Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well-documented publication, which meets basic scientific principles and was performed similar to OECD guideline 476, performed i.a. on the read-across substances glycidol und butylglycidyl ether

Data source

Reference
Reference Type:
publication
Title:
Mutagenicity of alkyl glycidyl ethers in three short-term assays
Author:
Thompson, E.D., Coppinger, W.J., Piper, C.E., McCarroll, N., Oberly, T.J., Robinson, D.
Year:
1981
Bibliographic source:
Mutation Research, 90 (1981) 213-231

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not applicable
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): glycidol; C-4 (butyl-GE), butyl glycidyl ether
- Substance type: pure active
- Analytical purity:
97.7% (glycidol)
99.1% (butyl glycidyl ether)
- Impurities (identity and concentrations):
0.4% C4 alcohol (butyl glycidyl ether)
- Other:
Glycidol and the butyl glycidyl ether were obtained in high purity by the following procedure: Glycidol, and butyl glycidyl ether were purchased and purified by fractional vacuum distillation.
The substances were characterized by gas liquid chromatography on a Hewlett Packard instrument equipped with a 2-m stainless steel column packed with SP2100 (Supelco). Oxirane levels were determined as described by Walker (Walker, R.O. (Ed.) (1978) Oxirane Oxygen, Official and Tentative Methods of the American Oil Chemists Society, Tentative Method Cd 9-57, 508 South Sixth Street, Champaign, IL 61820.). Free epichlorohydrin was determined by gas liquid chromatography of the head space.

Method

Target gene:
tk+
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The L5178Y mouse lymphoma cells were obtained from Dr. D. Clive, Burroughs Welcome Co.
Metabolic activation:
with and without
Metabolic activation system:
Liver homogenates prepared from Aroclor-1254-induced Sprague-Dawley rats; non-induced liver homogenate was prepared in the same manner from Sprague-Dawley rats injected with corn oil
Test concentrations with justification for top dose:
5 or more concentrations were used for the mutation assay. The highest dose was set at twice the level that killed 50% of the organisms in the toxicity assay.
Glycidol: 8, 15, 23, 30, 45, 60, 75, 94, 125, 187, 250 µg/mL
Butyl glycidyl ester: 84, 100, 130, 164, 200, 256, 300, 320, 400, 500, 640, 800 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
N-dimethylnitrosamine
ethylmethanesulphonate
Remarks:
Positive control without S9 fraction was ethyl methanesulfonate, 620 µg/ml; with induced S9 fraction, 2-acetylaminofluorene, 100 µg/ml; with uninduced S9 fraction, dimethylnitrosamine, 74 µg/ml.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 48h
- Selection time (if incubation with a selection agent): 10-12 days

SELECTION AGENT (mutation assays): 1 µg/ml trifluorothymidine

NUMBER OF REPLICATIONS: A single tube was prepared for each dose level, and the treated cells were cloned in triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; other: trypan blue exclusion
Evaluation criteria:
A compound was considered positive if dose-related increases in the mutation index over at least 3 concentrations with the highest response at least equal to 3.0 were obtained.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
both glycidol and butyl glycidyl ether
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The results of the mouse lymphoma assay are shown in Table 1. The assays were first performed over a wide range of concentrations, then rerun over the narrow ranges shown in the table to demonstrate dose response. The relative growth column is an index of the amount of toxicity to the cells. In most cases the range of concentrations was restricted to 5-fold or less due to toxicity. The mutation frequency was calculated by dividing the number of colonies appearing in the selective plates (TFT) by the number of surviving cells as determined by plating a sample of cells in the absence of the selective agent. The mutation index was calculated by dividing the mutation frequency of the test results by the mutation frequency of the solvent control. For this particular set of experiments, we considered a compound positive if dose-related increases in the mutation index over at least 3 concentrations with the highest response at least equal to 3.0 were obtained. The mouse lymphoma assays were performed without metabolic activation, with metabolic activation by non-induced rat liver microsome preparation, and with an Aroclor-1254-induced rat liver microsome preparation.

In general, the highest mutagenic responses were obtained in the assays without metabolic activation. Responses obtained were slightly reduced in the assays that used the non-induced S9 preparations, whereas much lower responses were obtained with the Aroclor-induced S9 preparations. Cell toxicity was reduced proportionally, i.e. the same dose of chemical produced a more toxic response in the absence of any S9 and a less toxic response in the presence of the S9.

Glycidol and the butyl glycidyl ether gave distinctly positive responses either with one of the S9 preparations or without activation. On a molar basis glycidol was considerably more potent than glycidyl ether.

Table 1: Results of the L5178Y TK+/- mouse lymphoma mutagenicity assay for glycidol and Butyl glycidyl ester ± S9 mix

Compound (µg/ml)

Relative growth (percent of control)

Mutation frequency per 10exp6 survivors

Mutation index (ratio of test/control)

No S9a

I S9b

UI S9c

No S9a

I S9b

UI S9c

No S9a

I S9b

UI S9c

Glycidol

250

-

9.5

-

-

497

-

-

13.1

-

187

-

34.9

-

-

302

-

-

7.9

-

125

-

69.5

-

-

148

-

-

3.9

-

94

-

99.4

-

-

113

-

-

3.0

-

75

-

-

46.1

-

-

507

-

-

9.1

60

-

-

48.7

-

-

301

-

-

5.4

45

-

-

44.8

-

-

287

-

-

5.1

30

6.5

-

42.5

727

-

176

21.4

-

3.1

23

25.1

-

-

581

-

-

17.1

-

-

15

67.4

-

77.7

277

-

78

6.7

-

1.4

8

80.1

-

-

141

-

-

4.1

-

-

Solvent control

100.0

100.0

100.0

34

38

56

1.0

1.0

1.0

Positive controle

38.5

65.0

50.0

818

140

1280

24.5

3.7

22.9

Butyl glycidyl ester

800

-

5.3

-

-

751

-

-

26.7

-

640

-

34.1

-

-

236

-

-

8.4

-

500

-

53.6

7.7

-

193

353

-

6.9

17.6

400

-

59.4

22.3

-

164

235

-

5.8

11.7

320

-

-

39.9

-

-

125

-

-

6.2

300

4.2

-

-

805

-

64

17.9

-

-

256

-

-

61.7

-

-

59

-

-

3.2

200

16.7

-

54.2

681

-

63

15.1

-

2.9

164

-

-

46.3

-

-

35

-

-

3.1

130

-

-

81.8

-

-

37

-

-

1.7

100

58.1

116.6

79.6

133

4

37

3.0

0.3

1.8

84

-

-

80.9

-

-

38

-

-

1.9

Solvent control

100.0

100.0

100.0

45

28

20

1.0

1.0

1.0

Positive controld

8.4

48.1

47.5

1410

90

84

31.3

3.2

4.1

a Without exogenous metabolic activation.

b With Aroclor-induced S9 fraction.

c With uninduced S9 fraction.

d Positive control without S9 fraction was ethyl methanesulfonate, 620 µg/ml; with induced S9 fraction, 2-acetylaminofluorene, 100 µg/ml; with uninduced S9 fraction, dimethylnitrosamine, 74 µg/ml.

e Positive control with S9 fraction for this compound only was benzo[a]pyrene, 3 µg/ml.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation glycidol
positive without metabolic activation glycidol
positive with metabolic activation butyl glycidyl ester
positive without metabolic activation butyl glycidyl ester

The mutagenic potential of the the read-across substances glycidol and butyl glycidyl ester for '1,2,3-propanetriol, glycidyl ethers' was performed in a study similar to OECD 476, assessed with Klimisch 2. Hence, the results are considered as sufficiently reliable to cover this endpoint.
In this assay, a compound was considered positive if dose-related increases in the mutation index over at least 3 concentrations with the highest response at least equal to 3.0 were obtained. These criteria are met for both glycidol and butyl glycidyl ester, without metabolic activation as well as with the induced and non-induced S9 fraction. Hence, both substances and hence '1,2,3-propanetriol, glycidyl ethers' has to be considered as mutagenic in the mouse lymphoma assay.
Executive summary:

In a mammalian gene mutation assay (Mouse lymphoma assay, similar to OECD 476). L5178Y cell cultures were exposed to the read-across substances glycidol and butyl glycidyl ester for '1,2,3-propanetriol, glycidyl ethers' at concentrations of 8, 15, 23, 30, 45, 60, 75, 94, 125, 187, 250 µg/ml (glycidol) or 84, 100, 130, 164, 200, 256, 300, 320, 400, 500, 640, 800 µg/ml with and without metabolic activation. The metabolic activation system was either liver homogenates prepared from Aroclor-1254-induced Sprague-Dawley rats or rats injected with corn oil (non-induced).

Glycidol and butyl glycidyl ester were both tested up to cytotoxic concentrations, i.e. the highest dose was set at twice the level that killed 50% of the organisms in the toxicity assay.

Positive controls (ethyl methanesulfonate, 620 µg/ml, –S9; 2-acetylaminofluorene, 100 µg/ml, + induced S9; dimethylnitrosamine, 74 µg/ml, + uninduced S9) induced the appropriate responses. For both glycidol and butyl glycidyl ester there was a concentration-related positive response as well as the stipulated at least three-fold increase of the mutation frequency over background without or with both available metabolic activation systems. So it can be concluded that '1,2,3-propanetriol, glycidyl ethers' has to be considered as mutagenic in the mouse lymphoma assay, too.

This study is classified as acceptable, reliable with restrictions and satisfies the requirements for OECD guideline 476 for in vitro mammalian cytogenicity data.