Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 1994-01-31 to 1994-02-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Performed under GLP, equivalent to OECD TG 471

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Ro 01-8300/000
- Physical state: dark orange-red powder
- Analytical purity: 98.2%
- Purity test date: 1993-12-15
- Lot/batch No.: 312131
- Expiration date of the lot/batch: 1994-12-15 (in sealed can at -20 °C)
- Storage condition of test material: protected from light, unopened container stored at -20°C; after opening, stored in exsiccator under nitrogen atmosphere in the refrigerator.

Method

Target gene:
Histidine for Salmonella
Species / strain
Species / strain:
other: TA1535, TA97, TA98, TA100, TA102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix prepared from the livers of phenobarbital/beta-naphthoflavone treated male albino rats
Test concentrations with justification for top dose:
Plate incorporation Assay: 25, 79, 250, 790, 2500 µg/plate
Preincubation Assay: 25, 79, 250, 790, 2500 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: Water containing 20% tetrahydrofurane and 8% TWEEN 80
- Justification for choice of solvent/vehicle: The test compound was highly insoluble in water. To enhance solubility a mixture of 1 Vol tetrahydrofurane (THF) and 4 Vol TWEEN 80 (10% in water) was chosen as solvent.
Controls
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9: sodium azide (TA1535, TA100), ICR 191 (TA97) 2-nitrofluorene (TA98), MMC (TA102). With and without S9: 2-aminoanthracenea (all strains)
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 30 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 (test compound and negative control), 2 (positive controls)

DETERMINATION OF CYTOTOXICITY
- Method: determination of the growth of the background lawn and/or frequency of spontaneous revertants
Evaluation criteria:
There is as yet no generally accepted statistical treatment of Ames test reversion system data. In most tests, the results are either clearly positive or clearly negative. A positive result is defined as a reproducible, dose-related increase in the number of his+ revertants. The increase should reach at least a doubling of the number of spontaneous revertants for Salmonella typhimurium strains TA1535 and TA98. For strains TA97, TA100 and TA102 a 1.5 - fold increase
over control values might be indicative of a mutagenic effect provided the negative control values fall within the historical control data. Other investigators have set higher limits for a mutagenic response (factor 3 and 2 for the respective groups of strains). These rules of thumb have a questionable scientific foundation and biological relevance should always be taken into account. A negative result is defined as the absence of a reproducible increase in the number of his+ revertant colonies.

Results and discussion

Test results
Species / strain:
other: TA1535, TA97, TA98, TA100, TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test compound was highly insoluble in water. To enhance solubility, a mixture of 1 Vol tetrahydrofurane (THF) and 4 Vol TWEEN 80 (10X in water) was chosen as solvent.
- Precipitation: Precipitation formed starting at concentrations of 250 µg/plate.

RANGE-FINDING/SCREENING STUDIES: Toxicity of the test substance was assessed in a preliminary toxicity assay by evaluating the growth on NB- and/or Vogel-Bonner minimal agar plates (determination of the growth of the background lawn and/or frequency of spontaneous revertants). Each test substance dose, as well as the appropriate solvent control, was evaluated in duplicate in strain TA100. The highest test dose for the main experiments is chosen to either produce signs of toxicity (reduction in the revertant colony number and/or observation of thinning or absence of the background lawn) or to be evidently insoluble in the aqueous medium.

COMPARISON WITH HISTORICAL CONTROL DATA: The mutant frequencies of the controls were in the range of the historical controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects was apparent for any of the tester strains.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative

Beta-Carotene was not mutagenic in the Ames test in the absence and presence of S9 mix.
Executive summary:

The mutagenic potential of beta-Carotene was tested under GLP in an Ames test according to OECD TG 471. The test compound was evaluated for genotoxic activity using two versions of the Ames test: the standard plate incorporation and the preincubation assays.

Five Salmonella tester strains were employed (TA1535, TA97, TA98, TA100, TA102). Metabolic activation was provided by an activation system (S9 mix) prepared from the livers of phenobarbital/beta-naphtoflavone treated rats. Activity of the enzymes and responsiveness of the strains was verified by including appropriate positive controls into each experiment. The test compound was dissolved in an aqueous solvent mixture with final concentrations of 20 % tetrahydrofurane and 8 % TWEEN 80. Upon addition of aliquots of the stock solution to the cell suspensions reddish precipitation formed starting at 250 µg/plate. A maximal test concentration of 2500 µg/plate was chosen due to precipitation.

Neither toxic nor genotoxic activity of the test compound was apparent under these test conditions. Thus it can be concluded that beta-Carotene is not mutagenic in the Ames test with and without metabolic activation.