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Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study performed with equivalent protocol to OECD guideline 471 but no certificate of analysis of the test substance and no data about its purity are reported.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
No certificate of analysis of test substance, no data about purity of test substance
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Mexoryl SAB
- Physical state: white fibrous powder
- Analytical purity: no data
- Lot/batch No.: OP T 4 E
- Storage condition of test material: at room temperature

Method

Target gene:
Not applicable
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Liver microsomal fraction (S9) from Fischer 344 rats induced with beta-naphthoflavone and sodium phenobarbitone
Test concentrations with justification for top dose:
- range finder experiment: 1.6, 8, 40, 200, 1000 and 5000 µg/plate in both the absence and presence of S9
- first experiment: 1.6, 8, 40, 200 and 1000 µg/plate in both the absence and presence of S9
- second experiment: 0.16, 0.8, 4, 20 and 100 µg/plate without S9; 0.32, 1.6, 8, 40 and 200 µg/plate with S9
Vehicle / solvent:
Vehicle: ethanol
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see table 1
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: about 65 h

SELECTION AGENT (mutation assays): histidine for Salmonella strains, tryptophane for Escherichia coli

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: appearance of a complete bacterial lawn
Evaluation criteria:
A positive result is indicated by a dose response with statistically significant increases in revertant numbers.
Statistics:
Mean, standard deviations and Dunnett's t-statistic were calculated for each group and bacterial strain.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA 98, TA 100, TA1535, TA 1537, TA 1538 and WP2 uvrA
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
for 100 µg/plate concentration in some of the strains (reduced background lawn)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
other: TA 98, TA 100, TA1535, TA 1537, TA 1538 and WP2 uvrA
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
for 200 µg/plate concentration in some of the strains (reduced background lawn)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: TA 98 and WP2 uvrA were used with and without S9 during 24 h; Mexoryl SAB had slight effects on bacterial growth for 1000 and 5000 µg/plate concentrations. Therefore the strains were exposed up to 1000 µg/plate in the main test.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2: results of the first experiment

Strain

Compound

Dose level (µg/plate)

S9 mix

Mean revertant colony counts

SD

Individual revertant colony counts

TA 1535

Ethanol

 

 

 

 

 

Sodium azide

 

Ethanol

 

 

 

 

 

2-aminoanthracene

 

1.6

8

40

200

1000

1

 

 

1.6

8

40

200

1000

2

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

7.3

7.3

13.3

10.3

-

-

96

 

13.3

15.3

14.3

19.0

15.7

-

48

1.53

3.79

2.31

1.15

-

-

10

 

3.06

4.51

1.53

6.24

5.03

-

11.36

6, 9, 7

9, 3, 10

16, 12, 12

9, 11, 11

a

b

96, 86, 106

 

10, 16, 14

20, 15, 11

13, 16, 14

26, 17, 14

21, 11, 15

a

40, 43, 61

TA 1537

Ethanol

 

 

 

 

 

9-aminoacridine

 

Ethanol

 

 

 

 

 

2-aminoanthracene

 

1.6

8

40

200

1000

50

 

 

1.6

8

40

200

1000

2

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

3.0

5.0

2.0

3.7

-

-

295.0

 

9.3

4.0

5.7

6.0

8.3

-

76.3

1.73

1.00

1.00

1.15

-

-

35.04

 

6.66

4.36

0.58

1.00

0.58

-

8.50

5, 2, 2

4, 5, 6

1, 3, 2

5, 3, 3

a

b

331, 261, 293

 

6, 17, 5

9, 2, 1

6, 5, 6

7, 5, 6

8, 8, 9

a

86, 70, 73

TA 1538

Ethanol

 

 

 

 

 

2-Nitrofluorene

 

Ethanol

 

 

 

 

 

2-aminoanthracene

 

1.6

8

40

200

1000

0.5

 

 

1.6

8

40

200

1000

2

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

10.7

10.7

15.7

9.3

-

-

232.7

 

24.3

19.3

24.3

26.7

19.3

-

1305.3

2.08

2.08

7.02

2.52

-

-

34.24

 

7.02

0.58

4.51

3.79

7.37

-

137.38

13, 9, 10

9, 10, 13

15, 9, 23

7, 9, 12

a

b

269, 201, 228

 

31, 17, 25

19, 20, 19

29, 24, 20

31, 24, 25

11, 25, 22

a

1393, 1376, 1147

TA 98

Ethanol

 

 

 

 

 

2-Nitrofluorene

 

Ethanol

 

 

 

 

 

2-aminoanthracene

 

1.6

8

40

200

1000

0.5

 

 

1.6

8

40

200

1000

2

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

19.0

17.7

12.0

21.7

-

-

249.0

 

25.0

19.0

20.0

22.0

25.3

-

923.3

2.65

4.62

1.73

7.57

-

-

3.61

 

7.55

1.73

2.65

5.57

9.29

-

50.90

18, 22, 17

15, 23, 15

14, 11, 11

25, 13, 27

a

b

250, 252, 245

 

26, 32, 17

17, 20, 20

18, 23, 19

17, 21, 28

33, 15, 28

b

937, 966, 867

TA 100

Ethanol

 

 

 

 

 

Sodium azide

 

Ethanol

 

 

 

 

 

2-aminoanthracene

 

1.6

8

40

200

1000

1

 

 

1.6

8

40

200

1000

2

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

68.7

67.7

74.0

72.3

-

-

466.0

 

83.3

82.0

85.7

77.0

61.3

-

951.3

13.32

8.50

6.00

12.22

-

-

60.51

 

12.22

14.00

8.39

9.17

4.62

-

122.68

72, 54, 80

59, 76, 68

74, 68, 80

59, 83, 75

a

a

441, 422, 535

 

86, 70, 94

66, 88, 92

91, 90, 76

69, 75, 87

64, 64, 56

a, b, b

861, 1091, 902

WP2 uvrA

Ethanol

 

 

 

 

 

 

4 -nitroquinoline-1-oxide

 

Ethanol

 

 

 

 

 

2-aminoanthracene

 

1.6

8

40

200

1000

 

 

1

 

 

1.6

8

40

200

1000

2

-

-

-

-

-

-

-

 

 

 

+

+

+

+

+

+

+

15.0

14.7

13.3

16.3

-

-

 

 

617.7

 

17.0

13.7

19.3

14.7

-

-

94.7

3.46

4.51

4.93

1.15

-

-

 

 

32.65

 

1.73

5.77

5.51

2.31

-

-

11.55

13, 13, 19

15, 10, 19

10, 11, 19

15, 17, 17

b

b

 

 

580, 638, 635

 

18, 18, 15

7, 17, 17

23, 22, 13

16, 16, 12

a

b

108, 88, 88

a = reduced background lawn

b = complete loss of lawn

Table 3: results of the second experiment

Strain

Compound

Dose level (µg/plate)

S9 mix

Mean revertant colony counts

SD

Individual revertant colony counts

TA 1535

Ethanol

 

 

 

 

 

Sodium azide

 

Ethanol

 

 

 

 

 

2-aminoanthracene

 

0.16

0.8

4

20

100

1

 

 

0.32

1.6

8

40

200

2

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

12.7

10.7

9.3

9.0

9.7

-

252.0

 

10.7

14.3

15.0

11.3

15.7

-

106.0

4.16

2.52

3.06

1.73

4.73

0.00

3.61

 

4.93

4.51

6.56

4.04

3.21

-

7.81

14, 8, 16

8, 11, 13

6, 10, 12

7, 10, 10

15, 8, 6

a

251, 256, 249

 

5, 14, 13

19, 14, 10

14, 22, 9

15, 7, 12

12, 17, 18

a

102, 101, 115

TA 1537

Ethanol

 

 

 

 

 

9-aminoacridine

 

Ethanol

 

 

 

 

 

2-aminoanthracene

 

0.16

0.8

4

20

100

50

 

 

0.32

1.6

8

40

200

2

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

4.7

2.0

2.0

3.7

4.3

-

852.7

 

11.3

6.3

6.7

6.0

8.7

-

101.3

2.52

1.00

1.00

2.08

1.53

0.00

8.14

 

1.53

0.58

0.58

1.73

1.53

-

22.19

2, 7, 5

1, 2, 3

1, 2, 3

2,3, 6

3, 4, 6

a

849, 847, 862

 

13, 10, 11

6, 7, 6

6, 7, 7

8, 5, 5

9, 10, 7

a

126, 83, 95

TA 1538

Ethanol

 

 

 

 

 

2-Nitrofluorene

 

Ethanol

 

 

 

 

 

2-aminoanthracene

 

0.16

0.8

4

20

100

0.5

 

 

0.32

1.6

8

40

200

2

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

17.7

18.3

18.3

17.3

16.7

-

90.0

 

22.3

21.0

22.3

25.3

26.0

20.0

1225.0

1.53

4.73

1.53

3.06

3.21

0.00

10.82

 

6.35

5.20

3.51

6.66

3.46

2.65

32.60

16, 18, 19

22, 20, 13

18, 17, 20

18, 14, 20

19, 18, 13

a

102, 87, 81

 

15, 26, 26

27, 18, 18

26, 19, 22

18, 27, 31

24, 24, 30

18, 23, 19

1191, 1228, 1256

TA 98

Ethanol

 

 

 

 

 

2-Nitrofluorene

 

Ethanol

 

 

 

 

 

2-aminoanthracene

 

0.16

0.8

4

20

100

0.5

 

 

0.32

1.6

8

40

200

2

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

14.0

15.7

12.0

10.0

14.3

14.3

81.0

 

24.0

22.0

23.3

21.0

22.3

17.3

1111.7

1.00

4.16

1.73

1.00

1.15

2.89

10.15

 

3.46

2.00

5.51

4.36

2.89

0.58

31.79

15, 14, 13

19, 17, 11

10, 13, 13

10, 9, 11

15, 13, 15

16, 11, 16

92, 79, 72

 

22, 28, 22

22, 24, 20

29, 23, 18

24, 23, 16

24, 19, 24

17, 18, 17

1098, 1148, 1089

TA 100

Ethanol

 

 

 

 

 

Sodium azide

 

Ethanol

 

 

 

 

 

2-aminoanthracene

 

0.16

0.8

4

20

100

1

 

 

0.32

1.6

8

40

200

2

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

67.0

70.0

76.0

70.7

67.0

 

421.0

 

81.7

80.7

89.0

81.0

73.0

66.0

1211.7

2.65

7.55

7.00

6.66

7.94

0.00

5.57

 

7.09

8.39

11.36

11.36

6.93

5.57

33.08

64, 68, 69

69, 63, 78

68, 79, 81

78, 65, 69

64, 61, 76

a

415, 426, 422

 

83, 88, 74

85, 71, 86

76, 94, 97

89, 86, 68

81, 69, 69

72, 65, 61

1249, 1186, 1200

WP2 uvrA

Ethanol

 

 

 

 

 

4 -nitroquinoline-1-oxide

 

Ethanol

 

 

 

 

 

2-aminoanthracene

 

0.16

0.8

4

20

100

 

1

 

 

0.32

1.6

8

40

200

2

-

-

-

-

-

-

 

-

 

+

+

+

+

+

+

+

15.3

14.7

14.0

15.7

15.3

-

 

832.3

 

18.7

14.3

16.7

16.0

16.3

-

127.7

1.53

4.62

3.46

3.21

4.04

-

 

55.82

 

6.66

4.73

1.15

2.65

0.58

-

4.62

15, 14, 17

20, 12, 12

16, 10, 16

17, 18, 12

13, 20, 13

a

 

861, 768, 868

 

26, 17, 13

18, 9, 16

16, 16, 18

13, 18, 17

16, 17, 16

a

133, 125, 125

a = reduced background lawn

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under these test conditions, Mexoryl SAB was found not to be mutagenic in TA 1535, TA 1537, TA 1538, TA 100, TA 98 and WP2 uvrA with and without metabolic activation.
Executive summary:

In a reverse gene mutation assay in bacteria, performed similarly to the OECD guideline 471, in compliance with GLP, strains of S. typhimurium (TA 1535, TA 1537, TA 1538, TA 100 and TA 98) and one Escherichia coli strain (WP2 uvrA) were exposed to Mexoryl SAB at concentrations of 1.6, 8, 40, 200, 1000 and 5000 µg/plate in both the absence and presence of S9 metabolic activation according to the direct plate incorporation method for a preliminary cytotoxicity test. In the main test, two experiments were performed at the following concentrations:

- first experiment: 1.6, 8, 40, 200 and 1000 µg/plate in both the absence and presence of S9

- second experiment: 0.16, 0.8, 4, 20 and 100 µg/plate without S9; 0.32, 1.6, 8, 40 and 200 µg/plate with S9.

These experiments were performed according to the direct plate incorporation method.

The positive controls induced the appropriate responses in the corresponding strains. Mexoryl SAB showed no substantial increases in revertant colony numbers over control count obtained with any of the tester strains at any concentrations in either presence or absence of S9 mix.

This study is classified as acceptable and satisfies the requirement for bacterial reverse gene mutation endpoint.

Under the test conditions, Mexoryl SAB is not considered as mutagenic in this bacterial system according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272-2008).