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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: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 03 July 2013 and 15 July 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
EC No. 440/2008 of 30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
other: 40 CFR 799.9510 TSCA bacterial reverse mutation test.
Deviations:
no
Qualifier:
according to
Guideline:
other: USA, EPA (TSCA) OCSPP harmonized guidelines.
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
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Identification: GL500
Test item (alternative names): LGflex GL500
GL500
GL520
CAS No: 1571954-81-8
Action of test item: Plasticizer
Appearance/Physical state: Clear colorless liquid
Batch: GLFG160607
Purity: 99.8%
Expiry date: 07 June 2017
Storage conditions: Room temperature in the dark
Specific details on test material used for the study:
Identification: Batch:vGL500 20130510
Purity: 99.81%
Expiry Date: 10 May 2018
Storage Conditions: Room temperature in the dark
No correction was made for purity was made

Method

Target gene:
All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB-bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TAl 00, the R-factor plasmid pK.MlOl enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).
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 applicable
Metabolic activation:
with and without
Metabolic activation system:
The S9 Microsomal fraction was prepared in-house from male rats induced with Phenobarbitone/ -Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4.
Test concentrations with justification for top dose:
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.

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 50 to 5000 µg/plate.

Vehicle / solvent:
The vehicle control used was acetone
Controls
Negative solvent / vehicle controls:
yes
Remarks:
Identity: Dimethyl sulphoxide Supplier: Fisher Scientific Batch number (purity): 1684307 (>99%) Expiry: 02/2022 1670196 (>99%) Expiry: 03/2022 (Experiment 2 only)
Positive controls:
yes
Positive control substance:
other: see section "Any other information on materials and methods incl. tables"
Details on test system and experimental conditions:
Microsomal Enzyme Fraction
Lot No. PB/13NF S9 14 April 2013 was used in this study. The S9 Microsomal fraction was prepared in-house from male rats induced with Phenobarbitone/ -Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenizing the liver in a 0.15M KCl solution (lg liver to 3 mL KCl) followed by centrifugation at 9000 g. The protein content of the resultant supernatant was adjusted to 20 mg/mL. Aliquots of the supernatant were frozen and stored at approximately -196 °C. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.

S9-Mix and Agar
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
S9 5.0 mL
1.65 M KCl/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADP 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 14.5 mL

A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Media
Top agar was prepared using 0.6% Bacto agar (lot number 2299373 09/17) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 34519 07/13, 34498 07/13 and 34520 07/13).

Test System
Tester strains
The five strains of bacteria used, and their mutations, are as follows:

Salmonella typhimurium
Strains Genotype Type of mutations indicated
TA1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA100 his G 46; rfa-; uvrB-;R-factor

Escherichia coli
Strain Genotype Type of mutations indicated
WP2uvrA trp-; uvrA-: base-pair substitution

All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).

The bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 or from the British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987. All of the strains were stored at approximately -196 °C in a Stateboume liquid nitrogen freezer, model SXR 34.

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot numbers 1218742 07/17 and 1253889 10/17) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Test and Control Item Preparation
Test Item
The test item was immiscible in dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at 100 mg/mL in solubility checks performed in-house. Acetone was therefore selected as the vehicle. Following solubility information provided by the Sponsor, sterile distilled water was not evaluated as a potential vehicle in this test system.
The test item was accurately weighed and approximate half-log dilutions prepared in acetone by mixing on a vortex mixer on the day of each experiment. Acetone is toxic to the bacterial cells at
0.1 mL (100 µL) after employing the pre-incubation modification; therefore all of the formulations for Experiment 2 were prepared at concentrations two times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 0.05 mL (50 µL) aliquots. 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. 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.

Control Items

Vehicle and positive controls were used in parallel with the test item. The vehicle control used was acetone.
The positive control items used in the series of plates without S9-mix were as indicated in "Any other information... (below):

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, vehicle or appropriate positive control was added to 2 mL of trace amino-acid supplemented media (at approximately 45 °C) 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. Each concentration of the test item, appropriate positive control, 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 trace amino-acid supplemented media instead of phosphate buffer.

All of the plates were incubated at 37 °C± 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).

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 50 to 5000 µg/plate.

Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.05 mL of the test item formulation or solvent or 0.1 mL of appropriate positive control were incubated at 37 °C± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. 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 °C ± 3 °C for 20 minutes (with shaking) and addition of amino-acid supplemented media. All testing for this experiment was performed in triplicate.

All of the plates were incubated at 37 °C± 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).

Acceptance Criteria
The reverse mutation assay may be considered valid if the following criteria are met:

All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).

All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the General Study Plan, Section 2.2.2 (negative controls).

All tester strain cultures should be in the range of 0.9 to 9 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 item dose levels.

There should be no evidence of excessive contamination.
Evaluation criteria:
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: All bacterial strains tested
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Remarks:
These data are for concurrent untreated control plates performed on the same day as the Mutation Test
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). 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. The test item formulation was also shown to be sterile.

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 and Table 4 and Table 5 for Experiment 2.

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, in the first mutation test and consequently the same maximum dose level was used in the second mutation test. 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, in the second mutation test. A test item precipitate (globular in appearance) was observed at and above 1500 µg/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 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 in Experiment 2 (pre-incubation 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.

Remarks on result:
other: GL500 was considered to be non-mutagenic

Any other information on results incl. tables

Table1      Spontaneous Mutation Rates (Concurrent NegativeControls)

Experiment 1

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TAlOO

TA1535

WP2uvrA

TA98

TA1537

99

 

20

 

21

 

25

 

9

 

87

(91)

19

(17)

12

(20)

21

(21)

13

(12)

87

 

11

 

27

 

16

 

13

 

 

Experiment 2 

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TAlOO

TA1535

WP2uvrA

TA98

TA1537

82

 

13

 

17

 

21

 

4

 

80

(81)

20

(18)

20

(21)

19

(17)

4

(5)

82

 

21

 

27

 

12

 

8

 

Table 2     Test Results:

Experiment 1 - Without MetabolicActivation

 

Test Period

From:05Julv2013

I

To: 08 Julv 2013

 

 

 

 

 

 

 

 

 

 

 

 

S9-Mix

(-)

Dose Level Per Plate

Number ofrevertants (mean)+/- SD

Base-oair substitution strains

Frameshift strains

TAlOO

TA1535

WP2uvrA

TA98

TAl537

Solvent Control (Acetone)

64       (84)

99     18.2#

90

23        (21)

17         3.8

24

29         (27)

25          2.1

28

23      (21)

16        4.7

25

12       (13)

12        2.3

16

 

1.5 µg

71       (93)

104      19.1

104

15        (17)

20        2.6

16

17        (22)

28          5.7

20

25       (28)

31        3.1

27

15       (15)

17        2.5

12

 

5µg

98       (89)

83        7.8

87

25        (22)

15         5.8

25

35        (25)

19          8.7

21

29      (21)

II        9.2

23

II        (16)

17        4.6

20

 

15 µg

87       (98)

110      11.6

96

19        (17)

17        1.5

16

23        (16)

12         6.1

13

31       (27)

31       6.4

20

13      (13)

13       0.6

12

 

50µg

100      (88)

86       11.1

78

25        (20)

15        5.0

21

16        (21)

27         5.6

20

21       (18)

16       2.6

17

21      (13)

9        7.2

8

 

150 µg

65      (83)

94       15.7

90

24        (19)

19        4.5

15

32        (28)

29         4.6

23

23      (19)

13       5.3

21

9       (11)

9        3.5

15

 

500µg

76      (77)

82       4.2

74

16        (16)

13        3.0

19

21        (25)

31         5.1

24

20      (20)

25      5.0

15

17      (14)

13       3.1

II

 

1500 µg

99P      (87)

88P     12.0

75 P

13p      (17)

12P       7.2

25P

28P        (29)

31P       1.7

28P

28P      (23)

21P     4.7

19 P

12P      (11)

II p      0.6

IIp

 

5000 µg

90P      (99)

96P     10.8

Ill  p

24P       (20)

16P       4.0

29P        (26)

29P       4.6

12P     (23)

24P     10.1

20P      (17)

15P     2.9

 

19P

21 P

32P

15 P

Positive controlsS9-Mix(-)

 

Name Dose Level

No. ofRevertants

ENNO

ENNO

ENNO

4NQO

9AA

3µg

5µg

2 µg

0.2 µg

80µg

373     (336)

318     31.8

184     (143)

134      37.3

330      (338)

357       16.9

195    (200)

195    9.2

211

843    (521)

314     282.6

 

 

318

Ill

326

406

Table3     Test Results:

Experiment 1 - With MetabolicActivation

 

Test Period

From: 05 July 2013

I

To: 08 July 2013

 

 

 

 

 

 

 

 

 

 

 

 

 

 

S9-Mix (+)

Dose Level Per Plate

Number of revertants (mean) +/- SD

Base-oair substitution strains

Frameshift strains

TAlOO

TA1535

WP2uvrA

TA98

TA1537

Solvent Control (Acetone)

74

103

96

(91)

15.1#

16

13

16

 

(15)

1.7

21

32

29

 

(27)

5.7

19

37

16

 

(24)

11.4

9       (11)

7        5.3

17

 

1.5 µg

87

104

(94)

8.7

12

13

 

(15)

4.4

29

25

 

(30)

5.6

25

17

 

(20)

4.2

7       (12)

13       4.6

 

92

 

20

 

 

36

 

 

19

 

 

16

 

5µg

102

96

(97)

4.2

13

19

 

(16)

3.1

27

27

 

(28)

2.3

21

28

 

(24)

3.5

7        (12)

19       6.1

 

94

 

15

 

 

31

 

 

24

 

 

11

 

15 µg

91

90

88

(90)

1.5

13

9

17

 

(13)

4.0

12

27

33

 

(24)

10.8

23

21

37

 

(27)

8.7

11       (14)

12       4.4

19

 

50 µg

79

87

82

(83)

4.0

16

20

 

(18)

2.1

33

24

 

(29)

4.5

17

21

 

(19)

2.0

5         (8)

11        3.1

 

17

 

 

29

 

 

19

 

 

7

 

150 µg

92

90

(93)

3.1

11

13

 

(12)

1.2

23

32

 

(27)

4.5

20

25

28

 

(24)

4.0

13        (14)

19        4.2

 

96

 

11

 

 

27

 

 

11

 

500 µg

94

115

95

(101)

11.8

11

11

 

(14)

4.6

25

27

 

(26)

1.2

13

23

 

(20)

6.4

13       (15)

8        7.6

 

19

 

 

27

 

 

25

 

 

23

 

1500 µg

95P

95P

(90)

9.2

16P

15P

 

(15)

0.6

31P

32P

 

(29)

4.9

15 P

19P

 

(15)

3.5

19P       (21)

21P        2.0

 

79P

 

15 P

 

 

23 P

 

 

12 P

 

 

23 P

 

5000 µg

78P

94P

95P

(89)

9.5

15P

12P

8P

 

(12)

3.5

17P

17P

20P

 

(18)

1.7

24P

20P

35P

 

(26)

7.8

19P      (15)

9P       5.3

17 P

Positive controls S9-Mix (+)

 

Name DoseLevel

No. ofRevertants

2AA

2AA

2AA

BP

2AA

1 µg

2µg

10 µg

5µg

2 µg

732

625

(739)

117.1

160

100

95

 

(118)

36.2

250

215

 

(230)

17.9

138

115

 

(123)

13.0

138     (135)

132    3.1

 

 

859

 

226

 

 

116

 

 

136

 

Table4     Test Results:

Experiment 2 - Without MetabolicActivation

 

Test Period

From: 11July2013                       I                   To: 14 July2013

 

 

 

 

 

 

 

 

S9-Mix

(-)

Dose Level Per Plate

Number ofrevertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TAlOO

TAI535

WP2uvrA

TA98

TA1537

Solvent Control (Acetone)

72        (81)

96       13.3#

74

19         (23)

28         4.5

23

16         (20)

29          7.8

15

23       (16)

12        6.4

12

7        (10)

8         3.8

14

 

50 µg

107        (94)

96        14.1

79

19         (20)

25         4.2

17

11         (19)

21         7.2

25

19        (19)

25        6.0

13

11       (12)

11        2.3

15

 

150 µg

88       (90)

82        9.2

100

13         (17)

17         3.5

20

13         (19)

25          6.0

19

9         (13)

12        4.0

17

5         (7)

8         2.1

9

 

500 µg

74       (77)

80        3.1

78

24         (21)

20         2.6

19

28         (23)

24          5.6

17

23        (22)

16        5.6

27

9        (10)

9         1.7

12

 

1500 µg

87P        (88)

91P         2.6

86P

15P         (18)

21P         3.1

17P

17P        (20)

21P         2.3

21 P

21P      (20)

21P        2.3

17 P

10P      (11)

11p         0.6

11 P

 

5000 µg

86P       (83)

99P      17.2

65 P

17P       (22)

23P        4.2

25 P

13p        (14)

15P        1.2

15 P

24P       (22)

17P       4.4

25 P

8P        (8)

8P         0.0

8P

Positive controlsS9-Mix(-)

 

Name DoseLevel

No. ofRevertants

ENNO

ENNO

ENNO

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

657      (575)

548      72.8

519

1447     (1423)

714      696.8

2107

298       (306)

330        21.2

290

83       (101)

112      15.5

107

441     (355)

323      74.9

302

Table5     Test Results:

Experiment 2 - With MetabolicActivation

 

Test Period

From: 11Julv2013                         I                          To: 14 July2013

 

 

 

 

 

 

 

 

 

 

S9-Mix

(+)

Dose Level Per Plate

Number of revertants (mean)+/-SD

Base-pair substitution strains

Frameshift strains

TAlOO

TA1535

WP2uvrA

TA98

TA1537

Solvent Control (Acetone)

88        (82)

87        9.5#

71

28         (22)

12          9.0

27

35        (33)

40          7.6

25

21        (22)

21         1.2

23

12         (9)

12        4.6

4

 

50 µg

71        (74)

68         7.4

82

13         (14)

11          4.2

19

33        (34)

33          1.7

36

19        (16)

9          6.1

20

3          (9)

13        5.5

12

 

150 µg

95        (89)

79         8.5

92

20         (22)

28          4.9

19

25        (29)

21         10.0

40

23        (18)

11         6.1

19

4          (4)

4         0.6

3

 

500 µg

79        (84)

96        10.1

78

20         (20)

28          8.0

12

23         (29)

33          5.3

31

23        (20)

13         6.1

24

5         (8)

9         3.1

11

 

1500 µg

92P        (91)

88P         3.1

94P

16P        (20)

24P         4.0

20P

41P        (32)

32P         9.0

23 P

16P       (20)

16P       6.4

27P

15P       (10)

8P         4.0

8P

 

5000 µg

82P       (75)

78P         8.9

65 P

23P        (23)

24P        0.6

23 P

27P       (27)

28P        1.5

25 P

23 P     (17)

13P       5.1

16 P

8P       (7)

4P         2.6

9P

Positivecontrols S9-Mix(+)

 

Name DoseLevel

No. ofRevertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

480      (491)

486

508       14.7

184        (195)

195

207        11.5

214       (215)

216

216        1.2

90       (126)

140

148      31.4

225      (207)

225

172       30.6

 

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

4NQO 4-Nitroquinoline-1-oxide

9AA 9-Aminoacridine

BP         Benzo(a)pyrene

2AA    2-Aminoanthracene

P      Test item precipitate

#            Standard deviation


Applicant's summary and conclusion

Conclusions:
GL500 was considered to be non-mutagenic under the conditions of this test.
Executive summary:

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 Bl3/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, 40 CFR 799.9510 TSCA bacterial reverse mutation test and the USA, EPA (TSCA) OCSPP harmonized guidelines.

  

Methods

Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TAlOO andEscherichia colistrainWP2uvrAwere 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 µg/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 50 to 5000µg/plate.

  

Results

The vehicle (acetone) 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 werevalidated.

 

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, in the first mutation test and consequently the same maximum dose level was used in the second mutation test. 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, in the second mutation test. A test item precipitate (globular in appearance) was observed at and above 1500 µg/plate, this observation did not prevent the scoring of revertantcolonies.

 

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 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 in Experiment 2 (pre-incubationmethod).

Conclusion

GL500 was considered to be non-rnutagenic under the conditions of this test.