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Developmental toxicity / teratogenicity

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developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Jun - 14 Jul 2015
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted Jan 2001
GLP compliance:
yes (incl. QA statement)
Health Care Inspectorate of the Ministry of Health, Welfare and Sport, Utrecht, The Netherlands
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
gas under pressure: liquefied gas
Details on test material:
- Name of test material (as cited in study report): Methyl Chloride, MeCl
- Physical state: colourless, odourless gas
- Storage condition of test material: 15-25ºC

Test animals

New Zealand White
Details on test animals or test system and environmental conditions:
- Source: Centre Lago, Vonnas, France
- Age at study initiation: 4 - 5 months
- Weight at study initiation: 3.36 - 4.46 kg
- Housing:individually in type I rabbit cages (65.3 x 65.3 x 45 cm). The cages and bedding were changed at least weekly and cage enrichment was supplied.
- Diet: rabbit diet, batch 1556 and 1788 (Stanrab, SDS Special Diets Services, Witham, England), ad libitum, and apples
- Water: tap water, ad libitum
- Acclimation period: 5 days

- Temperature (°C): 18 ± 3
- Humidity (%): 39.7 - 71
- Air changes (per hr): approximately 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16 Jun 2015 To: 14 Jul 2015

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
whole body
unchanged (no vehicle)
Details on exposure:
- Exposure apparatus: 2200 liter whole body exposure chamber (Hazleton Systems, Inc. Aberdeen, MD, USA), constructed of stainless steel, with glass doors on two sides which allow observation of the animals during exposure.
- Method of conditioning air: a controlled flow of gaseous test material was mixed with controlled flows of clean humidified air
- Temperature, humidity, pressure in air chamber: 22 ± 3˚C, relative humidity 40 - 70%
- Air flow rate: at least 22 m³/h or 367 L/min, monitored at least 3 times/day
- Air change rate: at least 10 times/h

- Brief description of analytical method used: the concentration of the test substance in the test atmospheres was measured by total carbon analysis (Sick Maihak EuroFID total carbon analyzer). Test atmosphere samples were taken continuously from the exposure chamber at the animals’ breathing zone and were passed to the total carbon analyzer (TCA) through a sample line. During the period that two exposure chambers per group were used, samples were taken intermittently from both chambers (automatic switch between chambers every 10 minutes). The response of the analyzer was recorded on a PC every minute using a CAN transmitter. The responses of the analyzers were converted to concentrations by means of calibration graphs. For each exposure day, the mean concentration was calculated from the values determined every minute. The test atmosphere flow was measured in the exhaust of the exposure chamber using a KIMO air velocity sensor.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The actual concentration of methyl chloride in the test atmosphere was measured by total carbon analysis or infrared analysis at least once each hour during exposure. The test atmosphere was sampled in the centre of the exposure chambers. The nominal concentration was determined by dividing the total amount of test material used (by weight) by the total volume of air passed through the inhalation chamber. The generation efficiency was calculated from the actual and the nominal concentration (efficiency = actual concentration as percentage of nominal concentration). The overall mean actual concentrations of the test substance in the test atmospheres as measured by total carbon analysis were 265 (± 5), 511 (± 12) and 1012 (± 25) ppm for the low-, mid- and high-concentration, respectively. The actual concentrations were close to the target concentrations (250, 500 and 1000 ppm).
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
Gestation day 6 - 28
Frequency of treatment:
6 h/day
Duration of test:
23 days
No. of animals per sex per dose:
22 females
Control animals:
Details on study design:
- Dose selection rationale: Dose selection was based on a 7-day range-finding study performed with 5 non-mated female New Zealand White rabbits exposed to 1000 ppm daily for 6 h/day. The dose selection for the range-finding study was based on literature (Smith W.W., von Oettingen W.F., 1947, The acute and chronic toxicity of methyl chloride. I Mortality resulting form exposures to methyl chloride in concentrations of 4000 to 300 parts per million., J. Ind. Hyg. Toxicol. 29, 47-52.) that compared various species regarding their sensitivity to methyl chloride. Based on this it was justified to consider the rabbit as sensitive as rats.


Maternal examinations:
- Time schedule: twice daily, before and after exposure. In addition, during exposure a group-wise observation was made halfway through each exposure day. Special attention was directed to breathing abnormalities and restlessness.
- Cage side observations included: abnormalities, signs of ill health, mortality


- Time schedule for examinations: gestation days 3, 6, 9, 12, 15, 18, 21, 24 and 29

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes, determined for the intervals gestation days 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-29
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


- Sacrifice on gestation day 29
- Organs examined: the dams were examined for gross abnormalities. Maternal tissues showing severe macroscopic abnormalities were retained and from all dams the heart was retained in a neutral, aqueous phosphate buffered 4% solution of formaldehyde.
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: empty uterus weight, ovary weight
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter. Fetal hearts were examined in situ based on the technique described by Staples (1974) in order to detect possible malformations on valves and papillary muscles as described in mice by Wolkowsky-Tyl et al (1983). After in situ examination, the fetal hearts were dissected and preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde (10% solution of formalin) for possible future histopathological examination.
- Skeletal examinations: Yes, all per litter. After the visceral examinations the intact and decapitated bodies were eviscerated, fixed in 96% alcohol, cleared in potassium hydroxide and stained with Alizarin Red S. All skeletons of each group were examined for skeletal abnormalities and then retained.
- Head examinations: Yes: half per litter. To examine the head for visceral abnormalities half of the number of foetuses in each litter were decapitated after visceral examination and their heads were fixed in Bouin’s fixative. Subsequently the heads were sectioned free-handed according to Van Julsinga and Bennett (1977). The sections obtained were examined for visceral anomalies.
Tests were generally performed as two-sided tests with results taken as significant where the probability of the results is p < 0.05 (*) or p < 0.01 (**). For incidences of histopathological changes Fisher’s exact probability test was used. The results obtained in non-pregnant animals were not included in the calculation of the means or in the statistical evaluation.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: non-adverse effects at 1000 ppm: decreased body weight gain and food consumption

Details on maternal toxic effects:
1/22 dams in the low-dose group died during exposure on gestation day 26. During the preceding days this animal showed a markedly decreased feed consumption and was recorded to be thin. The animal showed implantation sites in the uterus. The macroscopic examination did not reveal a cause of death, and the mortality was not considered to be exposure-related. Encrustations on the skin in the neck area was observed during the exposure period in 0/22, 3/22, 6/22 and 3/22 dams in the control, low-, mid- and high-dose group, respectively. This may be a result of the type of exposure, but is not considered to be toxicologically relevant.

A non-significant decrease in body weight (< 5%) was observed in all treatment groups in the period from gestation day 6 to 9 (see Table 1 under 'Any other information on results incl. tables'). This is considered to be related to the treatment starting on gestation day 6. In the high-dose group, significantly lower body weight and non-significantly lower body weight gain was observed on gestation day 15 - 21, compared with the control group. For the period from gestation day 24 to 29, the body weight gain in the high-dose group was significantly increased compared with the control group, and there was no significant difference in body weight (see Table 2 under 'Any other information on results incl. tables'). The effects are considered to be related to the reduced food intake noted in some of the high-dose rabbits. As the effect on body weight (gain) was transient, it was considered to be treatment-related, but not toxicologically relevant.

No significant effects on the overall food intake were observed during the exposure period. However, 2/19 animals in the control group, 2/19 in the low-dose group, 1/17 in the mid-dose group and 3/19 in the high-dose group showed markedly reduced feed intake on one or more days during gestation. Half an apple was given to these animals daily in order to stimulate feed intake. 1/19 animals in the low-dose group and 2/19 animals in the high-dose group showed prolonged decreased feed intake and received 40 mL Critical Care ®/day by oral gavage in order to avoid dehydration. Reduced feed intake is a common observation in rabbits and is not considered to be treatment-related.

The results of the post-mortem examinations were comparable between the dams in the control group and the treatment groups.

Out of 22 mated females per group, 19, 18, 17 and 19 females (for the control group, low-, mid- and high-dose group, respectively) were pregnant at necropsy on gestation day 29. One female in the low-dose group was found dead on gestation day 26 and excluded from the data. 1/19 females in the control group had a complete litter loss. There were no significant differences in ovary weight, gravid uterus weight, empty uterus weight, number of corpora lutea, number of implantation sites and early resorptions between the dams in the control group and the treatment groups.

Effect levels (maternal animals)

Key result
Dose descriptor:
Effect level:
>= 1 000 ppm
Based on:
test mat.
Basis for effect level:
other: Effects of a non adverse nature at the highest dose tested

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
A slightly higher number of dead fetuses in the high-dose group (12 fetuses (5.2%), compared with 2 fetuses (1.2%) in the control group) was caused by the complete litter loss (8) of one females. This was not considered to be treatment-related, but related to the poor condition of the dam ascribed to decreased feed consumption. The post-implantation loss was comparable between the control group and treatment groups (13.4% in the control group and 4.8, 6.6 and 10.6% in the low-, mid- and high-dose group, respectively). The mean placental weight and fetus weight was comparable for fetuses in the control and treatment groups. No significant difference in sex ratio was observed in the treatment groups, compared with the control group. The sex of one fetus of a dam in the high-dose group was not determined.
2/161, 2/171, 2/172 and 1/166 of the live fetuses in the control, low-, mid- and high-dose groups, respectively, showed external malformations or variations. Visceral malformations were observed in 0/161, 0/171, 1/172 and 0/166 of the live fetuses in the control, low-, mid- and high-dose groups, respectively.
The incidence of visceral variations was 45/161, 49/171, 46/172 and 48/166 of the live fetuses in the control, low-, mid- and high-dose groups, respectively
Based on the incidence and distribution of the external and visceral malformations and variations observed, these were not considered to be treatment-related effects. The visceral examination of the fetal heart revealed 1/161 fetuses in the control group with a blood-filled pericardium and 2/166 fetuses from one litter in the high-dose group with a fluid-filled pericardium. 1/171 fetuses in the low-dose group and 3/166 fetuses in the high-dose group showed indentation of the apex of the heart. These hearts showed no structural macroscopic changes in atria, ventricles and valves. No malformations were observed with respect to the valves of the heart, chordae tendineae and papillary muscles. Based on the limited number of animals showing these observations in the heart and based on the absence of a concentration-response relationship these observations were not considered to be related to treatment.

Effect levels (fetuses)

Key result
Dose descriptor:
Effect level:
>= 1 000 ppm (analytical)
Based on:
test mat.
Basis for effect level:
other: no effects on the offspring at the highest dose tested

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: Body weight of dams

Gestation day

kg (mean ± SD)



250 ppm

500 ppm

1000 ppm


3.74 ± 0.232

3.82 ± 0.214

3.84 ± 0.248

3.73 ± 0.279


3.89 ± 0.249

3.92 ± 0.196

3.95 ± 0.244

3.80 ± 0.319


3.80 ± 0.257

3.87 ± 0.208

3.87 ± 0.235

3.70 ± 0.270


3.83 ± 0.269

3.91 ± 0.208

3.89 ± 0.243

3.67 ± 0.264


3.91 ± 0.269

3.96 ± 0.219

3.94 ± 0.246

3.71 ± 0.274*


3.99 ± 0.259

4.00 ± 0.249

4.00 ± 0.256

3.78 ± 0.244*


4.07 ± 0.249

4.05 ± 0.258

4.07 ± 0.258

3.86 ± 0.232*


4.12 ± 0.255

4.10 ± 0.265

4.13 ± 0.279

3.94 ± 0.242


4.16 ± 0.230

4.16 ± 0.217

4.16 ± 0.290

4.05 ± 0.257

* p < 0.05

Table 2: Body weight change in dams

Gestation day

g (mean ± SD)



250 ppm

500 ppm

1000 ppm

3 - 6

147.9 ± 95.2

100.2 ± 55.6

113.4 ± 82.6

77.2 ± 97.8

6 - 9

-90.4 ± 58.1

-55.5 ± 66.9

-76.6 ± 58.2

-107.4 ± 98.5

9 - 12

31.3 ± 51.3

46.5 ± 54.2

18.6 ± 40.3

-8.4 ± 58.2

12 - 15

81.3 ± 69.5

46.3 ± 58.4

44.7 ± 61.3

25.4 ± 59.5

15 - 18

75.1 ± 42.8

40.2 ± 58.7

64.7 ± 68.8

64.1 ± 79.9

18 - 21

82.9 ± 57.5

50.2 ± 51.9

63.3 ± 39.8

84.5 ± 101.0

21 - 24

48.4 ± 67.9

44.6 ± 68.6

63.3 ± 62.6

77.3 ± 70.5

24 - 29

41.1 ± 90.1

34.6 ± 45.6

32.9 ± 61.7

109.2 ± 72.6*

3 - 29

417.6 ± 170.0

336.1 ± 135.6

324.4 ± 221.9

321.9 ± 138.2

* p < 0.05

Applicant's summary and conclusion

Methyl chloride had no effect on intrauterine development.