dichloromethane; methylene chloride

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1974

Report date:

1974

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

1975

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

see below

Principles of method if other than guideline:

This study was carried out before OECD guidelines existed. Only one concentration was tested, instead of three. However, this concentration showed limited signs of toxicity; in addition, two species (rats and mice) were tested. Therefore, sufficient data is available for interpretation of study results.

GLP compliance:

no

Remarks:

did not exist at that time

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dichloromethane

EC Number:

200-838-9

EC Name:

Dichloromethane

Cas Number:

75-09-2

Molecular formula:

CH2Cl2

IUPAC Name:

dichloromethane

Details on test material:

purity: 97.86%

Test animals

Species:

other: rats and mice

Strain:

other: Rat-Sprague-Dawley Mice-Swiss Webster

Details on test animals or test system and environmental conditions:

Sprague-Dawley female rats weighing approximately 250 g were used along with Swiss-Webster mice weighing 25 to 30 g. Both were obtained from the Spartan Research Animals, Incorporated, Hasslet, Michigan. Between exposures, animals were housed in wire-bottom cages in a room controlled for temperature, humidity and light cycle. Commercial laboratory rat chow and water were available free choice. Food and water were not provided during exposure to solvents.

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

Exposure was conducted in 3.7 cubic meter stainless steel, cubical exposure chamber. Vapors of the solvent, generated by metering the liquid at a known rate into a temperature controlled evaporator flask, were diluted with filtered room air at a rate calculated to give the desired concentration. The nominal concentration in each chamber atmosphere was calculated from the ratio of the rate of delivery of each solvent to the rate of total air flow through the chamber (350-400 L/min).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The actual concentration was analysed three times daily using a Beckman IR10 infrared spectrophotometer with a multipath gas cell. In addition, the concentration in the chamber was monitored continuously using a recording combustion analyzer to assure the absence of significant deviations from the desired level.

Details on mating procedure:

Natural mating; the day on which sperm were seen in a vaginal smear of rats or a vaginal plug was observed in mice was considered day zero of pregnancy.

Duration of treatment / exposure:

seven hours daily

Frequency of treatment:

on days 6-15 of gestation

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1250 ppm
Basis: target conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
1226 +/- 76 ppm
Basis: analytical conc.

No. of animals per sex per dose:

Control: 24 animals
1250 ppm group - 13 mice and 19 rats

Control animals:

yes

Details on study design:

Groups of nonpregnant female mice and rats were exposed simultaneously with the pregnant females. Blood samples for analysis were collected by orbital sinus puncture immediately following the third and tenth (last) exposure as well as 24 hours after the tenth exposure. Carboxyhemoglobin determinations were performed using the spectrophotometric method of Buchwald (1969).

Examinations

Maternal examinations:

Food consumption of each rat or cage of two rats was measured at 2-day intervals throughout the experimental period. The food consumption of mice was not measured. All rats and mice were observed daily throughout pregnancy and maternal body weights were recorded on days 6, 10 and 16 of gestation as well as on the day on which cesarean sections were performed, gestation days 21 and 18 in rats and mice, respectively. Prior to cesarean section, the dams were sacrificed by exposure to carbon dioxide.

Ovaries and uterine content:

After the uterine horns were exteriorized through a mid-line incision in the abdominal wall, the number and position of live, dead and resorbed fetuses were noted.

Fetal examinations:

Subsequently, the umbilical cord of each fetus was clamped and severed distally and the fetuses were removed. After being weighed, measured (crown-rumplength) and sexed, the fetuses were examined for external anomalies. Each litter was divided equally into two subgroups for preservation and subsequent examination. One subgroup, preserved in Bouin's solution, was examined by the method of Wilson (1965) for evidence of soft tissue anomalies. The second subgroup, preserved in alcohol, was cleared and stained with Alizarin red-S (Dawson, 1926) for examination for evidence of skeletal anomalies. One fetus randomly selected from each litter was preserved in buffered 10% formalin. Saggital sections (6 micron thickness) of the whole body were stained with hematoxylin and eosin for microscopic examination.

Statistics:

The Fisher Exact probability test (Siegel, 1956) was used to evaluate the incidence of anomalies and resorptions among litters. Maternal and fetal body weights and body measurements, food consumption values, liver weights and carboxyhemoglobin levels were analyzed statistically by an analysis of variance and Dunnett's test (Steel and Torrie, 1960).' In all cases, the chosen level of significance was p<0.05. The litter was considered the experimental unit of treatment and observation.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: increases in COHb

Details on maternal toxic effects:
The food consumption in rats was unaffected by exposure; food consumption of mice was not measured. Increased body weight (11-15%; p<0.05) was seen in mice exposed to 2500 ppm at GD 10, 16 and 21. However, a decreased body weight rather than an increased body weight would be considered a toxic effect. An increased absolute liver weight (p<0.05) was observed in exposed rats and mice, but as this was not accompanied by an increase in relative liver weight this increase in absolute weight was not considered to be toxicologically relevant. Elevated carboxyhemoglobin levels (p<0.05) in both mice and rats followed exposure to methylene chloride (after 3rd and 10th exposure). These levels were back to control 24 h after exposure.

Effect levels (maternal animals)

1

Dose descriptor:

LOAEC

Effect level:

1 226 other: ppm (4300 mg/m3)

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

LOAEC

Effect level:

1 226 other: ppm (4300 mg/m3)

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no teratogenic effects (no efefcts on averga enumber of implantation sites per litter, litter size, incidence of fetal resorptions, fetal sex ratios or detal body weight measurements. The incidence of lumbar ribs or spurs was significantly DECREASED compared to that of controls while the incidence of delayed ossification of sternebrae was significantly greater than in controls. Microscopic examination of sagittal sections of whole fetuses revealed no abnormalities of organs, tissues or cells as a result of maternal exposure to dichloromethane.
of organs, tissues or cells as a result of maternal exposure to any of the solvents

Effect levels (fetuses)

Dose descriptor:

NOAEC

Effect level:

>= 4 300 mg/m³ air

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Maternal body weights during gestation, liver weights and carboxyhemoglobin (mean ± SD)

		0 ppm	1250 ppm
mice
number of animals		24	13
bodyweight (g)	GD6	34 ± 3	36 ± 4
	GD 10	37 ± 4	41 ± 5*
	GD 16	53 ± 6	60 ± 7*
	GD 18	55 ± 7	63 ± 11*
liver weight (g)		2.8 ± 0.5	3.4 ± 0.5*
liver weight (mg/g bw)		52 ± 9	55 ± 10
carboxyHb (%) **	after 3rd	1.7 ± 2.1	12.6 ± 3.8*
	after 10th	1.1 ± 1.7	9.8 ± 2.2*
	24h after 10th	0.5 ± 1.0	0.5 ± 1.1
rats
number of animals		25	19
bodyweight (g)	GD 6	260 ± 13	262 ± 10
	GD 10	283 ± 16	281 ± 12
	GD 16	319 ± 21	317 ± 16
	GD 21	380 ± 31	383 ± 27
liver weight (g)		13.6 ± 2.9	14.7 ± 1.9*
liver weight (mg/g bw)		36 ± 5	38 ± 4
carboxyHb (%) ***	after 3rd	0.4 ± 0.7	10.3 ± 2.3*
	after 10th	0.4 ± 0.6	8.9 ± 1.7*
	24h after 10th	1.6 ± 1.8	1.0 ± 0.9
* p<0.05 ** n = 10 (control) or 9 (treatment) *** n = 8 (control and treatment)

Litter parameters (mean ± SD)

	Mice		Rats
	0 ppm	1250 ppm	0 ppm	1250 ppm
number litters	24	13	25	19
corpora lutea/dam	–	–	14 ± 2	13 ± 2
implantation sites/litter	14 ± 3	15 ± 3	12 ± 3	11 ± 4
live foetuses/litter	12 ± 4	13 ± 4	11 ± 3	10 ± 4
% resorptions/ implantation sites	14 (46/325)	14 (26/190)	6 (19/295)	8 (17/216)
% litters with resorptions	75 (18/24)	46 (6/13)	44 (11/25)	37 (7/19)
litters totally resorbed	1	0	0	0
resorptions/litter with resorptions	2.6 (46/18)	1.4 (14/10)	1.7 (19/11)	2.4 (17/7)
sex ratio (M:F)	54:46	46:54	52:48	54:46
foetal body weight (g)	1.34 ± 0.11	1.32 ± 0.08	5.81 ± 0.40	5.74 ± 0.36
foetal length (mm)	26.2 ± 0.7	26.1 ± 0.6	43.6 ± 1.0	43.6 ± 0.9

Foetal examinations (% (number) affected litters)

	Mice		Rats
	0 ppm	1250 ppm	0 ppm	1250 ppm
Number litters examined	22	12	25	19
GROSS
short tail	(0)	(0)	3 (1)	(0)
runts (wt < mean-3SD)	32 (7)	8 (1)	4 (1)	10 (2)
SOFT TISSUE
cleft palate	(0)	17 (2)	--	--
dilated renal pelvis	--	--	4 (1)	26 (5)*
rotated kidney	(0)	17 (2)	--	--
displaced kidney	--	--	4 (1)	(0)
subcutaneous oedema	45 (10)	42 (5)	12 (3)	(0)
dilated oesophagus	--	--	(0)	5 (1)
SKELETAL
delayed ossification – skull	36 (8)	25 (3)	20 (5)	21 (4)
lumbar ribs or spurs	68 (15)	58 (7)	32 (8)	5 (1)*
delayed ossification – sternebra	23 (5)	17 (2)	(0)	26 (5)*
split sternebra	18 (4)	8 (1)	(0)	16 (3)
extra sternebra	14 (3)	50 (6)*	--	--
misaligned sternebra	27 (6)	8 (1)	--	--

* p<0.05 by Fischer exact test

-- not reported, assumed (0)

Applicant's summary and conclusion

Conclusions:

The results of this study indicate that exposure of pregnant mice and rats to two times the maximum excursion limit of dichloromethane (1250 ppm; ACGIH, 1973) caused little or no maternal, embryonal or fetal toxicity. Dichloromethane did not cause a teratogenic response in either mice or rats. The level of 4300 mg/m3 was established to be a LOAEC for developmental toxicity (mild foetotoxicity) and for slight maternal toxicity.

Executive summary:

The results of this study indicate that exposure of pregnant mice and rats to 1250 ppm dichloromethane caused little or no maternal, embryonal or fetal toxicity. Elevated carboxyhemoglobin levels in both mice and rats were noted following exposure to dichloromethane. Dichloromethane did not cause a teratogenic response in either mice or rats.