Bis(2-chloroethyl) ether

Carcinogenicity

Description of key information

No study aiming at evaluating the carcinogenic potential of BCEE was conducted according to recognized guidelines. BCEE was found to induce an increased incidence of hepatomas in mice following repeated oral exposure to BCEE (Innes et al. 1969). This result was not confirmed in a similar study conducted on rats (Weisburg et al. 1981). BCEE did not induce an increased incidence of lung tumors following repeated i.p. injections (Theiss et al. 1977). BCEE was not tumorigenic but marginally sarcomagenic following repeated subcutaneous injections in female ICR/Ha Swiss mice (van Duuren, 1972) but did not increase the incidence of all malignant and benign tumours (e.g., mesenchymal, epithelial, sarcomas, carcinomas and unclassified) in male or female Sprague‐Dawley rats (Norpoth et al. 1986).

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Study limited owing to small number of BCEE-exposed animals, use of single dose level and inadequate reporting of tumour pathology. Amount of BCEE was not adjusted during initial period to account for weight gain. Increased incidence of mouse liver hepatomas has been questioned as a reliable indication of true carcinogenic potential (Maronpot et al. 1987).

Qualifier:

no guideline followed

Principles of method if other than guideline:

The Maximal Tolerable Dose was estimated following a sequence of studies, during which the maximal levels resulting in zero mortality were determined for a single dose, then for 6 daily doses, and finally for 19 daily doses. In the full study, mice (7 days of age) were first given a daily dose (corresponding to the MTD) of test substance by stomach tube. At weaning (4 weeks old), the chemicals were mixed directly with the diet, which was provided ad libitum until the mice were 18 months of age; no vehicle was used.

GLP compliance:

no

Remarks:

pre-GLP

Specific details on test material used for the study:

TEST MATERIAL
- Name (as cited): Bis(2-chloroethyl) ether
- Purity: commercial grade

Species:

mouse

Strain:

other: Two hybrid strains (C57BL/6 x C3H/Anf)F1 and (C57BL/6 x AKR)F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Cumberland View Farms (Clinton, Tennessee)
- Age at study initiation: 7 days
- Weight at study initiation: not specified
- Fasting period before study: not specified
- Diet: ad libitum
- Water: not specified
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
Not specified

Route of administration:

oral: feed

Vehicle:

other: water prior to weaning then no vehicle

Details on exposure:

Mice (7 days of age) were first given a daily dose (corresponding to the MTD) of test substance by stomach tube. At weaning (4 weeks old), the chemicals were mixed directly with the diet, which was provided ad libitum; no vehicle was used.

Before weaning: 100 mg/kg bw/day
After weaning: 300 ppm in diet

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

c.a. 18 months (see "Details on exposure")

Frequency of treatment:

Daily

Post exposure period:

None

Dose / conc.:

41.3 mg/kg bw/day (nominal)

Remarks:

The time-weighted average dose calculated to be 41.3 mg/kg bw/day (US EPA, 1987)

No. of animals per sex per dose:

18 animals/sex/strain

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: corresponding to the MTD

Positive control:

see "Any other information on materials and methods incl. tables"

Observations and examinations performed and frequency:

None reported

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
External examination and a thorough examination of thoracic and abdominal cavities.

HISTOPATHOLOGY: Yes
Histologic examination of major organs and of all grossly visible lesions. The cranium was not dissected. Thyroid glands were examined and sectioned in mice treated with Amitrol and in one negative control group, but not in other groups. The entire carcass and all internal organs were fixed and have been saved. Blood smears were made on all mice before they were killed, and then examined only in cases showing splenomegaly or lymphadenopathy.

Statistics:

NEGATIVE CONTROLS: Negative control groups differed with respect to their location within the experimental rooms, time of entry into the experiment, and length of time until necropsy. The variance among these groups in the incidence of hepatomas, pulmonary tumors, lymphomas, and total mice with tumors was analyzed using the "chi-square test for heterogeneity of proportions after adjustment of stratification (Armitage, 1966, J. Royal Stat. Soc (B)., 28:150-163) and ordinary chi-square tests. No statistically significant differences were found.

POSITIVE CONTROLS AND EXPERIMENT GROUPS: The individual positive controls and experimental groups were compared with the grouped negative controls. The relative risk (a measure of tumor incidence) was determined for four tumors groups: hepatomas, pulmonary tumors, lymphomas, and total mice with tumors. The significance test of each sex-strain subgroup and their various combinations were tested by the Mantel-Haenszel procedure (Snedecor & Cochran, 1967, Cancer Epidemiology: Methods of Study, pp. 253-6), and the combined relative risk used the weighted geometric mean (Gart, 1962, Biometrics, 18:601-10). All tests used the 1/2 corrections (Gart & Zweifel, 1967, Biometrika, 54:181-7; Snedecor & Cochran, 1967, Cancer Epidemiology: Methods of Study, pp. 497). Finally, to compare the tumor incidence between test animals and positive controls, the 7 positive controls were lumped together and the corrected relative risk was calculated for each experimental group compared to this positive baseline.

Clinical signs:

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Increased mortality in male of the (C57BL/6 x C3H/Anf)F1 strain (statistical significance not evaluated)

Body weight and weight changes:

not examined

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Increased relative risk of developping hepatomas in males of both strains

## Tumors among negative control mice:

Treatment	Strain	Number of mice at term		Total mice necropsied		Weeks at term		Mice with hepatomas		Mice with pulmonary tumors		Mice with lymphomas		Total mice with tumors
		M	F	M	F	M	F	M	F	M	F	M	F	M	F
Untreated Control-OD group	X Y	14 18	16 16	17 18	18 17	88 89	88 89	1 3	0 0	2 2	1 0	1 1	2 2	6 5	3 2
Untreated Control-OD group	X Y	15 18	17 13	15 18	18 15	78 78	78 78	3 0	0 0	2 3	1 0	0 0	1 1	5 3	2 1
Untreated Control-OD group	X Y	12 18	18 17	14 18	18 18	80 80	80 80	3 1	0 0	0 0	1 0	2 0	1 0	4 2	2 0
Untreated Control-OD group	X Y	16 17	16 14	17 18	17 15	81 81	81 81	1 0	0 1	1 3	0 2	2 0	0 0	7 3	1 2
Gelatin Control group	X Y	16 18	16 15	16 18	16 17	82 83	82 83	0 1	0 0	0 2	0 1	0 0	0 1	0 3	0 2
Total Negative control group	X Y	73 89	83 75	79 90	87 82	- -	- -	8 5	0 1	5 10	3 3	5 1	4 4	22 16	8 7

Strain X: (C57BL/6 x C3H/Anf)F1; Strain Y: (C57BL/6 x AKR)F1

## Tumors among positive control mice:

Treatment	Strain	Number of mice at term		Total mice necropsied		Weeks at term		Mice with hepatomas		Mice with pulmonary tumors		Mice with lymphomas		Total mice with tumors
		M	F	M	F	M	F	M	F	M	F	M	F	M	F
Ethyl carbamate*	X Y	9 15	13 18	20 22	23 19	71 73	69 74	8 14	12 5	6 15	6 17	1 6	3 1	13 20	19 18
Ethylene imine	X Y	4 1	14 3	17 16	15 11	78 77	77 77	15 9	11 2	15 12	15 10	0 0	0 2	16 16	15 11
Amitrol**	X Y	0 0	0 0	18 18	18 18	54 53	58 60	16 16	18 17	0 0	0 0	0 1	0 0	16 16	18 17
Aramite	X Y	16 17	16 16	16 17	17 16	81 78	78 81	6 1	1 0	0 0	1 3	1 0	1 0	7 2	8 4
D ihydrosafrole	X Y	16 15	16 15	17 17	17 18	82 82	82 82	10 8	0 1	4 5	5 4	1 0	1 1	12 11	7 6
Isosafrole	X Y	18 17	16 15	18 17	16 16	82 82	82 82	5 2	1 0	3 0	1 0	1 1	0 0	8 4	2 0
Safrole	X Y	14 17	14 16	17 17	16 17	82 82	82 82	11 3	16 16	0 0	0 0	0 0	0 0	11 3	16 16

Strain X: (C57BL/6 x C3H/Anf)F1; Strain Y: (C57BL/6 x AKR)F1

* Adenomas of the Harderian gland were found in 24 mice.

** Carcinomas of the thyroid were found in 64 mice.

## Tumors among mice receiving Bis(2 -chloroethyl) ether:

Compound

Strain

Number of mice at term

Total mice necropsied

Weeks at term

Mice with hepatomas

Mice with pulmonary tumors

Mice with lymphomas

Total mice with tumors

M

F

M

F

M

F

M

F

M

F

M

F

M

F

Bis (2-chloroethyl) ether

X Y

11 15

18 17

16 17

18 18

80 80

80 80

14 9

4 0

0 2

0 0

2 0

0 1

16 10

4 1

Strain X: (C57BL/6 x C3H/Anf)F1; Strain Y: (C57BL/6 x AKR)F1

Conclusions:

Exposure to Bis(2-chloroethyl) ether led to increased incidence of liver hepatomas, mainly in male mice of the two tested strains.

Executive summary:

This study aimed at evaluating the tumorigenicity of various chemical in mice. The Maximal Tolerable Dose was following a sequence of studies, during which the maximal levels resulting in zero mortality were determined for a single dose, then for 6 daily doses, and finally for 19 daily doses. In the full study, mice (7 days of age) were first given a daily dose (100 mg/kg bw/day) of test substance by stomach tube. At weaning (4 weeks old), the chemicals were mixed directly with the diet (300 ppm), which was provided ad libitum; no vehicle was used. The time‐weighted average dose for these studies was calculated to be 41.3 mg/kg body weight per day (US EPA, 1987). Exposure duration was 18 months and mice were necropsied at time of death or at the end of the experiment.

Increased mortality was observed in male of the (C57BL/6 x C3H/Anf)F1 strain. In addition the males of both strains showed an increased relative risk of developing hepatomas. Hepatomas were found in 88 and 53% of males from strain (C57BL/6 x C3H/Anf)F1 and strain (C57BL/6 x AKR)F1, respectively. In females, no hepatomas were found in animals from strain (C57BL/6 x AKR)F1, while they were found in 22% of females from strain (C57BL/6 x C3H/Anf)F1. No distinction was made between "hepatomas" and liver carcinomas, due to the impossibility of making the distinction between benign and malignant liver tumors in the mouse. However, the author noted that metastasizing hepatic cell tumors were rare but concluded that the great of tumors majority had malignant potentiality. No significant increase in other tumor types was detected.

This study is limited owing to small number of BCEE-exposed animals, the use of single dose level, inadequate reporting of tumour pathology, the fact that the amount of BCEE was not adjusted during initial period to account for weight gain, and that the exact dose received by the test animals can only be extrapolated. In addition, increased incidence of mouse liver hepatomas has been questioned as a reliable indication of true carcinogenic potential (Maronpot et al. 1987).

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

yes

Remarks:

Prior to the promulgation of the NCI guidelines: low number of animals, 18 months exposure, only two dose levels, twice-weekly exposure

GLP compliance:

not specified

Specific details on test material used for the study:

TEST MATERIAL
- Name (as cited): Bis(2-chloroethyl) ether
- Source: Aldrich Chemical Co.
- Purity: 100 % (NMR)

Species:

rat

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (North Wilmington, Mass.)
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: not specified
- Weight at study initiation: not specified
- Fasting period before study: not specified
- Housing: housed 2 per large plastic cage (12 inchesX14 inchesX7 inches). on corncob bedding (San-i-cel; Paxton Processing Co., Inc., Paxton, Ill.)
- Diet:Wayne Lab Blox meal ad libitum
- Water: ad libitum
- Acclimation period: 7-10 days of quarantine

ENVIRONMENTAL CONDITIONS
Animal rooms were air-conditioned and were in a facility constructed on the "clean-dirty" corridor principle.

Route of administration:

oral: gavage

Vehicle:

other: SSV=steroid suspending vehicle

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Suspension of the test compound in SSV was freshly prepared on the day of administration.

VEHICLE
- Justification for use and choice of vehicle: not specified why water was not used
- Concentration in vehicle: not specified
- Amount of vehicle: not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

18 months

Frequency of treatment:

twice-weekly

Post exposure period:

6 months observation period

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

26

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on toxicity response following an acute toxicity study and a sub-chronic (30 days) study

Positive control:

Two groups of positive control animals received the carcinogen N-2-fluorenylacetamide in the diet at 80 ppm (32 rats) or 250 ppm (20 rats)

Observations and examinations performed and frequency:

None reported in the publication.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Organs examined: cerebrum, cerebellum, pituitary gland, spinal cord plus vertebrae, lung, heart, mediastinum, thymus, thyroid gland, parathyroid gland, liver, spleen, pancreas, adrenal gland, kidney, urinary bladder, ovary, uterus or testis, accessory sex organ, esophagus, stomach, intestinal tract, and any abnormal tissue or mass.

Statistics:

Probabilities of survival were estimated by the product-limit procedure of Kaplan and Meier, which is commonly called the "life table method." For statistical analyses of a possible dose-related effect on survival, the method of Cox for testing 2 groups of equality and Tarone's extensions of Cox's methods for testing for a dose-related trend were used.

The one-tailed Fisher's exact probability test was used to determine whether animals receiving the test chemical developed a significantly higher proportion of tumors than did the control animals.The Cochran-Armitage test for linear trend in proportions, with continuity correction, was also used. Under the assumption of a linear trend, this test determined whether the slope of the dose-response curve is different from zero at the one-tailed 0.05 level of significance.

Clinical signs:

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

Survival at 52 weeks:
- Males: 96% at 25 mg/kg and 100% at 50 mg/kg
Mortality in male rats was not associated with treatment,

- Females: 96% at 25 mg/kg and 65% at 50 mg/kg
Significant association of mortality with treatment among the high-dose females

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a substantial difference between the mean weights of the treated females and the mean weights of the corresponding controls, and a reduction in mean weight in the high-dose males.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

no effects observed

Conclusions:

Under the conditions of this experiment bis(2-chloroethyl) ether was not carcinogenic in male or female rats.

Executive summary:

This study aimed at testing chemicals for carcinogenicity by oral administration in male and female Charles River CD rats. The method used was similar to OECD Technical Guideline 453. 26 males and 26 females were expose twice a week to Bis(2-chloroethyl) ether at 25 and 50 mg/kg bw/day for 18 months followed by a 6 months observation period. All rats were necropsied and examined histopathologically. However, detailed results of these examinations are not reported.

The dose administered to male rats did not significantly impact survival at 72 weeks. Female survival was however significantly reduced at 50 mg/kg bw/day (65% survival et 52 weeks). No gross pathology and histopathological findings were reported, which suggest that these examinations were unremarkable. Female body weight was significantly different from control at the two dose tested and male body weight was reduced at 50 mg/kg bw/day.

Although there is some question whether the dose levels for males were sufficiently high to provide maximum test sensitivity, under the conditions of this experiment bis(2-chloroethyl) ether was not carcinogenic in male or female rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Study duration:

chronic

Species:

mouse

Quality of whole database:

No study aiming at evaluating the carcinogenic potential of BCEE was conducted according to recognized guidelines.

Justification for classification or non-classification

Based on available data, there is limited evidence for the carcinogenic potential of BCEE in mammals. However, in Table 3.1. to Annex VI of the Regulation (EC) No 1271/2008 on the classification, labelling and packaging of substances and mixtures (CLP) a harmonized classification for Bis(2-chloroethyl) ether is provided and classifies the substance as Carcinogen Category II "Suspected human carcinogen".

Additional information

None of the available studies were conducted according to recognized guidelines. In most of time the period of treatment was shorter than 2 years, and a limited number of animals were used in most studies.

- Exposure route: oral

Innes et al.(1969) administered BCEE by gavage to two hybrid mouse strains, (C57Bl/6 x C3H/Anf)F1 and (C57Bl/6 x AKR)F1. Groups of 18 mice/sex/strain began treatment with 100 mg/kg at 7 days of age. At 4 weeks, after weaning, exposure was continued through the diet. Concentrations in diet were calculated so as to deliver the maximally tolerated dose (300 ppm estimated to be equivalent to daily intake of 100 mg/kg), and treatment was continued for 18 months. The time‐weighted average dose for these studies was calculated to be 41.3 mg/kg body weight per day (US EPA, 1987). There were multiple groups of controls consisting of animals of both strains and sexes. "Hepatomas" (representing benign hepatomas and malignant tumours), tumours of the pulmonary system (adenomas and adenocarcinomas) and lymphomas (Type‐B reticulum cell sarcomas and leukaemias) were the predominant types of tumours observed in these animals. Compared to unexposed controls, the incidence of "hepatomas" was significantly (p = 0.01) increased in the treated (C57BL/6 × C3H/Anf)F1 mice (in males, 8/79 versus 14/16; in females, 0/87 versus 4/18; in control and exposed animals, respectively) and in (C57BL/6 × AKR)F1 males (5/90 versus 9/17 in control and exposed animals, respectively). No distinction was made between "hepatomas" and liver carcinomas, due to the impossibility of making the distinction between benign and malignant liver tumors in the mouse. However, the author noted that metastasizing hepatic cell tumors were rare but concluded that the great of tumors majority had malignant potentiality. Theincidence of pulmonary tumours or lymphomas was not significantly increased in the BCEE‐exposed animals of eithersex. This study is limited owing to small number of BCEE-exposed animals, the use of single dose level, inadequate reporting of tumour pathology, the fact that the amount of BCEE was not adjusted during initial period to account for weight gain, and that the exact dose received by the test animals can only be extrapolated. In addition, increased incidence of mouse liver hepatomas has been questioned as a reliable indication of true carcinogenic potential (Maronpot et al. 1987).

Weisburger et al. (1981) reported that the oral administration of BCEE to rats had no significant carcinogenic effect. BCEE (dissolved in a solution containing sodium chloride, Polysorbate 80, carboxy‐methylcellulose and benzyl alcohol) was administered (by gavage) to groups of 26 male and 26 female Charles River CD rats (at doses of 50 and 25 mg/kg body weight) twice weekly for 78 weeks, after which time the animals were observed for a further 26‐week period. Control groups of each sex (the size of which was not clearly stated) were administered vehicle alone. The authors reported (although no data on tumour incidence were presented) that BCEE was not carcinogenic in these male or female rats.

 

- Exposure route: intra-peritoneal injection

Theiss et al. (1977) assessed the potential of BCEE to produce lung tumours in groups of 20 male A/St mice injected intraperitoneally three times per week with 8, 20 or 40 mg/kg body weight BCEE (dissolved in tricaprylin). Mice injected with 8 and 20 mg/kg received a total of 24 injections while animals administered 40 mg/kg only received 4 injections. Controls (n = 20) were injected with vehicle (tricaprylin) alone. The mice were killed 24 weeks after the initial injection and the number of surface lung tumours (adenomas) determined. The incidence of lung tumours (expressed as the number of lung tumours/mouse) in the BCEE‐exposed animals was less than that observed in animals injected with vehicle alone.

 

- Exposure route: subcutaneous injection

Van Duuren et al. (1972) investigated the potential of BCEE to induce tumours in a study in which groups of 30 female ICR/Ha Swiss mice were injected subcutaneously with 1 mg BCEE (suspended in 0.05 ml mineral oil) once per week for life (the median survival time of animals was 656 days). Compared to animals injected with vehicle alone, where no tumours developed at the site of injection, 2/30 animals injected with BCEE developed sarcomas at the site of injection.BCEE was thus considered marginally sarcomatogenic at s.c. injection sites in female ICR/Ha Swiss mice.

Norpoth et al. (1986) also examined the carcinogenicity of BCEE in a study in which groups of 50 male and 50 female Sprague‐Dawley rats were injected subcutaneously with either 4.36 μmole (0.62 mg) or 13.1 μmole BCEE (1.87 mg) (dissolved in 0.25 ml DMSO) once per week over a 2‐year period. Controls were injected with DMSO (alone) or left untreated. The incidence of all malignant and benign tumours (e.g., mesenchymal, epithelial, sarcomas, carcinomas and unclassified) in the BCEE‐exposed animals was not significantly different from that in the controls. The median survival time of the untreated control, vehicle‐treated control, and low‐ and high‐dose groups was 696, 605, 590 and 643 (for males), and 639, 668, 629 and 654 days (for females), respectively.

Conclusion:

No study aiming at evaluating the carcinogenic potential of BCEE was conducted according to recognized guidelines. BCEE was found to induce an increased incidence of hepatomas in mice following repeated oral exposure to BCEE (Innes et al. 1969). This results was not confirmed in a similar study conducted on rats (Weisburg et al. 1981). BCEE did not induced an increased incidence of lung tumors following repeated i.p. injections (Theiss et al. 1977). BCEE was not tumorigenic but marginally sarcomagenic following repeated subcutaneous injections in female ICR/Ha Swiss mice (van Duuren, 1972) but did not increase the incidence of all malignant and benign tumours (e.g., mesenchymal, epithelial, sarcomas, carcinomas and unclassified) in male or female Sprague‐Dawley rats (Norpoth et al. 1986). Taking into account the methodological limitations of these studies, these conclusions should be taken with caution. However, considering available data, there is limited evidence for the carcinogenic potential of BCEE in mammals.