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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:
8 October 1991 - 05 November 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD guideline and GLP. No CoA included in the report. One of the strains: E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 was not included in the study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
- One of the strains: E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 was not included in the study.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: Ca-Acetylacetonate
Batch No.: 106002
Physical state: Powder
Purity: 98%

No CoA included in the report.

Method

Target gene:
Histidine operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
11.9 µg; 59.5 µg; 119.0 µg; 595.0 µg and 1190.0 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene
Remarks:
Details see below
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: NA
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: triplicate colonies, duplicate experiment

NUMBER OF CELLS EVALUATED: the plates were scored by counting the number of revertant colonies on each plate.

DETERMINATION OF CYTOTOXICITY
- Method: bacterial background growth

Evaluation criteria:
The revertant colonies on each plate were counted. From each of 3 plates at each dose level the mean values together with standard deviations and enhancement factors as compared to the spontaneous reversion rates were determined.
A test article is considered as mutagenic, if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at anyone of the test points is considered non-mutagenic in this system.

A significant response is described as follows:
A test article is considered as mutagenic if in strain TA 100 the number of revertants will be at least twice as high and in strains TA 98, TA 1535 und TA 1537 it will be at least three times higher as compared to the spontaneous
reversion rate.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose will induce the above described enhancement factors or not.

The generally accepted conditions for the evalutation of the results will be: corresponding background growth on both negative control and test plates normal range of spontaneous reversion rates referred to the negative control groups without metabolic activation

Range of spontaneous reversion frequencies:
TA 98 15-60
TA 100 75-200
TA 1535 3-37
TA1537 4-31
These values refer to the negative control group without metabolic activation.
Statistics:
None

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

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

This study was performed to investigate the potential of calcium acetylacetonate to induce gene mutations according to the plate incorporation test using Salmonella typhimurium strains TA 98, TA-100, TA 1535 and TA 1537.

The assay was performed in two independent experiments using identical procedures, both with and without liver microsomal activation. Each

concentration, including the controls, was tested in triplicate. Calcium acetylacetonate was tested at the following concentrations: 11.9 µg; 59.5 µg; 119.0 µg; 595.0 µg and 1190.0 µg/plate

Precipitates were neither observed in the whole concentration range nor in the overlay agar. The plates incubated with the test article showed normal background growth up to 1190.0 µg/plate with and without S9-mix in all strains used. Up to the highest investigated dose, neither a significant and reproducible increase in the number of revertants was found in any strain as compared to the solvent control nor a concentration dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, calcium acetylacetonate is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.