Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Referenceopen allclose all

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study predates any guideline. In this study, animals were exposed to the test substance for 13 weeks prior to mating. Although this is not according to the current OECD testing guideline 415, the information of this test can be used as supporting information. This study was also peer-reviewed under auspices of WHO.
Qualifier:
no guideline available
Principles of method if other than guideline:
Exposure via the drinking-water at one dose level during a period of 13 weeks before mating. Parameters studied were the female fertility index, litter size, survival of pups at 4 weeks and the number of resorptions.
GLP compliance:
no
Species:
rat
Route of administration:
oral: drinking water
Duration of treatment / exposure:
13 weeks
Remarks:
Doses / Concentrations:
125 mg/L
Basis:

Dose descriptor:
NOAEC
Effect level:
>= 125 mg/L drinking water
Sex:
female
Basis for effect level:
other: no effects on female fertility index and litter size
When rats received methylene chloride in the drinking-water at a level of 125 mg/litre during a period of 13 weeks before mating, no effects were found on the female fertility index, litter size, survival of pups at 4 weeks or the number of resorptions.
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
>= 125 mg/L drinking water
Sex:
male/female
Basis for effect level:
other: no effects on pup survival (4 weeks) or number of resorptions
Reproductive effects observed:
not specified
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
OECD test guideline 1983
Principles of method if other than guideline:
Although prescribed by OECD guideline 416, DCM was not tested until a toxic level. However, the highest dose (1500 ppm = ca. 5300 mg/m3) is approximately equal to a limit dose of 1000 mg/kg bw/d (assuming a respiratory rate of the rat of 0.2 L/min, a body weight of 250 g (defaults listed in the REACh guidance) and correcting for a 5 days/week exposure in stead of 7 days/week). Therefore, the dose levels applied are not considered to deviate from the ones stipulated by the guideline.
Oestrus cycle, sperm parameters, organ weights, implantation sites, histopathology data were not collected, but were not routinely required under OECD TG 416 as conducted at the time.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female Fischer 344 rats (Charles River Breeding Laboratory,Kingston, PIY) approximately six weeks of age were purchased for the study. Upon arrival at the laboratory, the rats were examined for health status by a qualified professional and acclimated to the laboratory environment. For the randomization procedure, rats were weighed and ranked according to body weight and those from the extremes of the distribution were identified and removed from the population until only the number of rats required for the study remained. The remaining animals were randomly assigned by weight to the exposure groups.
Animals were housed singly in wire-mesh, stainless steel cages in racks. provided with DACB deotized cage board (Kal Shepherd Specialty Paper, Kalmazoo, MI) to minimize odor and provide a clean environment. During the non-exposure periods of late gestation and lactation , females were housed in plastic shoe-box cages with ground corn-cob nestingmaterial . The rooms were designed t o maintain humidi ty (approximately 40-60%) and temperature (approximately 22°C), a 12-hour photocycle and at least 12 air changes/hr. A pressure activated stainless steel water nipple (Automatic Water Supply, Edstrom Inc., Waterford, WI) was a component of all cages and water was available ad libitum including during exposure periods. A basal diet of Purina Certified Rodent Chow #5002 (Ral ston Purina Co., St. Louis, MO) was available ad libitum except during exposure periods.
Individual identification o f the animals was accompl.ished by inserting a numbered metal tag in one ear of each rat. In the event that an ear tag became dislodged during the course o f the study, a replacement ear tag with the same number was inserted.
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
Inhalation exposures were conducted in 14.5 cubic meter chambers with stainless steel pyramidal-shaped ceilings and epoxy-resin coated floors and walls. All chambers were operated under dynamic airflow conditions at a slight negative pressure relative to the surrounding area. Control animals were placed in an identical chamber. The air supplied to the chambers was controlled by a system designed to maintain temperature and relative humidity at approximately 21°C and 50%, respectively.
Methylene chloride test atmospheres were generated by metering the liquid test material at controlled rates into glass J-tubes. The liquid was vaporized in the tubes with preheated compressed air and was subsequently swept into the main chamber airstream where further dilution to the desired concentration occurred. The compressed air was preheated with a flameless heat torch (Master, Model FHT-4) at the lowest temperature necessary to facilitate complete vaporization of the liquid test material. Total chamber airflow was maintained at approximately 2200 liters per minute.
Details on mating procedure:
Breeding of F0 and Fl adults commenced after 14 and 17 weeks, respectively, of exposure. Each breeding program consisted of two 5-day cohabitation periods (1 ma1e: l female). For the f0 breeding period, the male was placed in the female cage. Due to a change in standard operating procedures, females were placed with the males for the fl breeding period. After the first 5-day mating period, the males (f0) or females (fl) were rotated and bred with a different rat from the respective exposure group. For the fl mating, habitation of male and female littermates was avoided. During the breeding period, vaginal smears were examined daily from each female to determine day 0 of gestation. After determining a female was sperm-positive, the animals were returned to their original cages and exposed to the appropriate level of methylene chloride.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test material in each chamber was determined at least once per hour with a Miran 1A infrared spectrometer at a wavelength of 7.95 microns. The nominal concentration (ratio of methylene chloride used to the total airflow through the chamber) was calculated for each chamber. The concentration of methylene chloride in the exposure chamber was determined by interpolation from a standard curve derived from vapor standards of known concentrations. Air standards were made by injecting calculated volumes of MC into Saran bags filled with a measured volume of filtered compressed air. Standard curves were run prior to the first exposure and at least monthly thereafter. A standard bag was run before each daily exposure to check the analytical system.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
5 day/week for 14 weeks
Details on study schedule:
The exposure to MC began when the f0 rats were approximately 7 weeks of age. After 14 weeks of exposure (5 days/week, excluding one holiday), F0 animals were allowed to mate using one male and one female of the respective treatment group to produce the F1 litters. Following weaning (4 weeks of age) of the last F1 litter, 30 males and 30 females from each treatment group were randomly selected (with the aid of a random number table) and assigned to the respective exposure groups. After approximately 17 weeks of exposure (5 days/week, excluding one holiday), the F1 adults were allowed to mate to produce the F2 litters. Exposure of F0 and Fl male and female adult rats to methylene chloride continued until they were sacrificed. Exposure of F0 and F1 rats, during the pre-mating period was conducted for 6 hr/day, 5 days/week, excluding holidays. During the mating, gestation and lactation periods, exposures were conducted 6 hr/day, 7 days/week. Animals were housed continuously in exposure chambers following the initial exposure to MC except during late gestation (after day 18 of gestation) and lactation when female rats were housed outside of exposure chambers in reproduction cages, containing corn-cob bedding during non-exposure periods. In addition, dams were not exposed from gestation day 21 (as calculated from day 0 of gestation following sperm positive vaginal smears) through the fourth day post-partum t o allow for delivery and rearing of the young. Non-pregnant females also were not exposed to methylene chloride vapors for a 4-day period to equalize the number of exposure days between pregnant and nonpregnant females. During the lactation period, neonates were not placed in exposure chambers, but remained in the nesting cages separated from the dams for 6 hr/day on lactation days 5 through 28. At weaning, adult rats were exposed to the respective level of MC until sacrificed.


Remarks:
100, 500 and 1500 ppm (target)
Remarks:
101 +/- 3, 500 +/- 8 and 1501 +/- 10 ppm (analytical)
No. of animals per sex per dose:
30/sex/concentration
Control animals:
yes
Parental animals: Observations and examinations:
Parental Data
Physical Observations. Each animal on study was observed for signs of toxicity and changes in demeanor at least once a day. Any pups found dead during lactation were examined for grossly visible effects including cleft palate and discarded.

Body Weight.
Body weights of all F0 and F1 animals were recorded prior to the initial exposure and at weekly intervals prior to mating. Weekly body weights for adult males were recorded after breeding had been completed. Body weights of sperm-positive females were recorded on days 0, 7, 14 and 21 of gestation.
Litter observations:
Litter Data.
All litters were examined as soon as possible after delivery. The following parameters were recorded for each of the litters: date of parturition, litter size on the day of parturition, number of live and dead pups on the day of parturition (day zero), number of live pups on day 1, 4, 7, 14, 21 and 28 after delivery, weight of the litter and lactating female on days 1, 4 (before and after culling) 7, 14, 21 and 28 of lactation, and individual body weights for each male and female pup (on day 28).
Postmortem examinations (parental animals):
The scheduled necropsy of the F0 and F1 adult rats was performed after the last litter of F1 and F2 pups, respectively, had been weaned. The adult males and females were fasted overnight, weighed, and anesthetized with methoxyflurane. The trachea was clamped and the rat sacrificed by decapitation. Lungs were infused with neutral phosphate-buffered 10% formalin to their approximate normal inspiratory volume. The nasal cavity was flushed with formalin via the pharyngeal duct to ensure rapid fixation of the tissue. The eyes were examined in-situ by gently pressing a glass slide against the cornea and observing the eyes under fluorescent light illumination. The rat which died spontaneously was necropsied as described above; however due t o the rapid development of postmortem corneal artifacts, a detailed eye examination was not performed. Tissues routinely collected were saved from these animals and preserved in neutral phosphate-buffered 10% formalin. Histologic examination of tissues was not-performed.

The following tissues were collected: liver, pancreas, heart, brain, spleen, pituitary, peripheral nerve, spinal cord, bone marrow, adrenal, kidney, stomach, small intestine, cecum, large intestine, mesenteric lymph node, mesenteric tissue, testicle, epididymis, seminal vesicle, coagulating gland,prostate, uterus, ovary, oviduct, cervix, vagina, urinary bladder, lungs, skeleta1 muscle,salivary gland, thymus, mediastinal tissue, mediastinal lymph node, aorta, esophagus, thyroid gland, parathyroid gland, trachea, larynx, skin, mammary gland, eye, tongue, nasal tissues, lacrimal gland, oral tissues, bone, auditory sebaceous glands
Postmortem examinations (offspring):
At the time of weaning, 10 pups/sex/exposure level from the F1 litters and 30 pups/sex/exposure level from the F2 litters were randomly selected for a complete gross necropsy examination by a veterinary pathologist . The pups were anesthetized with methoxyflurane, the trachea was clamped and the pups sacrificed by decapitation. Lungs were infused to their approximate normal inspiratory volume with neutral phosphate-buffered 10% formalin. Eyes were examined using the glass microscope slide technique. Tissues examined histologically were processed by conventional techniques, stained with hematoxylin and eosin and evaluated by light microscopy.
Statistics:
Body weights were evaluated by Bartlett's test for equality of variances (Winer, 1971). Based upon the outcome of Bartlett's test, a parametric or non-parametric analysis of variance (ANOVA) was performed. If the ANOVA was significant, a Dunnett's test (Steel and Torrie, 1960) or Wilcoxon's rank sum test with Bonferroni 's correction (Mil ler, 1966). The fertility indices were analyzed by the Fisher exact probability test (Seigel, 1956). Evaluation of the neonatal sex ratio was performed by the binominal distribution test (Steel and Torrie, 1960). Survival indices and other incidence data among neonates was analyzed using the litter as the experimental unit by the Wilcoxon test as modified by Haseman and Hoel (1974). Statistical out1iers for body weights were identified by a Sequential test (Grubbs , 1969). The statistical tests identified specific values requiring further scientific evaluation. The true level of statistical significance remains unknown because statistical tests on multiple, interrelated parameters increase alpha, the risk of Type-1 errors (false positive). The nominal alpha levels used are as follows:
Bart1ett' s Test for Variances alpha = 0.01
Analysis of Variance alpha = 0.10
Dunnett's Test alpha = 0.05 two-sided
Wilcoxon Rank-Sum Test alpha = 0.05 two-sided with Bonferroni Correction (Miller, 1966)
Outlier Determination alpha = 0.02 two-sided
Fisher's Test alpha = 0.05 one-sided
Censored Wilcoxon Test a = 0.05 one-sided (modified by Haseman and Hoel )
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
not required by TG in absence of gross findings
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Description (incidence and severity):
not required by TG
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
not required by TG
Reproductive performance:
no effects observed
No adverse effects.
Dose descriptor:
NOAEC
Effect level:
>= 1 500 other: ppm (5300 mg/m3)
Sex:
male/female
Basis for effect level:
other: No adverse effects observed in any of the dose groups.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Description (incidence and severity):
not required by TG
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
not required by TG in absence of gross findings
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Description (incidence and severity):
not required by TG in absence of gross findings
No adverse effects.
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
>= 1 500 other: ppm (5300 mg/m3)
Sex:
male/female
Basis for effect level:
other: No adverse effects observed in any of the dose groups.
Dose descriptor:
NOAEC
Generation:
F2
Effect level:
>= 1 500 other: ppm (5300 mg/m3)
Sex:
male/female
Basis for effect level:
other: No adverse effects observed in any of the dose groups.
Reproductive effects observed:
not specified
Conclusions:
Exposure of rats to concentrations as high as 1500 ppm (ca. 5300 mg/m3) methylene chloride, which has been shown in a 2-year study to produce treatment-related liver effects and increased incidence of benign mammary tumors, did not affect any of the reproductive parameters examined.
Executive summary:

Exposure of rats to concentrations as high as 1500 ppm methylene chloride (ca. 5300 mg/m3), which has been shown in a 2-year study to produce treatment-related liver effects and increased incidence of benign mammary tumors, did not affect any of the reproductive parameters examined.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Study duration:
subchronic
Species:
rat
Quality of whole database:
NOAEL is at least 125 mg/L drinking water per day. This study predates any guideline. In this study, animals were exposed to the test substance for 13 weeks prior to mating. Although this is not according to the current OECD testing guideline 415, the information of this test can be used as supporting information. This study was also peer-reviewed under auspices of WHO.
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Study duration:
subchronic
Species:
rat
Quality of whole database:
NOAEC is at least 5300 mg/m3. GLP compliant guideline study.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Humans (see also section 7.10)

One group of case studies reported reproductive effects in men who complained of central nervous system dysfunction following occupational

exposure to dichloromethane. The men had recent histories of infertility and complained of genital pain; the testes were atrophied in two workers.

Infectious disease was eliminated as a cause of these reproductive effects. Uncertainty regarding this study involves the small number of subjects,

the multiple exposure to other organic chemicals, the lack of blood or urine samples quantifying exposure to dichloromethane, and the lack of a

control group (Kelly, 1988). Contrary to this study, Wells et al. (1989) found no evidence of oligospermia in workers who had been exposed to levels

of dichloromethane that were twice as high as in the Kelly study.  

A study of 50 men working on aircraft maintenance at an US Air Force installation exposed to jet fuel and solvent concentrations below 6 ppm

(i.e. 21 mg/m3 for dichloromethane) for individual solvents found some effects on sperm generation but no clear association was found between

biological exposure measurements and spermatogenic changes (Lemasters et al., 1999).

 

In a retrospective study of pregnancy outcomes among Finnish pharmaceutical workers during the late 1970s, female workers at eight factories

had a higher rate of spontaneous abortions compared to the general population (Taskinen et al. 1986). It is likely that the women were exposed to

multiple solvents, including dichloromethane, prior to conception, as well as during pregnancy. In a corresponding case-control study of

pharmaceutical workers who had spontaneous abortions, exposure to various solvents, including dichloromethane, was associated with a

slightly higher abortion rate. Exposure to dichloromethane was associated with an odds ratio (OR) for spontaneous abortion of 2.3, but the

increase was of borderline significance. In view of the borderline statistical significance and because of the confounding multiple solvent exposure,

this study cannot be used to firmly establish the role of dichloromethane in induction of miscarriage. No other associations with dichloromethane

exposure were found (ATSDR, 2000; SCOEL, 2009), and thus there is no strong epidemiological evidence to suggest that dichloromethane

induces effects on fertility.

Animals

A two-generation reproductive toxicity study in rats exposed to dichloromethane by inhalation at concentrations of up to 5300 mg/m3, 6 h/day, 
5 days/week, did not show evidence of an adverse effect on any reproductive parameter, neonatal survival or neonatal growth in either the F0 or F1
generation. The parental and reproduction NOAEC in this respiratory two-generation study were >= 5300 mg/m3.

In the 2 -year inhalation study in mice (see also section 7.7) concentration-related increases were observed in the incidences of testicular atrophy in male mice and uterine and ovarian atrophy in female mice; these effects are considered to be secondary responses to neoplasia (NTP, 1986; Mennear, 1988).

When rats received dichloromethane in the drinking-water at a level of 125 mg/L during a period of 13 weeks before mating, no effects were found on the female fertility index, litter size,survival of pups at 4 weeks or the number of resorptions.

 

Short description of key information:

No reliable information in humans is available. In a well performed two-generation study with rats, inhalation exposure to dichloromethane concentrations as high as 5300 mg/m3 did not have any effect on fertility. In an oral study, dichloromethane did not induce changes in fertility at a level of 125 mg/L drinking water.

Justification for selection of Effect on fertility via oral route:

Oral study

Justification for selection of Effect on fertility via inhalation route:

Well performed inhalation study according to OECD 416 (1983)

Effects on developmental toxicity

Description of key information

In humans no adverse developmental effects were demonstrated. In the key developmental study, female rats and mice were exposed to 4,300 mg/m3 for 7 hours/day on gestation days 6-15. This level was shown to be a LOAEC for maternal toxicity based on increased carboxyhaemoglobin levels. Only minor visceral and skeletal variations were observed in the foetuses (Schwetz et al., 1975). These variations have not been confirmed in other oral or inhalation studies in rats exposed at higher dose or concentration levels, e.g. during exposure on gestation days 0-17 at a level of 15900 mg/m3 (Hardin & Manson, 1980).

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Only English abstract and tables/figures available. GLP status unclear, study comparable to guideline, published in peer reviewed literature, limitations in design and reporting. The information of this test can be used as supporting information.
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:
Part of the fetuses was examined on day 20 of pregnancy and in another part neonatal growth was measured for 8 weeks after birth. No further specifications, but the review documents by WHO (1996) and ATSDR (2000) considered the study results acceptable. Only 9-17 animals were used per group instead of 20.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Route of administration:
oral: feed
Details on mating procedure:
no data
Duration of treatment / exposure:
days 0-20 of pregnancy
Frequency of treatment:
daily
Duration of test:
ca. 11 weeks
Remarks:
Doses / Concentrations:
0, 0.04, 0.4 and 4.0 %
Basis:
nominal in diet
No. of animals per sex per dose:
9-17
Control animals:
yes
Fetal examinations:
Part of the fetuses was examined on day 20 of pregnancy and in another part neonatal growth was measured for 8 weeks after birth.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Maternal body weight was significantly reduced in the 4% group.
Dose descriptor:
NOAEL
Effect level:
0.4 other: % in diet
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 4 other: % in diet
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There was a slight but statistically significantly reduction (p<0.05) in the fetal weight of the females in the 0.4% group. As this was not seen in males of this group, and not seen in animals treated at 4% in the diet, this slight weight reduction was considered a coincidental finding. There were no differences in any of the groups in the number of implantations and resorptions. No external malformations were observed by fetal, skeletal and visceral examination, and no differences were observed in any group in the frequency of delayed ossifications or in the dilation of the renal pelvis. A slight decrease (p<0.05) in postnatal weight gain was observed in males of the 0.4% group from week 7 to week 8. No such change was seen in females. As the change was slight and occurring 7-8 weeks after birth (whils their mothers were treated from day 0 to day 20 of pregnancy), this change was considered to be toxicologically minor, if at all. Absolute liver and kidney weight were increased (p<0.05) in male offspring of the 0.4% group. However, relative weight was not increased and as such this finding was not considered to be toxicologically relevant. Relative kidney and liver weight were slightly increased (p<0.05) in males and females of the 0.04% group but as this was not seen at the higher dose group of 0.4%, this finding was not considered to be toxicologically relevant.
Dose descriptor:
NOAEL
Effect level:
>= 4 other: % in diet
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
No developmental effects were observed in rats up to and including a level of 4% dichloromethane in the diet.
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
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
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.

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
Remarks:
Doses / Concentrations:
1250 ppm
Basis: target conc.
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).
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.
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.
Dose descriptor:
LOAEC
Effect level:
1 226 other: ppm (4300 mg/m3)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEC
Effect level:
1 226 other: ppm (4300 mg/m3)
Basis for effect level:
other: developmental toxicity
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
Dose descriptor:
NOAEC
Effect level:
>= 4 300 mg/m³ air
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

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)

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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
rat
Quality of whole database:
NOAEL is at least 4% in the diet. Only English abstract and tables/figures available. GLP status unclear, study comparable to guideline, published in peer reviewed literature, limitations in design and reporting. The information of this test can be used as supporting information.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
4 300 mg/m³
Study duration:
subacute
Species:
other: rats and mice
Quality of whole database:
This study predates GLP but sufficient data is available for interpretation of study results.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Humans (see section 7.10)

In a study examining the relationship between birth weights and environmental exposures to dichloromethane, no significant adverse effects on birth weight were found among more than 90,000 single births from 1976 through. The highest predicted environmental concentration of dichloromethane was 0.01 ppm (Bell et al., 1991).

 

Animals

In a key study, DCM was not teratogenic in rats or mice at a concentration of 4300 mg/m3.

In this developmental study, foetal skeletal variations rather than malformations were observed which may have been caused by hypoxia as increased carboxyhaemoglobin levels were seen in the dams and hypoxia is known to affect the developing foetus. In addition, no skeletal malformations or soft tissue anomalies were observed in rats exposed to 15900 mg/m3 for 6 hours/day during 17 days of pregnancy, while the dams also showed increased blood carboxyhaemoglobin levels (7.1-10.1%). Therefore, foetal toxicity might have been mediated by maternal toxicity. A supporting study did not show developmental effects in rats at a level up to 4.0% dichloromethane in the diet. Taking into account a daily food intake of 20 g and a body weight of 300 g, the level of 4% would correspond to about 2500 mg/kg bw.

Although no recently carried out OECD 414 test guideline study is present, and the available studies have some shortcomings (which vary from study to study), overall evaluation of the available data (weight of evidence) does not indicate that dichloromethane causes effects on fertility and foetal development. A key developmental inhalation study has been carried out in two rodent species, viz. mice and rats. Although in this developmental study only one concentration has been tested, this concentration was high (viz.4300 mg/m3) and resulted in limited signs of toxicity but no developmental toxicity (Schwetz et al., 1975). The same observations were made in a second developmental toxiicity study in rats that had been exposed to 15900 mg/m3 on gestation days 0 -17. Changes observed consisted of increased maternal liver weights and COHb levels, and decreased fetal body weight, but there was no significant increase in the incidence of skeletal or soft tissue anomalies (Hardin & Manson, 1980). Also an oral study in rats at a high level of 2500 mg/kg bw did not show developmental toxicity (Nishio et al., 1984). Based on the other studies available, as a weight of evidence, there is no reason to believe, also based on human data, that dichloromethane would induce developmental toxicity. In addition, bioaccumulation is expected to be low; dichloromethane is rapidly and extensively absorbed from the lungs into the systemic circulation (uptake in humans 70-75%) and is well absorbed from the gastrointestinal tract of animals (uptake 97%). Dichloromethane is distributed to many organs, including liver, kidney, lungs, brain, muscle and adipose tissue, after respiratory and oral exposure. However, dichloromethane is quite rapidly excreted after oral exposure, mostly via the lungs in the exhaled air. It can cross the blood-brain barrier and be transferred across the placenta, and small amounts can be excreted in urine or in milk. At high doses, most of the absorbed dichloromethane is exhaled unchanged. The remainder is metabolized to carbon monoxide, carbon dioxide and inorganic chloride, whereby two routes of oxidative metabolism have been identified, one mediated by cytochrome P450 (predominantly in humans) and the other by glutathione-S-transferase (especially in mice). Dichloromethane can also be absorbed via the skin. However, due to its high volatility this route of exposure is of less relevance than other routes of exposure under non-occlusive conditions. Relevant uptake via the skin will only occur under accidental occlusive conditions.

Based on the rationale explained above, all developmental toxicity data combined are in compliance with pertinent REACH guidance. In addition, Janer et al. (2008) noted in their retrospective analysis that even though the combination of rat plus rabbit study will increase the probability of identifying developmental toxicity as compared to a single rat study, a developmental toxicity in other species (e.g. mice) might also lead to the indentification of developmentally toxic substances not identified in a particular rat study. With regard to dichloromethane both rats and mice were tested, and more than one rat study is available, using different routes of exposure. Moreover, recent evaluations by other bodies such as the SCOEL (2009) and OECD SIDS (2013) of the same data (and also the older evaluations by ATSDR (2000) and WHO (1996)), indicate that the available data are appropriate and sufficient to conclude about the absence of developmental toxicity. The extent of data available, also in humans, and the long and widespread use of DCM, also do not warrant an additional developmental toxicity study in rabbits. Therefore, an additional study on developmental toxicity in rabbits (Annex X, 8.7 column 2) will not provide any new information and is therefore scientifically and ethically (animal welfare) not justified.

Janer H, Slob W, Hakkert BC, Vermeire T & Piersma AH (2008) A retrospective analysis of developmental toxicity studies in rat and rabbit: What is the added value of the rabbit as an additional test species? Regul Toxicol Pharmacol 50, 206-217

Justification for selection of Effect on developmental toxicity: via oral route:

Oral OECD 414 study

Justification for selection of Effect on developmental toxicity: via inhalation route:

Inhalation study similar to OECD 414

Justification for classification or non-classification

The available data do not indicate that dichloromethane causes effects on fertility and foetal development. Classification is not warranted according to EU Directive 67/548/EEC and Regulation (EC) No. 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation.

Additional information