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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Comparable to OECD Guideline 471. Deficiencies were: Salmonella strains TA1535 and TA98 not tested (guideline recommends testing both); TA1537, TA97 and TA97a not tested (guideline recommends testing at least one of these)

Data source

Reference
Reference Type:
publication
Title:
The effectiveness of Salmonella strains TA100, TA102 and TA104 for detecting mutagenicity of some aldehydes and peroxides
Author:
Diilon, D., Combes, R., and Zeiger, E.
Year:
1998
Bibliographic source:
Mutagenesis vol.13 no.1 pp.19-26, 1998

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
guideline also recommends testing TA1535, TA98 and at least one of TA1537, TA97 and TA97a; 2-aminoanthracene not recommended as sole indicator of S-9 activity
GLP compliance:
not specified
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:
Test material is described using the generic CAS 101-39-3, however the name is given as α-Methyl-trans-cinnamaldehyde (sourced from Aldrich).

Method

Species / strain
Species / strain:
other: TA100, TA102, TA104
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat and mouse liver fractions (S9)
Test concentrations with justification for top dose:
No metabolic activation: 10 to 1000 μg/plate (TA 100 and TA102); 100 to 10,000 μg/plate (TA104)
With metabolic activation (mouse S9): 10 to 10,000 μg/plate (TA100); 100 to 10,000 μg/plate (TA102); 10 to 1000 μg/plate (TA104)
With metabolic activation (rat S9): 10 to 1000 μg/plate (TA100 and TA104); 10 to 10,000 μg/plate (TA102)
Vehicle:
- Vehicle(s)/solvent(s) used: no data
- Justification for choice of solvent/vehicle: no data
Controlsopen allclose all
Negative controls:
yes
Remarks:
dose of 0 μg/plate tested [no further details given]
Positive controls:
yes
Remarks:
with S9 (all strains)
Positive control substance:
other: 2-aminoanthracene
Negative controls:
yes
Remarks:
dose of 0 μg/plate tested [no further details given]
Positive controls:
yes
Remarks:
without S9 (TA100)
Positive control substance:
other: methylmethanesulphonate
Negative controls:
yes
Remarks:
dose of 0 μg/plate tested [no further details given]
Positive controls:
yes
Remarks:
without S9 (TA102)
Positive control substance:
mitomycin C
Negative controls:
yes
Remarks:
dose of 0 μg/plate tested [no further details given]
Positive controls:
yes
Remarks:
without S9 (TA104)
Positive control substance:
other: formaldehyde or crotonaldehyde
Details on test system and conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: performed to the protocol of Maron and Ames (1983) [20 min at 37 °C or 30 min at 30 °C]
- Exposure duration: performed to the protocol of Maron and Ames (1983) [48 h]

NUMBER OF REPLICATIONS: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: other: thinning of the background lawn and/or a reduction in the number of colonies on the plates
Evaluation criteria:
Responses were judged mutagenic (+) when there was a reproducible, dose-related response and weakly mutagenic (+w) when reproducible low-level increases were obtained or when both positive and equivocal responses were seen in repeat trials. Responses were judged equivocal (?) when the increases in revertants were not reproducible or were seen only at a single dose.
Statistics:
The significance of mean revertant counts at individual dose levels was assessed using Dunnett’s t-test and dose-response effects were analyzed by two methods, Wahrendorf ranking and linear regression (Mahon et al., 1989). The latter was used to obtain slope of the responses and both methods were only applied in the absence of a decline in revertant count at high dose levels. These analyses were used for guidance, but did not dictate the summary responses.

Results and discussion

Test results
Key result
Species / strain:
other: TA 100, TA102, TA104
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
other: highest dose (1000 or 10,000 μg/plate, depending on strain) apparently "limited by toxicity"
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data available
- Effects of osmolality: no data available
- Evaporation from medium: no data available
- Water solubility: no data available
- Precipitation: no data available
- Other confounding effects: no data available

RANGE-FINDING/SCREENING STUDIES: no data available

COMPARISON WITH HISTORICAL CONTROL DATA: no data available

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data available

OTHER: Test compound produced "inconsistent equivocal responses" in TA100 with mouse and rat S9, but was not considered mutagenic

Any other information on results incl. tables

Table 1. Mutagenicity of α-Methyl-trans-cinnamaldehyde

α-Methyl-trans-cinnamaldehyde

 

 

TA100

TA102

TA104

-S9

-

-

-

+S9

-

-

-

- non-mutagenic

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative

In a limited Ames test, similar to that recommended by OECD Guideline 471, α-methyl-trans-cinnamaldehyde showed no consistent evidence of mutagenic activity in three strains of Salmonella typhimurium, with or without S9.
Executive summary:

A publication briefly describes a bacterial reverse mutagenicity (Ames) assay, in a preincubation test conducted using a protocol similar to OECD Guideline 471.

Salmonella typhimurium strains TA100, TA102 and TA104 were tested with α-methyl-trans-cinnamaldehyde at up to 1,000 or 10,000 μg/plate (depending on strain, and apparently "limited by toxicity") in the presence or absence of a rat or mouse liver metabolic activation fraction (S9). Known mutagens were also tested as positive controls. Three plates were prepared for each concentration and incubated, after which the number of revertant colonies per plate was counted.

No reproducible, dose-related mutagenic response was observed. Under the conditions of this assay (which did not test the guideline recommended set of five S. typhimurium strains), α-methyl-trans-cinnamaldehyde showed no convincing evidence of mutagenic activity in S. typhimurium, with or without S9.