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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Source, RA-A, CAS 137-26-8, 2-generation reproductive toxicity study (rat, EPA OPP 83-4 similar to OECD 416, GLP, 399-104):

NOAEL (parental general toxicity): 30 ppm (corresponding to approx. 1.5 mg/kg bw/day)
LOAEL (parental general toxicity): 60 ppm (corresponding to approx. 3 mg/kg bw/day)
NOAEL (neonatal toxicity): 60 ppm (corresponding to approx. 3 mg/kg bw/day)
LOAEL (neonatal toxicity): 180 ppm (corresponding to approx. 9 mg/kg bw/day)
NOAEL (reproductive toxicity): 180 ppm (corresponding to approx. 9 mg/kg bw/day, highest dose tested)

After correction for molecular weight, the following NOAELs are set for the target substance (factor 1.23):

NOAEL (parental general toxicity): 30 ppm (corresponding to approx. 1.85 mg/kg bw/day)
LOAEL (parental general toxicity): 60 ppm (corresponding to approx. 3.69 mg/kg bw/day)
NOAEL (neonatal toxicity): 60 ppm (corresponding to approx. 3.69 mg/kg bw/day)
LOAEL (neonatal toxicity): 180 ppm (corresponding to approx. 11.07 mg/kg bw/day)
NOAEL (reproductive toxicity): 180 ppm (corresponding to approx. 11.07 mg/kg bw/day, highest dose tested)

Link to relevant study records

Referenceopen allclose all

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Jul 1989 - 2 Dec 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
adopted in 2001
Deviations:
yes
Remarks:
methodological limitations (missing examinations like anogenital distance, data on physical landmarks in pups and other postnatal developmental data, limited organ weights, and the oestrus cycle)
Qualifier:
according to guideline
Guideline:
EPA OPP 83-4 (Reproduction and Fertility Effects)
Deviations:
yes
Remarks:
Food intake dring mating period was not measured
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD® VAF/Plus®
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI, USA
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 49 days; (F1) x wks
- Weight at study initiation: ♂: 268-310 g; ♀: 176-205 g
- Fasting period before study: not applicable
- Housing: individually in stainless steel, wire-mesh cages, except during mating, gestation and lactation. Females were housed individually in plastic cages containing wood chip bedding during periods of gestation and lactation.
- Diet: Certified Rodent Chow #5002, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16.7 - 26.7
- Humidity (%): 26 - 76
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 Jul 1989 To: 2 Dec 1990
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with (basal diet): A pre-mix was prepared in a Hobart blender and then this pre-mix was added to appropriate amounts of the basal diet.
- Storage temperature of food: frozen until dispensed in daily aliquots

Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 21 days
- Proof of pregnancy: daily check, copulatory plug referred to as day 1 of pregnancy
- Due to poor conception rates in the F1b litter, the F0 parents were mated altogether three times. F1c litter was used to select the F1 parents, which were mated twice to produce the F2a and F2b litters.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test material was analyzed prior to initiation of treatment and after termination of the study and was found to be 97.6 and 97.84%, respectively.

Homogeneity of the test material was also evaluated before initiation of dosing and at study Week 20. Homogeneity was 79 – 84%, 86 – 95% and 89 – 172% of the target dietary concentration for 30, 60 and 180 ppm, respectively. The homogeneity was considered to be sufficient.

Stability of the test material in the diet was investigated for all dietary levels at the time of homogeneity testing. Samples were a) tested immediately, b) held at room temperature for 12 h before extraction, c) frozen for 7 days and then held at room temperature for 12 h before extraction or d) frozen for 12 days and then held at room temperature for 12 h before extraction. Concentration of the test substance in the diet decreased by up to 32% after 12 h at room temperature. However in most test, the test material dietary concentration decreased only by 9-18% after 12 h at room temperature. Freezing did not affect test item concentration in the diet.

Determination of test article concentration in the diet was analyzed from test article containing diets in Week 1 - 4 and every 4 weeks thereafter. The mean percent of target ppm found in all analyzed and administered diets was 93 ± 6.3%, 94 ± 6.6% and 98 ± 5.2% of the target dietary concentration for 30, 60 and 180 ppm, respectively.
Duration of treatment / exposure:
Duration of exposure before mating: F0 / F1 parents: 81/84 days
Duration of exposure in general F0, F1, F2 males, females: Continuously throughout the study
Frequency of treatment:
continously via the diet
Details on study schedule:
Study schedule:

F0 parents received the test diet for 81 days prior to initiation of mating (F1a).

Selection of parents from F1 generation when pups were 21 days of age. F1 parents received the test diet from weaning on for 81 days prior to initiation of mating (F2a). The F1c litters were used for mating F2 litters.
Dose / conc.:
30 ppm
Remarks:
corresponding to an actual ingested dose of 1.52 mg/kg bw/day (F0 males), 2.27 mg/kg bw/day (F0 females), 1.83 mg/kg bw/day (F1 males) and 2.39 mg/kg bw/day (F1 females)
Dose / conc.:
60 ppm
Remarks:
corresponding to an actual ingested dose of 2.94 mg/kg bw/day (F0 males), 4.61 mg/kg bw/day (F0 females), 3.82 mg/kg bw/day (F1 males) and 5.12 mg/kg bw/day (F1 females)
Dose / conc.:
180 ppm
Remarks:
corresponding to an actual ingested dose of 8.88 mg/kg bw/day (F0 males), 13.89 mg/kg bw/day (F0 females), 11.37 mg/kg bw/day (F1 males) and 16.21 mg/kg bw/day (F1 females)
No. of animals per sex per dose:
26
Control animals:
yes, concurrent no treatment
Positive control:
No
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to treatment (F1)/selection (F2), weekly throughout the pre-mating period. Body weights of female rats were determined on Days 0, 7, 14 and 20 of gestation and on Days 0, 4, 7, 14 and 21 of lactation, respectively.

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
Food consumption was recorded daily throughout the study except during the mating period.

WATER CONSUMPTION: No
Oestrous cyclicity (parental animals):
No
Sperm parameters (parental animals):
Parameters examined in all male parental generations:
testis weight, sperm count in epididymides
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Litter size was reduced by random selection to 8 pups of equal sex distribution on lactation day 4. An exception was made for the F1c litter due to too few pups.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring: litter size, stillbirths, live births and gross anomalies. Pups were observed for survival, behavioural abnormalities and dead pups at least twice daily. Such pups were subjected to a gross post mortem examination. Any abnormalities were recorded in pups killed or found dead before discarding.
Body weights of pups were recorded on lactation day 0, 4, 7, 14 and 21.


GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals , F0 generation: three weeks after the completion of the F1c mating period, F1 generation: three weeks after the completion of the F2b mating period.
- Maternal animals: All surviving animals, F0 generation: prior to the date on which it was decided to mate the F0 females a third time, 5, 7, 9 and 5 females of the control, low- mid and high-dose groups, respectively, where sacrificed as they did not produce litter. All other F0 females were euthanized after the selection of the F1 parents. F1 generation: sacrificed and necropsied after F2b litters were weaned.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
- Organ weights: testes
- Tissues: Coagulating gland, ovary, pituitary, prostate, seminal vesicle, testis with epididymis, uterus, cervix, vagina, gross lesions, tissue masses.
- How many animals: All animals from control and high-dose group (gross lesions also from low- and mid-dose group).
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age (following weaning).
- These animals were subjected to postmortem examinations as follows: culled pups for litter adjustment were examined externally and discarded. Any intact pups dying during lactation were necropsied, examined for abnormalities and preserved (10% neutral buffered formalin).

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

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues of gross abnormalities were prepared for microscopic examination and weighed, respectively. No other histopathology was performed.
Statistics:
All statistical analysis was performed to compare the test groups with the control group. Levels of significance were p < 0.05 and p < 0.01. All means were accompanied by standard deviation.

Statistical tests performed were the one-way analysis of variance (ANOVA), Bartlett's test for homogeneity of variance and the appropriate t-test with Dunnett's multiple comparison tables or Bonferroni correction, the chi-square test, Fisher's exact probability test and the Mann-Whitney U-test.
Reproductive indices:
Male and female viability indices were calculated.
Offspring viability indices:
Pup survival indices were calculated.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Treated F0 females showed an increased incidence of hair loss in a non-dose-related pattern.

Summarized results can be found in Attachment 1 in the attached background material.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two deaths occurred in the F0 generation but were not regarded as treatment-related.

Summarized results can be found in Attachment 1 in the attached background material.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant and treatment-related reductions in body weight in F0 females were observed during the F1a gestation period in the mid- and high-dose group. During the F1b and F1c gestation periods, reductions in body weight were observed in the high-dose group only.

Summarized results can be found in Attachment 1 in the attached background material.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reductions were noted for the F0 males at the high-dose level for three weeks and at the mid-dose level for two weeks. Food consumption of F0 females in the mid-dose group was reduced for one week and for several weeks in the high-dose group.
Dose-related reductions were additionally noted for F0 females in the mid- and high-dose group throughout the F1a gestation period. This treatment-related reduction continued throughout the F1b and, without reaching statistically significance, in the F1c gestation period in the high-dose group.

Summarized results can be found in Attachment 1 in the attached background material.
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 specified
Description (incidence and severity):
The following ED-related parameters were investigated in the study: histopathology of cervix, coagulating gland, epididymis, prostate, seminal vesicles, testis, thyroid, pituitary, uterus and vagina, organ weights were recorded for liver, coagulating gland, epididymis, prostate, seminal vesicles, uterus, testis and brain. Gestation length, litter size, viability, weight and number of implantation sites and live births were investigated as well as pre- and post-implantation loss, presence of anomalities, pup development and sex ratio. For details, please refer to the respective result fields and the endpoint summary.
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:
no effects observed
Description (incidence and severity):
All microscopic changes seen in the rats were considered to be spontaneous or agonal.

Summarized results can be found in Attachment 1 in the attached background material.
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:
not examined
Description (incidence and severity):
not applicable
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Sperm were present, motile and morphologically normal.

Summarized results can be found in Attachment 1 in the attached background material.
Reproductive performance:
no effects observed
Description (incidence and severity):
No treatment-related differences in the male and female copulatory and fertility indices, the gestation index, the mean copulatory interval or the mean gestation length were observed.

Summarized results can be found in Attachment 1 in the attached background material.
Key result
Dose descriptor:
NOAEL
Remarks:
parental general toxicity
Effect level:
30 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect was observed at this dose level.
Remarks on result:
other: corresponding to an actual ingested dose of approx. 1.5 mg/kg bw/day.
Key result
Dose descriptor:
LOAEL
Remarks:
parental general toxicity
Effect level:
60 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 an actual ingested dose of aprrox. 3 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Remarks:
reprotoxicity
Effect level:
180 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed up to and including the highest dose level.
Remarks on result:
other: corresponding to an actual ingested dose of approx. 9 mg/kg bw/day.
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
In F1 animals no clinical signs of toxicity were observed.

Summarized results can be found in Attachment 1 in the attached background material.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two deaths occurred in the F1 generation but were not regarded as treatment-related.

Summarized results can be found in Attachment 1 in the attached background material.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Decreases in the mean weekly body weight were observed in the F1 generation at the high dose level during the initial 10 weeks (males) or 13 weeks (females). These decreases were consistent with a reduced growth observed in these animals.
Statistically significant reductions were additionally noted for high-dose females during the F2a and F2b gestation period.

Summarized results can be found in Attachment 1 in the attached background material.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
During the F2a and F2b gestation and lactation period food consumption was significantly and treatment-related reduced in the high-dose group.

Summarized results can be found in Attachment 1 in the attached background material.
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:
no effects observed
Description (incidence and severity):
There was no treatment-related effect on testes weight in the F1 generation males.

Summarized results can be found in Attachment 1 in the attached background material.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All macroscopic changes seen in the rats were considered to be spontaneous or agonal.

Summarized results can be found in Attachment 1 in the attached background material.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
All microscopic changes seen in the rats were considered to be spontaneous or agonal.

Summarized results can be found in Attachment 1 in the attached background material.
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:
not examined
Description (incidence and severity):
not applicable
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Sperm were present, motile and morphologically normal.

Summarized results can be found in Attachment 1 in the attached background material.
Reproductive performance:
no effects observed
Description (incidence and severity):
No treatment-related differences in the male and female copulatory and fertility indices, the gestation index, the mean copulatory interval or the mean gestation length were observed.

Summarized results can be found in Attachment 1 in the attached background material.
Key result
Dose descriptor:
NOAEL
Remarks:
parental general toxicity
Effect level:
60 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed at this dose level.
Remarks on result:
other: corresponding to an actual ingested dose of approx. 3 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Remarks:
parental systemic toxicity
Effect level:
180 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 an actual ingested dose of approx. 9 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Remarks:
reprotoxicity
Effect level:
180 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed up to and including the highest dose level.
Remarks on result:
other: corresponding to an actual ingested dose of approx. 9 mg/kg bw/day
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
No dose-related trends were observed in the type and incidence of clinical observations in treated F1 offspring when compared to the untreated controls.

Summarized results can be found in Attachment 1 in the attached background material.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The mean number of stillborn and live pups in the treated groups was comparable with the one of the control litters at all dose levels.

Summarized results can be found in Attachment 1 in the attached background material.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean offspring body weights were consistently reduced to a significant degree in all litters at the 180 ppm level.

Summarized results can be found in Attachment 1 in the attached background material.
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):
No dose-related trends were observed in the type and incidence of clinical observations in treated F1 offspring when compared to the untreated controls.

Summarized results can be found in Attachment 1 in the attached background material.
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
Remarks:
neonatal toxicity
Generation:
F1
Effect level:
60 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed at this dose level.
Remarks on result:
other: corresponding to an actual digested dose of approx. 3 mg/kg bw/day.
Key result
Dose descriptor:
LOAEL
Remarks:
neonatal toxicity
Generation:
F1
Effect level:
180 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: corresponding to an actual digested dose of approx. 9 mg/kg bw/day.
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
No dose-related trends were observed in the type and incidence of clinical observations in treated F2 offspring when compared to the untreated controls.

Summarized results can be found in Attachment 1 in the attached background material.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The mean number of stillborn and live pups in the treated groups was comparable with the one of the control litters at all dose levels.

Summarized results can be found in Attachment 1 in the attached background material.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean offspring body weights were consistently reduced to a significant degree in all litters at the 180 ppm level.

Summarized results can be found in Attachment 1 in the attached background material.
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:
no effects observed
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):
No dose-related trends were observed in the type and incidence of clinical observations in treated F1 offspring when compared to the untreated controls.

Summarized results can be found in Attachment 1 in the attached background material.
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
Remarks:
neonatal toxicity
Generation:
F2
Effect level:
60 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed at this dose level.
Remarks on result:
other: corresponding to an actual digested dose of approx. 3 mg/kg bw/day.
Key result
Dose descriptor:
LOAEL
Remarks:
neonatal toxicity
Generation:
F2
Effect level:
180 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: corresponding to an actual digested dose of approx. 9 mg/kg bw/day.
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
The study was conducted similar to OECD guideline 416 and under GLP conditions. Several examinations are missing when the study protocol is compared to the current guideline, but the study was considered valid.

In conclusion, parental toxicity in the F0 and F1 generations was exhibited at a concentration of 60 and 180 ppm, respectively, by inhibition of body weight gain and reduced food consumption. No parental systemic toxicity was observed at concentrations of 30 ppm in the Fo or at 60 ppm in the F1 generation.
Reproductive performance was unaffected by test article administration at the 30, 60 and 180 ppm. Neonatal toxicity was expressed at a concentration of 180 ppm by reduced pup body weights (F1 and F2). No neonatal toxicity was observed at concentrations of 30 and 60 ppm. Based on the results of this study, in the P0 generation, a concentration of 30 ppm was considered to be the NOAEL (no observable adverse effect level) for parental systemic toxicity (corresponding to approx. 1.5 mg/kg bw/day), 60 ppm was considered to be the NOAEL for systemic toxicity in the P1 generation and for neonatal toxicity (corresponding to approx. 3 mg/kg bw/day) and 180 ppm was considered to be the NOAEL for reproductive and developmental neurotoxicity (corresponding to approx. 9 mg/kg bw/day).
Endpoint:
two-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Remarks:
parental general toxicity
Effect level:
30 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed at this dose level.
Remarks on result:
other: target substance, corresponding to 1.85 mg/kg bw/day after correction for molecular weight (factor 1.23).
Key result
Dose descriptor:
LOAEL
Remarks:
general parental toxicity
Effect level:
60 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: target substance, corresponding to 3.69 mg/kg bw/day after correction for molecular weight (factor 1.23).
Key result
Dose descriptor:
NOAEL
Remarks:
reprotoxicity
Effect level:
180 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed up to and including the highest dose level.
Remarks on result:
other: target substance, corresponding to 11.07 mg/kg bw/day after correction for molecular weight (factor 1.23).
Key result
Dose descriptor:
NOAEL
Remarks:
parental general toxicity
Effect level:
30 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect was observed at this dose level.
Remarks on result:
other: source substance, corresponding to approx. 1.5 mg/kg bw/day.
Key result
Dose descriptor:
LOAEL
Remarks:
parental general toxicity
Effect level:
60 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: source substance corresponding to aprrox. 3 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Remarks:
reprotoxicity
Effect level:
180 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed up to and including the highest dose level.
Remarks on result:
other: source substance, corresponding to approx. 9 mg/kg bw/day.
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Remarks:
parental general toxicity
Effect level:
60 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed at this dose level.
Remarks on result:
other: target substance, corresponding to 3.69 mg/kg bw/day after correction for molecular weight (factor 1.23).
Key result
Dose descriptor:
LOAEL
Remarks:
parental general toxicity
Effect level:
180 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: target substance, corresponding to 11.07 mg/kg bw/day after correction for molecular weight (factor 1.23).
Key result
Dose descriptor:
NOAEL
Remarks:
reprotoxicty
Effect level:
180 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed up to and including the highest dose level.
Remarks on result:
other: target substance, corresponding to 11.07 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Remarks:
parental general toxicity
Effect level:
60 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed at this dose level.
Remarks on result:
other: source substance, corresponding to approx. 3 mg/kg bw/day.
Key result
Dose descriptor:
LOAEL
Remarks:
parental general toxicity
Effect level:
180 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: source substance, corresponding to approx. 9 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Remarks:
reprotoxicity
Effect level:
180 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed up to and including the highest dose level.
Remarks on result:
other: source substance, corresponding to approx. 9 mg/kg bw/day.
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Remarks:
neonatal toxicity
Generation:
F1
Effect level:
60 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed at this dose level.
Remarks on result:
other: target substance, corresponding to 3.69 mg/kg bw/day after correction for molecular weight (factor 1.23).
Key result
Dose descriptor:
LOAEL
Remarks:
neonatal toxicity
Generation:
F1
Effect level:
180 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: target substance, corresponding to 11.07 mg/kg bw/day after correction for molecular weight (factor 1.23).
Key result
Dose descriptor:
NOAEL
Remarks:
neonatal toxicity
Generation:
F1
Effect level:
60 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed at this dose level.
Remarks on result:
other: source substance, corresponding to approx. 3 mg/kg bw/day.
Key result
Dose descriptor:
LOAEL
Remarks:
neonatal toxicity
Generation:
F1
Effect level:
180 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: source substance, corresponding to approx. 9 mg/kg bw/day.
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Remarks:
neonatal toxicity
Generation:
F2
Effect level:
60 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed at this dose level.
Remarks on result:
other: target substance, corresponding to an actual digested dose of approx. 3.69 mg/kg bw/day.
Key result
Dose descriptor:
LOAEL
Remarks:
neonatal toxicity
Generation:
F2
Effect level:
180 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: target substance, corresponding to an actual digested dose of approx. 11.07 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Remarks:
neonatal toxicity
Generation:
F2
Effect level:
60 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed at this dose level.
Remarks on result:
other: source substance, corresponding to an actual digested dose of approx. 3 mg/kg bw/day.
Key result
Dose descriptor:
LOAEL
Remarks:
neonatal toxicity
Generation:
F2
Effect level:
180 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: source substance, corresponding to an actual digested dose of approx. 9 mg/kg bw/day.
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Based on the read-across approach, a NOAEL for developmental toxicity of 11.1 mg/kg bw/day is derived by correcting the NOAEL of 9 mg/kg bw/day for thiram with the correction factor of 1.23 for molecular weight.

Conclusions:
The study was conducted with the source substance similar to OECD guideline 416 and under GLP conditions. Several examinations are missing when the study protocol is compared to the current guideline, but the study was considered valid.
In conclusion, parental toxicity in the F0 and F1 generations was exhibited at a concentration of 60 and 180 ppm, respectively, by inhibition of body weight gain and reduced food consumption. No parental systemic toxicity was observed at concentrations of 30 ppm in the Fo or at 60 ppm in the F1 generation.
Reproductive performance was unaffected by test article administration at the 30, 60 and 180 ppm. Neonatal toxicity was expressed at a concentration of 180 ppm by reduced pup body weights (F1 and F2). No neonatal toxicity was observed at concentrations of 30 and 60 ppm.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
11.1 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Toxicity to reproduction of the source substance was assessed in a 2-generation study conducted with the source substance in rats similar to OECD TG 416 (adopted 2001), under GLP conditions (RL2). 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

Due to the lack of available data, the potential of disulfiram to affect fertility is assessed using read-across from studies conducted with a surrogate substance, thiram (CAS 137 -26 -8). After correction for molecular weight (factor: 1.23) the NOAELs for thiram are used as NOAELs for disulfiram. (For detailed information on the justification of read-across, please refer to the analogue justification document attached in IUCLID section 13).

 

The reproductive toxicity of the read across source substance was assessed at dietary concentrations of 30, 60 and 180 ppm in Sprague Dawley rats (26 animals/sex/group) for two generations. The dietary concentrations corresponded to actual ingested doses of approx. 3, 6 and 9 mg/kg bw/day. Parental animals received the treatment for 81 days prior to mating. The F0 parents were mated two times to produce the F1a and F1b litters. Since conception rates were poor across all groups, including the controls, in the F1b litter, a third mating was held to produce an F1c litter. From the F1c litters, 26 male and 26 female rats were selected to become F1 parents. These animals were treated beginning at 22 days of age for a minimum of 84 days prior to their initial (F2a) mating. The F1 parents were mated twice to produce the F2a and F2b litters. At the parental level (F0 and F1), there were no treatment-related findings concerning mortality, antemortem observations and reproductive parameters.

Food consumption and body weight were statistically significantly decreased in the 60 and 180 ppm groups of both parental generations, mainly during gestation. Reductions in mean maternal body weight and/or food consumption were also observed at 180 ppm during the F1b and F1c gestation period and the F1a, F1b and F1c lactation periods. Similar reductions were observed for the F1 females at 180 ppm during the F2a and F2b gestation and lactation periods. Reductions in mean weekly food consumption of the F0 males and females were observed at 60 and 180 ppm. Thus, the no observable effect level with respect to parental systemic toxicity was judged to be 30 ppm for the F1a mating only and 60 ppm for all subsequent matings.

With regard to the litters, there were no treatment-related findings concerning the number of stillborn pups, survival indices, necropsy findings or malformations in both generations. Mean body weight was lower in the high-dose group in both generations. Erratic changes in pup body weights in the low-dose group without significant changes in the mid-dose group ruled out a dose response for this parameter. Thus, the no observable effect level with respect to filial systemic toxicity was judged to be 60 ppm and a NOAEL of > 180 ppm was established with respect to developmental toxicity.

No treatment-related differences in the male and female copulatory and fertility indices, the gestation index, the mean copulatory interval or the mean gestation length were observed for the F0 parents at the F1a, F1b or F1c matings and for the F1 parents at the F2a or F2b matings. Sperm were present, motile, and morphologically parents at the normal for all F0 and F1 males that underwent spermatogenesis evaluation. Thus the no observable effect level with respect to reproductive parameters was judged to be 180 ppm.

Under the conditions of the study, the NOAEL with respect to parental systemic toxicity was considered to be 30 ppm (corresponding to 1.52 mg/kg bw/day in male and 2.27 mg/kg bw/day in female) for the F1a mating and 60 ppm (corresponding to 3.82 mg/kg bw/day in male and 5.12 mg/kg bw/day in female) for all subsequent matings. With respect to reproductive parameters and developmental toxicity, the NOAEL was considered to be >180 ppm (corresponding to 8.88 mg/kg bw/day in F0 male and 13.89 mg/kg bw/day in F0 female and to 11.37 mg/kg bw/day in parental male and 16.21 mg/kg bw/day in parental female).

 

Based on the read-across from thiram, a NOAEL for reproductive toxicity of 11.1 mg/kg bw/day is derived by correcting the NOAEL of 9 mg/kg bw/day for thiram with the correction factor of 1.23 for molecular weight.

 

 An assessment on endocrine disruption was performed during the CoRAP substance evaluation of Thiram. The outcome of this evaluation in 2018 was that Thiram has no ED concern. 

Effects on developmental toxicity

Description of key information

Source, RA-A, CAS 137-26-8, Developmental toxicity (rat, EPA OPP 83-3 similar to OECD 414, GLP, 87/TRK002/179):

NOAEL (maternal general and developmental toxicity): <7.5 mg/kg bw/day

LOAEL (maternal general and developmental toxicity): 7.5 mg/kg bw/day

NOAEL (fetal developmental toxicity): 7.5 mg/kg bw/day

LOAEL (fetal developmental toxicity): 15 mg/kg bw/day

After correction for molecular weight, the following NOAELs are set for the target substance (factor 1.23):

NOAEL (maternal general and developmental toxicity): <9.23 mg/kg bw/day

LOAEL (maternal general and developmental toxicity): 9.23 mg/kg bw/day

NOAEL (fetal developmental toxicity): 9.23 mg/kg bw/day

LOAEL (fetal developmental toxicity): 18.45 mg/kg bw/day

 

Source, RA-A, CAS 137-26-8, Developmental toxicity (rabbit, EPA OPP 83-3 similar to OECD 414, GLP, 87/TRK004/541):

NOAEL (maternal general toxicity): 2.5 mg/kg bw/day

LOAEL (maternal general toxicity): 5 mg/kg bw/day

NOAEL (maternal and fetal developmental toxicity): > 5 mg/kg bw/day (highest dose level tested)

 After correction for molecular weight, the following NOAELs are set for the target substance (factor 1.23):

NOAEL (maternal general toxicity): 3.1 mg/kg bw/day

LOAEL (maternal general toxicity): 6.15 mg/kg bw/day

NOAEL (maternal and fetal developmental toxicity): > 6.15 mg/kg bw/day (highest dose level tested)

 

Source, RA-A, CAS 137-26-8, Developmental toxicity (rabbit, EPA OPP 83-3 similar to OECD 414, GLP, 399-121):

NOAEL (maternal general toxicity): > 10 mg/kg bw/day (highest dose level tested)

NOAEL (maternal developmental toxicity): > 10 mg/kg bw/day (highest dose level tested)

NOAEL (fetal developmental toxicity): 5 mg/kg bw/day

LOAEL (fetal developmental toxicity): 10 mg/kg bw/day

 After correction for molecular weight, the following NOAELs are set for the target substance (factor 1.23):

NOAEL (maternal general toxicity): > 12.3 mg/kg bw/day (highest dose level tested)

NOAEL (maternal developmental toxicity): > 12.3 mg/kg bw/day (highest dose level tested)

NOAEL (fetal developmental toxicity): 6.15 mg/kg bw/day

LOAEL (fetal developmental toxicity): 12.3 mg/kg bw/day

 

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 May - 21 Jun 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted in 2018
Deviations:
yes
Remarks:
methodological limitations (no examination of anogenital distance of fetuses)
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Hazleton Research Products, Inc., Kalamazoo, Michigan, USA
- Age at study initiation: 22 - 23 weeks
- Weight at study initiation: 3293 - 4238 g
- Fasting period before study: not applicable
- Housing: individually in suspended, stainless steel, wire mesh cages
- Diet: basal laboratory diet of Certified Rabbit Chow #5322 (Purina Mills, Inc., Missouri, USA), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 43 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 14.4 - 21.1
- Humidity (%): 46 - 89
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 8 Apr 1991 To: 21 Jun 1991 (insemination: 21 May 1991)
Route of administration:
oral: gavage
Vehicle:
other: Suspension of 0.5% Tween 80 and 0.5% low-viscosity carboxy-methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The appropriate amount of the test material was suspended in the vehicle. Individual dosages were determined from the most recently recorded individual body weight.

VEHICLE
- Concentration in vehicle: approx. 0.355 mg/mL, 1.80 mg/mL; 3.57 mg/mL for 1, 5 and 10 mg/kg bw/day
- Amount of vehicle: 3 mL
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability, homogeneity and test material concentration of the suspension were tested and considered satisfactory.
Details on mating procedure:
- Impregnation procedure: artificial insemination (no mating period)
- Sperm donors: eight proven male stock rabbits of the same breed an source
- Insemination: Semen was evaluated for motility (> 60 %), Within one hour after insemination, ovulation was induced by injection oh human chorionic gonadotropin
- The day of insemination was designated as Gestation Day 0.
Duration of treatment / exposure:
Day 7-19 post insemination
Frequency of treatment:
Daily
Duration of test:
Animals were sacrificed on Gestation Day 29
Dose / conc.:
1 mg/kg bw/day
Dose / conc.:
5 mg/kg bw/day
Dose / conc.:
10 mg/kg bw/day
No. of animals per sex per dose:
20 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were selected in a range finding study (Rep.No. 87-TRK004-541). Dosage levels of 1, 2.5 and 5 mg/kg bw/day were administered to 3 groups of 15 rabbits each, Significantly reduced body weight was observed during administration of 5 mg/kg bw/day. No treatment-related mortalities or clinical observations were observed in any treatment group. Cesarean section parameters and fetal morphology were comparable between the control and treatment groups.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: mortality twice daily, once daily for clinical signs on Days 7 - 29 of gestation

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 7, 13, 20, 24 and 29 of gestation.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Days 0-7, 7-13, 13-20, 20-24, 24-29, 7-20, and 0-29 of gestation

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: gravid uterus weight

Females not surviving to scheduled termination were necropsied. The fetuses from these does and maternal tissues were preserved in 10% neutral buffered formalin for possible histopathological examination.
At scheduled necropsy the abdominal and thoracic cavities and organs were examined for gross-pathological changes. Tissues of lesions were preserved for possible histopathological examination. Uterus from rabbits appeared to be non-gravid were examined for implantations.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Blood sampling:
- Plasma: No
- Serum: No
Fetal examinations:
- External examinations: Yes: all per litter
Each foetus was individually identified, weighed, sexed externally and given a gross examination for external malformations / variations including the head by multiple coronal slices. The heart was dissected.
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
After the visceral inspection, the foetuses were eviscerated and processed for staining of the ossified skeletal structures using the Alizarin Red S staining procedure.
- Head examinations: Yes: all per litter
- Anogenital distance of all live rodent pups: no
Statistics:
All statistical analyses compared the treatment groups with the control, with levels of significance at p < 0.05 and p < 0.01. Mean maternal body weights and body weight changes, mean food consumption, mean number of corpora lutea, total implantations, live foetuses and gravid uterine weights were compared by analysis of variance (one-way), Bartlett´s test for homogeneity of variance and the appropriate t-test as described by Steel and Torrie usinf Dunnett´s multiple comparison tables or pair wise comparisons with a Bonferroni correction to determine the significance of differences. Sex ratios and litter proportions were compared using Chi-square test and/or Fisher´s exact test.

Proportions of resorbed and dead foetuses and implantation losses were compared by the Kruskal-Wallis test. If the test was statistically significant (p < 0.05), then Dunn´s method of multiple comparisons was used.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related effects were noted in the animals surviving to scheduled necropsy.
The two control animals that died had reduced activity, loss of righting reflex, laboured breathing, abnormal vocalisation and/or no stool.
Prior to death the high-dose animal was observed with labored breathing, loss of righting reflex, red anogenital staining and prostration. As discussed below, the cause of such findings was considered to be a gavage injury.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
mortality observed, treatment-related
Description (incidence):
Mortalities were observed in the control (two) and high-dose (one) groups. The two animals in the control group died on gestation day 14 due to pneumonia. Prior to death, these rabbits showed reduced activity, loss of righting reflex, laboured breathing, abnormal vocalization and decreased defecation. The high-dose animal died on day 21 of gestation showed laboured breathing, loss of righting reflex, red anogenital staining and prostration. Necropsy revealed a scar in the oesophagus and a skeletal muscle abscess. The cause of death was considered to be a gavage injury due to dosing errors.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Statistical significant increase in body weight gain was noted in all treatment groups as compared to control animals. However, this effect was not considered test-substance related.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Statistical significant increase of food consumption was noted in all treatment groups as compared to control animals. However, this effect was not considered test- substance related.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
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 specified
Description (incidence and severity):
The following ED-related parameters were investigated in the study: gravid uterus weight, gestation length, litter size and litter/pup weight, number of implantations, corpera lutea, viable fetuses, pre- and post-implantation loss, presence of anomalities and sex ratio. For details, please refer to the respective result fields and the endpoint summary.
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):
Uterine weights of treated does were slightly increased when compared to control group. The increase was not considered an adverse effect of treatment.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related gross pathological findings were observed at necropsy.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable
Number of abortions:
no effects observed
Description (incidence and severity):
No treatment-related adverse effects were observed.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No treatment-related adverse effects were observed.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No treatment-related adverse effects were observed. Single whole-litter resorptions were observed in the low- and high-dose group.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Early or late resorptions:
no effects observed
Description (incidence and severity):
No treatment-related adverse effects were observed.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Dead fetuses:
no effects observed
Description (incidence and severity):
No treatment-related adverse effects were observed. Dead fetuses totaled 2, 2 and 1 in the control, low- and high-dose group, respectively.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
No treatment-related adverse effects were observed.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
No treatment-related adverse effects were observed.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Other effects:
not examined
Description (incidence and severity):
not applicable
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
>= 10 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No adverse effects were observed up to and including the highest dose level.
Key result
Dose descriptor:
NOAEL
Remarks:
maternal developmental toxicity
Effect level:
>= 10 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed at the highest dose level tested.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
No treatment-related adverse effects were observed.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No treatment-related adverse effects were observed.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No treatment-related effects were observed.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No treatment-related adverse effects were observed.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
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:
no effects observed
Description (incidence and severity):
No treatment-related adverse effects were observed. Most variations occurred at low incidences or at rates generally comparable to control.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Malformations:
No treatment-related differences in the proportions of litters with malformations were observed among the different test group. Malformations observed in the study either occured at low incidences or at rates that are comparable to those of the control group.

Variations:
Most variations occurred at low incidences or at rates generally comparable to control. However, there was a marked increase in the number of foetuses with 27 presacral vertebrae in the high-dose group when compared to control. This number was, however, within the IRDC historical control range and is not clearly dose related. Thus, the toxicological relevance of this finding remains unclear, especially as the historical control data indicate that the effect on presacral vertebrae may be covered by biological variation in this rabbit strain. Therefore, the toxicological relevance of this finding is equivocal. However, following a conservative approach, this variation was considered for the derivation of the LOAEL.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Visceral malformations:
no effects observed
Description (incidence and severity):
No treatment-related adverse effects were observed. Most variations occurred at low incidences or at rates generally comparable to control.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Other effects:
not examined
Description (incidence and severity):
not applicable
Key result
Dose descriptor:
NOAEL
Remarks:
fetotoxicity
Effect level:
5 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at this dose level.
Key result
Dose descriptor:
LOAEL
Remarks:
fetotoxicity
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: skeletal variation (increased number of fetuses with 27 presacral vertebrae)
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: vertebra
Description (incidence and severity):
At 10 mg/kg bw/day, an increased incidence of greater presacral vertebra was observed (considered as variation). However, the incidence is within the IRDC historical control range and is not clearly dose related, thereby indicating rather an incidental finding covered within the biological variation.
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
10 mg/kg bw/day
Treatment related:
not specified
Relation to maternal toxicity:
not specified
Dose response relationship:
no
Relevant for humans:
no

Maternal/foetal effects

Endpoint/dose   0   1 mg/kg bw/d   5 mg/kg bw/d   10 mg/kg bw/d    Historical control data
Dams data           
number gravid females  19  19  17  19  811
mortality  2   0 0  1  30 
food intake    ↑ (d7 -20)  ↑ (d7 -13)  NS  -
body weight gain    ↑ (d7 -13)  ↑ (d7 -13)  ↑ (d7 -13)  - 
uterine weight [mean in g] 392.2  470.7  417.8  478.0  - 
Foetal observation           
Greater than normal (26) presacral vertebrae [% incidence]  13.5 13.3  11.5  25.6 20.9 
dead foetuses [total number] 2  2  0  1   -

NS = not significant

Conclusions:
The current study was performed under GLP conditions and conducted similar to OECD TG 414 (adopted 2018) with some restrictions, such as no examination of anogenital distance of fetuses. Nevertheless, the study is reliable and valid.
According to these results, it is concluded that a dose level of >10 mg/kg bw/day is the No Observed Adverse Effect Level (NOAEL) for maternal systemic and developmental toxicity. The NOAEL for developmental toxicity is 5 mg/kg bw/day based on the increased incidence of foetuses with 27 presacral vertebra. Although this effect is considered a variations, and the incidence is in the range of historical control data, the increased incidence of foetuses with 27 presacral vertebrae was considered to define the LOAEL following a conservative approach. However, the observed skeletal variation of a supernumerary vertebra does not trigger classification for developmental toxicity.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 Jan - 29 Jun 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted in 2018
Deviations:
yes
Remarks:
methodological limitations (missing examinations, such as anogenital distance, blood sampling)
Qualifier:
according to guideline
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Protocol not fully in compliance with method B of the Directive 87/302/EEC
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Ranch Rabbits, Crawley Dawn, Sussex, England
- Age at study initiation: 16-24 weeks on arrival
- Weight at study initiation: 3.74 – 4.84 kg
- Fasting period before study: not applicable
- Housing: individually in galvanised steel caging with mesh floors.
- Diet: laboratory animal diet, S.Q.C. Standard Rabbit Diet (Special Diet Services Limited, Witham, England), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 23
- Humidity (%): 40 - 70
- Air changes (per hr): 17 - 20
- Photoperiod (hrs dark / hrs light): 14/10

IN-LIFE DATES: From: 6 Jan 1987 To: 24 Apr 1987
Route of administration:
oral: gavage
Vehicle:
other: Suspension of 0.5 % w/v aqueous CMC containing 0.5 % w/v Tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The appropriate amount of the test material was suspended in the vehicle. Individual dosages were determined from the most recently recorded individual body weight.

VEHICLE
- Concentration in vehicle: 0.02, 0.05, 0.1% (w/v) for 1, 2.5 and 5 mg/kg bw/day, respectively
- Amount of vehicle: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability, homogeneity and test material concentration of the suspension were tested and considered satisfactory.
Details on mating procedure:
No mating period. Females were artificially inseminated with semen from New Zealand White bucks of established fertility.
Duration of treatment / exposure:
Day 6 - 19 post insemination
Frequency of treatment:
Daily from Day 6 - 19 post insemination
Duration of test:
Sacrificed on Day 29 of gestation
Dose / conc.:
1 mg/kg bw/day
Dose / conc.:
2.5 mg/kg bw/day
Dose / conc.:
5 mg/kg bw/day
No. of animals per sex per dose:
15-20 pregnant females
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: once daily

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: Reproductive tract
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: placenta weight
Blood sampling:
- Plasma: No
- Serum: No
Fetal examinations:
- External examinations: Yes (including weight of individual foetuses, weight of individual placentae, external abnormalities): all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
After the visceral inspection, the foetuses were eviscerated and processed for staining of the ossified skeletal structures using the Alizarin Red S staining procedure.
- Head examinations: Yes: all per litter
- Anogenital distance of all live rodent pups: No
Statistics:
Significance of intergroup differences was tested using appropriate tests:

Multiple t-test or t-test: Bw, bw change, foetal weight, placental weight, litter size

Mann Whitney U-test: Corpora lutea, implantation and resporption count

Chi2 -test, Fishers Exact Probability test or Mann-U-test: Pre- or post-implantation loss.
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related effects were noted in the animals surviving to scheduled necropsy.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
mortality observed, treatment-related
Description (incidence):
- Control: 1/18 females
- 1 mg/kg bw/day: 1/15 females
- 2.5 mg/kg bw/day: 2/18 females
- 5 mg/kg bw/day: 4/20 females

Necropsy findings for the 8 decedents revealed evidence of respiratory or gastro-intestinal tract disorder, or accidental tracheal intubation.
Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The control group exhibited unusually poor pattern of body weight gain, especially due to 2 females. Therefore supplementary data from a comparable study control group (1A) was provided.

Body weight gain in low- and mid-dose group was superior to control group but essentially similar to supplementary control group. In the high-dose group, body weight gain was similar to control but inferior to supplementary control group. Hence, the possibility of an adverse effect on body weight gain due to treatment cannot be entirely excluded.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
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
Endocrine findings:
not specified
Description (incidence and severity):
The following ED-related parameters were investigated in the study: gravid uterus weight, gestation length, litter size and litter/pup weight, number of implantations, corpera lutea, viable fetuses, pre- and post-implantation loss, presence of anomalities and sex ratio. For details, please refer to the respective result fields and the endpoint summary.
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
not applicable
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related macroscopic changes were observed.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
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
Number of abortions:
no effects observed
Description (incidence and severity):
One female in each of control and high-dose group aborted. This was not considered treatment-related.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
One mid- and high-dose female exhibited total litter resorption loss. This was not considered as treatment related. Based on the low incidence, total litter loss is rather considered as incidental.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Early or late resorptions:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Dead fetuses:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Other effects:
no effects observed
Description (incidence and severity):
Placenta weights were comparable among groups.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
2.5 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No adverse effects were observed at this dose level.
Key result
Dose descriptor:
LOAEL
Remarks:
general toxicity
Effect level:
5 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Remarks:
maternal developmental toxicity
Effect level:
> 5 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed indicating maternal toxicity up to the highest dose tested.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed.

Summarized results can be found in Table 1 and Attachment 1 in the attached background material.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed.

Summarized results can be found in Attachment 1 in the attached background material.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed.

Summarized results can be found in Attachment 1 in the attached background material.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed.

Summarized results can be found in Attachment 1 in the attached background material.
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):
An apparent marginal increase in the incidence of small foetuses was observed at necropsy for females dosed with 1 and 5 mg/kg bw/day, compared with the concurrent controls, but the values were within the laboratory background control range (mean 13.6 % incidence, range 0 -32.3 %).

The apparent marginal increase in small foetuses was considered to be unrelated to treatment in view of the absence of dose- effect relationship or any corresponding reduction in group mean foetal weight.

Summarized results can be found in Attachment 1 in the attached background material.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed.

Summarized results can be found in Attachment 1 in the attached background material.
Visceral malformations:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Following freehand serial sectioning, a slight reduction was seen, in group 5 mg/kg bw/d, in the incidence of foetuses with both incisors erupted, and a corresponding increase in the incidence of foetuses with only the lower incisors erupted. Any treatment related effect was unlikely because of absence of correlation with foetal weight and because of the background control data.

Summarized results can be found in Attachment 1 in the attached background material.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: no effects
Key result
Dose descriptor:
NOAEL
Remarks:
fetotoxicity
Effect level:
> 5 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed up to the highest dose level.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Summarized results:

Table 1:         Teratogenicity study by the oral route in the rabbit.

Endpoint/dose (mg/kg bw/day)

0

1

2.5

5

 

Dams data:

 

 

          Body weight change         GD 0-6

                                                       GD 6-16

                                                       GD 6-28

0.08

0.12

0.14

0.11

0.18

0.19

0.12

0.15

0.26

0.11

0.07*

0.15

 

          Total number inseminated

18

15

18

20

 

          Mortality

1

1

2

4

 

          Not pregnant

5

1

3

2

 

          Abortion

1

0

0

1

 

          Number gravid females/group

13

14

15

18

 

          Total litter loss

0

0

1

1

 

          Pregnant at term with viable young

11

13

12

12

 

          Corpora lutea count

11.3

11.8

12.3

11.4

 

          Implantations

9.8

10.8

9.7

10.1

 

          Implantation loss pre- (%)

13.6

9.1

21.6

14.2

 

          Implantation loss post- (%)

9.3

10

14.7

12.4

 

          Early resorption

0.1

0.4

0.6

0.1

 

          Late resorption

0.8

0.7

0.8

1.2

 

          Placenta weight [mean in g]

5.1

5.3

5.5

5.4

 

          Foetal weight [mean in g]

38.2

38.2

39.2

37.3

 

Foetal observations

no toxic effects

 

          Incisor erupted

73.3(9)

67.5 (9)

70 (10)

54 (10)

 

          Lower incisor only erupted

3.3 (1)

7.5 (2)

3.3 (2)

20 (5)

 

 

 

 

 

 

 

 

Conclusions:
The current study was performed under GLP conditions and conducted similar to OECD TG 414 (adopted 2018) with some restrictions due to methodological limitations (missing examination of anogenital distance of fetuses,no blood sampling for analysis of T4, T3 and TSH). Nevertheless, the study is reliable and valid.
The NOAEL for maternal toxicity was 2.5 mg/kg bw/day based on decreased maternal body weight and body weight gain during treatment at the highest dose level. The NOAEL for developmental toxicity is >5 mg/kg bw/day.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Sep 1986 - 16 Oct 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted in 2018
Deviations:
yes
Remarks:
methodological limitations (missing examinations, such as food consumption, anogenital distance, blood sampling for analysis of thyroid hormones T4, T3 and TSH)
Qualifier:
according to guideline
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
CD®
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River U.K. Limited, Margate, Kent, England
- Age at study initiation: 9-11 weeks
- Weight at study initiation: 193-236 g
- Fasting period before study: Not applicable
- Housing: in groups of up to 5 female animals in RC1 and RB3-g cages (North Kent Plastics Limited, Dartford, England), with high density polypropylene bodies with lids and floors of stainless steel grid.
- Diet: laboratory animal diet (Labsure, Lavender Mill, England), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 Sep 1986 To: 16 Oct 1986
Route of administration:
oral: gavage
Vehicle:
other: aqueous 0.5 % carboxymethylcellulose containing 0.5 % (w/v) Tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The suspension was prepared daily by suspending the test material in aqueous 0.5% carboxymethylcellulose containing 0.5% (w/v) Tween 80.

VEHICLE
- Amount of vehicle: 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test substance was demonstrated with a sample from the container stored under the storage conditions of the study was returned to the sponsor for analysis after termination of the study. Results were considered satisfactory.

Homogeneityof the test substance in the vehicle was previously reported (Report Number 86/TRK001/704) but was also re-analyzed for samples of this study (first and last Week of treatment). Results were considered satisfactory.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: not reported. Virgin female rats were mated overnight individually with males. Each morning following pairing, the trays beneath the cages were checked for ejected copulation plugs and a vaginal smear was prepared from each female and examined for the presence of spermatozoa. The day on which a sperm positive vaginal smear or at least 3 vaginal plugs were found was designated as being day 0 of gestation.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: The day on which a sperm positive vaginal smear or at least 3 vaginal plugs were found was designated as being day 0 of gestation.
Duration of treatment / exposure:
Days 6-15 of gestation
Frequency of treatment:
once daily (mid-day)
Duration of test:
until sacrifice at Day 20 of gastation
Dose / conc.:
7.5 mg/kg bw/day
Dose / conc.:
15 mg/kg bw/day
Dose / conc.:
30 mg/kg bw/day
No. of animals per sex per dose:
25 female animals per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on a range finding experiment (Rep. No. 86/TRK001/704).
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3, 6-16, 18, 20 of gestation

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus

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: number of live and dead fetuses, fetus weight and sex
Blood sampling:
- Plasma: No
- Serum: No
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations:
Neck, thoracic and abdominal cavities: Yes: 2/3 of each litter
Details: All foetal abnormalities were recorded. Before fixation in industrial methylated spirit the offspring were eviscerated.
The remaining one-third of the foetuses of each litter were placed in Bouin´s fixative. After fixation they were examined by the Wilson free-hand serial section technique (Wilson, J.G., 1965).
- Skeletal examinations: Yes:2/3 per litter
Details: Skeletons of the eviscerated foetuses (see above) were examined after processing and Alizarin-red staining.
- Head examinations: No data
- Anogenital distance of all live rodent pups: not reported
Statistics:
Data were expressed as group means with standard deviations (S:D:) where apropriate. The significance of suggestive inter-group differences was tested using appropriate statistical tests. Differences with an associated probability of P < 0.05 were considered to be statistically significant.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The only treatment-related finding was a dose-related incidence in the number of dams showing areas of hair loss.

Summarized results can be found in Attachment 1 in the attached background material.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
no mortality observed
Description (incidence):
No deaths occurred.

Summarized results can be found in Attachment 1 in the attached background material.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 7.5 mg/kg bw/day: Body weight gain was marginally reduced during treatment at GD 6-16, but the subsequent rate of weight gain was unaffected (terminal body weight not significantly reduced). Post treatment body weight gain (GD 16-20) was identical in the low dose and control group, indicating the reversibility of the effect. This effect was of no biological relevance for the progress and outcome of pregnancy. Furthermore, the dose of 7.5 mg/kg bw/d had no effects on the offspring which demonstrates that even repeated dosing at 7.5 mg/kg bw did not cause a toxicologically relevant effect.
- At the 15 and 30 mg/kg bw/day, females showed a transient dose-related loss of body weight up to day 8 post-conception. Overall weight gain during treatment was significantly reduced compared to the control group.

Summarized results can be found in Attachment 1 in the attached background material.
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
Endocrine findings:
not specified
Description (incidence and severity):
The following ED-related parameters were investigated in the study: gravid uterus weight, gestation length, litter size and litter/pup weight, number of implantations, corpera lutea, viable fetuses, pre- and post-implantation loss, presence of anomalities and sex ratio. For details, please refer to the respective result fields and the endpoint summary.
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:
no effects observed
Description (incidence and severity):
Uterus weight was not affected by treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross treatment-related macroscopic abnormalities were observed.

Summarized results can be found in Attachment 1 in the attached background material.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable
Number of abortions:
no effects observed
Description (incidence and severity):
No abortions were reported.

Summarized results can be found in Attachment 1 in the attached background material.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No treatment-related effects were observed.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No treatment-related effects were observed.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Early or late resorptions:
no effects observed
Description (incidence and severity):
No treatment-related effects were observed.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Dead fetuses:
no effects observed
Description (incidence and severity):
No treatment-related effects were observed.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
No treatment-related effects were observed.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
No treatment-related effects were observed.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Other effects:
effects observed, treatment-related
Description (incidence and severity):
- 7.5 mg/kg bw/day: Placental weight was slightly lower than concurrent control values, but remained within the historical control range.
- 15 mg/kg bw/day: Placental weights were slightly lower as compared to controls, but remained within the historical control range.
- 30 mg/kg bw/day: Mean placenta weights were statistically significantly reduced (p < 0.001) compared to the control group.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
< 7.5 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
other: maternal general toxicity was observed at the lowest dose tested.
Key result
Dose descriptor:
LOAEL
Remarks:
general toxicty
Effect level:
7.5 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Remarks:
maternal developmental toxicity
Effect level:
< 7.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: placental weight was slightly reduced starting at the lowest dose tested
Key result
Dose descriptor:
LOAEL
Remarks:
maternal developmental toxicity
Effect level:
7.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
maternal abnormalities
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
placenta
other: placenta weight was slightly reduced
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 7.5 mg/kg bw/day: Foetal weight was unaffected.
- 15 mg/kg bw/day: Mean foetal weight was slightly below that of the concurrent control group (p = 0.0507), but within the historical control range..
- 30 mg/kg bw/day: Mean foetal weights were statistically significantly reduced (p < 0.001).

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No treatment-related effects were observed.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No treatment-related effects were observed.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related effects were observed regarding litter size but in fetal weights as described under the respective point.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Anogenital distance of all rodent fetuses:
not examined
Description (incidence and severity):
not applicable
Changes in postnatal survival:
not examined
Description (incidence and severity):
not applicable
External malformations:
effects observed, treatment-related
Description (incidence and severity):
- 7.5 mg/kg bw/day: No treatment-related effects were observed.
- 15 mg/kg bw/day: The incidence of small foetuses was slightly elevated compared to the control group, but both values were within the historical control range.
- 30 mg/kg bw/day: High incidence of small foetuses with weights of less than 2.7 g. There was an associated retardation of foetal development.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
- 7.5 mg/kg bw/day: No treatment-related effects were observed.
- 15 mg/kg bw/day: There was an increased incidence of foetuses with reduced 13th ribs compared to the control group (considered a variation).
- 30 mg/kg bw/day: There was an increased incidence of foetuses with reduced 13th ribs compared to the control group (considered a variation).
Reduction in the degree of skeletal ossification in the high dose group, most apparent in the supra-occipital bones, sternebrae, ribs, thoracic vertebrae, caudal vertebrae and metacarpals/metatarsals, slightly exceeded previously recorded background ranges. These observations were consistent with the incidence of small foetuses observed at necropsy in this group, and were considered to be indicative of slight developmental delay following treatment at 30 mg/kg bw/day.

Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
- 7.5 mg/kg bw/day: No treatment-related effects were observed.
- 15 mg/kg bw/day: No treatment-related effects were observed.
- 30 mg/kg bw/day: There was a slightly increased incidence of subcutaneous oedema (20.4%) compared to the control group (9.9%).


Summarized results can be found in Attachment 1 in the attached background material and in the overview Table under "Any other informations on results incl. tables".
Other effects:
not specified
Description (incidence and severity):
Hernia diaphragmatica was observed in two cases at 7.5 and in one case at 30 mg/kg bw/d. In this strain of rat, diaphragmatic hernia was a rare finding, but appears to be increasing in frequency as 4 of 2775 control foetuses (0.144%) have been affected in the past 2 years. The occurrence in 3 different treated litters was higher than might be expected, there was, however, no dose-dependency and group mean values were within previously recorded control ranges. The involvement of the test substance in the aetiology of this abnormality was considered to be equivocal.
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Effect level:
7.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed at this dose level.
Key result
Dose descriptor:
LOAEL
Remarks:
developmental
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
external malformations
skeletal malformations
other: effects indicative for developmental delay
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: small fetuses and developmental delay
Description (incidence and severity):
effects are considered to be affected by the maternal toxicity, occurring at all dose levels
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
15 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Summary of teratogenic/embryo toxic effects

Dose/endpoints Historical control data Recorded range  0 mg/kg bw/d  7.5 mg/kg bw/d  15 mg/kg bw/d  30 mg/kg bw/d
number pregnant females     25 25  25  25 
corpora lutea count [mean/dam]  16  13.9 -19  15 15.6  15  14.8 
implantations [mean/dam] 14.6  12 -16.7  13.9  14  13.6  13.5 
viable young (total)  13.7  11.1 -15.3  13.6  13.3  13.2  12.9 
resorptions (total)  0.86  0.25 -1.65  0.3  0.8  0.4  0.6 
Pre-implantation loss [%]  8.8  1.6 -16.5  8.4  10.0  9.6  9.7 
Post-implantation loss [%]  5.9  1.7 -12.7  2  5.4 3.2  4.2 
foetal weight [mean in g]  3.31  3 -3.55  3.24  3.26  3.13  2.89* 
placental weight [mean in g]  0.5  0.43 -0.57  0.50  0.47*  0.46*  0.41* 
Subcutaneus oedema [% incidence]  19.35 0 -63.2  9.9  5.1  11.3 20.4 
Reduced 13th ribs [% incidence] 1.77  0 -8.9  3.2  6.1  13.1  9.5 
Small foetuses (<2.7 g) [% incidence] 3.69   0 -16.9 1.8  1.8  8.2  23.8 

* significantly different from control: p<0.05 (nested analysis of variance and weighed t-test)

 

Conclusions:
The current study was performed under GLP conditions and conducted similar to OECD TG 414 (adopted 2018) with some restrictions, such as missing examination of anogenital distance of fetuses, and blood sampling for analysis of T4, T3 and TSH. Nevertheless, the study is considered reliable and valid.
According to these results, a NOAEL for maternal toxicity could not be determined, because of a decreased maternal body weight and body weight gain during treatment. The NOAEL for developmental toxicity is 7.5 mg/kg bw/day of thiram based on foetal weight and concomitant of small foetuses and developmental delay. These effects are considered to be affected by the maternal toxicity, occurring at all dose levels. Reduced body weights and body weight gains during sensitive periods of development are known to have an effects also on foetal development, especially weight and size. Thus, also small foetuses and small placenta are in general attributable to maternal toxicity.
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Remarks:
maternal general toxicity
Effect level:
3.1 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No adverse effects were observed at this dose level.
Remarks on result:
other: target substance after correction for molecular weight (factor 1.23)
Key result
Dose descriptor:
LOAEL
Remarks:
maternal general toxicity
Effect level:
6.15 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Remarks on result:
other: target substance after correction for molecular weight (factor 1.23)
Key result
Dose descriptor:
NOAEL
Remarks:
maternal developmental toxicity
Effect level:
> 6.15 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed indicating maternal toxicity up to the highest dose tested.
Remarks on result:
other: target substance after correction for molecular weight (factor 1.23)
Key result
Dose descriptor:
NOAEL
Remarks:
maternal general toxicity
Effect level:
2.5 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No adverse effects were observed at this dose level.
Remarks on result:
other: source substance
Key result
Dose descriptor:
LOAEL
Remarks:
maternal general toxicity
Effect level:
5 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Remarks on result:
other: source substance
Key result
Dose descriptor:
NOAEL
Remarks:
materal developmental toxicity
Effect level:
> 5 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed indicating maternal toxicity up to the highest dose tested.
Remarks on result:
other: source substance
Key result
Abnormalities:
no effects observed
Key result
Dose descriptor:
NOAEL
Remarks:
fetal developmental toxicity
Effect level:
>= 6.15 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed up to and including the highest dose level.
Remarks on result:
other: target substance after correction for molecular weight (factor 1.23)
Key result
Dose descriptor:
NOAEL
Remarks:
fetal developmental toxicity
Effect level:
>= 5 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed up to the highest dose level.
Remarks on result:
other: source substance
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
The current study was performed with the source substance under GLP conditions and conducted similar to OECD TG 414 (adopted 2018) with some restrictions due to methodological limitations (missing examination of anogenital distance of fetuses,no blood sampling for analysis of T4, T3 and TSH). Nevertheless, the study is reliable and valid.
The NOAEL for maternal toxicity was 2.5 mg/kg bw/day based on decreased maternal body weight and body weight gain during treatment at the highest dose level. The NOAEL for developmental toxicity is >5 mg/kg bw/day.
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13.
Reason / purpose for cross-reference:
assessment report
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
< 9.23 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
other: maternal general toxicity was observed at the lowest dose tested.
Remarks on result:
other: target substance after correction for molecular weight (factor 1.23)
Key result
Dose descriptor:
LOAEL
Remarks:
general toxicity
Effect level:
9.23 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Remarks on result:
other: target substance after correction for molecular weight (factor 1.23)
Key result
Dose descriptor:
NOAEL
Remarks:
maternal developmental toxicity
Effect level:
< 9.23 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
other: maternal developmental toxicity was observed at the lowest dose tested
Remarks on result:
other: target substance after correction for molecular weight (factor 1.23)
Key result
Dose descriptor:
LOAEL
Remarks:
maternal developmental toxicity
Effect level:
9.23 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Remarks on result:
other: target substance after correction for molecular weight (factor 1.23)
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
< 7.5 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
other: maternal general toxicity was observed at the lowest dose tested.
Remarks on result:
other: source substance
Key result
Dose descriptor:
LOAEL
Remarks:
general toxicty
Effect level:
7.5 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Remarks on result:
other: source substance
Key result
Dose descriptor:
NOAEL
Remarks:
maternal developmental toxicity
Effect level:
< 7.5 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: placental weight was slightly reduced starting at the lowest dose tested.
Remarks on result:
other: source substance
Key result
Dose descriptor:
LOAEL
Remarks:
maternal developmental toxicity
Effect level:
7.5 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
maternal abnormalities
Remarks on result:
other: source substance
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
placenta
other: placenta weight was slightly reduced
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Effect level:
9.23 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed at this dose level.
Remarks on result:
other: target substance after correction for molecular weight (factor 1.23)
Key result
Dose descriptor:
LOAEL
Remarks:
developmental
Effect level:
18.45 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
external malformations
skeletal malformations
other:
Remarks on result:
other: target substance after correction for molecular weight (factor 1.23)
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Effect level:
7.5 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed at this dose level.
Remarks on result:
other: source substance
Key result
Dose descriptor:
LOAEL
Remarks:
developmental
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
external malformations
skeletal malformations
other: effects indicative for developmental delay
Remarks on result:
other: source substance
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: small fetuses and developmental delay
Description (incidence and severity):
effects are considered to be affected by the maternal toxicity, occurring at all dose levels
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
18.45 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
The current study was performed with the source substance under GLP conditions and conducted similar to OECD TG 414 (adopted 2018) with some restrictions, such as missing examination of anogenital distance of fetuses, and blood sampling for analysis of T4, T3 and TSH. Nevertheless, the study is considered reliable and valid.
According to these results, a NOAEL for maternal toxicity could not be determined, because of a decreased maternal body weight and body weight gain during treatment. The NOAEL for developmental toxicity is 7.5 mg/kg bw/day of thiram based on foetal weight and concomitant of small foetuses and developmental delay. These effects are considered to be affected by the maternal toxicity, occurring at all dose levels. Reduced body weights and body weight gains during sensitive periods of development are known to have an effects also on foetal development, especially weight and size.
Thus, also small foetuses and small placenta are in general attributable to maternal toxicity.
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Remarks:
maternal general toxicity
Effect level:
>= 12.3 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No adverse effects were observed up to the highest dose level.
Remarks on result:
other: target substance after correction for molecular weight (factor 1.23)
Key result
Dose descriptor:
NOAEL
Remarks:
maternal developmental toxicity
Effect level:
>= 12.3
Based on:
test mat.
Basis for effect level:
other: No adverse effects were observed up to the highest dose level.
Remarks on result:
other: target substance after correction for molecular weight (factor 1.23)
Key result
Dose descriptor:
NOAEL
Remarks:
maternal general toxicity
Effect level:
>= 10 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No adverse effects were observed up to the highest dose tested.
Remarks on result:
other: source substance
Key result
Dose descriptor:
NOAEL
Remarks:
maternal developmental toxicity
Effect level:
>= 10 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No adverse effects were observed up to the highest dose tested.
Remarks on result:
other: source substance
Key result
Abnormalities:
no effects observed
Key result
Dose descriptor:
NOAEL
Remarks:
fetal developmental toxicity
Effect level:
6.15
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed at this dose level.
Remarks on result:
other: target substance after correction for molecular weight (factor 1.23)
Key result
Dose descriptor:
LOAEL
Remarks:
fetal developmental toxicity
Effect level:
12.3 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: skeletal variation (increased number of fetuses with 27 presacral vertebrae)
Remarks on result:
other: target substance after correction for molecular weight (factor 1.23)
Key result
Dose descriptor:
NOAEL
Remarks:
fetal developmental toxicity
Effect level:
5 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed up to this dose level.
Remarks on result:
other: source substance
Key result
Dose descriptor:
LOAEL
Remarks:
fetal developmental toxicity
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: skeletal variation (increased number of fetuses with 27 presacral vertebrae)
Remarks on result:
other: source substance
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: vertebra
Description (incidence and severity):
target substance, at 12.3 mg/kg bw/day, an increased incidence of greater presacral vertebra was observed (considered as variation). However, the incidence is within the IRDC historical control range and is not clearly dose related, thereby indicating rather an incidental finding covered within the biological variation.
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
12.3 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
The current study was performed with the source substance under GLP conditions and conducted similar to OECD TG 414 (adopted 2018) with some restrictions, such as no examination of anogenital distance of fetuses. Nevertheless, the study is reliable and valid.
According to these results, it is concluded that a dose level of >10 mg/kg bw/day is the No Observed Adverse Effect Level (NOAEL) for maternal systemic and developmental toxicity. The NOAEL for developmental toxicity is 5 mg/kg bw/day based on the increased incidence of foetuses with 27 presacral vertebra. Although this effect is considered a variations, and the incidence is in the range of historical control data, the increased incidence of foetuses with 27 presacral vertebrae was considered to define the LOAEL following a conservative approach. However, the observed skeletal variation of a supernumerary vertebra does not trigger classification for developmental toxicity.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
6.15 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
Developmental toxicity was assessed based on read across. The data available for the source substance were obtained in rodents (rat) and non-rodents (rabbit), according to EPA OPP 83-3 and/or similar to OECD TG 414 (2018). All three key studies were conducted under GLP conditions and were considered of reliable quality and validity (RL2) and thus, are suitable for the 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

Developmental toxicity studies via the oral route

Data on developmental toxicity following oral administration are available for the source substance to characterize the potential to induce toxicity after in utero exposure in rodents and non-rodents. Three key studies were considered to assess developmental toxicity in pregnant rats and rabbits after oral administration.

 

Rat

One oral developmental toxicity key study (Report number 87/TRK002/179) was conducted in rats with the source substance similar to OECD guideline 414 and under GLP conditions. The doses of 7.5, 15 and 30 mg/kg bw/day were selected based on the outcome of a pre-test (Report Number 86/TRK001/704). The doses were given to four groups of 25 pregnant rats on Days 6 through 15 of gestation at a constant volume of 10 mL/kg bw in 0.5% w/v CMC containing 0.5% w/v Tween 80. On Day 20, the animals were sacrificed, litter parameters were determined and fetuses were examined for visceral or skeletal defects.

 

At the 15 and 30 mg/kg bw/day, females showed a transient dose-related loss of body weight up to day 8 post-conception. Overall weight gain during treatment was significantly reduced compared to the control group.At 7.5 mg/kg bw/day, a slight but statistically significant reduction of body weight was observed during treatment at GD 6-16, but the subsequent rate of weight gain was unaffected (terminal body weight not significantly reduced).Post treatment body weight gain (GD 16-20) was identical in the low dose and control group, indicating the reversibility of the effect.Furthermore, the dose of 7.5 mg/kg bw/day had no effects on the offspring which demonstrates that even repeated dosing at 7.5 mg/kg bw/day did not cause a toxicologically relevant effect.

There was a dose-related increase in hair loss during gestation (alopecia on the neck or back on 25 dams, at 0, 7.5, 15, and 30 mg/kg bw/day, respectively).At necropsy, no treatment related effects were seen.

The numbers of implantations, resorptions and live young, and the extent of pre- and post-implantation losses, were unaffected by treatment.

Mean foetal weight was significantly reduced in 30 mg/kg bw/day treated females and there was a high incidence of small foetuses (less than 2.70 g). In 15 mg/kg bw/day treated females, mean foetal weight was slightly below that of the concurrent control group and the incidence of small foetuses was slightly elevated, but both values were within the range of previous control findings. Foetal weight in 7.5 mg/kg bw/day treated females was unaffected. A statistically significant and dosage-related reduction in mean placental weight was observed for all treated groups, but only in 30 mg/kg bw/day treated females was the value outside the previously recorded background control range.

Examination of foetuses at necropsy and following free-hand serial sectioning or skeletal processing revealed a small number of anomalies in all groups, the majority of which were of types and incidences previously recorded in control animals of this strain of rat in these laboratories and showed no association with treatment.

Hernia diaphragmatica was observed in two cases at 7.5 and in one case at 30 mg/kg bw/day. In this strain of rat, diaphragmatic hernia was a rare finding, but appears to be increasing in frequency as 4 of 2775 control foetuses (0.144%) have been affected in the past 2 years. The occurrence in 3 different treated litters was higher than might be expected, there was, however, no dose-dependency and group mean values were within previously recorded control ranges.

Reduction in the degree of skeletal ossification in the high dose group, most apparent in the supra-occipital bones, sternebrae, ribs, thoracic vertebrae, caudal vertebrae and metacarpals/metatarsals, slightly exceeded previously recorded background ranges.These observations were consistent with the incidence of small foetuses observed at necropsy in this group, and were considered to be indicative of slight developmental delay following treatment with 30 mg/kg bw/day.

In addition, in group 15 and 30 mg/kg bw/day, the incidence of 13th ribs of reduced size exceeded the previously recorded background range. Although this increase was greater in group 15 mg/kg bw/day than in group 30 mg/kg bw/day the possible involvement in the aetiology of this observation could not be excluded.

Based on the obtained results, a NOAEL for maternal toxicity could not be determined as decreased maternal body weight and body weight gain was already observed at the lowest dose during treatment. The NOAEL for developmental toxicity is 7.5 mg/kg bw/day based on foetal weight and concomitant of small foetuses and developmental delay. These effects are considered to be affected by the maternal toxicity, occurring at all dose levels. Reduced body weights and body weight gains during sensitive periods of development are known to have effects also on foetal development, especially weight and size. Thus, also small foetuses and small placenta are in general attributable to maternal toxicity.

Dose descriptors were adjusted to consider differences in molecular weights between the target and source substance and are provided in the target study record and under “Description of key information” in the technical dossier.

 

Rabbits

One range finding study study (Report number 87/TRK003/122) and two key studies were conducted with the source substance in pregnant rabbits to assess developmental toxicity (Report number 87/TRK004/541 and 399-121). The key studies were conducted similar to OECD guideline 414 and under GLP conditions. In each study, four groups of pregnant females received the test substance daily via gavage from Day 6 or 7 until Day 19 of gestation. The dose span was 1 -10 mg/kg bw/day. On Day 29, the animals were sacrificed, litter parameters were determined and fetuses were examined for visceral or skeletal defects.

 

In the first key study (Report number 87/TRK004/541), no abnormal clinical symptoms were detected. Necropsy findings for the 8 decedents revealed evidence of respiratory or gastro-intestinal tract disorder, or accidental tracheal intubation. An apparent marginal increase in the incidence of small foetuses was observed at necropsy for females in group 1 and 5 mg/kg bw/day. Litter parameters were unaffected by treatment.

The apparent marginal increase in small foetuses was considered to be unrelated to treatment in view of the absence of dose-effect relationship or any corresponding reduction in group mean foetal weight. With regard to the eruption of the scissors, any treatment related effect was unlikely because of absence of correlation with foetal weight and because of the background control data. Based on those results, a dose level of 2.5 mg/kg bw/day is considered as NOAEL for maternal toxicity based on reduced body weight gains. The NOAEL for developmental toxicity is >5 mg/kg bw/day.

Dose descriptors were adjusted to consider differences in molecular weights between the target and source substance and are provided in the target study record and under “Description of key information” in the technical dossier.

 

In the second key study (399 -121), deaths were observed in the control group, caused by pneumonia, and high dose group, considered as gavage injury.

Body weight gains of does in the treated groups were generally greater than that of does in the control group and correlated with an increase in food consumption. Uterine weights of does in the treated groups were slightly increased when compared with those of does in the control group. These increases were not considered as adverse.

Reproductive parameters evaluated at caesarian section examination were generally comparable between the control and treated group.

Foetal body weights, litter size and weights were comparable among all test groups, including the control. Further, no external or visceral malformations were observed indicating a treatment-related effect. Likewise, no treatment-related skeletal malformations were observed. There was a marked increase in the number of foetuses with 27 presacral vertebrae in the high-dose group when compared to control. This number was within the IRDC historical control range and is not clearly dose related. Thus, the biological relevance of this finding is considered low, especially as the historical control data clearly indicate that the effect on the number of presacral vertebrae lies within the biological variation under normal conditions in this rabbit strain. Therefore, the toxicological relevance of that finding is considered limited. However, following a conservative approach, this variation was considered for the derivation of the LOAEL.

 

 In conclusion, a dose level of >10 mg/kg bw/day is defined as NOAEL for maternal toxicity. The NOAEL for developmental toxicity is 5 mg/kg bw/day based an increased incidence of foetuses with 27 presacral vertebrae. Although this effect is considered a variation, and the incidence is in the range of historical control data, the increased incidence of foetuses with 27 presacral vertebrae was considered to define the LOAEL following a conservative approach. However, this skeletal variation is not severe enough to trigger classification for developmental toxicity. 

Dose descriptors were adjusted to consider differences in molecular weights between the target and source substance and are provided in the target study record and under “Description of key information” in the technical dossier.

 

 

Conclusion on developmental toxicity

Developmental effects associated with the treatment of the read across source substance, were observed in one rat study (Report number 87/TRK002/179). Maternal general toxicity started at the lowest dose group of 7.5 mg/kg bw/day, fetotoxicity was seen at the highest dose group of 30 mg/kg bw/day as decreased fetal weight and size. However, the reduced fetal body weights and the delayed fetal development can be regarded as secondary, non-specific effects related to maternal toxicity. In one developmental study conducted in rabbits, a marked increase in the number of fetuses with 27 presacral vertebrae was observed in the high dose group. This number was, however, within the IRDC historical control range and is not clearly dose related, clearly indicating that the effect on the number of presacral vertebrae lies within the biological variation under normal conditions in this rabbit strain. Therefore, the observed skeletal variation does not trigger classification for developmental toxicity.

Toxicity to reproduction: other studies

Additional information

An assessment on endocrine disruption was performed during the CoRAP substance evaluation of the source substance Thiram. The outcome of this evaluation in 2018 was that Thiram has no ED concern.

Mode of Action Analysis / Human Relevance Framework

Please refer to the respective Endpoint summary. 

Justification for classification or non-classification

The read across source substance did not affect fertility or reproductive performance in the rat. The available data do not meet the criteria for classification according to Regulation (EC) 1272/2008 and are therefore sufficient to warrant no classification of the test substance for reproductive toxicity.

All effects observed in the rat occurred at doses that caused marked maternal toxicity. In rabbits, a marked increase in the number of foetuses with supernumerary presacral vertebrae was determined which is covered by the historical control data range of the test facility. In addition, this finding is considered a variation and no dose-relationship was evident. Thus, the observed skeletal variation is not severe enough to trigger classification for developmental toxicity. Further, there was no evidence for a teratogenic potential of the test substance or the read across source substance. Based on the available data on developmental toxicity, the target substance does not require classification for developmental toxicity.

This is in line with the harmonised classification of the test substance (Annex VI of Regulation (EC) 1272/2008).

 

Additional information