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Developmental toxicity / teratogenicity

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developmental toxicity
Type of information:
experimental study
Adequacy of study:
other information
Study period:
year of publication: 1973
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well documented publication
Reason / purpose for cross-reference:
reference to same study

Data source

Reference Type:
Effects of o-chlorobenzylidene malononitrile (CS) and the stress of aerosol inhalation upon rat and rabbit embryonic development
Upshall DG
Bibliographic source:
Toxicology and Applied Pharmacology 24: 45-59

Materials and methods

Principles of method if other than guideline:
developmental toxicity
GLP compliance:
not specified
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): o-chlorobenzylidene malononitrile (CS)
- Molecular formula (if other than submission substance): C10H5ClN2
- Molecular weight (if other than submission substance): 188.62
- Smiles notation (if other than submission substance): #
- InChl (if other than submission substance): #
- Structural formula attached as image file (if other than submission substance): see Fig. #

Test animals

other: Porton strain SPF
Details on test animals or test system and environmental conditions:
- Housing: individually

- Photoperiod (hrs dark / hrs light): 12 hours
- soundproof environment

Administration / exposure

Route of administration:
Type of inhalation exposure (if applicable):
not specified
unchanged (no vehicle)
Details on exposure:
Inhalation exposure was against pure CS-aerosol with a particle size in the range of 1-2 µm mass median diameter. The aerosol was generated from a melt of pure crystallline substance, at approximately 120°C, using a Collision spray.
Additionally, rats were exposed intraperitoneally against the test item (test item dissolved in sterile polyethylene glycol 300, 20 mg/kg and 2 ml/kg).
Analytical verification of doses or concentrations:
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: over night
- Verification of same strain and source of both sexes: no data
- Proof of pregnancy: sperm in vaginal smear, referred to as day 1 of pregnancy
Duration of treatment / exposure:
inhalation: 5 minutes per day on 10 days
i.p.: single application on different days of pregnancy
Frequency of treatment:
inhalation: day 6-15 of pregnancy
i.p.: day 6, 8, 10, 12, or 14 of pregnancy
Duration of test:
till day 21 of pregnancy
Doses / concentrationsopen allclose all
Dose / conc.:
20 other: mg/kg bw
single i.p. on day 6, 8, 10, 12, or 14
Dose / conc.:
6 mg/m³ air
for 5 min on day 6-15
Dose / conc.:
20 mg/m³ air
for 5 min on day 6-15
Dose / conc.:
60 mg/m³ air
for 5 min on day 6-15
No. of animals per sex per dose:
up to 24 females per dose
Control animals:
yes, concurrent no treatment
yes, sham-exposed
other: positive control
Details on study design:
- sham-exposed ("stress-control groups"):
One control group was exposed to particulate aerosol for 5 minutes/day on days 6-15 of pregnancy (60 mg/m3 Neosil, generated from a suspension of 10% w/v Neosil in 0.1% v/v Triton X-100) and to a water vapor aerosol (generated from 0.1% v/v Triton X-100 in distilled water).

- untreated control groups:
The first was recaged and moved from its normal environment for the duration of aerosol exposure of the test goups, the second was left undisturbed apart from routine watering and feeding.

- positive control:
Trypan blue, 50 mg/kg in 4 ml isotonic saline, injected sc on day 8 of pregnancy

- Sacrifice:
Termination of pregnancy on day 21 of gestation either by cervical dislocation or nitrogen anoxy


Maternal examinations:
- body weight control: weekly
- recording of occurrence of vaginal bleeding and signs of ill health
Ovaries and uterine content:
- total uterus weight before and after revomal of fetuses
- weight of each placenta
- number of corpora lutea in each ovary
- number of resorptions
Fetal examinations:
- number, position, sex and weight of each fetus in each uterine horn
- number of fetuses with gross, externally visible malformations and the nature of these malformations
- rat fetuses from the preliminary CS, trypan blue and aerosol stress expseriments: 50% were assigned to Bouin fluid or 70% ethanol for subsequent visceral or skeletal examination. In all other studies one-third of the fetuses were assigned to Bouin Fluid and two third to 70% ethanol.
Internal development was evaluated using the freehand razor technique of Wilson. Skeletal abnormalities were examined after staining with Alizarin red S.
All measurements were compared statistically between control and test populations using Student's t-test. The number of dams was taken as the randomly assigned variable. The percent fetal loss, litter size, numbers of abnormal fetuses and incidence of anomalies were compared between test and control groups by ranking the percent incidence per litter of each condition and comparing using the Mann-Whitney U test.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Effect levels (maternal animals)

open allclose all
Dose descriptor:
Effect level:
20 other: mg/kg bw
Basis for effect level:
other: maternal toxicity
Dose descriptor:
Effect level:
20 other: mg/kg bw
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

No significant differences between treated and control animals were observed in the preliminary experiment, except a significantly lower (22.9%) weight gain of the maternal animals. This finding was not repeated in the dose-response experiment.

In the dose response experiment no dose related effects of the test substance were observed on the total number of pregnancies, total number of fetuses, mean litter size, percent fetal loss, or number of gross, externally visible malformations. The mean fetal weight decreased dose dependently in exposed rats (7.6%, 8.5%, 11.0% decrease in comparison to the control group in the low, mid and high dose group respectively). These differences were statistically significant but were similar to other control values.

Number of fetuses with visceral or skeletal anomalies were not significantly increased in treated rats compared to control animals. The predominant anomalies in rats treated with test item or control groups were hydroureter and hydronephrosis, but the incidence was not statistical significantly increased in treated rats in comparison to controls. Unilateral hydroureter predominated on the left side and there was a gradual increas with increasing CS concentration.

        % incidence of hydroureter and hydronephrosis                
            hydroureter           hydronephrosis
   BL  L  total  BL R  L  total
 control  6.1  3.0  6.1  15.2  4.5  6.1  6.1  16.7
 5 mg/m3  8.7  1.5  8.7  18.9  10.1  2.9  7.2  20.2
 20 mg/m3  0  0  9.4 9.4  1.3  2.7  6.6  10.6
 60 mg/m3  8.6  7.1  10.0  25.7  14.3  10.0  5.7  30.0

BL: bilateral; R, L: Right and left, unilateral

Single intraperitoneal injection of the test item during pregnancy did not result in significant differences betweent treated and control animals, except injection of the test substance on day 12 of pregnancy, which resulted in a 8% decreas of the mean fetal weight.

Neither exposure to Neosil or water vapour or recaging rats during the period of aerosol exposure of the other two groups on day 6-15 of pregnancy did result in significant effects on any of the measurements taken or the numbers of abnormalities noted.

The findings with the positive control were within the normal range (decreased mean litter size, increase in resorptions and malformed fetuses.

Applicant's summary and conclusion

No developmental toxicity was observed in rats treated with single intraperitoneal injections of the test substance (20 mg/kg bw) or repeated inhalative exposure (6, 20, or 60 mg/m3 for 5 min/day) on day 6-15 of pregnancy.
Executive summary:

Porton strain rats were exposed to [(2-chlorophenyl)methylene]malononitrile by single intraperitoneal injections at 20 mg/kg bw on day 6, 8, 10, 12, or 14 of pregnancy or to test substance aerosols at concentrations of 6, 20 and 60 mg/m3 for 5 minutes on days 6 -15 of pregnancy. No effects on number of litters, number of live fetuses, % fetal losses, mean litter size, mean fetal weight, mean placental weight, maternal weight increase and number of abnormal litters were observed. Abnormalities did not differ between treated and controls.

Assuming that about 90% of CS are converted to 2-chlorobenzaldehyde in the blood after intraperitoneal injection (see chapter 7.2) it can be concluded that a single intraperitoneal dose of 18 mg/kg 2-chlorobenzaldehyde on day 6, 8, 10, 12 or 14 of pregnancy does not result in developmental toxicity in rats. No conclusions on the effects of 2-chlorobenzaldehyde after inhalation exposure can drawn due to path specific differences (see chapter 7.1).