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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 December 2010 to 27 January 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to generally valid and/or internationally accepted testing guidelines and followed recognized GLP standards.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): ethylene glycol monopropyl ether
- Physical state: clear colorless liquid
- Analytical purity: 99.820 wt%
- Impurities (identity and amount): 0.012 wt%
- Purity test date: 15 November 2010 (COA)
- Lot/batch No.: TXEG
- Receipt date: 25 November 2010
- Expiration date of the lot/batch: 25 November 2011
- Storage condition of test material: room temperature in the dark
- Other: The integrity of supplied data relating to the identity, purity and stability of the test item was the responsibility of the Sponsor.

Method

Target gene:
Salmonella typhimurium:
TA 1535: his G 46; rfa-; uvrB-
TA 98: his D 3052; rfa-; uvrB-; R-factor
TA 1535: his G 46; rfa-; uvrB-
TA 100: his G 46; rfa-; uvrB-; R-factor

Escherichia coli (WP2uvrA/pKM101): trp-; uvrA-
contains the pKM101 plasmid
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 mix
Test concentrations with justification for top dose:
Preliminary Toxicity Test
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 microg/plate

Experiment 1
50, 150, 500, 1500 and 5000 microg/plate

Experiment 2 - with preincubation
50, 150, 500, 1500 and 5000 microg/plate
Vehicle / solvent:
Sterile distilled water (50 mg/mL)
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Pos. Control for TA98 at 5 microg/plate (+S9)
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA)
Remarks:
Pos. Control for TA100 at 1 microg/plate (+S9); TA1535 and TA1537, 2 microg/plate (+S9); and E. coli WP2uvrA- at 10 microg/plate (+S9)
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Pos. Control for E. coli WP2uvrA- at 2 microg/plate (-S9); TA100 at 3 microg/plate (-S9); TA1535 at 5 microg/plate (-S9)
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Pos. Control for TA1537 at 80 microg/plate (-S9)
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Pos. Control for TA98 at 0.2 microg/plate (-S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
A standard plate incorporation method was employed.

Preliminary Toxicity Test:
In order to select appropriate dose levels for use in the main test, a preliminary test were conducted in the presence or absence of S9. Ten dose levels and controls were tested up to and including 5,000 microg/plate. The assay was conducted by mixing 0.1 ml the bacterial culture (TA100 or WP2uvrA-), and 0.1 ml of the vehicle or test chemical mixture, 0.5 mL of S9-mix or phosphate buffer and 2.0 ml of molten agar supplemented with trace histidine or tryptophan and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). After approximately 48 hours incubation at 37 deg C, the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Experiment 1:
Five concentrations of the test material were assayed with or without S9-mix against each tester strain using the above described direct plate incorporation method. An additional dose level and expanded dose range were selected in order to achieve both four non-toxic doses and the toxic limit of the test material.

Experiment 2:
A second experiment was performed at the same concentrations as in Experiment 1 with fresh bacterial cultures, test material and control solutions. Preincubation was employed. Thus, measured aliquots (0.1 mL) of each bacterial culture were dispensed into sets of test tubes followed by 0.5 mL of S9-mix or phosphate buffer and 0.1 mL of vehicle or test material formulation and incubated for 20 minutes at 37 deg C with shaking at approximately 130 rpm prior to addition of 2 mL of molten trace histidine or tryptophan supplemented top agar. The contents of the tubes were mixed and poured onto the surface of Vogel-Bonner Minimal agar plates. This procedure was repeated for each bacterial strain either with or without S9.

NUMBER OF REPLICATIONS:
3 replicates/strain

DETERMINATION OF CYTOTOXICITY
Any toxic effects of the test substance would be detected by a substantial reduction in revertant colony counts or by the absence of a complete bacterial lawn.
Evaluation criteria:
Acceptance Criteria:

The following criteria must be met for acceptance:
- All tester strain cultures must exhibit a characteristic number of spontaneous revertants per plate in vehicle and untreated controls.
- The appropriate characteristics of each tester strain must be confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor.
- All tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria per mL.
- All positive control chemicals should induce marked increases in the frequency of revertant colonies, both with and without metabolic activation.
- The test should include a minimum of four non-toxic dose levels.
- There should be no evidence of excessive contamination.

Evaluation Criteria:

A dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain either with or without metabolic activation. Biological relevance of the response is to be considered first, as recommended by the UKEMS sub-committee on Guidelines for Mutagenicity Testing (1989). Statistical methods can be used as an aid to evaluation but may not be the only determining factor for a positive response. Fold increase greater than 2x the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response.

A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
Mean values with standard deviations were reported.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Please refer to Tables 1 to 5 for details of results.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Test

The test material was non-toxic at 5000 microg/plate to tester strains TA100 and WP2uvrA-. The results are summarized in Table 1.

Table 1: Numbers of revertant colonies in the preliminary toxicity test

With (+) or without (-) S9-mix

Strain

Dose (mg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

95

86

80

93

76

89

90

99

106

96

88

+

TA100

104

80

99

77

88

90

81

84

74

74

87

-

WP2uvrA-

41

45

40

38

44

37

33

40

33

41

33

+

WP2uvrA-

51

52

43

44

44

54

49

51

53

44

42

 

Table 2: Experiment 1 - without metabolic activation

Revertant colony counts (mean 3 replicates)

Addition (mg)

TA100

TA1535

WP2uvrA-

TA98

TA1537

0

100

24

47

21

11

50

106

26

43

24

12

150

100

22

43

22

13

500

104

20

47

27

12

1500

94

25

43

18

14

5000

99

24

40

19

11

ENNG (3)

403

 

 

 

 

ENNG (5)

 

285

 

 

 

ENNG (2)

 

 

290

 

 

4NQO (0.2)

 

 

 

184

 

9AA (80)

 

 

 

 

2993

Abreviations: ENNG, N-ethyl-Nā€™-nitro-N-nitrosoguanidine; 9AA, 2-aminoacridine; BP, benzo(a)pyrene; 2AA, 2-aminoanthracene; 4NQO, 4-nitroquinoline-1-oxide

 

Table 3: Experiment 1 - with metabolic activation

 

Revertant colony counts (mean 3 replicates)

Addition (mg)

TA100

TA1535

WP2uvrA-

TA98

TA1537

0

112

14

50

27

12

50

96

14

46

21

14

150

91

13

53

24

12

500

92

14

49

23

13

1500

97

11

52

22

13

5000

104

12

53

25

12

2AA (1)

472

 

 

 

 

2AA (2)

 

247

 

 

285

2AA (10)

 

 

327

 

 

BP (5)

 

 

 

188

 

 

Table 4: Experiment 2 - without metabolic activation

 

Revertant colony counts (mean 3 replicates)

Addition (mg)

TA100

TA1535

WP2uvrA-

TA98

TA1537

0

113

23

49

22

10

50

118

18

49

19

11

150

101

21

48

15

7

500

102

21

36

20

14

1500

104

21

51

23

12

5000

127

18

48

17

9

ENNG (3)

386

 

 

 

 

ENNG (5)

 

921

 

 

 

ENNG (2)

 

 

198

 

 

4NQO (0.2)

 

 

 

212

 

9AA (80)

 

 

 

 

1311

 

Table 5: Experiment 2 - with metabolic activation

 

Revertant colony counts (mean 3 replicates)

Addition (mg)

TA100

TA1535

WP2uvrA-

TA98

TA1537

0

122

12

49

37

13

50

91

15

41

37

12

150

103

16

46

35

13

500

120

13

46

42

14

1500

107

13

58

32

9

5000

130

15

57

33

16

2AA (1)

957

 

 

 

 

2AA (2)

 

344

 

 

247

2AA (10)

 

 

364

 

 

BP (5)

 

 

 

297

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9 -mix and the sensitivity of the bacterial strains.

The test material was considered to be non-mutagenic under the conditions of this test.