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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 July 2010 - 20 September 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Two test item flasks opened for two different uses instead of for only one use, but no major differences between the results obtained with a flask open for only one use and those obtained with a re-used flasks (study validity or integrity not compromised)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
Two test item flasks opened for two different uses instead of for only one use, but no major differences between the results obtained with a flask open for only one use and those obtained with a re-used flasks (study validity or integrity not compromised)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
Each strain derived from S. typhimurium LT2 contains one mutation in the histine operon, resulting in a requirement for histidine.
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:
other: additional mutations in rfa and uvrB genes. For the strains TA98 and TA100 presence of an additional plasmid pKM101 in order to enhance their sensitivity of detection of some mutagens. See Table 1.
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: mutated in rfa gene and presence of an additional plasmid pKM101 in order to enhance the sensitivity of detection of some mutagens. See Table 1.
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route.
Test concentrations with justification for top dose:
Preliminary test: First preliminary test: 10, 100, 500, 1000, 2500, 5000 µg/plate with and without S9 mix.
Second preliminary test: 1 to 2500 µg/plate with and without S9 mix.
Mutagenicity experiments: First experiment = 0.3 to 555.6 µg/plate without S9 mix (see details in Table 2); 6.9 to 5000 µg/plate with S9 mix (see details in Table 2). Second experiment = 0.3 to 185.2 µg/plate with and without S9 mix (see details in Table 2).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO), batch Nos. K39250750906 and K41063050021 (VWR, Fontenay Sous Bois, France).
- Justification for choice of solvent/vehicle: During the solubility assay, the test item was found soluble in DMSO at 50 mg/mL. Consequently, using a treatment volume of 100 µL/plate, it was possible to reach the dose-level of 5000 µg/plate which is the highest recommended dose-level in the international guidelines. DMSO was therefore selected as the vehicle to be used in this study.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-Aminoacridine, 2-Nitrofluorene, Mitomycin C for without S9 mix efficacy control. 2-Anthramine for with S9 mix efficacy control. See details in Table 3.
Remarks:
no remarks
Details on test system and experimental conditions:
METHOD OF APPLICATION:
direct plate incorporation: test item solution (0.1 mL), S9 mix when required or phosphate buffer pH 7.4 (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 50°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
or preincubation method: test item solution (0.1 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C, under shaking, before adding the overlay agar and pouring onto the surface of a minimum agar plate.
DURATION
- Preincubation period: 60 min at 37°C
- Exposure duration: 48 or 72 hours
SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable
NUMBER OF REPLICATIONS: Preliminary assays: two assays were performed: one with one petri dish per dose per strain and the other one with 2 petri dishes/dose/strain. Main tests: 2 independent tests with 3 plates/dose/strain.
NUMBER OF CELLS EVALUATED: not applicable
DETERMINATION OF CYTOTOXICITY
- Method: observation of the decrease in the number of revertant colonies and/or thinning of the bacterial lawn
OTHER EXAMINATIONS:
- Determination of polyploidy: not required
- Determination of endoreplication: not required
OTHER: All the Petri dishes obtained were placed in a sealed jar using one jar for each dose-level tested, one jar for the vehicle control and another jar for the positive controls. The jars were then incubated at 37°C. The precaution of using jars in this study was due to the volatile characteristic of the test item and to limit the oxidation of the test item.
The revertants were scored with an automatic counter (Cardinal counter, Perceptive Instruments, Suffolk CB9 7 BN, UK). Manual counting was used as needed.
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
Statistics:
no data

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See tables 4 to 8
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See tables 4 to 8
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See tables 4 to 8
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See tables 4 to 8
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See tables 4 to 7
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See tables 4 to 7
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See tables 4 to 8
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See tables 4 to 8
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See tables 4 to 7
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See tables 4 to 7
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: For the first preliminary test (Table 9), no precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels. Without S9 mix, a moderate to strong cytotoxicity was observed at dose-levels ≥ 100 µg/plate with the TA 98, TA 100 and TA 102 strains. With S9 mix, a moderate to strong cytotoxicity was observed at dose-levels ≥ 10 µg/plate with the TA 102 strain, at dose-levels ≥ 100 µg/plate with the TA 100 strain and at dose-levels ≥ 500 µg/plate with the TA 98 strain.
Based on the cytotoxicity results obtained with one plate/dose in the first preliminary test, a second test was performed using two plates/dose and a lower range of dose-levels (Table 10). No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels. Without S9 mix, a moderate to strong cytoxicity was observed at dose-levels ≥ 10 µg/plate with the TA 100 strain, at dose-levels ≥ 50 µg/plate with the TA 98 strain and at dose-levels ≥ 100 µg/plate with the TA 102 strain. With S9 mix, a moderate cytotoxicity (thinning of the bacterial lawn) was observed at dose-levels ≥ 750 µg/plate with the TA 98 strain, and a moderate to strong cytotoxicity was observed at dose-levels ≥ 250 µg/plate with the TA 100 strain and at 500 µg/plate with the TA 102 strain.

COMPARISON WITH HISTORICAL CONTROL DATA: The numbers of revertants for the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Without metabolic activation, in the first experiment (Table 4), a moderate to strong toxicity was noted at dose-levels  61.7 µg/plate in the TA 102 strain and a strong toxicity was noted at dose-levels  61.7 µg/plate in all the other strains.
In the second experiment (Table 6), a moderate toxicity was noted at 185.2 µg/plate in the TA 102 strain and a strong toxicity was noted at 61.7 µg/plate in all the other strains.
With metabolic activation, in the first experiment (Table 5), using the direct plate incorporation method, a moderate to strong toxicity was noted at dose-levels  555.6 µg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains and a moderate toxicity was noted at 1666.7 µg/plate in the TA 102 strain.
In the second experiment performed using the preincubation method, a moderate to strong toxicity was observed at dose-levels  20.6 µg/plate in all the tested strains (Table 8). Since this toxicity was noted at five out of six dose-levels, less than five analysable dose-levels were obtained in this experiment. This requirement of the international guidelines being not met, the results obtained were not retained and the treatment was repeated using lower ranges of dose levels (Table 7). In this second assay using the preincubation method with lower doses, a moderate toxicity was noted at 61.7 µg/plate in the TA 1535 and TA 100 strains, and a strong toxicity was observed at 61.7 µg/plate in the TA 98 strain. No noteworthy toxicity was induced in the TA 1537 and TA 102 strains up to the highest tested dose-levels of 61.7 and 185.2 µg/plate, respectively.

Any other information on results incl. tables

Table 4: Number of revertants per plate in the absence of metabolic activation in the first test (direct plate incorporation method)

 

Laevo Limonene Concentration
(µg/plate)

TA 1535

TA1537

TA 98

TA 100

TA 102

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

0*

15

6

11

6

31

8

124

39

380

25

0.3

18

2

-

-

-

-

144

18

-

-

0.8

22

3

10

5

26

4

159

59

-

-

2.3

21

4

7

2

22

4

114

8

349

25

6.9

24

7

16

3

23

7

114

13

430

56

20.6

14

3

11

2

25

4

101

22

426

19

61.7

7St

3

4St

2

12St

5

43St

21

199Mt

9

185.2

-

-

0St

1

7St

0

-

-

232St

6

555.6

-

-

-

-

-

-

-

-

209St

3

Positive control**

953

69

400

154

128

10

922

4

2009

329

$: Mean of triplicate

*Solvent control = negative control: DMSO

**Mutagens positive controls:

- NaN3(1 µg/plate) in TA1535 and TA100 strains

- 9AA (50 µg/plate) in TA1537 strain

- 2NF (0.5 µg/plate) in TA 98 strain

- MMC ( 0.5 µg/plate) in TA 102 strain

Mt: Moderate cytoxicity

St: Strong cytotoxicity 

 

Table 5:Number of revertants per plate in the presence of metabolic activation (S9 mix) in the first test (direct plate incorporation method) 

 

Laevo Limonene Concentration
(µg/plate)

TA 1535

TA1537

TA 98

TA 100

TA 102

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

0*

16

6

8

3

35

12

121

27

427

46

6.9

22

9

-

-

-

-

116

9

423

60

20.6

18

3

14

8

32

8

139

11

479

140

61.7

19

6

13

5

29

5

127

10

504

116

185.2

12

6

15

3

34

13

118

23

487

94

555.6

11Mt

2

9Mt

3

23Mt

6

111Mt

5

376

69

1666.7

12St

5

13Mt

4

22St

8

106St

15

511Mt

12

5000

-

-

3St

5

14St

16

-

-

-

-

Positive control**

410

50

126

10

855

196

242

13

2202

20

$: Mean of triplicate

*Solvent control = negative control: DMSO

**Mutagens positive controls:

- 2AM (2µg/plate) in TA1535, TA1537, TA98

- 2AM (10µg/plate) in TA102 strain

- BAP (5µg/plate) in TA100 strain

Mt: Moderate cytotoxicity

St: Strong cytotoxicity

  

Table 6:Number of revertants per plate in the absence of metabolic activation in the second test (direct plate incorporation method)

Laevo Limonene Concentration
(µg/plate)

TA 1535

TA1537

TA 98

TA 100

TA 102

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

0*

26

11

10

3

24

7

106

8

417

22

0.3

25

4

12

5

39

8

114

7

-

-

0.8

33

3

11

3

28

4

108

13

424

48

2.3

24

1

21

6

27

8

101

13

439

31

6.9

23

12

14

2

25

6

91

10

416

100

20.6

32

14

24

7

22

6

103

15

413

19

61.7

27St

9

6St

2

14St

7

103St

10

390

40

185.2

-

-

-

-

-

-

-

-

344Mt

30

Positive control**

706

99

193

87

141

20

778

61

1524

220

$: Mean of triplicate

*Solvent control = negative control: DMSO

- NaN3(1 µg/plate) in TA1535 and TA100 strains

- 9AA (50 µg/plate) in TA1537 strain

- 2NF (0.5 µg/plate) in TA 98 strain

- MMC ( 0.5 µg/plate) in TA 102 strain

Mt: Moderate cytotoxicity

St: Strong cytotoxicity 

 

Table 7: Number of revertants per plate in the presence of metabolic activation (S9 mix) in the second test (pre-incubation method)

 

Laevo Limonene Concentration
(µg/plate)

TA 1535

TA1537

TA 98

TA 100

TA 102

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

0*

21

4

11

5

34

8

108

12

428

71

0.3

19

5

14

2

32

5

121

8

-

-

0.8

18

2

13

6

38

10

142

9

527

115

2.3

20

5

8

6

30

7

129

6

571

34

6.9

16

5

14

4

36

5

138

21

511

54

20.6

22

2

13

3

34

5

130

14

480

93

61.7

18Mt

5

10

3

29St

7

127Mt

15

452

131

185.2

-

-

-

-

-

-

-

-

496

122

Positive control**

230

30

164

45

832

56

325

8

1577

175

$: Mean of triplicate

*Solvent control = negative control: DMSO

**Mutagens positive controls:

- 2AM (2µg/plate) in TA1535, TA1537, TA98 strains

- 2AM (10µg/plate) in TA102 strain

- BAP (5 µg/plate) in TA100 strain

Mt: Moderate cytotoxicity

St: Strong cytotoxicity

Table 8: Number of revertants per plate in the presence of metabolic activation (S9 mix) in the second test (pre-incubation method): this assay was considered non-valid as less than five analyzable dose-levels were obtained in this experiment (Remark: TA102 was not tested in this experiment).

 

Laevo Limonene Concentration
(µg/plate)

TA 1535

TA1537

TA 98

TA 100

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

0*

21

4

13

4

39

8

131

4

6.9

25

7

16

6

48

3

133

22

20.6

17 Mt

3

17 Mt

3

41 St

13

103 St

8

61.7

4 St

3

9 St

6

17 St

5

89 St

9

185.2

10 St

1

13 St

4

31 St

7

95 St

6

555.6

10 St

5

0 X+St

0

0 St

0

6 St

10

1666.7

0 St

0

1 St

1

5 St

2

1 St

1

Positive control**

189

9

170

24

1014

28

451

32

$: Mean of triplicate

*Solvent control = negative control: DMSO

**Mutagens positive controls:

- 2AM (2µg/plate) in TA1535, TA1537, TA98 strains

- BAP (5 µg/plate) in TA100 strain

Mt: Moderate cytotoxicity

St: Strong cytotoxicity

X: too many microcolonies

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the test item Laevo Limonene did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains.Laevo Limonene is not considered as mutagenic in this bacterial system according to the criteria of the Annex VI of the CLP Regulation (EC) N° (1272-2008).
Executive summary:

In a reverse gene mutation assay in bacteria (No. 36956 MMO), performed according to the OECD No.471 and EC No.B13/14 guidelines, Laevo Limonene (purity of 81.1%) diluted in Dimethylsulfoxide (DMSO) was tested in S. typhimurium TA1535, TA1537, TA100, TA98 and TA102 in the presence and the absence of mammalian metabolic activation (S9) using the direct plate incorporation or the preincubation method. All the concentrations and dose-levels were expressed as active item, taking into account the purity of the test item (81.1%).

Due to the volatile characteristic of the test item and in order to limit the oxidation of the test item, all the Petri dishes were placed in a sealed jar. One jar was used for each tested dose-level, one jar was used for the vehicle control and another jar for the positive controls. 

Six known mutagens, dissolved in dimethylsulfoxide (except for Mitomycin C which was dissolved in distilled water), were used to check the sensitivity of the test system. The positive controls induced the appropriate responses in the corresponding strains. The number of revertants in the vehicle controls was consistent with the historical data of the testing facility, and the number of revertants in the positive controls was higher than that of the vehicle controls (at least 2-fold increase for the TA 98, TA 100 and TA 102 strains and at least 3-fold increase for the TA 1535 and TA 1537 strains) and was consistent with the historical data of the testing facility. Therefore the study was considered valid.

Two independent preliminary tests were performed. Since the test item was cytotoxic (observation of a decrease in the number of revertant colonies and of thinning of the bacterial lawn) in these preliminary tests for all strains with or without metabolic activation, the choice of the highest dose-level to be used in the main test was based on the level of toxicity, according to the criteria specified in the international guidelines.

Two independent main experiments with and without metabolic activation were performed. In the second main experiment, two assays in presence of S9 mix according to the preincubation method were necessary. Indeed, in the first assay with S9 mix using this specific method, a moderate to strong toxicity was observed at dose-levels higher than 20.6 µg/plate in TA1535, TA1537, TA98 and TA100. Since this cytotoxicity was noted at five out of six dose-levels, less than five analyzable dose-levels were obtained in this assay. The results were therefore not retained and the treatment was repeated using lower ranges of dose-levels. In the second treatment with S9 mix using the preincubation method, there was sufficient analysable doses and the assay was considered as acceptable.

During the two main tests, no induced revertant over background was observed in any strains of S. typhimurium with or without metabolic activation whereas the cytotoxic dose-level was reached.

In conclusion, under the test conditions, Laevo Limonene did not show any mutagenic activity in the bacterial reverse mutation test using Salmonella typhimurium. Laevo Limonene is not considered as mutagenic in this bacterial system according to the criteria of the Annex VI of CLP Regulation (EC) N° (1272-2008) .

 

This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 471.