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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
Study period:
03 August 2015 to 20 August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-methylenebis[6-(1-methylcyclohexyl)-p-cresol]
EC Number:
201-044-5
EC Name:
2,2'-methylenebis[6-(1-methylcyclohexyl)-p-cresol]
Cas Number:
77-62-3
Molecular formula:
C29H40O2
IUPAC Name:
2-{[2-hydroxy-5-methyl-3-(1-methylcyclohexyl)phenyl]methyl}-4-methyl-6-(1-methylcyclohexyl)phenol
Test material form:
not specified
Details on test material:
Test Substance: Lowinox WSP
Batch No.: 5E049 (acc. to certificate of analysis)
Chem. Name (IUPAC): Methylen-bis-(2-hydroxy-3-a-methyl-cydohexyl-4-methyI-benzol) (acc. to Information Data Sheet)
CAS-No. (a.i.): 77-62-3 (acc. to safety data sheet)
Content of a.i.: 96.7 (%) (acc. to Information Data Sheet)
Stability: at maximum 6 months
Certificate of Analysis: dated May 23, 1995, revised May 30 and July 20, 1995
Safety Data Sheet: dated May 18, 1994
Information Data Sheet: dated May 23, 1995
Storage conditions specified by the Sponsor: room temperature,23 ± 5 (°C), protected from light (acc. to Information Data Sheet)
Storage conditions at the test facility: 23 ± 5 (°C), dark, protected from moisture.
Reserve sample on May 30, 1995 at least 1 (g) test substance has been stored

CHARACTERISTICS OF TEST SUBSTANCE
Water solubility: <100 (mg/L) (acc. to Information Data Sheet)
Volatility: not specified
Partition coefficient n-Octanol/Water: not specified
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

Method

Target gene:
histidine and tryptophan
Species / strain
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).
Controls
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.

Results and discussion

Test resultsopen allclose all
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.

Any other information on results incl. tables

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 ± 4NP

71 ± 8SP

74 ± 7n MP

1228± 61

41 ± 7

35 ± 5

42 ± 10

33 ± 5

40 ± 8

39 ± 7NP

42 ± 7SP

41 ± 9MP

25 ± 5n MP

With S9-mix1

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 ± 14NP

75 ± 7SP

64 ± 12n MP

442± 48

40 ± 8

41 ± 6

49 ± 6

46 ± 10

39 ± 7

46 ± 4NP

48 ± 1SP

35 ± 2MP

29 ± 9n 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 ± 3NP

11 ± 3n MP

503± 51

11 ± 1

4 ± 4

8 ± 8n

4 ± 1

3 ± 2NP

4 ± 2n MP

1144± 186

9 ± 1

12 ± 3

11 ± 5

10 ± 2

15 ± 5NP

6 ± 1n MP

With S9-mix1

Positive control

Solvent control

17

52

164

512

1600

267± 9

11 ± 4

8 ± 4

7 ± 4

4 ± 1

11 ± 3NP

15 ± 2n MP

400± 17

4 ± 1

7 ± 4

7 ± 3

9 ± 5

3 ± 2NP

4 ± 2n MP

1340± 72

17 ± 8

13 ± 3

17 ± 1

16 ± 2

13 ± 3NP

8 ± 3 nMP

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 ± 4NP

10 ± 2SP

7 ± 3n MP

737± 66

4 ± 1

4 ± 0

3 ± 1

4 ± 1NP

2 ± 3SP

2 ± 1n MP

1340± 236

17 ± 2

21 ± 9

18 ± 5

16 ± 4NP

18 ± 5SP

8 ± 4n MP

1031± 61

84 ± 12

78 ± 7

71 ± 6

105 ± 5NP

96 ± 10SP

77 ± 7n MP

1226± 126

19 ± 7

28 ± 6

17 ± 5

24 ± 6NP

35 ± 3SP

20 ± 5n MP

With S9-mix1

Positive control

Solvent control

154

275

492

878

1600

147± 2

9 ± 3

4 ± 4

6 ± 4

7 ± 5NP

12 ± 7SP

6 ± 1n MP

482± 44

5 ± 1

9 ± 2

6 ± 2

10 ± 4NP

9 ± 3SP

6 ± 1n MP

693± 30

23 ± 9

25 ±7

19± 7

21 ± 7NP

25 ± 3SP

16 ± 3n MP

1261± 72

63 ± 0

89 ± 3

72 ± 2

73 ± 6NP

81 ± 7SP

83 ± 8n MP

467± 12

37 ± 3

29 ± 6

30 ± 5

34 ± 10NP

33 ± 1SP

25 ± 2n 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.

Applicant's summary and conclusion

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.

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