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Diss Factsheets

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:
26 May - 19 Jun 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Deviations from current guideline version (e.g. ano-genital distance was not determined). For details please refer to materials and methods section.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 2018
Deviations:
yes
Remarks:
No blood sampling for analysis of T3, T4 and TSH, weight of thyroid gland not determined, no histopathology of thyroid gland. Anogenital distance of foetuses not determined. Temperature slightly out of range (18 to 26°C instead of 19 to 25°C).
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 1981
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
433-460-1
EC Name:
-
Cas Number:
210880-92-5
Molecular formula:
C6H8ClN5O2S
IUPAC Name:
(E)-N'-[(2-chloro-1,3-thiazol-5-yl)methyl]-N-methyl-N''-nitroguanidine

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD R BR VAF/PlusR
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan
- Age at study initiation: approx. 10 weeks
- Weight at study initiation: 201 - 231 g
- Housing: individually (except during cohabitation period)
- Diet: Certified Rodent Diet #5002 (PMI Feeds, St. Louis, Missouri), ad libitum
- Water: reverse osmosis water with chlorine added as a bacteriostat, ad libitum
- Acclimation period: six days

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

IN-LIFE DATES: From: 26 May To: 19 Jun 1997

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: aqueous 0.5% methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Suspensions of test substance were prepared weekly at the given concentrations. The test substance was considered 100% pure for the purpose of dosage calculations.

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

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Before and one hour after dosage (DGs 6-19) and on DG 20. Rats were also observed for viability at least twice daily.
- Cage side observations checked: clinical observation of effects of the test item, abortions, premature deliveries, and deaths

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

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Gross necropsy of the thoracic, abdominal and pelvic viscera was performed. To confirm pregnancy status, uteri from rats that appeared nonpregnant were stained with 10% ammonium sulfide. Tissues with gross lesions were preserved in neutral buffered 10% formalin for possible future evaluation. All other maternal tissues were discarded.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: distribution of implantation sites, live and dead foetuses
Blood sampling:
- Plasma: No
- Serum: No
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter (those used for soft tissue examination)
Statistics:
Clinical observation and other proportion data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution.
Continuous data (e.g., maternal body weights, body weight changes, feed consumption values and litter averages for percent male foetuses, percent resorbed conceptuses, foetal body weights, foetal anomaly data and foetal ossification site data) were analyzed using Bartlett's Test of Homogeneity of Variances and the Analysis of Variance, when appropriate [i.e., Bartlett's Test was not significant (p>=0.05)]|. If the Analysis of Variance was significant (p<=0.05), Dunnett's Test was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate [i.e., Bartlett's Test was significant (p<=0.05)], the Kruskal-Wallis Test was used, when less than or equal to 75% ties were present. In cases where the Kruskal-Wallis Test was statistically significant (p<=0.05), Dunn's Method of Multiple Comparisons was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher's Exact Test was used to analyze the data.
Count data obtained at Caesarean-sectioning of the dams were evaluated using the procedures described above for the Kruskal-Wallis Test.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical observations included localized alopecia on the limbs, underside, back and/or neck, chromorhinorrhea and missing/broken incisors. All observations were considered non-treatment related, since the incidences were not dosage-dependent or the observations occurred in only 1 or 2 rats and/or are common events in the laboratory environment.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
Not applicable
Mortality:
no mortality observed
Description (incidence):
Not applicable
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No effect on body weight or body weight changes was observed at the 10 mg/kg bw/day dosage level. In the 40 mg/kg bw/day group, body weight gain was significantly decreased (p<=0.01) compared to control. In the 125 mg/kg bw/day group a significant weight loss (p<= 0.01) was observed on DGs 6 to 9 and a significantly reduced weight gain (p<=0.01) in the entire dosage period (DGs 6 to 20) as well as through the entire gestation period (DGs 0 to 20). Therefore, average body weights were significantly reduced (p<=0.05 or p<=0.01) on DGs 7 through 20. Please refer to attachment 01.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Feed consumption (absolute+relative) was lower (p<=0.05 or p<=0.01) in 40 mg/kg bw/day dosed rats compared to control on DGs 6 to 9. In the 125 mg/kg bw/day group, feed consumption was decreased (p<=0.05 or p<=0.01) throughout the complete exposure period (calculated as DGs 6 to 20) and entire gestation period (calculated as DGs 0 to 20). No effect on food consumption was noted in the 10 mg/kg bw/day group. Please refer to attachment 02.
Food efficiency:
not examined
Description (incidence and severity):
Not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
Not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
Not applicable
Haematological findings:
not examined
Description (incidence and severity):
Not applicable
Clinical biochemistry findings:
not examined
Description (incidence and severity):
Not applicable
Endocrine findings:
not examined
Description (incidence and severity):
Not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
Not applicable
Behaviour (functional findings):
not examined
Description (incidence and severity):
Not applicable
Immunological findings:
not examined
Description (incidence and severity):
Not applicable
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Gravid uterine weights did not significantly differ among the four groups.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Necropsy findings consisted of a portion of the intermediate lobe of the liver adhered to the diaphragm, which was thin at the point of adhesion, for one 10 mg/kg/day dosage group dam (16230); slight dilation of the pelvis of each kidney for one 40 mg/kg/day dosage group dam (16261) and a dark red area on the dorsal surface of the left apical lobe of the lung for one 125 mg/kg/day dosage group darn (16292). All necropsy findings were considered unrelated to the test substance because each occurred in only one rat. The pregnancies of these dams were unaffected by the presence of these gross lesions.
Neuropathological findings:
not examined
Description (incidence and severity):
Not applicable
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
Not applicable
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
Not applicable
Other effects:
not examined
Description (incidence and severity):
Not applicable
Details on results:
Not applicable

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No abortions occurred during the study.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Not applicable
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Not applicable
Early or late resorptions:
no effects observed
Description (incidence and severity):
Not applicable
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead fetuses were observed.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Not applicable
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
23/25, 22/25, 24/25, and 25/25 rats (92%, 88%, 96% and 100%, respectively) were pregnant in the control, 10, 40, and 125 mg/kg bw/day dose groups, respectively.
Other effects:
not examined
Description (incidence and severity):
Not applicable

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
LOAEL
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
other: food consumption
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no adverse effect observed

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
not examined
Description (incidence and severity):
not applicable
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
not applicable
Changes in sex ratio:
no effects observed
Description (incidence and severity):
not applicable
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
not applicable
Anogenital distance of all rodent fetuses:
not examined
Description (incidence and severity):
not applicable
Changes in postnatal survival:
not examined
Description (incidence and severity):
not applicable
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Umbilical hernia occurred in one control group fetus. This foetus had no other soft tissue or skeletal alterations.
One control group foetus had an edematous body (anasarca). Skeletal examination of this foetus revealed a short fibula and tibia of each hindlimb and variations in thoracic vertebral ossification (bifid centra of the 9th, 11th and 12th thoracic vertebrae).
A depressed eye bulge occurred in one 10 mg/kg bw/day foetus and in one 40 mg/kg bw/day dosage group fetus. Skeletal examination of the 10 mg/kg bw/day dosage group fetus revealed a small right eye socket as the only alteration. Microphthalmia of the right eye was first identified at soft tissue examination of the 40 mg/kg bw/day dosage group foetus.
No other gross external fetal malformations or variations occurred in this study.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In individual cases fused, waved or additional ribs, short fibula and tibia in the hindlimbs, bifid centrum in thoracic vertebra, and affected ossifications of sternum or pelvis were noted. These events were considered to be not treatment-related, since no dose-dependency was observed and the findings were within normal variations.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In individual cases umbilical artery that descended to the left of the bladder, absent innominate artery or aortic arch dorsal to the trachea and esophagus were found. No dose-dependency was observed for these events and hence they are not considered treatment-related.
Other effects:
not examined
Description (incidence and severity):
not applicable
Details on embryotoxic / teratogenic effects:
Combination of malformations and variations resulted in 10 (43.5%), 10 (45.4%), 10 (41.7%) and 11 (44.0%) litters in the four respective dosage groups with fetuses with any alterations observed. In these same respective dosage groups, totals of 14 (4.7%), 10 (3.4%), 13 (3.9%) and 17 (5.0%) fetuses had any alterations observed, and averages of 4.7%, 3.4%, 3.9% and 4.9% of the fetuses per litter had an alteration.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 125 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of any developmental toxicity effect

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
external: eye
skeletal: sternum
skeletal: rib
skeletal: hindlimb
visceral/soft tissue: cardiovascular

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
The current study was performed under GLP conditions and conformed the US EPA OTS 798.4900 guideline. The study is also in accordance with OECD TG 414 (adopted 2018) with some restrictions, such as no examination of anogenital distance of fetuses, and no blood sampling for analysis of thyroid hormones T4, T3 and TSH. Nevertheless, the study is reliable and valid. The developmental toxicity NOAEL in Sprague-Dawley rats was determined to be greater than 125 mg/kg bw/day due to the absence of any developmental toxicity effect in this study. A NOAEL for maternal toxicity was determined to be 10 mg/kg bw/day due to decreased body weight gain and food consumption in the 40 mg/kg bw/day group.