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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted in a GLP facility according to OECD guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

1
Reference substance name:
Hydrogenated rosin alcohols
Cas Number:
2156595-41-2
IUPAC Name:
Hydrogenated rosin alcohols
Test material form:
liquid: viscous
Details on test material:
Sponsor's identification: ABITOL E
Description: colourless extremely viscous liquid
Batch number: SM-082
Date received: 13 November 2003
Storage conditions: room temperature in the dark

Method

Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
The Salmonella typhimurium strains were obtained from the University of California at Berkele in culture discs on 4 August 1995 whilst Escherichia coli strain WP2uvrA" was obtained from the British Industrial Biological Research Association on 17 August 1987. All of the strains were
stored at -196C. Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the sontaneous reversion rate.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
The Salmonella typhimurium strains were obtained from the University of California at Berkele in culture discs on 4 August 1995 whilst Escherichia coli strain WP2uvrA" was obtained from the British Industrial Biological Research Association on 17 August 1987. All of the strains were
stored at -196C. Prior to the master strains being used, characterisation checks
were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the sontaneous reversion rate.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver (induced) S9
Test concentrations with justification for top dose:
50, 150, 500, 1500 and 5000 ug/plate
Vehicle / solvent:
Dimethyl sulfoxide
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
Preliminary Toxicity Test

In order to select appropriate dose levels for use in the main test, a preliminary assay was
carried out to determine the toxicity of the test material. The concentrations tested were 0,
0.15, 0.5, 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 ug/plate. The assay was performed by mixing 0.1 ml of
bacterial culture (TA100 or WP2uvrA), 0.1 ml of test material formulation, 0.5 ml of S9-mix or
phosphate buffer and 2 ml of molten, trace histidine or tryptophan supplemented, top agar and
overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations
of the test material and a vehicle control (dimethyl sulphoxide) were tested. In addition, 0.1 ml
of the maximum concentration of the test material and 2 ml of molten, trace histidine or tryptophan
supplemented, top agar was overlaid onto a sterile Nutrient agar plate in order to
assess the sterility of the test material. After approximately 48 hours incubation at 37°C
the plates were assessed for numbers of revertant colonies using a Domino colony counter
and examined for effects on the growth of the bacterial bacterial background lawn.


Mutation Test- Experiment 1 (Range-finding Test)

Five concentrations of the test material (50, 150, 500, 1500 and 5000 ug/plate) were assayed in
triplicate against each tester strain, using the direct plate incorporation method.

Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test
tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml
of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or
phosphat buffer. The contents of each test tube were mixed and equally distributed onto
the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure
was repeated, in triplicate, for each bacterial strain and for each concentration of test
material both with and without S9-mix.

All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant
colonies assessed using a Domino colony counter.


Mutation Test-Experiment 2 (Main Test)

The second experiment was performed using methodology as described for the range-finding test
using fresh bacterial cultures, test material and control solutions. The test material dose range
was the same as the range-finding test (50 to 5000 ug/plate).


Evaluation criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate
in the vehicle and untreated controls.

The appropriate characteristics for each tester strain have been confirmed, eg rfa
cell-wall mutation and pKM101 plasmid R-factor etc.

All tester strain cultures should be in the approximate range of 1 to 9.9 x 10^9 bacteria per ml.

Each mean positive control value should be at least two times the respective vehicle control value
for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure
and the integrity of the S9 mix

There should be a minumum of 4 non-toxic test material dose levels

There should not be an excessive loss of plates due to contamination.

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a

rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 ug/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic

activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 ug/plate. A greasy film was observed at and above 1500 ug/plate, this did not prevent the scoring of revertant colonies. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

The test material was considered to be non-mutagenic under the conditions of this test.