Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Key information derived using EU Method B.17 and OECD guideline number 476. Negative with and without metabolic activation.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 July 2012 to 22 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with EU & OECD test guidelines in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
Detailed under Any other information
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
Detailed under Any other information
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase (TK)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
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 homogenate)
Test concentrations with justification for top dose:
Dose range finding test: 1, 3, 10, 33, 100 μg/ml
Experiment 1 & 2: 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 33 μg/ml
Vehicle / solvent:
dimethyl sulfoxide DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
Test substance preparation: No correction was made for the purity/composition of the test substance.
The test substance was dissolved in dimethyl sulfoxide (DMSO, SeccoSolv, Merck Darmstadt, Germany). 3,3’,3”,5,5’,5”-hexa-tert-butyl-α,α’,α”-(mesitylene-2,4,6-triyl) tri-p-cresol concentrations were used within 1 hour after preparation.
The final concentration of the solvent in the exposure medium was 0.8% (v/v).

Negative control: The solvent for the test article, i.e. dimethyl sulfoxide.
Positive controls
Without metabolic activation (-S9-mix): Methyl methanesulfonate (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. 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 10 μg/ml. 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 48 – 98%), containing 5.0 ± 0.5% CO2 in air, at a temperature of 37.0 ± 1.0°C (actual range 34.1 – 38.0°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 34.1 - 36.0°C), humidity (with a maximum of 30%) and CO2 percentage (with a maximum of 1%) that occurred were caused by opening and closing of the incubator door, the duration 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 deviation from the humidity in the dose range finding test is explained in protocol deviation 1.

Metabolic activation system: 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 of the above listed 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 in the second experiment 0.7 ml of the S9-fraction was added (70% (v/v) S9-fraction) to 0.3 ml of the S9-mix components to complete the S9-mix.
Appropriate metabolic activation conditions were achieved by adding 1.3 ml S9-mix to a total of 8 ml of the exposure medium. 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.

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 25 hours treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C (see protocol deviation 2). The test substance was tested in the absence and presence of 8% (v/v) S9-fraction.
Since 3,3’,3”,5,5’,5”-hexa-tert-butyl-α,α’,α”-(mesitylene-2,4,6-triyl) tri-p-cresol was poorly soluble in the exposure medium, the highest tested concentration was 100 μg/ml exposure medium.
Cell cultures were exposed to the test substance 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 which 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: 3,3’,3”,5,5’,5”-hexa-tert-butyl-α,α’,α”-(mesitylene-2,4,6-triyl) tri-p-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 the test substance in exposure medium in the absence and presence of S9-mix. In the second experiment, cell cultures were exposed to the test substance 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 (where 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: Eight doses of the test substance were selected for the mutation assay, both in the absence and presence of S9-mix. Except in the first experiment in which seven dose levels were tested in the absence of S9-mix.
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.
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 the incubation period, 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.

Determination of the mutant colonies: The colonies were divided into small and large colonies. Mutant cells that have suffered extensive genetic damage have prolonged doubling times and thus form small colonies. Less severe affected mutant cells have grown at rates similar to the parental cells and form large colonies. The small colonies can be associated with the induction of chromosomal mutations. The large colonies appeared to result from mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene.
The small colonies are morphological dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphological less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.
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:
None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
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 (ref. 12).
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Solubility: 3,3’,3”,5,5’,5”-hexa-tert-butyl-α,α’,α”-(mesitylene-2,4,6-triyl) tri-p-cresol precipitated in the exposure medium at concentrations of 33 μg/ml and above. The test substance 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 100 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 1 to 100 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.
Both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 100 μg/ml compared to the suspension growth of the solvent control.
In the absence of S9-mix, no toxicity in the relative suspension growth was observed up to highest tested concentrations of 100 μg/ml compared to the solvent control.

Mutation experiment
First mutagenicity test: No toxicity was observed up to and including the precipitating dose level of 100 μg/ml in the dose range finding test. Therefore, the highest concentration to be tested (33 μg/ml) was determined by the solubility in the culture medium. The following dose range was selected for the first mutagenicity test in the absence and presence of 8% (v/v) S9-mix: 0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 33 μg/ml exposure medium.

Evaluation of toxicity: No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix.

Evaluation of the mutagenicity: No significant increase in the mutation frequency at the TK locus was observed after treatment with 3,3’,3”,5,5’,5”-hexa-tert-butyl-α,α’,α”-(mesitylene-2,4,6-triyl) tri-p-cresol either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the 3,3’,3”,5,5’,5”-hexa-tert-butyl-α,α’,α”-(mesitylene-2,4,6-triyl) tri-p-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 3,3’,3”,5,5’,5”-hexa-tert-butyl-α,α’,α”-(mesitylene-2,4,6-triyl) tri-p-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: 0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 331) µg/ml exposure medium.
With 12% (v/v) S9-mix: 0.03, 0.1, 0.3, 1, 3, 10, 33 and 661) μg/ml exposure medium.
1) 3,3’,3”,5,5’,5”-hexa-tert-butyl-α,α’,α”-(mesitylene-2,4,6-triyl) tri-p-cresol precipitated in the exposure medium.

Evaluation of toxicity: No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix.

Evaluation of the mutagenicity: No significant increase in the mutation frequency at the TK locus was observed after treatment with 3,3’,3”,5,5’,5”-hexa-tert-butyl-α,α’,α”-(mesitylene-2,4,6-triyl) tri-p-cresol either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the 3,3’,3”,5,5’,5”-hexa-tert-butyl-α,α’,α”-(mesitylene-2,4,6-triyl) tri-p-cresol treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Chemical analysis of dose preparations: The concentrations analysed in the high formulations was in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
At the low concentration level, the peak of DMSO (i.e. the vehicle of the formulation), interfered with the HPLC determination so that accurate quantitative results could not be obtained. Since the low concentration formulation was prepared by dilution of the high concentration formulation and since the recovery of the low formulation was in agreement with the recovery of the procedural recovery samples, it was concluded that this formulation was also prepared accurately.
No test substance was detected in the vehicle.
Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 4 hours.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Dose range finding test: Cytotoxicity of 3,3’,3’’,5,5’,5’’-hexa-tert-butyl-α,α’,α’’-(mesitylene-2,4,6-triyl)tri-p-cresol (3 hours treatment

Dose

 

 

(μg/ml)

Cell count after 3 hours of treatment (cells/ml x 105)

Cell count after 24 hours of subculture (cells/ml x 105)

Cell count after 48 hours of subculture (cells/ml x 105)

SG(1)

 

 

(x105cells/ml)

RSG(2)

 

 

(%)

Without metabolic activation

SC

10.0

4.9

7.0

220

100

1

10.0

4.3

6.8

188

85

3

9.1

5.3

6.8

211

96

10

10.9

4.8

6.8

228

104

33(3)

10.3

4.7

7.3

227

103

100(3)

9.5

4.9

6.6

197

90

With metabolic activation

SC

11.5

4.7

7.1

246

100

1

10.0

5.1

7.3

238

97

3

10.5

5.4

7.7

281

114

10

10.7

5.2

7.0

250

101

33(3)

10.4

5.2

6.4

222

90

100(3)

9.9

4.9

8.0

248

101

Note: all calculations are made without rounding off

SC = solvent control = dimethyl sulfoxide

(1) = suspension growth

(2) = relative suspension growth

(3) = 3,3’,3’’,5,5’,5’’-hexa-tert-butyl-α,α’,α’’-(mesitylene-2,4,6-triyl)tri-p-cresol precipitated in the exposure medium

 

                                                                        Cell count after                        Cell count after

                                                                         24h subculture                         48h subculture

SG = Suspension growth = Cell count after x --------------------   x             ---------------------------

                                           3h treatment          Cell subcultured(at t=3h)      Cells subcultured(at t=24h)

                                                                              (1.25 x 105c/ml)                     (1.25 x 105c/ml)

 

RSG = [SG(test)/SG(control)] x 100

 

Table 2: Dose range finding test: Cytotoxicity of 3,3’,3’’, 5,5’,5’’-hexa-tert-butyl-α,α’,α’’-(mesitylene-2,4,6-triyl)tri-p-cresol (24 hours treatment)

Dose

 

 

 (μg/ml)

Cell count after 24 hours of treatment (cells/ml x 105)

Cell count after 24 hours of subculture (cells/ml x 105)

SG(1)

 

 

(x 105cells/ml)

RSG(2)

 

 

(%)

Without metabolic activation

SC

8.5

5.7

39

100

1

11.9

5.5

52

133

3

11.8

5.66

53

136

10

12.7

5.7

57

145

33(3)

12.3

5.4

54

137

100(3)

12.9

5.5

58

147

Note: all calculations were made without rounding off

SC = solvent control = dimethyl sulfoxide

(1) = suspension growth

(2) = relative suspension growth

(3) = 3,3’,3’’, 5,5’,5’’-hexa-tert-butyl-α,α’,α’’-(mesitylene-2,4,6-triyl)tri-p-cresol precipitated in the exposure medium

 

   Cell count after 24h subculture

SG = Suspension growth = Cell count after x --------------------------

                                             24h treatment     Cell subcultured

                                                                         After treatment

                                                                          (1.25 x 105c/ml)

RSG = [SG(test)/SG(control)] x 100

 

Table 3: Experiment 1: Cytotoxic and mutagenic response of 3,3’,3’’,5,5’5,’’-hexa-tert-butyl-α,α’,α’’-(mesitylene-2,4,6-triyl)tri-p-cresol in the mouse lymphoma L5178Y test system

Dose

RSG

CEday2

RSday2

RTG

Mutation frequency

Per 106survivors

(μg/ml)

(%)

(%)

(%)

(%)

Total

( small

large )

Without metabolic activation

3 hours treatment

SC1

100

110

100

100

53

( 25

26 )

SC2

111

70

( 28

39 )

0.01

104

85

77

80

74

( 44

28 )

0.03

101

123

111

113

55

( 25

28 )

0.3

108

85

77

83

55

( 19

35 )

1

112

86

78

87

64

( 27

35 )

3

107

107

97

103

49

( 18

30 )

10

117

98

89

104

46

( 11

34 )

33(1)

111

98

89

99

62

( 21

40 )

MMS

84

67

61

51

650

( 371

213 )

With 8% (v/v) metabolic activation

3 hours treatment

SC1

100

115

100

100

88

( 37

47 )

SC2

105

95

( 38

52 )

0.01

122

90

82

100

93

( 36

53 )

0.03

111

98

89

99

115

( 62

47 )

0.1

82

86

78

64

116

( 40

71 )

0.3

102

104

94

97

121

( 47

67 )

1

111

116

106

118

121

( 41

72 )

3

113

118

107

121

98

( 48

45 )

10

81

107

97

79

101

( 48

47 )

33(1)

106

97

88

94

143

( 53

80 )

CP

31

49

44

14

2067

( 862

708 )

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 = DMSO; MMS = Methylmethanesulfonate; CP = Cyclophospamide

(1) = 3,3’,3’’,5,5’,5’’-hexa-tert-butyl-α,α’,α’’-(mesitylene-2,4,6-triyl)tri-p-cresol precipitated in the exposure medium

 

Table 4: Experiment 2: Cytotoxic and mutagenic response of 3,3’,3’’,5,5’,5’’-hexa-tert-butyl-α,α’,α’’-(mesitylene-2,4,6-triyl)tri-p-cresol in the mouse lymphoma L5178Y test system

Dose

RSG

CSday2

RSday2

RTG

Mutation frequency

Per 106survivors

(μg/ml)

(%)

(%)

(%)

(%)

Total

( small

large )

Without metabolic activation

24 hours treatment

SC1

100

75

100

100

83

( 43

37 )

SC2

62

55

( 29

25 )

0.03

112

47

69

77

78

( 45

31 )

0.1

118

95

139

165

42

( 25

17 )

0.3

122

70

103

125

52

( 33

18 )

1

120

57

83

99

83

( 38

43 )

3

112

54

79

88

83

( 58

23 )

10

124

45

66

82

59

( 40

19 )

33

119

67

8

117

53

( 28

24 )

66(1)

121

43

63

76

109

( 52

55 )

MMS

105

28

41

43

870

( 570

254 )

With 12% (v/v) metabolic activation

3 hours treatment

SC1

100

115

100

100

65

( 25

37 )

SC2

139

59

( 29

27 )

0.01

113

98

77

87

53

( 31

21 )

0.03

102

116

92

94

50

( 25

24 )

0.1

104

97

76

79

73

( 33

37 )

0.3

114

93

73

83

57

( 23

32 )

1

107

110

87

93

41

( 19

20 )

3

121

94

74

90

59

( 26

31 )

10

97

99

79

77

48

( 22

25 )

33(1)

104

107

84

88

63

( 32

28 )

CP

70

48

38

26

1238

( 466

593 )

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 = DMSO; MMS = Methylmethanesulfonate; CP = Cyclophosphamide

(1) = 3,3’,3’’,5,5’,5’’-hexa-tert-butyl-α,α’,α’’-(mesitylene-2,4,6-triyl)tri-p-cresol precipitated in the exposure medium

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

In conclusion, 3,3’,3”,5,5’,5”-hexa-tert-butyl-α,α’,α”-(mesitylene-2,4,6-triyl) tri-p-cresol is not mutagenic in the TK mutation test system under the experimental conditions described in this report.
Executive summary:

Evaluation of the mutagenic activity of 3,3’,3”,5,5’,5”-hexa-tert-butyl-α,α’,α”-(mesitylene-2,4,6-triyl) tri-p-cresolin an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (with independent repeat).

 

This report describes the effects of 3,3’,3”,5,5’,5”-hexa-tert-butyl-α,α’,α”-(mesitylene-2,4,6-triyl) tri-p-cresolon 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 PTB2B0029 of 3,3’,3”,5,5’,5”-hexa-tert-butyl-α,α’,α”-(mesitylene-2,4,6-triyl) tri-p-cresol was a white powder with a purity of 99.1 Area %. The test substance was dissolved in dimethyl sulfoxide.

 

The concentrations analysed in the high formulations were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

 

At the low concentration level, the peak of DMSO (i.e. the vehicle of the formulation), interfered with the HPLC determination so that accurate quantitative results could not be obtained. Since the low concentration formulation was prepared by dilution of the high concentration formulation and since the recovery of the low formulation was in agreement with the recovery of the procedural recovery samples, it was concluded that this formulation was also prepared accurately.

 

In the first experiment, 3,3’,3”,5,5’,5”-hexa-tert-butyl-α,α’,α”-(mesitylene-2,4,6-triyl) tri-p-cresol was tested up to concentrations of 33 µg/ml in the absence and presence of 8% (v/v) S9-mix. The incubation time was 3 hours. In the second experiment, the test substance was tested up to concentrations of 33 and 66 µg/ml in the absence and presence of 12% (v/v) S9 -mix, respectively. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. No toxicity was observed up to and including the highest tested dose level in the absence and presence of S9-mix. The test substance precipitated in the culture medium at the highest tested dose level. This is the highest concentration recommended in the guidelines.

 

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.

 

Mutation frequencies in cultures treated with positive control chemicals were increased 11 - and 13 -fold for in the absence of S9-mix, and 23- and 20-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, the test substance 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, the test substance 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 3,3’,3”,5,5’,5”-hexa-tert-butyl-α,α’,α”-(mesitylene-2,4,6-triyl) tri-p-cresol is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

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

Additional information

Additional information from genetic toxicity in vitro:

The substance has been assessed for genetic toxicity by four in vitro assays with and without metabolic activation (S9-mix):

Ames assay, salmonella strains T98, T100, T1535, T1537, T1538

Chromosome aberration assay, Chinese hamster ovaries (CHO), read across to a close structural analogue.

Gene mutation assay, Chinese hamster lung fibroblasts (V79), by read across to a close structural analogue.

Gene mutation assay, mouse lymphoma L5178Y cells

 

All four assays produced a negative result in the presence and absence of metabolic activation demonstrating a lack of mutagenic response.


Justification for selection of genetic toxicity endpoint
Study performed in accordance with EU & OECD test guidelines in compliance with GLP.

Justification for classification or non-classification

All studies produced the same negative response, therefore the substance is not classified.