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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
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:
22 August 2012 and 21 September 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as an unpublished report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

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:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
- Physical state: Pale Yellow Solid
- Purity: Not applicable - UVCB
- Substance identity: Aluminum, benzoate C16-18-fatty acids complexes
- Batch number: 11074091 + Benzoic acid
- Carbon Content: 65.1%
- Analysis code: A118
- Date recieved: 07 June 2012
- Expiration date: 01 July 2013
- Storage of test material: Room temperature in the dark

Method

Target gene:
+Histidine for Salmonella.
Tryptophan for E.Coli
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:
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:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
main test:
Experiment one: 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile distilled water
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water (25 and 50 mg/ml), dimethyl sulphoxide, dimethyl formamide and acetonitrile (50 mg/ml), acetone (100 mg/ml) and tetrahydrofuran (200 mg/ml) in solubility checks performed in-house. The test item formed the best doseable suspension in sterile distilled water at 25 mg/ml; therefore, this solvent was selected as the vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 1 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 10 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix

Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitroquinoline-1-oxide: 0.2 µg/plate
Remarks:
without S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix

Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix

Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix

Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix

Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate
Details on test system and experimental conditions:
METHODS OF APPLICATION: in agar (plate incorporation) - Experiment 1 and pre-incubation - Experiment 2

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies using a colony counter and examined for effects on the growth of the bacterial background lawn. Manual counts were performed at 5000 µg/plate because of excessive test item precipitation.

Evaluation criteria:
Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.0 x 10+9 bacteria per ml.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
There should be a minimum of four non-toxic test material dose levels.
There should not be an excessive loss of plates due to contamination.

Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material 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 a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Statistics:
Standard deviation

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: The test item was insoluble in sterile distilled water (25 and 50 mg/ml), dimethyl sulphoxide, dimethyl formamide and acetonitrile (50 mg/ml), acetone (100 mg/ml) and tetrahydrofuran (200 mg/ml) in solubility checks performed in-house.
The test item formed the best doseable suspension in sterile distilled water at 25 mg/ml; therefore, this solvent was selected as the vehicle.
- Precipitation: A test item precipitate (particulate in appearance) was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment
were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: None
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

RESULTS

Preliminary ToxicityTest

The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile.

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-)

S9-mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

89

84

85

98

91

98

94

94

116

99P

81P

+

TA100

89

95

85

93

87

73

110

94

102

88

91P

-

WP2uvrA

24

31

28

27

18

21

30

19

29

31

39P

+

WP2uvrA

29

21

31

37

24

19

36

25

35

33P

33P

P: Precipitate

      

MutationTest

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in Table1(see below) 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 and Table 4 and Table 5 for Experiment 2.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.


All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.


Table1               Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

84

 

18

 

26

 

14

 

7

 

66

(77)

12

(15)

23

(23)

19

(15)

15

(10)

81

 

14

 

21

 

13

 

9

 

Experiment 2

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

93

 

29

 

18

 

12

 

11

 

103

(100)

18

(25)

21

(24)

18

(18)

10

(14)

103

 

27

 

32

 

25

 

20

 

 

Table 2: Test Results: Experiment 1– Without Metabolic Activation

With or without

S9-Mix

Dose Level Per Plate

Number of revertants (mean) ± SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

S9-Mix

(-)

Solvent Control (Water)

101

(90) 16.8#

23

(190) 4.6

23

(26) 4.2

20

(22) 3.2

9

(9) 0.6

99

20

31

26

10

71

14

25

21

9

50 μg

90

(85) 10.8

11

(16) 4.5

26

(20) 5.1

12

(21) 10.7

10

(13) 2.5

93

16

16

33

15

73

20

19

19

13

150 μg

103

(81) 19.1

19

(18) 2.3

29

(25) 4.0

15

(17) 2.9

12

(12) 2.5

70

19

24

20

14

70

15

21

15

9

500 μg

113

(103) 9.1

20

(20) 2.5

38

(31) 5.9

20

(21) 0.6

9

(10) 2.3

95

18

29

21

9

102

23

27

21

13

1500 μg

106p

(95) 10.6

24P

(21) 4.2

25P

(28) 4.6

19P

(24) 6.4

9P

(9) 2.5

93P

16P

25P

31P

12P

85P

22P

33P

21P

7P

5000 μg

98P

(86) 10.6

20P

(21) 1.5

21P

(24) 4.9

24p

(24) 4.9

9P

(11) 1.5

78P

21P

30P

25P

11P

82P

23P

22P

23P

12P

Positive

controls

S9-Mix

(-)

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentration

(μg/plate)

3 μg

5 μg

2 μg

0.2 μg

80 μg

No. colonies

per plate

577

(560) 48.1

284

(304) 63.4

977

(807) 147.5

103

(107) 3.2

1054

(94.9) 183.9

629

253

713

108

737

533

375

731

109

1057

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

4NQO 4-Nitroquinoline-1-oxide

9AA 9-Aminoacridine

P Precipitate

# Standard deviation

 

Table 3 Test Results: Experiment 1 – With Metabolic Activation

With or without

S9-Mix

Dose Level Per Plate

Number of revertants (mean) ± SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

S9-Mix

(+)

Solvent Control (Water)

106

(108) 11.6#

12

(130)2.3

35

(38) 7.4

22

(25) 4.6

14

(12) 2.0

97

12

32

22

10

120

16

46

30

12

50 μg

101

(96) 13.6

14

914) 0.6

38

(32) 10.1

29

(27) 2.5

13

(13) 1.5

107

14

37

27

15

81

15

20

24

12

150 μg

111

(108) 14.2

14

(14) 0.6

34

(39) 6.4

23

(27) 4.7

12

(12) 3.0

121

14

46

25

15

93

13

36

32

9

500 μg

118

(108) 20.3

19

(15) 5.1

29

(34) 5.7

14

(23) 8.3

11

(10) 3.1

122

16

32

26

13

85

9

40

30

7

1500 μg

113P

(111) 6.7

10p

(12) 1.5

31P

(32) 2.6

23P

(26) 5.2

12P

(12) 0.6

117P

13P

35P

23P

13P

104P

12P

30P

32P

12P

5000 μg

113P

(113)

3.0

12P

(13) 4.6

36P

(38) 5.3

31P

(270) 4.0

7p

(10) 3.0

110P

9P

34P

23P

13P

116P

18P

44P

26P

10P

Positive

controls

S9-Mix

(+)

Name

2AA

2AA

2AA

BP

2AA

Concentration

(μg/plate)

1 μg

2 μg

10 μg

5 μg

2 μg

No. colonies

per plate

827

(817) 10.5

167

(161) 6.0

400

(400) 10.5

207

(246) 34.1

203

(212) 15.6

806

155

390

268

230

818

162

411

264

203

2AA 2-Aminoanthracene

BP Benzo(a)pyrene

P Precipitate

# Standard deviation

Tables 4 and 5 below

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test item, aluminum, aluminum, benzoate C16-18-fatty acids complexes, was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction.

The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the, EPA (TSCA) OPPTS harmonised guidelines.

Method.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of the test item, Aluminium, benzoate iso-Pr alc.stearate complexes, using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test item formulations.

Results.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive controls 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 test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate (particulate in appearance) was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Conclusion.

The test item, aluminum, benzoate C16-18-fatty acids complexes, was considered to be non-mutagenic under the conditions of this test.