Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay / Ames test: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 1358: all negative with and without metabolic activation (analog OECD TG 471)
In Vitro Sister Chromatid Exchange Assay in Mammalian Cells: CHO cells: negative with and without metabolic activation (analog OECD TG 479)
In vitro Mammalian Cell Gene Mutation Test (HGPRT): CHO cells: negative with and without metabolic activation (analog OECD TG 476)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
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%
Target gene:
HGPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: antibiotic-free, Ham's Modified F12 Medium supplemented with 10% (v/v) heat-inactivated, fetal bovine sera (F12-lo). For chemical exposures of cells without metabolic activation, F12 medium with 5% (v/v) of dialyzed bovine serum (F12-D5) is used. For treatments incorporating an S9 metabolic activation system, identical medium, but without serum, is employed.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 metabolic activation system
Test concentrations with justification for top dose:
0.25 to 2.0 mg/ml without S-9 and 0.1 to 2.5 mg/ml with S-9
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: Dimethylnitrosamint (DMN) used as positive control mutagens for tests with an S9 metabolic activation system.
Details on test system and experimental conditions:
METHOD OF APPLICATION: not applicable

DURATION
- Preincubation period: not applicable
- Exposure duration:On the day of testing, appropriate concentrations of the test agent are added to duplicate cultures of cells and cultures are treated for 5 hr (without activation) and 2 hr (with activation) at 37°C. Following exposure, the medium and test agents are removed by suction, cells are rinsed once or twice with a physiological salt solution and fresh F12-D5 medium is added.

SELECTION AGENT (mutation assays): 6-Thioguanine (2 µg/mL)

NUMBER OF REPLICATIONS: Duplicate cultures of CHO cells were exposed for 2 and 5 hours to a minimum of five concentrations of DEGHE in tests both with and without the addition of a rat-liver S9 metabolic activation system, respectively.

NUMBER OF CELLS EVALUATED:The mutant fraction was assessed in selective medium with 2 x lo5 cells/plate in 5 plates/dosed culture (i.g. 1 x lo6 total cells/dosed culture). The plating efficiency of these cells was assessed in non-selective medium using 4 plates/dosed culture with 100 cells/plate.

DETERMINATION OF CYTOTOXICITY:The surviving fraction was determined at 18 to 24 hours after the removal of the test chemical using 4 plates/culture and 100 cell/plate.
-Cytotoxicity is determined by comparing the relative number of cells in control cultures (untreated cells) to the numbers of cells in cultures treated with various concentration8 of the test agent.

RANGE-FINDING/SCREENING STUDIES: Preliminary experiments were performed with CHO cells to determine an appropriate range of test concentrations in which the highest concentration would kill no more than (approximately) 90% of the treated cells.

COMPARISON WITH HISTORICAL CONTROL DATA: was done
Evaluation criteria:
Determination of mutant induction: At 2–3-day intervals after treatment, 5 × 105 cells from each culture were subcultured in 100-mm tissue culture dishes in F12-D5 medium and incubated at 37 °C in a 5–6% CO2 atmosphere. After at least 7 days, cells were dissociated with 0.075% trypsin, counted and plated at 2 × 105 cells per dish in five 100-mm culture dishes (i.e. 1 × 106 total cells), which each had 10 ml of F12-D5 medium containing 6-TG (2 μg ml−1). At the same time, and to assess viability (plating efficiency), cells from the same cultures were diluted and 100 cells per dish were added to four 60-mm culture plates containing F12-D5 medium (with 6-TG). The viable fraction is used to correct mutant frequency for cells with variable growth ability. The cultures then were incubated for a further 6–8 days, the medium discarded and the cells fixed and stained for counting with a Coulter Model F electronic cell counter. The numbers of mutants per 106 total cells and per 106 viable cells were calculated.
Statistics:
Mutation data were analyzed by the method of Irr and Snee (1979) after Box–Cox transformation (Box and Cox, 1964), before parametric analysis using Student’s t -test.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY: Test results with the test substance indicated that concentrations of 3 mg/mL or higher were lethal to CHO cells. The highest suitable concentrations tested were 2.0 mg/mL without S9 and 2.5 rng/mL with S9 activation for the CHO test.

Cell viability was not affected by test material concentration up to approximately 750 mg/ml in the absence of S-9 and 1 mg/ml in the presence of S-9. A concentration of 2.0 mg/ml caused 100% lethality. The plating efficiency for all concentrations was not affected by test material concentration (up to 2 mg/ml). Test material did not produce a dose-related increase in the number of mutants per 10E5 viable cells in the absence of S-9. In the presence of S-9, increases in the mutation frequency occurred that were not dose-related and were inconsistent between duplicate cultures. A linear regression analysis of the data showed a significant trend for the combined data from the two tests (r=0.82, p= 0.0035), but even the values for the highest DEGHE concentration was not significantly different from the control. Consequently, DEGHE is judged to non-mutagenic in this assay. Both positive controls produced highly significant increases in the number of mutants.

Table 1: Preliminary cytotoxicity data for CHO cells treated for 5 h with DEGHE or vehicle control DMSO

Materiala

Dose (mg/mL)

Initial cell concentration (cells per flask x105)

Final cell concentrationb(cells per plate x105)

Relative survival (% control)

 

 

 

Without activation

With activation

Without activation

With activation

DMSO

 

5

44.5

35.8

100

100

DEGHE

0.01

5

44.8

36.5

100.5

102.1

 

0.03

5

44.9

36.4

100.9

101.8

 

0.1

5

41.7

41.3

93.6

115.6

 

0.3

5

40.9

33.2

91.9

92.9

 

1.0

5

31.3

30.9

70.3

86.5

 

3.0

5

C

C

-

 

 

10.0

5

C

C

-

 

aDMSO = dimethylsulfoxide; DEGHE = diethylene glycol monohexyl ether;

bDetermined 18–14 h after removal of test agent; C = cytotoxic.

Table 2: Plating efficiencies and mutation frequencies in duplicate cultures in the CHO forward gene mutation test

Materiala

Dose

Plating efficiency of coloniesb

% solvent control

Mutation of coloniesb,c

Corrected mutation frequencyd(x106)

 

 

Total

Mean±SD

 

 

Total

Mean±SD

 

 

Without activation

DMSO (µL/mL)

20

263

65.8 ± 9.4

104

3

0.6 ± 0.5

4.6

 

20

244

61.0 ± 15.2

96

5

1.0 ± 1.0

8.2

Medium

 

311

77.8 ± 14.6

123

5

1.0 ± 1.0

6.4

EMS (µg/mL)

200

288

72.0 ± 4.5

114

196

39.2 ± 6.6

272.2

DEGHE

(mg/mL)

0.25

313

78.2 ± 4.7

123

0

0

0

 

0.25

334

83.5 ± 2.4

132

28

5.6 ± 1.5

33.5

 

0.75

319

81.5 ± 11.0

129

15

3.0 ± 1.2

18.4

 

0.75

321

80.2 ± 12.6

127

0

 

0

 

1.0

324

81.0 ± 5.4

128

0

 

0

 

1.0

313

78.2 ± 14.4

123

3

0.6 ± 0.5

3.8

 

1.5

214

53.5 ± 8.3

84

5

1.0 ± 1.2

9.3

 

1.5

238

59.5 ± 7.3

94

2

0.4 ± 0.5

3.4

 

2.0

C

C

 

C

 

 

 

2.0

C

C

 

C

 

 

With activation

DMSO (µL/mL)

20

400

100.0 ± 9.9

104

8

1.6 ± 0.9

8.0

 

20

362

93.2 ± 7.5

97

19

3.8 ± 1.6

20.4

Medium

 

353

88.2 ± 9.4

91

0

0

0

EMS (µg/mL)

200

370

92.5 ± 4.2

96

190

38.0 ± 5.4

205.4

DEGHE

(mg/mL)

0.1

363

90.8 ± 14.6

94

3

0.6 ± 0.9

3.3

 

0.1

405

101.2 ± 13.1

105

6

1.2 ± 0.8

5.9

 

0.25

408

121.8 ± 47.0

126

2

0.4 ± 0.5

1.6

 

0.25

388

97.0 ± 15.2

100

3

0.6 ± 0.5

3.1

 

0.75

449

112.2 ± 13.6

116

10

2.0 ± 2.3

8.9

 

0.75

330

82.5 ± 5.4

85

18

3.6 ± 1.9

21.8

 

1.0

374

93.5 ± 3.4

97

23

4.6 ± 1.5

24.6

 

1.0

393

98.2 ± 14.2

102

12

2.4 ± 1.8

12.2

 

1.5

357

89.2 ± 10.3

92

15

3.0 ± 1.0

16.8

 

1.5

380

90.0 ± 7.4

93

29

5.8 ± 1.9

32.2

aMA = metabolic activation; DMSO = dimethylsulfoxide (solvent); DEGHE = diethylene glycol monohexyl ether; EMS = ethylmethanesulfonate; DMN = dimethylnitrosamine; culture medium is negative control; results for 2 cultures each are shown.

b100 cells inoculated into each of four plates; C = cytotoxic.

c2 × 105cells inoculated into each of five plates (1 × 106total cells).

dTotal number of mutant colonies divided by viable fraction.

Conclusions:
The test substance produced slight increase in gene mutations in CHO cells in tests with rat-liver S9 activation system, but the effects were not dose dependent or repeatable in the duplicate cultures. There was no difference in the absence of S9.
DEGHE was not considered mutagenic in this assay by the registrant.
Executive summary:

The test substance was evaluated for potential genotoxic activity using the Chinese Hamster Ovary (CHO) Mutation test and the Sister Chromatid Exchange (SCE) test. Preliminary experiments were performed with CHO cells to determine an appropriate range of test concentrations in which the highest concentration would kill no more than (approximately) 90% of the treated cells. Test results with the test substance indicated that concentrations of 3 mg/ml or higher were lethal to CHO cells. The highest suitable concentrations tested were 2.0 mg/mL without S9 and 2.5 rng/mL with S9 activation for the CHO test.

Duplicate cultures of CHO cells were exposed for 5 hours to a minimum of five concentrations of the test substance in tests both with and without the addition of a rat-liver S9 metabolic activation system. Various dose levels of the test substance for testing were attained by direct addition of various aliquots of the diluted test agent into the cell culture medium. Dimethylsulfoxide was used as the diluent. All dilutions were freshly prepared prior to testing. The surviving fraction was determined at 18 to 24 hours after the removal of the test chemical using 4 plates/culture and 100 cells/plate. The mutant fraction was determined after a 9 to 12 day sub-culturing period to allow "expressionw of the mutant phenotype. The mutant fraction was assessed in selective medium with 2 x lo5 cells/plate in 5 plates/dosed culture (i.g. 1 x lo6 total cells/dosed culture). The plating efficiency of these cells was assessed in non-selective medium using 4 plates/dosed culture with 100 cells/plate. The mutagenicity/survival/plating efficiency data from at least the top five concentrations which allowed sufficient cell survival for assessment of survival and quantification of mutants were evaluated. The percentage of cells surviving the treatment, the numbers of mutant colonies, the percentage of clonable cells and the calculated number of mutants/l06 clonable cells were evaluated.

The results indicated that the test substace produced slight increases in gene mutations in CHO cells in tests with and without a rat-liver S9 activation system, but the effects were not repeatable in the duplicate cultures. A linear regression analysis of the dose-response data from the test with S9 indicated that the result represented a statistically significant, although very weak positive effect. Even the highest concentration of DEGHE did not lead to a significant difference compared to the control. Therefore, DEGHE is judged to be non-mutagenic in this assay.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Criteria for a positive response were not fully described.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
GLP compliance:
yes
Type of assay:
sister chromatid exchange assay in mammalian cells
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%
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: antibiotic-free, Ham's Modified F12 Medium supplemented with 10% (v/v) heat-inactivated, fetal bovine sera (F12-lo). For chemical exposures of cells without metabolic activation, F12 medium with 5% (v/v) of dialyzed bovine serum (F12-D5) is used. For treatments incorporating an S9 metabolic activation system, identical medium, but without serum, is employed.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 metabolic activation system
Test concentrations with justification for top dose:
0.75 to 2.0 mg/mL without S-9 and 1.0 to 2.5 mg/mL with S-9
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: For SCE assays, dimethylnitrosamine (+S9) (DMN)-CAS #62-75-9 and ethylmethanesulfonate (-S9) (EMS)-CAS #62-50-0 were used as positive control agents to assure the sensitivity and reliability of the test system.
Remarks:
none
Details on test system and experimental conditions:
METHOD OF APPLICATION: not applicable

DURATION
- Preincubation period: not applicable
- Exposure duration: For determination of direct genotoxic action, CHO cells were exposed to DEGHE and appropriate controls for 5 hours without S9 activation. Indirect activity, requiring metabolic activation by liver S9 homogenate, was studied with a 2-hour exposure period.

SELECTION AGENT (mutation assays): not applicable

SPINDLE INHIBITOR (cytogenetic assays): Colchicine (0.5 pg/ml) is added to culture flasks 1 to 2 hrs prior to harvesting to arrest cells in mitosis.
STAIN (for cytogenetic assays): Bromodeoxyuridine (BrdU), required to differentiate between the individual "sistern chromatids by SCE staining.
Chromosomes on the slides are stained for SCEs by treatment with 5.0 pglml of Hoechst 33258 dye for approximately 20 min, rinsed in distilled water, immersed in Sorenson's buffer and exposed to a high intensity sunlamp for 15 to 30 min., as required. Irradiated chromosomes are stained in Gurr's giemsa (diluted 1:25 with phosphate buffer), rinsed in water and dried before application of coverslips.

NUMBER OF REPLICATIONS: Duplicate cultures of CHO cells were exposed

NUMBER OF CELLS EVALUATED: A total of twenty-five cells/concentration was examined for SCE frequencies using duplicate cultures. At least 5 dose levels were tested both with and without metabolic activation.

DETERMINATION OF CYTOTOXICITY:The surviving fraction was determined at 18 to 24 hours after the removal of the test chemical using 4 plates/culture and 100 cell/plate.
-Cytotoxicity is determined by comparing the relative number of cells in control cultures (untreated cells) to the numbers of cells in cultures treated with various concentration8 of the test agent.
Evaluation criteria:
Interpretation of Data: The criteria for evaluation of a positive or negative response depend both on statistical analyses and scientific judgement. The key determinant is whether a dose-dependent increase in SCEs is induced by the test agent and if consistent responses are seen in duplicate cultures/dose. When no clear dose-response relationship is evident and when one or more responses of marginal indications of statistical differences are obtained, a careful examination of the data in comparison to the concurrent controls and the historical data base is necessary to
determine probable biological significance of the statistical indicators. Clearly positive responses will include any of the following:
(i) Doubling in the SCE frequency by one or more concentrations with both of the duplicate cultures/dose;
(ii) Statistically significant responses of p < 0.01 with one or more consecutive concentrations
(iii) Induction of a statistically significant, dose-related increase in the number of SCE.
Random statistical indications of positive increases which do not meet the criteria for a positive test result will be concluded to be negative indications of DNA damage potential.
Statistics:
Data were analyzed after Box–Cox transformation and those from the DEGHE and DMSO control groups were intercomposed for equality of variance by Levene’s test, analysis of variance (ANOVA) and t-tests. The t-tests were used when the F value from ANOVA was significant. When Levene’s test indicated similar variances and the ANOVA was significant, a pooled t-test was used for pairwise comparison. When Levene’s test indicated heterogeneous variances, all groups were intercomposed by an ANOVA for unequal variances, followed by a separate t-test for pairwise comparisons.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: Preliminary experiments were performed with CHO cells to determine an appropriate range of test concentrations in which the highest concentration would kill no more than (approximately) 90% of the treated cells.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Test results with the test substance indicated that concentrations of 3 mg/mL or higher were lethal to CHO cells. The highest suitable concentrations tested were 2.0 mg/mL without S9 and 2.5 Mg/ml with S9 activation for the CHO test.

There was no effect of treatment on the incidence of sister chromatid exchanges (SCEs) in the absence of metabolic activation.  

In the presence of metabolic activation, a significant  increase in SCEs was seen in one culture at 1.5 mg/ml (0.77 +/- 0.24 vs. 0.49 +/- 0.14 in DMSO control). 
This effect was not dose-related. There was no mitotic inhibition in the absence of S-9, and only the high dose (2 mg/ml) caused mitotic inhibition in the presence of S-9. The test was valid, as the positive controls induced significant increases in the frequency of SCEs.

Conclusions:
The SCE test results showed no significant increases of DNA damage in the test without S9 activation but one random statistically significant increase was observed in the test with S9. No pattern of a dose-related trend was evident and the single positive increase was not seen at higher doses or with the duplicate culture/dose. Because of the absence of consistent and dose related effects, the test substance was judged to possess at most, only a very weak genotoxic activity under the conditions of the present tests. However, the marginal increases seen in the CHO mutation test and SCE test, both with S9 activation, suggest that metabolism may play a critical role in biological effectiveness of the test substance. The present study suggests that the test substance lacks significant genotoxic potential but it must be considered a limited evaluation of possible metabolic effects upon biological activity. However, the presence of an active mutagenic contaminant responsible for the low level increases cannot be eliminated with current data.
Executive summary:

The test substace was evaluated for potential genotoxic activity using the Sister Chromatid Exchange (SCE) test.

Preliminary experiments were performed with CHO cells to determine an appropriate range of test concentrations in

which the highest concentration would kill no more than (approximately) 90% of the treated cells. Test concentrations for the SCE test were chosen in a range of 40 to 60% growth inhibition so that sufficient numbers of cells in the second division (M2) would be available for determination of SCEs. Test results with DEGHE indicated that concentrations of 3 mg/ml or higher were lethal to CHO cells. The highest suitable concentrations tested were 2.0 mg/ml without S9 and 2.5 rng/ml with S9 activation for the CHO test.

For the SCE test maximum concentrations tested were 2.0 mg/ml both with S9 and without S9 activation.

For determination of direct genotoxic action, CHO cells were exposed to DEGHE and appropriate controls for 5 hours without S9 activation. Indirect activity, requiring metabolic activation by liver S9 homogenate, was studied with a 2-hour exposure period. Bromodeoxyuridine (BrdU), required to differentiate between the individual "sistern chromatids by SCE staining, was present at a concentration of 3 pg/ml in the growth medium during treatment and during the culture period following exposure period. A total of twenty-five cells/concentration was examined for SCE frequencies using duplicate cultures. At least 5 dose levels were tested both with and without metabolic activation. SCE production was determined for the highest 3 doses which did not produce excessive cytotoxic inhibition of cell division. The number of SCEs/cell, mean number of SCEs/chromosome and the level of statistical significance of the increases above the concurrent solvent control values were scored.

The SCE test results showed no significant increases of DNA damage in the test without S9 activation but one random statistically significant increase was observed in the test with S9. No pattern of a dose-related trend was evident and the single positive increase was not seen at higher doses or with the duplicate culture/dose. Because of the absence of consistent and dose related effects, DEGHE was judged to possess at most, only a very weak genotoxic activity under the conditions of the present tests. However, the marginal increases seen in the CHO mutation test and SCE test, both with S9 activation, suggest that metabolism may play a critical role in biological effectiveness of DEGHE.

The present study suggests that the test substance lacks significant genotoxic potential but it must be considered a limited evaluation of possible metabolic effects upon biological activity. However, the presence of an active mutagenic contaminant responsible for the low level increases cannot be eliminated with current data.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
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%
Target gene:
Histidine location
Species / strain / cell type:
other: TA98, TA100, TA1535, TA1537, TA1538
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat-liver S9 metabolic activation system.
Test concentrations with justification for top dose:
0.03 to 3 mg/plate (without S-9); 0.1 to 10 mg/plate (with S-9)
Vehicle / solvent:
Solvent: DMSO;
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: The activation-independent controls are 4-nitro-o-phenylenediamine for TA98 and TA1538, sodium azide for TA100 and TA1535, and 9-aminoacridine for TA1537. The activation-dependent control in 2-aminoanthracene (2-anthramine) for all strains.
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incoporation

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: by observation of back ground lawn

OTHER:
plates are incubatedat 37°C for 48 hours. The plates are examined for the condition of their background lawns and growth is recorded as either confluent, sparse, or absent.
Evaluation criteria:
The spontaneous reversion for the solvent controls should be within this laboratory's historical range. The positive controls should demonstrate that the test systems are responsive with known mutagens. A test chemical is considered to be a bacterial mutagen if the number of revertant colonies is at least twice the solvent control for at least one dose level and there is evidence of a dose-related increase in the number of revertant colonies. If a test chemical produces a marginal or weak response that cannot be reproduced in a second test, the test result will be considered negative. If there is no evidence of a dose-related increase in the number of revertant colonies and the number of revertant colonies is not twice the solvent control, then the test chemical is not considered to be a bacterial mutagen.
Key result
Species / strain:
other: TA98, TA100, TA1535, TA1537, TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 3 mg/plate (without S-9), 10 mg/plate (with S-9)
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Test doses for the Ames test were chosen from data obtained in a preliminary study using strain TA100. Results of preliminary tests performed without an S9 metabolic activation system indicated that a concentration of 5 mg/plate of DEGHE produced complete absence of growth and a slightly lower dose of 3 mg/plate allowed only sparse growth of the bacterial lawn. With S9, doses of 30 and 93.5 mg/plate completely inhibited growth of the background lawn and a lower dose of 10 mg/plate allowed sparse growth but, reduced the number of revertant colonies to approximately 14% of the concurrent control value. On the basis of these results, five concentrations of DEGHE were tested ranging from 0.03 mg/plate to 3 mg/plate without metabolic activation and from 0.1 to 10 mg/plate with S9 activation using triplicate cultures for each dose level for each bacterial strain.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxicity was defined as either a reduction in the number of revertant colonies or an inhibition of growth of the background lawn.

Table 1: Cytotoxicity testing of DEGHE in S. typhimurium strain TA100

Test material

Dose (mg per plate)

Without MA

With MA

 

 

Lawn growth

Revertant colonies

Lawn growth

Revertant colonies

DMSO

110

C

109

C

93

DEGHE

0.01

C

76

C

81

 

0.03

C

78

C

70

 

0.1

C

48

C

70

 

0.3

C

58

C

91

 

1.0

C

35

C

99

 

3.0

S

27

C

72

 

5.0

A

0

C

79

 

10.0

A

0

S

13

 

30.0

A

0

A

0

 

93.5

A

0

A

0

DEGHE = diethylene glycol monohexyl ether; DMSO = dimethylsulfoxide; C = confluent;             S = sparse growth; A = absence of background lawn.

Table 2: Revertant mutant colonies in bacterial mutagenicity assay with DEGHE

S. typhimurium strain

Material

Dose (mg/plate)

Revertants (mean± SD)

 

 

 

Without activation

With activation

TA98

DMSO

110

24 ± 5.7

24 ± 3.6

 

DEGHE

0.03

21 ± 8.1

-

 

~

0.1

26 ± 3.6

35 ± 5.6

 

~

0.3

25 ± 8.7

26 ± 3.6

 

~

1.0

24 ± 6.1

30 ± 1.0

 

~

3.0

15 ± 4.2

21 ± 4.4

 

~

10.0

-

9 (T)

 

4-NPD

0.01

985 ± 95.0

-

 

2-AA

0.01

-

2578 ± 337

 

 

 

 

 

TA100

DMSO

110

97 ± 17.3

79± 6.7

 

DEGHE

0.03

95 ± 7.8

-

 

~

0.1

85 ± 8.2

92 ± 18.0

 

~

0.3

91 ± 19.2

89 ± 9.2

 

~

1.0

91 ± 11.7

80± 13.1

 

~

3.0

52 ± 7.9

72 ± 10.1

 

~

10.0

-

28 ± 5.0 (T)

 

NAZ

0.01

1965 ± 32.6

-

 

2-AA

0.01

-

1524 ± 337

 

 

 

 

 

TA1535

DMSO

110

18 ± 2.5

9± 3.8

 

DEGHE

0.03

18 ± 3.2

-

 

~

0.1

17 ± 1.0

11 ± 5.6

 

~

0.3

16 ± 1.2

8± 4.4

 

~

1.0

26 ± 5.9

9± 2.1

 

~

3.0

17 ± 2.9

13 ± 2.1

 

~

10.0

-

5 ± 2.1

 

NAZ

0.01

1763 ± 33.3

-

 

2-AA

0.01

-

61 ± 10.3

 

 

 

 

 

TA1537

DMSO

110

8 ± 2.3

8 ± 2.1

 

DEGHE

0.03

6 ± 3.1

-

 

~

0.1

7 ± 3.2

6 ± 0.0

 

~

0.3

8 ± 4.0

7 ± 3.8

 

~

1.0

9 ± 4.6

6 ± 1.5

 

~

3.0

3 ± 1.0 (T)

7 ± 1.2

 

~

10.0

-

2 ± 1.4 (T)

 

9-AA

0.01

240 ± 69.4

-

 

2-AA

0.01

-

166 ± 44.2

 

 

 

 

 

TA1538

DMSO

110

8 ± 3.0

22 ± 4.2

 

DEGHE

0.03

11 ± 1.2

-

 

~

0.1

8 ± 1.5

18 ± 6.4

 

~

0.3

10 ± 2.9

17 ± 1.2

 

~

1.0

9 ± 3.1

23 ± 4.6

 

~

3.0

T

18 ± 7.0

 

~

10.0

-

3 (T)

 

9-AA

0.01

1238 ± 55.4

-

 

2-AA

0.01

-

256 ± 71.6

DEGHE = diethylene glycol monohexyl ether; DMSO = dimethylsulfoxide; 4-NPD = nitro-o-phenylenediamine; 2-AA = 2-aminoanthracine; NaZ = sodium azide; 9-AA = 9-aminoacridine; T = toxic.

Conclusions:
Mutagenic activity was not observed with any of the five bacterial strains tested with or without the presence of an Aroclor 1254-induced rat-liver S9 metabolic activation system. The test substance was not considered to be mutagenic in this in vitro screening test.
Executive summary:

The test substance was tested for potential mutagenic activity using the Salmonella/microsome bacterial mutagenicity assay (Ames test). Test doses for the Ames test were chosen from data obtained in a preliminary study using strain TA100. Results of preliminary tests performed without an S9 metabolic activation system indicated that a concentration of 5 mg/plate of the test substance produced complete absence of growth and a slightly lower dose of 3 mg/plate allowed only sparse growth of the bacterial lawn. With S9, doses of 30 and 93.5 mg/plate completely inhibited growth of the background lawn and a lower dose of 10 mg/plate allowed sparse growth but, reduced the number of revertant colonies to approximately 14% of the concurrent control value. On the basis of these results, five concentrations of DEGHE were tested ranging from 0.03 mg/plate to 3 mg/plate without metabolic activation and from 0.1 to 10 mg/plate with S9 activation using triplicate cultures for each dose level for each bacterial strain. Mutagenic activity was not observed with any of the five bacterial strains tested with or without the presence of an Aroclor 1254-induced rat-liver S9 metabolic activation system. The test substance was not considered to be mutagenic in this in vitro screening test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Mammalian Erythrocyte Micronucleus Test: mouse: negative (analog OECD TG 474)
Mammalian Bone Marrow Chromosome Aberration Test: rat: negative (analog OECD TG 475)

Link to relevant study records

Referenceopen allclose all

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

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.

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.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
not specified
Remarks:
Likely to have been conducted under GLP
Type of assay:
other: Mammalian Erythrocyte Micronucleus Test
Specific details on test material used for the study:
- Analytical purity: 96.8% (by capillary gas chromatography)
Species:
mouse
Strain:
Swiss Webster
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Weight at study initiation: 23.8 - 26.9 g for males and 20.6 - 22.0 g for females
- Housing: five of each gender per cage in shoe-box-type plastic cages
- Assigned to test groups randomly: yes
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum): yes
- Acclimation period:5 - 6 days
Route of administration:
intraperitoneal
Vehicle:
- Vehicle used: corn oil
Details on exposure:
intraperitoneal
Duration of treatment / exposure:
single i.p. injection
Frequency of treatment:
once
Post exposure period:
30, 48 and 72 hours
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
Dose / conc.:
640 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 male and 5 females per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
triethylenemelamine
- Route of administration: i.p.
- Doses / concentrations: 0.3 mg/kg
Tissues and cell types examined:
erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
used doses represented 25%, 50% and 80% of LD50 determined in preliminary test (LD50 in males: 894 mg/kg; in females: 715 mg/kg)

DETAILS OF SLIDE PREPARATION:
at each time time period on or two drops of blood were collected from each animal by tail nicking and blood smears were prepared. Slides were stained with Gurr's R-66 Giemsa diluted with phosphate buffer and coded by animal number.

METHOD OF ANALYSIS:
blind reading, PCE/NCE ratio for 1000 total cells and the number of micronucleated polychromatic erythrocytes (MN-PCE) for 1000 PCE were determined.
Evaluation criteria:
statistically significant and dose-related increase in MN-PCE
Statistics:
Fisher's exact test
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 410-1000 mg/kg
- Harvest times: 48 and 72 h post-injection
- Other: intraperitoneal LD50 in males: 894 (664-1203) mg/kg; in females: 715 (623-821) mg/kg)

RESULTS OF DEFINITIVE STUDY
No positive or dose-related increases in the frequency of micronucleated PCE of the bone marrow were observed after intraperitoneal injection of the test substance to Swiss-Webster mice.
The test substance was considered to be inactive as a clastogenic agent.

Dosages, mortality and PCE/NCE ratios for mice given an acute i.p. injection of DEGHE or corn oil :

Materiala Dosage Group size Number
dyingb
Mortality (%) PCE/1000 NCEc      
48 h    72 h   
          Mean ± SD %Control Mean ± SD %Control
Males
DEGHE 410 mg/kg 5 0 0
DEGHE 512 mg/kg 5 0 0
DEGHE 640 mg/kg 5 1 20 33 ± 5.3 124 26.0 ± 3.7 88.4
DEGHE 800 mg/kg 5 4 80
DEGHE 1000 mg/kg 5 5 100
Corn oil 10 ml/kg 5 0 0 26.6 ± 4.0  - 29.4 ± 3.4
Females
DEGHE 410 mg/kg 5 0 0
DEGHE 512 mg/kg 5 0 0
DEGHE 640 mg/kg 5 0 0
DEGHE 800 mg/kg 5 2 40 24.3 ± 2.5d 79.9 24.0 ± 1.0 68.2
DEGHE 1000 mg/kg 5 3 60
Corn oil 10 ml/kg 5 0 0 30.4 ± 2.1  - 35.2 ± 8.1  -

aDEGHE = diethylene glycol monohexyl ether; corn oil is solvent control

bThree-day mortality value

cPCE = polychromatic erythrocyte; NCE = normochromatic erythrocyte

dP<0.05 compared with solvent control

Polychromatic erythrocyte to normochromatic erythrocyte ratios in peripheral blood samples from Swiss Webster mice at various times after an intraperitoneal injection of DEGHE or control substances:

Materiala Dosage Sex 30 hb  48 hb  70 hb 
PCE/1000 NCE % of controls PCE/1000 NCE % of controls PCE/1000 NCE % of controls
Corn oil 10 ml/kg M 37.8 ± 4.82 44.6 ± 6.02 39.4 ± 2.70
F 36.0 ± 3.39 41.4 ± 4.51 40.6 ± 6.80
TEM 0.3 mg/kg M 33.0 ± 7.07 87.3 NE NE
F 35.4 ± 3.05 98.3 NE NE
DEGHE 200 mg/kg M 37.0 ± 7.28 97.7 41.8 ± 4.32 93.7 38.4 ± 3.21 97.5
F 38.4 ± 3.85 106.7 42.4 ± 8.44 102.4 41.2 ± 6.02 101.5
DEGHE 400 mg/kg M 39.0 ± 8.51 103.2 41.0 ± 2.12 91.9 35.6 ± 3.21 90.4
F 42.6 ± 7.02 118.3 45.4 ± 4.98 109.7 41.5 ± 6.35 102.2
DEGHE 640 mg/kg M 35.6 ± 9.42 94.2 39.8 ± 6.53 89.2 33.0 ± 1.58 83.8
F 40.2 ± 6.34 111.7 40.2 ± 3.83 97.1 35.6 ± 2.97 87.7

aCorn oil was solvent control; TEM = triethylenemelamine; DEGHE = diethyl glycol monohexyl ether

bPCE = polychromatic erythrocyte; NCE = normochromatic erythrocyte; % change in ratio calculated against corn oil controls

cP<0.05 compared with corn oil control

Frequency of micronucleated polychromatophils in peripheral blood samples from Swiss Webster mice at various times after the intraperitoneal injection of DEGHE and control substances

Sample
time (h)
Materiala Dose Sex MN-PCE/1000 PCEb  Mean % MN-PCEc
Per animal Mean ± SD
30 Corn oil 10 ml/kg M 4,3,4,1,1 2.6 ± 1.52 0.26
F 1,2,2,1,0 1.2 ± 0.84 0.12
TEM 0.3 mg/kg M 27,28,31,13,39 27.6 ± 9.42 2.76c
F 44,35,30,30,15 30.8 ± 10.52 3.08c
DEGHE 200 mg/kg M 3,3,2,3,1 2.4 ± 0.89 0.24
F 2,4,1,1,1 1.8 ± 1.30 0.18
DEGHE 400 mg/kg M 5,1,1,3,1 2.2 ± 1.79 0.22
F 0,2,2,0,0 0.8 ± 1.10 0.08
DEGHE 640 mg/kg M 0,1,2,0,3 1.2 ± 1.30 0.12
F 2,1,1,2,0 1.2 ± 0.84 0.12
48 Corn oil 10 ml/kg M 3,5,3,4,2 3.4 ± 1.14 0.34
F 1,1,2,1,2 1.4 ± 0.55 0.14
DEGHE 200 mg/kg M 2,1,0,3,3 1.8 ± 1.30 0.18
F 2,1,1,1,3 1.6 ± 0.89 0.16
DEGHE 400 mg/kg M 5,2,4,1,2 2.8 ± 1.64 0.28
F 1,2,3,0,0 1.2 ± 1.30 0.12
DEGHE 640 mg/kg M 0,2,4,4,2 2.4 ± 1.67 0.24
F 0,0,1,0,0 0.2 ± 0.45 0.20
72 Corn oil 10 ml/kg M 6,0,2,2,1 2.2 ± 2.28 0.22
F 2,2,1,2,0 1.4 ± 0.89 0.14
DEGHE 200 mg/kg M 4,2,1,2,1 2.0 ± 1.22 0.20
F 3,3,2,2,8 3.6 ± 2.51 0.36
DEGHE 400 mg/kg M 2,0,1,4,3 2.0 ± 1.58 0.20
F 3,1,2,1,0 1.8 ± 0.96 0.18
DEGHE 640 mg/kg M 3,2,0,4,0 1.8 ± 1.79 0.18
F 1,1,1,1,0 0.8 ± 0.45 0.08

aCorn oil = solvent control; TEM = triethylenemelamine; DEGHE = diethylene glycol monohexy ether; MN-PCE = micronucleated polychromatophilic erythrocyte

b1000 PCE counted per animal (n = five of each sex per group)

cP<0.001 compared with corn oil control

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The test substance was not mutagenic in the Salmonella typhimurium assay (strains TA 1535, TA 1537, TA 98, TA 100 and TA 1358) or mammalian cells (HPRT assay), and did not produce an increase in SCEs in CHO cells (both tests with and without metablic activation). In vivo, the mouse peripheral blood micronucleus test and the rat femoral bone marrow chromosomal aberration test showed no evidence for a clastogenic potential.




Justification for classification or non-classification

Due to the fact that the test substance was not mutagenic in all in vitro and in vivo assays, classification is not warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.