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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Ames test: This study was performed to investigate the potential of the test substance to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA98, TA 100 and TA 102. The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore the test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay. Chromosom aberration test: The test item dissolved in DMSO, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments. The test item did not induce structural chromosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro. Therefore, Muguetalkohol is considered to be non-clastogenic in this chromosome aberration test when tested up to the highest evaluable concentrations. Mouse lymphoma assay: The study was performed to investigate the potential of the test item to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activatio. Therefore, the test substance is considered to be non-mutagenic in this mouse lymphoma assay.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-02-15 to 2008-02-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver S-9 mix
Test concentrations with justification for top dose:
3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
DMSO and deionised water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water deionised
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA1535, TA100 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 4-NOPD
Remarks:
TA 1537, TA98 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water deionised
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA102 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
All strains with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation), preincubation

2 experiments: Preincubation Test and Standard Plate test

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 hours

SELECTION AGENT: Histidine

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency
Evaluation criteria:
Considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice or thrice the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the trehsold at only one concnetration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependant increase in the number of revertant colonies below the treshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
other: negative controls valid, true negative controls not examined
Positive controls validity:
valid
Additional information on results:
Due to contamination of strain TA 1537 in experiment I not data could be obtained and this part was repeated under identical conditions (reported as experiment I). Each concnetration, including the contorls, was tested in triplicate.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation. There was aalso no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

TA1535

TA1537

TA98

TA100

TA102

Concentration (µg/Plate)

I

II

I

II

I

II

I

II

I

II

Negative

12

17

15

16

18

31

127

133

438

363

Solvent Control

13

15

17

11

28

31

121

112

463

365

Positive Control

2066

1848

88

124

364

578

2298

2175

4455

1937

3

15

14

15

9

23

31

125

119

386

315

10

11

14

14

11

25

27

132

111

463

365

33

13

13

20

16

21

26

142

114

466

366

100

16

15

15

13

29

27

120

108

464

352

333

16

18

14

10

23

27

110

107

302

265

1000

12

10

9

6

25

9

73

72

91

90

2500

1

2

0

0

0

0

0

0

1

0

5000

0

0

0

0

0

0

0

0

0

0

with S9 Mix

TA1535

TA1537

TA98

TA100

TA102

Concentration (µg/Plate)

I

II

I

II

I

II

I

II

I

II

Negative

16

20

21

21

35

45

150

133

509

435

Solvent Control

19

20

27

21

30

33

147

129

550

460

Positive Control

329

260

209

151

2292

1340

2969

1619

2938

2448

3

14

16

24

17

30

39

135

118

439

372

10

11

18

20

21

34

35

134

124

506

377

33

15

17

28

18

32

35

123

142

500

398

100

18

19

18

18

34

39

156

129

501

406

333

13

15

24

17

32

41

119

116

385

305

1000

12

4

20

2

27

14

99

6

189

7

2500

1

0

0

0

0

0

7

0

1

0

5000

0

0

0

0

0

0

0

0

0

0

Conclusions:
Interpretation of results (migrated information):
negative

It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore the test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of the test substance to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Due to contamination of strain TA 1537 in experiment I no data could be obtained and this part was repeated under identical conditions (reported as experiment I) Each concentration, including the controls was tested in triplicate. The test item was tested at the following concentrations:

3, 10, 33, 100, 33, 1000, 2500 and 5000 µg/plate.

Reduced background growth was observed in all strains with and without metabolic activation in both experiments.

Toxic effects, evident as reduction of the number of revertants (below the indicaiton factor of 0.5) were observed with and without metabolic activation in all strains used in both experiments. No substantial increase inrevertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 -Mix). There was also no tendency of higher mutaiton rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore the test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Ames test

A study was performed to investigate the potential of the test substance to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Due to contamination of strain TA 1537 in experiment I no data could be obtained and this part was repeated under identical conditions (reported as experiment I). Each concentration, including the controls was tested in triplicate. The test item was tested at the following concentrations:

3, 10, 33, 100, 33, 1000, 2500 and 5000 µg/plate.

Reduced background growth was observed in all strains with and without metabolic activation in both experiments. Toxic effects, evident as reduction of the number of revertants (below the indicaiton factor of 0.5) were observed with and without metabolic activation in all strains used in both experiments.Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore the test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Chromosome aberration test

The test item, dissolved in DMSO, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments. In each experimental group two parallel cultures were analysed. Per culture 100 metaphase plates were scored for structural chromosomal aberrations. In this study, either with or without metabolic activation no clastogenicity was observed at the concentrations evaluated. However, a dose-dependent increase of chromosomal aberrations could be observed in Experiment I in the presence of S9 mix after pulse treatment (1.0, 1.5, 2.5 % aberrant cells, excluding gaps). The values were within the range of the laboratory’s historical control data (0.0 – 4.0 % aberrant cells, excluding gaps) and considered as biologically irrelevant. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Mouse lymphoma assay

The study was performed to investigate the potential of the test item to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation. The concentration range of both main experiments was limited by cytotoxic effects of the test item. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximum concentration of the test item. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

Conclusion: Three OECD guideline studies were performed determining the genetic toxicity of the test item in an Ames test, one Chromosome aberration test and one Mouse lymphoma assay. No genetic toxicity was observed in all three studies.


Justification for selection of genetic toxicity endpoint
GLP and guideline study

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the test substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008.