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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:
May 1985 - January 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1986
Report date:
1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
no exposure through the entire period of gestation
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Chloroethane
EC Number:
200-830-5
EC Name:
Chloroethane
Cas Number:
75-00-3
Molecular formula:
C2H5Cl
IUPAC Name:
chloroethane
Details on test material:
- Name of test material (as cited in study report): ethyl chloride
- Analytical purity: 99.9%

Test animals

Species:
mouse
Strain:
CF-1
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. (Portage, MI, USA)
- Weight at study initiation: females: 25-30 g
- Housing: in wire bottom cages
- Diet (e.g. ad libitum): Certified Laboratory Animal Chow No. 5002, Ralston Purina Company, St. Louis, MO
- Water (e.g. ad libitum): municipal tap water
- Acclimation period: at least two weeks

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
not specified
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 157 L stainless steel and glass Rochester-type inhalation chambers
- Method of conditioning air: Test atmospheres of chloroethane were generated by pumping from Tedlar gas sampling bags containing 100% chloroethane into a chamber inlet duct. Chloromethane was mixed with air in this duct, and the mixture was drawn into the chambers at approximately 30 L/min. An FMI pump was used to pump the chloroethane gas into the inlet duct.
- Temperature, humidity in air chamber: 19-24 °C, 49 - 68%
- Air flow rate: 30 L/min


TEST ATMOSPHERE
- Brief description of analytical method used: Samples were taken at least once/two hours from chamber and analysis were performed with an infrared analyzer. Chamber chloromethane concentrations were determined by interpolation from a standard curve. Calibration check was performed daily.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The mean time averages were: 491 +/- 37 ppm, 1504 +/- 84 ppm, 4946 +/- 159 ppm
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1:2
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
day 6 - 15 of gestation
Frequency of treatment:
6 hours/day
Duration of test:
Animals were sacrificed on day 18 of gestation.
Doses / concentrationsopen allclose all
Dose / conc.:
500 ppm
Remarks:
corresponding to 1338 mg/m³
Dose / conc.:
1 500 ppm
Remarks:
corresponding to 4015 mg/m³
Dose / conc.:
5 000 ppm
Remarks:
corresponding to 13385 mg/m³
No. of animals per sex per dose:
30 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The high exposure concentration was chosen on the basis of the results of a pilot study. At 5000 ppm increased activity during exposure and decreased maternal body weights were observed.

Examinations

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

BODY WEIGHT: Yes
- Time schedule for examinations: on gestation days 6,9, 12, 15 and 18

FOOD CONSUMPTION: Yes
Time schedule for examinations: in 3-day intervals beginning on day 6 of gestation

WATER CONSUMPTION: Yes
- Time schedule for examinations: in 3-day intervals beginning on day 6 of gestation

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 18
- Organs examined: Maternal liver weights were recorded at the time of caesarean section. Sections of liver were saved in neutral, phosphate-buffered 10% formalin, but were not examined histopathologically.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number and position of resorption sites: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter]

The heart of all foetuses was examined for cardiac anomalies.
Statistics:
Bartlett's test for equality of variance (α = 0.01) was used for body weights and body weight gains and organ weights. Based on the outcome of Bartlett's test, a parametric or nonparametric analysis of variance (ANOVA) was performed. If the ANOVA (α = 0.1) was significant, analysis was performed by Dunnett's test (α = 0.05, two sided) or the Wilcoxon Rank Sum test (α = 0.05, two sided) with Bonferroni's correction. A censored Wilcoxon test ((α = 0.05, one sided) with Bonferroni's correction was used for statistical evaluation of resorptions among litters and the fetal fetal population and frequency of fetal alterations. Number of implants and litter size were analyzed with a non-parametric ANOVA followed by the Wilcoxon Rank-Sum test with Bonferroni's correction. The pregnancy rate was analyzed by the Fisher (α = 0.05, one-sided) exact probability test. Statistica outliers were indentified by a sequential outlier test (Grubb's test, α = 0.02, two sided).
Indices:
pregnancy index

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
No maternal toxicity was recorded in this study. No significant effects were noted in clinical signs, body weight, body weight gain, liver weight, food and water consumption at any of the exposure concentrations tested. However, an earlier pilot study with non-pregnant female mice at the same concentrations showed an exposure-related decrease in body weight gain.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEC
Effect level:
13 385 mg/m³ air (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
No effects on the number of live and dead foetuses or on the number and position of resorption sites were observed in mice exposed up to and including 5000 ppm chloroethane 6 hours/day on gestation days 6-15.
There was no indication of a teratogenic response observed in any of the exposure groups when compared to controls. In all exposure groups including the control group a low incidence of malformations with no indication of a consistent pattern of effects was observed. Among the control mice one foetus had a cleft palate and another foetus from a different litter exhibited microphthalmia. In the 500 ppm group, three foetuses from separate litters had cleft palates, with one foetus also exhibiting dilateral lateral ventricles of the brain and forked and fused ribs. Among litters in the 1500 ppm exposure group, three foetuses from different litters had malformations. A single foetus exhibited a clear cleft palate. Exencephaly was seen in the other two malformed foetuses, one of which also had an omphalocele and fused cervical vertebrae. At the highest dose a single foetus with cleft palate and micrognathia was the only malformed foetus observed.
A number of minor visceral and skeletal alterations were observed scattered among all exposure groups including the controls. A statistical significant increase was seen in the incidence of foramina (small centers of unossified bone) in the skulls of foetal mice exposed to 5000 ppm chloroethane. At this concentration, 5 foetuses were affected in a total of 5 litters vs. 1 foetus in 1 litter in the controls and in each lower exposure group (the skull bones were examined in 22 to 25 litters in the controls and at each exposure level). Historically, the incidence of foramina in the skull bones of CF-1 mice is low (mean = 0.2% of foetuses, range = 0 - 1.2%); however, as cited by the authors the toxicological significance of a minor delay in ossification such this is questionable.
There were no other changes to suggest any treatment-related effects in any of the exposed groups.




Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEC
Remarks:
teratogenicity
Effect level:
13 385 mg/m³ air (nominal)
Based on:
test mat.
Basis for effect level:
other: no teratogenicity noted up to and including 13385 mg/m³
Key result
Dose descriptor:
NOAEC
Remarks:
embryo-fetal toxixity
Effect level:
4 015 mg/m³ air (nominal)
Based on:
test mat.
Basis for effect level:
other: delayed ossification (increased incidence of foramina in the skull bones)

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
13 385 mg/m³ air
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes

Any other information on results incl. tables

Incidence of fetal skeletal alterations among litters in mice

 

Chloroethane [ppm]

0

500

1500

5000

Number of foetuses (litters) examined for alterations in the skull bones

257 (22)

299 (25)

311 (26)

242 (22)

Foramina of the skull bones; percent (number) of foetuses affected

1 (1)

1 (1)

1 (1)

4 (5)*

Foramina of the skull bones; percent (number) of litters affected

5 (1)

4 (1)

4 (1)

23 (5)

 *: Different from control value by a censored Wilcoxon test, α = 0.05

Applicant's summary and conclusion

Conclusions:
Exposure of pregnant CF-1 mice to chloroethane vapors for 6 hours per day on days 6 through 15 of gestation at levels up to 5000 ppm produced no indication of any teratogenic effects. However, a slight delay in ossification was noted at 5000 ppm.
Executive summary:

The objective of this study was to evaluate the teratogenic potential of inhaled ethyl chlorid in CF-1 mice. Groups of 30 bread mice were exposed to EtCl vapors at concentrations of 0, 500, 1500 or 5000 ppm (0, 1338, 4015 and 13385 mg/m³) for 6 hours per day on days 6 through 15 of gestation.

No significant effects on maternal body weights, liver weights, reproductive parameters or fetal body weights were observed at any of the exposure levels tested. Examination of fetal mice for external, visceral and skeletal malformations failed to produce any indication of a teratogenic response. A small increase in the incidence of foramina of the skull bones (small centers of unossified bone) suggestive of, at most, very slight fetotoxicity was observed only at the high exposure concentration (5000 ppm).

In conclusion, exposure to ethyl chloride at concentrations up to 5000 ppm during the period of major organogenesis did not produce a teratogenic response in fetal mice.