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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:
11 Jul 1995 - 09 May 1996
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:
1997
Report date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 1981
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 2018
Deviations:
yes
Remarks:
thyroid weight, thyroid histopathology and thyroid hormones (T4, T3 and TSH) not investigated, anogential distance not measured, justification for choice of vehicle not provided
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(2-chlor-5-pyridyl-methyl)-cyanimino-1,3-thiazolidin
EC Number:
601-147-9
Cas Number:
111988-49-9
Molecular formula:
C10H9ClN4S
IUPAC Name:
3-(2-chlor-5-pyridyl-methyl)-cyanimino-1,3-thiazolidin

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Age at study initiation: not specified
- Weight at study initiation: more than 300 g for males and 182 - 239 g for females
- Housing: During adaptation, females were kept in groups in Type III Makrolon® cages. Starting from gestation day 0 they were individually accommodated in Type II Makrolon® cages on low-dust wood shavings. The males were kept individually in Type III Makrolon® cages.
- Diet standard rat diet (Altromin® 1324, produced by the Altromin company in Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.0 ± 3.0
- Humidity (%): approximately 50
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 11 Jul 1995 To: 09 May 1996

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% aqueous carboxymethyl cellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was formulated in 0.5% aqueous carboxymethyl cellulose suspension. Animals received a uniform volume of 10 mL/kg bw per oral gavage.

OTHER:
The animals of the control group received vehicle only (0.5% carboxymethyl cellulose) at the same volume. After insemination was ascertained, 28 females each were allocated to four experimental groups according to a computer-generated randomization plan. Due to inadequate skeletal and cartilage staining for unknown reasons in single fetuses from several litters of the different dose groups 7 additional inseminated females were allocated non-randomized to the study at each dose level in order to obtain a minimum of 20 litters for complete skeletal and cartilage evaluation in this study. The animals were treated daily from day 6 to 19 post coitum (p.c.).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability of the test article/vehicle mixtures concentration were determined by HPLC. The analytical data verified that the test material content in the toxicology test mixture agreed with the target concentration within defined limits. The concentration were 101.0 - 108.0% (for 0.2 mg/mL), 81.1 - 111.0% (for 1.0 mg/mL) and 104.0 - 112.0% (for 5.0 mg/mL). Furthermore, the analytical data verified that the test material was homogeneously distributed and chemically stable within the concentration range of 0.02 mg/mL to 20.0 mg/mL. Under current sample preparation and handling conditions stability and homogeneity was assured at room temperature for a period of at least 8 days.
Details on mating procedure:
The animals were mated by placing two females overnight into a Type III cage together with one male rat. If sperm was detected in the vaginal smear taken on the morning following mating, this day was regarded as day 0 of gestation.
Duration of treatment / exposure:
The animals were treated daily from day 6 to 19 p.c.
Frequency of treatment:
daily
Duration of test:
14 days
Doses / concentrationsopen allclose all
Dose / conc.:
2 mg/kg bw/day
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
50 mg/kg bw/day
No. of animals per sex per dose:
35
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels used were selected according to a preceding developmental toxicity dose-range finding study. In this study, dose level of 2, 10 and 50 mg/kg bw were tested. Effects on maternal animals were only observed at 50 mg/kg bw/day shown as reduced food consumption and body weight gain. The same dose level caused decreased fetal weights and increased number of fetuses with skeletal retardations. The external, visceral and skeletal examination of the fetuses revealed no treatment-related malformations. Thus, the test substance revealed embryotoxicity (retardation) in a maternally toxic dose range, therefore, the same dose levels were proposed for the main developmental toxicity study.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: From day 0 to 20 p.c. all animals were inspected twice daily.
- Cage side observations checked: general condition of the rats (appearance, behavior), any alterations concerning their excretory products

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: The body weights of the animals were determined on day 0 p.c. and daily from day 6 to 20 p.c.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, food consumption was calculated as g per animal per day
The feed consumption of the animals on gestation days 0 - 6, 6 - 11, 11 - 16 and 16 - 20 was determined based on the differences in weight of feed provided and feed which remained unconsumed.

WATER CONSUMPTION: Yes
Water consumption was determined by estimation of the remaining quantities in the water bottles.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 p.c.
- Organs examined: the uterus, uterine contents, number of corpora lutea
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: Individual weight and appearance of the placentas, number of live fetuses, sex of live fetuses, individual weight of fetuses
Blood sampling:
- Plasma: No data
- Serum: No data
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter], however, during skeletal and cartilage evaluation it turned out that inadequate staining had occurred in fetuses from several litters. Therefore, 7 additional inseminated females per group were assigned to the study.
- Head examinations: No data
- Anogenital distance of all live rodent pups: no
- Methods:
For visceral malformations and other findings deviating from normal, evaluation of about half of the fetuses were done by razor blade sectioning according to the modified WILSON technique. For findings in abdominal, pelvic, thoracic organs and skeletal and cartilage, DAWSON technique modified by the addition of cartilage staining (method described by INOUYE, modified) was used. Furthermore, the skin was viscerated and removed, the cartilage was stained with alcian blue GX, the fetuses were cleared with dilute potassium hydroxide solution, the skeletal system was stained with alizarin red S, and the cartilage and skeletal system were assessed.
Statistics:
Animals without implantation sites were excluded from statistical evaluation. Animals with total resorption were not taken into account for calculation of group mean values of body weights, body weight gains and feed intakes. Differences between the control and treated groups were considered significant when p<0.05. Statistical significance was tested using the following methods:
- Analysis of Variance (ANOVA) and in case of significant results Dunnett's t-Test as posthoc test for: feed intakes, body weights and body weight gains, uterus weights, corrected body weight gains, number of corpora lutea per dam, number of implantations per dam, number of live fetuses per dam and as percentage of implantations per dam, placental weights, fetal weights
- CHI2 test (correction according to Yates) for: fertility rate, gestation rate, number of fetuses or litters with malformations
- 2 by N CHI2 test; in case of significant differences Fisher's exact test with Bonferroni correction for: number of implantations per group, number of preimplantation losses per group, number of postimplantation losses, early resorptions, late resorptions or dead fetuses per group, number of live fetuses per group in percent of implantations, number of male or female fetuses or fetuses with undeterminable sex per group, number of fetuses or litters with skeletal findings
- Kruskall-Wallis test and in case of significant differences Dunn's test for: number of preimplantation losses per dam, number of postimplantation losses, early resorptions, late resorptions or dead fetuses per dam, number of male or female fetuses or fetuses with undeterminable sex per dam
Indices:
No indices were calculated.
Historical control data:
Yes, available in the study report and in an expert statement. For details, please refer to the attached background material (attachment 8).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No relevant treatment-related clinical observations were made in this study up to and including 50 mg/kg bw/day except for urinary and fecal excreta as follows:
2 and 10 mg/kg bw/day: No treatment-related changes were noted after treatment up to and including 10 mg/kg bw/day.
50 mg/kg bw/day: Urinary and also fecal excreta were affected by the treatment at 50 mg/kg bw/day. Towards and during the second half of gestation animals displayed increased urine excretion, which can be explained by the increased water consumption occurring at the same time. Decreased amounts of feces were observed mainly during the first few days after initiation of treatment which is highly correlated with the decreased feed intake detected during the same time period at this dose level. Six dams of the high dose group also displayed light discoloration of the feces, a finding which could be due to the elimination of test material which is a pale-yellow powder.
For details, please refer to the attached background material (attachment 1).
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
no mortality observed
Description (incidence):
No mortality occurred in this study up to and including 50 mg/kg bw/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Severe transient body weight loss was observed during days 6 to 9 p.c. at 50 mg/kg bw/day resulting in significantly decreased absolute body weight during the entire treatment period. Furthermore, mean maternal body weight gain was significantly decreased during days 6 to 9, 14 to 15 and 17 to 18 p.c. at 50 mg/kg bw/day. No effect on body weight development of the females was observed at 2 and 10 mg/kg bw/day.
For details, please refer to the attached background material (attachment 2).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
2 and 10 mg/kg bw/day: No treatment-related changes in food consumption were noted after treatment up to and including 10 mg/kg bw/day.
50 mg/kg bw/day: Feed consumption was decreased throughout the entire treatment period at the high dose level (50 mg/kg bw/day) with pronounced effect during day 6 to 11 p.c. whereas during day 11 to 20 p.c. the observed effect on feed consumption was less severe.
For details, please refer to the attached background material (attachment 3).
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
2 and 10 mg/kg bw/day: No treatment-related changes in water consumption were noted after treatment up to and including 10 mg/kg bw/day.
50 mg/kg bw/day: Water consumption was affected by the treatment at 50 mg/kg bw/day. During the first days after initiation of treatment an increased incidence of animals with decreased water consumption was observed at the high dose level whereas more animals displayed increased water consumption towards and during the second half of gestation at this dose level. For details, please refer to the attached background material (attachment 1).
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:
not examined
Description (incidence and severity):
not applicable
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
2, 10 and 50 mg/kg bw/day: No treatment related gross pathological findings were evident at necropsy up to and including the dose of 50 mg/kg bw/day.
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:
not specified
Description (incidence and severity):
not applicable
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
2 and 10 mg/kg bw/day: No alterations in the rate of post-implantation loss was noted at dose levels of up to and including 10 mg/kg bw/day.
50 mg/kg bw/day: The elevated incidence of late resorptions in animals with viable fetuses resulted in a significantly increased rate of post-implantation loss in the high dose group (50 mg/kg bw/day).
For details, please refer to the attached background material (attachment 4).
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
2 and 10 mg/kg bw/day: The resorption rate was unaffected at dose levels of up to and including 10 mg/kg bw/day.
50 mg/kg bw/day: Total resorption of the conceptus was observed in one female at the 50 mg/kg bw/day dose level. However, in the rat strain used single total resorptions occur spontaneously as demonstrated by historical data collected during 1991 to 1994 in the laboratory performing this study. Data from those studies indicate that single total resorptions can occur by chance, apparently independent of treatment. Since marked maternal toxicity was visible at the high dose level, it can not be excluded that the single total resorption observed at this dose level is related to the treatment with the test compound.
For details, please refer to the attached background material (attachment 4).
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
2 and 10 mg/kg bw/day: The resorption rate was unaffected at dose levels of up to and including 10 mg/kg bw/day.
50 mg/kg bw/day: Incidence of late resorptions (total number per group) in animals with viable fetuses was elevated in the high dose group (50 mg/kg bw/day). Since the incidence of late resorptions at this dose level was above the range of historical control data from this rat strain, it is considered to be related to the treatment with the test compound.
For details, please refer to the attached background material (attachment 4).
Dead fetuses:
no effects observed
Description (incidence and severity):
The number of dead fetuses was not affected by the treatment.
Changes in pregnancy duration:
not specified
Description (incidence and severity):
not applicable
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The number of pregnant animals were not affected by the treatment.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Weight and appearance of placentas:
The weight of the placentas was unaffected by the treatment at doses of up to and including 50 mg/kg bw/day. Necrotic placental borders were observed in all dose groups including the control animals without dose-dependency. The incidence of necrotic placental borders appeared to be increased at dose levels of 2 mg/kg bw/day and above when compared to control animals. However, this is a common finding, displayed no dose-dependency and was at all dose levels within the incidence range of untreated control animals of former studies, therefore, this observation is considered incidental and not related to the treatment with the test compound. It is therefore concluded that the weight and appearance of the placentas was not affected up to and including 50 mg/kg bw/day.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed at this 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 this dose level
Key result
Dose descriptor:
LOAEL
Remarks:
general toxicity
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
Key result
Dose descriptor:
LOAEL
Remarks:
maternal developmental toxicity
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
pre and post implantation loss

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
2 and 10 mg/kg bw/day: The mean fetal weight was unaffected by the treatment at dose levels of up to and including 10 mg/kg bw/day.
50 mg/kg bw/day: The mean fetal weight recorded at cesarean section was statistically significantly decreased.
For details, please refer to the attached background material (attachment 4).
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The number of live fetuses was not affected by the treatment.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No changes in sex ratio were observed in any treatment group when compared to the control group.
Changes in litter size and weights:
not examined
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, treatment-related
Description (incidence and severity):
50 mg/kg bw/day: At cesarean section forelimb deviations were observed in 2 fetuses at 50 mg/kg bw/day. Although these limb anomalies were observed in a single litter, this finding is considered to be related to treatment with the test compound since these findings were confirmed by skeletal evaluation (limb bone dysplasia) and since similar findings were also observed during skeletal evaluation of other fetuses in several litters at the 50 mg/kg bw dose level. All other observations made during external examination are not considered to be compound-related findings because of lack of dose-dependency or correspondingly frequent incidences in historical control animals of other studies with similar study design. At the high dose level one fetus with multiple malformations (gastrochisis, shortened mandible, cleft palate, limb deviations, etc.) was observed. Historical control data for malformations demonstrated that single fetuses with multiple malformations including those observed in this fetus may also occur spontaneously in untreated control animals of this rat strain. But, considering the increased incidence of late resorptions and the fetal skeletal findings (see below) together with the distinct maternal toxicity at the high dose level (50 mg/kg bw/day) it can not be excluded that the single multiple malformation observed in this dose group might be related to treatment with the test compound.
For details, please refer to the attached background material (attachment 5 and 9).
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
It has to be noted, that during skeletal and cartilage evaluation, an inadequate staining had occurred in fetuses from several litters. Therefore, 7 additional inseminated females per group were assigned to the study and with regards to skeletal and cartilage examination, the data generated were evaluated and reported separately for completely evaluated litters and for litters where complete evaluation was not possible due to insufficient staining.

2 and 10 mg/kg bw/day: Some statistically significant deviations were noticed at the 2 and 10 mg/kg bw/day dose levels, which should not be regarded to be of toxicological relevance because of the lack of dose-dependency or comparable rate of occurrence in control groups of previously conducted studies with similar study design in the same rat strain.
50 mg/kg bw/day: Several statistically significant observations were recorded with respect to ossification and skeletal variations. In all these cases statistically significant findings were made due to cases of impaired ossification (retardations) in different skeletal localizations. These observations were apparent in an individual and on a per litter basis. The findings considered toxicologically relevant were cases of retardations (impaired ossification) or variations (wavy ribs, asymmetrical sternebrae) at the high dose level. Further, an increased total incidence of malformations observed was especially due to an increased incidence of extremital bone dysplasia (tubular bones, clavicula and scapula). These limb bone malformations are prevalent spontaneous malformations in the rat strain used. The incidence at which they were observed in this study was however elevated at the high dose level.
For details, please refer to the attached background material (attachment 6 and 7).

Additional information on dysplasia of limb bones were given in an expert statement (M-031344-01-1) and the observed effects in the present rat developmental toxicity study were further assessed in a position paper (M-278407-01-1). With regards to the increased incidence of dysplastic limb bones in the fetuses of the highest dose group, it was noted, that these incidences were within the historical control range. Further it was stated, that if a treatment relationship would be assumed, then it would most likely be related to the observed maternal toxicity. In addition, a clear dose-response relationship was not visible: The next lower level (10 mg/kg bw/day) did not cause maternal toxicity and hence dysplastic limb bones were not observed. At the low dose of 2 mg/kg bw/day this finding was seen at an incidence within the historical control range. In total, the incidence showed a high variability so that a treatment relationship is questionable, especially since this finding occurs spontaneously in the rat strain used at a rather high and variable rate. This was supported by the results of further toxicity studies: In a study, which was conducted at the same time period, 3.7% of the fetuses and 23.1% of the litters in the control group were affected. In another study, the highest incidence of this finding was detected in the control animals, with a fetal incidence of 2.7% and a litter incidence of 14.3%. This demonstrates that even the effects in the highest dose group with a fetal incidence of 3.0% and a litter incidence of 20.7% were covered by, or were comparable to control incidences. Furthermore, if litters from dams mated with certain males (for which an association to an increased malformation rate could be suspected), are excluded, the incidence of findings would be smaller. In the years 1994 up to 1999, the historical control incidence was 0-3.45% in the fetuses and from 0-23.07% in the litters, which shows that the incidences observed in all groups, even in the 50 mg/kg bw group, did not exceed historical control ranges. For details, please refer to the attached background material (attachment 8) and to the respective endpoint summary.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
10 and 50 mg/kg bw/day: Slightly increased incidence of renal pelvis dilatation were noted at the 10 and 50 mg/kg bw dose levels. However, renal pelvis is not considered to be a compound-related finding because of the lack of dose-dependency or correspondingly frequent incidences in historical control animals of other studies with similar study design.
For details, please refer to the attached background material (attachment 5 and 9).
Other effects:
not examined
Description (incidence and severity):
not applicable
Details on embryotoxic / teratogenic effects:
not reported

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed at this dose level
Key result
Dose descriptor:
LOAEL
Remarks:
developmental toxicity
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
external malformations
skeletal malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: skeletal: limb, ribs, sternebrae
Description (incidence and severity):
Incidence of extremital bone dysplasia together with multiple malformations (one fetus) as well as an increased incidence of skeletal retardations (impaired ossification) and variations (wavy ribs, asymmetrical sternebrae) at the highest dose tested.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
50 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

Applicant's summary and conclusion

Conclusions:
The study was performed under GLP conditions and according to OECD TG 414 (adopted 1981). Notable deviations from the OECD guideline mainly concerned thyroid parameters, which were not examined. However, the study is considered reliable and valid. Under the conditions of this study, no clear and indisputable evidence for a specific developmental toxicity potential of the test substance was determined since all developmental effects observed were manifest only at dose levels which also induced severe maternal toxicity and/or are common spontaneous malformations in the rat strain used. The NOAEL was 10 mg/kg bw/day for maternal toxicity and developmental toxicity based on maternal and fetal body weight, maternal food consumption and embryotoxic effects at 50 mg/kg bw/day.