Bis(2-chloroethyl) ether

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-chloroethyl) ether

EC Number:

203-870-1

EC Name:

Bis(2-chloroethyl) ether

Cas Number:

111-44-4

Molecular formula:

C4H8Cl2O

IUPAC Name:

1-chloro-2-(2-chloroethoxy)ethane

Test material form:

liquid

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

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

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

Examinations

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.

Results and discussion

Results of examinations

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

Effect levels

1

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

2

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

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.