4,4'-methylenedi-2,6-xylenol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from authoritative database

Data source

Reference

Reference Type:

other: J-check

Title:

Gene mutation toxicity study of the test chemical

Author:

NITE

Year:

2018

Bibliographic source:

J-check

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Bacterial gene mutation assay was performed to determine the mutagenic nature of the test chemical

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol

EC Number:

204-327-1

EC Name:

6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol

Cas Number:

119-47-1

Molecular formula:

C23H32O2

IUPAC Name:

2,2'-methylenebis(6-tert-butyl-4-methylphenol)

Test material form:

solid

Details on test material:

- Name of test material: 2 ,2'-Methylenebis (6-tert-butyl-p-cresol)
- Molecular formula: C23H32O2
- Molecular weight: 340.5038 g/mol
- Substance type: Organic
- Physical state: white crystalline powder
- Purity: ≥98%
- Impurities (identity and concentrations): No data

Method

Target gene:

Histidine

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

S9 metabolic activation system isolated from 7 week old male Sprague-Dawley male rats administered concomitant administration of phenobarbital (PB) and 5,6-benzoflavone (BF).

Test concentrations with justification for top dose:

0, 313, 625, 1250, 2500 or 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test chemical was soluble in acetone

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2- (2-furyl) -3- (5-nitro-2-furyl) acrylamide, and 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No data
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy:Yes
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

When the increase in the number of revertants/plate increased more than 2 times and the increase was found to be reproducible or dose dependent, it was decided that the test substance has mutagenicity (positive) in this test system.

Statistics:

No data

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 100, TA 1535, TA 98, TA 1537,

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Additional information on results
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: When the test was carried out with a common ratio of about 3 in the range of 50 to 5000 μg / plate for the test chemical, it was confirmed that in all test bacteria, no antibacterial activity was observed in any of the addition tests.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No muatgenic potential

Any other information on results incl. tables

Table: Mutation data for the test chemical (Test 1)

With or without S9 mix	Dose (µg/plate)	Number of revertants/plate
		Base pair substitution type			Frameshift type
		TA100	TA1535	WP2uvrA	TA98	TA1537
- S9 mix	0	125±10.5	13±3.6	23±4.7	23±5.7	7±1.0
	313	137±6.0	16±2.0	24±5.9	24±5.0	8±2.6
	625	145±6.5	15±3.0	18±3.8	32±1.7	11±0.0
	1250	130±6.5	13±2.6	16±5.1	21±0.6	10±1.5
	2500	129±4.7	14±3.0	22±2.6	16±2.6	6±1.2
	5000	144±17.8	17±0.6	18±4.2	21±2.5	10±2.0
+ S9 mix	0	148±2.3	14±4.9	26±12.1	39±7.6	12±4.5
	313	141±2.6	15±4.0	29±6.1	38±3.5	19±3.1
	625	136±8.5	19±4.5	16±1.2	28±4.0	16±5.5
	1250	139±16.3	11±3.5	20±3.5	32±6.7	14±3.1
	2500	147±14.6	13±5.6	24±2.1	32±1.5	17±4.5
	5000	133±14.5	14±2.6	19±4.6	22±3.5	9±2.9
Positive control –S9 mix	Chemical	AF	SA	AG2	AF2	9AA
	Dose (µg/plate)	0.01	0.5	0.01	0.1	80
	No. Of colonies/plate	833±41.4	188±17.2	184±22.5	915±22.6	1427±77.6
Positive control +S9 mix	Chemical	2AA	2AA	2AA	2AA	2AA
	Dose (µg/plate)	1	2	10	0.5	2
	No. Of colonies/plate	1385±98.4	266±6.1	1231±60.5	401±20.0	309±32.0

 

Table: Mutation data for the test chemical (Test 2)

With or without S9 mix	Dose (µg/plate)	Number of revertants/plate
		Base pair substitution type			Frameshift type
		TA100	TA1535	WP2uvrA	TA98	TA1537
- S9 mix	0	124±8.5	15±4.2	22±2.6	26±2.5	9±1.5
	313	114±3.1	15±1.0	26±5.9	19±6.2	8±4.2
	625	128±13.3	18±0.6	25±4.2	25±1.5	9±1.2
	1250	133±15.5	17±4.9	27±2.0	20±7.2	7±0.0
	2500	129±8.6	15±5.5	22±1.0	18±1.2	6±1.2
	5000	122±6.1	16±4.5	20±2.5	18±3.0	10±4.0
+ S9 mix	0	138±6.8	15±2.0	27±1.5	29±6.0	14±5.7
	313	118±17.0	16±3.5	29±12.5	33±5.0	17±1.2
	625	133±14.9	13±2.9	25±6.1	28±4.9	10±3.1
	1250	139±30.4	15±2.6	24±3.1	30±6.7	8±3.2
	2500	157±8.7	15±4.0	18±3.1	25±3.8	9±4.2
	5000	126±13.4	14±3.5	16±1.2	31±3.6	13±1.2
Positive control –S9 mix	Chemical	AF	SA	AG2	AF2	9AA
	Dose (µg/plate)	0.01	0.5	0.01	0.1	80
	No. Of colonies/plate	721±110	151±8.5	172±16.1	884±21.7	1138±88.1
Positive control +S9 mix	Chemical	2AA	2AA	2AA	2AA	2AA
	Dose (µg/plate)	1	2	10	0.5	2
	No. Of colonies/plate	1652±69.3	301±2.6	1558±43.3	479±33.3	381±30.6

Applicant's summary and conclusion

Conclusions:

The test chemical did not induce gene mutation in Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537, and Escherichia coli WP 2 uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Bacterial gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537, and Escherichia coli WP 2 uvrA in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in acetone and used at dose level of 0, 313, 625, 1250, 2500 or 5000µg/plate. No increase in the number of mutant colonies that were more than twice the solvent control value in the S9 mix non-addition test and addition test of the five test bacteria used was observed. Based on the observations made, the test chemical did not induce gene mutation in Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537, and Escherichia coli WP 2 uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.