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Toxicological information

Toxicity to reproduction

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Administrative data

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

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1985
Report Date:
1985
Reference Type:
publication
Title:
Methylene chloride: two-generation inhalation reproductive study in rats
Author:
Nitschke KD, Eisenbrandt DL, Lomax LG, Rao KS
Year:
1988
Bibliographic source:
Toxicol Sci 11 (1), 60-67

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Mehtylene chloride, purity 99.86%

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals 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.

Administration / exposure

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.


Doses / concentrationsopen allclose all
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

Examinations

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 )

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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

Details on results (P0)

No adverse effects.

Effect levels (P0)

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.

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

No adverse effects.

Effect levels (F1)

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.

Results: F2 generation

Effect levels (F2)

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.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

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.