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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2001

Materials and methods

Test guideline
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

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%

Method

Target gene:
HGPRT
Species / strain
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
Controls
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.

Results and discussion

Test results
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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.