chloromethane; methyl chloride

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented publication which meets basic scientific principles

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Reproduction in Fischer-344 rats exposed to methyl chloride by inhalation for two generations

Author:

Hamm, T.E. et al.

Year:

1985

Bibliographic source:

Fundamental and Applied Toxicology; 5(3):568-577

Reference 2

Reference Type:

secondary source

Title:

Chloromethane CAS: 74-87-3

Author:

OECD SIDS

Year:

2004

Bibliographic source:

SIDS Initial Assessment Report for SIAM 15 ; www.iccahpv.com

Reference 3

Reference Type:

secondary source

Title:

Methyl Chloride

Author:

Löf, A. et al.

Year:

2000

Bibliographic source:

Concise International Chemical Assessment Document 28

Reference 4

Reference Type:

secondary source

Title:

Toxicological review of methyl chloride (CAS No. 74-87-3)

Author:

Greenberg, M. and Riddle, J.

Year:

2001

Bibliographic source:

U.S. Environmental Protection Agency, EPA/635/R01/003

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

females (P and F1) not exposed gestation day 18 to postnatal day 4, influence on oestrous cycle length not reported, only brain, liver and testes weight recorded.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Chloromethane

EC Number:

200-817-4

EC Name:

Chloromethane

Cas Number:

74-87-3

Molecular formula:

CH3Cl

IUPAC Name:

chloromethane

Details on test material:

no further data
- Name of test material (as cited in study report): methyl chloride
- Physical state: gaseous
- Analytical purity: 99.98%
- Impurities (identity and concentrations): only detected impurity was water
- Lot/batch No.: L24-0095

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY, USA
- Weight at study initiation: (P) males: 100-186 g; females: 73-127 g
- Housing: individually in hanging stainless steel mesh cages (Hazleton)
- Diet: ad libitum NIH-07 open formula pelleted chow except during exposure
- Water: ad libitum purified tap water
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 +/- 1.1
- Humidity (%): 50 +/- 5
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 8m³ glass and stainless steel inhalation chamber
- Source and rate of air: air supply was filtered using HEPA Type-A filters
- Method of conditioning air: chloromethane was metered with a calibrated flowmeter and diluted with filtered chamber supply air
- Air flow rate: 2000 L/min


TEST ATMOSPHERE
- Brief description of analytical method used: chloromethane analysis was done using an infrared gas analyzer
pathlenght: 20.25 m, analytical wavelength: 9.991 µm, slit opening: 1 mm
- Samples taken from breathing zone: yes, hourly throughout exposure

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: until copulation plug was visible or up to 1 week after that the male was placed individually with a second female
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- Further matings after one unsuccessful attempt: no
- After successful mating each pregnant female was caged (how): in polycarbonate cages with hardwood chip bedding

10 males were necropsied after mating. The remaining 30 males per group were bred during the next 2 weeks to two unexposed females and all data recorded as described for the preceding mating. These males were housed for 10 additional weeks.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

analyses were done using an infrared gas analyzer
time weighted average analytical concentrations in the 4 chambers were:
0 ppm (0 mg/m³)
151 +/- 4 ppm (312 +/-8 mg/m³)
472 +/- 7 ppm (975 +/- 14 mg/m³)
1502 +/- 19 ppm (3102 +/- 39 mg/m³)

Duration of treatment / exposure:

females (P):
10 weeks prior to mating (5 days/week); then 7 days/week for weeks during mating and to gestation day (GD 18); exposure discontinued from GD18 to postnatal day (PND) 4; and exposure resumed from PND 4 to PND 28.

males (P):
10 weeks prior to mating (5 days/week); then 7 days/week for 2 weeks during mating

males and females (F1):
F1 pups were exposed starting from day PND 28 to the same concentration as their parents for 10 weeks and then mated.

Frequency of treatment:

males and females: 6h/d

Details on study schedule:

10 males were necropsied after mating. The remaining 30 males per group were bred during the next 2 weeks to two unexposed females and all data recorded as described for the preceding mating. These males were housed for 10 additional weeks.

Doses / concentrations

Remarks:

Doses / Concentrations:
150, 475 and 1500 ppm (310, 981 or 3098 mg/m³)
Basis:
nominal conc.

No. of animals per sex per dose:

P: 40 exposed males/group, 80 exposed females/group, 60 non-exposed females/group
F1: 40 males and 80 females in the control group, 150 and 1500 ppm dose group; 23 males and 46 females in the 475 ppm dose group

Control animals:

yes, sham-exposed

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
Drom GD 20 until parturition every hour from 7 am to 7 pm

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (except for females during pregnancy)

Sperm parameters (parental animals):

Parameters examined in [P/F1] male parental generations:
testis weight, sperm count in testes, sperm count in epididymides, sperm morphology

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible). Excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 ] offspring:
PND 0: number and sex of pups, number and sex of live pups, combined weight of male pups and combined weight of female pups
PND 4, 14, 21 and 28: number and sex of pups, combined weight by sex


GROSS EXAMINATION OF DEAD PUPS: no data

Postmortem examinations (parental animals):

SACRIFICE
- Male animals:
F0: 10 males/dose at the end of the breeding period, recovery group: 10 males/dose after 12 weeks after first breeding, 10 males/dose 30 weeks after first breeding, 10 males/dose 32 weeks after first breeding
F1: 15 males/dose at the end of weaning
- Maternal animals:
P: 25 females/dose at the end of weaning
F1: 15 females/dose at the end of weaning


GROSS NECROPSY/ORGAN WEIGHTS
- Gross necropsy consisted of external examination of carcass, weighing of whole animal, gross examination

ORGAN WEIGHTS:
weighing of brain, liver and testes

HISTOPATHOLOGY
collected tissues were: brain, liver, adrenals, kidneys, testes, epididymides, ovary, oviduct, uterine horn, cervix and any lesion found

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at the end of weaning
- These animals were subjected to postmortem examinations as described above (parental animals)

GROSS NECROPSY
- Gross necropsy as described above (parental animals)


HISTOPATHOLOGY / ORGAN WEIGTHS
as described above (parental animals)

Statistics:

one tailed Fisher test with Bonferroni correction or Arcsin sqare root transformation and ANOVA
Normality: Kolmogrov-Smirnov test (n>50) or Wilk-Shapiro test (n<=50)
If normality was not achieved data were ranked prior to ANOVA.
If ANOVA was significant Dunnetts test was applied to compare those groups to controls.
Significance: p<0.05

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

BODY WEIGHT (PARENTAL ANIMALS):
3098 mg/m³ (1500 ppm): Both males (after day 15 of exposure) and females (after day 7 of exposure) developed a significant body weight gain depression of 10-20% with the time of exposure.

981 mg/m³ (475 ppm): Both males (after day 57 of exposure) and females (after day 57 of exposure) developed a significant body weight gain depression of 5-7% with the time of exposure.

310 mg/m³ (150 ppm): No significant effects on body weight gain at any time in males were observed. Females had a 3% body weight gain depression (after day 57 of exposure).

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS):
3098 mg/m³ (1500 ppm):
treatment related lesions: minimal to severe atrophy of 60-80 % of the seminiferous tubulus (10/10 males) and granulomas in the epididymides (3/10 males)
severly affected seminiferous tubuli: lined by Sertoli's cells and by stem cell spermatogonia
less severly affected tubuli: decreased number of spermatogonia primary spermatocytes, and/or secondary spermatocytes
epididymides: few sperm, sloughed spherical cells

981 mg/m³ (475 ppm):
no treatment related lesions were found
normal tubuli: spermatids, sperm

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
3098 mg/m³ (1500 ppm):
no litters born to exposed or unexposed females mated with exposed males

981 mg/m³ (475 ppm):
significantly fewer litters born to exposed or unexposed females mated with exposed males

310 mg/m³ (150 ppm):
no data reported

Number of females with copulation plugs was not statistically different in all dose groups, therefore mating behaviour was not influenced by chloromethane.

ORGAN WEIGHTS (PARENTAL ANIMALS):
3098 mg/m³ (1500 ppm):
testes weight was reduced about 50% in males

981 mg/m³ (475 ppm):
no data reported

310 mg/m³ (150 ppm):
no data reported


OTHER FINDINGS:
recovery group males mated after a period of 10 weeks without exposure to unexposed females
3098 mg/m³ (1500 ppm): body weight became comparable to control group 9 weeks after stop of exposure; partial recovery of fertility; histopathological findings were still evident; no epididymal granulomas were found.

981 mg/m³ (475 ppm):
body weight increased, no difference to control animals; fertility not affected

recovery group males mated after a period of 80 weeks without exposure to unexposed females:
no further improvement in fertility

Effect levels (P0)

Key result

Dose descriptor:

NOAEC

Effect level:

310 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

reproductive performance

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING): Litter size, sex ratio, pup viability, pup survival and pup growth were not affected by chloromethane among the 475, 150 ppm or control group.

SEXUAL MATURATION (OFFSPRING):
981 mg/m³ (475 ppm): tendency toward decreased fertility (the animals had fewer litters than the control group), significantly decreased percentage of males in the F2 generation, significantly decreased male and female pup (F2) growth from day 14 to 21

310 mg/m³ (150 ppm): no significant effect on reproduction 

Effect levels (F1)

Dose descriptor:

NOAEC

Effect level:

310 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

sexual maturation

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

	Number (percent) of exposed males proven fertile
	0 mg/m³	310 mg/m³	981 mg/m³	3098 mg/m³
F0 generation: mated to exposed females	18/40 (45%)	20/39 (51%)	12/40 (30%)	0/40 (0%)
F0 generation: mated to unexposed females	23/28 (82%)	21/28 (75%)	12/28 (43%)	0/26 (0%)
F0 generation (recovery 10 weeks): mated to unexposed females	15/20 (75%)	-	13/20 (65%)	5/20 (25%)
F1 generation of both sexes exposed: mated to exposed females	31/40 (78%)	26/40 (65%)	14/23 (61%)	-