Bis(2-chloroethyl) ether

Endpoint summary

Description of key information

For Bis(2-chloroethyl) ether, a NOAEL of 15 mg/kg bw/day for oral exposure was established based on a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test performed in rat according to OECD Guideline N°422 under GLP (reliability 1).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

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. Amount of BCEE was not adjusted during initial period to account for weight gain.

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: è 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

Details on route of administration:

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.

Vehicle:

other: water prior to weaning then no vehicle

Details on oral exposure:

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

Frequency of treatment:

Daily

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

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.

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

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

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 examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Increased relative risk of developping hepatomas in males of both strains

Survival data at study termination:

Strain	N° of surviving mice a study termination
	Male	Female	Negative control (Male)	Negative control (Female)
(C57BL/6 x C3H/Anf)F1	11/18 (61%)	18/18 (100%)	73/90 (81%)	83/90 (92%)
(C57BL/6 x AKR)F1	15/18 (83%)	17/18 (94%)	89/90 (99%)	75/90 (83%)

Relative risk for development of tumors among mice treated with Bis(2-chloroethyl) ether when compared with negative controls:

Starin	Hepatomas		Pulmonary tumors		Lymphomas
	Male	Female	Male	Female	Male	Female
(C57BL/6 x C3H/Anf)F1	48.8	N.A.	0	0	2.3	0
(C57BL/6 x AKR)F1	17.4	0	1.2	0	0	1.5

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 chemical was mixed directly with the diet (300 ppm), which was provided ad libitum; no vehicle was used. 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 developping hepatomas.

This study is limited owing to small number of BCEE-exposed animals, the use of single dose level and inadequate reporting of tumour pathology. In addition, the amount of BCEE was not adjusted during initial period to account for weight gain and the exact dose received by the test animals can only be extrapolated. Considering the lack of detailed information and the methodological deficiencies, no NOAEL can be derived.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted on behalf of the Japanese Ministry of Health, Labour and Welfare

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

TEST MATERIAL
- Name (as cited): 2,2'-Dichlorodiethyl ether
- Purity: 99.50% (GC)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at room temperature in a dark place

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan (Hino Breeding Center)
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: 9 weeks old
- Weight at study initiation: males = 333.0–370.1g ; females = 205.3–250.1g
- Housing: biotron barrier system
- Diet: ad libitum
- Water ad libitum
- Acclimation period: eight days, including six days of quarantine

DETAILS OF FOOD AND WATER QUALITY:
- Feed: Oriental Kobo Kogyo solid feed, MF, lot nos. 060306 and 060509
- Water: Hita City tap water (chlorine added)
Feed and water confirmed to be within the reference values set down by the "Contaminant Limits in Feed and Media" in the EPA’s harmful substance guidelines

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21–25°C
- Humidity (%): 40–70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 19h dark / 7h light

Route of administration:

oral: gavage

Vehicle:

olive oil

Remarks:

Fujimi Seiyakusho, lot no. 0380HS

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was prepared by weighing a known amount of 2,2'-dichloroethyl ether in olive oil to reach a final concentration of 0.3% (w/v). Solutions at 0.06 and 0.012% (w/v) were diluted from the 0.3 w/v% solution with olive oil. New stock solution were prepared weekly and stored in a cool dry place before dosing.

VEHICLE
- Justification for use and choice of vehicle: the substance was considered to be insoluble in pure water
- Concentration in vehicle: 0.06, 0.012 and 0.3% (w/v)
- Amount of vehicle: 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The uniformity of the test substance in the prepared solution was measured by taking n = 1 samples from the top, middle, and bottom layers of the prepared 0.3 and 0.12 w/v% solutions, pretreating the samples, and measuring the test substance concentration once each using gas chromatography (GC).
Stability of the test substance in the prepared solution was measured by storing the prepared solutions whose uniformity was confirmed in a cool, dark place, taking n = 1 samples from the middle layer after storage for seven and 14 days, pretreating these, and then using GC to measure the test substance concentrations once each.
Concentration of the test substance in the prepared solution was measured by taking n = 1 samples from the middle layer after preparation of the initially prepared 0.3, 0.06, and 0.012 w/v% solutions, pretreating these, and then using GC to measure the test substance concentrations once each.
The 0.3 and 0.012 w/v% solutions were confirmed to be uniform and stable after storage for 14 days in a cool, dark place. The test substance concentration in the prepared solution used for the initial dosing was confirmed to be 100±10% for all concentrations in the set values for 0.3, 0.06, and 0.012 w/v%.

Duration of treatment / exposure:

Males: 42 days
Females: 42-45 days

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose selection was based on a range finding study.
Three male and three female rats from each group were used to conduct a repeated oral dosing for a period of 14 days at doses of 0, 1, 5, 20, and 100 mg/kg bw/day.
Observations: Increase in hematocrit at 5 mg/kg bw/day and higher. At 20 mg/kg bw/day and higher, low spontaneous locomotion and low respiratory rate were observed. At 100 mg/kg bw/day, weight loss, staggering gait, abnormal gait, coma, feeding problems, low stool amount, low body temperature, lid-closure, deep breathing, mucous stool, prone and recumbent positions, and staining of the lower abdomen and around the anus. All test animals died at this dose.
Dose selection for the full test: Based on observations from the range finding study, the maximum dose was set at 15 mg/kg bw/day with two other doses at 3 and 0.6 mg/kg/day. The doses were slected based on the fact that low spontaneous locomotion and low respiratory rate was observed at 20 mg/kg bw/day and that the dosing period would be longer.

- Rationale for animal assignment: randomized extraction by weight, so that members of each group were of approximately the same weight

- Rationale for selecting satellite groups: 5 males and females (unmated) from the 0 and 15 mg/kg bw/day groups

- Post-exposure recovery period in satellite groups: 14 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Males: twice a day from the first day of dosing until the day before necropsy
Females: twice a day from the first day of dosing until the fourth day of delivery, including the delivery state and nursing state
Recovery period: once a day during the recovery period until the day before necropsy

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: observed in a blind fashion once prior to dosing and once a week after dosing had started

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were measured once a week during the habituation period and on the day of grouping. After grouping, males were measured on dosing day 1, 3, 7, 14, 21, 28, 35, 42, and 43 (day of removal). The females were measured on dosing day 1, 3, 7, and 14, and gestation day 0, 7, 14, 17, and 20 during the gestation period, and, after delivery, postpartum day 0 (day of delivery), 4, and 5 (day of removal). During the recovery period, both males and females were measured on day 1, 3, 7, 14, and 15 of recovery.

FOOD CONSUMPTION AND COMPOUND INTAKE :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: the last day of the dosing period and the last day of the recovery period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: 5 animals/sex/dose group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: the last day of the dosing period and the last day of the recovery period
- Animals fasted: Yes
- How many animals: 5 animals/sex/dose groupd.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule: observed in a blind fashion once prior to dosing and once a week after dosing had started
- Dose groups that were examined: all dose groups
- Battery of functions tested: reactivity testing, posture, motor activity, respiration, lid closure, gait, tremor/twitch/convulsion, and presence/absence of stereotypic behaviour and abnormal behaviour

IMMUNOLOGY: No

OTHER: Oestrous cycles, Fertility test, Delivery and nursing, examination of pups

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (Naked eye observation following necropsy / Organ weight)
Organs weighted: Trachea and lungs, stomach, intestine (duodenum to rectum, including Peyer’s patches), liver, heart, kidneys, bladder, testes, epididymis, prostate, seminal vesicles, ovaries, uterus, vagina, brain (cerebrum, including the cerebellum and pons), spinal cord, sciatic nerve, bone marrow (femur), lymph nodes (axillary and mesenteric lymph nodes, spleen, thymus, pituitary, thyroid (including parathyroid), adrenal.

HISTOPATHOLOGY: Yes
For the control groups and the 15 mg/kg bw/day groups, the trachea, lung, stomach, intestine (duodenum-rectum, Peyer's patches included), liver, heart, kidney, bladder, testis, epididymis, prostate, seminal vesicles, ovary, uterus, vagina, brain (cerebrum, including the cerebellum and pons), spinal cord, sciatic nerve, bone marrow (femur), axillary lymph nodes, mesenteric lymph nodes, spleen, thymus, pituitary, thyroid (including parathyroid), and the adrenal gland of five males each in ascending order of animal number and five females each closest to their delivery dates were examined. Effects of test substance dosing were suspected, so the stomachs of five females each closest to delivery in the 0.6 and 3 mg/kg bw/day group and of the females in the recovery group were examined. Effects of test substance dosing were suspected due to changes in organ weight, so the adrenal glands of the males in the control recovery group and the 15 mg/kg group were examined. The spleen of one male in the control group of the recovery group which displayed gross lesions was examined, as were the glandular stomach in one male and the epididymis in another male in the 0.6 mg/kg group, the glandular stomach and epididymis of one male in the 3 mg/kg bw/day group, and the liver of one female and the kidney and thymus of another female in the 3 mg/kg bw/day group.

Statistics:

Bartlett testing was used for parent weight (not including when removed), food intake, haematology examination items, blood biochemistry examination items, organ weight, average days in oestrous cycle, number of days for mating, gestation period, number of corpora lutea and implantation marks, grip strength, spontaneous locomotion grade, number of pups, number of live pups, day zero live pup ratio, day 4 live pup ratio, and weight, and if variance was seen at the 5% significance level, a one-way analysis of variance was used. If a significant difference was seen in the analysis of variance, the Dunnett method was used between the control groups and the various dose groups. If no equal variance was seen, a Kruskal-Wallis rank sum test method was used to test significance among all groups, followed by a test of significant difference with the control groups using the Dunnett multiple comparison method.
Kruskal-Wallis was used for the number of defecations and urinations, and if a significant difference was seen, the significant difference with the control groups was tested using the Dunnett multiple comparison method.
Significant differences with the control group in the parents’ mating rate, insemination rate, conception rate, implantation rate, pre-implantation embryo loss rate, post-implantation embryo loss rate, birth rate, delivery rate, day 0 live birth rate and day 4 live birth rate were tested using the chi-square test (or Fisher's exact test if the peripheral frequency was 10 or less).
The Kruskal-Wallis rank sum test method was used to test significance of the birth rate, live birth rate, out of table abnormality rate, and day 4 survival rate of pups in all groups, followed by a test of significant difference with the control groups using the Dunnett multiple comparison method.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

DOSING PERIOD
A missing upper incisor and reddish tearing was seen in one male in the 0.6 mg/kg group, but this was a variation whose relation to dose was not clear. No abnormalities were seen in the 3 and 15 mg/kg groups.
Salivation was observed in one female each in the 3 and 15 mg/kg groups before mating was confirmed. Besides this, low spontaneous locomotion, respiratory rate and body temperature were seen on dosing day 22 in one animal in the 0.6 mg/kg group during the gestation period. This animal died during delivery the next day. Reddish urine was observed in one animal in the 15 mg/kg group.

RECOVERY PERIOD
No abnormalities were seen in any of the males or females.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female in the 0.6 mg/kg group died during delivery.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

DOSING PERIOD
No significant differences were seen in the test substance dosing groups among males or females.

RECOVERY PERIOD
No significant differences were seen in the 15 mg/kg group among males or females.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

DOSING PERIOD
No significant differences were seen in the test substance dosing groups among males. No significant differences were seen in the test substance dosing groups among females before copulation, during gestation and postpartum period.

RECOVERY PERIOD
No significant differences were seen in the 15 mg/kg group among males or females.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

AT THE END OF DOSING PERIOD
Among males, low mean corpuscular volume and mean corpuscular haemoglobin were observed in the 3 mg/kg and higher groups. No significant differences were seen in the 0.6mg/kg group.
Among females, a high lymphocyte ratio was seen in the 0.6 mg/kg group, but no related changes were observed, so this was not thought to be a toxicologically important change. No significant differences were seen in the 3 and 15 mg/kg groups.

AT THE END OF RECOVERY PERIOD
No significant differences were seen in the male groups receiving the test substance.
Among females, high red blood cell and platelet counts were observed in the 15 mg/kg group, and a low reticulocyte count ratio and neutrophil ratio were observed.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

AT THE END OF DOSING PERIOD
No significant differences were seen in the test substance dosing groups among males or females.

AT THE END OF RECOVERY PERIOD
Among males, high chlorine values were seen in the 15 mg/kg group. Among females, high urea nitrogen values were observed in the 15 mg/kg group, but no relevant changes were seen, so this was thought to be an incidental change.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

DOSING PERIOD
No abnormalities were seen in any of the males or females.

RECOVERY PERIOD
No examinations were performed, since no changes were observed suggesting that dosing of the test substance had an effect during the dosing period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

DOSING PERIOD
Males: lower absolute adrenal weight at 15 mg/kg bw/day
Females: no significant differences observed in absolue and relative organ weights

RECOVERY PERIOD
Males: higher absolute and relative adrenal weight at 15 mg/kg bw/day
Females: higher absolute thymus weight at 15 mg/kg bw/day

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

END OF DOSING PERIOD
Males: a blackish part of the mucous membrane of the glandular stomach was observed in one animal in the 0.6 mg/kg group, a whitish part of the epididymis was observed in another animal in the same group, a blackish part of the mucous membrane of the glandular stomach and whitish part of the epididymis were observed in one animal in the 3 mg/kg group, and an apparent spotty pattern was observed in the kidney and a whitish part of the epididymis were observed in one animal in the 15 mg/kg group.
Females: recessed regions of the mucosa in the forestomach, discoloration of the kidneys, oedema-like changes and small size in the thymus, and pleural effusion were observed in one animal in the control group, recessed regions of the mucosa in the forestomach were observed in another animal in the same group, abnormal lobulation of the liver was seen in one animal in the 3 mg/kg group, paleness of the kidneys and small size of the thymus were observed in another animal in the same group, recessed regions of the mucosa in the forestomach were observed in two animals in the 15 mg/kg group, and small size of the thymus were observed in one animal in the same group. No abnormalities were seen in the 0.6mg/kg group, except recessed regions of the mucosa in the forestomach and adrenal enlargement were observed in one female which died in the 0.6 mg/kg group.
The findings were observed in single animals with no connection to dose. They were thus considered to be incidental.

AT THE END OF RECOVERY PERIOD:
No abnormalities were seen in any of the males or females

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

AT THE END OF DOSING PERIOD:
No abnormalities were seen in any of the males or females of the dose groups

AT THE END OF RECOVERY PERIOD:
No abnormalities were seen in any of the males or females of the recovery groups

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

AT THE END OF DOSING PERIOD:
No abnormalities were seen in any of the males or females of the dose groups

AT THE END OF RECOVERY PERIOD:
No abnormalities were seen in any of the males or females of the recovery groups

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects at the highest dose tested

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects at the highest dose tested

Critical effects observed:

no

The test substance is an ether, so it may be expected to have depressing effect to the central nervous system. In the range finding 14-day study, drop in spontaneous locomotion, low respiratory rate, staggering gait, abnormal gait, eating disorder, decreased defecation, weight loss, temperature drops, lid closure, deep breathing, and prone and recumbent positions were observed in the 100 mg/kg bw/day group. All test animal exposed to this dose died. Central nervous system depression was also observed at the 20 mg/kg bw/day (drop in spontaneous locomotion and low respiratory rate). However, in the full study, no such effects were in the 15 mg/kg bw/day group (highest dose tested).

Reddish urine was observed in one female in the 15 mg/kg bw/day group during gestation. While degeneration and necrosis of the renal tubules was observed in the kidneys of this animal, no similar changes were seen other animals, so the connection between the change in the kidneys and the reddish urine was therefore considered independent of the test substance. In one female in the 0.6 mg/kg bw/day group which died during delivery, a drop in spontaneous locomotion, low respiratory rate, and a drop in body temperature were observed, and histopathological changes were seen in the glandular stomach, the kidneys, the spleen, the thymus, and the adrenal glands. Nevertheless, the connection with the dose was not supported by observation made on other test animals. Regarding organs weight, high absolute and relative weights of the adrenal glands were seen in the 15 mg/kg bw/day group of the male recovery group, and high absolute weights of the thymus were seen in females from the same dose group. However, the adrenal glands did not have any other accompanying organic changes, nor were any changes seen in the relative weights of the thymus, so these were judged to be incidental changes caused by low body weight. In the haematology examinations, low mean corpuscular volume and low mean corpuscular haemoglobin were observed in the 3 mg/kg groups and higher for the males, but no abnormalities were observed in the red blood cell count, so this was deemed an incidental change.

Conclusions:

Following the oral (gavage) administration of Bis(2-chloroethyl) ether at doses of 0.6, 3 and 15 mg/kg/Day, the only effect observed was a higher absolute and relative weights of the adrenal glands in the 15 mg/kg group of the male recovery group, and high absolute weights of the thymus in the same female group. However, as these findings were not associated with any other organic changes, they were considered incidental.
The No Observed Adverse Effect Level (NOAEL) for parental toxicity is therefore 15 mg/kg/Day.

Executive summary:

The objectives of this study, conducted on behalf of the Japanese Ministry of Health, Labour and Welfare, were to evaluate the potential toxic effects of the test item, Bis(2-chloroethyl) ether, when exposed for a minimum of 28 Days and the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. Parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No-Observed-Adverse-Effect-Levels (NOAELs) were evaluated. This GLP study was carried out according to OECD test guideline No. 422.

Three groups of 12 males and 12 females Crl:CD (SD) rats were given the test item, Bis(2-chloroethyl) ether, by daily oral (gavage) administration at dose levels of 0.6, 3 and 15 mg/kg/day. The male animals received dosing starting two weeks prior to the start of the mating period, throughout the mating period, and up to the day before the necropsy, for a total of 42 days. The female animals received dosing starting two weeks prior to the start of the mating period, throughout the mating, gestation, and nursing periods, and up to the day before the necropsy (fourth day of delivery), for a total of 42 to 45 days. Males and females were subjected to general and detailed observation throughout dosing, including functional assessments, weight and food intake measurements, necropsy, histopathological examinations of major organs and blood tests.

High absolute and relative weights of the adrenal glands were seen in the 15 mg/kg group of the male recovery group, and high absolute weights of the thymus were seen in the same female group. However, the adrenal glands did not have any other accompanying organic changes, nor were any changes seen in the relative weights of the thymus, so these were judged to be incidental changes.

Accordingly, the exposure to doses up to 15 mg/kg bw/day was not considered to induce any significant change in any of the investigated parameters. The NOAEL and NOEL related to repeated dose toxicity of Bis(2-chloroethyl) ether under the conditions of this study was 15 mg/kg bw/day. In a range-finding test, higher dose where found to significantly impact the health of test animal including central nervous system depression at 20 mg/kg bw/day and death of all animals at 100 mg/kg bw/day.

Reference 3

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Technical Guideline 451

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 oral 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 followed by a 6 months observation period

Frequency of treatment:

twice-weekly

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.

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 consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

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

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Conclusions:

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. Based obn reduced body weight, the NOEL is < 25 mg/kg bw/day in female and 25 mg/kg bw/day in male rats.

Executive summary:

This study was originally aiming at testing chemicals for carcinogenicity by oral administration in male and female Charles River CD rats. The method used was similar to OECD Guideline No. 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. Accordingly, the NOEL is < 25 mg/kg bw/day in female and 25 mg/kg bw/day in male rats.

Endpoint conclusion

Dose descriptor:

NOAEL

15 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Two pre-GLP chronic oral studies are available (Innes et al. 1969, Weisburger et al. 1981). However, the objective of these studies was to evaluate the carcinogenic potential of Bis(2-chlorethyl) ether and very limited information on gross pathology and histological examination is provided. Apart from these two studies, the only information aivailable originates from a sub-chronic GLP study based on OECD Guideline N°422. However, no standard 90-days repeated dose toxicity study is available.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

no guideline followed

Principles of method if other than guideline:

Study report from Dow chemical (1958). Exposure to a fixed dose 7hr/day, 5dys/week for 130 days. No access to the full study report.

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: not specified

Species:

rat

Strain:

not specified

Sex:

not specified

Route of administration:

inhalation

Type of inhalation exposure:

not specified

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

130 days

Frequency of treatment:

7h/day; 5 days/week

Dose / conc.:

69 ppm

Remarks:

equivalent to 0.43 mg/L

No. of animals per sex per dose:

Not specified

Control animals:

not specified

Observations and examinations performed and frequency:

BODY WEIGHT: Yes

HAEMATOLOGY: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Organ weight (lungs)

HISTOPATHOLOGY: Yes
- Lungs; Kidneys; Liver; Heart

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduced body weight was observed.

Haematological findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No significant changes in lung/body weight ratios.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No histological changes in lungs, heart, liver and kidneys.

Dose descriptor:

NOAEC

Effect level:

69 ppm

Based on:

test mat.

Sex:

not specified

Basis for effect level:

mortality

Key result

Dose descriptor:

LOAEC

Effect level:

69 ppm

Based on:

test mat.

Sex:

not specified

Basis for effect level:

body weight and weight gain

Dose descriptor:

NOAEC

Effect level:

69 ppm

Based on:

test mat.

Sex:

not specified

Basis for effect level:

haematology

Dose descriptor:

NOAEC

Effect level:

69 ppm

Based on:

test mat.

Sex:

not specified

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

no

Conclusions:

In the conditions of this test, following repeated exposure via the inhalation route for 130 days, the LOAEC of Bis(2-chloroethyl) ether in rats is 69 ppm.

Executive summary:

In this review on the toxicology of Bis(2-chloroethyl) ether, the author cites an unpublished study from Dow Chemical (1958), where rats were exposed for 130 days to the substance (concentration: 69 ppm; 7h/days; 5 days/week). The publication does not detail the result obtained from this study. The significance of the findings is questionable as only one test concentration was studied and the number of test animals is not specified. However, it has been subjected to the formal review process of U.S. EPA and it is thus judged acceptable for the assessment of the substance.

Regarding the results of this study, it is stated that the following parameters were examined: mortality, body weight, histopathology of main organs (including heart, lungs, kidneys, liver), haematology, and neurological parameters (unspecified). Exposure to the substance did not result in significant changes in lung/body weight ratios, and did not lead to histological changes in lungs. The only finding observed following the 130 days exposure period was a decrease in body weight. Accordingly, based on this study the LOAEC of Bis(2-chloroethyl) ether in rats is 69 ppm.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

no guideline followed

Principles of method if other than guideline:

Study report from Dow chemical (1958). Exposure to a fixed dose 7hr/day, 5dys/week for 130 days. No access to the full study report.

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: not specified

Species:

guinea pig

Strain:

not specified

Sex:

not specified

Route of administration:

inhalation

Type of inhalation exposure:

not specified

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

130 days

Frequency of treatment:

7h/day; 5 days/week

Dose / conc.:

69 ppm

Remarks:

equivalent to 0.43 mg/L

No. of animals per sex per dose:

Not specified

Control animals:

not specified

Observations and examinations performed and frequency:

BODY WEIGHT: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Organ weight (lungs)

HISTOPATHOLOGY: Yes
- Lungs; Kidneys; Liver

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduced body weight was observed.

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No significant changes in lung/body weight ratios.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No histological changes in lungs, heart, liver and kidneys.

Dose descriptor:

NOAEC

Effect level:

69 ppm

Based on:

test mat.

Sex:

not specified

Basis for effect level:

mortality

Key result

Dose descriptor:

LOAEC

Effect level:

69 ppm

Based on:

test mat.

Sex:

not specified

Basis for effect level:

body weight and weight gain

Dose descriptor:

NOAEC

Effect level:

69 ppm

Based on:

test mat.

Sex:

not specified

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

no

Conclusions:

In the conditions of this test, following repeated exposure via the inhalation route for 130 days, the LOAEC of Bis(2-chloroethyl) ether in guinea pigs is 69 ppm.

Executive summary:

In this review on the toxicology of Bis(2-chloroethyl) ether, the author cites an unpublished study from Dow Chemical (1958), where guinea pigs were exposed for 130 days to the substance (concentration: 69 ppm; 7h/days; 5 days/week). The publication does not detail the result obtained from this study. The significance of the findings is questionable as only one test concentration was studied and the number of test animals is not specified. However, it has been subjected to the formal review process of U.S. EPA and it is thus judged acceptable for the assessment of the substance.

Regarding the results of this study, it is stated that the following parameters were examined: mortality, body weight, histopathology of main organs (including heart, lungs, kidneys, liver), and neurological parameters (unspecified). Exposure to the substance did not result in significant changes in lung/body weight ratios, and did not lead to histological changes in lungs. The only finding observed following the 130 days exposure period was a decrease in body weight. Accordingly, based on this study the LOAEC of Bis(2-chloroethyl) ether in guinea pigs is 69 ppm.

Endpoint conclusion

Dose descriptor:

LOAEC

403.6 mg/m³

Study duration:

chronic

Species:

rat

Quality of whole database:

Only limited data (Dow Chemical 1958) are available on the effects of repeated inhalation exposure to BCEE. This study employed only one exposure level (69 ppm), so thresholds were not established for any adverse effects following repeated exposures. For this reason, further studies using modern histological and biochemical tests would be useful in determining the NOAEC and LOAEC values for injury to lung and other tissues.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

No data available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

No data available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

No data available

Additional information

3 studies on repeated oral exposure to Bis(2-chloroethyl) ether (BCEE) via the oral route on rats and mice were available. Exposure duration ranged from 42 days to 18 months. Two studies were originally designed to evaluate the carcinogenicity of the substances (Innes et al. 1969; Weisburg et al. 1981). These pre-GLP studies does not report gross pathological and histological observations in sufficient details to assess the systemic toxicity induced by repeated oral exposure to Bis(2-chloroethyl) ether.

In Innes et al. (1969), mice (7 days of age) from two hybrid strains were first given a daily dose (100 mg/kg bw/day) of test substance by stomach tube. At weaning (4 weeks old), the chemical was mixed directly with the diet (300 ppm), which was provided ad libitum. The time‐weighted average dose for these studies was calculated to be 41.3 mg/kg body weight per day (US EPA, 1987). Increased mortality was observed in male of the (C57BL/6 x C3H/Anf)F1 strain but no information on the post necropsy examinations are reported. Owing to small number of BCEE-exposed animals, the use of single dose level and inadequate reporting, the study was considered as unreliable for the assessment of the impact of repeated exposure to BCEE.

In weisburg et al. (1981). Accrodingly, no NOAEL was derived. In Weisburg et al. (1981), 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. 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. Accordingly, the NOEL is < 25 mg/kg bw/day in female and 25 mg/kg bw/day in male rats.

The NITE Chemical Risk Information Platform includes a report on a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD Guideline N°422 under GLP (reliability 1). This study was selected as Key study. Three groups of 12 males and 12 females Crl:CD (SD) rats were given BCEE by daily oral (gavage) administration at dose levels of 0.6, 3 and 15 mg/kg/day. The male animals received dosing starting two weeks prior to the start of the mating period, throughout the mating period, and up to the day before the necropsy, for a total of 42 days. The female animals received dosing starting two weeks prior to the start of the mating period, throughout the mating, gestation, and nursing periods, and up to the day before the necropsy (fourth day of delivery), for a total of 42 to 45 days. High absolute and relative weights of the adrenal glands were seen in the 15 mg/kg group of the male recovery group, and high absolute weights of the thymus were seen in the same female group. However, the adrenal glands did not have any other accompanying organic changes, nor were any changes seen in the relative weights of the thymus, so these were judged to be incidental changes. Accordingly, the exposure to doses up to 15 mg/kg bw/day was not considered to induce any significant change in any of the investigated parameters. The NOAEL and NOEL related to repeated dose toxicity of Bis(2-chloroethyl) ether under the conditions of this study was 15 mg/kg bw/day. In a range-finding test, higher dose where found to significantly impact the health of test animal including central nervous system depression at 20 mg/kg bw/day and death of all animals at 100 mg/kg bw/day.

The only information available regarding repeated exposure to BCEE via the inhalation route are reported in a review on the toxicology of Bis(2-chloroethyl) ether published by U.S EPA (1989). The results originate from unpublished reports by Dow Chemical (1958). Rats and Guinea pigs rats were exposed for 130 days to the substance (concentration: 69 ppm; 7h/days; 5 days/week). The significance of the experimental findings is questionable as only one test concentration was studied and the number of test animals is not specified. However, it has been subjected to the formal review process of U.S. EPA and it is thus judged acceptable for the assessment of the substance. The following parameters were examined: mortality, body weight, histopathology of main organs (including heart, lungs, kidneys, liver), haematology (rats only), and neurological parameters (unspecified). The only finding observed following the 130 days exposure period was a decrease in body weight in rats and guinea pigs. Accordingly, based on this study the LOAEC of Bis(2-chloroethyl) ether in rats is 69 ppm.

Justification for classification or non-classification

3 studies on repeated oral exposure via the oral route on rats and mice were available. Only one study was conducted according to current recommended guideline (NITE-CHRIP, 2007) and was selected as key study. In this study, following a c.a. 42 days daily oral exposure to BCEE no effect was observed at the highest dose tested (15 mg/kg bw/day). In a range-finding test, higher dose where found to significantly impact the health of test animal including central nervous system depression at 20 mg/kg bw/day and death of all animals at 100 mg/kg bw/day. However, insufficient details are provided in order to judge the validity of this observation.

Reagarding studies on repeated exposure via the inhalation route, 2 unpublished reports are cited in a review on the toxicology of Bis(2-chloroethyl) ether published by U.S EPA (1989). While limited information are reported, U.S. EPA publication are subjected to a formal review process and the information provided was judged acceptable for this assessment. Exposure for 130 days to the substance (concentration: 69 ppm; 7h/days; 5 days/week) had no effect on mortality, body weight, histopathology of main organs (including heart, lungs, kidneys, liver), haematology (examined on rats only), and neurological parameters (unspecified) of rats and guinea pigs. The only finding observed following the 130 days exposure period was a decrease in body weight. Accordingly, based on this study the LOAEC of Bis(2-chloroethyl) ether in rats is 69 ppm (equivalent to 403.6 mg/m3)

According to the classification criteria of EC regulation 1272/2008, Bis(2-chloroethyl) ether should not be classified for repeated dose toxicity.