
not examined
Description (incidence and severity):
not applicable
Details on maternal toxic effects:
Reflecting the described mortality, premature deliveries and abortions, Caesarean-sectioning observations were based on 18, 23, 20, 17 and 10 pregnant rabbits with live litters in the five respective dose groups. These numbers exclude does with litters of three or fewer foetuses (two and one in the 25 and 100 mg/kg bw/day dosage groups, respectively).
Key result
Dose descriptor:
LOAEL
Effect level:
25 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
mortality
other: food consumption, abortions
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
mortality
other: food consumption, abortion: no adverse effects observed
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced foetal body weights were observed in the 100 mg/kg bw/day dosage group (36.66 +- 5.81 grams) compared to control (43.85 +- 5.08 grams) (p<= 0.01). Please refer to attachment 05.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
not applicable
Changes in sex ratio:
no effects observed
Description (incidence and severity):
not applicable
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The observed post-implantation loss was not sufficient to result in a statistically significant reduction in live litter size in the 100 mg/kg bw/day dose group. However, the mean litter size and number of live foetuses at 100 mg/kg bw/day was 7.4 as compared to 8.3 in the control (-10.8%). Please refer to attachment 04.
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):
External malformations (e.g. short snout, umbilical hernia, oedema, short or kinked tail) were only observed in individual cases and thus not considered treatment-related.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
A significant reduction in the litter average for ossified sternal centra per foetus was observed in the 100 mg/kg bw/day dosage group. This small retardation in ossification was considered to be a reversible variation associated with the tendency for reduced foetal body weights in this dose group. An increased incidence of small kidneys at 100 mg/kg bw/day was attributed to 3 foetuses from the same litter and these foetuses also had absent intermediate lung lobe in addition to other alterations. The statistically significant increase in absent intermediate lung lobe in 3 foetuses from 3 litters at 75 mg/kg bw/day and in 7 foetuses from 4 litters at 100 mg/kg bw/day is a common finding in this strain of rabbit.
The incidences in high dose group animals were within the historical control range (0-5 litters and 0-7 fetuses per control group). According to RAC in most of the cases, these increased incidences are still within the historical control data and moreover, the top dose caused 13% mortality (includes 2 dead and 1 sacrificed animal out of 23 animals); which is above the limit value of 10% of mortality established in the CLP Regulation to be considered as excessive maternal toxicity.
Please refer to attachment 06.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
An increased incidence of small kidneys at 100 mg/kg bw/day was attributed to 3 foetuses from the same litter and these foetuses also had absent intermediate lung lobe in addition to other alterations. The statistically significant increase in absent intermediate lung lobe in 3 foetuses from 3 litters at 75 mg/kg bw/day and in 7 foetuses from 4 litters at 100 mg/kg bw/day is a common finding in this strain of rabbit.
The incidences in high dose group animals were within the historical control range (0-5 litters and 0-7 fetuses per control group). According to RAC in most of the cases, these increased incidences are still within the historical control data and moreover, the top dose caused 13% mortality (includes 2 dead and 1 sacrificed animal out of 23 animals); which is above the limit value of 10% of mortality established in the CLP Regulation to be considered as excessive maternal toxicity.
The incidence of visceral intermediate lung lobe absence in rabbits of the 74 mg/kg bw/day dose group was statistically significantly higher than the concurrent control and above the historical control data.
However, lobation of the lung in rabbits is similar to that for humans and absence of the intermediate lung lobe occurs due to failure in formation of a lung fissure and does not involve the absence of lung tissue or alter lung functionality. Moreover, RAC notes that these effects appear at a dose causing 9% mortality (borderline with the 10% limit that would avoid considering this effect as supportive of developmental toxicity). Therefore, and according to the RAC opinion the absence of visceral intermediate lung lobe does not warrant a classification for developmental toxicity. Please refer to attachments 07 to 09 for details.
Other effects:
not examined
Description (incidence and severity):
not applicable
Details on embryotoxic / teratogenic effects:
Combination of malformations and variations resulted in the following incidences for foetal alterations. In the 0 (Vehicle), 10, 25, 75 and 100 mg/kg/day dosage groups, respectively, 4 (22.2%), 10 (43.5%), 10 (45.4%), 6 (35.3%) and 7 (63.6%) litters had fetuses with one or more alterations observed. In these same respective dosage groups, the total numbers of fetuses with identified alterations were 4 (2.7%) 13 (6.5%), 20 (11-2%; p<=0.01), 8 (6.0%) and 12 (14.1%; p<=0.01). One or more alterations occurred in averages of 2.6%, 6.7%, 15.2%, 6.5% and 20.5% of the foetuses per litter in the five respective dosage groups.

Foetuses with any malformation observed resulted in the following incidences: 0 (0.0%), 6 (3.0%; p<=0.01), 3 (1.7%), 2 (1.5%) and 5 (5.9%; p<=0.01) in the 0 (Vehicle), 10, 25, 75 and 100 mg/kg/day dosage groups, respectively. One or more malformations occurred in averages of 0.0%, 2.9%, 1.5%, 1.6% and 9.0% of the foetuses per litter in the five respective dosage groups.

Foetuses with any variation observed resulted in the following incidences: 4 (2.7%), 7 (3.5%), 17 (9.5%; p<=0.01), 6 (4.5%) and 7 (8.2%; p<=0.01) in the 0 (Vehicle), 10, 25, 75 and 100 mg/kg/day dosage groups, respectively. One or more variations occurred in averages of 2.6%, 3.8%, 13.6%, 5.0% and 11.5% of the foetuses per litter in the five respective dosage groups.

Except for the small but significant (p<=0.01) reduction in the litter average for ossified sternal centra per foetus, all other foetal alterations were considered non-related to treatment by the study director as 1) the incidences were within ranges of HCD of the testing laboratory, 2) did not follow a dose-response pattern, 3) the alterations were not observed in the litters from does that died or were sacrificed, 4) the incidences did not significantly differ from the concurrent control and 5) the alteration occurred in only one high-dose litter.
Key result
Dose descriptor:
LOAEL
Remarks:
developmental
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
visceral malformations
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Effect level:
25 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduced foetal body weights
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
25 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: sternum
skeletal: vertebra
skeletal: hindlimb
visceral/soft tissue: urinary
Description (incidence and severity):
Slightly delayed ossification (reduced sternal ossification centres) was noted at 75 mg/kg bw/day. Furthermore, at 100 mg/kg bw/day absent hindlimb phalanges, fused caudal vertebrae and small kidneys were observed. Developmental toxicity observed at the top dose is attributed to severe maternal toxicity at this dose level.
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
75 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:
not specified
Relevant for humans:
not specified
Conclusions:
The current study was performed under GLP conditions and according to OECD TG 414 (adopted 1981). The study is reliable and valid. The developmental toxicity NOAEL in New Zealand White Rabbits was determined to be 25 mg/kg bw/day due to an increase of postimplantation loss, reduced fetal body weights and retarded fetal sternal ossification at a dose level of 75 mg/kg bw/day. The NOAEL for maternal toxicity was determined to be 10 mg/kg bw/day due to reduced faecal output and discoloured urine observed at 25 mg/kg bw/day. Severe maternal toxicity at the two highest dose levels of 75 and 100 mg/kg bw/day was characterised by increased mortality, reduced body weight and increased incidences of abortions and premature deliveries. Significantly reduced foetal weight, embryo-foetotoxicity and an increase in the incidence of some malformations at these dose levels was attributed to the excessive maternal toxicity.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
10.2 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Developmental toxicity was assessed in two prenatal toxicity studies, one conducted with male and female rats and the other one with male and female rabbits, respectively. Both studies were performed according to OECD TG 414 (1981), under GLP conditions. The studies are considered of reliable quality and validity, fulfill the criteria of key studies and thus, 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 the effect on development, two developmental toxicity key studies, one conducted with the rat as rodent and one conducted with the rabbit as non-rodent species, are available. For each study, also a dose-range finding study was conducted.

 

Developmental toxicity

To assess the developmental toxicity of the test item, two developmental toxicity key studies were conducted, one in rats as a rodent species and one in rabbits as a non-rodent species. Furthermore, for each key study one preliminary study was performed for dose-range finding purposes.

In the dose-range finding study with Sprague-Dawley rats (M-027430-02-1, GLP conform, EPA OPPTS 870.3700), 8 presumably pregnant females per group were assigned to the dose levels of 0, 125, 250, 500 and 1000 mg/kg bw/day. Treatment was performed daily via gavage on days 6 through 19 of presumed gestation. Viability, clinical signs, body weights and feed consumption were monitored during the administration period. Surviving rats were sacrificed on day 20 of presumed gestation, a gross necropsy was performed and number and distribution of corpora lutea, implantation sites and uterine contents noted. Foetuses were examined for external alterations, gender and body weight.

In the top dose group all eight dams died. Compared to control, terminal body weights were reduced by 10, 19 and 43% for the 125, 250 and 500 mg/kg bw/day dosage groups, respectively. Gravid uterine weights were reduced by 7.3, 25.8 and 95.1% of control for the 125, 250 and 500 mg/kg bw/day groups, respectively. Reduced feed consumption (absolute and relative) was evident for the 125 mg/kg bw/day and higher dosage groups. There were 8, 7, 6, 8 and 0 pregnant rats at Caesarean sectioning on gestation day 20 in the respective dosage groups. The average numbers of early resorptions were increased in the 250 and 500 mg/kg bw/day groups and resulted in reduction of litter sizes. There were six totally resorbed litters in the 500 mg/kg bw/day group. The average fetal weights were decreased when compared to control (3.50 g) at the 125 (3.22 g), 250 (2.57 g) and 500 (1.04 g) mg/kg bw/day dose levels. A total of 116, 103, 70, 3 and 0 foetuses were examined from the respective five dosage groups and a total of 4 foetuses were malformed, thereof one foetus in the 250 mg/kg bw/day (e.g. short snout, curled tail, exencephaly, short trunk) and three foetuses of the 500 mg/kg bw/day (e.g. whole body oedema, short trunk, short tail) dosage group.

Based on these data, dosage groups of 0, 10, 40 and 125 mg/kg bw/day were suggested for the key developmental toxicity study in Sprague-Dawley rats, with 125 mg/kg bw/day to be expected to induce maternal toxicity, while the 10 mg/kg bw/day to be expected to be the NOAEL for maternal and developmental toxicity.

 

In the key developmental toxicity study (M-027416-01-1), conducted under GLP conditions and in accordance with OECD TG 414, 25 presumed pregnant female Sprague-Dawley rats per dose were treated with the test item at levels of 0, 10, 40 and 125 mg/kg bw/day via gavage once daily. Administration was performed on gestation days 6 to 19. Rats were regularly observed for viability, clinical signs, abortions, premature deliveries and deaths. Body weights and food consumption were recorded, too. On gestation day 20, rats were subjected to Caesarean-sectioning. Gravid uterine weight was recorded as well as the number of corpora lutea, number and distribution of implants, live and dead foetuses and early/late resorptions. Each foetus was weighed and examined for sex and gross external alterations. One half of the foetuses per litter was examined for tissue alterations and the other for skeletal alterations.

No deaths, abortions or premature deliveries occurred during the study. All clinical and necropsy observations were considered unrelated to treatment. Maternal body weight gain was significantly reduced in the 40 mg/kg bw/day group on gestation days 6 to 9 and the 125 mg/kg bw/day group exhibited significant weight loss on gestation days 6 to 9, resulting in significantly reduced weight gains and body weights. Food consumption (absolute and relative) were significantly reduced in the 40 mg/kg bw/day group on gestations days 6 to 9 and throughout the dose period in the 125 mg/kg bw/day group. In each group, pregnancy occurred in 22 to 25 rats. No treatment-related effects on Caesarean-sectioning or litter parameters were observed. No dam had litters with all conceptuses resorbed and there were no dead foetuses. Foetal evaluations showed no treatment-related effects.

In conclusion the available study is considered reliable and valid and can be used as a key study. The study was also conducted according to OECD TG 414 with minor deviations to the current version (2018). The deviations observed were not requested by the previous OECD guideline 414 (1981) and considered not so seriously compromise the outcome of the study as some information like the ano-genital distance at birth was investigated in the 2-generation reproduction toxicity study. Based on the data presented, the maternal NOAEL for the test item was 10 mg/kg bw/day due to reduced maternal body weight gain and food consumption values during the gestation days 6 to 9 in the 40 mg/kg bw/day group. There were no adverse effects observed on pup development in this study and the developmental NOAEL was therefore set to ≥ 125 mg/kg bw/day.

 

In another study (M-027436-02-1, GLP-conform, EPA OPPTS 870.3700), a dose-range finding experiment was performed to assess developmental toxicity of the test item in rabbits. Per group, five timed-pregnant female New Zealand White rabbits were used. Administration was performed once daily via stomach tube on presumed gestation days 6 through 28 at dose levels of 0, 62.5, 125, 250 and 500 mg/kg bw/day. Animals were checked for viability, clinical signs, body weights and feed consumption during the administration period. Surviving rabbits were sacrificed on gestation day 29 and examined for number and distribution of corpora lutea, implantation sites and uterine contents. A gross necropsy was also performed. Foetuses were weighed and examined for gross alterations and sex.

All five does in each, the 125, 250 and 500 mg/kg bw/day, dosage groups either died during study or were sacrificed due to abortion or moribund condition. The remaining five does in each the control and 62.5 mg/kg bw/day groups survived until the day of scheduled sacrifice. Clinical observations included dark urine at the 62.5 and 125 mg/kg bw/day dosages, red substance, mucoid, scant or no faeces in cage pan at 125 mg/kg bw/day and higher dosages, decreased motor activity and labored breathing at 250 mg/kg bw/day and higher dosages nd impaired or lost righting reflex, lacrimation, red substance in vagina and head tilt at the top dose (500 mg/kg bw/day). There were no treatment-related maternal necropsy observations at Caesarean-sectioning. Maternal body weight losses and reduced feed consumption (absolute and relative) were evident at doses at 125 mg/kg bw/day and higher dosages over gestation days 6 to 9, 9 to 12 and 12 to 15, compared to control figures. All rabbits were pregnant, but only 5, 4, 0, 0 and 0 were Caesarean-sectioned on gestation day 29 in the five respective dosage groups due to death, premature delivery, abortion or moribund sacrifice. When compared to control, the number of corpora lutea, implantations, litter sizes early/late resorptions, sex ratio and foetal weights were generally comparable in the 62.5 mg/kg bw/day dosage group. There were no dead foetuses and all live foetuses appeared normal at gross external examination.

From these data it can be concluded, that the highest dosage selected for the key developmental toxicity study in New Zealand White rabbits should exceed 62.5 mg/kg bw/day but be less than 125 mg/kg bw/day of the test substance. The dose levels of 0, 10, 25, 75 and 100 mg/kg bw/day were suggested with the 100 mg/kg bw/day dose being expected to produce maternal toxicity and minimal developmental toxicity, while the 10 mg/kg bw/day is expected to be the NOAEL for maternal and developmental toxicity.

 

In the key developmental toxicity study (M-027442-01-1), conducted under GLP conditions and in accordance with OECD TG 414, 23 presumed pregnant female New Zealand White rabbits per dose were treated with the test item at levels of 0, 10, 25, 75 and 100 mg/kg bw/day via gavage once daily. Administration was performed on days 6 through 28 of presumed gestation. All rabbits were regularly observed for viability, clinical signs, abortions, premature deliveries and deaths. Body weights and food consumption were recorded, too. On gestation day 29, all surviving rabbits were subjected to Caesarean-sectioning and sacrificed afterwards for gross necropsy. Gravid uterine weight was recorded as well as the number of corpora lutea, number and distribution of implants, live and dead foetuses and early/late resorptions. Each foetus was weighed and examined for sex and gross external alterations. Placentae were evaluated for abnormalities in size, shape and colour. Each foetus was weighed and examined for gross external alterations, sex and visceral alterations. For about one half of the foetuses per litter the brain was examined in situ and the remaining half of the foetuses were decapitated, and the heads examined using Wilson’s technique. All foetuses were eviscerated and examined for skeletal alterations.

At the dose levels of 75 and 100 mg/kg bw/day, maternal toxicity was observed, evident as mortality (2/23 and 3/23, respectively). Body weight gain was reduced for the entire dosage period and entire gestation period in the 75 and 100 mg/kg bw/day groups, with statistically significant values for the 100 mg/kg bw/day group when compared to control. Furthermore,  feed consumption (absolute and relative) was also significantly lower in the 75 and 100 mg/kg bw/day dosage groups compared to the control figures. Clinical signs attributable to 25 mg/kg bw/day and higher dosages included scant or no faeces or red substance in the cage pan, orange urine, decrease of motor activity and loss of righting reflex. The test item also induced premature deliveries on gestation day 29 in two does in each of the 75 and 100 mg/kg bw/day groups. Abortions were observed in 3, 1 and 6 cases in the control, 75 and 100 mg/kg bw/day groups, respectively, with the latter being statistically significant when compared to control. In the top dose group also increased postimplantation loss and significantly lower foetal body weights were found. A significant reduction in the litter average for ossified sternal centra per foetus and absent intermediate lung lobes were noted for the 75 and 100 mg/kg bw/day dose level as well as small kidneys at 100 mg/kg bw/day. However, it should be noted, that the absence of intermediate lung lobes observed in the 75 mg/kg bw/day group was within historical control ranges and may therefore be considered unrelated to treatment.

In conclusion, the study is considered reliable and valid and can be used as a key study. The maternal NOAEL was 10 mg/kg bw/day based on clinical signs at 25 mg/kg bw/day. Developmental toxicity characterised primarily by increased post implantation loss, reduced fetal weight and delayed ossification was noted at 100 mg/kg bw/day. These findings were attributed to the severe maternal toxicity at this dose level. The NOAEL for developmental toxicity was 25 mg/kg bw/day based on minor developmental effects (reduced sternal ossification sites) at 75 mg/kg bw/day.

In regard to classification purposes, the effects observed on in utero development are not considered to warrant classification and labelling for developmental toxicity as those effects were only observed in the presence of maternal toxicity and/or were within ranges of historical control data of the testing laboratory. This is in line with the conclusion taken by RAC (RAC opinion, 2021) stating that “none of the developmental effects reported in the CLH report is strong and robust enough for supporting a classification for developmental toxicity

References not included in IUC:

M-027430-02-1, York, R.G.: 1998: Oral (Gavage) Dosage-Range Developmental Toxicity Study of TI-435 in Rats (study report), Testing laboratory: Argus Research Laboratories, Inc., Horsham, Pennsylvania, USA, Report no: 1120-001P, Owner company; Bayer AG, Study number: M-027430-01-1, Report date: Jan 15, 1998

M-027430-02-1, York, R.G.: 1999: Oral (Gavage) Dosage-Range Developmental Toxicity Study of TI-435 in Rats (report amendment), Testing laboratory: Argus Research Laboratories, Inc., Horsham, Pennsylvania, USA, Report no: 1120-001P, Owner company; Bayer AG, Study number: M-027430-02-1, Report date: Nov 11, 1999

M-027436-02-1, York, R.G.: 1998: Oral (Stomach Tube) Dosage-Range Developmental Toxicity Study of TI-435 in Rabbits (study report), Testing laboratory: Argus Research Laboratories, Inc., Horsham, Pennsylvania, USA, Report no: 1120-002P, Owner company; Bayer AG, Study number: M-027436-01-1, Report date: Jan 15, 1998

M-027436-02-1, York, R.G.: 1999: Oral (Stomach Tube) Dosage-Range Developmental Toxicity Study of TI-435 in Rabbits (report amendment), Testing laboratory: Argus Research Laboratories, Inc., Horsham, Pennsylvania, USA, Report no: 1120-002P, Owner company; Bayer AG, Study number: M-027436-02-1, Report date: Nov 11, 1999

RAC opinion, adopted 16 Sept, 2021: Committee for Risk Assessment - RAC - Opinion proposing harmonised classification and labelling at EU level of clothianidin (ISO); (E)-1-(2-chloro-1,3-thiazol-5-ylmethyl)-3-methyl-2-nitroguanidine, EC Number: 433-460-1, CAS Number: 210880-92-5

Mode of Action Analysis / Human Relevance Framework

No data available.

Justification for classification or non-classification

According to the RAC opinion, classification of the test substance for fertility and sexual function as Repr. 2 (H361f) is warranted according to CLP Regulation (EC) No. 1272/2008 mainly based on delayed male sexual maturation supported by alterations in sperm motility. No classification in regard to developmental toxicity is warranted.

Additional information