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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
publication
Title:
In vitro and in vivo|genetic toxicology studies with diethylene glycol monohexyl|ether.
Author:
Ballantyne B and Vergnes JS
Year:
2001
Bibliographic source:
published in . J Appl Toxicol 21:449-460

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
other: Chromosome aberration assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report):Diethylene glycol monohexyl ether (DEGHE)
- Analytical purity: 96.8%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s): corn oil
- Justification for choice of solvent/vehicle: not specified in the report
- Concentration of test material in vehicle: not specified in the report
- Amount of vehicle (if gavage or dermal): not specified in the report
- Type and concentration of dispersant aid (if powder): not specified in the report
- Lot/batch no. (if required): not specified in the report
- Purity: not specified in the report
Duration of treatment / exposure:
up to 48 hours
Frequency of treatment:
once
Post exposure period:
Animals treated with test material were terminated at preassigned time intervals of 12 hr, 24 hr or 48 hours for the first study, and 24 or 48 hours for the second study. Vehicle controls were terminated at 24 hours in the first study and at 24 and 48 hours in the second study. Positive control animals were killed at 24 hours in both studies.
Doses / concentrationsopen allclose all
Dose / conc.:
450 mg/kg bw/day (actual dose received)
Remarks:
first study / females
Dose / conc.:
900 mg/kg bw/day (actual dose received)
Remarks:
first study / females
Dose / conc.:
1 500 mg/kg bw/day (actual dose received)
Remarks:
first study / females
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
first study / males
Dose / conc.:
1 500 mg/kg bw/day (actual dose received)
Remarks:
first study / males
Dose / conc.:
2 400 mg/kg bw/day (actual dose received)
Remarks:
first study / males
Dose / conc.:
375 mg/kg bw/day (actual dose received)
Remarks:
second study / males
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Remarks:
second study / males
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
second study / males
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
second study / males
Dose / conc.:
1 500 mg/kg bw/day (actual dose received)
Remarks:
second study / males
No. of animals per sex per dose:
In the first test, groups of 5 animals/sex/test interval were dosed with test material by gavage at 25% (750 mg/kg for males, 450 mg/kg for females), 50% (1500 mg/kg for males and 900 mg/kg for females), and 80% (2400 mg/kg for males and 1500 mg/kg for females) of the LD50 values.
An additional dose of 375 mg/kg (12.5% of LD50 value) was given to 5 males/ test interval (two of these rats were not within 2 standard deviations of the mean) due to high mortality at the high dose. Groups of 5/sex/interval were administered corn oil vehicle by gavage.
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): historical control
- Route of administration: i. p.
- Doses / concentrations:30 mg/kg

Examinations

Tissues and cell types examined:
When possible, 50 metaphase cells per animal were evaluated blindly for chromosome number, specific type of chromosome or chromatid-type aberrations, deletions and exchanges, gaps, endoreplicated chromosomes and polyploid cells were noted but were not included as statistically evaluated aberrations. Severely damaged cells (> = 10 breakage events) and pulverized cells were recorded as severely damaged, but no attempt was made to classify the types of damage in these cells.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A preliminary test acute peroral test was conducted as follows: males, 3016 (2496–3616) mg kg−1; females, 1823 (1927–2295) mg kg−1. Dose levels, selected on the basis of 25%, 50% and 80% of the LD50, were 750, 1500 and 2400 mg kg−1 for males and 450, 900 and 1500 mg kg−1 for females. Because of mortality at the high dose with males, an additional dose group of 375 mg kg−1 was added to the male groups.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Animals treated with test material were terminated at preassigned time intervals of 12 hr, 24 hr or 48 hours for the first study, and 24 or 48 hours for the second study. Vehicle controls were terminated at 24 hours in the first study and at 24 and 48 hours in the second study. Positive control animals were killed at 24 hours in both studies. Colchicine (4 mg/kg) was injected i.p. 2-3 hours prior to euthanization.

DETAILS OF SLIDE PREPARATION: A femur was removed from each animal. Bone marrow cells were collected and fixed. Slides were prepared and chromosomes were stained with Giemsa.

METHOD OF ANALYSIS: When possible, 500 cells per animal were scored for the proportion of mitotic cells. When possible, 50 metaphase cells per animal were evaluated blindly for chromosome number, specific type of chromosome or chromatid-type aberrations, deletions and exchanges, gaps, endoreplicated chromosomes and polyploid cells were noted but were not included as statistically evaluated aberrations. Severely damaged cells (> = 10 breakage events) and pulverized cells were recorded as severely damaged, but no attempt was made to classify the types of damage in these cells.
Evaluation criteria:
A positive effect will be detected by production of a statistically significant, dose-related increase in the frequency of structural chromosome aberrations. Alternatively, production of statistically significant increase for at least one dose level will be considered an equivocal effect if there is no evidence of a dose-related response.
For test with such random statistical indication, effects will not be considered to be biologically significant if increases are within the range of variation of the historical control data. Additional testing may be required to clarify suspect findings. A test substances which does not produce positive increases as described in the preceding paragraph will be considered inactive as a clastogenic agent in vivo.
Statistics:
Data were analyzed by Fisher’s Exact Test to determine any statistical increase of aberrant cells between the DEGHE and CP groups compared with the corn oil controls.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
None of the the test substance dose levels tested produced statistically significant or dose-related increases in relative numbers of chromosomal aberrations compared to control values among female Sprague-Dawley rats.
Similarly, male Sprague-Dawley rats in the 12 hr and 24 hr post-treatment sacrifice groups had no statistically significant or dose related increases in the incidence of chromosomal aberrations. However, male Sprague-Dawley rats in the 48 hr post-treatment sacrifice group treated at 375 mg/kg or at 750 mg/kg test substance did have statistically significant increases in the incidence of chromosomal aberrations. The incidence of chromosomal aberrations increased with increasing concentrations over these two dose levels. Due to the magnitude of the increases, their biological significance could not be ruled out.
Therefore, the study was repeated in part. Male Sprague-Dawley rats (5/dose/sacrifice) were dosed at 375, 600, 750, 1000 or 1500 mg/kg test substance and sacrificed at either 24 hr or 48 hr post-treatment. There were no statistically significant or dose-related increases in the incidence of chromosomal aberrations among male Sprague-Dawley rats in the repeat test. Consequently, the test substance was not  considered clastogenic to Sprague-Dawley rats under the conditions of this in vivo test.

Any other information on results incl. tables

Table 1: Summary of chromosomal aberrations in femoral bone marrow of Sprague-Dawley rats at various times after dosing perorally with DEGHE and corn oil and following an intraperitoneal injection of cyclophosphamide

Sample time (h)

Materiala

Dose (mg/kg)

Mitotic index (%)b

Total aberrant cells for whole groupc

Aberrant cells as mean± SD

 

First study - males

12

DEGHE

375

1.7 ± 0.4

8

3.2 ± 2.7

 

~

750

1.7 ± 0.9

4

1.6 ± 1.7

 

~

1500

3.1 ± 0.8

9

3.6 ± 3.0

24

DEGHE

375

4.1 ± 1.5

4

1.6 ± 1.70

 

~

750

5.8 ± 1.4

3

1.2 ± 1.8

 

~

1500

6.7 ± 2.8

3

1.2 ± 1.1

 

Corn oil

 

4.8 ± 1.7

3

1.2 ± 1.1

 

CP

30

2.3 ± 0.4

63

25.2 ± 3.6d

48

DEGHE

375

4.6 ± 2.2

17

6.8 ± 4.6e

 

~

750

6.3 ± 1.4

58

34.3 ± 30.7f

 

~

1500

4.8 ± 1.3

5

2.0 ± 2.0

First study - females

12

DEGHE

450

5.8 ± 1.2

4

1.6 ± 2.2

 

~

900

6.0 ± 1.0

0

0

 

~

1500

5.5 ± 0.7

9

3.6 ± 2.6

24

DEGHE

450

5.4 ± 1.6

13

5.2 ± 2.3

 

~

900

5.2 ± 1.7

13

5.2 ± 4.8

 

~

1500

6.4 ± 2.1

10

4.0 ± 2.8

 

Corn oil

 

3.7 ± 1.0

8

3.2 ± 1.8

 

CP

30

2.5 ± 1.4

49

24.2 ± 11.0

48

DEGHE

450

4.1 ± 1.9

6

2.4 ± 0.9

 

~

900

3.7 ± 1.4

6

2.4 ± 0.9

 

~

1500

4.5 ± 2.0

10

4.0 ± 3.2

Second study - males

24

DEGHE

375

5.9 ± 0.7

3

1.2 ± 1.8

 

 

600

6.8 ± 1.6

4

1.6 ± 1.7

 

 

750

6.2 ± 1.2

4

1.6 ± 2.6

 

 

1000

5.9 ± 2.9

4

1.6 ± 1.7

 

 

1500

6.4 ± 2.3

2

0.8 ± 1.1

 

Corn oil

 

6.8 ± 2.3

3

1.2 ± 1.8

 

CP

30

2.0 ± 0.5

41

16.4 ± 7.3d

48

DEGHE

375

7.2 ± 1.3

0

0

 

 

600

6.7 ± 1.7

2

0.8 ± 1.1

 

 

750

8.4 ± 1.2

0

0

 

 

1000

7.2 ± 1.5

3

1.2 ± 1.8

 

 

1500

8.0 ± 1.6

0

0

 

Corn oil

 

6.7 ± 1.9

2

0.8 ± 1.1

aDEGHE = diethylene glycol monohexyl ether; CP = cyclophosphamide.

bMean ± SD.

c50 cells scored for each animal; n = five of each gender per group.

dP < 0.001 compared with corn oil controls.

eP < 0.01 compared with corn oil controls.

Applicant's summary and conclusion

Conclusions:
The test substance was not considered clastogenic to Sprague-Dawley rats under the conditions of this in vivo test.
Executive summary:

The test substance was evaluated for the chromosomal abberation test in rats. The test substance was given to both male and female Sprague-Dawley rats as a single oral dose by gavage. Bone marrow cells were harvested and evaluated for potential chromosomal damage. Preliminary testing (LD50) indicated that the substance produced mortality in all of the female rats at dose levels between 3200 and 5000 mg/kg, in 4 out of 5 male rats at 4000 mg/kg and in all of the male rats dosed at 5000 mg/kg. The LD50 was 3016 mg/kg (2496 to 3616, 95% confidence interval) for the male rats and 1823 mg/kg (1297 to 2295, 95% confidence interval) for the female rats. Dose levels for the definitive bone marrow cytogenetics assay were approximately 80% (2400 mg/kg for males, 1500 mg/kg for females), 50% (1500 mg/kg for males, 900 mg/kg for females) and 25% (750 mg/kg for males, 450 mg/kg for females) of the LD50. An additional dose level of 12.5% of the LD50 (male rats only) was added to the study on the day of dosing since 9 out of 15 male rats dosed at 80% of the LD50 (2400 mg/kg) died within 4.5 hours of dosing.
None of the test substance dose levels tested produced statistically significant or dose-related increases in relative numbers of chromosomal aberrations compared to control values among female Sprague-Dawley rats.
Similarly, male Sprague-Dawley rats in the 12 hr and 24 hr post-treatment sacrifice groups had no statistically significant or dose related increases in the incidence of chromosomal aberrations. However, male Sprague-Dawley rats in the 48 hr post-treatment sacrifice group treated at 375 mg/kg or at 750 mg/kg the test substance did have statistically significant increases in the incidence of chromosomal aberrations. The incidence of chromosomal aberrations increased with increasing the test substance concentrations over these two dose levels. Due to the magnitude of the increases, their biological significance could not be ruled out.
Therefore, the study was repeated in part. Male Sprague-Dawley rats (5/dose/sacrifice) were dosed at 375, 600, 750, 1000 or 1500 mg/kg the test substance and sacrificed at either 24 hr or 48 hr post-treatment. There were no statistically significant or dose-related increases in the incidence of chromosomal aberrations among male Sprague-Dawley rats in the repeat test. Consequently, the test substance was not considered clastogenic to Sprague-Dawley rats under the conditions of this in vivo test.