Registration Dossier

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 01 March 2017 and 04 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted and well described study in accordance with GLP and OECD guideline 471 without any deviation

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)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
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
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Identification: Lyrame
Chemical Name: Reaction mass of methyl 2-({(E)-[4-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1-ylidene]methyl}amino)benzoate and methyl 2-({(E)-[3-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1-ylidene]methyl}amino)benzoate and methyl 2-({(Z)-[4-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1-ylidene]methyl}amino)benzoate and methyl 2-({(Z)-3(4-hydroxy-4-methylpentyl)cyclohex-3-en-1-ylidene]methyl}amino)benzoate
Physical state/Appearance: Yellow viscous liquid
Batch: SM16066125
Purity: Mixture, tested as supplied (100%)
Expiry Date: 07 June 2019
Storage Conditions: Approximately 4 °C in the dark under nitrogen
No correction was required for purity because the test item was considered a mixture.

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No. 17 February 2017 was used in this study.
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 g/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 g/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Test for Mutagenicity: Confirmatory Experiment 1 – Plate Incorporation Method
Dose selection
The dose range was the same as Experiment 1 (1.5 to 5000 µg/plate).

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 15 to 5000 µg/plate.

Vehicle / solvent:
The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in house. Dimethyl sulphoxide was selected as the vehicle
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
The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No. 17 February 2017 was used in this study.

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 6147883 03/21) 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 43934 04/17, 43923 04/17 and 44018 05/17).

Test System and Supporting Information
Bacteria
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:
• University of California, Berkeley, on culture discs, on 04 August 1995.
• 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 Statebourne 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 number 1865318 05/21) 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.

Experimental Design and Study Conduct

Test Item Preparation and Analysis
The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in house. Dimethyl sulphoxide was selected as the vehicle.
The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 10 minutes at 40 °C on the day of each experiment. No correction was required for purity because the test item was a mixture and was tested as supplied. 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.

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method
Dose selection
The test item was tested using the following method. The maximum concentration was 5000 g/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 0.1 mL of 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 5000 µg/plate (absence of S9-mix only) because of a test item film. A number of further manual counts were also performed due to colonies spreading, thus distorting the actual plate count.

Test for Mutagenicity: Confirmatory Experiment 1 – Plate Incorporation Method
A confirmatory experiment was performed, in triplicate, to attain reproducibility in response to small but statistically significant increases TA1537 revertant colony frequency (absence of S9-mix only) noted in the first mutation test. The experiment was performed using the plate incorporation method.

Dose selection
The dose range was the same as Experiment1 (1.5 to 5000 µg/plate).

Without Metabolic Activation
The procedure was the same as described previously

With Metabolic Activation
The procedure was the same as described previously .
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 5000 µg/plate because of a test item film.

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 15 to 5000 µg/plate.
Six test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of 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, solvent vehicle or 0.1 mL of 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 (see 3.3.3.2) 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). A number of manual counts were performed due to colonies spreading, thus distorting the actual plate count.

Acceptability 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), Mortelmans and Zeiger (2000), Green and Muriel (1976) and Mortelmans and Riccio (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:
TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60

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
Positive controls validity:
valid
Additional information on results:
Mutation Test
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), Table 4 for Experiment 1 (confirmatory, plate incorporation) and Table 5 and Table 6 for Experiment 2 (pre-incubation).

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 was used in the second mutation test. The test item did, however, induce toxicity to the bacterial background lawns in the second mutation test after employing the pre-incubation modification with weakened bacterial background lawns noted in the absence of S9-mix from 1500 µg/plate (TA100, TA1537 and TA1535) and at 5000 µg/plate (TA98). No visible reductions in the growth of the bacterial background lawns were noted to any of the Salmonella strains dosed in the presence of S9 mix and Escherichia coli strain WP2uvrA dosed in the presence and absence of S9-mix. In the first mutation test (plate incorporation method) a test item film (creamy in appearance) was noted by eye at 5000 µg/plate to all of the strains dosed in the absence of S9-mix only. In the second mutation test (pre-incubation method) a test item precipitate rather than a film was noted (globular in appearance) by eye at 5000 µg/plate in the absence of S9-mix only. The film/precipitate observations in each experiment did not prevent the scoring of revertant colonies.

There were no toxicologically meaningful 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). Small, statistically significant increases in revertant colony frequency were observed in the first mutation test at 1500 µg/plate for TA100 (absence of S9-mix) and at 500 and 1500 µg/plate for TA1537 (absence of S9-mix). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility (a confirmatory test was performed for TA1537 which showed no evidence of a mutagenic response). Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase only just achieved a twofold response (TA1537 at 1500 µg/plate) over the concurrent vehicle controls.

Similarly, no toxicologically meaningful 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). A small but statistically significant increase in revertant colony frequency was observed in the second mutation test at 5000 µg/plate for WP2uvrA (absence of S9-mix). Again, this increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 5000 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.3 times the concurrent vehicle control.

The vehicle (dimethyl sulphoxide) 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 thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.


Any other information on results incl. tables

Table1            Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1 Plate-Incorporation

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535†

WP2uvrA

TA98

TA1537

109

 

16

 

35

 

26

 

10

 

96

(109)

20

(16)

25

(27)

26

(27)

7

(8)

122

 

11

 

21

 

30

 

8

 

 

Confirmatory Test – Plate-Incorporation

Number of revertants (mean number of colonies per plate)

Frameshift type

TA1537

9

 

11

(9)

8

 

Experiment 2 Pre-Incubation

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

88

 

30

 

27

 

26

 

19

 

81

(82)

17

(24)

35

(35)

26

(28)

14

(15)

77

 

26

 

43

 

33

 

13

 

 

Table2            Test Results: Experiment 1 – Without Metabolic Activation(Plate incorporation)

Test Period

From: 10 March 2017

16 March 2017†

To: 13 March 2017

19 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

(DMSO)

107

94

123

(108)

14.5#

9

15

15

(13)

3.5

15

25

30

(23)

7.6

27

19

19

(22)

4.6

6

10

11

(9)

2.6

1.5 µg

114

102

106

(107)

6.1

11

19

8

(13)

5.7

21

21

29

(24)

4.6

18

16

22

(19)

3.1

9

15

8

(11)

3.8

5 µg

98

109

117

(108)

9.5

14

11

12

(12)

1.5

26

18

19

(21)

4.4

17

11

20

(16)

4.6

7

13

8

(9)

3.2

15 µg

104

105

97

(102)

4.4

15

14

14

(14)

0.6

12

24

19

(18)

6.0

14

28

22

(21)

7.0

11

9

11

(10)

1.2

50 µg

109

103

113

(108)

5.0

15

16

17

(16)

1.0

28

22

25

(25)

3.0

15

19

18

(17)

2.1

11

10

5

(9)

3.2

150 µg

100

88

117

(102)

14.6

15

12

13

(13)

1.5

42

29

13

(28)

14.5

14

15

15

(15)

0.6

13

11

8

(11)

2.5

500 µg

125

109

123

(119)

8.7

17

13

16

(15)

2.1

22

22

29

(24)

4.0

22

18

20

(20)

2.0

15

17

19

*

(17)

2.0

1500 µg

148

127

133

*

(136)

10.8

14

12

12

(13)

1.2

26

27

27

(27)

0.6

25

30

20

(25)

5.0

25

15

17

**

(19)

5.3

5000 µg

105 F

118 F

131 F

(118)

13.0

14 F

12 F

11 F

(12)

1.5

18 F

19 F

18 F

(18)

0.6

16 F

21 F

17 F

(18)

2.6

12 F

11 F

15 F

(13)

2.1

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

617

683

784

(695)

84.1

328

301

307

(312)

14.2

1051

1015

1037

(1034)

18.1

87

107

117

(104)

15.3

211

337

193

(247)

78.5

ENNG4NQO9AAF***#

Table3            Test Results: Experiment 1 – With Metabolic Activation(Plate incorporation)

Test Period

From: 10 March 2017

16 March 2017†

To: 13 March 2017

19 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

(DMSO)

106

126

127

(120)

11.8#

9

11

10

(10)

1.0

37

36

31

(35)

3.2

21

21

33

(25)

6.9

14

17

13

(15)

2.1

1.5 µg

114

112

105

(110)

4.7

10

10

11

(10)

0.6

34

20

35

(30)

8.4

28

29

30

(29)

1.0

14

23

11

(16)

6.2

5 µg

97

114

95

(102)

10.4

10

11

12

(11)

1.0

44

30

30

(35)

8.1

27

23

18

(23)

4.5

11

13

15

(13)

2.0

15 µg

109

111

110

(110)

1.0

9

10

11

(10)

1.0

28

32

31

(30)

2.1

25

27

26

(26)

1.0

17

15

18

(17)

1.5

50 µg

109

91

97

(99)

9.2

11

9

10

(10)

1.0

16

46

31

(31)

15.0

35

23

23

(27)

6.9

9

15

12

(12)

3.0

150 µg

105

91

96

(97)

7.1

13

10

12

(12)

1.5

15

22

17

(18)

3.6

38

13

26

(26)

12.5

11

5

11

(9)

3.5

500 µg

108

94

113

(105)

9.8

10

12

9

(10)

1.5

30

22

11

(21)

9.5

23

35

30

(29)

6.0

13

14

16

(14)

1.5

1500 µg

96

120

147

(121)

25.5

7

10

10

(9)

1.7

16

19

21

(19)

2.5

23

25

20

(23)

2.5

13

19

16

(16)

3.0

5000 µg

138

112

115

(122)

14.2

10

8

9

(9)

1.0

33

33

24

(30)

5.2

21

29

29

(26)

4.6

18

14

11

(14)

3.5

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

2380

2324

2451

(2385)

63.6

277

269

258

(268)

9.5

284

289

310

(294)

13.8

268

222

249

(246)

23.1

492

438

468

(466)

27.1

BP2AA #

 


 

Table4            Confirmatory Experiment – Without Metabolic Activation (Plate Incorporation)

Test Period

From: 21 March 2017

To: 24 March 2017

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Frameshift Strains

Without S9-mix

TA1537

Solvent Control

(DMSO)

5

10

13

(9)

4.0

1.5 µg

4

14

7

(8)

5.1

5 µg

14

10

12

(12)

2.0

15 µg

13

9

9

(10)

2.3

50 µg

8

7

11

(9)

2.1

150 µg

7

8

4

(6)

2.1

500 µg

8

7

10

(8)

1.5

1500 µg

13

12

12

(12)

0.6

5000 µg

14 F

12 F

12 F

(13)

1.2

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

9AA

80 µg

304

198

259

(254)

53.2

9AAF#

 

 

Table5            Test Results: Experiment 2 – Without Metabolic Activation(Pre-Incubation)

Test Period

From: 31 March 2017

To: 03 April 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

(DMSO)

109

112

100

(107)

6.2#

22

15

15

(17)

4.0

33

30

30

(31)

1.7

17

25

26

(23)

4.9

11

12

13

(12)

1.0

15 µg

79

92

114

(95)

17.7

30

24

9

(21)

10.8

41

36

34

(37)

3.6

17

13

33

(21)

10.6

10

8

19

(12)

5.9

50 µg

94

98

104

(99)

5.0

18

13

23

(18)

5.0

32

44

41

(39)

6.2

17

20

26

(21)

4.6

20

6

16

(14)

7.2

150 µg

100

100

96

(99)

2.3

14

8

14

(12)

3.5

32

38

43

(38)

5.5

30

23

32

(28)

4.7

7

9

22

(13)

8.1

500 µg

90

105

101

(99)

7.8

20

12

16

(16)

4.0

38

40

38

(39)

1.2

17

26

22

(22)

4.5

11

7

4

(7)

3.5

1500 µg

118 S

101 S

97 S

(105)

11.2

16 S

18 S

9 S

(14)

4.7

37

39

40

(39)

1.5

35

28

25

(29)

5.1

7 S

7 S

10 S

(8)

1.7

5000 µg

71 PS

63 PS

72 PS

(69)

4.9

0 PV

0 PV

0 PV

(0)

0.0

44 P

42 P

33 P

*

(40)

5.9

19 PS

19 PS

28 PS

(22)

5.2

0 PV

0 PV

0 PV

(0)

0.0

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

919

722

612

(751)

155.5

700

583

726

(670)

76.2

640

651

531

(607)

66.3

238

270

223

(244)

24.0

643

601

308

(517)

182.5

ENNG4NQO9AAPSV*#

 

Table6            Test Results: Experiment 2 – With Metabolic Activation

Test Period

From: 31 March 2017

To: 03 April 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

(DMSO)

77

90

79

(82)

7.0#

14

17

15

(15)

1.5

44

36

37

(39)

4.4

23

34

22

(26)

6.7

13

14

9

(12)

2.6

15 µg

81

75

98

(85)

11.9

20

9

12

(14)

5.7

52

37

39

(43)

8.1

22

18

26

(22)

4.0

4

12

3

(6)

4.9

50 µg

92

77

88

(86)

7.8

8

12

11

(10)

2.1

46

42

51

(46)

4.5

16

21

19

(19)

2.5

24

12

12

(16)

6.9

150 µg

91

102

76

(90)

13.1

9

7

7

(8)

1.2

43

43

27

(38)

9.2

35

18

20

(24)

9.3

15

16

13

(15)

1.5

500 µg

83

87

91

(87)

4.0

9

11

12

(11)

1.5

28

40

43

(37)

7.9

27

24

33

(28)

4.6

2

2

7

(4)

2.9

1500 µg

103

87

110

(100)

11.8

20

14

9

(14)

5.5

34

44

45

(41)

6.1

30

26

36

(31)

5.0

10

5

8

(8)

2.5

5000 µg

88

103

107

(99)

10.0

12

9

7

(9)

2.5

50

35

35

(40)

8.7

31

28

34

(31)

3.0

9

11

12

(11)

1.5

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

561

1219

978

(919)

332.9

203

180

189

(191)

11.6

206

233

231

(223)

15.0

137

139

131

(136)

4.2

314

313

281

(303)

18.8

BP2AAP


             Experimental procedure repeated at a later date (with and without S9-mix) due to contamination in the original test

†                         Experimental procedure repeated at a later date due to contamination in the original test

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

4NQO         4-Nitroquinoline-1-oxide

9AA           9-Aminoacridine

F              Test Item Film

*               p£0.05

**              p£0.01

#               Standard deviation

             Experimental procedure repeated at a later date due to contamination in the original test

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

#            Standard deviation

9AA           9-Aminoacridine

F              Test Item Film

#               Standard deviation

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

4NQO         4-Nitroquinoline-1-oxide

9AA           9-Aminoacridine

P               Test item precipitate

S                        Sparse bacterial background lawn

V                       Very weak bacterial background lawn

*               p£0.05

#               Standard deviation

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

P            Test item precipitate

Applicant's summary and conclusion

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

The test method was designed to be compatible with 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 and Escherichia coli strain WP2uvrA were 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. Sixtest item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

A confirmatory experiment was also performed, in triplicate, in response to small increases in TA1537 revertant colony frequency (absence of S9-mix only) noted in the first mutation test. The experiment utilised a dose range of 1.5 to 5000 µg/plate using the plate incorporation method.

 Results

The vehicle (dimethyl sulphoxide) 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 was used in the second mutation test. The test item did, however, induce toxicity to the bacterial background lawns in the second mutation test after employing the pre-incubation modification with weakened bacterial background lawns noted in the absence of S9-mix from 1500 µg/plate (TA100, TA1537 and TA1535) and at 5000 µg/plate (TA98). No visible reductions in the growth of the bacterial background lawns were noted to any of theSalmonellastrains dosed in the presence of S9‑mix andEscherichia colistrain WP2uvrAdosed in the presence and absence of S9-mix. In the first mutation test (plate incorporation method) a test item film (creamy in appearance) was noted by eye at 5000 µg/plate to all of the strains dosed in the absence of S9-mix only. In the second mutation test (pre-incubation method) a test item precipitate rather than a film was noted (globular in appearance) by eye at 5000 µg/plate in the absence of S9-mix only. The film/precipitate observations in each experiment did not prevent the scoring of revertant colonies.

There were no toxicologically meaningful 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). Small, statistically significant increases in revertant colony frequency were observed in the first mutation test at 1500 µg/plate for TA100 (absence of S9-mix) and at 500 and 1500 µg/plate for TA1537 (absence of S9-mix). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility (a confirmatory test was performed for TA1537 which showed no evidence of a mutagenic response). Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase only just achieved a twofold response (TA1537 at 1500 µg/plate) over the concurrent vehicle controls. 

Similarly, no toxicologically meaningful 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). A small but statistically significant increase in revertant colony frequency was observed in the second mutation test at 5000 µg/plate for WP2uvrA(absence of S9-mix). Again, this increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 5000 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.3 times the concurrent vehicle control.

Conclusion

Lyramewas considered to be non-mutagenic under the conditions of this test.