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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Although GLP status is not known (not indicated in the publication), the study is a guideline study, published in peer reviewed literature; there are no restrictions, therefore fully adequate for assessment

Data source

Reference
Reference Type:
publication
Title:
Methylene chloride: A 2-year inhalation toxicity and oncogenicity study in rats
Author:
Nitschke KD, Burek JD, Bell TJ, Kociba RJ, Rampy LW, McKenna MJ
Year:
1988
Bibliographic source:
Fundam Appl Toxicol 11, 48-59

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Technical-grade methylene chloride (Lot No. TA 05038 C) from Dow Chemical Co., Plaquemine, Louisiana, USA, 99.5 % purity

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Spartan Research Animals, Inc., Haslett, Michigan
- Age at purchase: 6-8 weeks old
- Housing: 3/cage in wire-bottom stainless-steel cages
- Diet (e.g. ad libitum): Purina Laboratory Chow, Ralston-Purina Co., St. Louis, MO, ad libitum, except during exposure periods
- Water (e.g. ad libitum): ad libitum, except during exposure periods
- Acclimation period: at least 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): ca. 50
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks:
unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: Not applicable (vapour)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless-steel and glass chambers of 4.1 m3 volume.
- System of generating particulates/aerosols: exposure concentrations of methylene chloride were generated by metering the liquid at a constant rate with a precision pump into a warmed vaporization flask (ca. 60 C). Vapours frim the flask were swept with compressed air into the main chamber airstream where further mixing and dilution occured.
- Temperature, humidity, pressure in air chamber: 22 C, 50%
- Air flow rate: 800 l/min
- Air change rate: 12/hr

TEST ATMOSPHERE
- Brief description of analytical method used: IR spectroscopy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of methylene chloride in each chamber was determined 1-2 times each hour by IR spectroscopy using a Miran I IR spectrophotometer (Foxboro/Wilks, South Norwalk, CT) at a wave length of 7.95 to 13.35 microm.
Duration of treatment / exposure:
2 years
Frequency of treatment:
6 hours/day, 5 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50, 200, and 500 ppm
Basis:
other: target concentrations
Remarks:
Doses / Concentrations:
50+/-3, 199+/-5, 499+/-10 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
90 (male), 108 (female)
Control animals:
yes
Details on study design:
- Rationale for selecting satellite groups: In addition to the main experiment, 30 female rats, identified as 500con, were exposed to 500 ppm methylene chloride for the first 12 months and to room air for the last 12 months of the study. Thirty additional female rats, identified as con500, were exposed to room air for the first 12 months and 500 ppm methylene chloride for the last 12 months of the study. These animals were added to assess the temporal relationship between the initial exposure to methylene chloride and expression of the liver and mammary lesions. A subgroup of 18 female rats/exposure level was used to measure liver DNA synthesis. All remaining animals (70/sex/exposure group and 25 female rats from 500con and con500 exposure groups) were the major portion intended for oncogenic evaluation.
A separate, predesignated sub-group of 18 female rats/exposure level was used to mea­sure the rate of liver DNA synthesis. Groups of 4 female rats/exposure level were selected by a computer-derivedrandomization procedure from this subgroup after 6 and 12 months of exposure to methylene chloride. Approximately 18 hr after the last designated exposure to methylene chloride, each rat was injected ip with 0,0054 mg/kg [3H]thymidine. All rats were sacrificed 6 hr following thep injection. At the time of termination the liver was removed, the DNA was extracted, and [3H]thymidine in-corporation into the DNA was determined. All remaining animals in this subgroup were terminated without a gross examination after 12 months.

- Section schedule rationale (if not random): Subgroups of 5 rats/sex/exposure level were scheduled for interim terminations after 6, 12, 15, and 18 months of exposure to methylene chloride. Subgroups of 5 female rats from each of the 500con and con500 exposure groups were sacrificed at the 18 month interim necropsy.
Male rats were necropsied after 20 months due to the relatively limited lifespan of this substrain of male rat. All surviving female rats were necropsied after 24 months of exposure to methylene chloride.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: each day after exposure for signs of toxicity, also prior to exposure and on weekends and holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: examinations of all rats for palpable masses were conducted prioir to the intial exposure to DCM, and at monthly intervals after the first 12 months, except for the subgroup designated for measuring liver DNA synthesis.

BODY WEIGHT: Yes
- Time schedule for examinations: all rats were weighed prior to the initial exposure, twice a month for the first 3 months of the study and monthly thereafter.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at various interim necropsies, also at multiple time intervals for determining carboxyhemoglobin levels
- How many animals: 4-5 for determining carboxyhemoglobin levels
- Parameters examined: total birubin (except at 6 months), cholesterol, triglyceride, potassium, estradiol (except at 6 months), FSH and LH levels.
Sacrifice and pathology:
All rats were subjected to a complete gross necropsy examination.
An extensive set of tissues intended to include all those collected at the interim and terminal kills were embedded in paraffin, sectioned and stained for microscopy.
Other examinations:
Liver DNA synthesis: a separate, predesignated subgroup of 18 female rats/exposure level was used to measure the rate of DNA liver synthesis.
Statistics:
Carboxyhemoglobin levels, rate of thymidine incorporation into the liver, absolute and relative organ weights, body weights, and clinical chemistry determinations were evaluated by analysis of variance and Dunnett's test (Steel and Torrie, 1960). Palpable mass data was analyzed by the Wilcoxon test (Haseman and Hoel, 1974). Gross pathologic observations and histopathologic data from interim kills lvere not evaluated statistically. Incidences of mofality and remaining histopathologic observations were evaluated by Fisher's Exact Probability Test (Siegel, 1956). Because numerous measurements were statistically compared on the same group of animals, the frequency of false positive (type I ) errors is unknown but is greater than the nominal a level of 0.05 used for Dunnett's and Fisher's Exact Probability Test. In addition to results of statistical evaluation, the final toxicologic interpretation of the data considered other factors such as dose-response relationships, biological plausibility, consistency, and historical values.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
No exposure-related gross or histopathologic changes were observed in animals from interim sacrifice groups. At terminal sacrifice, the incidences of both hepatocellular cytoplasmic vacuolization consistent with fatty changes and multinuceated hepatocytes were statistically elevated in female rats exposed to 200 and 500 ppm of methylene chloride; a slight increase in the incidence of hepatocellular vacuolization was also observed in male rats exposed to 500 ppm. Histopathological changes in the liver were not found in the male rats exposed to 50 or 200 ppm of methylene chloride. No other pathologic or histopathologic nontumor findings were reported.

Effect levels

Dose descriptor:
NOAEC
Effect level:
200 ppm
Sex:
male/female
Basis for effect level:
other: Histopathological changes in liver of male and female rats

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Mortality and Observations

 

The mortality rates for the various groups of male and female rats exposed to methylene chloride were comparable to control values. However, due to the early onset of geriatric changes which is characteristic of this substrain of rat and which occurred in all groups of male rats, exposure of male rats was terminated after 20 months of exposure to methylene chloride and the remaining survivors were necropsied. Exposure to 50, 200, or 500 ppm methylene chloride had no discernable adverse effect on the animals' appearance during the course of the study. Group mean body weights of male and female rats exposed to methylene chloride were not adversely affected throughout the 24-month study.

 

 

Absolute and Relative Organ Weights, Clinical Chemistry, Plasma Hormone Levels and Carboxyhaemoglobin

 

Absolute and relative organ weights, clinical chemistry values, and plasma hormone levels were not altered in rats exposed to methylene chloride (data not shown). Occasional statistically significant differences between control and exposed group mean values were considered to be sporadic occurrences unrelated to exposure. Blood carboxyhaemoglobin levels of rats exposed to methylene chloride were elevated in an apparent dose-dependent relationship above control values (Table). The percentage carboxyhaemoglobin was similar within exposure groups after 6, 12, or 20 -24 (terminal kill) months, indicating a lack of accumulation with repeated exposure.

 

Table - Carboxyhaemoglobin values° of rats exposed to methylene chloride

ppm

Sex

Time interval

6months

12months

Terminal

kill

0

male

4.8 ± 2.6°

0.3 ± 0.7

2.2 ± 1.3

50

male

8.8 ± 2.0

2.8 ± 0.3*

6.5 ± 1.1*

200

male

14.3 ± 1.3*

9.6 ± 1.2*

12.5 ± 0.8*

500

male

16.7±2.4*

12.7±1.6*

13.7±0.6*

0

female

1.1 ± 0.4

2.6 ± 1.1

5.1 ± 3.4

50

female

6.3 ± 2.7*

8.2 ± 3.1*

5.9 ± 0.7

200

female

13.9 ± 1.1*

13.1 ± 1.4*

13.8 ± 1.0*

500

female

17.9 ± 2.9*

17.5 ± 1.5*

17.6 ± 2.9*

° %, mean ± standard deviation for four to five rats/ sex/exposure level.

* Significantly different from control value by Dun­nett's or Wilcoxon's test, a < 0.05.

 

 

DNA Synthesis

 

The incorporation of [3H]thymidine into hepatic DNA of female rats exposed to 50, 200, or 500 ppm methylene chloride for either 6 or 12 months was comparable to control animals. There was no detectable alteration in the rate of DNA synthesis in the liver of rats exposed to concentrations as high as 500 ppm methylene chloride.

 

 

Pathology and Palpable Mass Data

 

No gross or histopathological lesions were observed in rats from the interim kill groups which were attributed to exposure to methylene chloride. Exposure-related histopathological changes observed in rats that died spontaneously or at the terminal kill were confined to the liver and mammary tissue. For the liver, the incidence of both hepatocellular cytoplasmic vacuolization (consistent with fatty change) and multinucleated hepatocytes was statistically increased in female rats exposed to 500 ppm methylene chloride. A slight increase in the incidence of hepatocellular vacuolization was observed in malerats exposed to 500 ppm methylene chloride (22 of 70 control animals vs. 28 of 70 animals exposed to 500 ppm methylene chloride). However, these liver effects were not observed in male or female rats exposed to 50 or 200 ppm methylene chloride. In female rats exposed to 200 ppm methylene chloride, the incidence of multinucleated hepatocytes was numerically but not statistically increased above the rate for control animals; this effect was considered to be of doubtful biologic significance.

See for a summary on palpable masses and tumours section 7.7 (Nitschke et al., 1988).

Applicant's summary and conclusion

Conclusions:
Histopathological changes were seen in liver of male and female rats at 500 ppm. The NOAEC was 200 ppm.