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Key value for chemical safety assessment

Effects on fertility

Description of key information

Oral exposure:

Based on a GLP study carried out according to OECD test guideline No. 422 (reliability 1), the No Observed Adverse Effect Level (NOAEL) for P0 reproductive performance is 3 mg/kg/day based on reduced delivery index at 15 mg/kg bw/day. The NOEL for P0 reproductive performance and fetal toxicity is < 0.6 mg/kg/day based on slightly higher post-implantation loss and lower birth index at 0.6 mg/kg bw/day.

Inhalation exposure:

Only low reliability (reliability 4) data are available. The data originate from a review on the toxicology of Bis(2-chloroethyl) ether by USEPA (1989), which cites the results of unpublished studies by Dow Chemical. Rats and guinea pigs were exposed for 130 days to the substance (concentration: 0.43 mg/L; 7h/days; 5 days/week). Exposure to Bis(2-chloroethyl) ether did not produce any gross or histological changes to reproductive tissues, but no tests of reproductive function or success were performed.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
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 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
Details on mating procedure:
- M/F ratio per cage: one-to-one
- Length of cohabitation: until mating but no longer than 14 days
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged alone
Analytical verification of doses or concentrations:
not specified
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
Parental animals: Observations and examinations:
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
Oestrous cyclicity (parental animals):
For all females, vaginal plaque was taken from the day dosing started until the day mating was confirmed. Giemsa staining was used for optical microscopy. The average number of days in the oestrous cycle was calculated between oestrus days between the first and 14th days of dosing. The incidence of abnormal oestrous cycles (individuals not exhibiting regular oestrous cycles) was also calculated.
Statistics:
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.
Reproductive indices:
FERTILITY TEST:
Mating was checked daily during the mating period. If sperm was found in the vaginal plug or the vaginal plaque, mating was assumed to have been performed, and that day was established as day 0 of pregnancy. The following indices were calculated:
- Mating rate: (number of copulating animals/number of animals placed together) × 100
- Insemination rate: (number of males that impregnated a female/number of mating males) × 100
- Conception rate: (number of pregnant females/number of mating males) × 100
- Days required for mating
- Implantation rate: (number of implantation marks/number of corpora lutea of pregnancy) × 100
- Pre-implantation embryo loss rate: [(number of corpora lutea of pregnancy – number of implantation marks)/number of corpora lutea of pregnancy] × 100
- Post-implantation embryo loss rate: [(number of implantation marks – number of pups born)/number of implantation marks] × 100

DELIVERY AND NURSING:
The following indices were calculated:
- Gestation period (number of days from the day mating was confirmed until the day delivery was complete)
- Birth rate: (number of females birthing live pups/number of pregnant females) × 100
- Delivery rate: (number of birthed pups/number of implantation marks) × 100
Offspring viability indices:
Number of births, number of dead pups, number of live births, sex and number of live births outside the table were examined on the day of birth (calculated starting on day 0 after birth). The general state, the number of pups alive and the number of pups who died was observed through day 3 after birth, and on the fourth day after birth (slaughter date) the number of pups, the sex, the number of live pups outside the table, the number of dead pups, and the number of dead pups outside the table were examined, and the following indices were calculated:
- Birth rate: (number of live births/number of implantation marks) × 100
- Live birth rate: (number of live births/number of births) × 100
- Day 0 live pup ratio: number of live male pups on day 0/number of live pups on day 0
- Out of table abnormality rate: (number of live births having out of table abnormalities/number of examined live births) × 100
- Day 4 survival rate: (number of live pups four days after birth/number of births on day of birth) × 100
- Day 4 live pup ratio: number of live male pups on day 4/number of live pups on day 4
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
DOSING PERIOD
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 animal in the 0.6 mg/kg group died during delivery.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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, non-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
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Although continuous resting was observed in one animal in the 3 mg/kg group, the animal was normal until collection of the vaginal plaque on day 16, so this was deemed to be caused by a phantom pregnancy. No abnormalities were seen in any other animals.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
All females were pregnant in the control and treated groups. There was no effect on gestation length, the number of corpora lutea or the number of implantation sites. No differences were observed on the gestation index (Number of pregnant females with live pups / Number of pregnant females ×100), on the implantation index (Number of implantation sites / Number of corpora lutea × 100) or on preimplantation loss.

A lower delivery index (Number of pups born / Number of implantation sites × 100) was observed in test animals treated with 0.6 and 15 mg/kg bw/day. However, no effect was observed at 3 mg/kg bw/day and the connection with dose is thus unclear. The effect on delivery rate was considered incidental in the 0.6 and 3 mg/kg bw/day, while it was considered to be an effect of toxicity of the test substance at 15 mg/ kg bw/day.

Higher post-implantation loss ((Number of implantation sites - Number of live pups on postnatal day 0) / Number of implantation sites x 100) was observed at 0.6 mg/kg bw/day and above (7.2, 15.6, 17.0 and 18.7%, in the control, 0.6, 3, 15 mg/kg bw/day) group, respectively). Similarly, lower birth index (Number of live pups on postnatal day 0 / Number of implantation sites × 100) was observed at 0.6 mg/kg bw/day and above (92.8, 84.4, 83.0 and 81.3%, in the control, 0.6, 3, 15 mg/kg bw/day) group, respectively). However, it is these observation originates from toxicity towards germ cells of the P0 generation, from embryotoxicity or, more generally, from maternal toxicity. In addition, no effect was observed on measured parameters and significant differences were only observed on derived indices.

See "Any other informations on results incl. tables" for details
Key result
Dose descriptor:
NOAEL
Remarks:
Parental toxicity
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect at the highest dose
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive performance
Effect level:
3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: reduced delivery index at 15 mg/kg bw/day
Key result
Dose descriptor:
LOEL
Effect level:
< 0.6 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: slighlty higher post-implantation loss and lower birth index at 0.6 mg/kg bw/day
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
A drop in body temperature in three litters was observed in the 3 mg/kg group, a wound to the end of the tail was observed in one animal in nine in one litter in the 15 mg/kg group, and a drop in body temperature was observed in another litter in the same group. These observations were considered incidental.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The live birth index (number of pups born alive/number of pups born x 100) was slightly lower in the 3 mg/kg bw/day (88.8%) compared with the concurrent contrl (96%). However, without any dose relationship and any similar finding at the high dose of 15 mg/kg bw/day, this was considered incidental.

Higher (98.1%) day 4 survival rates (i.e. viability index corresponding to the number of pups alive on PND 4/number of pups born alive x 100) as compared to control (89.9%) was observed in the 0.6 mg/kg bw/day group. At the opposite, a significant decrease in the viability index was found in the 3 mg/kg group (80.1%), while no significant effects were detected at 15 mg/kg bw/day (87.8%). Without any dose relationship and without any significant effect observed at 15 mg/kg bw/day, these observations were considered as incidental.

Higher post-implantation loss ((Number of implantation sites - Number of live pups on postnatal day 0) / Number of implantation sites x 100) was observed at 0.6 mg/kg bw/day and above (7.2, 15.6, 17.0 and 18.7%, in the control, 0.6, 3, 15 mg/kg bw/day) group, respectively). However, it is unclear if post-implantation originates from toxicity towards germ cells of the P0 generation, from embryotoxicity or, more generally, from maternal toxicity.

Significant differences related to pups mortality parameters were only observed on derived indices. No significant differences were observed on measured parameters (number of live pups (male or female) at birth or at PND 4). The mortality observed is not considered to be treatment related.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant differences were seen in the weights on the day of birth and four days later in groups receiving the test substance.
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
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Key result
Dose descriptor:
LOEL
Generation:
F1
Effect level:
< 0.6 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: slighlty higher post-implantation loss that may originate from embryotoxicity.
Reproductive effects observed:
yes
Lowest effective dose / conc.:
0.6 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
yes

## Reproductive parameters in parental rats treated orally with 2,2'-dichlorodiethylether in the combined repeated dose and reproductive/developmental toxicity screening test:

  Dose (mg/kg bw/day)
  0 0.6 3 15
 Number of mated pairs 12 12  12  12 
 Number of of copulated pairs 12 12  12  12 
 Number of of pregnant females 12 12  12  12 
 Copulation index (%)* 100  100  100  100 
 Fertilization index (%)** 100 100  100  100 
 Conception index (%)*** 100 100  100  100 
 Pairing days until copulation (Mean ± SD) 2.7 ± 1.0 2.2  ± 1.0 2.0 ± 0.9 2.0 ± 0.9

* (Number of copulated pairs /Number of mated pairs) × 100

** (Number of impregnated males /Number of males in copulated pairs) × 100

*** (Number of pregnant females /Number of females in copulated pairs) × 100

## Postnatal course of rat litters whose parents treated orally with 2,2'-dichlorodiethylether in the combined repeated dose and reproductive/developmental toxicity screening test:

  Dose (mg/kg bw/day)
  0 0.6 3 15
Number of pregnants 12 12 12 12
Gestation length in days (Mean ± SD) 21.9 ± 0.5 21.9 ± 0.3 22.1 ± 0.5 22.2 ± 0.4
Number of dams with live newborns:
- on postpartum day 0 12 11 12 12
- on postpartum day 4 11 11 9 11
Number of corpora lutea (Mean ± SD) 15.3 15.9 15.7 15.7
Number of implantation sites (Mean ± SD) 15.1 15.5 15.2 15.2
- Gestation index (%)1 100 (12/12) 100 (12/12) 100 (12/12) 100 (12/12)
- Implantation index (%)2 98.9 (181/183) 97.4 (186/191) 96.8 (182/188) 96.8 (182/188)
- Delivery index (%)3 96.7 (175/181) 87.1 (162/186)** 93.4 (170/182) 88.5 (161/182)**
Postnatal day 0        
  - Number of pups born (Mean ± SD) 14.6 ± 1.6 14.7 ± 1.7 14.2 ± 1.6 13.4 ± 3.1
  - Number of live pups (Mean ± SD) 14.0 ± 2.2 14.3 ± 2.2  12.6 ± 2.9 12.3 ± 3.1
  - Number of live males (Mean ± SD) 6.9 ± 2.1 7.3 ± 2.1 6.9 ± 2.9 6.3 ± 2.6
  - Number of live females (Mean ± SD) 7.1 ± 2.7 7.0 ± 2.1 5.7 ± 2.1 6.1 ± 2.4
  - Birth index (%)4 92.8 (168/181) 84.4(157/186)* 83.0(151/182)** 81.3(148/182)**
  - Live birth index (%)5 96.0 (168/175) 96.9(157/162) 88.8(151/170)* 91.9(148/161)
  - Sex ratio6 83/168 80/157 83/151 75/148
   - Sex ratio per dam (Mean ± SD) 0.50 ± 0.15 0.51 ± 0.12 0.54 ± 0.15 0.51 ± 0.15
  - Preimplantation loss (%)7 1.1 (2/183) 2.6 (5/191) 3.2 (6/188) 3.2 (6/188)
  - Postimplantation loss8 (%)  7.2 (13/181) 15.6 (29/186)* 17.0 (31/182)** 18.7 (34/182)**
  - Pups weight (Mean ± SD, g)        
  Male 6.4 ± 0.5 6.6 ± 0.5 6.6 ± 0.6 6.5 ± 0.7
  Female 6.2 ± 0.5 6.3 ± 0.5 6.0 ± 0.6 6.3 ± 0.7
 Postnatal day 4      
  - Number of live pups (Mean ± SD) 13.7 ± 1.8 14.0 ± 2.1 13.4 ± 2.1 11.8 ± 3.0
  - Number of live males (Mean ± SD) 6.5 ± 1.9  7.1 ± 1.9  7.8 ± 2.5  5.9 ± 2.2
  - Number of live females (Mean ± SD) 6.7 ± 3.1  7.0 ± 2.1  4.3 ± 3.0  5.4 ± 2.8
  - Viability index (%)9 89.9 (151/168)  98.1 (154/157)**  80.1 (121/151)*  87.8 (130/148)
- Sex ratio10 71/151  78/154  70/121  65/130
  - Sex ratio per dam (Mean ± SD) 0.48 ± 0.13 0.51 ± 0.12 0.57 ± 0.13 0.57 ± 0.13
  - Pups weight (Mean ± SD, g)        
  Male 9.6 ± 1.7 10.3 ± 1.3 9.8 ± 2.1 11.0 ± 1.2
  Female 9.3 ± 1.6 9.9 ± 1.2 9.1 ± 2.1 10.5 ± 1.6

1 Number of pregnant females with live pups / Number of pregnant females ×100

2 Number of implantation sites / Number of corpora lutea × 100

3 Number of pups born / Number of implantation sites × 100

4 Number of live pups on postnatal day 0 / Number of implantation sites × 100

5 Number of live pups on postnatal day 0 / Number of pups born × 100

6 Number of live males on postnatal day 0 / Number of live pups on postnatal day 0

7 Number corpora lutea - Number of implantation sites) / Number of corpora lutea × 100

8 (Number of implantation sites - Number of live pups on postnatal day 0) / Number of implantation sites × 100

9 Number of live pups on postnatal day 4 / Number of live pups on postnatal day 0 × 100

10 Number of live males on postnatal day 4 / Number of live pups on postnatal day 4

* 　Significantly different from vehicle control at p <0.05

** Significantly different from vehicle control at p <0.01

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.

There were no test item-related pathological changes in the male or female genital system and no effect on the oestrus cycle. here was no effect on gestation length, the number of corpora lutea or the number of implantation sites. No differences were observed on the gestation index, on the implantation index or on preimplantation loss. A lower delivery index was observed in test animals treated with 0.6 and 15 mg/kg bw/day. However, no effect was observed at 3 mg/kg bw/day and the connection with dose is thus unclear. The effect on delivery rate was considered incidental in the 0.6 and 3 mg/kg bw/day, while it was considered to be an effect of toxicity of the test substance at 15 mg/ kg bw/day.

Higher post-implantation loss was observed at 0.6 mg/kg bw/day and above (7.2, 15.6, 17.0 and 18.7%, in the control, 0.6, 3, 15 mg/kg bw/day group, respectively). Similarly, lower birth index (Number of live pups on postnatal day 0 / Number of implantation sites × 100) was observed at 0.6 mg/kg bw/day and above (92.8, 84.4, 83.0 and 81.3%, in the control, 0.6, 3, 15 mg/kg bw/day group, respectively).

Under the conditions of this study he No Observed Adverse Effect Level (NOAEL) for P0 reproductive performance is 3 mg/kg/day based on reduced delivery index at 15 mg/kg bw/day. The NOEL for P0 reproductive performance and foetal toxicity is < 0.6 mg/kg/day based slighlty higher post-implantation loss and lower birth index at 0.6 mg/kg bw/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 femalesCrl: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. No ignificant change in any other the investigated parameters was observed at 15 mg/kg bw/day, which was considered to be the No Observed Adverse Effect Level (NOAEL) for parental toxicity.

P0 Generation:

There were no test item-related pathological changes in the male or female genital system and no effect on the oestrus cycle. All females were pregnant in the control and treated groups. There was no effect on gestation length, the number of corpora lutea or the number of implantation sites. No differences were observed on the gestation index, on the implantation index or on preimplantation loss. A lower delivery index was observed in test animals treated with 0.6 and 15 mg/kg bw/day. However, no effect was observed at 3 mg/kg bw/day and the connection with dose is thus unclear. The effect on delivery rate was considered incidental in the 0.6 and 3 mg/kg bw/day, while it was considered to be an effect of toxicity of the test substance at 15 mg/ kg bw/day.

Higher post-implantation loss was observed at 0.6 mg/kg bw/day and above (7.2, 15.6, 17.0 and 18.7%, in the control, 0.6, 3, 15 mg/kg bw/day group, respectively). Similarly, lower birth index (Number of live pups on postnatal day 0 / Number of implantation sites × 100) was observed at 0.6 mg/kg bw/day and above (92.8, 84.4, 83.0 and 81.3%, in the control, 0.6, 3, 15 mg/kg bw/day group, respectively). However, it is unclear if these observations originate from toxicity towards germ cells of the P0 generation, from embryotoxicity or, more generally, from maternal toxicity. In addition, no effect was observed on measured parameters and significant differences were only observed on derived indices.

F1 generation:

The live birth index (number of pups born alive/number of pups born x 100) was slightly lower in the 3 mg/kg bw/day (88.8%) compared with the concurrent contrl (96%). However, without any dose relationship and any similar finding at the high dose of 15 mg/kg bw/day, this was considered incidental.

Higher (98.1%) day 4 survival rates (i.e. viability index corresponding to the number of pups alive on PND 4/number of pups born alive x 100) as compared to control (89.9%) was observed in the 0.6 mg/kg bw/day group. At the opposite, a significant decrease in the viability index was found in the 3 mg/kg group (80.1%), while no significant effects were detected at 15 mg/kg bw/day (87.8%). Without any dose relationship and without any significant effect observed at 15 mg/kg bw/day, these observations were considered as incidental.

In conclusion, under the conditions of this study the No Observed Adverse Effect Level (NOAEL) for P0 reproductive performance is 3 mg/kg/day based on reduced delivery index at 15 mg/kg bw/day. The NOEL for P0 reproductive performance and fetal toxicity is < 0.6 mg/kg/day based slightly higher post-implantation loss and lower birth index at 0.6 mg/kg bw/day.

Endpoint:
reproductive toxicity, other
Remarks:
repeated inhalation exposure
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
no tests of reproductive function or success were performed
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
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
Positive control:
Not specified
Postmortem examinations (parental animals):
GROSS NECROPSY
- Gross necropsy of reproductive tissues

HISTOPATHOLOGY / ORGAN WEIGHTS
- Histopathological examination of reproductive tissues
Reproductive indices:
Reproductive function or success not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced body weight.
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No effects on lungs, heart, liver, kidneys and reproductive tissues.
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Conclusions:
Inhalation exposure to Bis(2-chloroethyl) ether (7h/days; 5 days/week) for 130 days at 0.43 mg/L did not produce any gross or histological changes to reproductive tissues.
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.

Inhalation exposure to Bis(2-chloroethyl) ether (7h/days; 5 days/week) for 130 days at 0.43 mg/L did not produce any gross or histological changes to reproductive tissues, but no tests of reproductive function or success were performed.

Endpoint:
reproductive toxicity, other
Remarks:
repeated inhalation exposure
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
no tests of reproductive function or success were performed
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
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
Positive control:
Not specified
Postmortem examinations (parental animals):
GROSS NECROPSY
- Gross necropsy of reproductive tissues

HISTOPATHOLOGY / ORGAN WEIGHTS
- Histopathological examination of reproductive tissues
Reproductive indices:
Reproductive function or success not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced body weight.
Haematological findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No effects on lungs, heart, liver, kidneys and reproductive tissues.
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Conclusions:
Inhalation exposure to Bis(2-chloroethyl) ether (7h/days; 5 days/week) for 130 days at 0.43 mg/L did not produce any gross or histological changes to reproductive tissues.
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.

Inhalation exposure to Bis(2-chloroethyl) ether (7h/days; 5 days/week) for 130 days at 0.43 mg/L did not produce any gross or histological changes to reproductive tissues, but no tests of reproductive function or success were performed.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
3 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP study conducted according to a relevant guideline (OECD TG 422).
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
430 mg/m³
Study duration:
chronic
Species:
rat
Quality of whole database:
Non guideline study. No detailed methodology or results are provided. The reliability of existing data is considered to be low.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

3 studies were used to assess toxicity to reproduction. Two studies, assigned in reliability 4, originate from a review on the toxicology of Bis(2-chloroethyl) ether by USEPA (1989), which cites the results of unpublished studies by Dow Chemical. Rats and guinea pigs were exposed for 130 days to the substance (concentration: 0.43 mg/L; 7h/days; 5 days/week). Exposure to Bis(2-chloroethyl) ether did not produce any gross or histological changes to reproductive tissues, but no tests of reproductive function or success were performed.

The third study, from the NITE Chemical Risk Information Platform, consists in 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. There were no test item-related pathological changes in the male or female genital system and no effect on the oestrus cycle. here was no effect on gestation length, the number of corpora lutea or the number of implantation sites. No differences were observed on the gestation index, on the implantation index or on preimplantation loss. A lower delivery index was observed in test animals treated with 0.6 and 15 mg/kg bw/day. However, no effect was observed at 3 mg/kg bw/day and the connection with dose is thus unclear. The effect on delivery rate was considered incidental in the 0.6 and 3 mg/kg bw/day, while it was considered to be an effect of toxicity of the test substance at 15 mg/ kg bw/day. Higher post-implantation loss was observed at 0.6 mg/kg bw/day and above (7.2, 15.6, 17.0 and 18.7%, in the control, 0.6, 3, 15 mg/kg bw/day group, respectively). Similarly, lower birth index (Number of live pups on postnatal day 0 / Number of implantation sites × 100) was observed at 0.6 mg/kg bw/day and above (92.8, 84.4, 83.0 and 81.3%, in the control, 0.6, 3, 15 mg/kg bw/day group, respectively).

Under the conditions of this study he No Observed Adverse Effect Level (NOAEL) for P0 reproductive performance is 3 mg/kg/day based on reduced delivery index at 15 mg/kg bw/day. The NOEL for P0 reproductive performance and foetal toxicity is < 0.6 mg/kg/day based slighlty higher post-implantation loss and lower birth index at 0.6 mg/kg bw/day.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on a GLP study carried out in rat according to OECD test guideline No. 422 (reliability 1) and selected as key study, the No Observed Adverse Effect Level (NOAEL) for P0 reproductive performance is 3 mg/kg/day based on reduced delivery index at 15 mg/kg bw/day. The NOEL for P0 reproductive performance and fetal toxicity is < 0.6 mg/kg/day based on slightly higher post-implantation loss and lower birth index at 0.6 mg/kg bw/day.

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

Additional information