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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:
From the 28th of August to the 14th of September, 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted according to internationally accepted guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Environmental fate/Ecotoxicological/Toxicological studies:
Storage: Room temperature, 20-24 °C, in the dark
Stability: pure, for 5 years; in solvent > 24 hours in water, DMSO, DMF, stable for at least 2 hours in aqueous medium.
Evidence of chemical instability at pH 6: none
Safety precaution: routine hygienic procedures were sufficient to assure personnel health and safety.
Toxicological studies:
Preparation: On the day of the experiment, the test article was suspended in Carboxymethylcellulose (1 %).
The vehicle was chosen to its nontoxicity for the animals. All animals received a single standard dose volume of 10 mL/kg body weight orally.

Method

Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without metabolic activation, TA1535, TA100
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 4-NOPD
Remarks:
Without metabolic activation, TA1537, TA1538, TA98
Positive controls:
yes
Positive control substance:
other: 2-amminoanthracene 2-AA
Remarks:
With metabolic activation, TA1535, TA1537, TA1538, TA98, TA100
Details on test system and experimental conditions:
Characterization of the Salmonella Typhimurium Strains
Salmonella typhimurium
TA 1537
his C 3076; rfa-; uvrB-; frame shift mutations
TA 1538
his D 3052; rfa-; uvrB-: frame shift mutations
TA 98
his D 3052; rfa-; uvrB-;R-factor: frame shift mutations
TA 1535:
his G 46; rfa-; uvrB-; base-pair substitutions
TA 100
his G 46; rfa-; uvrB-; R-factor; base-pair substitutions
Regular checking of the properties of the strains with regard to membrane permeability and ampicillin resistance as well as normal spontaneous mutation rates is performed in C C R according to Ames et al . . In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

Storage
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSQ in liquid nitrogen.

Precultures
From the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. This nutrient medium contains per litre: 8 g Difco Nutrient Broth, 5 g NaCl.
The bacterial culture was incubated in a shaking water bath for 6 hours at 37 °C.

Selective agar
2.0 % Vogel-Bonner-Glucose-Minimal-Agar was used as selective agar. Each petri dish was filled with 20 mL of this nutrientvmedium. Sterilizations were performed at 121 °C in an autoclave.

Overlay agar
The overlay agar contains per litre:
6.0 g Difco Bacto Agar
6.0 g NaCl
10.5 mg L-histidine x HC1 x H20
12.2 mg biotin
Sterilizations were performed at 121 °C in an autoclave.


PRE-EXPERIMENT FOR TOXICITY
To evaluate the toxicity of the test article a prestudy is performed with strains TA 98 and TA 100. 8 concentrations are tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment are the same as described below forthe experiment. Toxicity of the test article may be evidenced by a reduction in the nunther of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

DOSE SELECTION
According to the results of this pre-experiment the concentrations applied in the main experiments are chosen. The concentration range covers at least two decadic logarithms.
The maximum concentration is 5000.0 µg/plate, unless limited by toxicity or solubility of the test article. The concentration range includes at least two decadic logarithms. In this study at least five adequate spaced concentrations are tested. Two independent experiments are performed.
In case the results of the pre-experiment are in accordance with the criteria described above, these data are reported as a part of the main experiments.

EXPERIMENTAL PERFORMANCE
For each strain and dose level, including the controls, a minimum of three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL: test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control)
500 ul: S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation)
100 ul: Bacteria suspension (cf. test system, pre—culture of the strains)
2000 ul: overlay agar

After solidification the plates were incubated upside down for 72 hours at 37 °C in the dark.

DATA RECORDING
The colonies were counted using the BIOTRAN III counter (BIOTRO- NIK, D-6000 Frankfurt, F.R.G.). The counter was connected to an IBM XT compatible PC with printer which printed out the individual values and the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. If precipitation of the test article precluded automatic counting the revertant colonies were counted by hand.

Evaluation criteria:
The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates.

A test article is considered as positive 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 any one of the test points is considered non-mutagenic in this system.

A test article is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, TA 1538, and TA 98 it is 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 induced the above described enhancement factorsor not.
Statistics:
Due to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time.

Results and discussion

Test resultsopen allclose all
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:
cytotoxicity
Remarks:
A decrease in the number of spontaneous revertants indicating bacteriotoxicity was observed
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A decrease in the number of spontaneous revertants indicating bacteriotoxicity was observed
Additional information on results:
PRE-EXPERIMENT
The plates with the test article showed normal background growth up to 5000.0 µg/plate in strain TA 98 and TA 100, respectively.
According to the dose selection criteria, the test article was tested at the following concentrations: 10.0; 100.0; 333.3; 1000.0 and 5000.0 µg/plate.

MAIN EXPERIMENT
A decrease in the number of spontaneous revertants indicating bacteriotoxicity was observed in the following strains at the highest investigated dose: TA 1537 (exp. I), TA 1538 (exp. I and II), TA 98 (exp. I and II) all with metabolic activation and TA 100 (exp. II) without metabolic activation. In strain TA 98 the number of spontaneous revertants was already reduced at 1000.0 µg/plate with metabolic activation in experiment I.
background growth up to 5000.0 p.g/plate with and without S9 mix in all strains used, except in strain TA 1538 in experiment I without S9 mix at 5000.0 µg/p1Ltes where the background growth was slightly reduced.
Up to the highest investigated dose, no significant and reproducible dose-dependent increase in revertant colony numbers was obtained in any of theSalmonella typhimurium strains used. 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.
The test article did not induce point mutations by base pair substitutions or frameshifts in the genome of the strains used.

Applicant's summary and conclusion

Conclusions:
Negative.
The test article did not induce point mutations by base pair substitutions or frameshifts in the genome of the strains used.
The substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

Materials and methods

This study was performed to investigate the potential of the substance to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, according to the OECD 471 and EU Method B.13/14.

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. The test article was tested at the following concentrations: 10.0; 100.0; 333.3; 1000.0 and 5000.0 µg/plate.

Observations

Up to the highest investigated doses, no significant and reproducible dose-dependent increase in revertant colony numbers was obtained in any of the Salmonella typhimurium strains used. The presence of liver microsomal activation did not influence these findings.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Results

The substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.