Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 February 2018 to 21 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
D,L- Menthol / D,L-Isomenthol
Molecular formula:
C10H20O
IUPAC Name:
D,L- Menthol / D,L-Isomenthol

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 Liver homogenates from rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Experiment I (plate incorporation test):
All strains with and without S9: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment II (pre-incubation test):
All strains without S9: 3, 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Strains TA 1535, TA 1537, and TA 100 with S9 mix: 3; 10; 33; 100; 333; and 1000 μg/plate
Strain TA 98 and WP2 uvrA with S9 mix: 10; 33; 100; 333; 1000; and 2500 μg/plate

Top dose concentrations based on results of the initial toxicity test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen based on its solubility properties and its nontoxicity to the bacteria
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (-S9 at 10ug/plate in TA98 and 50 ug/plate in TA1537); methyl methane sulfonate (-S9 at 2.0uL/plate in WP2 uvrA); 2-aminoanthracene (+S9 at 2.5 ug/plate in all strains)
Remarks:
The positive control 2-aminoanthracene was only tested with metabolic activation.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)
- Cell density at seeding: 108-109 cells/mL

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable



NUMBER OF REPLICATIONS: 3 replicate plates were kept in the dark for 48 hours at 37 ºC


DETERMINATION OF CYTOTOXICITY
- Method: manually counting cells and observing the reduction in the number of revertants.
- Any supplementary information relevant to cytotoxicity: Not specified

OTHER EXAMINATIONS: Precipitation of the test item was observed in the incubated overlay agar plate in both experiments. The undissolved particles had no influence on the results.

Rationale for test conditions:
Following standard guidelines
Evaluation criteria:
The number of revertant colonies on the plates, with and without the test compound, were compared to evaluate mutagenicity. A reduction in the number of revertant colonies and/or a diminution of the background lawn was taken as an indication of cytotoxicity.
Statistics:
Not reported

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not specified
- Effects of osmolality: Not specified
- Evaporation from medium: Not specified
- Water solubility: Not specified
- Precipitation: The test item showed signs of precipitation in experiment I (with metabolising system) and II (with metabolising system). The undissolved particulates had no effect on the recorded data.
- Definition of acceptable cells for analysis: Not specified
- Other confounding effects: Not specified


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The data set without S9 are: TA 1535 (334-1816); TA 1537 (43-157); TA 98 (211-627); TA 100 (498-2767); WP2 uvrA. The data set with S9 are: TA 1535 (176-668); TA 1537 (83-434); TA 98 (360-6586); TA 100 (536-6076); WP2 uvrA (167-1265).

- Negative (solvent/vehicle) historical control data: The data set without S9 are: TA 1535 (6-25); TA 1537 (6-19); TA 98 (13-43); TA 100 (78-209); WP2 uvrA (27-63). The data set with S9 are: TA 1535 (7-26); TA 1537 (7-30); TA 98 (15-58); TA 100 (73-208); WP2 uvrA (28-72).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Revertant colonies

Any other information on results incl. tables

Table 1- Induction of revertants in Salmonella typhimurium and Escherichia coli by the test item in the absence of a metabolising system (Experiment I)

Strain

Dose level per plate (µg)

Mean revertants per plate

Standard deviation

Ratio treated/solvent

Individual revertant colony counts

TA 1535

3

9.7

2.5

0.9

7,10, 12

10

10.0

3.6

0.9

14,9,7

33

14.7

1.2

1.4

14,16,14

100

9.0

4.4

0.8

6,7,14

333

4.7

0.6

0.4

5,4,5

1000

8.3

1.2

0.8

7 R, 9 R, 9 R

2500

8.7

1.5

0.8

7 M R,9 M R, 10 MR

5000

6.7

1.2

0.6

6 M R, 6 M R, 8 M R

Solvent control

10.7

1.5

 

9,12,11

Negative control

8.3

1.2

 

9,9,7

TA 1537

3

7.7

1.2

0.9

9,9,7

10

10.7

0.6

1.2

11,10,11

33

5.3

0.6

0.6

6,5,5

100

8.3

2.1

0.9

6,9,10

333

6.7

2.1

0.7

6,5,9

1000

3.7

1.5

0.4

5 R, 2 R, 4 R

2500

6.0

1.0

0.7

5 R, 7 R, 6R

5000

4.3

2.1

0.5

6 R, 5 R, 2 R

Solvent control

9.0

0.0

 

9, 9, 9

Negative control

7.3

2.3

 

6, 6, 10

TA 98

3

19.3

4.7

0.9

14, 23, 21

10

20.3

5.5

0.9

26, 15, 20

33

19.3

4.0

0.9

15, 20, 23

100

24.7

7.5

1.1

25, 17, 32

333

18.7

3.5

0.9

22, 15, 19

1000

21.0

6.2

1.0

14 R, 26 R, 23 R

2500

15.0

5.3

0.7

17 R, 19 R, 9 R

5000

10.0

1.0

0.5

11 R, 10 R, 9 R

Solvent control

21.7

5.8

 

25, 25, 15

Negative control

29.3

7.1

 

37, 28, 23

TA 100

3

136.7

22.9

0.9

163, 121, 126

10

148.3

15.6

1.0

150, 163, 132

33

137.3

15.5

0.9

150, 142, 120

100

154.7

8.7

1.0

145, 157, 162

333

107.0

5.2

0.7

104, 104, 113

1000

32.3

10.0

0.2

40 R, 36 R, 21 R

2500

34.3

8.0

0.2

42 R, 26 R, 35 R

5000

26.7

4.7

0.2

32 R, 25 R, 23 R

Solvent control

153.7

28.3

 

146, 130, 185

Negative control

181.7

3.2

 

183, 178, 184

WP2 uvrA

3

22.0

3.0

0.7

19, 22, 25

10

25.0

4.4

0.8

23, 22, 30

33

30.0

5.3

1.0

26, 28, 36

100

31.0

4.0

1.0

27,31,35

333

27.0

1.0

0.9

26,27,28

1000

23.7

8.3

0.8

21,33,17

2500

22.0

1.0

0.7

21, 33, 22

5000

14.0

3.5

0.5

16, 10, 16

Solvent control

29.7

3.5

 

33, 26, 30

Negative control

37.0

6.1

 

41, 40, 30

M= Manuel count

R= Reduced background growth

P= Precipitate

Table 2- Induction of revertants inSalmonella typhimuriumandEscherichia coliby the test item in the presence of a metabolising system (Experiment I)

Strain

Dose level per plate (µg)

Mean revertants per plate

Standard deviation

Ratio treated/solvent

Individual revertant colony counts

TA 1535

3

11.3

4.0

1.0

7,10, 15

10

12.3

2.9

1.1

14,14,9

33

9.0

3.0

0.8

9,12,6

100

11.7

2.5

1.0

12,9,14

333

10.7

5.5

0.9

16,5,11

1000

3.3

1.5

0.3

3 M R, 5 M R, 2 M R

2500

0.0

0.0

0.0

0 R,0 R, 0 R

5000

0.0

0.0

0.0

0 R P, 0 R P, 0 R P

Solvent control

11.3

5.1

 

7,17,10

Negative control

10.0

1.0

 

10,9,11

TA 1537

3

14.0

1.7

1.2

15,15,12

10

8.7

1.5

0.7

7,10,9

33

10.0

1.0

0.9

9,10,11

100

9.3

3.8

0.8

5,12,11

333

12.0

2.0

1.0

12,14,10

1000

2.3

0.6

0.2

3 M R, 2 M R, 2 M R

2500

0.7

1.2

0.1

0 R, 2 R, 0 R

5000

0.0

0.0

0.0

0 R P, 0 R P, 0 R P

Solvent control

11.7

2.5

 

9, 14, 12

Negative control

9.7

3.2

 

12,11,6

TA 98

3

33.0

8.9

1.3

36, 40,23

10

30.0

2.0

1.2

28,30,32

33

34.3

6.4

1.3

38,38,27

100

26.7

4.7

1.0

23,32,25

333

28.0

11.5

1.1

40,27,17

1000

11.7

2.5

0.5

12 M R, 9 M R, 14 M R

2500

2.3

1.2

0.1

1 M R, 3 M R, 3 M R

5000

0.0

0.0

0.0

0 R P, 0 R P, 0 R P

Solvent control

25.7

5.5

 

31,20,26

Negative control

32.0

4.0

 

36,28,32

TA 100

3

142.3

16.7

1.0

161, 137, 129

10

116.0

4.4

0.8

121, 113, 114

33

123.3

10.1

0.9

122, 134, 114

100

135.3

7.1

1.0

143, 129, 134

333

100.3

 

21.0

0.7

79, 121, 101

1000

27.0

8.2

0.2

36 M R, 25 M R, 20 M R

2500

0.7

0.6

0.0

1 R, 1 R, 0 R

5000

0.0

0.0

0.0

0 R P, 0 R P, 0 R P

Solvent control

137.7

8.6

 

136, 147, 130

Negative control

157.7

14.2

 

173, 155, 145

WP2 uvrA

3

33.0

2.0

1.1

35,33,31

10

30.7

2.3

1.0

28,32,32

33

39.7

8.1

1.3

47,31,41

100

33.7

9.5

1.1

41,23,37

333

27.3

8.1

0.9

36,26,20

1000

23.0

3.6

0.7

22 R, 27 R, 20 R

2500

6.7

0.6

0.2

7 M R, 7 M R, 6 M R

5000

7.3

0.6

0.2

8 M R P, 7 M R P, 7 M R P

Solvent control

31.3

4.6

 

26,40,28

Negative control

40.3

3.8

 

43, 42, 36

M= Manuel count

R= Reduced background growth

P= Precipitate

Table 3- Induction of revertants in Salmonella typhimurium and Escherichia coli by the test item in the absence of a metabolising system (Experiment II)

Strain

Dose level per plate (µg)

Mean revertants per plate

Standard deviation

Ratio treated/solvent

Individual revertant colony counts

TA 1535

3

9.3

4.5

0.8

9,5,14

10

8.7

3.2

0.8

5,10,11

33

10.7

0.6

1.0

10,11,11

100

9.3

2.1

0.8

10,11,7

333

4.0

3.0

0.4

7 R,4 R,1 R

1000

0.3

0.6

0.0

1 R, 0 R, 0 R

2500

0.0

0.0

0.0

0 R, 0 R, 0 R

5000

0.0

0.0

0.0

0 R, 0 R, 0 R

Solvent control

11.0

1.0

 

12,10,11

Negative control

12.0

4.4

 

10,17,9

TA 1537

3

8.7

3.5

1.0

9,12,5

10

7.3

1.5

0.8

9,6,7

33

8.3

3.1

0.9

11,5,9

100

8.7

3.5

1.0

9,5,12

333

5.7

2.9

0.6

9,5,4

1000

0.3

0.6

0.0

0 R, 1 R, 0 R

2500

0.0

0.0

0.0

0 R, 0 R, 0 R

5000

0.0

0.0

0.0

0 R, 0 R ,0 R

Solvent control

9.0

1.7

 

10,10,7

Negative control

10.0

3.6

 

7,14,9

TA 98

3

20.7

1.2

0.8

20,20,22

10

29.0

3.6

1.2

28,33,26

33

24.3

6.4

1.0

28,17,28

100

26.7

2.9

1.1

25,30,25

333

14.7

4.6

0.6

20,12,12

1000

0.7

1.2

0.0

2 R, 0 R, 0 R

2500

0.0

0.0

0.0

0 R, 0 R, 0 R

5000

0.0

0.0

0.0

0 R, 0 R, 0 R

Solvent control

24.3

3.2

 

28,22,23

Negative control

28.0

5.3

 

32,22,30

TA 100

3

120.0

5.2

0.9

126,117,117

10

134.7

16.8

1.0

120,131,153

33

137.0

1.7

1.0

135,138,138

100

120.0

9.5

0.9

111,130,119

333

47.3

4.5

0.3

52,47,43

1000

9.3

2.5

0.1

12 R, 9 R, 7 R

2500

0.3

0.6

0.0

1 R, 0 R, 0 R

5000

0.0

0.0

0.0

0 R,0 R, 0 R

Solvent control

137.3

8.0

 

129,138,145

Negative control

203.7

35.0

 

169,239,203

WP2 uvrA

3

24.0

3.5

0.9

28,22,22

10

24.3

4.0

0.9

25,20,28

33

29.0

2.6

1.1

31,26,30

100

29.3

9.3

1.1

19,32,37

333

18.3

4.2

0.7

15,17,23

1000

0.7

0.6

0.0

0 R, 1 R, 1 R

2500

0.0

0.0

0.0

0 R, 0 R, 0 R

5000

0.0

0.0

0.0

0 R, 0 R, 0 R

Solvent control

27.3

0.6

 

27,27,28

Negative control

31.0

1.0

 

31,32,30

 R= Reduced background growth

Table 4- Induction of revertants in Salmonella typhimurium and Escherichia coli by the test item in the absence of a metabolising system (Experiment II)

Strain

Dose level per plate (µg)

Mean revertants per plate

Standard deviation

Ratio treated/solvent

Individual revertant colony counts

TA 1535

3

10.3

1.5

1.0

12,9,10

10

9.7

3.2

1.0

11,6,12

33

11.3

5.5

1.1

15,5,14

100

11.7

2.9

1.2

15,10,10

333

10.3

4.7

1.0

14,5,12

1000

1.0

1.0

0.1

0 R, 2 R, 1 R

Solvent control

10.0

1.0

 

10,9,11

Negative control

9.7

3.2

 

11,12,6

TA 1537

3

14.0

2.0

0.9

14,16,12

10

16.0

2.6

1.1

15,19,14

33

11.7

0.6

0.8

11,12,12

100

13.0

2.6

0.9

12,11,16

333

7.0

2.0

0.5

9,7,5

1000

0.0

0.0

0.0

0 R, 0 R, 0 R

Solvent control

15.0

2.6

 

12,17,16

Negative control

16.3

4.6

 

19,11,19

TA 98

10

40.7

5.1

1.1

42,35,45

33

42.3

4.0

1.2

47,40,40

100

36.0

7.9

1.0

30,33,45

333

13.3

2.9

0.4

10,15,15

1000

1.3

0.6

0.0

1 R, 1R, 2 R

2500

0.3

0.6

0.0

0 R, 0 R, 1 R

Solvent control

36.7

3.5

 

40, 33, 37

Negative control

46.3

3.8

 

48, 49, 42

TA 100

3

112.0

12.1

0.9

105, 126, 105

10

125.3

18.6

1.0

146, 120, 110

33

127.7

7.1

1.0

129, 134, 120

100

96.3

14.5

1.7

96, 111, 82

333

17.0

3.6

0.1

14, 16, 21

1000

0.3

0.6

0.0

1 R, 0 R, 0 R

Solvent control

130.7

6.1

 

136, 132, 124

Negative control

189.0

1.0

 

190, 188, 189

WP2 uvrA

10

31.7

9.0

0.8

42,27,26

33

36.3

11.5

0.9

36,48,25

100

36.7

5.0

0.9

36,32,42

333

38.3

11.0

0.9

31,33,51

1000

18.0

3.6

0.4

14,21,19

2500

0.0

0.0

0.0

0 R, 0 R, 0 R

Solvent control

41.0

7.0

 

36,38,49

Negative control

46.7

1.5

 

45,48,47

R= Reduced background growth

 

 

Table 5- Concentrations where the test item showed reduced background growth

Strain

Experiment 1

Experiment 2

 

Without S9 mix

With S9 mix

Without S9 mix

With S9 mix

TA 1535

1000-5000

1000-5000

333-5000

1000

TA 1537

1000-5000

1000-5000

1000-5000

1000

TA 98

1000-5000

1000-5000

1000-5000

1000-2500

TA 100

1000-5000

1000-5000

1000-5000

1000

EP2 uvrA

/

1000-5000

1000-5000

2500

 

Table 6- Concentrations where the test item showed toxic effects based on the reduction in the number of revertants 

Strain

Experiment 1

Experiment 2

 

Without S9 mix

With S9 mix

Without S9 mix

With S9 mix

TA 1535

333

1000-5000

333-5000

1000

TA 1537

1000

1000-5000

1000-5000

1000

TA 98

/

2500-5000

1000-5000

333-2500

TA 100

1000-5000

1000-5000

333-5000

333-1000

EP2 uvrA

/

2500-5000

1000-5000

1000-2500

 

Applicant's summary and conclusion

Conclusions:
Under the reported experimental conditions, no biological relevant increase in the number of revertant colonies were observed. Therefore, the test item is considered to not induce mutagenic effects via the processes of base pair changes or frameshifts within the genome of the tested amino stains used up to the highest concentration (5000 µg/plate). 
Executive summary:

The potential mutagenicity of the test item was investigated in an Ames test with the Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102) and Escherichia coli strain (WP2uvrA) in the presence and absence of a metabolising system in the concentration range of 3-5000 µg/plate. Each assay was observed for precipitation, normal background lawn and for the number of revertant colonies. Precipitation was observed in both experiments.

Under the reported experimental conditions, no biological relevant increase in the number of revertant colonies were observed. Therefore, the test item is considered to not induce mutagenic effects via the processes of base pair changes or frameshifts within the genome of the tested amino stains used up to the highest concentration (5000 µg/plate). 

This study (2018) is GLP-compliant and follows the OECD Test Guideline 471 and is therefore reliable without restrictions (Klimisch 1).