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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 April 2017 to 28 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD Guidelines for Testing of Chemicals, No. 471, “Bacterial Reverse Mutation Test”, adopted July 21, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
EPA Health Effects Test Guidelines, OPPTS 870.5100, “Bacterial Reverse Mutation Test”, EPA 712-C-98-247, August 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Commission Regulation (EC) No 440/2008, part B:
Methods for the Determination of Toxicity and other health effects, Guideline B.13/14 “Mutagenicity: Reverse Mutation Test using Bacteria”. Official Journal of the European Union No. L142, 31 May 2008.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
6,6'-di-tert-butyl-2,2'-thiodi-p-cresol
EC Number:
202-009-7
EC Name:
6,6'-di-tert-butyl-2,2'-thiodi-p-cresol
Cas Number:
90-66-4
Molecular formula:
C22H30O2S
IUPAC Name:
6,6'-di-tert-butyl-2,2'-thiodi-p-cresol
Test material form:
solid: particulate/powder
Details on test material:
Name: 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol
Other name: LOWINOX® TBP-6
CAS number: 90-66-4
Batch/Lot Number: C034J0059 / C036K0111
Description: White powder
Purity*: 99.8%
Expiry date: 12 May 2017
Storage condition: Controlled room temperature (15-25 ºC, below 70 RH%)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.
* No correction for purity of the test item was applied.
Specific details on test material used for the study:
No further details specified in the study report.

Method

Target gene:
histidine & tryptophan
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
Concentrations were selected on the basis of the Preliminary Solubility Test and Preliminary Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test and Confirmatory Mutation Test, the different concentrations were used.
Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation.
Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate, in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.
Vehicle / solvent:
Based on the results of the Compatibility Test, the test item was dissolved in N,N-Dimethylformamide (DMF) at a concentration of 100 mg/mL.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
N,N-Dimethylformamide (DMF); Dimethyl sulfoxide (DMSO); Distilled water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylenediamine (NPD); 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
DESCRIPTION OF THE TEST PROCEDURE
The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test and a Confirmatory Mutation Test. In the Preliminary Concentration Range Finding Test as well as in the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used.

Preliminary Compatibility Test
The solubility of the test item was examined using Distilled water, N,N-Dimethylformamide (DMF) and Dimethyl sulfoxide (DMSO). Test item was insoluble at 100 mg/mL concentration using Distilled water. Partial dissolution was observed at the same concentration using DMSO. The test item was soluble at this concentration using DMF. Therefore DMF was selected as vehicle (solvent) for the study. The obtained stock solution (50 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension.

Preliminary Concentration Range Finding Test (Informatory Toxicity Test)
Based on the solubility test, a 100 mg/mL stock solution was prepared in Distilled water. Seven test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98 and TA100) were determined at concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item, in the absence and presence of metabolic activation. In the Preliminary Concentration
Range Finding Test the plate incorporation method was used.

Test Item Concentrations in the Mutagenicity Tests (Initial Mutation Test and Confirmatory Mutation Test)
Based on the results of the preliminary tests, a 100 mg/mL stock solution was prepared in Distilled water. Maximum seven test concentrations were prepared by successive dilutions of the stock solution, to obtain lower doses. The maximum test concentration was 5000 μg test item/plate.
Examined concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate.
Examined concentrations in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.

Control Groups Used in the Tests
Strain-specific positive and negative (solvent) controls, both with and without metabolic activation were included in each test. In addition, an untreated control was used demonstrating that the chosen vehicle induced no deleterious or mutagenic effects.

Procedure for Exposure in the Initial Mutation Test
The Initial Mutation Test followed the standard plate incorporation procedure.
Bacteria (cultured in Nutrient Broth No.2) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).

The content of the tubes:
top agar: 2000 μL
vehicle or test item formulation (or reference controls): 50 μL
overnight culture of test strain: 100 μL
phosphate buffer (pH 7.4) or S9 mix: 500 μL

This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37 °C for 48 ± 1 hours.
Examined concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50, and 15.81 μg/plate in the absence and presence of metabolic activation.

Procedure for Exposure in the Confirmatory Mutation Test
The Confirmatory Mutation Test followed the standard pre-incubation procedure since no biologically relevant increase in the number of revertant colonies was observed in the Initial Mutation Test.
Bacteria (cultured in Nutrient Broth No.2) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45 °C.
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37 °C in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37 °C for 48 ± 1 hours.
Examined concentrations in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate in the absence and presence of metabolic activation.
Rationale for test conditions:
The experimental methods were conducted according to the methods described by Ames et al. and Maron and Ames, Kier et al., Venitt and Parry, OECD Guideline No. 471, 1997, Commission Regulation (EC) No. 440/2008, 2008, EPA Guidelines, OPPTS 870.5100, 1998, 1996 [7][8] and according to the relevant SOPs of CiToxLAB Hungary Ltd.
Evaluation criteria:
The colony numbers on the untreated / negative (solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported.

Criteria for Validity:
The study was considered valid if:
-the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
-at least five analysable concentrations are presented in all strains of the main tests;

Criteria for a Positive Response:
A test item was considered mutagenic if:
-a concentration-related increase in the number of revertants occurs and/or;
-a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
-the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
-the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

Criteria for a Negative Response:
A test article was considered non-mutagenic if:
-the total number of revertants in tester strain Salmonella typhimurium TA98, TA100 or Escherichia coli WP2 uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strain Salmonella typhimurium TA1535 or TA1537 is not greater than three times the concurrent vehicle control;
-the negative response should be reproducible in at least one follow up experiment.
Statistics:
The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.
According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitate was observed at the highest concentration.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitate was observed at the highest concentration.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitate was observed at the highest concentration.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitate was observed at the highest concentration.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitate was observed at the highest concentration.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY TEST)
In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. Each sample (including the controls) was tested in triplicate.
Following concentrations were examined: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate.
In the preliminary experiment, the numbers of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system).
Precipitate/slight precipitate was observed in the Preliminary Concentration Range Finding Test in both bacterial strains with and without metabolic activation at 5000 and 2500 μg/plate concentrations.
Inhibitory or toxic effects of the test item were not detected in the preliminary experiment.
Based on the results of the Range Finding Test and the solubility findings, the maximum final concentration to be tested in the main experiments was 5000 μg/plate.

INITIAL AND CONFIRMATORY MUTATION TESTS
In the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used. The Initial Mutation Test and Confirmatory Mutation Test were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and the Escherichia coli WP2 uvrA strain. The Initial Mutation Test and Confirmatory Mutation Test were performed in the presence and absence of a metabolic activation system. Each test was performed with appropriate untreated, negative (solvent) and positive controls. In the main tests each sample (including the controls) was tested in triplicate.
Based on the results of the preliminary experiment, the examined test concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate and in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.
Precipitate was observed in the main tests in all examined strains with and without metabolic activation at 5000 μg/plate concentration.
Inhibitory, cytotoxic effect of the test item was not detected in the Initial Mutation Test and Confirmatory Mutation Tests.
In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.
In the Initial Mutation Test (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 15.81 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.63). However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.
In the Confirmatory Mutation Test (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 5 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.61). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.
Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

VALIDITY OF THE TESTS
Untreated, negative (vehicle/solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains. At least five analysable concentrations were presented in all strains with and without metabolic activation.
The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid.

Any other information on results incl. tables

Summary Table of the Range Finding Test

Concentrations

(μg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

TA98

TA100

-S9

+S9

-S9

+S9

Untreated control

Mean

20.7

26.3

88.0

92.3

MF

0.93

1.01

0.99

0.98

Distilled water control

Mean

-

-

91.3

-

MF

-

-

1.02

-

DMSO control

Mean

20.3

29.7

-

92.0

MF

0.91

1.14

-

0.98

DMF control

Mean

22.3

26.0

89.3

94.0

MF

1.00

1.00

1.00

1.00

5000

Mean

28.7

38.0

95.3

98.0

MF

1.28

1.46

1.07

1.04

2500

Mean

27.7

27.7

87.7

94.7

MF

1.24

1.06

0.98

1.01

1000

Mean

21.7

25.7

86.0

94.0

MF

0.97

0.99

0.96

1.00

316

Mean

20.3

37.7

79.7

95.0

MF

0.91

1.45

0.89

1.01

100

Mean

24.0

30.7

93.0

98.3

MF

1.07

1.18

1.04

1.05

31.6

Mean

20.3

32.0

107.7

101.3

MF

0.91

1.23

1.21

1.08

10

Mean

19.3

37.0

101.1

104.0

MF

0.87

1.42

1.13

1.11

NPD (4μg)

Mean

373.3

-

-

-

MF

18.36

-

-

-

2AA (2μg)

Mean

-

2440.0

-

2396.0

MF

-

82.25

-

26.04

SAZ (2μg)

Mean

-

-

11963.3

-

MF

-

-

13.07

-

 

Summary Table of the Initial Mutation Test

Concentration

(μg/plate)

Mean values of revtants / Mutation factor (MF)

Salmonella typhimuriumtester stains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated control

Mean

19.0

21.7

93.0

104.3

11.3

12.0

9.0

8.3

29.7

40.7

MF

1.06

1.07

1.02

1.20

0.87

1.00

1.23

1.04

0.79

0.98

Distilled water control

Mean

-

-

83.7

-

13.7

-

-

-

34.0

-

MF

-

-

0.92

-

1.05

-

-

-

0.90

-

DMSO control

Mean

20.0

20.7

-

85.3

-

12.7

7.0

9.3

-

36.3

MF

1.11

1.02

-

0.98

-

1.06

0.95

1.17

-

0.87

DMF control

Mean

18.0

20.3

91.3

87.0

13.0

12.0

7.3

8.0

37.7

41.7

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

5000

Mean

24.3

25.0

70.0

82.7

16.0

13.3

10.7

12.7

40.3

37.0

MF

1.35

1.23

0.77

0.95

1.23

1.11

1.45

1.58

1.07

0.89

1581

Mean

22.3

20.3

68.0

71.0

13.3

8.3

9.3

10.3

37.3

35.7

MF

1.23

1.00

0.74

0.82

1.03

0.69

1.27

1.29

0.99

0.86

500

Mean

21.0

25.7

73.0

82.3

14.3

9.3

8.7

10.7

37.0

31.7

MF

1.17

1.26

0.80

0.95

1.10

0.89

1.18

1.33

0.98

0.76

158.1

Mean

22.3

23.0

72.0

73.0

11.0

9.0

8.0

9.3

33.0

36.3

MF

1.24

1.13

0.79

0.84

0.85

0.75

1.09

1.17

0.88

0.87

50

Mean

19.3

24.7

72.3

84.3

13.3

8.0

9.3

8.7

36.0

33.7

MF

1.07

1.21

0.79

0.97

1.03

0.67

1.27

1.08

0.96

0.81

15.81

Mean

19.3

24.0

77.0

83.0

14.0

9.3

9.0

13.0

35.7

34.3

MF

1.07

1.18

0.84

0.95

1.08

0.78

1.23

1.63

0.95

0.82

NPD (4μg)

Mean

379.3

-

-

-

-

-

-

-

-

-

MF

18.97

-

-

-

-

-

-

-

-

-

2AA (2μg)

Mean

-

2366.7

-

2486.7

-

213.3

-

197.0

-

-

MF

-

114.52

-

29.14

-

16.84

-

21.11

-

-

2AA (50μg)

Mean

-

-

-

-

-

-

-

-

-

255.3

MF

-

-

-

-

-

-

-

-

-

7.03

SAZ (2μg)

Mean

-

-

1033.3

-

1190.7

-

-

-

-

-

MF

-

-

12.35

-

87.12

-

-

-

-

-

9AA (50μg)

Mean

-

-

-

-

-

-

396.0

-

-

-

MF

-

-

-

-

-

-

56.57

-

-

-

MMS (2μL)

Mean

-

-

-

-

-

-

-

-

956.0

-

MF

-

-

-

-

-

-

-

-

28.12

-

 

Summary Table of the Confirmatory Mutation Test

Concentration

(μg/plate)

Mean values of revtants / Mutation factor (MF)

Salmonella typhimuriumtester stains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated control

Mean

21.3

21.0

98.7

97.7

13.7

13.0

13.3

12.0

35.3

48.0

MF

1.12

0.79

1.03

1.10

1.03

1.03

1.14

0.90

0.94

1.06

Distilled water control

Mean

-

-

93.0

-

15.3

-

-

-

42.3

-

MF

-

-

0.97

-

11.5

-

-

-

1.12

-

DMSO control

Mean

18.7

24.3

-

92.3

-

13.0

11.7

11.3

-

42.3

MF

0.97

0.91

-

1.04

-

1.03

1.00

0.85

-

0.93

DMF control

Mean

19.0

26.7

95.7

89.0

13.3

12.7

11.7

13.3

37.7

45.3

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

5000

Mean

13.7

22.7

86.7

92.7

11.7

14.0

13.7

13.0

35.0

40.3

MF

0.72

0.85

0.91

1.04

0.88

1.11

1.17

0.98

0.93

0.89

1581

Mean

15.7

28.3

80.3

100.3

13.0

12.0

13.0

13.0

35.0

39.7

MF

0.82

1.06

0.84

1.13

0.98

0.95

1.11

0.98

0.93

0.88

500

Mean

18.7

28.7

90.7

98.7

11.7

10.0

13.0

12.7

32.7

39.7

MF

0.98

1.08

0.95

1.11

0.88

0.79

1.11

0.95

0.87

0.88

158.1

Mean

19.3

24.3

96.3

105.3

14.0

12.3

12.0

12.7

32.7

40.0

MF

1.02

0.91

1.01

1.18

1.05

0.97

1.03

0.95

0.87

0.88

50

Mean

22.7

25.0

93.7

109.0

15.3

14.3

13.0

13.3

33.7

39.3

MF

1.19

0.94

0.98

1.22

1.15

1.13

1.11

1.00

0.89

0.87

15.81

Mean

22.3

23.7

95.3

110.7

14.0

12.0

12.0

13.7

32.3

36.0

MF

1.18

0.89

1.00

1.24

1.05

0.95

1.03

1.03

0.86

0.79

5

Mean

24.3

25.3

98.7

115.7

11.0

20.3

11.3

12.7

32.3

39.36

MF

1.28

0.95

1.03

1.30

0.83

1.61

0.97

0.95

0.86

0.87

NPD (4μg)

Mean

340.0

-

-

-

-

-

-

-

-

-

MF

18.21

-

-

-

-

-

-

-

-

-

2AA (2μg)

Mean

-

2477.3

-

2410.7

-

208.0

-

199.3

-

-

MF

-

101.81

-

26.11

-

16.00

-

17.59

-

-

2AA (50μg)

Mean

-

-

-

-

-

-

-

-

-

300.3

MF

-

-

-

-

-

-

-

-

-

7.09

SAZ (2μg)

Mean

-

-

1184.0

-

1176.0

-

-

-

-

-

MF

-

-

12.73

-

76.70

-

-

-

-

-

9AA (50μg)

Mean

-

-

-

-

-

-

413.3

-

-

-

MF

-

-

-

-

-

-

35.43

-

-

-

MMS (2μL)

Mean

-

-

-

-

-

-

-

-

1025.3

-

MF

-

-

-

-

-

-

-

-

24.22

-

 

Applicant's summary and conclusion

Conclusions:
The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.
The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.
The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test and a Confirmatory Mutation Test. In the Preliminary Concentration Range Finding Test as well as in the Initial Mutation Test the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used.
The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol (Batch Number: C034J0059) has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.
Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

 

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone induced rats.

 

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

 

Based on the results of the Compatibility Test, the test item was dissolved in N,N-Dimethylformamide (DMF) at a concentration of 100 mg/mL. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate, in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.

 

Precipitate was observed in the main tests in all examined strains with and without metabolic activation at the highest concentration.

 

Inhibitory, cytotoxic effect of the test item was not detected in the Initial Mutation Test and Confirmatory Mutation Tests.

 

In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect.

 

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

 

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

 

In conclusion, the test item 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol (Batch Number: C034J0059) has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.