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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
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 Type:
publication
Title:
Unnamed
Year:
1986

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: in drinking water
Details on test material:
Food-grade DCM supplied by Diamond Shamrock Industries (Cleveland, OH, USA). Specifications published by Krishman et al. (1986): Kirschman JC, Brown NM, Coots RH, & Morgareidge K (1986) Review of investigations of dichloromethane metabolism and subchronic oral toxicity study as the basis for the design of chronic oral studies in rats and mice. Food Chem Toxicol, 24: 943-949.

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, MI
- Age at study initiation: ca. 7 weeks old
- Housing: individually in stainless-steel hanging wire-mesh cages
- Diet (e.g. ad libitum): Purina Rodent Laboratory Chow (No. 5001), ad libitum
- Water (e.g. ad libitum): deionized water provide from water bottles with stainless steel ballpoints, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 70.5 +/- 1.75 F
- Humidity (%): 53.9 +/- 5.82
- Photoperiod (hrs dark / hrs light): 12/12


Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
An appropriate amount of DCM was added to deionized water on a volume/volume basis to yeld a 1% stock solution. A sample was analysed prospectively to ensure that the concentration was within +/- 5% of the desired target level. Volumes of the stock solution were added to glass carboys and diluted with deionized water to yield the desired concentratios of DCM. During the first 13 weeks, dosage formulations were adjusted to the most recently recorded body weight and water consumption data; thereafter, they were adhysted every 4 weeks. Fresh dilutions were prepared and presented four times/week, twice for each sex. Samples from three randomly selevted bottles, which had been on the animal cages for a 4-day period, were anlyzed for DCM concentration weekly for the first 12 weeks and at least every 4 weeks thereafter.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In order to ensure that the animals received the proper dosages of the test material, samples of the drinking-water administered to all groups were routinely analysed throughout the course of the study. Quantitative measurements of DCM concentrations in the aqueous delivery system were made utilizing headspace analysis by gas chromatography (Perkin-Elmer Sigma II gas chromatograph using a glass column, 6 ft x 4 mm ID packed with 0.2% Carbowas 1500 on Carbopack C, 00/80 mesh). All standards and test-solution analyses were averaged from at least duplicate injections.
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 6, 52, 125, 235, 232 (recovery, 18 months exposure) mg/kg bw/day (males)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
0, 6, 58, 136, 263, 269 (recovery, 18 months exposure) mg/kg bw/day (females)
Basis:
actual ingested
No. of animals per sex per dose:
85 (main experiment), 25 (high dose, recovery), 50 (recovery control group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were selected on the basis of findings from subchronic and pharmacokinetic studies of DCM as described by Kirschmanm, Brown, Coots and Margareidge (1986). The findings from a 90-day drking-water study in ratws revealed histopathological effects on the livers of males and females dosed at levels of 166 and 209 mg/kg/day, respectively. Pharmacokinetic studies revealed a significant change in the slope of DCM equivalents expired as carbonmonoxied and carbon dioxide at dose levels of approx. 1000 mg/kg/day, indicating a saturation of the metabolic process at higher dose levels. Therefore, the dose levels for this study were selected to induce liver toxicity at the highest level and to have two dose levels above and two below the apparent metabolic saturation point.

- Rationale for selecting satellite groups: an additional group of rats (the recovery group) was exposed to 250 mg/kg bw/day for 78 weeks followed by 26 weeks without DCM treatment, to determine whether any toxicity was reversible with time.

- Section schedule rationale (if not random): after treatment for 26, 52 and 78 weeks, 5, 10 and 20 animals, respectively, of each sex, preselected prior to initiation of the study, were killed from control group 1 and from each of the DCM-treated groups (except the recovery group) to monitor the effects of DCM treatment over time.
Positive control:
Not used.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily throughout the study for deaths and signs of moribund condition. During the second year of the study, a late-night observation was also performed on 5 nights/week.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations were recorded on all rats weekly throughout the study.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to treatment and weekly throughout the study. Fasted body weights were also recorded on all rats killed as scheduled for organ/body weight ration determinations.

FOOD CONSUMPTION:
- Food consumption was measured weekly for the first 13 weeks for all rats

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations:
- Water consumption was measured twice weekly (after 3- and 4-day periods) throughout the study for all rats, adn the values were combined to yeld the weekly water consumption

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to treatment and after treatment for 26, 52, 78 and 103 weeks, all rats were subjected to an ophthalmoscopic examination. Examinations were performed using an indirect ophthalmoscope after instillation of a 1% tropicamide ophthalmic solution (Mydriacyl, Alcon Kaboratories, Inc., For Worth, TX) to induce mydriasis.
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after treatment for 52 and 78 weeks, blood samples were collected from rats scheduled for the interim kill from control group 1 and from groups receiving DCM at levels of 5, 50, 125 and 250 mg/kg/day.
- Anaesthetic used for blood collection: not used
- Animals fasted: yes, overnight
- How many animals: 10 of each sex
- Parameters examined: haematocrit and haemoglobin determinations, total erythrocyte count, total and differential leucocyte counts, calculation of mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration, estimation of prothrombin time and examination of erythrocyte and leucocyte morphology.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after treatment for 52 and 78 weeks, blood samples were collected from rats scheduled for the interim kill from control group 1 and from groups receiving DCM at levels of 5, 50, 125 and 250 mg/kg/day.
- Animals fasted: yes, overnight
- How many animals: 10 of each sex
- Parameters examined: glutamic-pyrubic transaminase, glutamic-oxalacetic transaminase, alkaline phosphatase, total cholsterol, creatinine, blood urea nitrogen, total protein and glucose.

URINALYSIS: Yes
- Time schedule for collection of urine: after treatment for 52 and 78 weeks, urine samples were collected from rats scheduled for the interim kill from control group 1 and from groups receiving DCM at levels of 5, 50, 125 and 250 mg/kg/day.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes, overnight
- Parameters examined: protein, pH, ketones, glucose and occult blood


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All rats received a complete necropsy.

HISTOPATHOLOGY: Yes
The following tissues were taken and preserved in 10% neutral buffered formalin: brain, spinal cord (three levels), pituitary, femur, pancrease, mesenteric lymph node, thyroid and parathyroids, adrenals, heart, lung, spreen, liver, kidneys, stomach, duodenum, jejunum, ileum, colon, caecum, rectum, sternum marrow, costochondral rib junction, tongue, Zymbal gland, testes, ovaries, prostate, urinary bladder, uterus, epididymides, seminal cesicles, salicary glands, thymus, oesophagus, trachea, larynx, aorta, thigh muscle, sciatic nerve, eyes, mammary gland, skin and lesions. In addition, the hard and soft palates, and the nasal turbinates were taken from rats killed by design after treatment for 26 and 52 weeks.
Statistics:
Body weight, food and water consumption, haem¬atology (except differential leucocyte counts), serum chemistry and organ weight data were evaluated using a preliminary test for homogeneity of variance (Bartlett, 1937) followed by a one-way classification analysis of variance-ANOVA (Winer, 1971). Fisher's least significant difference (LSD) test (Miller, 1981) was used to perform control versus treatment mean comparisons for data with homogeneous vari¬ances and significant ANOVA. In the case of significant ANOVA of data with heterogeneous vari¬ances, Wilcoxon's two-sample nonparametric rank-sum test (Hollander & Wolfe, 1973) was used for the same purpose. Linear regression analysis and lack of fit tests (Draper & Smith, 1966) were performed on all data by sex against each control group or com¬bined controls, where appropriate. A significance level of P = 0.05 was used for all tests, with the group comparisons being two-tailed. Survival was evaluated for trend and heterogeneity by the Gehan-Breslow (Generalized Kruskal-Wallis) test. Additional graph¬ical evaluation of the survival data was performed using the Kaplan-Meier method. All of the above analyses were performed with the National Cancer Institute Package (Thomas, Breslow & Gart, 1977).
All tumour data were analysed in two phases. The unadjusted tumour incidences were analysed by the Cochran-Armitage test for trend and chi-square and Fisher-Irwin exact tests for heterogeneity (Thakur, Berry & Mielke, 1985). Intercurrent mortality-adjusted tumour data (for `incidental' tumours) were analysed by the prevalence method of Dinse & Lagakis (1983). All other binary incidence data were analysed by the methods used in the case of unadjusted tumour analysis.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
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:
no effects observed
Details on results:
Survival, clinical observations, body weights and food and water consumption
Overall survival rates among male and female groups were 76 and 71%, respectively, with no significant treatment-related trend or heterogeneity in
any of the groups in the study. No treatment-related clinical observations were noted. Incidental observations common to the rat were seen with similar frequency among control and treated groups of both sexes. Palpable masses were noted occasionally and were generally described as subcutaneous masses in the axillary or inguinal regions. Lower body weights and body-weight gains were observed generally throughout the study in both sexes treated with DCM at levels of 125 or 250 mg/kg/day, the differences from the controls being small but statistically significant. Small but statistically significant decreases in water consumption values were observed in both sexes treated with DCM at levels of 125 or 250 mg/kg/day generally throughout the study. Concomitant decreases in food consumption values were noted in the same groups during the first 13 wk of treatment.

Ophthalmology
No treatment-related ophthalmoscopic findings were noted during the study.

Haematology, serum chemistry and urinalysis
Increases in mean haematocrit, haemoglobin and erythrocyte count were observed in both sexes treated with DCM at levels of 50, 125 or 250 mg/kg/day for 52 or 78 wk. Approximately half of these increased mean values were determined to be statistically significant. Results of all other mean haematology and erythrocyte indices were consistent among all groups. Decreases in alkaline phosphatase, creatinine, blood urea nitrogen, total protein and cholesterol values were generally noted in both sexes of the higher-dose groups. The majority of these mean values were determined to be statistically significant at one or both of the intervals evaluated. Results of all other parameters were consistent among all groups. No treatment-related effects on urinalysis were noted during the study.

Organ weights and organ/body weight ratios
No treatment-related effects on organ weights or organ/body weight ratios were observed during the study. Instances of statistically significant positive and negative differences were noted in several organs, but were considered to be unrelated to treatment because of the lack of meaningful and distinct dose-related trends.

Pathology
Scattered incidental findings in gross pathology were noted across all groups and were considered to be unrelated to treatment. In the histopathological examinations, no treatment-related effects were noted in any of the tissues examined, with the exception of the liver. Histomorphological alterations of the liver were observed. A dose-related positive trend in the incidences of foci/areas of celIular alteration was noted in both sexes treated with
DCM. Group comparisons were statistically significant at all levels except 5 mg/kg/day. This finding was first noted after treatment for 78 wk and
progressed until wk 104. In addition, an increased incidence of fatty change in the liver was observed in tissues stained with haematoxylin and eosin, and was confirmed by staining with Oil Red O. This effect was generally observed in both sexes treated with DCM at levels of 50, 125 or 250 mg/kg/day at both wk 78 and wk 104. An increase in the incidence of foci/areas of cellular alteration, similar to that noted in the 250 mg/kg/day group, was observed in the recovery group receiving 250 mg/kg/day. However, the fatty change was less severe in the recovery group than in the group receiving 250 mg DCM/kg/day for 104 wk.

Effect levels

Dose descriptor:
NOAEL
Effect level:
6 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Increases in mean haematocrit, haemoglobin erythrocyte count. Increased incidences of foci/areas of cellular alteration and fatty change in the liver at 52/58 mg/kg bw.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1. Estimates of mean daily consumption of dichloromethane (DCM) by groups of rats given DCM in the drinking-water for 2 yr

Group

Target level (mg/kg/day)

Mean intake (mg/kg/day)

Males

Females

low

5

6

6

mid 1

50

52

58

mid 2

125

125

136

high

250

235

263

high (recovery)*

250

232

269

*this group did not receive DCM after 18 months, so the mean for this group is for the first 18 months only.

Survival,clinical observations, body weights and food and water consumption

Overall survival rates among male and female groups were 76 and 71%, respectively, with no significant treatment-related trend or heterogeneity in any of the groups in the study. No treatment-related clinical observations were noted. Lower body weights and body-weight gains were observed generally throughout the study in both sexes treated with DCM at levels of 125 or 250 mg/kg/day, the differences from the controls being small but statistically significant. Small but statistically significant decreases in water consumption values were observed in both sexes treated with DCM at levels of 125 or 250 mg/kg/day generally throughout the study. Concomitant decreases in food consumption values were noted in the same groups during the first 13 wk of treatment.

 

Haematology, serum chemistry and urinalysis

Increases in mean haematocrit, haemoglobin and erythrocyte count were observed in both sexes treated with DCM at levels of 50, 125 or 250 mg/kg/day for 52 or 78 wk. Approximately half of these increased mean values were determined to be statistically significant. Results of all other mean haematology and erythrocyte indices were consistent among groups.

Decreases in alkaline phosphatase, creatinine, blood urea nitrogen, total protein and cholesterol values were generally noted in both sexes of higher-dose groups. The majority of these mean values were determined to be statistically significant at one or both of the intervals evaluated. Results for all other parameters were consistent among groups.

No treatment-related effects on urinalysis were noted during the study.

Organ weights and organ/body weight ratios

No treatment-related effects on organ weights organ/body weight ratios were observed during the study. Instances of statistically significant positive and negative differences were noted in several organs but were considered to be unrelated to treatment because of the lack of meaningful and distinct do related trends.

Pathology

Scattered incidental findings in gross pathology were noted across all groups and were considered be unrelated to treatment.

In the histopathological examinations, no treatment-related effects were noted in any of the tissues examined, with the exception of the liver. Histomorphological alterations of the liver were observed and are presented in Table 2. A dose-related positive trend in the incidences of foci/areas of cellular alteration was noted in both sexes treated with DCM. Group comparisons were statistically significant at all levels except 5 mg/kg/day. This finding was first noted after treatment for 78 wk and progressed until wk 104. In addition, an increased incidence of fatty change in the liver was observed in tissues stained with haematoxylin and eosin, and was confirmed by staining with Oil Red O. This effect was generally observed in both sexes treated with DCM levels of 50, 125 or 250 mg/kg/day at both wk 78 and wk 104. An increase in the incidence of foci/areas of cellular alteration, similar to that noted in the 50-250 mg/kg/day group, was observed in the recovery group receiving 250 mg/kg/day. However, the fatty change was less severe in the recovery group than in group receiving 250 mg DCM/kg/day for 104 wk.

Table 2. Incidence of liver foci/areas of alteration in rats given dichloromethane (DCM) in the drinking-water for up to 2 yr.

 

 

Incidence of lesion* in rats given a DCM target dose (mg/kg/day) of:

Sex

0

0

5

50

125

250

250**

Wk78

Males

7/20

--

3/20

15/20

13/20

20/20

--

Females

3/20

--

11/20

14/20

16/20

17/20

--

Wk 104

Males

27/36

25/40

22/34

35/38

34/35

40/41

15/15

Females

17/31

17/36

12/29

30/41

34/38

31/34

17/20

*No. of rats affected/no. examined. **Recovery group. Bold: statistically significant differences with control group.

See for a summary on tumour data, section 7.7 (Serota et al., 1986, rat study)

Applicant's summary and conclusion

Conclusions:
The 2-year NOAEL for oral toxicity was 6 mg/kg bw/day in rats, based on increased values of haematological parameters and increased foci/areas of cellular alteration and fatty changes in the liver. It was, however, noted that (a) approximately half of the increased mean values of the haematology parameters was statistically significant and no dose-response relationship was mentioned, and (b) increased foci/areas of cellular alteration and fatty acid change in the liver were based on incidence; nothing was mentioned on severity.