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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Well documented bacterial reverse mutation assay, conducted not in accordance with current guideline. However, it is in accordance with OECD guideline 471 as adopted 1983-05-26. There were only four strains tested, and spacing of test concentration was greater than 10exp(1/2). However, these deficiencies are considered to be minor as the fifth strain was added to the guideline because the former common four strains may not detect certain oxidising mutagens, cross-linking agents and hydrazines. Based on the chemical structure, this mode of action is not to be expected from the test item. There was no dose-dependent and biologically relevant increase of the mutant figures to the double of the negative controls. Actually, there was no increase in the mutant frequency in any of the tester strains at all, leading to the conclusion that any variation of the testing method could not lead to the observation of any mutagenic effect.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
as adopted 1983-05-26
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
B14, as set out in EEC Directive 84/449/EEC
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): 2,2'-Methylene-bis-(6-cyclohexyl-4-methylphenol) or Phenol, 2,2'-methylenebis(6-cyclohexyl-4-methyl)-
- Substance type: pure substance
- Analytical purity: 97.2%
- Purity test date: 1990-08-22
- Lot/batch No.: H7208886 (sample No. 019900/1990)
- Expiration date of the lot/batch: 1992-01-25
- Stability under test conditions: A stability test in the solvent did not detect a relevant change in the percent active ingredient.
- Storage condition of test material: refrigerator

Method

Target gene:
his-
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
other: pKM 101 in TA100 and TA98
Metabolic activation:
with and without
Metabolic activation system:
induced S9
Test concentrations with justification for top dose:
0, 8, 40, 200, 1000, 5000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: ethanol (test item); DMSO (positive controls)
- Justification for choice of solvent/vehicle: The used solvent was chosen out of the following solvents, in the order given: water, methanol, ethanol, acetone, DMSO, DMF, and ethylene glycol dimethylether according to information given by the internal sponsor.
Controls
Negative controls:
yes
Remarks:
vehicle
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: nitrofuranthion; 4-nitro-1,2-phenylene diamine, 2-aminoanthracene
Remarks:
sodium azide 10 µg/plate (only TA1535), nitrofuranthion 0.2 µg/plate (only TA100), 4-nitro-1,2-phenylene diamine 10 µg/plate (only TA1537) / 0.5 µg/plate (only TA98), 2-aminoanthracene 3 µg/plate
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 30 sec
- Exposure duration: 48 h
- Fixation time (start of exposure up to fixation or harvest of cells): 48 h

SELECTION AGENT (mutation assays): his-negative agar

NUMBER OF REPLICATIONS: 4 plates per strain, dose, ± S9

DETERMINATION OF CYTOTOXICITY
- Method: The toxicity of the substance was assessed in three ways. The first was a gross appraisal of background growth on the plates for mutant determination. If a reduction in background growth was observed, it was indicated in the tables by the letter "b" after the mutant count. Where only a single "b", without any other values, is noted for a concentration, this "b" represents four plates with background growth. (The same applies to the signs "c", "v", "p", "n" or "%", which may also be used in the tables.) Secondly, a toxic effect of the substance was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls. Thirdly, the titer was determined. Total bacterial counts were taken on two plates for each concentration studied with S9 mix. However, if an evaluation was performed only without S9 mix, the bacterial count was taken without S9 mix.
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice the amount of negative controls, whereas for TA 1537, at least a threefold increase should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At 1000 µg/plate, the substance started to precipitate.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Doses >200 µg/plate had a weak, strain-specific bacteriotoxic effect. Nevertheless, these doses could be used for assessment up to and including 5000 µg/plate.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative

Testing for mutagenicity in bacteria was performed according to OECD guideline 471 as adopted 1983-05-26. Although not being conducted to recent guidelines, the test was conducted scientifically reasonable with negligible deficiencies. Also, the testing was sufficiently documented, positive and negative controls gave the appropriate response. Hence, the results can be considered as sufficiently relieable to assess the mutagenic potential of 2,2'-Methylene-bis-(6-cyclohexyl-4-methylphenol) in bacteria. A dose-dependent and biologically relevant increase of the mutant figures to the double of the negative controls could not be observed with any of the four strains used. This applied to the examinations with and without the S-9 mix. In consequence, 2,2'-Methylene-bis-(6-cyclohexyl-4-methylphenol) is considered to be non-mutagenic under the conditions of this test.
Executive summary:

In a reverse gene mutation assay in bacteria (OECD 471), histidine-auxotrophic strains TA1535, TA100, TA1537 and TA98 of S. typhimurium were exposed to 2,2'-Methylene-bis-(6-cyclohexyl-4-methylphenol) in ethanol at concentrations of 0, 8, 40, 200, 1000, 5000 µg/plate in the presence and absence of mammalian metabolic activation (induced S9) via plate co-incubation.

2,2'-Methylene-bis-(6-cyclohexyl-4-methylphenol) was tested up to limit concentration 5000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence or a concentration related positive response of induced mutant colonies over background.

Doses up to and including 200 µg/plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a weak, strain-specific bacteriotoxic effect, so that this range could nevertheless be used for assessment purposes. Substance precipitation occurred at the dose 1000 µg/plate and above.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 471 (as adopted 1983-05-26) for in vitro mutagenicity (bacterial reverse gene mutation) data with minor deviations from the recent Guideline.