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Carcinogenicity

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Description of key information

Oral:
Two long-term carcinogenicity studies in rat and mice showed tumours in a number of organs (mammary gland adenocarcinomas, squamous cell carcinomas of the forestomach and hemangiosarcomas) after oral 1,2-dichloroethane administration by gavage.
Inhalation:
A 2-yr inhalation exposure of rats and mice to 1,2-dichloroethane produced a dose-dependent increase in incidences of benign and malignant tumors.
Thus the test substance is regarded as carcinogenic after oral and inhalation exposure.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented, but with deviations from the current OECD test guideline method. Acceptable for assessment of a principal carcinogenic potential.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
Deviations:
yes
Principles of method if other than guideline:
Limitations of the study:
Experimental design differs largely from current testprocedure and requirements: Limitations include questionable, unclear TS purity with contaminants not being characterized, potential of influence from other chemicals being tested in the same room (e.g. 1,1-dichloroethane, dibromopropane, trichloroethylene, and carbon disulfide), only 2 dose levels tested, poor survival at the high dose (top dose was distinctly too high contrary to requirements), lack of a third non-toxic lower dose, adjusted and intermittent dosage including prolonged higher doses than the average makes believe, low number of controls, risk irrelevant application mode (gavage) with poor practical relevance. Technical grade TS was used in this study. Data on degree of purity given in this study and in the publication of Ward (1980) are conflicting. According to Maltoni et al. (1980; page 4), technical product was found to contain up to 7% of bis(2-chloroethyl)ether amongst other impurities. However, this study has been regarded as valid key study and the results were used by EPA to derive carcinogenicity potency factors (e.g. unit risk values).
GLP compliance:
not specified
Species:
rat
Strain:
Osborne-Mendel
Sex:
male/female
Details on test animals and environmental conditions:
The Osborne-Mendel rat was selected on the basis of a comparative study of the tumorigenic responsiveness to carbon tetrachloride of five different strains of rats (Reuber and Glover, 1970).
Rats of both sexes were obtained through contracts of the Division of Cancer Treatment, National Cancer Institute. The Osborne-Mendel rats were obtained from the Charles River Breeding Laboratories, Inc., Wilmington, Massachusetts. Upon receipt, animals were quarantined for at least 10 days, observed for visible signs of disease or parasites, and assigned to the various dosed and control groups.
All animals were housed by species in temperature- and humidity controlled rooms. The temperature range was 20° to 24°C and the relative humidity was maintained between 45 and 55 percent. The air conditioning system in the laboratory provided filtered air at a rate of 12 to 15 complete changes of room air per hour. Fluorescent lighting was provided on a 12-hour-daily cycle.
The rats were individually housed in suspended galvanized-steel wire-mesh cages with perforated floors. Rats received sanitized cages with no bedding with the same frequency. Food hoppers were changed and heat-sterilized once a week for the first 10 weeks and once a month thereafter. Fresh heat-sterilized glass water bottles were provided three times a week. Food (Wayne Lab-Blox , Allied Mills, Inc., Chicago, Illinois) and water were available ad libitum.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Intubation was performed for five consecutive days per week on a mg/kg body weight basis utilizing the most recently observed group mean body weight as a guide for determining the dose. Mean body weights for each group were recorded at weekly intervals for the first 10 weeks and at monthly intervals thereafter. All animals of one sex within a treated group received the same dose. Animals were gavaged with the test solution under a hood to minimize extraneous exposure of other animals and laboratory personnel to the chemical.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Two analyses, performed about 14 and 18 months later, suggested no significant decomposition.
Duration of treatment / exposure:
78 wk, including 9 weeks without treatment (wk 36 and every 5th week thereafter)
Frequency of treatment:
5 d/wk
Post exposure period:
Post-exposure period: 15 - 32 wk
Remarks:
Doses / Concentrations:
47 and 95 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
50 male and 50 female animals
Control animals:
other: yes, concurrent vehicle and concurrent no treatment
Details on study design:
The treated and vehicle control rats were all approximately 9 weeks old at the time the experiment began. The initial doses utilized for the first 7 weeks of the experiment for rats of both sexes were 100 and 50 mg/kg/day. Throughout this report those rat groups receiving initial dosages of 100 mg/kg/day are referred to as the high dose groups and those receiving initial dosages of 50 mg/kg/day are referred to as the low dose groups. The high and low doses were increased to 150 and 75 mg/kg/day, respectively, for the next 10 weeks and then decreased again, to 100 and 50 mg/kg/day for the last 61 weeks of chemical administration. In week 36, intubation ceased for all treated animals for 1 week, followed by 4 weeks of dose administration. This cyclic pattern of dosage administration continued for the remainder of the dosing period. The last high dose male rat died during week 23 of the observation period following chemical administration and the last high dose female rat died during week 15 of the observation period. Low dose and vehicle control rats were observed for 32 weeks after dose administration.
The untreated controls received no 1,2-dichloroethane or corn oil, while the vehicle controls were intubated with pure corn oil.
Positive control:
no positive control
Observations and examinations performed and frequency:
Animals were weighed immediately prior to initiation of the experiment. From the first day, all animals were inspected daily for mortality. Body weights, food consumption, and data concerning appearance, behavior, signs of toxic effects, and incidence, size, and location of tissue masses were recorded at weekly intervals for the first 10 weeks and at monthly intervals thereafter. The presence of tissue masses was determined by observation and palpation of each animal.
Sacrifice and pathology:
A necropsy was performed on each animal regardless of whether it died, was killed when moribund, or was sacrificed at the end of the bioassay. The animals were euthanized by exsanguination under sodium pentobarbital anesthesia, and were immediately necropsied. The histopathologic examination consisted of gross and microscopic examination of major tissues, organs, or gross lesions taken from sacrificed animals and, whenever possible, from animals found dead. Slides were prepared from the following tissues: skin, subcutaneous tissue, lungs and bronchi, trachea, bone marrow, spleen, lymph nodes, thymus, heart, salivary gland, liver, gallbladder and bile duct (mice), pancreas, esophagus, stomach, small intestine, large intestine, kidney, urinary bladder, pituitary, adrenal, thyroid, parathyroid, testis, prostate, brain, tunica vaginalis, uterus, mammary gland, and ovary.
Tissues for which slides were prepared were preserved in 10 percent buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin prior to microscopic examination. An occasional section was subjected to special staining techniques for more definitive diagnosis.
A few tissues were not examined for some animals, particularly for those that died early. Also, some animals were missing, cannibalized, or judged to be in such an advanced state of autolysis as to preclude histopathologic interpretation. Thus, the number of animals for which particular organs, tissues, or lesions were examined microscopically varies and does not necessarily represent the number of animals that were placed on experiment in each group.
Other examinations:
none.
Statistics:
- Probabilities of survival were estimated by the product-limit procedure of Kaplan and Meier (1958).
- Statistical analyses for a possible dose-related effect on survival used the method of Cox (1972) for testing two groups for equality and used Tarone's (1975) extensions of Cox's methods for testing a dose-related trend.
- The Cochran-Armitage test for linear trend in proportions, with continuity correction (Armitage, 1971, pp. 362-365), was also used.
- A time-adjusted analysis was applied when numerous early deaths resulted from causes that were not associated with the formation of tumors.
- When appropriate, life-table methods were used to analyze the incidence of tumors.
- The approximate 95 percent confidence interval for the relative risk of each dosed group compared to its control was calculated from the exact interval on the odds ratio (Gart, 1971).
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
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:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
Clinical observation
From week 6 several treated rats showed hunched appearance and transient labored respiration. The incidence of signs was higher in treated animals during the first year. Respiratory signs (labored respiration, wheezing, nasal discharge) were observed in all groups in the second year, and were predominant observations in all survivors at termination of the study. Chronic murine pneumonia was identified in 60-95% of all control and test group rats. Body weight development was not influenced. 

Mortality
Mortality was early and severe especially in high dose animals. In high-dose groups, 50% of males were dead by week 55 and 50% of females by week 57; by week 75, 84% of males and 80% of females were dead. The last high-dose male rat died during week 23 and the last high-dose female rat died during week 15 of the observation period.  In low-dose group, 52% of males survived over 82 week, and 50% of females survived over 85 week. Thus at the low dose survival was similar to the matched vehicle controls.  Mean survival was approx. 90, 74, 75, and 55 weeks for male animals (untreated, low dose, vehicle control, high dose, resp.). Mean survival of females was approx. 90, 76, 55 weeks (vehicle control, low dose, high dose, resp.).  Animals dying early had a variety of lesions, including bronchopneumonia and endocardial thrombosis, but no tumors (cf. Ward 1980). According to Ward (1980) the early deaths were usually not due to cancer. Susceptibility to pneumonia may have been aggravated by toxicity of the test substance.

Tumor formation
Tumors seen in male rats (low and high dose, resp.)
Subcutaneous fibroma: 5/50 (p=0.017) and 6/50 (p=0.007) Forestomach; squamous-cell carcinomas: 3/50 (NS) and 9/50 (p=0.001) Hemangiosarcomas(spleen and other sites): 9/50 (p=0.003) and 7/50 (p=0.016) Vehicle controls: 0/20 for each of the listed tumors.
Tumors seen in female rats (low and high dose, resp.)
Mammary gland, adenocarcinomas 1/50 (NS) and 18/50 (p<0.001) Hemangiosarcomas (spleen and other sites): 4/50 (p=0.041) and 4/50 (p=0.041) Vehicle controls: 0/20 for each of the listed tumors. Subcutaneous fibroma: 1/50 and 2/50. Vehicle control 1/20 Mammary gland, fibroadenomas 14/50 (p=0.007) and 8/50 (NS).
Untreated controls 2/20 (mammary fibroadenomas)
Note: P-values give the level of probability for the Fisher exact test for the comparison with the pooled vehicle control group. NS=not significant. Additionally, 7 other cases of unusual tumors were seen in kidney, stomach and small intestine. 9 Rats developed metastatic tumors, predominantly in the high dose groups.
Relevance of carcinogenic effects / potential:
Result (carcinogenicity): positive
Dose descriptor:
LOAEL
Effect level:
47 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: formation of tumors
Remarks on result:
other: Effect type: carcinogenicity (migrated information)
Conclusions:
Oral administration of 1,2-dichloroethane to male and female Osborne-Mendel rats over a period of 78 wk (including 9 treatment-free weeks) caused severe mortality in high dose animals of both sexes receiving 95 mg/kg bw/d. At the low dose (47 mg/kg bw/d) survival was similar to control animals.
Significantly enhanced tumor formation was seen in both high- and low-dose males as evidenced by subcutaneous fibroma; squamous-cell carcinomas in forestomach; hemangiosarcomas in spleen and other sites. Significantly enhanced tumor formation in female rats was seen as increased numbers of adenocarcinomas in the mammary gland (high-dose animals) and hemangiosarcomas low- and high-dose rats).

There are, however, limitations of the study due to methodological deficiencies. Amongst several others, poor degree of TS purity (>90%) should be mentioned.
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Executive summary:

A bioassay of technical-grade 1,2 -dichloroethane for possible carcinogenicity was conducted using Osborne-Mendel rats. 1,2-Dichloroethane in corn oil was administered by gavage, at either of two dosages, to groups of 50 male and 50 female animals. During the exposure period of 78 -weeks treatment ceased for 9 weeks (wk 36 and every 5th week thereafter). The exposure period was followed by an observation period of 32 weeks for the low dose rats of both sexes. The last high dose male rat died after 23 weeks of observation and the last high dose female rat died after 15 weeks of observation.

Initial dosage levels for the chronic bioassay were selected on the basis of a preliminary subchronic toxicity test. Subsequent dosage adjustments were made during the course of the chronic bioassay. The time-weighted average high and low doses of 1,2-dichloroethane in the chronic study were 95 and 47 mg/kg/day, respectively,

for rats of both sexes. 20 animals of each sex were placed on test as vehicle controls. These animals were gavaged with corn oil at the same times that dosed animals were gavaged with the 1,2-dichloroethane mixtures. Twenty animals of each sex were placed on test as untreated controls. These animals were not intubated.

A statistically significant positive association between dosage and the incidence of squamous-cell carcinomas of the forestomach and hemangiosarcomas of the circulatory system occurred in the male rats, but not in the females. There was also a significantly increased incidence of adenocarcinomas of the mammary gland in female rats.

Under the conditions of this study, 1,2-dichloroethane was carcinogenic to Osborne-Mendel rats, causing squamous-cell carcinomas of the forestomach, hemangiosarcomas, and subcutaneous fibromas in male rats and causing mammary adenocarcinomas in female rats.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
47 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Several limitations such as dosage adjustment, intermittent, higher than- average dosing, poor survival in the top-dose group (in particular in rats) with a reasonable non-toxic low–dose group missing, unclear quality of the test substance, and treatment time too short. In addition, the impact of the concurrent chronic pneumonia is unknown. ). Significantly increased tumour incidences were seen in males as substantiated by haemangiosarcomas of the circulatory system, fibromas of subcutaneous tissue, and squamous cell carcinomas of the forestomach (significant only high dose). In females, haemangiosarcomas of the circulatory system, mammary gland adenocarcinomas (significant only high dose), and mammary gland fibroadenomas (significantly at low dose) were seen. In addition 7 cases of unusual tumours were seen in various organs, and rats bearing metastatic tumours especially in the high dose groups

Carcinogenicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to OECD guideline and GLP
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
no
GLP compliance:
yes
Species:
rat
Strain:
Fischer 344/DuCrj
Sex:
male/female
Details on test animals and environmental conditions:
F344/DuCrj (SPF) rats of both sexes were obtained at 4 wk of age from Charles River Japan, Inc. (Kanagawa, Japan). The animals were quarantined and acclimated for 2 wk, and then divided by stratified randomization into 4 body weight matched groups, each comprising 50 rats and 50 mice of both sexes. The animals were housed individually in stainlesssteel wire hanging cages (150 mm [W] × 220 mm [D] × 176 mm [H] for rats and 100 mm [W] × 120 mm [D] × 120 mm [H] for mice) in stainless steel inhalation exposure chambers maintained at a temperature of 23 ± 2°C and at a relative humidity of 55 ± 10% with 12 ± 1 air changes/h. Fluorescent lighting was controlled automatically to give a 12-h light/dark cycle. All rats had free access to sterilized water and a γ-irradiation-sterilized commercial pellet diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan). The average body weight measured immediately before the first exposure to 1,2-dichloroethane or clean air was 120 ± 5 (mean ± SD) g for male rats, 100 ± 3 g for female rats.
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
Airflow containing DCE vapor at a target concentration for rats of 10, 40 or 160 ppm was prepared by a vaporization technique. The saturated vapor-air mixture was generated by bubbling clean air through liquid 1,2-dichloroethane in a temperature-regulated glass flask (25°C), and by cooling it through a thermostatted condenser at 18°C. The airflow containing the saturated vapor was diluted with clean air, and then warmed to 25°C in a thermostatted circulator which served to stabilize the vapor concentration by complete gasification of 1,2-dichloroethane. The flow rate of vapor-air mixture was regulated with a flow meter, further diluted with humidity- and temperature-controlled clean air in a spiraling line mixer, and then supplied to the inhalation exposure chambers. Four inhalation exposure chambers of 7,600 L in volume for rats were used in this study. Each exposure chamber accommodated 100 individual cages for 50 males and 50 females.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations of 1,2-dichloroethane were monitored by gas chromatography every 15 min, and maintained constant at 10.0 ± 0.1 (mean ± SD), 39.8 ± 0.6 and 159.7 ± 2.1 ppm for the exposure of rats throughout the 2-yr exposure period.
Duration of treatment / exposure:
104 weeks, 6 hours per day
Frequency of treatment:
5 days per week
Post exposure period:
none
Remarks:
Doses / Concentrations:
0, 10, 40, or 160 ppm (0, 41.1, 164.5 or 658.1 mg/m3)
Basis:
nominal conc.
No. of animals per sex per dose:
50
Control animals:
yes, concurrent no treatment
Details on study design:
Groups of 50 male and 50 female rats were exposed to airflow containing 1,2-dichloroethane vapor at a target concentration of 10, 40, or 160 ppm for rats for 6 h/d, 5 d/wk and for 104 wk (2 yr). Fifty rats of both sexes, serving as respective controls, were handled in the same manner as the 1,2-dichloroethane-exposed groups, but were exposed to clean air in the inhalation exposure chambers. The lowest exposure concentration of 10 ppm was selected in consideration of the OEL of 10 ppm for 1,2-dichloroethane. Selection of the highest concentrations of 160 ppm for rats was based on both subchronic toxicity and body weight decrement from a preliminary 13-wk inhalation exposure study conducted at the JBRC. All rats died during the first week of 13-wk exposure to 320 ppm 1,2-dichloroethane, but 13-wk exposure of rats to 160 ppm 1,2-dichloroethane did not cause any deaths, overt toxic signs or body weight decrements. Therefore, the highest exposure concentrations of 160 ppm for rats were predicted not to exceed the MTD from the results of the 13-wk inhalation exposure study.
Positive control:
none
Observations and examinations performed and frequency:
The animals were observed daily for clinical signs and mortality. Body weights and food consumption were measured once a week for the first 14 wk, and every 4 wk thereafter.
Sacrifice and pathology:
All the rats which died or were killed in a moribund state during the 2-yr exposure period, or survived to the end of the 2-yr period received complete
necropsy. Urinary parameters were measured from urine sampled with Ames Reagent Strips (Multistix for rats, Bayer Corporation, NY, USA) in the last week of the 2-yr exposure period. For hematology and blood biochemistry, the surviving animals were bled under ether anesthesia at the terminal necropsy after they were fasted overnight. The blood samples were analyzed on an automatic blood cell analyzer Coulter Counter SP (Coulter Electronics, Luton, UK) for hematology, and an automatic analyzer Hitachi 705 and a flame analyzer Hitachi 750 (Hitachi, Tokyo, Japan) for blood biochemistry. Organs were removed, weighed, and examined for macroscopic lesions at the necropsy. Tissues for microscopic examinations were fixed in 10% neutral buffered formalin and embedded in paraffin. Tissue sections 5 µm thick were prepared and stained with hematoxylin and eosin.
Other examinations:
none
Statistics:
Incidences of neoplastic lesions were analyzed for a dose response relationship indicated by a significant positive trend by Peto’s test, and for a significant difference from the concurrent control group by Fisher’s exact test. Incidences of non-neoplastic lesions and urinary parameters were analyzed by Chi-square test. Survival curves were plotted according to the Kaplan- Meier method, and the log-rank test was used to test statistical significance of the difference between any 1,2-dichloroethane-exposed rat group of either sex and the respective control. Body weight, organ weight, hematological and blood biochemical parameters were analyzed by Dunnett’s test.
When tumor incidences were increased in a dose related manner as indicated by Peto’s test and when the tumor incidence in each of the DCE-exposed groups was increased but not statistically significant as compared with the concurrent, matched-control group by Fisher’s exact test, the borderline increase in the tumor incidence was tested as to whether or not it was biologically meaningful, using a range of minimum and maximum tumor incidences in the JBRC historical control data.
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):
no effects observed
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:
no effects observed
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
Survival, body weight, food consumption and clinical observations and analyses:
There was no significant difference in the survival rate at any time point of the 2- yr exposure period between any 1,2-dichloroethane-exposed group of either sex and the respective control. At the end of 2-yr exposure period, the survival rates of the 0 (clean air as control), 10, 40 and 160 ppm 1,2-dichloroethane exposure groups were 74, 70, 64 and 74% for males, and 70, 82, 74 and 76% for females, respectively. Neither growth rate nor food consumption was suppressed in any 1,2-dichloroethane-exposed group of either sex as compared with the respective control. The body weights of the 0, 10, 40 and 160 ppm 1,2-dichloroethane exposure groups at the end of 2-yr exposure period were 434 +/- 46, 459 +/-59, 448 +/- 41 and 467 +/- 85 g for males, and 317 +/- 46, 329 +/- 41, 33 +/- 45 and 336 +/- 56 g for females, respectively.
Incidences of subcutaneous masses, which were found in the breast, back, and abdominal and perigenital areas by clinical observation, tended to increase in the 1,2-dichloroethane exposed groups of both sexes. No exposure-related change in any hematological, blood biochemical or urinary parameter was found in any 1,2-dichloroethane-exposed group of either sex.

Pathology:
Macroscopic examination at necropsy revealed that incidences of subcutaneous masses were increased in an exposure concentration-related manner (for males: 8 /50 cases in control, 13/50 at 10 ppm, 20/50 at 40 ppm and 18/50 at 160 ppm, and for females: 12/50 in control, 12/50 at 10 ppm, 14/50 at 40 ppm and 30/50 at 160 ppm). There was no significant difference in organ weight between any 1,2-dichloroethane-exposed group of either sex and the respective control.
In male rats, incidences of fibromas in the subcutis, fibroadenomas in the mammary gland and mesotheliomas in the peritoneum showed a significant positive trend. The incidences of subcutaneous fibromas in both the 40 and 160 ppm 1,2-dichloroethane exposed male groups exceeded the maximum tumor incidence of the historical control data, although those tumor incidences were not statistically increased as compared with the concurrent control. The fibromas were non-invasive and expansile benign tumors composed of fibroblasts and abundant collagenous stroma. The incidence of mammary gland fibroadenomas in the 160 ppm 1,2-dichloroethane exposed male group was statistically increased as compared with the concurrent control (p <=0.05), and also exceeded the maximum tumor incidence of the historical control data. The mammary fibroadenomas were benign tumors composed of both glandular epithelium and fibrous connective tissue. Mammary gland adenomas were also found as benign tumors mainly composed of glandular epithelium, but their tumor incidence was not increased in any 1,2-dichloroethane-exposed male group. However, the combined incidences of mammary adenomas and fibroadenomas exhibited a significant positive trend, and the combined mammary tumor incidence in the 160 ppm 1,2-dichloroethane exposure group was statistically increased (p <= 0.05), and exceeded the maximum tumor incidence of the historical control data. These mammary gland tumors were diagnosed according to the criteria of Boorman et al. The incidence of peritoneal mesotheliomas in the 160 ppm 1,2-dichloroethane exposed male group exceeded the maximum tumor incidence of the historical control data, although the tumor incidence was not statistically increased as compared with the concurrent control. The mesotheliomas were malignant tumors involving the surface of the peritoneal cavity, especially the scrotal sac, and were composed of one to several layers of neoplastic mesothelial cells covering pedunculated fibrovascular stalks.
In female rats, incidences of fibromas in the subcutis and adenomas, fibroadenomas and adenocarcinomas in the mammary gland showed a significant positive trend. The incidence of subcutaneous fibromas in the 160 ppm 1,2-dichloroethane exposed female group was statistically increased (p <= 0.05), and also exceeded the maximum tumor incidence of the historical control data. The incidences of mammary gland adenomas and fibroadenomas in the 160 ppm 1,2-dichloroethane exposed female group and the combined incidence of those two benign mammary tumors were statistically increased (p <= 0.05), and also exceeded the maximum tumor incidences of the historical control data.
Besides, the combined incidence of mammary gland adenomas and fibroadenomas in the 40 ppm 1,2-dichloroethane exposed female group exceeded the maximum tumor incidence of the historical control data, but did not attain statistical significance as compared with the concurrent control. Furthermore, the incidence of mammary gland adenocarcinomas in the 160 ppm 1,2-dichloroethane exposed female group exceeded the maximum tumor incidence of the historical control data without significant increase as compared with the concurrent control. The neoplastic epithelia of the adenocarcinomas were arranged in a ductular structure, forming multiple layers indicative of malignancy, but did not metastasize to any other organ. The combined incidence of all the mammary gland tumors showed a significant positive trend. The combined incidence of those three tumors in the 160 ppm 1,2-dichloroethane exposed female group was statistically increased (p <= 0.01), and also exceeded the maximum tumor incidence of the historical control data.
No exposure-related, non-neoplastic lesions were histopathologically observed in any 1,2-dichloroethane-exposed rat group of either sex.
Relevance of carcinogenic effects / potential:
Development of tumors in rats after inhaltion exposure of 1,2-dichloroethane.
Dose descriptor:
NOAEL
Effect level:
658.1 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
other: no adverse effects observed expect of carcinogenicity
Remarks on result:
other: Effect type: toxicity (migrated information)
Dose descriptor:
BMC: BMC10
Effect level:
42 ppm
Sex:
male/female
Remarks on result:
other: Effect type: carcinogenicity (migrated information)
Dose descriptor:
other: T25
Effect level:
99 ppm
Sex:
male/female
Remarks on result:
other: Effect type: carcinogenicity (migrated information)
Conclusions:
1,2-dichloroethane was shown to be carcinogenic after inhalation exposure to rats during 2 years.
A BMC10 and T25 value of 42 ppm and 99 ppm, repsectively, have been derived (obtained for the modelling of combined fibroadenoma and adenoma and combined fibroadenoma, adenoma and adenocarcinoma in female rats, respectively).
Executive summary:

Carcinogenicity and chronic toxicity of 1,2- dichloroethane were examined by inhalation exposure of groups of 50 F344 rats of both sexes to 1,2- dichloroethane vapor or clean air as control for 6 h/d, 5 d/wk and 104 wk. The rats were exposed to 0, 10, 40 or 160 ppm (v/v) (equivalent to 0, 41.1, 164.5 or 658.1 mg/m3) 1,2- dichloroethane. The 2-yr exposure to 1,2- dichloroethane produced a dose-dependent increase in incidences of benign and malignant tumors, including subcutaneous fibroma, mammary gland fibroadenoma and peritoneal mesothelioma in male rats; subcutaneous fibroma and mammary gland adenoma, fibroadenoma and adenocarcinoma in female rats. No exposure-related change in the incidence of non-neoplastic lesions or in any hematological, blood biochemical or urinary parameter occurred in any 1,2- dichloroethane -exposed rat group. Selection of the exposure concentrations was considered appropriate with reference to the maximum tolerated dose for the highest doses and an occupational exposure limit of 1,2 - dichloroethane for the lowest dose. A BMC10 and T25 value of 42 ppm and 99 ppm, repsectively, have been derived (obtained for the modelling of combined fibroadenoma and adenoma and combined fibroadenoma, adenoma and adenocarcinoma in female rats, respectively).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
T25
406 mg/m³
Study duration:
chronic
Species:
rat

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral:

In the oral gavage study by NCI (1978), Osborne-Mendel rats of both sexes received 47 and 95 mg/kg bw/d (time-weighted doses, intermittent, 78 wk, 5d/wk) dissolved in corn oil. Two control animal groups (20 animals/sex each) were included, one group being vehicle treated and the other one untreated. Evaluation of the results included comparison of tumour incidences in treated animals against matched control animals, and against pooled vehicle control animals from experiments with different chemicals which were conducted in parallel in the same room. The only clinical symptom during the first year of treatment was respiratory impairment while body weights, general appearance and behaviour were comparable to controls. Chronic pneumonia aggravated during the second year and was identified in 60-95% of all control and test animals. Mortality rates were increased in the high dose groups at 50% of males by week 55 and of females by week 57. By week 75, 84% of males and 80% of females were dead. Survival of the low dose rats was similar to that of the vehicle controls (males: 52% survived until week 82; females: 50% until week 85). Significantly increased tumour incidences were seen in males as substantiated by haemangiosarcomas of the circulatory system, fibromas of subcutaneous tissue, and squamous cell carcinomas of the forestomach (significant only high dose). In females, haemangiosarcomas of the circulatory system, mammary gland adenocarcinomas (significant only high dose), and mammary gland fibroadenomas (significantly at low dose) were seen. In addition 7 cases of unusual tumours were seen in various organs, and rats bearing metastatic tumours especially in the high dose groups (National Cancer Institute 1978; Ward 1980).

In the corresponding second chronic oral study (NCI, 1978) using B6C3F1 mice receiving time-weighted doses of 97 and 195 mg/kg bw/d (males) and 149 and 299 mg/kg bw /d (females), a significant increase in mortality rates was seen only in high dose females (32%); mortality of low dose females (72%) was similar to vehicle controls (20%) after 80 weeks. Clinical symptoms from study week six were abscesses on body and extremities as well as generalised and local alopecia. Behaviour of treated groups was comparable to controls throughout the study period. Significant tumour increases in high dose males were located in lung (alveolar/bronchiolar adenoma) and liver (hepatocellular carcinoma). Significant differences in both low- and high-dose females were seen in incidences of mammary gland carcinomas and of alveolar/bronchiolar adenomas. Also, a trend suggesting substance-related increases in the incidence of uterine carcinomas and squamous cell carcinomas of the forestomach in females and hepatocellular carcinomas in males was noted (National Cancer Institute 1978; Ward 1980).

 

However, both studies share several limitations such as dosage adjustment, intermittent, higher than- average dosing, poor survival in the top-dose group (in particular in rats) with a reasonable non-toxic low–dose group missing, unclear quality of the test substance, and treatment time too short. In addition, the impact of the concurrent chronic pneumonia is unknown.

Inhalation:

The most recent and well-conducted bioassay on 1,2-dichloroethanae is the study by Nagano et al. (2006). The carcinogenicity and chronic toxicity of 1,2 -dichloroethane were examined by inhalation exposure of groups of 50 F344 rats and 50 BDF1 mice of both sexes to 1,2-dichloroethane vapor or clean air as control for 6 h/d, 5 d/wk and 104 wk. The rats were exposed to 0, 10, 40 or 160 ppm (v/v) (0, 41.1, 164.5 or 658.1 mg/m3) 1,2-dichloroethane, while the mice were exposed to 0, 10, 30 or 90 ppm (0, 41.1, 123.4 or 370.2 mg/m3). According to the authors, the 2-year exposure to 1,2-dichloroethane produced a dose-dependent increase in incidences of benign and malignant tumors, including subcutaneous fibroma, mammary gland fibroadenoma and peritoneal mesothelioma in male rats; subcutaneous fibroma and mammary gland adenoma, fibroadenoma and adenocarcinoma in female rats; and bronchiolo-alveolar adenoma and carcinoma, endometrial stromal polyp, mammary gland adenocarcinoma and hepatocellular adenoma in female mice. No exposure-related change in the incidence of non-neoplastic lesions or in any hematological, blood biochemical or urinary parameter occurred in any DCE-exposed rat or mouse group. The types of tumors and their target organs found in this study were consistent with those observed in rats and mice administered 1,2-dichloroethane by gavage in a NCI study. Selection of the exposure concentrations was considered appropriate with reference to the maximum tolerated dose for the highest doses and an occupational exposure limit of 1,2- dichloroethane for the lowest dose.

 

In the Nagano et al. study (2006), there were no statistically significant increases in tumors at the low or mid doses of DCE in rats, except for sub-cutis fibromas in male rats (12 tumors/24% at 40 ppm) and combined mammary adenomas plus fibroadenomas in female rats (11 tumors/22% at 40 ppm) and combined mammary adenomas plus fibroadenomas plus adenocarcinomas in female rats (11 tumors/22% at 40 ppm). These mid-dose responses are not far outside of the historical control. The authors used an 18-year period for the historical control data, which seems to be extraordinarily long. None of the mid-dose responses were statistically identified: it was the trend only that was statistically identified. Also, they state that no dose-related, non-tumor effects were noted. For the mammary tumour data, the maximum for adenomas was 9/50 and for fibroadenomas it was 8/50 and for adenocarcinomas it was 2/50, but for the combined (both types of combinations) it was only 10/50. Somehow one would have expected a higher max number of tumor-bearing animals for the combined counts.

 

The responses in the mouse were a bit different, with liver haemangiosarcomas statistically identified at mid-dose 30 ppm (6/50; 12%) in males. Lymphomas were statistically identified, but there was not dose-response in females (22/50; 44%) and they fall within historical control of maximum 23/50. In fact the authors state that both tumour types are unlikely to be causally related to 1,2-dichloroethane exposure.

 

In another long-term rat inhalation study with sole exposure to 50 (206 mg/m3) ppm 1,2-dichloroethane (2 yr, 5d/wk, 7 h/d) no tumor formation was noted in either sex (Cheever et al. 1990, see section 7.5.3). At that time 50 ppm was the occupational standard. In this study, blood levels were determined to be in the range of 0.2-0.3 μg/ml immediately after the 7-h inhalation exposure. In animals receiving a combined 1,2-dichloroethane/disulfiram treatment blood levels were approx. 5-fold increased, and tumour incidence was significantly increased in several organs (liver, skin, testes). Thus inhibition of the ethanol metabolism pathway enzymes increased both blood levels and tumour incidence.

 

No differences in tumour formation were seen in two inhalation studies with groups of 90 animals, Sprague-Dawley rats and Swiss mice of both sexes (Maltoni et al., 1980/Spreafico et al., 1980). The animals were exposed to 1,2-dichloroethane concentrations of 5, 10, 50 and 150 – 250 ppm (corresponding to 21, 41, 206 and 617 – 1028 mg/m3) for 78 consecutive weeks, 7 hr/d, 5 d/wk. Due to pronounced signs of toxicity especially in mice, the highest concentration was reduced to 617 mg/m3 (150 ppm) after a few weeks. Apart from toxicity at 250 ppm, no clinical signs were noted in any group. In mice, survival rates were slightly reduced in the two highest dose groups (43.9 and 38.9% vs. 47.4% in controls), while in rats survival rates were not changed even in the high dose group (17.2% vs. 18.9% in controls).

 

In neither study were specific types of tumours or relevant changes in the incidence of the tumours normally occurring in the strain of rats and mice noted. An apparent, slight increase of mammary fibromas and fibroadenomas was statistically significant in the 250-150, 50 and 5 ppm female rat groups, but is to be considered incidental. In mice, no differences between treated and control groups as to the type and the number of tumours was noted in any of the dose groups. In conclusion, based on toxicokinetic data, 150 ppm can be assumed to be the reasonable upper tolerable exposure concentration in such a long-term study (Spreafico et al., 1980). Inhaled 1,2-dichloroethane was not carcinogenic in male and female Sprague Dawley rats nor in male and female Swiss mice under the conditions of the experiment including a chronic but less-than-lifetime exposure time of 78 weeks (Maltoni et al., 1980). However, due to the above mentioned short-comings of the study, a final evaluation cannot be drawn from these data.

Mode of Action for Mammary Tumours:

The potential mode of action (MoA) of 1,2-dichloroethane (DCE)-induced mammary tumors in was investigated in female F344/DuCrl rats in a GLP study. Groups of 28 female F344/DuCrl rats were exposed to target concentrations of 0 or 200 ppm of DCE vapors (six hours/day, seven days/week) for at least 28 exposures. Exposures occurred under dynamic airflow conditions in whole-body inhalation exposure chambers. An additional group of three animals were administered N-Nitroso-N-methylurea (MNU) at 100 mg/kg, via oral gavage, approximately three hours prior to the scheduled necropsy. This group of animals served as the positive control group for the Comet Assay. An additional group of six animals were administered 750 mg/kg diethyl maleate (DEM) via intraperitoneal (i.p.) injection approximately two hours prior to necropsy to serve as a positive control for depletion of glutathione (GSH) in mammary and liver tissue. Study parameters measured included cage-side and clinical observations, feed consumption, body weights/body weight gains, estrous evaluations, serum prolactin levels, measurement of reduced (GSH) and oxidized (GSSG) glutathione, DCE-glutathione conjugates S-(2-Hydroxyethyl)glutathione hydrochloride (HESG) and S,S’-Ethylene-bis-glutathione (EBG), DNA adducts 8-Hydroxy-2’-deoxyguanosine (8-OH dG) and S-(2-guanylethyl) glutathione (GEG) in mammary and liver tissue, Comet assay (mammary tissue), morphometric evaluation of mammary gland structure, cell proliferation (Ki-67), and histopathology (mammary tissue). All rats were sacrificed immediately after exposure on the first diestrus after a minimum of 28 consecutive days of exposure. This resulted in a range of 28-31 days of exposure to average chamber concentrations of 0.0 ± 0.0 (control) or 205.1 ± 4.2 ppm DCE (study mean ± standard deviation). The range in exposure days had no measurable impact on any study parameter. Animals exposed to 200 ppm DCE had treatment-related decreases in body weight gain from test day (TD) 1-15 with correlating decreases in feed consumption from TD 1-8; however, body weight gain and feed consumption were similar to control for the remainder of the study.

Under the conditions of this study, repeated inhalation exposure to a high concentration of DCE vapors had no effect on body weights, clinical observations, serum prolactin levels, mammary epithelial cell proliferation/numeric density, or mammary gland morphology or histopathology. Based on the results of the Comet assay, no exposure-related DNA damage was detected in mammary epithelial cells (MEC) isolated from DCE-exposed rats, compared to control rats. Compared to control rats, repeated inhalation of DCE had no effect on GSH or GSSG levels in mammary tissue; however, DCE exposure decreased liver (non-target tissue) GSH and GSSG levels by approximately 72 and 62%, respectively. The GSH/GSSG ratio remained essentially unchanged. No HESG or EBG was measured at levels greater than the lower limit of quantitation (LLQ) in mammary or liver tissue samples (LLQ = 10 and 10 ng/g tissue for both mammary and liver tissue) isolated from control or DCE-exposed rats. Compared to control rats, DCE exposure had no effect on 8-OHdG adduct levels in mammary tissue; however, liver 8-OHdG levels in DCE-exposed rats were significantly less than control rats.

 

In contrast to the 8-OHdG adduct, endogenous GEG adduct, the predominant adduct formed following exposure to DCE, was not quantifiable in mammary or liver tissue isolated from control rats, with a lower limit of quantitation of 0.6 ngGEG/mL(equivalent to 42.8 and 89.3 GEG adducts/106dG,respectively) for both mammary and liver tissue. Compared to control rats a statistically significant DCE-dependent increase in GEG levels in both mammary (~2.4-fold; 103 ± 16 GEG adducts/106dG residues) and liver (4.3-fold; 222 ± 41 GEG adducts/106dG residues) was observed. Thus the GEG adduct levels in the non-target tissue (liver) were approximately ~54% higher than in the mammary (target) tissue from the same DCE-exposed rats.

 

In conclusion, repeated inhalation exposure to 200 ppm DCE vapor, a concentration approximately 20% higher than the concentration reported to induce mammary tumors in rats (Nagano et al., 2006) had no statistically significant effect on serum prolactin levels, GSH/GSSG levels, cell proliferation, or DNA damage in mammary tissue. The N7-guanylethyl glutathione (GEG) cross-link adduct was identified as a biomarker of exposure, with higher levels of the GEG adduct measured in (non-target) liver tissue compared to (target) mammary tissue isolated from the same rats. The results of this sub-acute inhalation-exposure study do not support a specific known MoA for DCE-induced mammary tumors in rats. In particular, the lack of any exposure-related genotoxic effects in the Comet assay or relevant target-tissue specific DNA adducts does not support a genotoxic mode of action.

Overall conclusion

The suspected relationship between the carcinogenic response of 1,2-dichloroethane and the associated risk has been carefully considered. There is one long-term inhalation study with 1,2-dichloroethane (in rats and mice) available in which mammary tumours were observed. These findings were not confirmed in two other long-term inhalation studies. A recent, well-conducted Mode of Action study indicates that there is no support for a non-threshold mechanism for the induction of mammary tumours. In particular, noexposure-related genotoxic effects (Comet assay) were observed in mammary epithelial cells after repeatedin vivoinhalation exposure of female rats. The available epidemiological data are not supportive of 1,2-dichloroethane exerting a carcinogenic effect in humans.

 


Carcinogenicity: via inhalation route (target organ): glandular: mammary gland

Justification for classification or non-classification

Based on the results 1,2-dichloroethane has to be regarded as suspected human carcinogen and classified carc. cat. 2 (R45) according to 67/548/EEC and Carc. 1B (H350) according to Regulation 1272/2008 (CLP, GHS).