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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:
July - August 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Bond.Ease™ Topical Skin Adhesive (polymerized)
- Batch no.: 10312101
- Physical state: Liquid
- Purity: >96%

Method

Target gene:
S. typhimurium: Histidine gene
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535
Metabolic activation:
with and without
Metabolic activation system:
S-9
Vehicle / solvent:
0.9% NaCl (saline) and PEG 400
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoflourene and 2-aminoanthracene
Details on test system and experimental conditions:
TEST ARTICLE PREPARATION: The amount of material tested was based on ISO extraction ratios. An aliquot of the solvents used were included and incubated in parallel with the test articles to serve as solvent controls (negative controls).
Extract solvent 1: 0.9% NaCl (saline) – Extraction ratio : 6cm2/mL – Test Article/Extraction Solvent amount : 42 cm2/7 mL – Extraction parameters : Temp = 37 ± 1ºC, time: 72 ± 2 hours (with agitation).
Extract solvent 2: PEG 400 – Extraction ratio : 6cm2/mL – Test Article/Extraction Solvent amount : 42 cm2/7 mL – Extraction parameters : Temp = 37 ± 1ºC, time: 72 ± 2 hours (with agitation).

PROCEDURE:
BROTH CULTURE PREPARATION: Commercial culture discs were used to inoculate nutrient broth for testing. The cultures were incubated at 37 ± 2ºC for 10-14 hours on an orbital shaker until when measured spectrophotometrically at 660nm, an absorbance reading of approximately 1.0 to 2.0 was obtained. Validation data of the cultures showed absorbance readings in the range resulted in concentrations of approximately 10E9 CFU/mL.

STRAIN GENOTYPE VERIFICATION: The culture disc lot number is checked for presence of appropriate strain genotype characteristics. These tests included verification of the following:
- Presence of uvrB mutation
- Presence or absence of R-factor plasmid
- Presence of rfa mutation
- Requirement for histidine
The uvrB mutation was verified by demonstrating UV sensitivity (lack of repair system). The R-factor was checked by determining sensitivity or resistance to ampicillin (0.08% in 0.02 NaOH). The presence if the rfa mutation was verified by demonstrating sensitivity to crystal violet (0.1% in water) on nutrient agar plates. The histidine requirement was assured by plating onto minimal glucose agar plates spread with 0.1 mL of 0.5 mM biotin and both with and without 0.1 mL of 0.1 M histidine.

METABOLIC ACTIVATION SYSTEM: The S-9 activation system was used to screen for the presence of mutagens from byproducts of the test article. Rat liver S-9 homogenate was obtained from Molecular Toxicology, Inc. The homogenate was kept frozen at ≤ -60ºC upon receipt. Plates requiring activation contained approximately 20 µL rat liver S-9 per plate. When working with soft agar plates did not exceed 47ºC.

TOP AGAR PREPARATION: Aliquots of top agar were melted and maintained at 45 ± 2ºC. Each 100 mL aliquot of top agar was fortified with 5-10mL of 0.5 mM histidine prior to use.

PLATE INCORPORATION TESTS: Each test article extract and solvent control was tested both with and without S-9 metabolic activation. The S-9 specific chemical controls (2-aminofluorene and 2-aminoanthracene) were also tested both with and without S-9 metabolic activation. Strain specific non-metabolic chemical controls were also included (Sodium Azide, Mitomycin-C and 4-nitro-0-phenylene-diamine. The non-metabolic chemical controls were tested without S-9 activation only.
Sterile 13 x 100 mm test tubes were transferred to a waterbath held at 45 ± 2ºC. Two mL aliquots of top agar were transferred to each test tube. Three replicates for each test article or control were prepared.
1) Solvent controls: 2mL top agar, 100 µL test organism and 100 µL of each solvent control.
2) Test article Extracts: 2mL top agar, 100 µL test organism and 100 µL of each test article extract.
3) Chemicals controls: 2mL top agar, 100 µL test organism and 10 µL of the specified chemical.
Each replicate requiring S-9 metabolic activation has 0.5 mL of the prepared S-9 mix added.
The replicates were vortexed, poured onto MGPA plates, swirled to form an even layer and allowed to solidify. The plates were incubated for growth 37± 2ºC for 48-72 hours.

SPOT TESTS: The test article extracts were also analysed using the spot method on plates with and without the S-9 activation system. Two mL aliquots of the top agar mixture and 0.1 mL of the appropriate test organism was added to minimal glucose agar plates. The plates were allowed to harden then 10 µL of the test article extract was added as a spot on the surface of the plate. The plates were incubated for growth of the organisms at 37± 2ºC ºC for 48-72 hours.

CHEMICAL CONTROL MATERIALS: The following chemical controls were used. The concentrations listed are the amount added per plate: 1.5 µg Sodium Azide, 2.5 µg Mitomycin-C, 20 µg 4-nitro-0-phenylene-diamine (NPD), 20 µg 2-aminofluorene (2-AF) and 7 µg 2-aminoanthracene (2-AA). The chemical controls were tested using the plate incorporation method only.
Evaluation criteria:
The criteria for acceptance of the test and criteria for determination of a mutagen are listed below.
1) Tested strains for genotype verification and achieved the appropriate responses.
2) All chemical controls included in the test gave the appropriate responses.
3) The reversion rates for each tester strains were within the historical ranges as outlined in the standard protocol.
Criteria for a Mutagen: A greater than two-fold increase in the number of revertants in strains TA97a, TA100 and TA102 or a greater than three-fold increase in strains TA98 and TA1535 when compared to the solvent control (percent of control >200% or >300%).
Criteria for a Non-Mutagen: A less than two-fold increase in the strains TA97a, TA100 and TA102 or a less than three-fold increase in strains TA98 and TA1535 when compared to the solvent control (percent of control <200% or <300%).

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535
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

Applicant's summary and conclusion

Conclusions:
The test article extracts did not produce a two-fold or a three-fold increase in the number of revertants in any of the 5 tester strains. The spot tests showed no zone of increased reversion or of toxicity. In summary, the extracts tested against the five strains did not meet the criteria for a potential mutagen.
Executive summary:

The Salmonella typhimurium reverse mutation assay (Ames test) is used to determine the potential mutagenic activity of a solid test article extract by exposing a large number of the test organisms to the extract fluid in agar plates. The agar plates are monitored for growth of revertants (organisms mutating to the wild type) which are counted and used to estimate the mutagenic potential of the test article.

The Ames test employs several histidine dependent (His+) strains of S. typhimurium which require the amino acid histidine for growth. The test detects mutations which cause the bacterial strains to revert to histidine independent (His-) bacteria which are capable of synthesizing histidine and can grow in the absence of histid ine. The assay uses tester strains TA97a, TA98, TA100, TA102 and TA1535 which were selected to detect various types of mutagens. The test is performed both with and without metabolic

activation using an S-9 activation system. The S-9 activation system is designed to simulate mammalian liver enzyme systems and is used to detect substances which undergo metabolic activation from nonmutagenic forms.

All test method acceptance criteria were met.

Results: The results are calculated using a validated computer program. Manual calculations may differ slightly due to rounding. All results greater than 300 colony forming units (CFU) are considered estimates.

The test article extracts did not produce a two-fold or a three-fold increase in the number of revertants in any of the 5 tester strains. The spot tests showed no zone of increased reversion or of toxicity. In summary, the extracts tested against the five strains did not meet the criteria for a potential mutagen.