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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Thiram
EC Number:
205-286-2
EC Name:
Thiram
Cas Number:
137-26-8
Molecular formula:
C6H12N2S4
IUPAC Name:
tetramethylthiuram disulfide

Test animals

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)

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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

Effect levels (maternal animals)

open allclose all
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.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

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

Effect levels (fetuses)

open allclose all
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)

Fetal abnormalities

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.

Overall developmental toxicity

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.