4,4'-cyclohexylidenedi-o-cresol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-cyclohexylidenedi-o-cresol

EC Number:

219-110-7

EC Name:

4,4'-cyclohexylidenedi-o-cresol

Cas Number:

2362-14-3

Molecular formula:

C20-H24-O2

IUPAC Name:

4-[1-(4-hydroxy-3-methylphenyl)cyclohexyl]-2-methylphenol

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Dimethyl cyclohexyl bisphenol (DMBPC; CAS No. 2362-14-3)
- Synonyms: Dimethyl bisphenolcyclohexane; 4,4’-cyclohexylidene di-o-cresol; 1,1-Bis(4-hydroxy-3-ethyl)cyclohexane; Bis-OC-Z
- Appearance: fine white powder

Method

Species / strain

Species / strain / cell type:

bacteria, other: Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 micrograms per plate with and w/out S9 activation (Preliminary toxicity and initial mutagenicity assay)
5.0, 15, 50, 150, 500, 1500 and 5000 micrograms per plate (Independent repeat/confirmatory mutagenicity assay)

Vehicle / solvent:

DMSO (CAS No. 67-68-5); from Fisher Scientific

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

for all strains; 1.0 µg/plate for all S. typhimurium; 10 µg/plate E. coli

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 - Without S9 activation; 1.0 µg/plate

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide

Remarks:

TA100, TA1535 - Without S9 activation; 1.0 µg/plate

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 - Without S9 activation; 75 µg/plate

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA - Without S9 activation; 1000 µg/plate

Details on test system and experimental conditions:

Preliminary Toxicity/Initial Mutagenicity Assay: The preliminary toxicity/initial mutagenicity assay was used to establish the dose range over which the test article would be assayed and to provide a preliminary mutagenicity evaluation. A vehicle control, positive controls and eight dose levels of the test article were plated, two plates per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor induced rat liver S9.

Confirmatory Mutagenicity Assay: The confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test article. Six dose levels of test article along with vehicle control and appropriate positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA in the presence and absence of Aroclor induced rat liver S9. All dose levels of test article, vehicle control and positive controls were plated in triplicate.

Plating and Scoring Procedures: The test system was exposed to the test article via the plate incorporation method. On the day of its use, minimal top agar was melted and supplemented. Top agar, not used with S9 or Sham mix, was supplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, all references to water imply sterile, deionised water produced by the Milli Q Reagent Water System. Bottom agar was supplemented Vogel Bonner minimal medium E.

Each plate was labelled with a code system that identified the test article, test phase, dose level, tester strain and activation.

Test article dilutions were prepared immediately before use. One half (0.5) millilitre of S9 or Sham mix, 100 µL of tester strain and 50 µL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45 ± 2 °C. After vortexing, the mixture was 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 µL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37 ± °C. Plates that were not counted immediately following the incubation period were stored at 2 - 8 °C until colony counting could be conducted.

The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification. Toxicity and degree of precipitation was scored relative to the vehicle control plate.

Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity. Plates with sufficient test article precipitate to interfere with automatic colony counting were counted manually.

Evaluation criteria:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and reported.
For the test article to be evaluated positive, it must have caused 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 article. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean 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 equal to or greater than 2.0-times the mean vehicle control value.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Generally observed beginning at 150, 500 or 1500 µg/plate in the confirmatory assay.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Generally observed beginning at 150, 500 or 1500 µg/plate in the confirmatory assay.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Generally observed beginning at 150, 500 or 1500 µg/plate in the confirmatory assay.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Generally observed beginning at 150, 500 or 1500 µg/plate in the confirmatory assay.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Generally observed beginning at 150, 500 or 1500 µg/plate in the confirmatory assay.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In the preliminary toxicity/initial mutagenicity assay (Experiments B1 and B2), the doses tested were 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 µg/plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 1800 µg/plate. Toxicity was generally observed beginning at 200, 600 or 1800 µg/plate. Due to an unacceptable vehicle control value, tester strain WP2 uvrA in the absence of S9 activation was not evaluated in Experiment B1 but was successfully retested in Experiment B2. Based on the preliminary toxicity findings the doses tested for the confirmatory mutagenicity assay were 5, 15, 50, 150, 500 and 1500 µg/plate without metabolic activation and 15, 50, 150, 500, 1500 and 5000 µg/plate with metabolic activation.
For the Confirmatory Mutagenicity Assay (Experiment B3), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 5000 µg/plate with S9 activation. Toxicity was generally observed beginning at 150, 500 or 150 µg/plate.

Any other information on results incl. tables

Initial Mutagenicity Assay (without activation)

Mean Number of Revertants Per Plate

 

Dose (µg/plate)	TA98	TA100	TA1535	TA1537	WP2uvrA
Vehicle (DMSO)	14 ± 1	162 ± 1	12 ± 1	5 ± 2	12 ± 1
2.5	9 ± 2	156 ± 15	10 ± 6	4 ± 1	9 ± 4
7.5	9 ± 3	169 ± 2	8 ± 2	3 ± 1	8 ± 1
25	11 ± 3	160 ± 18	10 ± 0	9 ± 1	10 ± 1
75	10 ± 1	112 ±3	11 ± 1	5 ± 4	7 ± 3
200	7 ± 2	87 ± 6	12 ± 4	2 ± 2	5 ± 1
600	4 ± 1	30 ± 2	5 ± 1	1 ± 1	2 ± 1
1800	1 ± 1	0 ± 0	1 ± 0	0 ± 0	1 ± 0
5000	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Positive Control	169 ± 25	747 ± 148	207 ± 0	453 ± 42	93 ± 1

 

 

 

Initial Mutagenicity Assay (with activation)

Mean Number of Revertants Per Plate

 

Dose (µg/plate)	TA98	TA100	TA1535	TA1537	WP2uvrA
Vehicle (DMSO)	12 ± 4	138 ± 24	9 ± 1	4 ± 1	10 ± 0
2.5	11 ± 0	176 ± 11	11 ± 4	4 ± 2	10 ± 5
7.5	12 ± 3	151 ± 5	9 ± 1	4 ± 3	8 ± --
25	13 ± 4	160 ± 35	9 ± 5	3 ± 2	8 ± 1
75	10 ± 0	154 ± 17	7 ± 2	5 ± 2	8 ± 1
200	6 ± 0	123 ± 14	8 ± 4	7 ± 1	8 ± 0
600	2 ± 0	100 ±18	7 ± 1	2 ± 0	5 ± 4
1800	2 ± 1	16 ± 7	3 ± 1	0 ± 0	0 ± 0
5000	0 ± 0	2 ± 1	0 ± 0	0 ± 0	0 ± 0
Positive Control	1102 ± 265	1205 ± 39	141 ± 24	130 ± 21	227 ± 133

 

 

 

Confirmatory Mutagenicity Assay (without activation)

Mean Number of Revertants Per Plate

 

Dose (µg/plate)	TA98	TA100	TA1535	TA1537	WP2uvrA
Vehicle (DMSO)	12 ± 3	155 ± 15	12 ± 5	4 ± 2	11 ± 2
5.0	10 ± 1	163 ± 5	14 ± 1	5 ± 2	10 ± 1
15	13 ± 3	204 ± 10	9 ± 4	4 ± 1	11 ± 1
50	8 ± 1	106 ±9	12 ± 1	5 ± 3	9 ± 1
150	6 ± 2	36 ± 3	6 ± 1	2 ± 1	6 ± 3
500	1 ± 1	1 ± 2	2 ± 1	0 ± 0	0 ± 1
1500	0 ± 1	0 ± 0	0 ± 0	0 ± 0	0 ± 1
Positive Control	87 ± 15	571 ± 21	135 ± 28	476 ± 145	70 ± 5

 

 

 

Confirmatory Mutagenicity Assay (with activation)

Mean Number of Revertants Per Plate

 

Dose (µg/plate)	TA98	TA100	TA1535	TA1537	WP2uvrA
Vehicle (DMSO)	14 ± 3	133 ± 21	6 ± 1	5 ± 2	11 ±
15	14 ± 3	142 ± 6	5 ± 1	4 ± 2	11 ± 2
50	14 ± 3	140 ±21	7 ± 2	6 ± 1	11 ± 1
150	10 ± 1	79 ± 8	5 ± 1	3 ± 1	9 ± 2
500	3 ± 2	43 ± 2	4 ± 1	2 ± 1	4 ± 2
1500	1 ± 1	5 ± 1	1 ± 1	0 ± 1	1 ± 1
5000	0 ± 0	0 ± 1	0 ± 1	0 ± 0	0 ± 0
Positive Control	1578 ± 365	1691 ± 552	122 ± 13	227 ± 21	138 ± 53

 

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test material was negative in both the presence and absence of metabolic activation.

Executive summary:

The potential of the test material to cause genetic toxicity was investigated in a bacterial reverse mutation assay conducted in accordance with the standardised guideline OECD 471 under GLP conditions.

Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA were exposed to the test material in DMSO via the plate incorporation method at dose levels of 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 micrograms per plate with and without S9 activation in a preliminary toxicity and initial mutagenicity assay. The strains were then exposed to 5.0, 15, 50, 150, 500, 1500 and 5000 micrograms per plate in an independent repeat/confirmatory mutagenicity assay.

In the preliminary toxicity/initial mutagenicity assay (Experiments B1 and B2) no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 1800 µg/plate. Toxicity was generally observed beginning at 200, 600 or 1800 µg/plate. Due to an unacceptable vehicle control value, tester strain WP2 uvrA in the absence of S9 activation was not evaluated in Experiment B1 but was successfully retested in Experiment B2. 

For the confirmatory mutagenicity assay (Experiment B3), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 5000 µg/plate with S9 activation. Toxicity was generally observed beginning at 150, 500 or 150 µg/plate. All criteria for a valid study were met. 

Under the conditions of this study, the test material was negative in both the presence and absence of metabolic activation.