Reaction mass of 4-(2-methylbutan-2-yl)phenol and 2,4-bis(2-methylbutan-2-yl)phenol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 September 2015 to 30 October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Tester strain TA1535 was tested both in the absence and presence of S9-mix in the additional direct plate assay. The study integrity was not adversely affected by the deviation.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Tester strain TA1535 was tested both in the absence and presence of S9-mix in the additional direct plate assay. The study integrity was not adversely affected by the deviation.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1639131-79-5

Cas Number:

1639131-79-5

IUPAC Name:

1639131-79-5

Constituent 2

Reference substance name:

Phenol, 1,1-dimethylpropyl derivs.

IUPAC Name:

Phenol, 1,1-dimethylpropyl derivs.

Test material form:

other: pale yellow solid

Details on test material:

Identification: Phenol, 1,1-dimethylpropyl derivs.Appearance: Colourless to pale yellow solidBatch: OP: C605E003.1Purity/Composition: 100% Unknown or Variable Composition, Complex Reaction Products and Biological Materials (UVCB)Test item storage: At room temperature protected from light container flushed with nitrogenStable under storage conditions until: 31 July 2016 (expiry date)Purity/composition correction factor: No correction factor requiredTest item handling: Use amber glassware or wrap container in aluminum-foilStability at higher temperatures: Yes, maximum temperature: 130°C maximum duration: multiple hours if stored under nitrogen. If heated only minutes.Chemical name (IUPAC), synonym or trade name: Phenol, 1,1-dimethylpropyl derivs.CAS Number: 1639131-79-5Molecular formula: Reaction mass so not availableMolecular weight: Reaction mass so not available

Method

Target gene:

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.

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 were 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 was 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:

Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested.

Vehicle / solvent:

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:

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 rpm), 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 a test.

Agar plates : Agar plates (ø 9 cm) contained 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). 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 36.1 – 39.3°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 S9-fraction: 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). Before use, all S9 batches were characterised with the mutagens benzo-(a)-pyrene (Sigma) 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 and 15.2 mg glucose-6-phosphate in 5.5 ml Milli-Q water; 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution; 1 ml 0.33 M KCl solution. 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.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 the WP2uvrA, both with and without 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 the level at which the test item inhibited bacterial growth. At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain in the absence and presence of S9-mix. The first experiment was a direct plate assay and the second experiment was a pre-incubation assay. Initially in the direct plate assay, tester strain TA98 was rejected due to technical reasons, this part of the study was repeated. An additional direct plate assay was performed with tester strain TA1535. The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.

First experiment: direct plate assay The above mentioned dose range finding study with two tester strains is reported as a part of the direct plate assay. In the second part of this experiment, the test item was tested both in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98. 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. Second experiment: pre-incubation assayThe test item was tested both in the absence and presence of S9-mix in all tester strains. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were pre-incubated for 30 minutes by 70 rpm at 37°C, 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), 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. After the pre-incubation period the solutions were added to 3 ml molten top agar. 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.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if: a) The total number of revertants in 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 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 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 or TA98 is greater than three (3) times the concurrent control. b) In case a repeat 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 results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

First experiment: Direct plate assay The test item was initially tested in the tester strains TA100 and WP2uvrA as a dose range finding test with 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 in the absence and presence of S9-mix: 1.7, 5.4, 17, 52 and 164 μg/plate with the tester strains TA1535 and TA1537 and 1.7, 5.4, 17, 52, 164 and 512 μg/plate with tester strain TA98. Precipitate Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 1600 μg/plate and upwards and at 5000 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 observed. The reduction of the bacterial background lawn and the reduction in the number of revertants is presented in Table form (See Any other information).Mutagenicity In the direct plate test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested. First experiment (additional): Direct plate assay Since in the presence of S9-mix, insufficient toxicity without precipitate on the plates was observed in tester strain TA1535, an additional experiment was performed. The following dose range was selected for the mutation assay in the absence and presence of S9-mix: 1.7, 5.4, 17, 52, 164 and 512 μg/plate. Precipitate Precipitation of Phenol, 1,1-dimethylpropyl derivs.on the plates was not observed at the start or at the end of the incubation period. Toxicity The reduction of the bacterial background lawn and the reduction in the number of revertants is presented in table form (see Any other information). Mutagenicity In the direct plate test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested. Second experiment: Pre-incubation assay To obtain more information about the possible mutagenicity of the test item, a pre-incubation experiment was performed in the absence and presence of S9-mix. Based on the results of the first mutation assay, the test item was tested up to the dose level of 164 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and up to 1600 μg/plate in tester strain WP2uvrA. Precipitate Precipitation of the test item on the plates was observed at the start of the incubation period at the concentration of 1600 μg/plate and no precipitation was observed at end of the incubation period.Toxicity The reduction of the bacterial background lawn and the reduction in the number of revertants is presented in table form (see Any other information).Mutagenicity In the pre-incubation test, no increase in the number of revertants was observed upon treatment with Phenol, 1,1-dimethylpropyl derivs. under all conditions tested.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Toxicity in the dose range finding test/first experiment

Strain	Without S9-mix			With S9-mix
	Dose (μg/plate)	Bacterial background lawn	Revertant colonies	Dose (μg/plate)	Bacterial background lawn	Revertant colonies
Dose range finding test
TA 100	52 164 512 1600 5000	Slight Extreme Absent Absent Absent	^-1 Microcolonies Complete Complete Complete	52 164 512 1600 5000	Normal Slight Absent Absent Absent	Slight Moderate Complete Complete Complete
WP2uvrA	512 1600 5000	Slight Extreme Absent	^-2 Microcolonies Complete	1600 5000	Extreme Absent	Microcolonies Complete
First mutation experiment
TA 1535	164	Moderate	^-2	164	Normal	^-2
TA 1537	52 164	Slight Moderate	^-2 ^-2	164	Slight	^-2
TA 98	164 512	Slight Absent	^-2 Complete	164 512	Slight Extreme	^-2 Microcolonies

^-1No reduction in the number of revertant colonies less than 20% compared to the concurrent vehicle control

^-2No reduction in the number of revertant colonies less than the minimal value of the historical control data range

 

Toxicity in the additional experiment

Strain	Without S9-mix			With S9-mix
	Dose (μg/plate)	Bacterial background lawn	Revertant colonies	Dose (μg/plate)	Bacterial background lawn	Revertant colonies
TA 1535	164 512	Moderate Absent	Extreme Complete	164 512	Slight Extreme	^-1 Microcolonies

^-1No reduction in the number of revertant colonies less than the minimal value of the historical control data range.

 

Toxicity in the second mutation experiment

Strain	Without S9-mix			With S9-mix
	Dose (μg/plate)	Bacterial background lawn	Revertant colonies	Dose (μg/plate)	Bacterial background lawn	Revertant colonies
TA 1535	17 52 164	Extreme Extreme Absent	Microcolonies Microcolonies Complete	52 164	Slight Extreme	^-1 Microcolonies
TA 1537	17 52 164	Extreme Extreme Extreme	Microcolonies Microcolonies Microcolonies	164	Extreme	Microcolonies
TA 98	52 164	Extreme Extreme	Microcolonies Microcolonies	164	Extreme	Microcolonies
TA 100	17 52 164	Extreme Extreme Absent	Microcolonies Microcolonies Complete	164	Absent	Complete
WP2uvrA	164 512 1600	Extreme Extreme Absent	Microcolonies Microcolonies Complete	512 1600	Extreme Absent	Microcolonies Complete

^-1No reduction in the number of revertant colonies less than the minimal value of the historical control data range.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):negative with and without metabolic activationBased on the results of this study it is concluded that Phenol, 1,1-dimethylpropyl derivs. is not mutagenic in the Salmonella typhimurium reverse gene mutation assay and in the Escherichia coli reverse gene mutation assay.

Executive summary:

Evaluation of the mutagenic activity of Phenol, 1,1-dimethylpropyl derivs. in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (plate incorporation and pre-incubation methods).

 

Phenol, 1,1-dimethylpropyl derivs. was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) 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, at first a direct plate assay was performed and secondly a pre-incubation assay both in the absence and presence of S9-mix (rat liver S9-mix induced Aroclor 1254). An additional direct plate assay was performed with tester strain TA1535.

 

The study procedures described in this report were based on the most recent OECD and EC guidelines.

 

Batch OP: C605E003.1 of the test item was a colourless to pale yellow solid. The test item was dissolved in dimethyl sulfoxide.

 

In the dose range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item precipitated on the plates at the top dose of 5000 μg/plate. Toxicity was observed in both tester stains. In the first mutation experiment, the test item was tested up to concentrations of 164 μg/plate in the tester strains TA1535 and TA1537 and up to 512 μg/plate in tester strain TA98. Toxicity was observed in all three tester strains, except in tester strain TA1535 in the presence of S9-mix. Since in the presence of S9-mix, insufficient toxicity without precipitate on the plates was observed in tester strain TA1535, an additional experiment was performed. The test item was tested up to concentrations of 512 μg/plate. Toxicity was observed in tester strain TA1535.

 

In the second mutation experiment, the test item was tested up to concentrations of 164 μg/plate in the tester strains TA1535, TA1537, TA98andTA100 and up to 1600μg/plate in tester strain WP2uvrA in the pre-incubation assay. Toxicity was observed in all tester strains.

 

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

Phenol, 1,1-dimethylpropyl derivs. 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 tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments.

 

Based on the results of this study it is concluded that Phenol, 1,1-dimethylpropyl derivs. is not mutagenic in the Salmonella typhimurium reverse gene mutation assay and in the Escherichia coli reverse gene mutation assay.