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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:
13 Jul 1987 - 24 Nov 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Referenceopen allclose all

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 2018
Deviations:
yes
Remarks:
no weight and histopathology of thyroid gland; thyroid hormones not checked in blood, no gross pathology; treatment was terminated on Day 16 instead of one day prior to sacrifice (Day 20)
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 1981
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 1984
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
428-040-8
EC Name:
-
Cas Number:
138261-41-3
Molecular formula:
C9H10ClN5O2
IUPAC Name:
2-chloro-5-{[2-(nitroimino)imidazolidin-1-yl]methyl}pyridine
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: KFM, Kleintierfarm Madoesrin AG, Fuellinsdorf, Switzerland
- Age at study initiation: 11 weeks, minimum
- Weight at study initiation: 184 - 240 g
- Housing: individually in Makrolon cages (type-3) with wire mesh tops and standardized granulated softwood bedding (Lignocel, Schill AG, Muttenz, Switzerland).
- Diet: Pelleted standard Kliba 343 rat/mouse maintenance diet ("Kliba", Klingentalmuehle AG, Kaiseraugst, Switzerland, Batch nos. 73/87 and 75/87), ad libitum
- Water: tap water, ad libitum
- Acclimation period: minimum 7 days prior to mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + /- 3
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 Jul To: 12 Aug 1987

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other:
Remarks:
distilled water with 0.5 % Cremophor EL
Mass median aerodynamic diameter (MMAD):
other: not applicable
Remarks on MMAD:
not applicable
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The mixtures of the test article and vehicle were prepared daily before administration.
The test material was weighed into a glass beaker on a tared precision balance (Mettler PK 300) and the vehicle added (w/w). The mixtures were prepared using a homogenizer. During the daily administration period, homogeneity was maintained using a magnetic stirrer.

VEHICLE
- name: distilled water with 0.5 % Cremophor EL
- Amount of vehicle: 10 mL/kg bw
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 were determined on one occasion before the dosing period. Samples were taken immediately after preparation and again 2 hours later. During the dosing period, samples were taken for confirmation of concentration and homogeneity on one occasion. Analysis was done using a HPLC.

It was shown that the test material was stable in the vehicle for up to two hours. The mean concentrations found were in the range of 81.9 % to 96.6 % of the nominal concentration. Homogenity was also confirmed.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: no
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 post coitum (Day 0 of pregnancy)
Duration of treatment / exposure:
from Day 6 through to Day 15
Frequency of treatment:
daily
Duration of test:
from Day 0 of pregnancy to Day 21
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosages were based on the results of the dose range-finding study (RCC Project 083507). In this study, effects on maternal animals were observed at 50, 100 and 150 mg/kg bw/day (all doses tested) shown as reduced food consumption and body weight gain. Developmental toxicity and teratogenicity was not observed up to the highest dose. Therefore, 10, 30 and 100 mg/kg bw/day of the test substance were used in the main study.
- Fasting period before blood sampling for (rat) dam thyroid hormones: not examined
- Time of day for (rat) dam blood sampling: not examined

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule: not reported

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: all internal organs, with emphasis upon the uterus, uterine contents, position of fetuses in the uterus and 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
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations (visceral and brain): Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
- Anogenital distance of all live rodent pups: no
Statistics:
For data about body weights, food consumption, reproduction and skeletal examination:

One-way analysis of variance (ANOVA) to test for significance between tretment groups
If a normal distribution could be assumed, a Dunnett's test was performed for the comparison between the treated groups and the control group. When no normal distribution was assumed, the Steel-test (many-one rank test) was applied. Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.
Indices:
Pre implantation loss (%) was calculated as
(number of corpora lutea - number of implantations)/(number of corpora lutea) x 100

Post implantation loss (%) was calculated as
(number of implantations - number of live fetuses)/(number of implantations) x 100

sex ratio of males/females (%) was calculated as
(number of males/females)/(number of fetuses) x 100
Historical control data:
Yes, available in the study report

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
In one female of the control group and in one female of the high dose group, bald areas in the dorsal thoracic region were noted from days 15 and 11 post coitum, respectively. This is a common finding in pregnant animals of this strain and age.
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):
- 30 mg/kg bw/day: reduced body weight gain throughout the study (corrected bw gain of 5.6 g instead of 7.9 g in controls from Day 6 - Day 21 (non-adverse))
- 100 mg/kg bw/day: statistically significantly reduced body weight gain throughout the study (corrected bw gain of 4.2 g instead of 7.9 g in controls from Day 6 - Day 21)

Details can be found in Table 1 in Attachment 1.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 10 mg/kg bw/day: reduced food consumption from Day 6-11 compared to controls (-9.5%), increased food consumption from Day 11-16 (+6.8% compared to controls ), the increase in food consumption was also found on Day 16-21 (post-treatment, +10.0% compared to controls )
- 30 mg/kg bw/day: reduced food consumption compared to controls from Day 6-11 (-10.0%)
- 100 mg/kg bw/day: reduced food consumption compared to controls from Day 6-11 (-9.5%) and from Day 11-16 (-19.9%), after the end of treatment (Day 16-21), food consumption was increased (+20.5%), all compared to controls.

Details can be found in Table 2 in Attachment 2.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
not examined
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No abnormal findings were noted in any female of any group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No abnormal findings were noted in any female of any group.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
no effects observed
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:
Reproduction data

No test article-related or statistically significant differences between the mean values for the reproduction data of all dose-groups and the vehicle control group were noted. All differences observed were within the normal range of variation for animals of this strain and age.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
not applicable
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):
not applicable
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
not applicable
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
not applicable

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed at this dose level.
Key result
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
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:
effects observed, non-treatment-related
Description (incidence and severity):
-100 mg/kg bw/day: sex ratio was m/f 58.6/41.4% compared to 51.0/49.0% in controls. However, this was seen as incidental since In the dose range finding study (RCC Project 083507) to this study, the sex ratio of the high dose group was also shifted but in favour of the female fetuses. Further, in this dose range finding study, a sex ratio of 50:50 was noted at a higher dose level of 150 mg/kg bw/day.
Changes in litter size and weights:
no effects observed
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):
not applicable
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
- 100 mg/kg bw/day: wavy ribs were found more frequently, there were higher stages of skeletal ossification for example for both forelimbs, hind limbs, and lower ossification for example for cervical vertebrae and the sternum. As wavy ribes constitute a slight, transient alteration, which is fully reversed if pups are raised to age, the occurrence of wavy ribs is not rated as an adverse effect

Summarized Data can be found in Table 3 and 4 in Attachment 3 and 4 in the attached background material
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- 10 mg/kg bw/day: one fetus was noted with visceral malformations in form of severe haemorrhage around the brain and dilated lateral ventricles of the brain; the heart was enlarged - the auricles were dilated with blood and the walls of the ventricles were thickened. Since this was a single occurrence and was not found in higher doses, this finding was considered to be incidental and not related to treatment. The internal bleeding had caused increased body weight (7.5 grams cf. group mean of 4.8 grams) and was possibly the consequence of an injury.
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:
systemic
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed at this dose
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Key result
Dose descriptor:
NOAEL
Remarks:
teratogenicty
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at the highest dose tested

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
skeletal: rib
Description (incidence and severity):
Wavy ribs, higher stages of skeletal ossification for example for both forelimbs, hind limbs, and lower ossification for example for cervical vertebrae and the sternum at the highest dose level of 100 mg/kg bw/day, regarded as secondary effects to maternal toxicity and therefore not considered directly treatment-related.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
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:
no

Applicant's summary and conclusion

Conclusions:
In the current study, developmental toxicity was evaluated in rats. Dams received 10, 30 or 100 mg/kg bw test substance per day via gavage (25 dams/dose) from Day 6 of pregnancy to Day 16. They were sacrificed on Day 21. Appearance, behaviour, and mortality of the dams were unchanged up to and including 100 mg/kg bw/day. A NOAEL for maternal toxicity of 30 mg/kg bw/day was derived based on initial body weight loss, reduced food consumption, and decreased body weight gain at 100 mg/kg bw/day (as discussed in the biocidal assessment report, Assessment Report, 2011). Fetuses of the control and treatment groups did not differ for external and visceral examination. Slight effects on skeletal development of fetuses, described as increased incidence of wavy ribs, were reported for the highest dose level of 100 mg/kg bw/day. However, these findings clearly correlated with obvious maternal toxicity. There was no evidence for teratogenicity in rat at doses up to including the highest tested level of 100 mg/kg bw/day.

The study was conducted under GLP conditions, according to according to OECD TG 414, adopted 1981. Therefore, examinations that are required by the current guideline, namely thyroid gland weight and histopathology were not performed, neither was blood analyzed for T4, T3 and TSH assessment. Nevertheless, the study is considered reliable and fully acceptable.