Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer-reviewed journal.

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Gene mutation toxicity study of the test chemical
Author:
Sekizawa, J., Shibamoto, T.
Year:
1982
Bibliographic source:
Mutation Research, 1982
Reference Type:
publication
Title:
Gene mutation toxicity study of the test chemical
Author:
D. Bickers et. al.
Year:
2005
Bibliographic source:
Food and Chemical Toxicology (2005)

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
In vitro bacterial reverse gene mutation assay for test chemical was studied in S. typhimurium and Escherichia coli strains. The mutagenicity assay was conducted as described by Ames et al. with slight modifications. All tester strains were examined periodically for the markers indicated by Ames et al.



GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
Oily liquid
Details on test material:
- Name of test material (as cited in study report): Cinnamyl alcohol
- IUPAC name : 3-Phenylprop-2-en-1-ol
- Molecular Formula: C9H10O
- Molecular Weight: 134.177 g/mol
- Smiles: c1(ccccc1)/C=C/CO
- InChI: 1S/C9H10O/c10-8-4-7-9-5-2-1-3-6-9/h1-7,10H,8H2/b7-4+
- Substance type: Organic
- Physical state: Crystalline solid

Method

Target gene:
Histidine for Salmonella strains and tryptophan for E. coli strains
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium, other: TA100, TA1535, TA98, TA1537, TA1538
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: S9 mix (500 µM) consisted of 100µL of S9, 50 µmoles sodium phosphate buffer (pH 7.4), 4 µmoles MgCl2, 16.5 µmoles KCl, 2.5 µmoles G-6-P, 2 µmoles NADH and 2 µmoles NADPH.
- source of S9 : PCB-treated male Sprague-Dawley rats
- method of preparation of S9 mix : No data
- concentration or volume of S9 mix and S9 in the final culture medium : No data
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data
Test concentrations with justification for top dose:
0, 250, 750, 1500 or 3000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: No data available
Controlsopen allclose all
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
9-aminoacridine
2-nitrofluorene
sodium azide
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
For Salmonella strains:
The assays without S9 were performed by the plate-incorporation method.
The assays with S9 were conducted by the pre-incubation method described by Yahagi et al. (1975). The pre-incubation method is useful in detecting weak mutagenicity in samples (Yahagi et al., 1977).. Histidine-independent colonies were scored after incubation at 37°C for 48-72 h.

For E. coli strains:
The assay was performed in the same manner as with the Salmonella assay except that the supplement of 0.1 µmole histidine plus 0.1 mole biotin in the soft agar was replaced with a supplement of 0.1 µmole of tryptophan. Tryptophan-independent revertant colonies were scored with E. coli.

DURATION
- Preincubation period: 37°C
- Exposure duration: 48-72 h.


Rationale for test conditions:
No data available
Evaluation criteria:
A bacterial cell line was observed for biologically relevant increase in the number of revertants. Revertants per plate represent average values from 3 to 5 replications.
Statistics:
No data available

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA100, TA1535, TA98, TA1537, TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
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:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
All test compounds showed killing on bacteria at the highest doses used.
Remarks on result:
other: No mutagenic potential

Any other information on results incl. tables

Table: RESULTS OF MUTAGENICITY TEST OF THE TEST CHEMICAL


Chemical

Dose (µg/plate)

Revertants/plate (mean±S.D.)

Salmonella typhimuriumstrains

Escherichia coli

Without S9

 

DMSO

50µL

99±6

8±2

24±4

4±2

15±3

55±6

Positive control

 

769±54

266±34

660±30

822±88

301±33

452±68

Test chemical

250

98±5

10±4

28±1

3±1

9±1

50±9

750

81±6

5±1

24±1

3±1

10±2

57±6

1500

81±13

7±1

22±8

5±0

14±0

46±3

3000

24±13

2±1

20±8

2±2

6±1

33±6

With S9

 

 

 

 

 

 

 

DMSO

50µL

106±9

10±3

32±4

7±2

21±4

59±5

Positive control

 

431±92

158±45

180±7

125±37

154±17

753±95

Test chemical

250

87±1

7±2

31±3

9±2

24±8

58±6

 

750

130±3

10±1

27±3

7±2

18±4

62±3

 

1500

123±12

9±2

24±3

8±3

21±3

42±4

 

3000

72±11

12±2

21±2

4±2

11±0

39±8

Applicant's summary and conclusion

Conclusions:
The test chemical was negative in Ames test carried out using Salmonella typhimurium strains TA100, TA1535, TA98, TA1537, TA1538 as well in Escherichia coli WP2 uvr A in the presence and absence of S9 metabolic activation. Hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

In vitro bacterial reverse gene mutation assay for test chemical was studied using S. typhimurium and Escherichia coli WP2 uvrA strains. The mutagenicity assay was conducted as described by Ames et al. with slight modifications. Ames test was performed using Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 and TA1538 and Escherichia coli WP2 uvrA strains in the presence and absence of S9 metabolic activation system at dose level of 0, 250, 750, 1500 or 3000 µg/plate. For Salmonella strains, the assays without S9 were performed by the plate-incorporation method and the assays with S9 were conducted by the pre-incubation method described by Yahagi et al. (1975). The pre-incubation method is useful in detecting weak mutagenicity in samples (Yahagi et al., 1977). Histidine-independent colonies were scored after incubation at 37°C for 48-72 h. For E. coli strains, the assay was performed in the same manner as with the Salmonella assay except that the supplement of 0.1 µmole histidine plus 0.1 mole biotin in the soft agar was replaced with a supplement of 0.1 µmole of tryptophan. Tryptophan-independent revertant colonies were scored with E. coli. No mutagenicity was noted at the tested dose levels. Based on the observations made, the test chemical was negative in Ames test carried out using Salmonella typhimurium strains TA100, TA1535, TA98, TA1537, TA1538 as well in Escherichia coli WP2 uvr A in the presence and absence of S9 metabolic activation. Hence it is not likely to classify as a gene mutant in vitro.