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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:
2016 - 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
O-isopropyl ethylthiocarbamate
EC Number:
205-517-7
EC Name:
O-isopropyl ethylthiocarbamate
Cas Number:
141-98-0
Molecular formula:
C6H13NOS
IUPAC Name:
N-ethyl(propan-2-yloxy)carbothioamide
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Cheminova A/S Batch No. 0001026602
- Expiration date of the lot/batch: October 13th, 2017
- Purity test date: October 13th , 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle:Soluable and stable in the vehicle


Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: females : approx. 11-12 weeks old, males:Age of the males at the start of pairing: between 12 weeks and not older than 17 weeks
- Weight at study initiation: Body weight before initiation of pairing: males: 320 – 350 g (mean: 334.40 g, ± 20% = 267.52 – 401.28 g) females: 203 - 242 g (mean: 222.76 g, ± 20% = 178.21 – 267.31 g)
- Fasting period before study:
- Housing: Full barrier in an air-conditioned room. The animals were kept individually in IVC cages (type III H, polysulphone cages) on Altromin saw fibre bedding (except during the pre-mating period when females were kept in groups of two animals and during mating period when two females were paired with one male)
- Diet (e.g. ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum):Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period:t 5-7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 17th February 2016 To:17th June 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0, 0.78, 2.6 and 7.8 mg/l
- Amount of vehicle (if gavage): 4 ml/kg bw/day
- Lot/batch no. (if required): Manufacturer: Sigma, Batch No.: MKBS6944V
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: 1-6 days
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear; referred to as day 0 of pregnancy
- Any other deviations from standard protocol:
Duration of treatment / exposure:
20 days
Frequency of treatment:
daily
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control group (group C)
Dose / conc.:
3 mg/kg bw/day
Remarks:
Group name LD.
Purity level of 95,7 w/w % of test item solution taking into consideration, so dosage concentration is in mg AI/ kg bw /day
Dose / conc.:
10 mg/kg bw/day
Remarks:
Group name MD.
Purity level of 95,7 w/w % of test item solution taking into consideration, so dosage concentration is in mg AI/ kg bw /day
Dose / conc.:
30 mg/kg bw/day
Remarks:
Group name HD.
Purity level of 95,7 w/w % of test item solution taking into consideration, so dosage concentration is in mg AI/ kg bw /day
No. of animals per sex per dose:
24-25 females per dose (males were not exposed, but removed from cages after positive vaginal smear
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, except on weekends and public holidays when observations were made once daily
- Cage side observations included: morbidity and mortality, spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once before initiation of pairing to ensure that the body weights are within + 20% variation.
The sperm positive females were weighed during gestations days 0, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study except once before initiation of pairing.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, although this was not a feeding study; Food consumption of sperm positive females was measured on gestations days 5, 8, 11, 14, 17 and 20.
Food consumption was not measured for males during the entire study or for both male and females during the mating period.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
On gestation day 20, sperm positive females were subjected to a caesarean section after sacrificing the animals using an overdose of pentobarbital injected intraperitoneally at a dosage of approximately 8 mL/kg bw.
At the time of termination, the dam (presumed pregnant female) was examined macroscopically for any structural abnormalities or pathological changes, which may have influenced the pregnancy. Unusual growth in fat tissue attached to the uterus (with oviduct and cervix) of female no. 60 of MD group was preserved in 4% neutral-buffered formaldehyde for potential histopathological examination.
Immediately after the termination, the uteri were removed, and the pregnancy status of the dams was confirmed. Uteri that appear non-gravid were further examined by staining with 10 % ammonium sulphide solution to confirm the non-pregnant status.
Each gravid uterus with the cervix was weighed. The number of corpora lutea was counted for pregnant animals. The uterine contents were examined for embryonic or fetal deaths as well as the number of viable fetuses. The degree of resorption (late and early) was confirmed in order to help estimate the relative time of death of the conceptus. The position and number of fetuses in each uterine horn was also recorded.
Males were used for other studies.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes / No / No data
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
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [ half per litter ]
- Skeletal examinations: Yes: [half per litter ]
- Head examinations: Yes: [half per litter]

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no adverse clinical signs in the animals of the control and test-tem treated groups. Alopecia (on various parts- hindlimb, forelimb, abdomen and dorsal back of the body) was noted in a few animals of control and dose groups. There was no dose-response relationship for this finding and it was considered spontaneous.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no effects on body weight and body weight gain. Slight, statistically significantly higher mean body weights were noted in LD and HD groups on GD 0, 5, 8 and 11 and also on GD 14 in LD group. These changes were not dose-dependent and as noted already prior to dosing not considered test item related. The mean body weights on GD 17 and GD 20 in dose groups were comparable to the corresponding control group. No statistically significant changes in body weight gain were noted between treated and control groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption was not affected in dose group animals when compared to control group. No statistically significant differences were noted between the dose and control groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
At necropsy, macroscopic findings relevant to test item treatment were not observed. One female (no. 60) of MD group had a high amount of fat tissue attached to the uterus (with oviduct and cervix). This finding was spontaneous in nature as the finding occurred in a single isolated female.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically, no significant differences noted in prenatal parameters including uterus weight, the number of corpora lutea, the number of implantation sites, the number of resorptions (early, late and total), the number of live fetuses, the number of dead fetuses, the number of female fetuses, sex ratio, percent pre-implantation and post-implantation losses. A statistically significantly higher number of mean male fetuses was observed in LD group compared to the control, which in the absence of dose-response relation was considered incidental. Higher mean post-implantation losses in MD and HD groups compared to the control, were in the absence of dose-response relationship and absence of statistical significance were not considered test item related. Further, all mean values for percent pre- and postimplantation loss observed in control and test item treated groups were within the range of historical control data (0 to 51.20 % and 0 to 32.15 %, respectively).
There was a single dead fetus in female no. 100 of HD group, which was an isolated incident and was considered incidental.
Slightly higher numbers of early and total resorptions were noted in the HD group compared to the control group. However, the values in test-item groups were not statistically significantly different compared to the control and the mean values were also within the range of the historical control data (0 to 2.7 %). Therefore, the findings were not considered an adverse effect of test item treatment.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically, no significant differences noted in prenatal parameters including uterus weight, the number of corpora lutea, the number of implantation sites, the number of resorptions (early, late and total), the number of live fetuses, the number of dead fetuses, the number of female fetuses, sex ratio, percent pre-implantation and post-implantation losses. A statistically significantly higher number of mean male fetuses was observed in LD group compared to the control, which in the absence of dose-response relation was considered incidental. Higher mean post-implantation losses in MD and HD groups compared to the control, were in the absence of dose-response relationship and absence of statistical significance were not considered test item related. Further, all mean values for percent pre- and postimplantation loss observed in control and test item treated groups were within the range of historical control data (0 to 51.20 % and 0 to 32.15 %, respectively).
There was a single dead fetus in female no. 100 of HD group, which was an isolated incident and was considered incidental.
Slightly higher numbers of early and total resorptions were noted in the HD group compared to the control group. However, the values in test-item groups were not statistically significantly different compared to the control and the mean values were also within the range of the historical control data (0 to 2.7 %). Therefore, the findings were not considered an adverse effect of test item treatment.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically, no significant differences noted in prenatal parameters including uterus weight, the number of corpora lutea, the number of implantation sites, the number of resorptions (early, late and total), the number of live fetuses, the number of dead fetuses, the number of female fetuses, sex ratio, percent pre-implantation and post-implantation losses. A statistically significantly higher number of mean male fetuses was observed in LD group compared to the control, which in the absence of dose-response relation was considered incidental. Higher mean post-implantation losses in MD and HD groups compared to the control, were in the absence of dose-response relationship and absence of statistical significance were not considered test item related. Further, all mean values for percent pre- and postimplantation loss observed in control and test item treated groups were within the range of historical control data (0 to 51.20 % and 0 to 32.15 %, respectively).
There was a single dead fetus in female no. 100 of HD group, which was an isolated incident and was considered incidental.
Slightly higher numbers of early and total resorptions were noted in the HD group compared to the control group. However, the values in test-item groups were not statistically significantly different compared to the control and the mean values were also within the range of the historical control data (0 to 2.7 %). Therefore, the findings were not considered an adverse effect of test item treatment.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically, no significant differences noted in prenatal parameters including uterus weight, the number of corpora lutea, the number of implantation sites, the number of resorptions (early, late and total), the number of live fetuses, the number of dead fetuses, the number of female fetuses, sex ratio, percent pre-implantation and post-implantation losses. A statistically significantly higher number of mean male fetuses was observed in LD group compared to the control, which in the absence of dose-response relation was considered incidental. Higher mean post-implantation losses in MD and HD groups compared to the control, were in the absence of dose-response relationship and absence of statistical significance were not considered test item related. Further, all mean values for percent pre- and postimplantation loss observed in control and test item treated groups were within the range of historical control data (0 to 51.20 % and 0 to 32.15 %, respectively).
There was a single dead fetus in female no. 100 of HD group, which was an isolated incident and was considered incidental.
Slightly higher numbers of early and total resorptions were noted in the HD group compared to the control group. However, the values in test-item groups were not statistically significantly different compared to the control and the mean values were also within the range of the historical control data (0 to 2.7 %). Therefore, the findings were not considered an adverse effect of test item treatment.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically, no significant differences noted in prenatal parameters including uterus weight, the number of corpora lutea, the number of implantation sites, the number of resorptions (early, late and total), the number of live fetuses, the number of dead fetuses, the number of female fetuses, sex ratio, percent pre-implantation and post-implantation losses. A statistically significantly higher number of mean male fetuses was observed in LD group compared to the control, which in the absence of dose-response relation was considered incidental. Higher mean post-implantation losses in MD and HD groups compared to the control, were in the absence of dose-response relationship and absence of statistical significance were not considered test item related. Further, all mean values for percent pre- and postimplantation loss observed in control and test item treated groups were within the range of historical control data (0 to 51.20 % and 0 to 32.15 %, respectively).
There was a single dead fetus in female no. 100 of HD group, which was an isolated incident and was considered incidental.
Slightly higher numbers of early and total resorptions were noted in the HD group compared to the control group. However, the values in test-item groups were not statistically significantly different compared to the control and the mean values were also within the range of the historical control data (0 to 2.7 %). Therefore, the findings were not considered an adverse effect of test item treatment.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
not specified
Other effects:
not specified

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
act. ingr.
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result
Abnormalities:
no effects observed
Localisation:
not specified

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean fetal weight in the HD group was statistically significantly lower compared to the control group value (3.38 g in HD Vs 3.70 g in C). However, the numbers of live fetuses and total litter weight were not statistically different in HD group compared to the control. Further, the mean fetal weight in HD group was within the range of historical control data (2.63 g to 4.71 g). However in light of other findings potentially indicative of embryotoxicity (delayed ossification of bones) a test item-related effect on fetal weights in the HD group cannot be ruled out.
The mean male litter weight in the LD group was statistically significantly higher compared to the control value (26.23 g in LD Vs 20.80 g in C). In the absence of a dose- response relationship, this finding was considered incidental.
There were no statistically significant differences noted for total litter weight and female litter weight of treated groups compared to the corresponding control.
There were comparable numbers of fetuses in both (left and right) uterine horns of control and test item treated groups.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The mean fetal weight in the HD group was statistically significantly lower compared to the control group value (3.38 g in HD Vs 3.70 g in C). However, the numbers of live fetuses and total litter weight were not statistically different in HD group compared to the control. Further, the mean fetal weight in HD group was within the range of historical control data (2.63 g to 4.71 g). However in light of other findings potentially indicative of embryotoxicity (delayed ossification of bones) a test item-related effect on fetal weights in the HD group cannot be ruled out.
The mean male litter weight in the LD group was statistically significantly higher compared to the control value (26.23 g in LD Vs 20.80 g in C). In the absence of a dose- response relationship, this finding was considered incidental.
There were no statistically significant differences noted for total litter weight and female litter weight of treated groups compared to the corresponding control.
There were comparable numbers of fetuses in both (left and right) uterine horns of control and test item treated groups.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Hematoma was noted in few isolated fetuses from all test item treated and control groups without any dose- response relationship. This finding was concluded as not related to treatment with the test item. Malformations were noted in two fetuses of the HD group. Fetus No. 7 (dam no. 81) had an absent left eye and misshapen snout. Fetus no. 5 (dam no. 82) had an umbilical hernia. However, these malformations occurred in single isolated fetuses of the HD group and additionally, the finding absent eye was within the historical control range. Therefore, these findings were considered spontaneous in origin. One fetus of the control group was noted with a malformation (right hindlimb rotated).
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
The fetal skeletal examinations found a statistically significantly increased incidence of findings in the HD group, including incomplete ossification of skull frontal bone (bilateral) (35% in HD and 0% in C), zygomatic arch (right) (35% in HD and 5% in C) and supraoccipital bone (80% in HD and 35% in C); statistically significantly higher incidence of unossified metacarpals (60% in HD and 10% in C); statistically significantly higher incidences of rudimentary right cervical rib (7th) (25% in HD and 0% in C); statistically significantly higher incidences of wavy ribs (80% in HD and 45% in C); and a statistically significantly higher incidence of misshapen humerus (25% in HD and 0% in C). In addition, a statistically significantly increased incidence of incomplete ossification of parietal bone (bilateral) was noted in the in MD group (65% in MD and 25% in C), and a statistically significantly increased incidence of incomplete ossification of pelvic girdle pubis in LD group (23% in LD and 0% in C).
Moreover, increased incidences of incomplete ossification of frontal (bilateral) (20% in MD group and 0% in C) , hyoid body (25% in HD and 5% in C), interparietal (75% in MD, 85% in HD and 55% in C), bent scapula and spine (right sided) (15% in HD and 0% in C), bent scapula (right sided and bilateral) (25% and 20% in HD and 5% and 0% in C, respectively). These findings did not attain the statistical significance compared to the corresponding control.
When compared to historical control data (HCD), the majority of these skeletal findings occurred at a higher incidence than HCD mean value, but for most observations within HCD ranges. However, there were incidences which were either outside the range of HCD or the HCD incidence was 0.

The findings of incomplete ossification of various bones, including frontal, zygomatic, supraoccipital, metacarpals and hyoid body are variations associated with delayed ossification and transient in nature. Also wavy ribs are conformational bone changes that are transient and reversible [11] [17] and of minor toxicological significance. In combination with the observed lower fetal weight these findings are considered a sign of test item related embryotoxicity in the HD group. In the absence of associated signs of adverse effects, the increased incidence of incomplete ossification of parietal bone, rudiementary 14th rib, bent scapula and long bones observed in the MD group is not considered an adverse effect of the test item [17]. The increased incidence of incomplete ossification of pelvic girdle pubis in the LD group did not show dose-response relationship and was therefore concluded not to be related to treatment with the test item. The other skeletal findings listed in the above table were statistically not significantly different compared to the corresponding control and/or were not dose response related.

The statistically significantly higher incidences of rudimentary cervical rib (7th) in HD group are considered adverse effects of treatment with the test item because the cervical ribs causes considerable adverse effects in human [11], but in rats it is a minor variation [16]. The misshapened bones represent malformations, irrespective of the structure concerned [11], thus the statistically significantly higher incidences of misshapen humeri in HD group was an adverse effect of test-item treatment.
Visceral malformations:
no effects observed
Other effects:
not specified

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day
Based on:
act. ingr.
Sex:
not specified
Basis for effect level:
fetal/pup body weight changes
skeletal malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: forelimb
skeletal: rib

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
30 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

Applicant's summary and conclusion

Conclusions:
On the basis of this prenatal developmental toxicity study in Wistar pregnant female rats with IPETC at dose levels of 3, 10, and 30 mg/ kg body weight/ day administered on gestation days 5 to 19, the following conclusions can be made: There were no test item related findings in maternal animals.
There were increased incidences of rudimentary cervical rib (7th) and misshapen humerus in high dose (HD) group compared to the corresponding control and were considered to be adverse effects. In addition, in the same group, increased incidences of reversible skeletal variations and reduced fetal weights were considered signs of test item related embryotoxicity. Thus, the NOAEL for the maternal toxicity is 30 mg/ kg body weight/ day and the NOAEL for the embryo-fetal developmental toxicity is 10 mg/ kg body weight/ day.
Executive summary:

Developmental toxicity test was done according to OECD Guideline 414. Wistar pregnant female rats were exposed to test item concentration of 3, 10 and 30mg/kg bw/day for 20 days. There were no test item related findings in maternal animals. There were increased incidences of rudimentary cervical rib (7th) and misshapen humerus in high dose (HD) group compared to the corresponding control and were considered to be adverse effects. In addition, in the same group, increased incidences of reversible skeletal variations and reduced fetal weights were considered signs of test item related embryotoxicity. Thus, the NOAEL for the maternal toxicity is 30 mg/ kg body weight/ day and the NOAEL for the embryo-fetal developmental toxicity is 10 mg/ kg body weight/ day.