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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
14 May 2012 to 24 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP. Tested on a close structural analogue. Justification for read across is detailed below.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
6,6'-di-tert-butyl-4,4'-butylidenedi-m-cresol
EC Number:
201-618-5
EC Name:
6,6'-di-tert-butyl-4,4'-butylidenedi-m-cresol
Cas Number:
85-60-9
IUPAC Name:
4,4'-butane-1,1-diylbis(2-tert-butyl-5-methylphenol)
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identification: 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol
Molecular formula: C26H38O2
Molecular weight: 382.58
CAS Number: 85-60-9
Description: White powder
Purity: 99.4%
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 31 May 2013
Stability in vehicle: Ethanol: Unknown
Solubility in vehicle: Ethanol: Not indicated

Method

Target gene:
thymidine-kinase locus (TK-locus).
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Test System: L5178Y/TK+/--3.7.2C mouse lymphoma cells.
Rationale: Recommended test system in international guidelines (e.g. OECD, EC) and literature.
Source: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
Stock cultures of the cells were stored in liquid nitrogen (-196°C). The cultures were checked for mycoplasma contamination. Cell density was preferably kept below 1 x 106 cells/ml.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat liver microsomal enzymes (S9-mix)
Test concentrations with justification for top dose:
Based on the results of the dose range finding test, the following dose range was selected for the first mutagenicity test:
Without S9-mix: 0.03, 0.1, 0.3, 1, 3, 10, 15, 20, 25, 30 and 35 μg/ml exposure medium.
With 8% (v/v) S9-mix: 0.3, 1, 3, 10, 20, 30, 50, 65, 85, 100 and 125 μg/ml exposure medium.
The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 0.03, 0.1, 0.3, 1, 3, 10 and 15 μg/ml exposure medium.
With 8% (v/v) S9-mix: 0.3, 1, 3, 10, 20, 30 and 50 μg/ml exposure medium.

Based on the results of the dose range finding test and experiment 1, the following dose levels were selected for mutagenicity testing.
Without S9-mix: 0.03, 0.1, 0.3, 1, 3, 6.6, 10, 12.5, 15, 17.5, 20 and 22.5 μg/ml exposure medium.
With 12% (v/v) S9-mix: 1, 3, 10, 20, 30, 50, 60, 65, 70 and 75 μg/ml exposure medium.
The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 0.3, 1, 3, 6.6, 10, 12.5, 15 and 17.5 μg/ml exposure medium.
With S9-mix: 1, 3, 10, 30, 50, 60, 65 and 70 μg/ml exposure medium.
Vehicle / solvent:
The test substance was dissolved in ethanol (Merck, Darmstadt, Germany). 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol concentrations were used within 1 hour after preparation. The final concentration of the solvent in the exposure medium was 0.4% (v/v).
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
Negative control: The solvent for the test article, i.e. ethanol.
Positive controls
Without metabolic activation (-S9-mix): Methyl methane sulfonate (MMS); CAS no. 66-27-3 (purity 98%, Sigma, Zwijndrecht, The Netherlands). MMS was used as a direct acting mutagen at a concentration of 15 and 5 μg/ml for a 3 and 24 hours treatment period, respectively. MMS was dissolved in dimethyl sulfoxide (Merck Darmstadt, Germany). The stock solutions of MMS were prepared immediately before use.
With metabolic activation (+S9-mix): Cyclophosphamide (CP); CAS no. 50-18-0 (purity 100%, Endoxan, Asta-Werke, Germany). CP was used as an indirect acting mutagen, requiring metabolic activation, at a final concentration of 7.5 and 10 μg/ml in the first and second experiment, respectively. CP was dissolved in Hanks’ balanced salt solution (HBSS) (Invitrogen Corporation, Breda, The Netherlands) without calcium and magnesium.
The stock solutions of CP were stored in aliquots at ≤-15C in the dark and one sample was thawed immediately before use.

Cell culture
Horse serum: Horse serum (Invitrogen Corporation) was inactivated by incubation at 56°C for at least 30 minutes.
Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) (Invitrogen Corporation) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively) (Invitrogen), 1 mM sodium pyruvate (Sigma) and 2 mM L-glutamin (Invitrogen Corporation).
Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium
For 3 hour exposure: Cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
For 24 hour exposure: Cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 μg/ml trifluorothymidine (TFT) (Sigma).
Non-selective medium: Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).
Environmental conditions: All incubations were carried out in a controlled environment in the dark, in which optimal conditions were a humid atmosphere of 80 – 100% (actual range 36 – 97%), containing 5.0 ± 0.5% CO2 in air, at a temperature of 37.0 ± 1.0°C (actual range 35.7 – 37.6°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature (in the range of 35.7 - 36.0°C), humidity (with a maximum of 25%) and CO2 percentage (with a maximum of 1%) occurred that were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 4 hours. Based on laboratory historical data these deviations are considered not to affect the study integrity. The temporary deviations from the humidity during the subculturing period are explained in protocol deviation 1.

Metabolic activation system
Preparation of S9-fraction: In the dose range finding test and the first mutation experiment, rat liver microsomal enzymes were routinely prepared in the house and adult male Wistar rats were used, which were obtained from Charles River, Sulzfeld, Germany.
The animals were housed at WIL Research Europe in a special room under standard laboratory conditions, as described in the Standard Operating Procedures. The rats were orally dosed at three consecutive days with a suspension of phenobarbital (Bufa B.V., IJsselstein, The Netherlands) (80
mg/kg body weight) and ß-naphthoflavone (Sigma Aldrich Chemie) (100 mg/kg body weight) in corn oil (Roth, Karlsruhe, Germany) (they were denied access to food for 3 to 4 hours preceding each dosing). One day after the final exposure (24 h), the rats were sedated using oxygen/carbon dioxide and then killed by decapitation. The rats received a limited quantity of food during the night before sacrifice. The livers of the rats were removed aseptically, and washed in cold (0°C) sterile 0.1 M sodium phosphate buffer (pH 7.4, Merck) containing 0.1 mM Na2-EDTA (Merck). Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9-fraction) was transferred into sterile ampules, which were stored in liquid nitrogen (-196°C) for a maximum of 1 year.
In the second mutation experiment, rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

Preparation of S9-mix: S9-mix was prepared immediately before use and kept on ice. S9-mix contained per ml: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 4 μmol HEPES (Invitrogen).The above solution was filter (0.22 μm)-sterilized. To 0.5 ml S9-mix components 0.5 ml S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 0.3 ml S9-mix components 0.7 ml S9-fraction was added (70% (v/v) S9-fraction) to complete the S9-mix in the second experiment.
Metabolic activation was achieved by adding 1.3 ml S9-mix to a total of 8 ml, the concentration of the S9-fraction in the exposure medium was 8% (v/v) in the dose range finding test and the first experiment and 12% (v/v) in the second experiment. The S9-batches used were 12-4, 12-5, 2836, 2930 and 2879.

Study design
Cleansing: Prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10 medium containing 10-4 M hypoxanthine (Sigma), 2 x 10-7 M aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 1.6 x 10-5 M thymidine (Merck) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 medium containing hypoxanthine and thymidine only. After this period cells were returned to R10 medium for at least 1 day before starting the experiment.

Dose range finding test: In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating 8 x 106 cells (106 cells/ml for 3 hours treatment) or 5 x 106 cells (1.25 x 105 cells/ml for 24 hours treatment) with a number of test substance concentrations increasing with approximately half log steps. The cell cultures for the 3 hours treatment were placed in sterile 30 ml centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 spm. The cell cultures for the 24 hours treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. 6,6’-di-tert-butyl-4,4’- butylidenedi-m-cresol was tested in the absence and presence of 8% (v/v) S9-fraction.
Since 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was poorly soluble in culture medium, the highest tested concentration was 333 μg/ml exposure medium.
Cell cultures were exposed to 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol in exposure medium for 3 hours in the presence of S9-mix and for 3 and 24 hours in the absence of S9-mix. After exposure, the cells were separated from treatment solutions by 2 centrifugation steps (216 g, 8 min) each followed by removal of the supernatant. The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the final centrifugation step the cells were resuspended in R10 medium. The cells in the final suspension were counted with the coulter particle counter.
For determination of the cytotoxicity, the surviving cells of the 3 hours treatment were subcultured twice. After 24 hours of subculturing, the cells were counted and subcultured again for another 24 hours, after that the cells were counted. The surviving cells of the 24 hours treatment were subcultured once. After 24 hours of subculturing, the cells were counted. If less than 1.25 x 105 cells/ml were counted no subculture was performed.
The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose range for the mutagenicity tests.

Mutagenicity test: 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was tested both in the absence and presence of S9-mix in two independent experiments. Per culture 8 x 106 cells (106 cells/ml for 3 hours treatment) or 5 x 106 cells (1.25 x 105 cells/ml for 24 hours treatment) were used. The cell cultures for the 3 hours treatment were placed in sterile 30 ml centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 spm. The cell cultures for the 24 hours treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. Solvent and positive controls were included and the solvent control was tested in duplicate.
In the first experiment, cell cultures were exposed for 3 hours to 6,6’-di-tert-butyl-4,4’-butylidenedi-mcresol in exposure medium in the absence and presence of S9-mix. In the second experiment, cell cultures were exposed to 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol in exposure medium for 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix.
After exposure, the cells were separated from treatment solutions by 2 centrifugation steps (216 g, 8 min) each followed by removal of the supernatant. The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the final centrifugation step the cells were resuspended in R10 medium. The cells in the final suspension were counted with the coulter particle counter.

Expression period: For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 106 cells (if possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test substance the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF).

Determination of the mutation frequency: Seven or eight doses of the test substance were selected for the mutation assay in the first and second experiment, respectively (see protocol deviation 2).
For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. 1 cell was added per well (2 x 96-well microtiter plates/concentration) in non selective medium. At the mid dose of 1 μg/ml in the first experiment (presence of S9-mix) a total number of 184 wells was used for determination of the CEday2 (see protocol deviation 3).
For determination of the MF a total number of 9.6 x 105 cells/concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 105 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After that, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.
Evaluation criteria:
A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.
Statistics:
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Solubility: 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol precipitated in the exposure medium at concentrations of 100 μg/ml and above. 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range finding test was 333 μg/ml.

Dose range finding test: In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 3 to 333 μg/ml in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period.
In the absence of S9-mix, the relative suspension growth was 12% at the test substance concentration of 33 μg/ml compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at test substance concentrations of 100 μg/ml and above.
In the presence of S9-mix, the relative suspension growth was 10% at the test substance concentration of 333 μg/ml compared to the relative suspension growth of the solvent control.
In the absence of S9-mix, the relative suspension growth was 63% at the test substance concentration of 10 μg/ml compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at test substance concentrations of 33 μg/ml and above.

Mutation experiment: Further investigation showed that at a concentration of 35 μg/ml 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol already precipitated in the exposure medium. 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was tested beyond the limit of the solubility to obtain adequate mutagenicity data.

First mutagenicity test: Based on the results of the dose range finding test, the following dose range was selected for the first mutagenicity test:
Without S9-mix: 0.03, 0.1, 0.3, 1, 3, 10, 15, 20, 25, 30 and 35 μg/ml exposure medium.
With 8% (v/v) S9-mix: 0.3, 1, 3, 10, 20, 30, 50, 65, 85, 100 and 125 μg/ml exposure medium.

Evaluation of toxicity: In the absence of S9-mix, the dose levels of 20 to 35 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. In the presence of S9-mix, the dose levels of 65 to 125 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 0.03, 0.1, 0.3, 1, 3, 10 and 15 μg/ml exposure medium.
With 8% (v/v) S9-mix: 0.3, 1, 3, 10, 20, 30 and 50 μg/ml exposure medium.
In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 74% compared to the total growth of the solvent controls (see protocol deviation 2).
In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 46% compared to the total growth of the solvent controls (see protocol deviation 2).

Evaluation of the mutagenicity: No significant increase in the mutation frequency at the TK locus was observed after treatment with 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Second mutagenicity test: To obtain more information about the possible mutagenicity of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol, a second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period and in the presence of 12% (v/v) S9-mix with a 3 hour treatment period. Based on the results of the dose range finding test and experiment 1, the following dose levels were selected for mutagenicity testing.
Without S9-mix: 0.03, 0.1, 0.3, 1, 3, 6.6, 10, 12.5, 15, 17.5, 20 and 22.5 μg/ml exposure medium.
With 12% (v/v) S9-mix: 1, 3, 10, 20, 30, 50, 60, 65, 70 and 75 μg/ml exposure medium.

Evaluation of toxicity: In the absence of S9-mix, the dose levels of 0.03 to 10 μg/ml showed no cytotoxicity. Therefore, the dose levels of 0.03 and 0.1 μg/ml were not regarded relevant for mutation frequency measurement. The dose levels of 20 to 22.5 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
In the presence of S9-mix, the dose levels of 1 to 20 μg/ml showed no cytotoxicity. Therefore, the dose level of 20 μg/ml was not regarded relevant for mutation frequency measurement. The dose level of 75 μg/ml was not used for mutation frequency measurement, since this dose level was too toxic for further testing.

The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 0.3, 1, 3, 6.6, 10, 12.5, 15 and 17.5 μg/ml exposure medium.
With S9-mix: 1, 3, 10, 30, 50, 60, 65 and 70 μg/ml exposure medium.
In the absence of S9-mix, the relative total growth of the highest test substance was reduced by 94% compared to the total growth of the solvent controls (see protocol deviation 4).
In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 83% compared to the total growth of the solvent controls.

Evaluation of mutagenicity: No significant increase in the mutation frequency at the TK locus was observed after treatment with 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol treated cultures were comparable to the numbers of small and large colonies of the solvent controls.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Experiment 1; Cytotoxic and mutagenic response of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol in the mouse lymphoma L5178Y test system

Dose

RSG

CE day2

RS day2

RTG

Mutation frequency

per 106survivors

(μg/ml)

(%)

(%)

(%)

(%)

Total

( small

Large )

Without metabolic activation

3 hours treatment

SC1

 

120

 

 

64

( 24

37 )

 

100

 

100

100

 

 

 

SC2

 

89

 

 

85

( 26

55 )

0.03

91

105

101

92

69

( 24

42 )

0.1

103

97

93

96

79

( 18

59 )

0.3

92

97

93

85

70

( 33

35 )

1

97

99

95

93

76

( 36

37 )

3

106

101

97

102

69

( 22

44 )

10

91

120

115

105

64

( 21

40 )

15

29

93

89

26

66

( 25

38 )

MMS

59

53

51

30

887

( 332

453 )

With 8% (v/v) metabolic activation

3 hours treatment

SC1

 

77

 

 

107

( 25

79 )

 

100

 

100

100

 

 

 

SC2

 

84

 

 

90

( 11

77 )

0.3

93

113

141

131

87

( 26

58 )

1

95

94

117

111

96

( 38

54 )

3

92

91

114

105

116

( 30

81 )

10

75

133

165

123

83

( 19

61 )

20

88

99

124

109

85

( 28

54 )

30

85

102

127

108

90

( 28

58 )

50(1)

40

110

137

54

97

( 31

61 )

CP

61

52

65

39

1150

( 443

539 )

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent Control = Ethanol; MMS = Methylmethanesulfonate; CP = Cyclophosphamide

(1)= 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol precipitated in the exposure medium

 

Table 2: Experiment 2; Cytotoxic and mutagenic response of 6,6’-di-tert-butyl-4,4’-butylidenedi-mcresol in the mouse lymphoma L5178Y test system

Dose

RSG

CE day2

RS day2

RTG

Mutation frequency per 106survivors

(μg/ml)

(%)

(%)

(%)

(%)

Total

( small

Large )

Without metabolic activation

24 hours treatment

SC1

 

95

 

 

73

( 27

43 )

 

100

 

100

100

 

 

 

SC2

 

66

 

 

64

( 14

49 )

0.3

103

88

108

111

34

( 7

27 )

1

106

63

78

83

55

( 17

37 )

3

99

74

91

91

76

( 29

45 )

6.6

93

89

110

102

35

( 11

24 )

10

84

83

102

86

34

( 5

28 )

12.5

73

84

104

75

57

( 21

35 )

15

73

80

99

73

47

( 14

32 )

17.5

7

62

77

6

61

( 17

43 )

MMS

125

52

65

80

751

( 343

335 )

With 12% (v/v) metabolic activation

3 hours treatment

SC1

 

97

 

 

200

( 97

84 )

 

100

 

100

100

 

 

 

SC2

 

101

 

 

138

( 71

58 )

1

100

94

95

95

131

( 68

55 )

3

106

105

107

113

124

( 70

46 )

10

108

94

95

103

130

( 66

56 )

30

73

110

111

81

123

( 51

64 )

50(1)

47

123

125

59

100

( 32

63 )

60(1)

44

90

91

40

108

( 40

64 )

65(1)

26

102

104

27

126

( 55

63 )

70(1)

16

104

105

17

128

( 52

68 )

CP

61

61

62

38

1650

( 649

601 )

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent Control = ethanol; MMS = Methylmethanesulfonate; CP = Cyclophosphamide

(1) = 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol precipitated in the exposure medium

Applicant's summary and conclusion

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

The read across substance, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol is not mutagenic in the TK mutation test system under the experimental conditions described in this report.
Executive summary:

This study is read across to the structural analogue, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol.

Evaluation of the mutagenic activity of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (with independent repeat).

 

This report describes the effects of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in two independent experiments in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

 

The study procedures described in this report were based on the most recent OECD and EC guidelines. The study was performed in accordance with the Principles of Good Laboratory Practice (GLP).

 

Batch W4B9F0001 of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was a white powder with a purity of 99.4%. The test substance was dissolved in ethanol.

 

In the first experiment, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was tested up to concentrations of 15 and 50 μg/ml in the absence and presence of 8% (v/v) S9-mix, respectively. The incubation time was 3 hours. 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was tested up to cytotoxic levels of 74 and 46% in the absence and presence of S9-mix, respectively. 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol precipitated in the culture medium at dose levels of 35 μg/ml and above.

 

In the second experiment, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was tested up to concentrations of 17.5 and 70 μg/ml in the absence and presence of 12% (v/v) S9-mix, respectively. The incubation times were 24 hours in the absence of S9-mix and 3 hours for incubations in the presence of S9-mix. The test substance tested up to cytotoxic levels of 94 and 83% in the absence and presence of S9-mix, respectively. 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol precipitated in the culture medium at dose levels of 50 μg/ml and above.

 

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Except the response of one of the solvent control cultures in the second experiment in the absence of S9-mix. However since this response was just above the upper limit of the range and clear negative results were obtained, the validity of the test was considered to be not affected.

 

Mutation frequencies in cultures treated with positive control chemicals were increased by 12- and 11-fold for MMS in the absence of S9-mix, and by 12- and 10-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

 

In the absence of S9-mix, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

 

In the presence of S9-mix, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.

 

It is concluded that 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.