Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date 30 January 2017 Experimental completion date 03 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: • Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Identification: 2,2’-[propane-1,3-diylbis(oxy)]bis(3”,5,5”-tri-tert-butyl-5’-methyl-1,1’:3’,1”-terphenyl-2’-ol)
CAS Number: 1042662-40-7
Physical state/Appearance: White powder
Batch: 1502501005
Purity: 100%
Expiry Date: 01 February 2025
Storage Conditions: Room temperature in the dark

Method

Target gene:
Histidine operon for Salmonellla
Tryptophan operon for Escherichia
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Liver microsomal preparation (S9-mix)
Test concentrations with justification for top dose:
Esperiment 1 - With and without metabolic activation
1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Experiment 2 - With and without metabolic activation
15, 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
Dimethyl formamide
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
(-S9 mix) 2 µg/plate fpr WP2uvrA, 3 µg/plate for TA 100 and 5 µg/plate for TA1535
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
(-S9 mix) 80 µg/plate for TA1537
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
(-S9 mix) 0.2 µg/plate for TA98
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
(+ S9 mix) 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537 10 µg/plate for WP2uvrA
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
(+ S9 mix) 5 µg/plate for TA98
Details on test system and experimental conditions:
Test for Mutagenicity: Experiment 1 - Plate Incorporation Method

Dose selection
The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9 mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
All of the plates were incubated at 37 ± 3°C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were performed at and above 1500 µg/plate because of test item precipitation.

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As the result of Experiment 1 was deemed negative, Experiment 2 was performed using the pre incubation method in the presence and absence of metabolic activation.

Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was as 15 to 5000 µg/plate.
Six test item concentrations were selected in Experiment 2 in order to achieve both four non toxic dose levels and the potential toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.

Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation or solvent vehicle or appropriate positive control were incubated at 37 ± 3°C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9 mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3°C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37 ± 3°C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were again performed at and above 1500 µg/plate because of test item precipitation.
Rationale for test conditions:
In solubility checks performed in house, the test item was noted as insoluble in sterile distilled water, acetonitrile and dimethyl sulphoxide at 50 mg/mL and acetone at 100 mg/mL but was fully soluble in dimethyl formamide at 50 mg/mL and tetrahydrofuran at 200 mg/mL. Dimethyl formamide was selected as the vehicle.

The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl formamide by mixing on a vortex mixer and sonication for 30 minutes at 40 °C on the day of each experiment. No correction for purity was required. Dimethyl formamide is considered an acceptable vehicle for use in this test system (Maron et al., 1981). Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino silicate pellets with a nominal pore diameter of 4 x 10-4 microns.

All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 (plate incorporation) and Table 4 and Table 5 for Experiment 2 (pre-incubation).
A history profile of vehicle, untreated and positive control values (reference items) is presented in Appendix 1.
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level (5000 µg/plate) was selected in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawns of any of the tester strains (except TA100 dosed in the absence of S9-mix) at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). Slightly weakened bacterial background lawns were noted to TA100 in the absence of S9-mix only at 5000 µg/plate after the incorporation of the pre-incubation modification in the second mutation test. A test item precipitate (powdery in appearance) was noted at and above 500 g/plate, this observation did not prevent the scoring of revertant colonies.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre incubation method).
The vehicle (dimethyl formamide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Any other information on results incl. tables

Test Results: Experiment 1 – Without Metabolic Activation (Plate Incorporation)

Test Period

From: 16 February 2017

To: 19 February 2017

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMF)

98

83

69

(83)

14.5#

24

31

29

(28)

3.6

19

27

15

(20)

6.1

20

20

15

(18)

2.9

8

10

7

(8)

1.5

1.5 µg

83

88

96

(89)

6.6

18

9

16

(14)

4.7

21

21

13

(18)

4.6

21

12

22

(18)

5.5

10

11

9

(10)

1.0

5 µg

74

80

78

(77)

3.1

33

7

18

(19)

13.1

16

15

22

(18)

3.8

20

16

8

(15)

6.1

11

12

10

(11)

1.0

15 µg

68

100

99

(89)

18.2

17

16

20

(18)

2.1

19

19

12

(17)

4.0

14

22

22

(19)

4.6

7

17

8

(11)

5.5

50 µg

75

87

76

(79)

6.7

12

20

20

(17)

4.6

16

24

29

(23)

6.6

33

9

20

(21)

12.0

9

13

22

(15)

6.7

150 µg

91

94

79

(88)

7.9

21

24

23

(23)

1.5

7

18

12

(12)

5.5

30

14

23

(22)

8.0

9

10

9

(9)

0.6

500 µg

111 P

84 P

93 P

(96)

13.7

16 P

15 P

31 P

(21)

9.0

20 P

16 P

20 P

(19)

2.3

23 P

16 P

29 P

(23)

6.5

10 P

10 P

17 P

(12)

4.0

1500 µg

75 P

85 P

75 P

(78)

5.8

16 P

24 P

16 P

(19)

4.6

19 P

17 P

18 P

(18)

1.0

22 P

24 P

24 P

(23)

1.2

10 P

9 P

8 P

(9)

1.0

5000 µg

72 P

91 P

90 P

(84)

10.7

20 P

13 P

16 P

(16)

3.5

19 P

26 P

21 P

(22)

3.6

19 P

28 P

26 P

(24)

4.7

7 P

7 P

10 P

(8)

1.7

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

353

319

300

(324)

26.9

137

149

220

(169)

44.9

489

524

417

(477)

54.6

129

134

134

(132)

2.9

257

339

332

(309)

45.5

ENNG4NQO9AAP#

  Test Results: Experiment 1 – With Metabolic Activation(Plate Incorporation)

Test Period

From: 16 February 2017

To: 19 February 2017

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMF)

87

72

120

(93)

24.6#

10

7

10

(9)

1.7

29

17

22

(23)

6.0

13

15

24

(17)

5.9

12

15

6

(11)

4.6

1.5 µg

75

96

105

(92)

15.4

6

9

6

(7)

1.7

19

32

32

(28)

7.5

23

14

24

(20)

5.5

12

8

14

(11)

3.1

5 µg

105

113

91

(103)

11.1

8

8

17

(11)

5.2

28

16

21

(22)

6.0

24

20

24

(23)

2.3

14

11

15

(13)

2.1

15 µg

120

100

92

(104)

14.4

12

9

25

(15)

8.5

26

28

21

(25)

3.6

14

24

22

(20)

5.3

14

12

18

(15)

3.1

50 µg

113

104

103

(107)

5.5

19

11

18

(16)

4.4

21

23

15

(20)

4.2

13

18

20

(17)

3.6

6

11

17

(11)

5.5

150 µg

105

104

101

(103)

2.1

7

11

7

(8)

2.3

27

34

27

(29)

4.0

18

25

15

(19)

5.1

11

10

17

(13)

3.8

500 µg

76 P

111 P

95 P

(94)

17.5

25 P

11 P

7 P

(14)

9.5

31 P

26 P

26 P

(28)

2.9

18 P

16 P

23 P

(19)

3.6

12 P

12 P

15 P

(13)

1.7

1500 µg

83 P

90 P

96 P

(90)

6.5

8 P

9 P

12 P

(10)

2.1

19 P

23 P

20 P

(21)

2.1

17 P

14 P

19 P

(17)

2.5

8 P

6 P

12 P

(9)

3.1

5000 µg

90 P

75 P

70 P

(78)

10.4

15 P

13 P

14 P

(14)

1.0

17 P

22 P

21 P

(20)

2.6

17 P

12 P

14 P

(14)

2.5

13 P

13 P

8 P

(11)

2.9

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

1865

1945

1919

(1910)

40.8

271

252

259

(261)

9.6

237

202

196

(212)

22.1

96

123

132

(117)

18.7

178

207

162

(182)

22.8

2AABPP#

 

Test Results: Experiment 2 – Without Metabolic Activation
(Pre-Incubation)

Test Period

From: 28 February 2017

To: 03 March 2017

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMF)

85

80

69

(78)

8.2#

13

17

14

(15)

2.1

23

32

20

(25)

6.2

18

15

15

(16)

1.7

7

14

14

(12)

4.0

15 µg

71

81

86

(79)

7.6

19

18

8

(15)

6.1

25

31

39

(32)

7.0

10

11

11

(11)

0.6

13

19

9

(14)

5.0

50 µg

73

75

67

(72)

4.2

19

15

12

(15)

3.5

25

36

19

(27)

8.6

12

18

18

(16)

3.5

12

14

24

(17)

6.4

150 µg

67

67

75

(70)

4.6

13

11

11

(12)

1.2

31

24

28

(28)

3.5

12

10

13

(12)

1.5

12

13

13

(13)

0.6

500 µg

72 P

68 P

71 P

(70)

2.1

10 P

11 P

19 P

(13)

4.9

38 P

37 P

21 P

(32)

9.5

16 P

17 P

18 P

(17)

1.0

11 P

14 P

7 P

(11)

3.5

1500 µg

64 P

65 P

73 P

(67)

4.9

12 P

8 P

8 P

(9)

2.3

27 P

31 P

32 P

(30)

2.6

10 P

16 P

12 P

(13)

3.1

12 P

11 P

8 P

(10)

2.1

5000 µg

66 PS

65 PS

50 PS

(60)

9.0

12 P

10 P

7 P

(10)

2.5

17 P

17 P

24 P

(19)

4.0

19 P

18 P

14 P

(17)

2.6

20 P

13 P

11 P

(15)

4.7

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

1295

1677

1576

(1516)

197.9

1879

1910

1911

(1900)

18.2

495

562

473

(510)

46.4

223

269

211

(234)

30.6

199

168

213

(193)

23.0

 

ENNG4NQO9AASP #

 

Test Results: Experiment 2 - With Metabolic Activation (Pre-Incubation)

Test Period

From: 28 February 2017

To: 03 March 2017

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMF)

107

85

102

(98)

11.5#

15

10

10

(12)

2.9

28

26

37

(30)

5.9

15

19

12

(15)

3.5

16

18

12

(15)

3.1

15 µg

82

122

100

(101)

20.0

12

9

20

(14)

5.7

30

36

37

(34)

3.8

14

16

9

(13)

3.6

10

18

12

(13)

4.2

50 µg

114

92

87

(98)

14.4

16

7

14

(12)

4.7

32

27

34

(31)

3.6

23

14

18

(18)

4.5

23

15

16

(18)

4.4

150 µg

80

105

88

(91)

12.8

14

9

15

(13)

3.2

39

26

43

(36)

8.9

27

16

26

(23)

6.1

17

15

19

(17)

2.0

500 µg

82 P

83 P

90 P

(85)

4.4

15 P

10 P

14 P

(13)

2.6

21 P

38 P

40 P

(33)

10.4

14 P

19 P

18 P

(17)

2.6

21 P

18 P

10 P

(16)

5.7

1500 µg

93 P

92 P

93 P

(93)

0.6

11 P

8 P

8 P

(9)

1.7

33 P

37 P

37 P

(36)

2.3

17 P

15 P

18 P

(17)

1.5

15 P

10 P

7 P

(11)

4.0

5000 µg

88 P

64 P

99 P

(84)

17.9

13 P

9 P

12 P

(11)

2.1

29 P

31 P

29 P

(30)

1.2

18 P

11 P

12 P

(14)

3.8

8 P

5 P

10 P

(8)

2.5

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

1016

890

914

(940)

66.9

289

276

246

(270)

22.1

215

214

256

(228)

24.0

126

155

123

(135)

17.7

309

324

312

(315)

7.9

 

BP2AAP


ENNG      N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO       4-Nitroquinoline-1-oxide

9AA         9-Aminoacridine

P                    Test item precipitate

#             Standard deviation

2AA  2-Aminoanthracene

BP           Benzo(a)pyrene

P                Test item precipitate

#             Standard deviation

ENNG         N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO          4-Nitroquinoline-1-oxide

9AA            9-Aminoacridine

S                         Sparsebacterial background lawn

P                         Test Item precipitate

#                Standard deviation

BP           Benzo(a)pyrene

2AA        2-Aminoanthracene

P                    Test Item precipitate

Applicant's summary and conclusion

Conclusions:
2,2’-[propane-1,3-diylbis(oxy)]bis(3”,5,5”-tri-tert-butyl-5’-methyl-1,1’:3’,1”-terphenyl-2’-ol) was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Summary

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 andEscherichia colistrain WP2uvrAwere treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 mg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 µg/plate. Six test item concentrations were selected in Experiment 2 in order to achieve both four non‑toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

Results

The vehicle (dimethyl formamide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level (5000 µg/plate) was selected in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawns of any of the tester strains (except TA100 dosed in the absence of S9-mix) at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). Slightly weakened bacterial background lawns were noted to TA100 in the absence of S9-mix only at 5000 µg/plate after the incorporation of the pre-incubation modification in the second mutation test. A test item precipitate (powdery in appearance) was noted at and above 500 mg/plate, this observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre‑incubation method). 

Conclusion

2,2’-[propane-1,3-diylbis(oxy)]bis(3”,5,5”-tri-tert-butyl-5’-methyl-1,1’:3’,1”-terphenyl-2’-ol)was considered to be non-mutagenic under the conditions of this test.