Bis(2-chloroethyl) ether

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 August 1989 to 5 September 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: dichloroethylether, Lot# 641

Test animals

Species:

mouse

Strain:

ICR

Details on species / strain selection:

Standard strain / species

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague-Dawley, Inc., Frederick, MD.
- Age at study initiation: 8.5 weeks
- Weight at study initiation: 29.0 - 38.0 grams (males), 22.4 - 30.5 grams (females)
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: Polycarbonate cages, up to 5 animals per cage.
- Diet (e.g. ad libitum): Purina Certified Laboratory Chow® #5002
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least seven days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72±6°F
- Humidity (%): 50±20%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 15 August 1989 To: 5 September 1989

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Corn oil

Details on exposure:

Single dose

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Once

Post exposure period:

The test article dosed animals were euthanised at 24, 48 and 72 hours after administration of the test article. The positive and vehicle control animals were euthanatized 24 hours after the administration of the control articles.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

18 mg/kg bw/day (nominal)

Remarks:

Single dose

Doses / concentrations 3

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

Single dose

Doses / concentrations 4

Dose / conc.:

180 mg/kg bw/day (nominal)

Remarks:

Single dose

No. of animals per sex per dose:

Five

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: dose of 80 mg/kg bw

Examinations

Tissues and cell types examined:

Tibial bone marrow PCEs

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The dose levels used in this assay were based upon the results of a previously conducted dose range finding assay.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): The test article dosed animals were euthanised 24, 48 and 72 hours after administration of the test article. The positive and vehicle control animals were euthanised 24 hours after the administration of the control articles.

DETAILS OF SLIDE PREPARATION: At the appropriate harvest time, the animals were euthanised with CO2 and the adhering soft tissue and epiphyses of both tibiae were removed. The marrow was flushed from the bone and transferred to centrifuge tubes containing 3 mL fetal calf serum (one tube for each animal). Following centrifugation to pellet the tissue, most of the supernatant was drawn off, the cells were resuspended, and the suspension spread on slides and air-dried. The slides were then fixed in methanol, stained in May Gruenwald solution followed by Giemsa, and rinsed in deionized water. After being air-dried, the slides were coverslipped using Depex® mounting medium.

METHOD OF ANALYSIS: The coded slides were then scored for micronuclei and the polychromatic (PCE) to normochromatic (NCE) cell ratio. Standard forms were used to record these data. One thousand PCEs per animal were scored. The frequency of micronucleated cells as expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The normal frequency of micronuclei in this mouse strain is about 0.0-0.4%. The frequency of PCEs versus NCEs was determined by scoring the number of NCEs observed in the optic fields while scoring the 1000 PCEs for micronuclei.

Evaluation criteria:

The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond and ring-shaped micronuclei occasionally occur. Micronuclei had sharp borders and were generally between 1/20 and 1/5 the size of the PCE. The unit of scoring was the micronucleated cell, not the micronucleus; thus the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei. The staining procedure permitted the differentiation by colour of PCEs and NCEs (bluish-grey and red, respectively).
The criteria for determining a positive response involved a statistically significant dose-related increase in micronucleated PCEs, or the detection of a reproducible and statistically significant positive response for at least one dose level. A test article that induced neither a statistically significant dose response nor a statistically significant and reproducible increase at one dose level was considered negative. In either case, the final decision was based on scientific judgment.

Statistics:

Data were tabulated with individual animal results and these data were summarized by sex and dose group at the different time points. The analysis of the data was performed using an Analysis of Variance on the square root arcsine transformation which was performed on the proportion of cells with micronuclei per animal (square root arcsine proportion). Once the Analysis of Variance had been performed, Tukey's Studentized range test (HSD) with adjustment for multiple comparisons was used at each harvest time to determine which dose groups, if any, were significantly different from the vehicle control. Analyses were performed separately for each harvest time and sex combination, and also at each harvest time for the sexes combined.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

All animals were observed immediately after dosing and periodically throughout the duration of the assay for toxic symptoms and/or mortalities. All animals appeared normal after dosing and remained healthy until the appropriate harvest times. The test article, DCEE, induced no significant increases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times. The positive control, CP, induced significant increases in micronucleated PCEs in both sexes, with means and standard errors of 0.68% ± 0.21% and 0.96% ± 0.28% for the males and females, respectively. The individual animal data are found in Tables 1 through 3 and are summarized by dose group in Table 4.

Any other information on results incl. tables

MICRONUCLEUS DATA SUMMARY TABLE

 

TREATMENT	DOSE	HARVEST TIME (HR)	% MICRONOCLEATED PCEs MEAN OF 1000 PER ANIMAL ± S .E.			RATIO PCE:NCE MEAN ± S.E.
			MALES	FEMALES	TOTAL	MALES	FEMALES
VEHICLE CONTROL CORN OIL	10.0 ml/kg	24	0.04 ± 0.02	0.02 ± 0.02	0.03 ± 0.02	0.58 ± 0.06	0.73 ± 0.08
POSITIVE CONTROL CP	80.0 mg/kg	24	0.68 ± 0.21*	0.96 ± 0.28*	0.82 ± 0.17*	0.75 ± 0.10	0.79 ± 0.14
TEST ARTICLE	18.0 Mg/kg	24	0.04 ± 0.02	0.10 ± 0.05	0.07 ± 0.03	0.50 ± 0.04	0.71 ± 0.04
		48	0.04 ± 0.02	0.20 ± 0.08	0.12 ± 0.05	0.48 ± 0.04	0.86 ± 0.09
		72	0.00 ± 0.00	0.06 ± 0.04	0.03 ± 0.02	0.77 ± 0.07	0.68 ± 0.12
	60.0 mg/kg	24	0.06 ± 0.04	0.02 ± 0.02	0.04 ± 0.02	0.56 ± 0.07	0.78 ± 0.13
		48	0.02 ± 0.02	0.16 ± 0.07	0.09 ± 0.04	0.50 ± 0.03	0.70 ± 0.13
		72	0.08 ± 0.04	0.04 ± 0.02	0.06 ± 0.02	0.51 ± 0.04	0.95 ± 0.09
	180 mg/kg	24	0.04 ± 0.02	0.12 ± 0.06	0.08 ± 0.03	0.56 ± 0.14	0.82 ± 0.15
		48	0.12 ± 0.06	0.06 ± 0.04	0.09 ± 0.03	0.51 ± 0.06	0.76 ± 0.12
		72	0.04 ± 0.02	0.10 ± 0.05	0.07 ± 0.03	0.51 ± 0.08	0.75 ± 0.08
* Significantly greater than the corresponding negative control, p<0.05

Applicant's summary and conclusion

Conclusions:

The test material, DCEE, did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse bone marrow micronucleus test.

Executive summary:

The objective of this in vivo assay was to evaluate the ability of the test article, DCEE, to induce micronuclei in bone marrow polychromatic erythrocytes of ICR mice. The test article was solubilized in corn oil and dosed by oral gavage at 18, 60, and 180 mg/kg, based upon the results of a previously conducted dose range finding assay. Ten animals (five males and five females) were randomly assigned to each dose/harvest time group. Vehicle and positive control groups euthanatized 24 hours after dosing were included in the assay. The animals were dosed with the test article and were euthanaised 24, 48 and 72 hours after dosing for extraction of the bone marrow. The test material, DCEE, did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse bone marrow micronucleus test.