2,2'-methylenebis[6-(1-methylcyclohexyl)-p-cresol]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In Vitro Genetic Toxicity

Bacterial reverse mutation assay

Bacterial reverse mutation assay, performed in accordance with OECD Guideline 471 and EU Method B13/14: Negative in four strain of Salmonella typhimurium (TA1535, TA1537, TA98 & TA100) and in Escherichia colia (WP2uvrA) with and without metabolic activation.

Chromosome aberration

Due to study data being not available prior to the registration deadline, the following studies are used for read across purposes:

2,2'-(2-methylpropylidene)bis[4,6-xylenol]; CAS 33145-10-7; EC 251-394-8

It is concluded that this test is valid and that this substance is not clastogenic in human lymphocytes.

6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol; CAS 119-47-1; EC 204-327-1

It is concluded that this test is valid and that this substance is not clastogenic in CHL/IU cell lines.

6,6'-di-tert-butyl-4,4'butylidenedi-m-cresol; CAS 85-60-9; EC 201-618-5  

It is concluded that this test is valid and that this substance is not clastogenic in human lymphocytes.

Mammalian Gene Cell Mutation

Due to study data being not available prior to the registration deadline, the following studies are used for read across purposes:

2,2'-(2-methylpropylidene)bis[4,6-xylenol]; CAS 33145-10-7; EC 251-394-8

Not mutagenic in the TK mutation test system under the experimental conditions described in this report.

6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol; CAS 119-47-1; EC 204-327-1

Did not induce gene mutations at the HPRT locus in V79 cells under the experimental conditions used.

6,6'-di-tert-butyl-4,4'butylidenedi-m-cresol; CAS 85-60-9; EC 201-618-5  

Not mutagenic in the TK mutation test system under the experimental conditions described.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 August 2015 to 20 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals; Guideline no. 471: "Genetic Toxicology: Bacterial Reverse Mutation Test" (Adopted July 21, 1997).

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.13/14: "Mutagenicity: Reverse Mutation Test using Bacteria”. Official Journal of the European Union No. L142, 31 May 2008.

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Test item: 206533/A
Identification: 2,2'-methylenebis[6-(1-methylcyclohexyl)-p-cresol]
Appearance: White to cream powder
Batch: WWP4J0001
Purity/Composition: 92.2%
Test substance storage: At room temperature
Stable under storage conditions until: 03 September 2018 (retest date)
Purity/composition correction factor: Yes, correction factor is according to purity
Test substance handling: No specific handling conditions required
Stability at higher temperatures: Not available
Chemical name (IUPAC), synonym or trade name: 2,2'-methylenebis[6-(1-methylcyclohexyl)-p-cresol] Trade name (LOWINOX® WSP)
CAS Number: 77-62-3
Molecular formula: C29H40O2
Molecular weight: 420.63

Target gene:

histidine and tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Test system: Salmonella typhimurium bacteria and Escherichia coli bacteria
Rationale: Recommended test system in international guidelines (e.g. OECD, EC).
Source: Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames (TA1535: 2006, TA1537: 2009, TA98: 2006, TA100: 2006; and Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK (WP2uvrA: 2008)]
The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)
Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)

The Salmonella typhimurium strains are regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment.
The strain is regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.

Stock cultures of the five strains were stored in liquid nitrogen (-196°C).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate. The highest concentration of the test item used in the subsequent mutation assay was 1600 μg/plate.

Vehicle / solvent:

The vehicle of the test item, which was dimethyl sulfoxide (SeccoSolv, Merck, Darmstadt, Germany).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191; 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

Test item preparation
A correction factor of 1.08 for the purity/composition of the test item was applied in this study. The test item was dissolved dimethyl sulfoxide.

Cell culture
Preparation of bacterial cultures
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1°C, 150 spm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for testing.
Agar plates
Agar plates (ø 9 cm) containing 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Merck) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate biotin (Merck) and 15 μg/plate histidine (Sigma) and the agar plates for the test with the Escherichia coli strain contained 15 μg/plate tryptophan (Sigma).
Top agar
Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v) sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.
Environmental conditions
All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 34.7 – 38.9°C). The temperature was continuously monitored throughout the experiment. Due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door, temporary deviations from the temperature may occur. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Metabolic activation system
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg).
Each S9 batch is characterised with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.

Preparation of S9-mix
S9-mix was prepared immediately before use and kept on ice. S9-mix contained per 10 ml: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 ml or 5.0 ml Milli-Q water (first or second experiment respectively) (Millipore Corp., Bedford, MA., USA); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution (Merck); 1 ml 0.33 M KCl solution (Merck). The above solution was filter (0.22 μm)-sterilized. To 9.5 ml of S9-mix components 0.5 ml S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 ml of S9-mix components 1.0 ml S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.

Study design
Dose range finding test
Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate. The highest concentration of the test item used in the subsequent mutation assay was 1600 μg/plate.
Mutation assay
At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain. The above mentioned dose range finding study with the two tester strains TA100 and WP2uvrA, is reported as a part of the first mutation experiment. In the second part of this experiment, the test item was tested both in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment with additional parameters, the test item was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.
Colony counting
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually.
Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Rationale for test conditions:

The objective of this study was to evaluate 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains resulting in histidine-independent strains, and in a gene of tryptophan-requiring Escherichia coli bacterial strain resulting in a tryptophan-independent strain.

Background of the test system
The Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay have been shown to be rapid and adequate indicators for the mutagenic activity of a wide range of chemical compounds.
The assay was conducted in the absence and presence of a metabolizing system (S9-mix).
The Salmonella typhimurium strains used in this study were TA1535, TA1537, TA98 and TA100. The Escherichia coli strain used was WP2uvrA. The strains TA1537 and TA98 are capable of detecting frameshift mutagens, strains TA1535, TA100 and WP2uvrA are capable of detecting base-pair substitution mutagens

Evaluation criteria:

In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Dose range finding test/first mutation experiment
2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] was tested in the tester strains TA100 and WP2uvrA at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 17, 52, 164, 512 and 1600 μg/plate.

Precipitate
In the dose range finding, precipitation of the test item on the plates was observed at the start and at the end of the incubation period at concentrations of 1600 μg/plate and upwards. In tester strain WP2uvrA, precipitation was also observed at the dose level of 512 μg/plate.
In the first mutation assay, precipitation the test item on the plates was observed at the start of the incubation period at concentrations of 512 and 1600 μg/plate and at 1600 μg/plate at the end of the incubation period.

Toxicity
To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.
No reduction of the bacterial background lawn was observed. A slight decrease in the number of revertants was observed in tester strains TA100 (presence of S9-mix) and TA98 (absence of S9-mix) at the highest concentration tested. A moderate decrease in the number of revertants was observed in tester strain TA98 (presence of S9-mix) at the highest concentration tested.

Mutagenicity
No increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Experiment 2
To obtain more information about the possible mutagenicity the test item, a second mutation experiment was performed in the absence of S9-mix and in the presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, the test item was tested up to the dose level of 1600 μg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

Precipitate
Precipitation of the test item on the plates was observed at the start and at the end of the incubation period at concentrations of 878 and 1600 μg/plate.

Toxicity
No reduction of the bacterial background lawn was observed. A moderate decrease in the number of revertants was only observed in tester strain TA1537 (absence of S9-mix) at concentrations of 878 and 1600 μg/plate.

Mutagenicity
In the second mutation assay, no increase in the number of revertants was observed upon treatment with 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] under all conditions tested.

Formulation analysis
The concentrations analysed in the second mutation experiment in the low, intermediate and high formulations were in agreement with target concentrations (i.e. mean accuracies were 99%, 98% and 91%, respectively).
Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 4 hours.

SUMMARY TABLES

 

Dose range finding test: Mutagenic response in theSalmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay

Dose (μg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with one strain ofSalmonella typhimuriumand oneEscherichia colistrain
	TA100	WP2uvrA
Without S9-mix
Positive control Solvent control 1.7 5.4 17 52 164 512 1600 5000	1007± 102 78 ± 12 82 ± 11 73 ± 13 77 ± 13 92 ± 10 94 ± 6 101 ± 4^NP 71 ± 8^SP 74 ± 7^n MP	1228± 61 41 ± 7 35 ± 5 42 ± 10 33 ± 5 40 ± 8 39 ± 7^NP 42 ± 7^SP 41 ± 9^MP 25 ± 5^n MP
With S9-mix^1
Positive control Solvent control 1.7 5.4 17 52 164 512 1600 5000	1362± 55 91 ± 7 79 ± 14 76 ± 17 84 ± 4 93 ± 15 88 ± 8 93 ± 14^NP 75 ± 7^SP 64 ± 12^n MP	442± 48 40 ± 8 41 ± 6 49 ± 6 46 ± 10 39 ± 7 46 ± 4^NP 48 ± 1^SP 35 ± 2^MP 29 ± 9^n MP

¹Plate incorporation assay (5% S9)

MP Moderate Precipitate

NP No precipitate

SP Slight Precipitate

n Normal bacterial background lawn

 

Experiment 1: Mutagenic response in theSalmonella typhimuriumreverse mutation assay

Dose (μg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains ofSalmonella typhimurium
	TA1535	TA1537	TA98
Without S9-mix
Positive control Solvent control 17 52 164 512 1600	665± 37 8 ± 3 5 ± 3 9 ± 4 9 ± 6 11 ± 3^NP 11 ± 3^n MP	503± 51 11 ± 1 4 ± 4 8 ± 8^n 4 ± 1 3 ± 2^NP 4 ± 2^n MP	1144± 186 9 ± 1 12 ± 3 11 ± 5 10 ± 2 15 ± 5^NP 6 ± 1^n MP
With S9-mix^1
Positive control Solvent control 17 52 164 512 1600	267± 9 11 ± 4 8 ± 4 7 ± 4 4 ± 1 11 ± 3^NP 15 ± 2^n MP	400± 17 4 ± 1 7 ± 4 7 ± 3 9 ± 5 3 ± 2^NP 4 ± 2^n MP	1340± 72 17 ± 8 13 ± 3 17 ± 1 16 ± 2 13 ± 3^NP 8 ± 3 n^MP

¹Plate incorporation assay (5% S9)

MP Moderate Precipitate

NP No Precipitate

n Normal bacterial background lawn

 

Experiment 2: Mutagenic response in theSalmonella typhimuriumreverse mutation assay and in the

Escherichia colireverse mutation assay

Dose (μg/plate)	Mean number revertant colonies/3 replicate plates (± S.D.) with different strains ofSalmonella typhimuriumand oneEscherichia colistrain
	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9-mix
Positive control Solvent control 154 275 492 878 1600	738± 37 8 ± 6 5 ± 5 10 ± 2 12 ± 4^NP 10 ± 2^SP 7 ± 3^n MP	737± 66 4 ± 1 4 ± 0 3 ± 1 4 ± 1^NP 2 ± 3^SP 2 ± 1^n MP	1340± 236 17 ± 2 21 ± 9 18 ± 5 16 ± 4^NP 18 ± 5^SP 8 ± 4^n MP	1031± 61 84 ± 12 78 ± 7 71 ± 6 105 ± 5^NP 96 ± 10^SP 77 ± 7^n MP	1226± 126 19 ± 7 28 ± 6 17 ± 5 24 ± 6^NP 35 ± 3^SP 20 ± 5^n MP
With S9-mix^1
Positive control Solvent control 154 275 492 878 1600	147± 2 9 ± 3 4 ± 4 6 ± 4 7 ± 5^NP 12 ± 7^SP 6 ± 1^n MP	482± 44 5 ± 1 9 ± 2 6 ± 2 10 ± 4^NP 9 ± 3^SP 6 ± 1^n MP	693± 30 23 ± 9 25 ±7 19± 7 21 ± 7^NP 25 ± 3^SP 16 ± 3^n MP	1261± 72 63 ± 0 89 ± 3 72 ± 2 73 ± 6^NP 81 ± 7^SP 83 ± 8^n MP	467± 12 37 ± 3 29 ± 6 30 ± 5 34 ± 10^NP 33 ± 1^SP 25 ± 2^n MP

¹Plate incorporation assay (10% S9)

MP Moderate Precipitate

NP No Precipitate

SP Slight Precipitate

n Normal bacterial background lawn

 

HISTORICAL CONTROL DATA OF THE SOLVENT CONTROL

	TA1535		TA1537		TA98		TA100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	6 – 33	3 – 34	3 – 25	3 – 28	7 – 50	10 – 52	64 – 153	70 – 156	13 – 68	12 – 70
Mean	19	14	8	10	19	27	109	109	29	35
SD	5	5	3	4	6	7	16	15	7	7
n	1185	1248	1023	1041	1299	1324	1284	1259	996	1026

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between July 2013 and May 2015.

 

HISTORICAL CONTROL DATA OF THE POSITIVE CONTROL ITEMS

	TA1535		TA1537		TA98		TA100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	78 – 1932	78 – 1332	51 – 2126	46 – 1112	347 – 1634	210 – 1885	542 – 1436	620 – 2995	118 – 1958	85 – 1390
Mean	792	232	611	381	900	766	911	1435	1324	256
SD	302	97	243	142	148	283	130	381	358	158
n	1270	1278	992	1032	1289	1317	1269	1257	1004	1032

SD = Standard deviations

n = Number of observations

Historical control data from experiments performed between July 2013 and May 2015.

Conclusions:

Based on the results of this study it is concluded that 2,2’-methylenebis[6-(1-methylcyclohexyl)-pcresol] is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Evaluation of the mutagenic activity of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay.

 

The test item was tested in the Salmonella typhimurium reverse mutation assay with four histidine requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

 

The study procedures described in this report are in compliance with the following guidelines:

- Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals; Guideline no. 471: "Genetic Toxicology: Bacterial Reverse Mutation Test" (Adopted July 21, 1997).

- European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.13/14: "Mutagenicity: Reverse Mutation Test using Bacteria”. Official Journal of the European Union No. L142, 31 May 2008.Batch WWP4J0001 of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] was a white to cream powder.

 

A correction factor of 1.08 was used to correct for the purity (92.2%). The test item was dissolved in dimethyl sulfoxide.

 

The concentrations analysed in the samples prepared for use during the second mutation experiment were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).

No test substance was detected in the vehicle. Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 4 hours.

 

In the dose range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test item precipitated on the plates at dose levels of 1600 and 5000 μg/plate in tester strain TA100 and at 512 μg/plate and above in tester strain WP2uvrA. Cytotoxicity, as evidenced by a biologically relevant decrease in the number of revertants, was only observed in tester strain TA100 in the presence of S9-mix at the highest tested concentration.

 

Based on the results of the dose range finding test, the test item was tested in the first mutation assay at a concentration range of 17 to 1600 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test tem precipitated on the plates at the top dose of 1600 μg/plate. Cytotoxicity, as evidenced by a biologically relevant decrease in the number of revertants, was only observed in tester strain TA98 in the absence and presence of S9-mix at the highest tested concentration.

 

In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 154 to 1600 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item precipitated on the plates at dose levels of 878 and 1600 μg/plate. Cytotoxicity, as evidenced by a biologically relevant decrease in the number of revertants, was only observed in tester strain TA1537 in the absence of S9-mix at the two highest tested concentrations.

 

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9 metabolic activation. These results were confirmed in a follow-up experiment.

 

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

Based on the results of this study it is concluded that 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

3 substances are available for read across

Adequacy of study:

weight of evidence

Justification for type of information:

The substance is a member of a group of substituted phenols which share similar characteristics across the group. Please see attached justification for read across.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Read Across to Category source.

Deviations:

no

Key result

Species / strain:

other: From read across

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: Not mutagenic on the basis of read across.

Conclusions:

The substance, CAS 77-62-3; EC 201-044-5, is analogous to the substances to be read across to, in terms of basic form, and the degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of mutagenicity. The substance is considered to be not mutagenic for the defined endpoints on the basis of read across. This will be confirmed by appropriate study data as soon as this is available.

Executive summary:

The substance, CAS 77-62-3; EC 201-044-5, is analogous to the substances to be read across to, in terms of basic form, and the degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of mutagenicity. The substance is considered to be not mutagenic for the defined endpoints on the basis of read across. This will be confirmed by appropriate study data as soon as this is available.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

3 substances are available for read across

Adequacy of study:

weight of evidence

Justification for type of information:

The substance is a member of a group of substituted phenols which share similar characteristics across the group. Please see attached justification for read across.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

Read Across to Category source.

Deviations:

no

Key result

Species / strain:

other: From read across

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: Not mutagenic on the basis of read across.

Conclusions:

The substance, CAS 77-62-3; EC 201-044-5, is analogous to the substances to be read across to, in terms of basic form, and the degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of mutagenicity. The substance is considered to be not mutagenic for the defined endpoints on the basis of read across. This will be confirmed by appropriate study data as soon as this is available.

Executive summary:

The substance, CAS 77-62-3; EC 201-044-5, is analogous to the substances to be read across to, in terms of basic form, and the degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of mutagenicity. The substance is considered to be not mutagenic for the defined endpoints on the basis of read across. This will be confirmed by appropriate study data as soon as this is available.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro Genetic toxicity - Bacterial Reverse Mutation Assay

Evaluation of the mutagenic activity of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay.

The test item was tested in the Salmonella typhimurium reverse mutation assay with four histidine requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

 In the dose range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test item precipitated on the plates at dose levels of 1600 and 5000 μg/plate in tester strain TA100 and at 512 μg/plate and above in tester strain WP2uvrA. Cytotoxicity, as evidenced by a biologically relevant decrease in the number of revertants, was only observed in tester strain TA100 in the presence of S9-mix at the highest tested concentration.

 Based on the results of the dose range finding test, the test item was tested in the first mutation assay at a concentration range of 17 to 1600 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test tem precipitated on the plates at the top dose of 1600 μg/plate. Cytotoxicity, as evidenced by a biologically relevant decrease in the number of revertants, was only observed in tester strain TA98 in the absence and presence of S9-mix at the highest tested concentration.

 In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 154 to 1600 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item precipitated on the plates at dose levels of 878 and 1600 μg/plate. Cytotoxicity, as evidenced by a biologically relevant decrease in the number of revertants, was only observed in tester strain TA1537 in the absence of S9-mix at the two highest tested concentrations.

 The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9 metabolic activation. These results were confirmed in a follow-up experiment.

 Based on the results of this study it is concluded that 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

 

Chromosome aberration

Due to study data being not available prior to the registration deadline, the following studies are used for read across purposes:

2,2'-(2-methylpropylidene)bis[4,6-xylenol]; CAS 33145-10-7; EC 251-394-8

Evaluation of the potential of the test substance to induce chromosome aberrations in cultured peripheral human lymphocytes.

 

This report describes the effect of The test substance on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity of The test substancewas tested in two independent experiments.

 The study procedures described in this report are in compliance with the following guidelines:

Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no. 473: In Vitro Mammalian Chromosome Aberration Test (adopted September 26, 2014).

 Batch WB44L0016 of the test substance was a white to cream coloured powder with a purity of 99.7%. The test substance was soluble in dimethyl sulfoxide at concentrations of 20 mg/ml and below but formed a suspension at concentrations of 25 mg/ml and higher.

 In the first cytogenetic assay, the test substance was tested up to 50 and 70 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction, respectively. Appropriate toxicity was reached at these dose levels.

 In the second cytogenetic assay, the test substance was tested up to 45 μg/ml for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

The test substance did not induce a statistically significant and biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.

 No biologically relevant effects of the test substance on the number of polyploid cells and cells with endo-reduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

 Finally, it is concluded that this test is valid and that the test substance is not clastogenic in human lymphocytes under the experimental conditions described in this report.

6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol; CAS 119-47-1; EC 204-327-1

Results:

Cytotoxic concentration: The concentrations of 50% growth inhibition were as follows:

·       -S9 mix (continuous treatment): 0.008 mg/ml

·       -S9 mix (short-term treatment): 0.002 mg/ml

·       +S9 mix (short-term treatment): 0.03 mg/ml

No genotoxic effects were note with and without metabolic activation in any study design evaluated (short-term and continuous treatment).

The author concluded that cytogenetic effects were not seen under the conditions of this experiment.

6,6'-di-tert-butyl-4,4'butylidenedi-m-cresol; CAS 85-60-9; EC 201-618-5  

This report describes the effect of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was tested in two independent experiments.

The study procedures described in this report were based on the most recent OECD and EC guidelines. The study was performed in accordance with the Principles of Good Laboratory Practice (GLP).

Batch W4B9F0001 of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was a white powder with a purity of 99.4%. The test substance was dissolved in ethanol.

In the first cytogenetic assay, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was tested up to 30 and 40 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction, respectively. 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol precipitated in the culture medium at concentrations of 40 μg/ml and higher. Appropriate toxicity was reached at these dose levels.

In the second cytogenetic assay, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was tested up to 6 μg/ml for a 24 h continuous exposure time with a 24 and 48 h fixation time in the absence of S9-mix. In the presence of S9-mix 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was tested up to 35 μg/ml for a 3 h exposure time with a 48 h fixation time. Appropriate toxicity was reached at these dose levels. 6,6’-ditert-butyl-4,4’-butylidenedi-m-cresol precipitated in the culture medium at a concentration of 35 μg/ml.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.

No effects of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Finally, it is concluded that this test is valid and that 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Mammalian Gene Cell Mutation

Due to study data being not available prior to the registration deadline, the following studies are used for read across purposes:

2,2'-(2-methylpropylidene)bis[4,6-xylenol]; CAS 33145-10-7; EC 251-394-8

Evaluation of the mutagenic activity of the test substance in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells.

 This report describes the effects oftThe test substance on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the absence of S9-mix with 3 and 24-hour treatment periods and in the presence of S9-mix with a 3 hours treatment period (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

 The study procedures described in this report were based on the most recent OECD guideline.

 Batch WB44L0016 of the test substance was a white to cream coloured powder with a purity of 99.7%. The test item was dissolved in dimethyl sulfoxide.

In the first experiment, the test substance was tested up to concentrations of 15 and 17.5 μg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was 32 and 24% in the absence and presence of S9-mix, respectively.

In the second experiment, the test substance was tested up to concentrations of 22.5 μg/ml in the absence of S9-mix. The incubation time was 24 hours. The RTG was 11%.

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, the test substance did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

 In the presence of S9-mix, the test substance did not induce a significant increase in the mutation frequency.

 It is concluded that the test substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol; CAS 119-47-1; EC 204-327-1

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

The author stated that the test substance did not induce gene mutations at the HPRT locus in V79 cells under the experimental conditions used.

6,6'-di-tert-butyl-4,4'butylidenedi-m-cresol; CAS 85-60-9; EC 201-618-5  

Evaluation of the mutagenic activity of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (with independent repeat).

This report describes the effects of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in two independent experiments in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

The study procedures described in this report were based on the most recent OECD and EC guidelines. The study was performed in accordance with the Principles of Good Laboratory Practice (GLP).

Batch W4B9F0001 of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was a white powder with a purity of 99.4%. The test substance was dissolved in ethanol.

In the first experiment, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was tested up to concentrations of 15 and 50 μg/ml in the absence and presence of 8% (v/v) S9-mix, respectively. The incubation time was 3 hours. 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was tested up to cytotoxic levels of 74 and 46% in the absence and presence of S9-mix, respectively. 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol precipitated in the culture medium at dose levels of 35 μg/ml and above.

In the second experiment, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was tested up to concentrations of 17.5 and 70 μg/ml in the absence and presence of 12% (v/v) S9-mix, respectively. The incubation times were 24 hours in the absence of S9-mix and 3 hours for incubations in the presence of S9-mix. The test substance tested up to cytotoxic levels of 94 and 83% in the absence and presence of S9-mix, respectively. 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol precipitated in the culture medium at dose levels of 50 μg/ml and above.

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Except the response of one of the solvent control cultures in the second experiment in the absence of S9-mix. However since this response was just above the upper limit of the range and clear negative results were obtained, the validity of the test was considered to be not affected.

Mutation frequencies in cultures treated with positive control chemicals were increased by 12- and 11-fold for MMS in the absence of S9-mix, and by 12- and 10-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.

 It is concluded that 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Justification for classification or non-classification

In Vitro Genetic Toxicity

Based on the results of the studies and read across it is concluded that 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] is not mutagenic and therefore does not fulfil the requirements for classification. The test substance is "NOT CLASSIFIED".