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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In Vitro Genetic Toxicity - Bacterial Reverse Mutation Assay.

Negative in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA with an without metabolic activation.

In Vitro Genetic Toxicity- Mammalian Cell Gene Mutation Assay (Mouse Lymphoma)

No clear mutagenic effect of 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol was observed in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

In Vitro Genetic Toxicity - Chromosome Aberration Assay

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol] did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation.

Link to relevant study records

Referenceopen allclose all

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
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
Specific details on test material used for the study:
No further details specified in the study report.
Target gene:
histidine & tryptophan
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.
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.
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.

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

-

 

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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 August 2017 to 09 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
OECD Guidelines for the Testing of Chemicals No.490. "In Vitro Mammalian Cell Gene Mutation Test using the Thymidine Kinase Gene" (adopted 29 July 2016)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Commission Regulation (EC) No. 440/2008 of 30 May 2008, B.17. "Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test" (Official Journal L 142, 31/05/2008)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: In vitro Mammalian Cell Gene Mutation Test (Mouse Lymphoma Assay)
Specific details on test material used for the study:
No further details specified in the study report.
Target gene:
thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Cell line: L5178Y TK+/- 3.7.2 C mouse lymphoma
Product No.: CRL-9518
Lot No.: 1661603 / 60797977
Supplier: American Type Culture Collection (Manassas, Virginia, USA)
Date of receipt: 22 January 2004 / 27 February 2014
Date of working lot: 21 January 2016 (MP13) / 17 May 2017 (MP14)*
*Note: MP13 was used in the preliminary experiment, MP14 was used in the main tests.

The original L5178Y TK+/- 3.7.2 C mouse lymphoma cell line was obtained from the American Type Culture Collection. Cells were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of TK-/--mutants and checked for the absence of mycoplasma. For each experiment, one or more vials was thawed rapidly, cells were diluted in RPMI-10 medium and incubated at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5% CO2 in air. When cells were growing well, subcultures were established in an appropriate number of flasks (after thawing, the cells were subcultured no more than 5 times before used in the main tests (in the preliminary experiment cells were subcultured 8 times).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
The test item was examined up to 2000 μg/mL (the recommended maximum concentration) in the Preliminary Toxicity Test. Based on the results of the preliminary experiment (the test item had a special solubility and cytotoxicity profile), the following test item concentrations were examined in the mutation assays:
Assay 1, 3-hour treatment with metabolic activation: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625 and 7.813 μg/mL,
Assay 1, 3-hour treatment without metabolic activation: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625 and 7.813 μg/mL,
Assay 2, 3-hour treatment with metabolic activation: 2000, 1000, 500, 250, 125, 100, 80, 62.5, 50, 40, 31.25, 15.625 and 7.813 μg/mL,
Assay 2, 24-hour treatment without metabolic activation: 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5 and 2.5 μg/mL.
Vehicle / solvent:
Acetone was selected for vehicle of the study as the test item was insoluble in other, most frequently used vehicles (Distilled water or Dimethyl sulfoxide).
Name: Acetone
Supplier: Sigma-Aldrich Co.
Lot No.: 15J060514
Appearance: Clear colourless liquid
Expiry date: 31 October 2020
Storage conditions: Room temperature

Dimethyl sulfoxide was used for vehicle (solvent) of the positive control chemicals.
Name: Dimethyl sulfoxide (anhydrous)
Abbreviation: DMSO
Supplier: Sigma-Aldrich Co.
Lot No.: STBG8411
Appearance: Clear colourless liquid
Expiry date: 29 February 2020
Storage conditions: Room temperature, under N2
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
cyclophosphamide
Details on test system and experimental conditions:
Formulation
Based on the available information (trial formulations for in vitro chromosome aberration study performed at the Test Facility, CiToxLAB study code:17/080-020C), Acetone was selected for vehicle of the study as the test item was insoluble in other, most frequently used vehicles (Distilled water or Dimethyl sulfoxide). Based on those data, the 200 mg/mL concentration formulation was achievable using the selected vehicle. The selected vehicle was compatible with the survival of the cells and the S9 activity.
For the treatments in the study, stock formulations (400 mg/mL) were prepared in the testing laboratory as follows. The necessary amount of test item was weighed into a calibrated volumetric flask; approximately 80% of the required volume of the vehicle was added and the resulting formulation was stirred by a magnetic stirrer until homogeneity was reached. Then it was filled up to the final volume with the vehicle to form a stock formulation and stirred again. From the stock formulation several dilutions were prepared using the selected vehicle for dosing formulations. In each case, the stock formulation and the vehicle was filtered sterile using a 0.22 μm syringe filter (Supplier: Millipore, Lot No.: R6NA19212, Expiry date: October 2019) before the preparation of the dosing formulations. The stock formulations and all the dilutions were prepared immediately before the treatment of the cells in a sterile hood. No purity conversion was applied during formulation as agreed by the Sponsor.
Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.

TEST PROCEDURE
Preliminary Toxicity Test
A preliminary toxicity test was performed to select dose levels for the main assays.
During the preliminary test, a 3-hour treatment in the presence and absence of S9-mix and a 24-hour treatment in the absence of S9-mix were performed with a range of test item concentration to determine toxicity.
The procedures were performed as described in Main Mutation Assays, however, the test used single cultures and positive controls were not included. The highest test concentration in the preliminary test was 2000 μg/mL (the recommended maximum concentration).
Following treatments, cell concentrations were determined using a haemocytometer.
Visual examination for any kind of insolubility in the final culture medium was conducted at the beginning and end of treatment. Measurement of pH and osmolality was performed at the end of the treatment period. Then cells were transferred for the expression period for two extra days and repeated cell counting was performed.

Main Mutation Assays
In Assay 1, cells were treated for 3 hours in the presence and absence of S9-mix. In Assay 2, cells were treated for 3 hours in the presence of S9-mix and for 24 hours in the absence of S9-mix.
A suitable volume* of RPMI-5 medium, vehicle (solvent), test item formulations or positive control solutions, and 1.0 mL of S9-mix (in experiments with metabolic activation) or 1.0 mL of 150 mM KCl (in case of 3-hour treatment without metabolic activation) were added to a final volume of 20 mL per culture in each experiment. For the 3-hour treatments, 107 cells were placed in each of a series of 75 cm2 sterile flasks.
For the 24-hour treatment, 6x106 cells were placed in each of a series of 25 cm2 sterile flasks. The treatment medium contained a reduced serum level of 5% (v/v) RPMI-5.
*Note: Treatment volume of 0.1 mL for a final volume of 20 mL (5 μL/mL) was used for Acetone vehicle (solvent) control and test item formulations; while treatment volume of 0.2 mL for a final volume of 20 mL (10 μL/mL) was used for RPMI-5 medium (untreated control), DMSO vehicle (solvent) control and positive control solutions.
Duplicate cultures were used for each treatment. Cultures were visually examined at the beginning and end of treatments. During the treatment period, cultures were incubated at 37 °C ± 1 °C (approximately 5% CO2 in air). Gentle shaking was used during the treatments. Measurement of pH and osmolality was also performed after the treatment period.
Then cultures were centrifuged at 2000 rpm (approximately 836 g) for 5 minutes, washed with tissue culture medium and suspended in at least 20 mL RPMI-10. The number of viable cells in the individual samples was counted manually using a haemocytometer.
Where sufficient cells survived, cell density was adjusted to a concentration of 2E+5 cells/mL. Cells were transferred to flasks for growth through the expression period (maximum 30 mL of suspension) or diluted to be plated for survival.

Plating for Survival
Using a multi-channel pipette, 0.2 mL of the final concentration of each culture were placed into each well of two, 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37 °C ± 0.5 °C containing approximately 5% (v/v) CO2 in air for approximately two weeks. Wells containing viable clones were identified by eye using background illumination and counted.

Expression Period
To allow expression of TK- mutations, cultures were maintained in flasks for 2 days.
During the expression period, subculturing was performed daily. On each day, cell density was adjusted to a concentration of 2E+5 cells/mL (whenever possible) and transferred to flasks for further growth.
On completion of the expression period, six test item treated samples, untreated, negative (vehicle) and positive controls were plated for determination of viability and 5-trifluorothymidine (TFT) resistance.

Plating for Viability
At the end of the expression period, the cell density in the selected cultures was determined and adjusted to 1E+4 cells/mL with RPMI-20 for plating for a viability test.
Using a multi-channel pipette, 0.2 mL of the final concentration of each culture was placed into each well of two, 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37 ºC ± 0.5 °C containing approximately 5% (v/v) CO2 in air for 12 days. Wells containing viable clones were identified by eye using background illumination and counted.

Plating for -trifluorothymidine (TFT) resistance
At the end of the expression period, the cell concentration was adjusted to 1E+4 cells/mL. TFT (300 μg/mL stock solution) was diluted 100-fold into these suspensions to give a final concentration of 3 μg/mL. Using a multi-channel pipette, 0.2 mL of each suspension was placed into each well of four, 96-well microplates (384 wells) at 2E+3 cells per well.
Microplates were incubated at 37 °C ± 0.5 °C containing approximately 5% (v/v) CO2 in air for approximately two weeks (12 days) and wells containing clones were identified by eye and counted. In addition, scoring of large and small colonies was performed to obtain information on the possible mechanism of action of the test item, if any.
Rationale for test conditions:
In accordance with test guidelines.
Evaluation criteria:
The test item was considered to be clearly positive (mutagenic) in this assay if all the following criteria were met:
1. At least one concentration exhibited a statistically significant increase (p<0.05) compared with the concurrent negative (vehicle) control and the increase was biologically relevant (i.e. the mutation frequency at the test concentration showing the largest increase was at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle/solvent) control value).
2. The increases in mutation frequency were reproducible between replicate cultures and/or between tests (under the same treatment conditions).
3. The increase was concentration-related (p < 0.05) as indicated by the linear trend analysis.
The test item was considered clearly negative (non-mutagenic) in this assay if in all experimental conditions examined there was no concentration related response or, if there is an increase in MF, but it did not exceed the GEF. Then, test item was considered unable to induce mutations in this test system.
Results, which only partially satisfied the acceptance and evaluation criteria, were evaluated on a case-by-case basis. Positive results seen only at high levels of cytotoxicity were interpreted carefully when assessing their biological significance.
Caution was exercised with positive results obtained at levels of cytotoxicity lower than 10% (as measured by RTG).
Statistics:
Statistical significance of mutant frequencies (total wells with clones) was performed using Microsoft Excel software.
The negative (vehicle/solvent) control log mutant frequency (LMF) was compared to the LMF of each treatment concentration, based on Dunnett's test for multiple comparisons and the data were checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Key result
Species / strain:
mouse lymphoma L5178Y cells
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:
Treatment concentrations for the mutation assay were selected based on the results of a short Preliminary Toxicity Test. 3-hour treatment in the presence and absence of metabolic activation system (S9-mix) and 24-hour treatment in the absence of metabolic activation system was performed with a range of test item concentrations to determine toxicity immediately after the treatments. The highest concentration tested in the preliminary experiment was 2000 μg/mL (the recommended maximum concentration).

Insolubility and cytotoxicity was detected in the preliminary experiments. Therefore, concentrations were selected for the main experiments to cover the range from insolubility to no or little insolubility and/or from cytotoxicity to no or little cytotoxicity according to the criteria of the OECD No. 490 guideline. At least nine concentrations were selected for the main experiments in each assay (the test item had a special solubility and cytotoxicity profile).

Note, this test item displayed an abnormal concentration-solubility relationship, based on the scientific basis that cytotoxicity is related to the concentration of test item available (dissolved). At higher concentrations where precipitation was evident, there was a lower cytotoxicity, indicating a lower dissolved concentration (this can occur when the precipitate particles result in dissolved test item partitioning to the particles and coming out of solution). This phenomenon causes the data interpretation to require careful consideration.

Results of the Preliminary Toxicity Test

(3-hour treatment in the presence of metabolic activation)

Test item (or solvent) concentration

Total cell number (Relative Survival*, %) after treatment, Day 0

Total cell number (Relative Survival*, %) on Day 1

Total cell number (Relative Survival*, %) on Day 2

Observations at the beginning / after treatment

Untreated control

8.20E+06 (115)

1.97E+07 (100)

3.54E+07 (125)

Normal / normal (pH: 7.0, osmolality: 284 mmol/kg)

Negative (vehicle) control)

7.15E+06 (100)

1.97E+07 (100)

2.84E+07 (100)

Normal / normal (pH: 7.0, osmolality: 280 mmol/kg)

2000 μg/mL

4.50E+06 (63)

1.20E+06 (6)

5.00E+05 (2)

Precipitate, oily film / precipitate, oily film

(pH: 7.0, osmolality: 279 mmol/kg)

1000 μg/mL

3.15E+06 (44)

3.08E+06 (16)

9.40E+06 (33)

Precipitate, oily film / precipitate, oily film

(pH: 7.0, osmolality: 279 mmol/kg)

500 μg/mL

3.25E+06 (45)

2.63E+06 (13)

4.80E+06 (17)

Precipitate, oily film / precipitate, oily film

(pH: 7.0, osmolality: 291 mmol/kg)

250 μg/mL

3.20E+06 (45)

1.95E+06 (10)

1.60E+06 (6)

Precipitate, oily film / precipitate#, oily film

(pH: 7.0, osmolality: 281 mmol/kg)

125 μg/mL

3.60E+06 (50)

2.40E+06 (12)

8.00E+06 (28)

Precipitate, oily film / precipitate#, oily film

(pH: 7.0, osmolality: 282 mmol/kg)

62.5 μg/mL

4.55E+06 (64)

6.60E+06 (34)

3.28E+07 (115)

Precipitate#, oily film / precipitate#, oily film#

(pH: 7.0, osmolality: 279 mmol/kg)

31.25 μg/mL

5.00E+06 (70)

1.01E+07 (51)

2.92E+07 (103)

Precipitate#, oily film#/ precipitate#

(pH: 7.0, osmolality: 279 mmol/kg)

15.625 μg/mL

5.50E+06 (77)

1.34E+07 (68)

3.40E+07 (120)

Normal / normal (pH: 7.0, osmolality: 280 mmol/kg

7.813 μg/mL

7.50E+06 (105)

1.85E+07 (94)

2.56E+07 (90)

Normal / normal (pH: 7.0, osmolality: 278 mmol/kg)

3.906 μg/mL

6.80E+06 (95)

1.80E+07 (92)

2.22E+07 (78)

Normal / normal (pH: 7.0, osmolality: 281 mmol/kg)

*: compared to the relevant negative control (Acetone)                             #: minimal amount

 

Results of the Preliminary Toxicity Test

(3-hour treatment in the absence of metabolic activation)

Test item (or solvent) concentration

Total cell number (Relative Survival*, %) after treatment, Day 0

Total cell number (Relative Survival*, %) on Day 1

Total cell number (Relative Survival*, %) on Day 2

Observations at the beginning / after treatment

Untreated control

1.10E+07 (94)

1.86E+07 (109)

2.98E+07 (106)

Normal / normal (pH: 7.0, osmolality: 271 mmol/kg)

Negative (vehicle) control)

1.17E+06 (100)

1.71E+07 (100)

2.80E+07 *100)

Normal / normal (pH: 7.0, osmolality: 272 mmol/kg)

2000 μg/mL

5.10E+06 (44)

1.01E+07 (59)

2.50E+07 (89)

Precipitate, oily film / precipitate#, oily film#

(pH: 7.0, osmolality: 272 mmol/kg)

1000 μg/mL

5.20E+06 (44)

6.15E+06 (36)

7.90E+06 (28)

Precipitate, oily film / precipitate#, oily film#

(pH: 7.0, osmolality: 275 mmol/kg)

500 μg/mL

3.40E+06 (29)

1.58E+06 (9)

2.00E+06 (7)

Precipitate, oily film / precipitate#, oily film

(pH: 7.0, osmolality: 280 mmol/kg)

250 μg/mL

4.35E+06 (37)

7.50E+05 (4)

6.00E+06 (2)

Precipitate, oily film / precipitate#, oily film

(pH: 7.0, osmolality: 270 mmol/kg)

125 μg/mL

4.55E+06 (39)

1.28E+06 (7)

5.00E+06 (2)

Precipitate, oily film / oily film

(pH: 7.0, osmolality: 273 mmol/kg)

62.5 μg/mL

4.85E+06 (41)

3.90E+06 (23)

1.01E+07 (36)

Precipitate#, oily film / normal

(pH: 7.0, osmolality: 272 mmol/kg)

31.25 μg/mL

5.95E+06 (51)

1.20E+07 (70)

2.42E+07 (86)

oily film#/ normal (pH: 7.0, osmolality: 274 mmol/kg)

15.625 μg/mL

7.40E+06 (63)

1.65E+07 (96)

2.14E+07 (76)

Normal / normal (pH: 7.0, osmolality: 282 mmol/kg

7.813 μg/mL

9.10E+06 (78)

1.67E+07 (97)

2.62E+07 (94)

Normal / normal (pH: 7.0, osmolality: 270 mmol/kg)

3.906 μg/mL

9.60E+07 (82)

2.00E+07 (117)

2.26E+07 (81)

Normal / normal (pH: 7.0, osmolality: 270 mmol/kg)

*: compared to the relevant negative control (Acetone)                             #: minimal amount

 

Results of the Preliminary Toxicity Test

(24-hour treatment in the absence of metabolic activation)

Test item (or solvent) concentration

Total cell number (Relative Survival*, %) after treatment, Day 0

Total cell number (Relative Survival*, %) on Day 1

Total cell number (Relative Survival*, %) on Day 2

Observations at the beginning / after treatment

Untreated control

1.61E+07 (107)

1.09E+7 (100)

2.30E+07 (100)

Normal / normal (pH: 7.0, osmolality: 288 mmol/kg)

Negative (vehicle) control)

1.50E+07 (100)

2.09E+07 (100)

2.30E+07 (100)

Normal / normal (pH: 7.0, osmolality: 290 mmol/kg)

2000 μg/mL

0.00E+00 (0)

--

--

Precipitate, oily film / discoloured medium, precipitate, oily film

(pH: 7.0, osmolality: 294 mmol/kg)

1000 μg/mL

0.00E+00 (0)

--

--

Precipitate, oily film / discoloured medium, precipitate, oily film

(pH: 7.0, osmolality: 288 mmol/kg)

500 μg/mL

0.00E+00 (0)

--

--

Precipitate, oily film / discoloured medium, precipitate#, oily film#

(pH: 7.0, osmolality: 290 mmol/kg)

250 μg/mL

0.00E+00 (0)

--

--

Precipitate, oily film / discoloured medium, precipitate#

(pH: 7.0, osmolality: 291 mmol/kg)

125 μg/mL

0.00E+00 (0)

--

--

Precipitate#, oily film / discoloured medium, precipitate#

(pH: 7.0, osmolality: 289 mmol/kg)

62.5 μg/mL

4.00E+05 (3)

0.00E+00 (0)

--

oily film / discoloured medium (pH: 7.0, osmolality: 288 mmol/kg)

31.25 μg/mL

2.85E+06 (19)

6.75E+06 (32)

2.22E+07 (97)

oily film / discoloured medium (pH: 7.0, osmolality: 294 mmol/kg)

15.625 μg/mL

8.20E+06 (55)

2.16E+07 (104)

2.02E+07 (88)

Normal / discoloured medium#(pH: 7.0, osmolality: 279 mmol/kg

7.813 μg/mL

1.23E+07 (82)

2.46E+07 (118)

2.18E+07 (95)

Normal / normal (pH: 7.0, osmolality: 280 mmol/kg)

3.906 μg/mL

1.52E+07 (101)

2.34E+07 (112)

2.34E+07 (102)

Normal / normal (pH: 7.0, osmolality: 283 mmol/kg)

*: compared to the relevant negative control (Acetone)                             #: minimal amount

 

Summary Table of Survival Data

 

Survival Results of Assay 1 (I)

(3-hour treatment in the presence of S9-mix)

S9-mix

Treatment period (hours)

Study phase

Test item of control concentration

Number of empty wells/total number of wells

Plating Efficiency (PE)

Relative Survival§(%RS)

Relative Total Growth (%RTG)

+

3

A1

2000 μg/mL

177/384

0.484

30

17

1000 μg/mL

174/384

0.495

29

13

500 μg/mL

268/384

0.225

10

<2

250 μg/mL

322/384

0.110

3

ND

125 μg/mL

135/384

0.653

29

<1

62.5 μg/mL

175/384

0.491

35

18

31.25 μg/mL

65/384

1.110

79

74

15.625 μg/mL

57/384

1.192

111

91

7.813 μg/mL

74/384

1.0259

90

92

Vehicle control

88/384

0.921

100

100

Vehicle control for CP

98/384

0.854

95

117

Untreated control

66/384

1.101

135

138

Positive control (CP)

141/384

0.626

71

35

A1 = Assay 1

+ = in the presence of S9-mix

Negative (vehicle) control = Acetone

Negative (vehicle) control for CP = DMSO

DMSO = Dimethyl sulfoxide

CP = Cyclophosphamide (4 μg/mL)

ND = No data (Cells did not survive the treatment or expression period)

§ = Relative survival values (%) corrected with the post treatment cell concentrations.

 

Survival Results of Assay 1 (II)

(3-hour treatment in the absence of S9-mix)

S9-mix

Treatment period (hours)

Study phase

Test item of control concentration

Number of empty wells/total number of wells

Plating Efficiency (PE)

Relative Survival§(%RS)

Relative Total Growth (%RTG)

-

3

A1

2000 μg/mL

66/384

1.101

54

6

1000 μg/mL

166/384

0.524

26

12

500 μg/mL

322/384

0.110

3

<1

250 μg/mL

360/384

0.040

1

<1

125 μg/mL

362/384

0.037

1

<1

62.5 μg/mL

355/384

0.049

1

<2

31.25 μg/mL

167/384

0.520

26

9

15.625 μg/mL

79/384

0.988

79

95

7.813 μg/mL

74/384

1.029

89

78

Vehicle control

86/384

0.935

100

100

Vehicle control for NQO

71/384

1.055

113

96

Untreated control

44/384

1.354

131

145

Positive control (NQO)

126/384

0.696

58

47

A1 = Assay 1

- = in the absence of S9-mix

Negative (vehicle) control = Acetone

Negative (vehicle) control for NQO = DMSO

DMSO = Dimethyl sulfoxide

NQO = 4-Nitroquinoline-N-oxide (0.15 μg/mL)

§ = Relative survival values (%) corrected with the post treatment cell concentrations.

 

Survival Results of Assay 2 (I)

(3-hour treatment in the presence of S9-mix)

S9-mix

Treatment period (hours)

Study phase

Test item of control concentration

Number of empty wells/total number of wells

Plating Efficiency (PE)

Relative Survival§(%RS)

Relative Total Growth (%RTG)

+

3

A2

2000 μg/mL

378/384

0.010

0

ND

1000 μg/mL

368/384

0.027

1

ND

500 μg/mL

354/384

0.051

3

ND

250 μg/mL

243/384

0286

12

ND

125 μg/mL

111/384

0.776

46

ND

100 μg/mL

103/384

0.822

54

10

80 μg/mL

116/384

0.748

39

16

62.5 μg/mL

97/384

0.860

56

25

50 μg/mL

90/384

0.907

60

37

40 μg/mL

62/384

1.140

79

59

31.25 μg/mL

60/384

1.160

100

68

15.625 μg/mL

52/384

1.250

117

83

7.813 μg/mL

74/384

1.029

100

62

Vehicle control

57/384

1.192

133

100

Vehicle control for CP

60/384

1.160

117

95

Untreated control

48/384

1.300

142

109

Positive control (CP)

374/384

0.016

1

<1

A2 = Assay 2

+ = in the presence of S9-mix

Negative (vehicle) control = Acetone

Negative (vehicle) control for CP = DMSO

DMSO = Dimethyl sulfoxide

CP = Cyclophosphamide (4 μg/mL)

ND = No data (Cells did not survive the treatment or expression period, or the total cell number was less than requested for plating)

§ = Relative survival values (%) corrected with the post treatment cell concentrations.

 

Survival Results of Assay 2 (II)

(24-hour treatment in the absence of S9-mix)

S9-mix

Treatment period (hours)

Study phase

Test item of control concentration

Number of empty wells/total number of wells

Plating Efficiency (PE)

Relative Survival§(%RS)

Relative Total Growth (%RTG)

-

24

A2

70 μg/mL

ND

ND

ND

ND

60 μg/mL

ND

ND

ND

ND

50 μg/mL

ND

ND

ND

ND

45 μg/mL

ND

ND

ND

ND

40 μg/mL

ND

ND

ND

ND

35 μg/mL

384/384

0.00

0

ND

30 μg/mL

384/384

0.00

0

ND

25 μg/mL

311/384

0.132

2

1

20 μg/mL

143/384

0.617

19

14

15 μg/mL

71/384

1.055

68

75

10 μg/mL

86/384

0.935

86

66

5 μg/mL

91/384

0.900

99

76

2.5 μg/mL

93/384

0.886

105

112

Vehicle control

96/384

0.866

100

100

Vehicle control for NQO

100/384

0.841

106

95

Untreated control

74/384

1.029

143

176

Positive control (NQO)

167/384

0.520

50

53

A2 = Assay 2

- = in the absence of S9-mix

Negative (vehicle) control = Acetone

Negative (vehicle) control for NQO = DMSO

DMSO = Dimethyl sulfoxide

NQO = 4-Nitroquinoline-4-N-oxide (0.1 μg/mL)

ND = No data (Cells did not survive the treatment or expression period)

§ = Relative survival values (%) corrected with the post treatment cell concentrations.

 

Summary Table of Viability Data

 

Viability Results of Assay 1 (I)

(3-hour treatment in the presence of S9-mix)

S9-mix

Treatment period (hours)

Study phase

Test item of control concentration

Number of empty wells/total number of wells

Plating Efficiency (PE)

+

3

A1

2000 μg/mL

144/384

0.613

1000 μg/mL

121/384

0.722

500 μg/mL

253/384

0.261

250 μg/mL

ND

ND

125 μg/mL

371/384

0.022

62.5 μg/mL

66/384

1.101

31.25 μg/mL

72/384

1.046

15.625 μg/mL

88/384

0.921

7.813 μg/mL

81/384

0.973

Vehicle control

74/384

1.029

Vehicle control for CP

65/384

1.110

Untreated control

68/384

1.082

Positive control (CP)

148/384

0.596

A1 = Assay 1

+ = in the presence of S9-mix

Negative (vehicle) control = Acetone

Negative (vehicle) control for CP = DMSO

DMSO = Dimethyl sulfoxide

CP = Cyclophosphamide (4 μg/mL)

ND = No data (Cells did not survive the treatment or expression period)

 

Viability Results of Assay 1 (II)

(3-hour treatment in the absence of S9-mix)

S9-mix

Treatment period (hours)

Study phase

Test item of control concentration

Number of empty wells/total number of wells

Plating Efficiency (PE)

-

3

A1

2000 μg/mL

106/384

0.805

1000 μg/mL

121/384

0.722

500 μg/mL

312/384

0.130

250 μg/mL

382/384

0.003

125 μg/mL

383/384

0.002

62.5 μg/mL

246/384

0.278

31.25 μg/mL

78/384

0.996

15.625 μg/mL

64/384

1.120

7.813 μg/mL

76/384

1.012

Vehicle control

76/384

1.012

Vehicle control for NQO

82/384

0.965

Untreated control

64/384

1.120

Positive control (NQO)

93/384

0.886

A1 = Assay 1

- = in the absence of S9-mix

Negative (vehicle) control = Acetone

Negative (vehicle) control for NQO = DMSO

DMSO = Dimethyl sulfoxide

NQO = 4-Nitroquinoline-N-oxide (0.15 μg/mL)

 

Viability Results of Assay 2 (I)

(3-hour treatment in the presence of S9-mix)

S9-mix

Treatment period (hours)

Study phase

Test item of control concentration

Number of empty wells/total number of wells

Plating Efficiency (PE)

+

3

A2

2000 μg/mL

ND

ND

1000 μg/mL

ND

ND

500 μg/mL

ND

ND

250 μg/mL

ND

ND

125 μg/mL

ND

ND

100 μg/mL

91/384

0.900

80 μg/mL

54/384

1.226

62.5 μg/mL

63/384

1.130

50 μg/mL

80/384

0.980

40 μg/mL

101/384

0.835

31.25 μg/mL

96/384

0.866

15.625 μg/mL

105/384

0.810

7.813 μg/mL

123/384

0.712

Vehicle control

89/384

0.914

Vehicle control for CP

97/384

0.860

Untreated control

92/384

0.893

Positive control (CP)

360/384

0.040

A2 = Assay 2

+ = in the presence of S9-mix

Negative (vehicle) control = Acetone

Negative (vehicle) control for CP = DMSO

DMSO = Dimethyl sulfoxide

CP = Cyclophosphamide (4 μg/mL)

ND = No data (Cells did not survive the treatment or expression period)

 

Viability Results of Assay 2 (II)

(24-hour treatment in the absence of S9-mix)

S9-mix

Treatment period (hours)

Study phase

Test item of control concentration

Number of empty wells/total number of wells

Plating Efficiency (PE)

-

24

A2

70 μg/mL

ND

ND

60 μg/mL

ND

ND

50 μg/mL

ND

ND

45 μg/mL

ND

ND

40 μg/mL

ND

ND

35 μg/mL

ND

ND

30 μg/mL

ND

ND

25 μg/mL

15/192

1.593

20 μg/mL

92/384

0.893

15 μg/mL

84/384

0.950

10 μg/mL

119/384

0.732

5 μg/mL

153/384

0.575

2.5 μg/mL

99/384

0.847

Vehicle control

125/384

0.701

Vehicle control for NQO

126/384

0.696

Untreated control

98/384

0.854

Positive control (NQO)

94/384

0.880

A2 = Assay 2

- = in the absence of S9-mix

Negative (vehicle) control = Acetone

Negative (vehicle) control for NQO = DMSO

DMSO = Dimethyl sulfoxide

NQO = 4-Nitroquinoline-4-N-oxide (0.1 μg/mL)

ND = No data (Cells did not survive the treatment or expression period)

Note: In case of 25 μg/mL concentration only one replicate survived the treatment, calculation was made using data of that replicate only.

 

 

Summary Tables of Mutagenicity Data

 

Mutagenicity Results of Assay 1 (I)

(3-hour treatment in the presence of S9-mix)

S9-mix

Treatment period (hours)

Test item of control concentration

Number of empty wells/total number of wells

Number of large colonies/total number of wells

Number of small colonies/total number of wells

Dn2/var(Dn)

Mutation frequency

+

3

2000 μg/mL

585/768

127/768

56/768

--

NE

1000 μg/mL

564/768

143/768

61/768

--

NE

500 μg/mL

289/384

68/384

27/384

--

NE

250 μg/mL

ND

ND

ND

ND

ND

125 μg/mL

365/384

9/384

10/384

--

NE

62.5 μg/mL

549/768

131/768

88/768

6.451

155.1*

31.25 μg/mL

649/768

75/768

44/768

0.126

80.5

15.625 μg/mL

650/768

56/768

62/768

0.016

90.6

7.813 μg/mL

643/768

71/768

54/768

0.026

91.3

Vehicle control

641/768

64/768

63/768

--

87.8

Vehicle control for CP

628768

85/768

55/768

--

90.9

Untreated control

631/768

75/768

62768

--

90.8

Positive control

(CP: 4 μg/mL)

213/768

171/768

384/768

◊◊

6.19E-13

1076.1*

In linear trend analysisβ2/var (β) = 4.09, significant(at p<0.05)

* = Statistically significant

◊ = Evaluated by Dunnett’s test for multiple comparisons. Significant id Dn2/var(Dn) >4.67(p<0.05).

◊◊ = Evaluated nu T-test for independent samples (compared to the DMSO vehicle control).

Dn= Difference of log mutant frequency of dose “n” and that of the vehicle control

Var(Dn) = variamce of Dn  β = slope of the curve        var(β) = variance of the slope

+ = in the presence of S9-mix

Negative (vehicle) control = Acetone

Negative (vehicle) control for CP = DMSO

DMSO = Dimethyl sulfoxide

CP = Cyclophosphamide

ND = No data (Cells did not survive the treatment or expression period)

NE = Not evaluated (due to extreme level of cytotoxicity or suboptimal level of solubility)

Note: Mutation frequency refers to 106viable cells.

 

Mutagenicity Results of Assay 1 (II)

(3-hour treatment in the absence of S9-mix)

S9-mix

Treatment period (hours)

Test item of control concentration

Number of empty wells/total number of wells

Number of large colonies/total number of wells

Number of small colonies/total number of wells

Dn2/var(Dn)

Mutation frequency

-

3

2000 μg/mL

604/768

97/768

67/768

--

NE

1000 μg/mL

562/768

146/768

60/768

--

NE

500 μg/mL

510/628

88/628

30/628

--

NE

250 μg/mL

279/280

0/280

1/280

--

NE

125 μg/mL

536/548

10/548

2/548

--

NE

62.5 μg/mL

465/768

265/768

38/768

89.002

901.4*

31.25 μg/mL

464/768

244/768

60/768

19.597

252.9*

15.625 μg/mL

646/768

67/768

44/768

0.159

84.0

7.813 μg/mL

645/768

67/768

56/768

0.091

86.2

Vehicle control

636/768

73/768

59/768

--

93.1

Vehicle control for NQO

648/768

60/768

60/768

--

88.0

Untreated control

616/768

82/768

70/768

--

98.5

Positive control

(NQO: 0.15 μg/mL)

205/768

229/768

334/768

◊◊

1.84E-14

745.1*

In linear trend analysisβ2/var (β) = 21.73, significant(at p<0.05)

* = Statistically significant

◊ = Evaluated by Dunnett’s test for multiple comparisons. Significant id Dn2/var(Dn) >4.67(p<0.05).

◊◊ = Evaluated nu T-test for independent samples (compared to the DMSO vehicle control).

Dn= Difference of log mutant frequency of dose “n” and that of the vehicle control

Var(Dn) = variamce of Dn  β = slope of the curve        var(β) = variance of the slope

- = in the absence of S9-mix

Negative (vehicle) control = Acetone

Negative (vehicle) control for NQO = DMSO

DMSO = Dimethyl sulfoxide

NQO = 4-Nitroquinoline-N-oxide

NE = Not evaluated (due to extreme level of cytotoxicity or suboptimal level of solubility). In case of 2000 μg/mL concentration the calculated mutation frequency is 149.3, in case of 1000 μg/mL the calculated mutation frequency is 216.3.

Note: Mutation frequency refers to 106viable cells.

 

Mutagenicity Results of Assay 2 (I)

(3-hour treatment in the presence of S9-mix)

S9-mix

Treatment period (hours)

Test item of control concentration

Number of empty wells/total number of wells

Number of large colonies/total number of wells

Number of small colonies/total number of wells

Dn2/var(Dn)

Mutation frequency

+

3

2000 μg/mL

ND

ND

ND

ND

ND

1000 μg/mL

ND

ND

ND

ND

ND

500 μg/mL

ND

ND

ND

ND

ND

250 μg/mL

ND

ND

ND

ND

ND

125 μg/mL

ND

ND

ND

ND

ND

100 μg/mL

674/768

61/768

33/768

0.019

72.5

80 μg/mL

610/768

98/768

60/768

1.361

93.9

62.5 μg/mL

637/768

79/768

52/768

0.425

82.8

50 μg/mL

648/768

68/768

52/768

0.666

86.6

40 μg/mL

673/768

65/768

30/768

0.196

79.1

31.25 μg/mL

688/768

45/768

35/768

0.111

63.5

15.625 μg/mL

667/768

41/768

50/768

0.648

87.0

7.813 μg/mL

689/768

43/768

36/768

0.091

76.3

Vehicle control

676/768

55/768

37/768

--

69.8

Vehicle control for CP

691/768

39/768

38/768

--

61.4

Untreated control

676/768

397/768

53/768

--

71.4

Positive control

(CP: 4 μg/mL))

633/768

25/768

110/768

◊◊

5.29E-03

2396.3*

In linear trend analysisβ2/var (β) = 0.35, not significant

* = Statistically significant

◊ = Evaluated by Dunnett’s test for multiple comparisons. Significant id Dn2/var(Dn) >5.66(p<0.05).

◊◊ = Evaluated nu T-test for independent samples (compared to the DMSO vehicle control).

Dn= Difference of log mutant frequency of dose “n” and that of the vehicle control

Var(Dn) = variamce of Dn  β = slope of the curve        var(β) = variance of the slope

+ = in the presence of S9-mix

Negative (vehicle) control = Acetone

Negative (vehicle) control for CP = DMSO

DMSO = Dimethyl sulfoxide

CP = Cyclophosphamide

ND = No data (Cells did not survive the treatment or expression period)

Note: Mutation frequency refers to 106viable cells.

 

Mutagenicity Results of Assay 2 (II)

(24-hour treatment in the absence of S9-mix)

S9-mix

Treatment period (hours)

Test item of control concentration

Number of empty wells/total number of wells

Number of large colonies/total number of wells

Number of small colonies/total number of wells

Dn2/var(Dn)

Mutation frequency

-

24

70 μg/mL

ND

ND

ND

ND

ND

60 μg/mL

ND

ND

ND

ND

ND

50 μg/mL

ND

ND

ND

ND

ND

45 μg/mL

ND

ND

ND

ND

ND

40 μg/mL

ND

ND

ND

ND

ND

35 μg/mL

ND

ND

ND

ND

ND

30 μg/mL

ND

ND

ND

ND

ND

25 μg/mL

289/384

67/384

28/384

--

NE

20 μg/mL

658/768

73/768

37/768

0.134

86.6

15 μg/mL

677/768

47/768

44/768

0.428

65.3

10 μg/mL

658/768

60/768

50/768

1.211

105.6

5 μg/mL

689/768

50/768

29/768

0.413

94.4

2.5 μg/mL

701/768

40/768

27/768

1.600

53.9

Vehicle control

688/768

51/768

29/768

--

78.4

Vehicle control for NQO

677/768

52/768

39/768

--

90.5

Untreated control

677/768

39/768

52/768

--

73.9

Positive control

(NQO: 0.1 μg/mL)

333/768

264/768

171/768

◊◊

3.38E-07

475.0*

In linear trend analysisβ2/var (β) = 0.61, not significant

* = Statistically significant

◊ = Evaluated by Dunnett’s test for multiple comparisons. Significant id Dn2/var(Dn) >4.97(p<0.05).

◊◊ = Evaluated nu T-test for independent samples (compared to the DMSO vehicle control).

Dn= Difference of log mutant frequency of dose “n” and that of the vehicle control

Var(Dn) = variamce of Dn  β = slope of the curve        var(β) = variance of the slope

- = in the absence of S9-mix

Negative (vehicle) control = Acetone

Negative (vehicle) control for NQO = DMSO

DMSO = Dimethyl sulfoxide

NQO = 4-Nitroquinoline-N-oxide

ND = No data (Cells did not survive the treatment of expression period)

NE = Not evaluated (due to extreme level of cytotoxicity).

Note: Mutation frequency refers to 106viable cells.

 

Historical Control Data

(updated on 11 January 2017)

Mutation Frequency of the Negative Controls (2006-2016)

 

Culture medium

Distilled water

Treatments

3h,S9+

3h,S9-

24h,S9-

3h,S9+

3h,S9-

24h,S9-

Average

SD

Min.

Max.

n

94.3

26.9

39.3

198.5

84

103.6

35.3

52.6

235.6

43

106.4

27.4

41.7

179.1

44

90.4

22.7

33.4

121.8

26

96.6

19.0

55.1

125.0

13

96.3

24.6

43.2

141.1

13

 

Dimethyl sulfoxide (DMSO)

 

Treatments

3h,S9+

3h,S9-

24h,S9-

 

 

 

Average

SD

Min.

Max.

n

97.3

33.7

44.2

269.9

101

97.3

38.5

33.7

261.6

57

98.9

26.8

47.1

159.4

50

 

 

 

Mutation Frequency of the Positive Controls (2006-2016)

 

Cyclophosphamide

4-Nitroquinoline-N-oxide

Treatments

3h,S9+

 

 

 

3h, S9-

24h,S9-

Average

SD

Min.

Max.

n

1178.7

524.7

196.1

2642.5

106

 

 

 

722.2

330.0

223.5

1687.3

58

831.9

337.2

245.0

1577.6

52

h = hour

SD = Standard Deviation

S9+ = experiment with metabolic activation

S9- = experiment without metabolic activation

n = number of cases

Conclusions:
The Mouse Lymphoma Assay with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol on L5178Y TK +/- 3.7.2 C cells was considered to be valid and to reflect the real potential of the test item to cause mutations in the cultured mouse cells used in this study.

In the Mouse Lymphoma Assay, treatment with the test item did not result in a statistically significant and biologically relevant increase in the mutation frequency both in the presence and absence of a rat metabolic activation system (S9 fraction) at concentration with acceptable level of cytotoxicity. In case of short treatment without metabolic activation, substantial increases in mutation frequency were seen at concentrations with an unacceptably high level of cytotoxicity (<10% measured by RTG), those results were not reproducible in the experiment with long treatment without metabolic activation. Therefore, no mutagenic activity of the test item was concluded in the performed experiments.

In conclusion, no mutagenic effect of 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol was observed in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.
Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol test item to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix).

 

Acetone was used as vehicle of the test item in this study. The test item was examined up to 2000 μg/mL (the recommended maximum concentration) in the Preliminary Toxicity Test. Based on the results of the preliminary experiment (the test item had a special solubility and cytotoxicity profile), the following test item concentrations were examined in the mutation assays:

Assay 1, 3-hour treatment with metabolic activation: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625 and 7.813 μg/mL,

Assay 1, 3-hour treatment without metabolic activation: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625 and 7.813 μg/mL,

Assay 2, 3-hour treatment with metabolic activation: 2000, 1000, 500, 250, 125, 100, 80, 62.5, 50, 40, 31.25, 15.625 and 7.813 μg/mL,

Assay 2, 24-hour treatment without metabolic activation: 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5 and 2.5 μg/mL.

 

In Assays 1-2, there were no large changes in pH or osmolality after treatment.

Insolubility / minimal amount of insolubility was observed in the final treatment medium at the end of the treatment in both assays (in the 2000-31.25 μg/mL concentration range in Assay 1 with metabolic activation; in the 2000-125 μg/mL concentration range in Assay 1 without metabolic activation, in the 2000-125 μg/mL concentration range Assay 2 with metabolic activation and in the 70-45 μg/mL concentration range in Assay 2 without metabolic activation).

 

In Assay 1, following a 3-hour treatment with metabolic activation, cytotoxicity of the test item was observed. No cells survived the expression period in the samples of 250 μg/mL concentration, furthermore the total number of surviving cells was much less than requested for plating in one replicate of 125 μg/mL concentration. Thus, an evaluation was made using data of the next concentration of 62.5 μg/mL (relative total growth of 18%) and three lower concentrations (a total of four concentrations).

Statistically significant increase was noted at 62.5 μg/mL, however the observed values was biologically not relevant (the difference compared to the negative (vehicle) control value was not larger than the global evaluation factor). Therefore, the slight dose response relationship was considered as not relevant. In overall, this experiment was considered as being negative.

 

In Assay 1, following a 3-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed in the 500-125 μg/mL concentration range, the total cell number of one or two replicates was lower than required for plating.

An evaluation was made using data of the next concentration 62.5 μg/mL (relative total growth of <2%) and three lower concentrations (a total of four concentrations).

Statistically significant and biologically relevant increase in the mutation frequency was observed 62.5 and 31.25 μg/mL concentrations, although the levels of cytotoxicity at both concentrations were lower than 10%. A significant dose-response to the treatment was also indicated by the linear trend analysis. Based on the fact that the substantial increases were seen only at high level of cytotoxicity (additional concentrations of 2000 and 1000 μg/mL - above the level of optimal solubility, but with acceptable level of cytotoxicity, 6% and 12%, respectively – did not show biologically relevant increases in the mutation frequency). This experiment gave apparently positive results, but the degree of cytotoxicity in these concentrations was out of the acceptability range, in all cases of acceptable cytotoxicity there was no relevant increase in the mutation frequency. Hence the result of this experiment was considered as being negative.

 

In Assay 2, following a 3-hour treatment with metabolic activation, excessive cytotoxicity of the test item was observed in the 2000-250 μg/mL concentration range, cells of one or two replicates of these samples did not survive the treatment or expression period. At the following concentration of 125 μg/mL, the total number of surviving cells was less than minimal, thus this sample was not used for viability / mutagenicity plating. An evaluation was made using data of the highest concentration of 100 μg/mL (relative total growth of 10%) and seven lower concentrations (a total of eight concentrations). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. This experiment showed clear negative result.

 

In Assay 2, following a 24-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed in the 70-30 μg/mL concentration range, cells of these samples did not survive the treatment or expression period. The following concentration of 25 μg/mL still showed marked cytotoxicity (relative total growth: <1%) and it was excluded from the evaluated concentration range. Thus, an evaluation was made using data of the next concentration of 20 μg/mL (relative total growth of 14%) and four lower concentrations (a total of five concentrations). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. This experiment was clearly negative.

 

The experiments were performed using appropriate untreated, negative (vehicle) and positive control samples in all cases. The spontaneous mutation frequency of the negative (vehicle) controls was in the appropriate range. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the negative (vehicle) controls at the end of the expression period were acceptable in all assays. The evaluated concentration ranges were considered to be adequate. The number of test concentrations met the acceptance criteria. Therefore, the study was considered to be valid.

 

In conclusion, no clear mutagenic effect of 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol was observed in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 August 2017 to 29 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
OECD Guidelines for Testing of Chemicals, Section 4, No. 473, “In Vitro Mammalian Chromosome Aberration Test”, 29 July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Commission Regulation (EU) No 440/2008 B.10.as amended by Commission Regulation 2017/735 (2017)
Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), (Official Journal L 112 p23-39)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro mammalian chromosome aberration test (migrated information)
Specific details on test material used for the study:
No further details specified in the study report.
Target gene:
structural chromosome aberrations in somatic and/or germ cells
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
V79: Chinese hamster lung, male
ECACC Cat. No.: 86041102
Lot No.: 10H016
Date of working lot: 31 July 2015
Supplier: ECACC (European Collection of Cells Cultures) Morphology: Fibroblast
The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for genetic toxicity assays with low background aberrations. These cells are chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12-14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male). This cell line was purchased from ECACC (European Collection of Cells Cultures). The cell stocks were kept in a freezer at -80 ± 10°C (for short-term storage) or in liquid nitrogen (long-term storage).
The stock was checked for mycoplasma infection. No infection of mycoplasma was noted.
Trypsin-EDTA (0.25% Trypsin, 1mM EDTA) solution was used for cell detachment to subculture (cells were rinsed with 1X PBS before detachment). The laboratory cultures were maintained in 150 cm2 plastic flasks at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5% CO2 in air. The V79 cells for this study were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 1% (v/v) Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and 10% (v/v) heat-inactivated fetal bovine serum (DMEM-10, culture medium). When cells were growing well, subcultures were established in an appropriate number of flasks (after thawing, the cells were subcultured no more than 5 times before used in the study). During the treatments, the serum content of the medium was reduced to 5% (v/v) (DMEM-5).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Chromosome Aberration Assay 1: 250, 83.33, 27.78, 9.26, 3.09 and 1.03 μg/mL (experiment without metabolic activation); and 500, 166.67, 55.56, 18.52, 6.17 and 2.06 μg/mL (experiment with metabolic activation).
Chromosome Aberration Assay 3: 10, 8, 7, 6, 5, 2.5 and 1.25 μg/mL (experiment without metabolic activation); and 500, 166.67, 55.56, 18.52, 6.17 and 2.06 (experiment with metabolic activation).
Treatment concentrations for the mutation assay were selected based on the results of a short preliminary test.
Vehicle / solvent:
Based on the available solubility information (trial formulations of the test item performed at the Test Facility) and the available information (Citoxlab study code: 17/080-007M), Acetone was selected for vehicle (solvent) of the study. The vehicle was compatible with the survival of the cells and the S9 activity.
Data of the chemicals used for preparation of the vehicle are shown below:
Name: Acetone
Supplier: VWR
Lot No.: 15J060514
Expiry date: 31 October 2020
Storage conditions: Room temperature
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
EXTERNAL METABOLIC ACTIVATION SYSTEM
An advantage of using in vitro cell cultures is the accurate control of the concentration and exposure time of cells to the test item under the study. However, due to the limited capacity of cells growing in vitro for metabolic activation of potential mutagens, an exogenous metabolic activation system is necessary.
Many substances only develop mutagenic potential after they are metabolised.
Metabolic activation of substances can be achieved by supplementing the cell cultures with liver microsome preparations (S9 mix).
In the experiments with metabolic activation in this study, a cofactor-supplemented post-mitochondrial S9 fraction prepared from activated rat liver was used as an appropriate metabolic activation system.
The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of Citoxlab Hungary Ltd. according to Ames et al. and Maron and Ames. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.

TEST PROCEDURE
Toxicity and Concentration Selection
Treatment concentrations for the mutation assay were selected based on the results of a short preliminary test.
In this Preliminary Toxicity Test, two assays were performed. In Assay A, cells were treated for 3-hours in the presence and absence of S9-mix with a 20-hour harvesting time. In Assay B, cells were treated for 20 hours in the absence of S9-mix with a 20-hour harvesting time.
The assays were performed with a range of test item concentrations to determine cytotoxicity. Treatment was performed as described for the main test. However, single cultures were used and positive controls were not included. Visual examination of the final culture medium was conducted at the beginning and end of the treatments.
Measurement of pH and osmolality was also performed at the end of the treatment period.
At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (vehicle) control as RICC (Relative Increase in Cell Counts).

Chromosome Aberration Assays
The Chromosome Aberration Assays were conducted as two independent experiments (Assay 1 and Assay 3) in the presence and in the absence of metabolic activation. In Assay 1, 3-hour treatment was performed with and without metabolic activation (in the presence and absence of S9 mix); cells were harvested 20-hour after the beginning of the treatment.
In Assay 3, a 3-hour treatment was performed with metabolic activation (in the presence of S9 mix) and 20-hour without metabolic activation (in the absence of S9 mix) in duplicate cultures; cells were harvested 20-hour after the beginning of the treatment.

Treatment of the Cells
For the cytogenetic experiments, 1-3 day old cultures (more than 50 % confluency) were used. Cells were seeded into 92 x 17 mm tissue culture dishes at 5e+5 cells/dish concentration and incubated for approximately 24 hours at 37°C in 10 mL of culture medium (DMEM-10). Duplicate cultures were used for each test item concentration or controls.
After the seeding period, the medium was replaced with 9.90 or 9.95 mL treatment medium (DMEM-5) in case of experiments without metabolic activation or with 9.40 or 9.45 mL treatment medium (DMEM-5) + 0.5 mL S9-mix in case of experiments with metabolic activation.
Cells were treated with different concentration test item solutions, untreated, negative (vehicle) or positive control solution (treatment volume: 100 μL/dish in case of the untreated and positive control; 50 μL/dish in case of the test item and negative control) for the given period of time at 37°C in the absence or presence of S9-mix. After the exposure period, the cultures were washed with DMEM-0 medium (Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine and 1% (v/v) Antibiotic-antimycotic solution). Then, 10 mL of fresh culture medium were added into the dishes and cells were incubated further until the scheduled harvesting time.
Harvesting was performed after 20 hours (approximately 1.5 normal cell cycles) from the beginning of treatment.
Solubility of the test item in the final treatment medium was visually examined at the beginning and end of the treatment in each case. Measurement of pH and osmolality was also performed at the end of the treatment period in both main tests.
For concurrent measurement of cytotoxicity an extra dish was plated for each sample and treated in the same manner. At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (vehicle) control as RICC (Relative Increase in Cell Counts).

Preparation of Chromosomes
2-2.5 hours prior to harvesting, cell cultures were treated with Colchicine (0.2 μg/mL).
The cells were swollen with 0.075 M KCl hypotonic solution for 4 minutes, then were washed in fixative (Methanol : Acetic-acid 3 : 1 (v : v) mixture) until the preparation became plasma free (4 washes). Then, a suspension of the fixed cells* was dropped onto clean microscope slides and air-dried. The slides were stained with 5% Giemsa solution, air-dried and coverslips were mounted. At least three slides were prepared for each culture.
*Note: Fixed cells were stored frozen in case if any additional action was required (as documented in the raw data and reported). After the finalization of the report, the remaining frozen cell suspension samples will be discarded.

Examination of Slides
The stained slides were given random unique code numbers at the Test Facility by a person who was not involved in the metaphase analysis. The code labels covered all unique identification markings on the slides to ensure that they were scored without bias.
The metaphase analysis was conducted under the control of the Principal Investigator.
When the metaphase analysis was completed for each test, the slide codes were broken and the number of metaphases with aberrations (excluding gaps) and the types of aberrations for each culture were presented in tables.
At least 150* metaphases with 22±2 chromosomes (centromeres) from each culture (replicate) will be examined for the presence or absence of chromosomal aberrations (approximately 1000x magnification), where possible. Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.
*Note: The examination of slides from a culture was halted when 25 or more metaphases with aberrations (excluding gaps) have been recorded for that culture.

The aberrations are defined in the following way:
Gap: small unstained lesion smaller than the width of a chromatid and with minimal misalignment of the chromatid(s)
Break: unstained lesion larger than the width of a chromatid, or with clear misalignment
Exchange: breakage and reunion of chromatids within a chromosome, or between chromosomes
Chromatid-type: structural chromosome damage expressed as breakage of single chromatids or breakage and reunion between chromatids
Chromosome-type: structural chromosome damage expressed as breakage, or breakage and reunion, of both chromatids at an identical site.
Fragments could arise from breakage and exchange events. When the origin of a fragment was clear, it was recorded under that category (e.g. a dicentric chromosome with a fragment was recorded as one chromosome exchange event). When the origin of the fragment was not clear, it was recorded as a chromatid break. Metaphases with more than five aberrations (excluding gaps) were recorded as showing multiple damage.
Additionally, the number of polyploid and endoreduplicated cells was scored. Polyploid metaphases are defined as metaphases with approximate multiples of the haploid chromosome number (n), other than the diploid number (i.e. ca. 3n, 4n etc).
Endoreduplicated metaphases have chromosomes with 4, 8, etc. chromatids. Marked reductions in the numbers of cells on the slides were recorded if needed.
The vernier co-ordinates of at least five metaphases (with aberrations, where possible) were recorded for each culture.
Rationale for test conditions:
In accordance with test guidelines
Evaluation criteria:
The assay is considered valid, if the following criteria are met:
- The negative (vehicle) control data are within the laboratory’s normal range for the spontaneous aberration frequency.
- The positive controls induce increases in the aberration frequency, which are significant.

The test item is considered to have shown clastogenic activity in this study if all of the following criteria are met:
- Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
- The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases are statistically significant.
- The increases are not associated with large changes in pH or osmolality of the treated cultures.

The historical control data for this laboratory were also considered in the evaluation.
Evidence of a dose-response relationship (if any) was considered to support the conclusion.
The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed.
Statistics:
For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
VEHICLE AND CONCENTRATION SELECTION
Based on the available solubility information (trial formulations of the test item performed at the Test Facility) and the available information (Citoxlab study code: 17/080-007M), Acetone at 400 mg/mL concentration was selected for vehicle (solvent) of the study. The highest examined concentration in the preliminary test was 2000 μg/mL.
Two Concentration Selection Cytotoxicity Assays (Assay A: 3-hour treatment with and without metabolic activation, 20-hour harvesting time; and Assay B: 20-hour treatment without metabolic activation, 20-hour harvesting time) were performed as part of the study to establish an appropriate concentration range for the Chromosome Aberration Assays.
A total of ten test concentrations between 2000 and 3.906 μg/mL were used to evaluate toxicity in the presence and absence of metabolic activation in each cytotoxicity assay. Treatment concentrations for the chromosome aberration assays were selected on the basis of results of the performed Concentration Selection Cytotoxicity Assays according to the OECD guideline instructions (up to the solubility limit or cytotoxicity limit).

CHROMOSOME ABERRATION ASSAYS
In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 250, 83.33, 27.78, 9.26, 3.09 and 1.03 μg/mL (experiment without metabolic activation); and 500, 166.67, 55.56, 18.52, 6.17 and 2.06 μg/mL (experiment with metabolic activation).
In Assay 1, insolubility (precipitate) was detected at the end of the treatment period in the final treatment medium in the 500-166.67 μg/mL concentration range with metabolic activation and in the 250-83.33 μg/mL concentration range without metabolic activation furthermore insolubility (minimal amount of precipitate) was detected at the end of the treatment period in the final treatment medium in the 55.56 μg/mL concentration with metabolic activation and in the 27.78 μg/mL concentration without metabolic activation. There were no large changes in the pH and osmolality.
Marked cytotoxicity was also observed in the experiment without metabolic activation at 250, 83.33 and 27.78 μg/mL concentrations (cytotoxicity values of 63%, 69% and 89%, respectively). Concentrations of 166.67, 55.56 and 18.52 μg/mL were chosen for evaluation in the experiment with metabolic activation and concentrations of 83.33, 27.78, 9.26 and 3.09 μg/mL were chosen for evaluation in the experiment without metabolic activation.
Due to the technical reason in the Assay 2 (a decrease in the number of seeded cells was observed before the treatment) an additional experiment was performed for validity of the study using the same experimental conditions as in the Assay 2.
In Chromosome Aberration Assay 3, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 10, 8, 7, 6, 5, 2.5 and 1.25 μg/mL (experiment without metabolic activation); and 500, 166.67, 55.56, 18.52, 6.17 and 2.06 (experiment with metabolic activation).
In Assay 3, similarly to the first experiment, insolubility (precipitate) was detected at the end of the treatment period in the final treatment medium in the 500-166.67 μg/mL concentration range and insolubility (minimal amount of precipitate) in the 55.56 μg/mL concentration with metabolic activation. And no insolubility detected without metabolic activation. There were no large changes in the pH and osmolality. Marked cytotoxicity was observed in the experiment without metabolic activation at 10, 8, 7 and 6 μg/mL concentrations (cytotoxicity values of 96%, 59%, 64% and 65%, respectively). Concentrations of 166.67, 55.56 and 18.52 μg/mL were chosen for evaluation in the experiment with metabolic activation and concentrations of 8, 5, and 2.5 μg/mL were chosen for evaluation in the experiment without metabolic activation.
None of the treatment concentrations caused a biologically or statistically significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation when compared to the appropriate negative (vehicle) control values.
Polyploid metaphases (1-4) were found in some cases in the negative (vehicle) control, positive control or test item treated samples in the performed experiments, but their incidence was not related to treatment with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol. Endoreduplicated metaphases (1-3) were detected in the performed experiments.

VALIDITY OF THE STUDY
The tested concentrations in the chromosome aberration assays were selected based on the results of the preliminary experiments. Insolubility was detected in all experiments with and/or without metabolic activation; while cytotoxicity was detected in any experiment without metabolic activation. The evaluated concentration ranges of Assay 1 and Assay 3 were considered to be adequate, as they covered the range from insolubility to no insolubility and/or from toxicity to no or little toxicity*.
*Note:
Comment 1.: In Assay 1 and Assay 3 with metabolic activation the evaluated concentrations were chosen based on the observed insolubility because the observed maximum cytotoxicity values were 42 % (in Assay 1) and 43 % (in Assay 3) which did not achieve the recent OECD guideline recommended cytotoxicity parameters (50-60% range). Moreover, in the preliminary experiment almost the same cytotoxicity value (41 %) was observed at the maximum recommended concentration.
Comment 2.: In Assay 1 without metabolic activation the evaluated concentrations were chosen based on the observed cytotoxicity and insolubility. Regarding cytotoxicity, the recent OECD guideline requires that the highest evaluated concentration should have cytotoxicity in the 50-60% range. In the performed experiments higher values were detected even using a closely spaced concentration range, however as the expected cytotoxic range was covered in all the cases, this fact was considered as acceptable and did not adversely affect the results of the study. To support the cytotoxicity data, an additional concentration was evaluated in the Assay 1 without metabolic activation to provide additional information for cytotoxicity. The cytotoxicity values were 69 % and 89 % at the two upper evaluated concentration.
Comment 3.: In Assay 3 without metabolic activation the evaluated concentrations were chosen based on the observed cytotoxicity (the cytotoxicity value was 59 % at the maximum evaluated concentration).
At least three test item concentrations were evaluated in each experiment.
Historical control data are presented in Appendix 6. The spontaneous aberration frequencies of the negative (vehicle) controls in the performed experiments were in harmony the general historical control range* of the testing laboratory.
*Note: In the Assay 1 with metabolic activation the mean% aberrant cells of the negative (vehicle) control was higher (7) than the maximum mean% aberrant cells of the general historical control range. This fact had no impact on the results or integrity of the study since this experiment was repeated in the Assay 3 and then the mean% aberrant cells of the negative (vehicle) control was in the historical range.
In the performed experiments, the positive control substances (Cyclophosphamide (CP) in the experiments with metabolic activation and Ethyl methanesulfonate (EMS) in the experiments without metabolic activation) caused the expected statistically significant increase in the number of cells with structural chromosome aberrations demonstrating the sensitivity of the test system in each assay.
The study was considered to be valid.

 Summarized results of the Concentration Selection Cytotoxicity Assay A experiment without metabolic activation

Test group

Dose (μg/mL)

S9-mix

Treatment / sampling time

Cell number (total)

RICC (%)*

Observations

beginning / end of treatment

Untreated control

-

-

3/20

8.70E+06

103

normal / normal (pH: 7.0; osm: 349 mmol/kg)

Negative (vehicle) control

-

-

3/20

8.50E+06

100

normal / normal (pH: 7.0; osm: 346 mmol/kg)

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol

2000

-

3/20

6.38E+06

73

oily film / oily film (pH: 7.2; osm: 345 mmol/kg)

1000

-

3/20

5.55E+06

63

oily film / oily film (pH: 7.2; osm: 345 mmol/kg)

500

-

3/20

5.45E+06

62

oily film / oily film (pH: 7.2; osm: 347 mmol/kg)

250

-

3/20

4.00E+06

44

oily film / oily film (pH: 7.2; osm: 348 mmol/kg)

125

-

3/20

1.85E+06

17

oily film / oily film (pH: 7.2; osm: 348 mmol/kg)

62.5

-

3/20

1.50E+06

17

oily film / oily film (pH: 7.2; osm: 346 mmol/kg)

31.25

-

3/20

3.53E+06

38

oily film / oily film (pH: 7.2; osm: 344 mmol/kg)

15.625

-

3/20

4.38E+06

48

oily film / oily film#(pH: 7.2; osm: 346 mmol/kg)

7.813

-

3/20

6.85E+06

79

oily film#/ oily film (pH: 7.2; osm: 347 mmol/kg)

3.906

-

3/20

6.95E+06

81

normal / normal (pH: 7.2; osm: 347 mmol/kg)

*: compared to the negative (vehicle) control (0.5% Acetone)                                                 #: minimal amount

RICC: Relative Increase in Cell Counts                                                                               osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Summarized results of the Concentration Selection Cytotoxicity Assay A experiment with metabolic activation

Test group

Dose (μg/mL)

S9-mix

Treatment / sampling time

Cell number (total)

RICC (%)*

Observations

beginning / end of treatment

Untreated control

-

+

3/20

6.45E+06

111

normal / normal (pH: 7.0; osm: 346 mmol/kg)

Negative (vehicle) control

-

+

3/20

5.85E+06

100

normal / normal (pH: 7.0; osm: 345 mmol/kg)

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol

2000

+

3/20

3.65E+06

59

oily film / oily film (pH: 7.2; osm: 347 mmol/kg)

1000

+

3/20

3.90E+06

64

oily film / oily film (pH: 7.2; osm: 341 mmol/kg)

500

+

3/20

4.08E+06

67

oily film / oily film (pH: 7.2; osm: 343 mmol/kg)

250

+

3/20

4.83E+06

81

oily film / oily film (pH: 7.2; osm: 341 mmol/kg)

125

+

3/20

3.90E+06

64

oily film / oily film (pH: 7.2; osm: 342 mmol/kg)

62.5

+

3/20

1.55E+06

20

oily film / oily film#(pH: 7.2; osm: 342 mmol/kg)

31.25

+

3/20

4.45E+06

74

oily film#/ normal (pH: 7.2; osm: 343 mmol/kg)

15.625

+

3/20

6.28E+06

108

oily film#/ normal (pH: 7.2; osm: 343 mmol/kg)

7.813

+

3/20

6.35E+06

109

oily film#/ normal (pH: 7.2; osm: 342 mmol/kg)

3.906

+

3/20

7.13E+06

124

normal / normal (pH: 7.2; osm: 343 mmol/kg)

*: compared to the negative (vehicle) control (0.5% Acetone)                                                  #: minimal amount

RICC: Relative Increase in Cell Counts                                                                                 osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Summarized results of the Concentration Selection Cytotoxicity Assay B experiment without metabolic activation

Test group

Dose (μg/mL)

S9-mix

Treatment / sampling time

Cell number (total)

RICC (%)*

Observations

beginning / end of treatment

Untreated control

-

-

20/20

7.30E+06

101

normal / normal (pH: 7.0; osm: 342 mmol/kg)

Negative (vehicle) control

-

-

20/20

7.23E+06

100

normal / normal (pH: 7.0; osm: 342 mmol/kg)

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol

2000

-

20/20

0.00E+00

NA

oily film / oily film (pH: 7.2; osm: 348 mmol/kg)

1000

-

20/20

0.00E+00

NA

oily film / oily film (pH: 7.2; osm: 347 mmol/kg)

500

-

20/20

0.00E+00

NA

oily film / oily film (pH: 7.2; osm: 346 mmol/kg)

250

-

20/20

0.00E+00

NA

oily film / oily film (pH: 7.2; osm: 348 mmol/kg)

125

-

20/20

0.00E+00

NA

oily film / oily film (pH: 7.2; osm: 348 mmol/kg)

62.5

-

20/20

0.00E+00

NA

oily film / oily film#(pH: 7.2; osm: 346 mmol/kg)

31.25

-

20/20

0.00E+00

NA

oily film / oily film#(pH: 7.2; osm: 349 mmol/kg)

15.625

-

20/20

0.00E+00

NA

oily film / normal (pH: 7.2; osm: 348 mmol/kg)

7.813

-

20/20

2.53E+06

30

oily film#/ normal (pH: 7.2; osm: 346 mmol/kg)

3.906

-

20/20

5.90E+06

80

normal / normal (pH: 7.2; osm: 347 mmol/kg)

*: compared to the negative (vehicle) control (0.5% Acetone)                                                  #: minimal amount

RICC: Relative Increase in Cell Counts                                                                              osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Summarized results of Chromosome Aberration Assay 1 experiment without metabolic activation

Test group

Dose (μg/mL)

S9-mix

Treatment / sampling time

Cell number (total)

RICC (%)*

Observations

beginning / end of treatment

Untreated control

-

-

3/20

6.00E+06

91

normal / normal (pH: 7.2; osm: 341 mmol/kg)

Negative (vehicle) control

-

-

3/20

6.38E+06

100

normal / normal (pH: 7.2; osm: 342 mmol/kg)

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol

250

-

3/20

3.78E+06

37

oily film / oily film (pH: 7.2; osm: 346 mmol/kg)

83.33

-

3/20

3.53E+06

31

oily film / oily film (pH: 7.2; osm: 342 mmol/kg)

27.78

-

3/20

2.70E+06

11

oily film / oily film#(pH: 7.2; osm: 348 mmol/kg)

9.26

-

3/20

5.38E+06

76

oily film / normal (pH: 7.2; osm: 361 mmol/kg)

3.09

-

3/20

5.93E+06

89

normal / normal (pH: 7.2; osm: 340 mmol/kg)

1.03

-

3/20

6.10E+06

93

normal / normal (pH: 7.2; osm: 341 mmol/kg)

Positive control (1 μL/mL EMS)

-

-

3/20

5.08E+06

69

normal / normal (pH:7.2; osm: 358 mmol/kg)

*: compared to the negative (vehicle) control (0.5% Acetone)                                                  #: minimal amount

RICC: Relative Increase in Cell Counts                                                                               osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Summarized results of Chromosome Aberration Assay 1 experiment with metabolic activation

Test group

Dose (μg/mL)

S9-mix

Treatment / sampling time

Cell number (total)

RICC (%)*

Observations

beginning / end of treatment

Untreated control

-

+

3/20

5.88E+06

102

normal / normal (pH: 7.2; osm: 349 mmol/kg)

Negative (vehicle) control

-

+

3/20

5.80E+06

100

normal / normal (pH: 7.2; osm: 343 mmol/kg)

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol

500

+

3/20

4.30E+06

58

oily film / oily film (pH: 7.2; osm: 340 mmol/kg)

166.67

+

3/20

4.85E+06

73

oily film / oily film (pH: 7.2; osm: 346 mmol/kg)

55.56

+

3/20

4.93E+06

75

oily film / oily film#(pH: 7.2; osm: 343 mmol/kg)

18.52

+

3/20

5.33E+06

87

oily film / normal (pH: 7.2; osm: 349 mmol/kg)

6.17

+

3/20

5.60E+06

94

normal / normal (pH: 7.2; osm: 337 mmol/kg)

2.06

+

3/20

5.85E+06

101

normal / normal (pH: 7.2; osm: 342 mmol/kg)

Positive control (6 μg/mL CP)

-

+

3/20

4.85E+06

73

normal / normal (pH: 7.2; osm: 343 mmol/kg)

*: compared to the negative (vehicle) control (0.5% Acetone)                                                  #: minimal amount

RICC: Relative Increase in Cell Counts                                                                                osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Summarized results of Chromosome Aberration Assay 3 experiment without metabolic activation

Test group

Dose (μg/mL)

S9-mix

Treatment / sampling time

Cell number (total)

RICC (%)*

Observations

beginning / end of treatment

Untreated control

-

-

20/20

7.43E+06

123

normal / normal (pH: 7.4; osm: 345 mmol/kg)

Negative (vehicle) control

-

-

20/20

6.33E+06

100

normal / normal (pH: 7.4; osm: 341 mmol/kg)

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol

10

-

20/20

1.70E+06

4

oily film / normal (pH: 7.4; osm: 342 mmol/kg)

8

-

20/20

3.48E+06

41

oily film / normal (pH: 7.4; osm: 347 mmol/kg)

7

-

20/20

3.23E+06

36

oily film / normal (pH: 7.4; osm: 350 mmol/kg)

6

-

20/20

3.20E+06

35

oily film#/ normal (pH: 7.4; osm: 349 mmol/kg)

5

-

20/20

4.45E+06

61

normal / normal (pH: 7.4; osm: 343 mmol/kg)

2.5

-

20/20

5.53E+06

83

normal / normal (pH: 7.4; osm: 347 mmol/kg)

1.25

-

20/20

5.80E+06

89

normal / normal (pH: 7.4; osm: 352 mmol/kg)

Positive control (0.4 μL/mL EMS)

-

-

20/20

4.10E+06

54

normal / normal (pH:7.2; osm: 355 mmol/kg)

*: compared to the negative (vehicle) control (0.5% Acetone)                                                  #: minimal amount

RICC: Relative Increase in Cell Counts                                                                              osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Summarized results of Chromosome Aberration Assay 3 experiment with metabolic activation

Test group

Dose (μg/mL)

S9-mix

Treatment / sampling time

Cell number (total)

RICC (%)*

Observations

beginning / end of treatment

Untreated control

-

+

3/20

6.20e+06

127

normal / normal (pH: 7.4; osm: 343 mmol/kg)

Negative (vehicle) control

-

+

3/20

5.20e+06

100

normal / normal (pH: 7.4; osm: 339 mmol/kg)

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol

500

+

3/20

3.60E+06

57

oily film / oily film (pH: 7.4; osm: 341 mmol/kg)

166.67

+

3/20

4.55E+06

82

oily film / oily film (pH: 7.4; osm: 346 mmol/kg)

55.56

+

3/20

5.68E+06

113

oily film / oily film#(pH: 7.4; osm: 350 mmol/kg)

18.52

+

3/20

5.18E+06

99

oily film / normal (pH: 7.4; osm: 346 mmol/kg)

6.17

+

3/20

5.90E+06

119

oily film#/ normal (pH: 7.4; osm: 341 mmol/kg)

2.06

+

3/20

5.63E+06

112

normal / normal (pH: 7.4; osm: 349 mmol/kg)

Positive control (6 μg/mL CP)

-

+

3/20

3.35E+06

50

normal / normal (pH: 7.4; osm: 347 mmol/kg)

*: compared to the negative (vehicle) control (0.5% Acetone)                                                  #: minimal amount

RICC: Relative Increase in Cell Counts                                                                               osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Summary table of Chromosome Aberration Assay 1 without metabolic activation

Concentration (μg/mL)

[Number of analyzed cells]

Time of Treatment / Sampling

RICC#(%)

Precipitate (oily film)##

Mean % aberrant cells###

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol without metabolic activation (-S9)

Untreated control

3h / 20h

91

-

NE

Negative (vehicle) control [300]

3h / 20h

100

-

3.7

250 μg/mL

3h / 20h

37

+

NE

83.33 μg/mL [300]

3h / 20h

31

+

3.7

27.78 μg/mL [300]

3h / 20h

11

+a

7.3

9.26 μg/mL [300]

3h / 20h

76

-

5.3

3.09 μg/mL [300]

3h / 20h

89

-

7.3

1.03 μg/mL

3h / 20h

93

-

NE

Positive control [300]

3h / 20h

69

-

13.7**

Negative (vehicle) control: (0.5% Acetone)

Positive control (-S9): Ethyl methanesulfonate, 1 μL/mL

NE: not evaluated

RICC: Relative Increase in Cell Counts

a: minimal amount of oily film

#: compared to the negative (vehicle) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

**: p<0.01 comparing number of aberrant cells excluding gaps with corresponding negative control.

 

Summary table of Chromosome Aberration Assay 1 with metabolic activation

Concentration (μg/mL)

[Number of analyzed cells]

Time of Treatment / Sampling

RICC#(%)

Precipitate (oily film)##

Mean % aberrant cells###

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol without metabolic activation (+S9)

Untreated control [300]

3h / 20h

102

-

6.0

Negative (vehicle) control [300]

3h / 20h

100

-

7.0

500 μg/mL

3h / 20h

58

+

NE

166.67 μg/mL

3h / 20h

73

+

7.7

55.56 μg/mL [300]

3h / 20h

75

+a

7.3

18.52 μg/mL [300]

3h / 20h

87

-

9.0

6.17 μg/mL [300]

3h / 20h

94

-

NE

2.06 μg/mL

3h / 20h

101

-

NE

Positive control [98]

3h / 20h

73

-

52.0***

Negative (vehicle) control: (0.5% Acetone)

Positive control (+S9): Cyclophosphamide, 6 μg/mL

NE: not evaluated

RICC: Relative Increase in Cell Counts

a: minimal amount of oily film

#: compared to the negative (vehicle) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

***: p<0.001 comparing number of aberrant cells excluding gaps with corresponding negative control.

 

Summary table of Chromosome Aberration Assay 3 without metabolic activation

Concentration (μg/mL)

[Number of analyzed cells]

Time of Treatment / Sampling

RICC#(%)

Precipitate (oily film)##

Mean % aberrant cells###

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol without metabolic activation (-S9)

Untreated control

20h / 20h

123

-

NE

Negative (vehicle) control [300]

20h / 20h

100

-

1.0

10 μg/mL

20h / 20h

4

-

NE

8 μg/mL [300]

20h / 20h

41

-

1.0

7 μg/mL

20h / 20h

36

-

NE

6 μg/Ml

20h / 20h

35

-

NE

5 μg/mL [300]

20h / 20h

61

-

2.3

2.5 μg/mL [300]

20h / 20h

83

-

0.7

1.25 μg/mL

20h / 20h

89

-

NE

Positive control [228]

20h / 20h

54

-

20.6***

Negative (vehicle) control: (0.5% Acetone)

Positive control (-S9): Ethyl methanesulfonate, 0.4 μL/mL

NE: not evaluated

RICC: Relative Increase in Cell Counts

a: minimal amount of oily film

#: compared to the negative (vehicle) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

**: p<0.001 comparing number of aberrant cells excluding gaps with corresponding negative control.

 

Summary table of Chromosome Aberration Assay 3 with metabolic activation

Concentration (μg/mL)

[Number of analyzed cells]

Time of Treatment / Sampling

RICC#(%)

Precipitate (oily film)##

Mean % aberrant cells###

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol without metabolic activation (+S9)

Untreated control

3h / 20h

127

-

NE

Negative (vehicle) control [300]

3h / 20h

100

-

2.7

500 μg/mL

3h / 20h

57

+

NE

166.67 μg/mL [300]

3h / 20h

82

+

3.3

55.56 μg/mL [300]

3h / 20h

113

+a

2.0

18.52 μg/mL [300]

3h / 20h

99

-

3.7

6.17 μg/mL

3h / 20h

119

-

NE

2.06 μg/mL

3h / 20h

112

-

NE

Positive control [68]

3h / 20h

50

-

73.5***

Negative (vehicle) control: (0.5% Acetone)

Positive control (+S9): Cyclophosphamide, 6 μg/mL

NE: not evaluated

RICC: Relative Increase in Cell Counts

a: minimal amount of oily film

#: compared to the negative (vehicle) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

***: p<0.001 comparing number of aberrant cells excluding gaps with corresponding negative control.

 

HISTORICAL CONTROL DATA

3/20h treatment/sampling time without S9-mix

 

Aberration rate

(phases with aberration in %)

Negative control

Positive control

(EMS)

Incl. Gaps

Excl. Gaps

Incl. Gaps

Excl. Gaps

Mean

2.71

1.20

22.67

18.02

SD

1.65

0.82

12.43

8.16

Range

0-7

0-3

4-63

4-40

n

46

46

40

40

3/20h treatment/sampling time with S9-mix

 

Aberration rate

(phases with aberration in %)

Negative control

Positive control

(CP)

Incl. Gaps

Excl. Gaps

Incl. Gaps

Excl. Gaps

Mean

3.24

1.45

73.00

69.50

SD

1.57

0.95

23.51

25.64

Range

0-8

0-4

21-100

21-100

n

43

43

21

21

SD = standard deviation

Range – min.-max. values

n = number of experiments

EMS = Ethyl methanesulfonate

CP = Cyclophosphamide

Notes:

1. In the period 2008-2009, NNDA (N-Nitrosodimethylamine) was used as positive control substance in the experiments with metabolic activation. Mean aberration frequency for NNDA was 22.91 (including gaps) and 18.07 (excluding gaps) in 22 experiments.

2. In studies performed before the update OECD guideline (2014) 200 metaphases were scored for chromosomal aberration per samples. Minimum and maximum values reflect the total number of aberrant cells in 200 metaphases. Furthermore, in those studies counting for a positive control sample halted when 15 aberrant cells were counted.

 

20/28h treatment/sampling time without S9-mix

 

Aberration rate

(phases with aberration in %)

Negative control

Positive control

(EMS)

Incl. Gaps

Excl. Gaps

Incl. Gaps

Excl. Gaps

Mean

2.58

1.13

34.97

30.96

SD

1.81

0.86

10.94

9.80

Range

0-8

0-4

5-76

5-68.2

n

43

43

43

43

3/28h treatment/sampling time with S9-mix

 

Aberration rate

(phases with aberration in %)

Negative control

Positive control

(CP)

Incl. Gaps

Excl. Gaps

Incl. Gaps

Excl. Gaps

Mean

3.07

1.47

52.63

48.80

SD

1.64

1.07

23.98

24.49

Range

0-8

0-5

7-93.8

6-93.8

n

43

43

21

21

SD = standard deviation

Range – min.-max. values

n = number of experiments

EMS = Ethyl methanesulfonate

CP = Cyclophosphamide

Notes:

1. In the period 2008-2009, NNDA (N-Nitrosodimethylamine) was used as positive control substance in the experiments with metabolic activation. Mean aberration frequency for NNDA was 22.34 (including gaps) and 18.36 (excluding gaps) in 22 experiments.

2. In studies performed before the update OECD guideline (2014) 200 metaphases were scored for chromosomal aberration per samples. Minimum and maximum values reflect the total number of aberrant cells in 200 metaphases. Furthermore, in those studies counting for a positive control sample halted when 15 aberrant cells were counted.

3. These historical controls were used for this study due to the lack of the 20h/20h treatment/sampling time without S9-mix historical control.

Conclusions:
The test item 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol was tested for potential clastogenic activity using the Chromosome Aberration Assay. The study included two Concentration Selection Cytotoxicity Assays and two Chromosome Aberration Assays.
The performed experiments were considered to be valid and to reflect the real potential of the test item to cause structural chromosomal aberrations in the cultured V79 Chinese hamster cells used in this study.
Treatment with the test item did not result in a statistically and biologically significant, reproducible, dose-dependent increase in the frequency of the cells with structural chromosome aberrations without gaps either in the presence or 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.
In conclusion, 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. Therefore, 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol was considered as not clastogenic in this test system.
Executive summary:

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol was tested in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells. The test item was formulated in Acetone and it was examined up to cytotoxic concentrations according to the OECD guideline recommendations. In independent Chromosome Aberration Assays using duplicate cultures, at least 300 well-spread metaphase cells (or until a clear positive response was detected) were analysed for each evaluated test item treated, negative (vehicle) and positive control sample.

 

In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 250, 83.33, 27.78, 9.26, 3.09 and 1.03 μg/mL (experiment without metabolic activation); and 500, 166.67, 55.56, 18.52, 6.17 and 2.06 μg/mL (experiment with metabolic activation).

 

In Assay 1, insolubility (precipitate) was detected at the end of the treatment period in the final treatment medium in the 500-166.67 μg/mL concentration range with metabolic activation and in the 250-83.33 μg/mL concentration range without metabolic activation furthermore insolubility (minimal amount of precipitate) was detected at the end of the treatment period in the final treatment medium in the 55.56 μg/mL concentration with metabolic activation and in the 27.78 μg/mL concentration without metabolic activation. There were no large changes in the pH and osmolality.

Marked cytotoxicity was also observed in the experiment without metabolic activation at 250, 83.33 and 27.78 μg/mL concentrations (cytotoxicity values of 63%, 69% and 89%, respectively). Concentrations of 166.67, 55.56 and 18.52 μg/mL were chosen for evaluation in the experiment with metabolic activation and concentrations of 83.33, 27.78, 9.26 and 3.09 μg/mL were chosen for evaluation in the experiment without metabolic activation.

 

Due to a technical reason in the Assay 2 (a decrease in the number of seeded cells was observed before the treatment) an additional experiment was performed for validity of the study using the same experimental conditions as in the Assay 2.

 

In Chromosome Aberration Assay 3, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 10, 8, 7, 6, 5, 2.5 and 1.25 μg/mL (experiment without metabolic activation); and 500, 166.67, 55.56, 18.52, 6.17 and 2.06 (experiment with metabolic activation).

 

In Assay 3, similarly to the first experiment, insolubility (precipitate) was detected at the end of the treatment period in the final treatment medium in the 500-166.67 μg/mL concentration range and insolubility (minimal amount of precipitate) in the 55.56 μg/mL concentration with metabolic activation. No insolubility was detected without metabolic activation. There were no large changes in the pH and osmolality. Marked cytotoxicity was observed in the experiment without metabolic activation at 10, 8, 7 and 6 μg/mL concentrations (cytotoxicity values of 96%, 59%, 64% and 65%, respectively). Concentrations of 166.67, 55.56 and 18.52 μg/mL were chosen for evaluation in the experiment with metabolic activation and concentrations of 8, 5, and 2.5 μg/mL were chosen for evaluation in the experiment without metabolic activation.

 

None of the treatment concentrations caused a biologically or statistically significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation when compared to the appropriate negative (vehicle) control values.

 

Polyploid metaphases (1-4) were found in some cases in the negative (vehicle) control, positive control or test item treated samples in the performed experiments, but their incidence was not related to treatment with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol. Endoreduplicated metaphases (1-3) were detected in the performed experiment in some test item treated samples.

 

The negative (vehicle) control data were within the acceptable range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system. The evaluated concentration range was considered to be adequate; at least three test item treated concentrations were evaluated in each assay. The tests were considered to be valid.

 

In conclusion, 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol] did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. Therefore, 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol was considered as not clastogenic in this test system.

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

Additional information

In Vitro Genetic Toxicity - 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.

 

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.

In Vitro Genetic Toxicity - Mammalian Cell Gene Mutation Assay

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol test item to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix).

 

The test item was examined up to 2000 μg/mL (the recommended maximum concentration) in the Preliminary Toxicity Test. Based on the results of the preliminary experiment (the test item had a special solubility and cytotoxicity profile), the following test item concentrations were examined in the mutation assays:

Assay 1, 3-hour treatment with metabolic activation: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625 and 7.813 μg/mL,

Assay 1, 3-hour treatment without metabolic activation: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625 and 7.813 μg/mL,

Assay 2, 3-hour treatment with metabolic activation: 2000, 1000, 500, 250, 125, 100, 80, 62.5, 50, 40, 31.25, 15.625 and 7.813 μg/mL,

Assay 2, 24-hour treatment without metabolic activation: 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5 and 2.5 μg/mL.

 

Insolubility / minimal amount of insolubility was observed in the final treatment medium at the end of the treatment in both assays (in the 2000-31.25 μg/mL concentration range in Assay 1 with metabolic activation; in the 2000-125 μg/mL concentration range in Assay 1 without metabolic activation, in the 2000-125 μg/mL concentration range Assay 2 with metabolic activation and in the 70-45 μg/mL concentration range in Assay 2 without metabolic activation).

 

In Assay 1, following a 3-hour treatment with metabolic activation, cytotoxicity of the test item was observed. No cells survived the expression period in the samples of 250 μg/mL concentration, furthermore the total number of surviving cells was much less than requested for plating in one replicate of 125 μg/mL concentration. Thus, an evaluation was made using data of the next concentration of 62.5 μg/mL (relative total growth of 18%) and three lower concentrations (a total of four concentrations).

Statistically significant increase was noted at 62.5 μg/mL, however the observed values was biologically not relevant (the difference compared to the negative (vehicle) control value was not larger than the global evaluation factor). Therefore, the slight dose response relationship was considered as not relevant. In overall, this experiment was considered as being negative.

In Assay 1, following a 3-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed in the 500-125 μg/mL concentration range, the total cell number of one or two replicates was lower than required for plating.

An evaluation was made using data of the next concentration 62.5 μg/mL (relative total growth of <2%) and three lower concentrations (a total of four concentrations).

Statistically significant and biologically relevant increase in the mutation frequency was observed 62.5 and 31.25 μg/mL concentrations, although the levels of cytotoxicity at both concentrations were lower than 10%. A significant dose-response to the treatment was also indicated by the linear trend analysis. Based on the fact that the substantial increases were seen only at high level of cytotoxicity (additional concentrations of 2000 and 1000 μg/mL - above the level of optimal solubility, but with acceptable level of cytotoxicity, 6% and 12%, respectively – did not show biologically relevant increases in the mutation frequency). This experiment gave apparently positive results, but the degree of cytotoxicity in these concentrations was out of the acceptability range, in all cases of acceptable cytotoxicity there was no relevant increase in the mutation frequency. Hence the result of this experiment was considered as being negative.

 

In Assay 2, following a 3-hour treatment with metabolic activation, excessive cytotoxicity of the test item was observed in the 2000-250 μg/mL concentration range, cells of one or two replicates of these samples did not survive the treatment or expression period. At the following concentration of 125 μg/mL, the total number of surviving cells was less than minimal, thus this sample was not used for viability / mutagenicity plating. An evaluation was made using data of the highest concentration of 100 μg/mL (relative total growth of 10%) and seven lower concentrations (a total of eight concentrations). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. This experiment showed clear negative result.

In Assay 2, following a 24-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed in the 70-30 μg/mL concentration range, cells of these samples did not survive the treatment or expression period. The following concentration of 25 μg/mL still showed marked cytotoxicity (relative total growth: <1%) and it was excluded from the evaluated concentration range. Thus, an evaluation was made using data of the next concentration of 20 μg/mL (relative total growth of 14%) and four lower concentrations (a total of five concentrations). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. This experiment was clearly negative.

 

In conclusion, no clear mutagenic effect of 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol was observed in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

In Vitro Genetic Toxicity - Chromosome Aberration Assay

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol was tested in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells. The test item was formulated in Acetone and it was examined up to cytotoxic concentrations according to the OECD guideline recommendations.

 

In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 250, 83.33, 27.78, 9.26, 3.09 and 1.03 μg/mL (experiment without metabolic activation); and 500, 166.67, 55.56, 18.52, 6.17 and 2.06 μg/mL (experiment with metabolic activation).

In Assay 1, insolubility (precipitate) was detected at the end of the treatment period in the final treatment medium in the 500-166.67 μg/mL concentration range with metabolic activation and in the 250-83.33 μg/mL concentration range without metabolic activation furthermore insolubility (minimal amount of precipitate) was detected at the end of the treatment period in the final treatment medium in the 55.56 μg/mL concentration with metabolic activation and in the 27.78 μg/mL concentration without metabolic activation. There were no large changes in the pH and osmolality.

Marked cytotoxicity was also observed in the experiment without metabolic activation at 250, 83.33 and 27.78 μg/mL concentrations (cytotoxicity values of 63%, 69% and 89%, respectively). Concentrations of 166.67, 55.56 and 18.52 μg/mL were chosen for evaluation in the experiment with metabolic activation and concentrations of 83.33, 27.78, 9.26 and 3.09 μg/mL were chosen for evaluation in the experiment without metabolic activation.

 

Due to a technical reason in the Assay 2 (a decrease in the number of seeded cells was observed before the treatment) an additional experiment was performed for validity of the study using the same experimental conditions as in the Assay 2.

 

In Chromosome Aberration Assay 3, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 10, 8, 7, 6, 5, 2.5 and 1.25 μg/mL (experiment without metabolic activation); and 500, 166.67, 55.56, 18.52, 6.17 and 2.06 (experiment with metabolic activation).

In Assay 3, similarly to the first experiment, insolubility (precipitate) was detected at the end of the treatment period in the final treatment medium in the 500-166.67 μg/mL concentration range and insolubility (minimal amount of precipitate) in the 55.56 μg/mL concentration with metabolic activation. No insolubility was detected without metabolic activation. There were no large changes in the pH and osmolality. Marked cytotoxicity was observed in the experiment without metabolic activation at 10, 8, 7 and 6 μg/mL concentrations (cytotoxicity values of 96%, 59%, 64% and 65%, respectively). Concentrations of 166.67, 55.56 and 18.52 μg/mL were chosen for evaluation in the experiment with metabolic activation and concentrations of 8, 5, and 2.5 μg/mL were chosen for evaluation in the experiment without metabolic activation.

 

None of the treatment concentrations caused a biologically or statistically significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation when compared to the appropriate negative (vehicle) control values.

 

In conclusion, 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol] did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. Therefore, 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol was considered as not clastogenic in this test system.

Justification for classification or non-classification

In Vitro Genetic Toxicity - Bacterial Reverse Mutation Assay

The test item 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol has no mutagenic activity on the growth of the bacterial strains and therefore does not fulfill the requirements for classification.

In Vitro Genetic Toxicity - Mammalian Cell Gene Mutation Assay (mouse lymphoma)

No clear mutagenic effect of 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol was observed in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay and therefore does not fulfill the requirements for classification.

In Vitro Genetic Toxicity - Chromosome Aberration Assay

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol was considered as not clastogenic in the test system and therefore does not fulfill the requirements for classification.