2,2'-(2-methylpropylidene)bis[4,6-xylenol]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 April 2016 to 12 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study performed in accordance with OECD, MITI & EU test guidelines in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-(2-methylpropylidene)bis[4,6-xylenol]

EC Number:

251-394-8

EC Name:

2,2'-(2-methylpropylidene)bis[4,6-xylenol]

Cas Number:

33145-10-7

Molecular formula:

C20H26O2

IUPAC Name:

2-[1-(2-hydroxy-3,5-dimethylphenyl)-2-methylpropyl]-4,6-dimethylphenol

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification: Lowinox® 22IB46IUPAC: 2,2’-(2-methylpropylidene)bis[4,6-xylenol]Appearance: White to cream coloured powderBatch: WB44L0016Purity/Composition: 99.7%Test item storage: At room temperatureStable under storage conditions until: 18 November 2018 (retest date)

Method

Target gene:

Salmonella typhimurium (uvrB-)Escherichia coli (WP2uvrA)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-Mixture

Test concentrations with justification for top dose:

Lowinox® 22IB46 was tested up to concentrations of 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. Lowinox® 22IB46 precipitated on the plates at the dose levels of 1600 and 5000 μg/plate.

Vehicle / solvent:

The vehicle of the test article, being ethanol (Extra pure, Merck, Darmstadt, Germany).

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

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°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 (Oxoid LTD) 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 35.0 – 38.0°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) was obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that had been injected intraperitoneal with Aroclor 1254 (500 mg/kg body weight). 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
First experiment: Seven concentrations of the test item, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA.
Second experiment: Based on the results of the first mutation assay, five doses (increasing with approximately half-log steps) of the test item were selected and tested in triplicate in each strain in the second experiment. The highest concentration of Lowinox® 22IB46 used in the second mutation assay was 5000 μg/plate.

Experimental procedure: The test item was tested both in the absence and presence of S9-mix in each strain, in two independent experiments. The vehicle control 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 ethanol, 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 h. After this period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli) were counted.

Colony counting: The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually. Evidence of test article 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:

Recommended test system in international guidelines (e.g. OECD, EC and MITI).Trinova Biochem GmbH, Germany (Master culture from Dr. Bruce N. Ames) (TA1535: 2006, TA1537: 2009, TA98: 2015, TA100: 2015) and (Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK) (WP2uvrA, 2008)

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 vehicle 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 independently repeated 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 vehicle control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.

Statistics:

No formal hypothesis testing was done.

Results and discussion

Test results

Test results 1

Key result

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

Test results 2

Key result

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

Test results 3

Key result

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

Test results 4

Key result

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

Test results 5

Key result

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:

First mutation experiment: Lowinox® 22IB46 was tested in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA with concentrations of 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix.
Precipitate: Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards and at 1600 and 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 examined.No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
Mutagenicity: No increase in the number of revertants was observed upon treatment with Lowinox® 22IB46 under all conditions tested.

Second mutation assay: To obtain more information about the possible mutagenicity of the test item, a second mutation experiment was performed in the absence and presence of 10% (v/v) S9-mix. Based on the results of the first mutation experiment, the test item was tested up to concentrations of 5000 μg/plate.
Precipitate: Precipitation of Lowinox® 22IB46 on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards and at 1600 and 5000 μg/plate at the end of the incubation period.
Toxicity: With the exception of tester strain TA98 (absence of S9-mix) and TA1535 (presence of S9-mix) where an extreme reduction of the revertant colonies was observed at the highest tested concentration), there was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
Mutagenicity: No increase in the number of revertants was observed upon treatment with Lowinox® 22IB46 under all conditions tested.

Any other information on results incl. tables

Experiment 1: Mutagenic response of Lowinox®22IB46 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 different strains ofSalmonella typhimuriumand oneEscherichia colistrain
	TA 1535		TA 1537		TA 98		TA 100		WP2uvrA
Without S9-mix
Positive control Solvent control 5.4 17 52 164 512 1600 5000	1158 ± 6 ± 10 ± 10 ± 10 ± 10 ± 14 ± 9 ± 19 ±	16 2 5 6 5 2 5^NP 2^MP 2^n HP	844 ± 4 ± 5 ± 5 ± 4 ± 4 ± 7 ± 6 ± 5 ±	54 3 1 2 3 1 4^NP 5^MP 1^n HP	1972 ± 16 ± 21 ± 23 ± 24 ± 25 ± 22 ± 19 ± 20 ±	153 5 3 1 5 2 1^NP 3^MP 4^n HP	1076 ± 102 ± 96 ± 108 ± 111 ± 111 ± 84 ± 119 ± 69 ±	37 14 14 8 5 5 5^NP 20^MP 7^n HP	1811 ± 19 ± 20 ± 21 ± 20 ± 22 ± 27 ± 22 ± 18 ±	55 5 9 2 8 7 6^NP 1^MP 10^n HP
With S9-mix^1
Positive control Solvent control 5.4 17 52 164 512 1600 5000	411 ± 8 ± 7 ± 8 ± 4 ± 13 ± 9 ± 6 ± 8 ±	8 3 4 2 3 4 4^NP 2^MP 3^n HP	370 ± 4 ± 7 ± 6 ± 6 ± 4 ± 5 ± 6 ± 6 ±	96 5 3 1 2 2 2^NP 1^MP 3^n HP	1297 ± 25 ± 28 ± 23 ± 22 ± 29 ± 28 ± 19 ± 26 ±	66 6 2 1 7 2 3^NP 2^MP 1^n HP	1731 ± 99 ± 119 ± 115 ± 96 ± 83 ± 110 ± 122 ± 91 ±	113 2 10 11 6 16 19^NP 4^MP 7^n HP	624 ± 25 ± 26 ± 26 ± 28 ± 25 ± 28 ± 21 ± 25 ±	17 8 3 6 5 1 6^NP 6^MP 2^n HP

¹Plate incorporation assay (5% S9)

^HPHeave Precipitate

^MPModerate Precipitate

^NPNo precipitate

ⁿNormal bacterial background lawn

 

Experiment 2: Mutagenic response of Lowinox®22IB46 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 different strains ofSalmonella typhimuriumand oneEscherichia colistrain
	TA 1535		TA 1537		TA 98		TA 100		WP2uvrA
Without S9-mix
Positive control Solvent control 52 164 512 1600 5000	229± 6 ± 7 ± 10 ± 7 ± 5 ± 5 ±	13 2 2 4 2^NP 1^MP 1^n MP	686 ± 5 ± 7 ± 7 ± 7 ± 6 ± 4 ±	56 5 3 6 3^NP 2^MP 2^n MP	1407 ± 17 ± 20 ± 22 ± 23 ± 16 ± 6 ±	74 8 3 3 7^NP 7^MP 2^n MP	1139 ± 119 ± 133 ± 119 ± 128 ± 108 ± 114 ±	51 11 9 16 16^NP 12^MP 22^n MP	1190 ± 24 ± 24 ± 23 ± 22 ± 17 ± 18 ±	40 5 5 6 11^NP 3^MP 7^n MP
With S9-mix^1
Positive control Solvent control 52 164 512 1600 5000	861 ± 10 ± 7 ± 9 ± 11 ± 5 ± 3 ±	57 2 5 2 4^NP 2^MP 1^n MP	545 ± 8 ± 7 ± 7 ± 10 ± 7 ± 8 ±	44 2 3 3 4^NP 3^MP 2^n MP	683 ± 25 ± 26 ± 27 ± 25 ± 14 ± 19 ±	69 6 6 7 6^NP 5^MP 7^n MP	1453 ± 110 ± 92 ± 98 ± 71 ± 77 ± 81 ±	90 6 11 15 6^NP 3^MP 10^n MP	502 ± 34 ± 31 ± 34 ± 23 ± 24 ± 24 ±	13 12 10 11 4^NP 8^MP 6^n MP

¹Plate incorporation assay (10% S9)

^MPModerate Precipitate

^NPNo precipitate

ⁿNormal bacterial background lawn

 

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that Lowinox® 22IB46 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 Lowinox® 22IB46 in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay.

 

Lowinox® 22IB46 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 in the presence and absence of S9-mix (rat liver S9-mix induced by 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.

- Guideline stipulated by the Japanese Ministry of Health, Labour and Welfare, Ministry of Economy, Trade and Industry and Ministry of the Environment (revised March 31st, 2011).

 

Batch WB44L0016 of Lowinox® 22IB46 was a white to cream coloured powder with a purity of 99.7%. The test item was dissolved in ethanol.

 

In the first mutation assay, Lowinox® 22IB46 was tested up to concentrations of 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. Lowinox® 22IB46 precipitated on the plates at the dose levels of 1600 and 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed.

 

In the second mutation assay, Lowinox® 22IB46 was tested up to concentrations of 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix. Lowinox® 22IB46 precipitated on the plates at the dose levels of 1600 and 5000 μg/plate. Cytotoxicity, as evidenced by a biologically decrease in the number of revertants, was observed in tester strain TA98 in the absence of S9-mix and TA1535 in the presence of S9-mix at the highest tested concentration.

 

Lowinox® 22IB46 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 an independently repeated 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 Lowinox® 22IB46 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.