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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: The study was conducted according to the O.E.C.D. test guideline 471 with GLP compliance.

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)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
As per IUCLID Sections 1.1. 1.2. and 4.

Method

Target gene:
Histidine synthesis
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Rat liver derived S9 fraction.
Test concentrations with justification for top dose:
The dose levels tested were 5.0, 15, 50, 150, 500 and 1500 ug per plate with tester strains TA100, TA1535 and TA1537 and 15, 50, 150, 500, 1500 and 5000 jig per plate with tester strains TA98 and WP2 uvrA.
Vehicle / solvent:
Ethanol
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene with S9 metabolic activation preparation.
Remarks:
Positive controls without S9 metabolic activation were: 2-nitrofluorene, sodium azide, 9-aminoacridine, and methyl methane sulfonate.
Details on test system and experimental conditions:
Overnight cultures of the bacterial tester strains were grown up in nutrient broth at approximately 37C with shaking to late log phase. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x109 cells per milliliter.

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 fraction was prepared by and purchased from Moltox (Boone, NC). Upon arrival at BioReliance, the S9 was stored at -60°C or colder until used. The S9 mix was prepared immediately before its use and contained 10% S9, 5 mM glucose-6-phosphate, 4 mM fi-nicotinamide-adenine dinucleotide phosphate, 8 mM MgC12 and 33 mM KC1 in a 100 mM phosphate buffer at pH 7.4.

The mutagenicity assay was conducted With a mimum of five dose levels of test article along with appropriate vehicle control and positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective agar in the presence and absence of Aroclor-induced rat liver S9 fraction. All dose levels of test article, vehicle control and positive controls were plated in triplicate. The test system was exposed to the test article via the preincubation methodology described by Yahagi et a?. (1977).

On the day of its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaC1 (W/V), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a fmal concentration of 50 uM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of water for each 100 mL of minimal top agar. Bottom agar was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5 % (W/V) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder).

Exposure was conducted by adding 0.5 milliliter of S9 or sham mix (PBS), 100 gL of tester strain (cells seeded) and 25 uL of vehicle or test article dilution were added to 13 X 100 mm glass culture tubes pre-heated to 37+/-2°C. After vortexing, these mixtures were incubated with shaking for 60+/-2 minutes at 37+/-2°C. Following the preincubation, 2.0 mL of selective top agar was added to each tube and the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 utL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37+2°C.

Evaluation criteria:
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance Data sets for tester strains TA 1535 and TA 1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean concurrent vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.

Statistics:
None

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
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
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:
Interpretation of results (migrated information):
negative with and without rat liver S9 metabolic activation.

The test substance did not induce a gene-mutation responce in any of the bacterial tester strains with and without a rat liver derived S9 metabolic activation fraction.
Executive summary:

The test substance, 4,4'-Isopropylidenedicyclohexanol, oligomeric reaction products with 1 -chloro-2,3 -epoxypropane, did not induce a gene-mutation responce in an O.E.C.D. 471 bacterial mutation assay by the preincubation method in any of the bacterial tester strains with and without a rat liver derived S9 metabolic activation fraction.