2,2'-methylenebis(4,6-di-tert-butyl-phenyl)-2-ethylhexyl phosphite

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:

3 April 1989 to 18 May 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: "Standards to be observed by Mutagenicity Testing Institutions" issued by the Japanese Ministry of Labour,

Principles of method if other than guideline:

The study was conducted in compliance with "Good Laboratory Practice (GLP) Standards as set forth in Article 4 relating to Test Items of New Chemical Substances and Survey Items of Toxicity of Designated Chemical Substances'' issued by the Japanese Ministry of International Trade and Industry (MITI) etc. and "Standards to be observed by Mutagenicity Testing Institutions" issued by the Japanese Ministry of Labour, and the report presents a full and accurate account of the results of the study.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Name of the new chemical substance: 2,4,8,10-Tetrakis(1,1-dimethyl ethyl)-6-(2-ethylhexyloxy)-12H-dibenzo [d,g] [1,3,2]dioxaphosphocin
Other name: 2,2'-Methylenebis(4.6-di-tert-butylphenyl)2-ethylhexyl phosphate
Lot No. (Batch No.): 102
Purity of the new chemical substance tested: 100 %
Molecular weight: 583
Appearance at ordinary temperature: White powder
Stability
Heat: Stable below 200'C
Light: Stable
Water: Stable(until 10 days)
DMSO: Stable(until 10 days)
Acetone: Stable(until 10 days)
Degree of solubility
Water: Less soluble
DMSO: 0.8%
Acetone: 1.3%

Method

Target gene:

Histidene

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 applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Dose determination test: 1, 5, 10, 50, 100, 500, 1000, 5000 μg/plate
Main test: 156, 313, 625, 1250, 2500, 5000 μg/plate

Vehicle / solvent:

Dimethylsulfoxide (DMSO)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2- (2- Fury I)-3- (5-nitro-2-furyl)- acrylamide (AF- 2); 2- Aminoanthracene ( 2- AA)

Details on test system and experimental conditions:

Test substance: 2,4,8,10-Tetrakis (1,1-dimethyl ethyl)-6-(2-ethylhexyloxy)-12H-dibenzo[d.g] [1,3,2] dioxaphosphocin [other name: 2,2'-methylenebis(4,6-di-tertbutylphenyl)2-ethylhexyl phosphite] was suspended in dimethylsulfoxide (DMSO) by uitrasonification.
Bacterial strains: Salmonella typhimurium stranis TA98, TA100. TA1535 and TA1537 and Escherichia coli WP2uvrA were supplied by Dr. T. Matsushima, Department of Molecular Oncology, Institute of Medical Science, University of Tokyo.
Bacterial cultures in Oxoid nutrient broth #2 were freshly prepared before use by inoculating bacteria from frozen stock cultures (kept at -80°C).
Fifty μl stock solution was inoculated into the 15ml broth and incubated for 10 hours at 37°C and 180rpm using rotary shaker.
S9 and S9Mix;: S9, post-mitochondrial supernatant of rat-liver homogenates was obtained from Kikkoman Corporation. Chiba (JAPAN). This S9 was prepared by slight modifications of method of Ames [1] :5.6-Benzoflavone and Phenobarbital were used as inducers of drug-metabolizing enzyme system [2]. The S9mix contained 4mM NADPH, 4mM NADH. SmM G-6-P. 8mM MgCl2, 33mM KCI. 100mM sodium phosphate buffer (pH 7.4) and 10% S9 (50 μl S9 per plate).

Evaluation criteria:

Not specified in the study report.

Statistics:

Not specified in the study report.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

2,4,8,10-Tetrakis(1,1-dimethyl ethyl)-6-(2-ethylhexyloxy)-12H-dibenzo[d,g] [1 ,3,2] dioxaphosphocin did not induce revertant colonies more than twice of that of the solvent control on the S. typhimurium TA98, TA100, TA1535 and TA1537, and E. coli WP2uvrA with or without metabolic activation.
Positive control substances showed increases in revertant colony count more than twice of the solvent control values with bacterial strains in various tests, and these suggested that susceptibilities of test bacterial strains were adequately maintained and the test was adequately carried out.
Based on the above results, the present test substance was assessed to be negative.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Based on the results, the present test substance was assessed to be negative.

Executive summary:

Purpose of this test is an evaluation of the mutagenicity of 2,4,8,10-tetrakis(1,1-dimethyl ethyl)-6-(2-ethylhexyloxy)-12H-dibenzo [d.g] [1,3,2]dioxaphosphocin [other name: 2,2'-Methylenebis(4,6-di-tert-butylphenyl)2-ethylhexyl phosphite] by the microbial mutagenicity test.

 

The study was conducted in compliance with "Good Laboratory Practice (GLP) Standards as set forth in Article 4 relating to Test Items of New Chemical Substances and Survey Items of Toxicity of Designated Chemical Substances'' issued by the Japanese Ministry of International Trade and Industry (MITI) etc. and "Standards to be observed by Mutagenicity Testing Institutions" issued by the Japanese Ministry of Labour, and the report presents a full and accurate account of the results of the study.

2,4,8,10-Tetrakis(1,1-dimethyl ethyl)-6-(2-ethylhexyloxy)-12H-dibenzo[d,g] [1,3,2] dioxaphosphocin did not induce revertant colonies morethan twice of that of the solvent control on the S. typhimurium TA98,TA100, TA1535 and TA1537, and E. coli WP2uvrA with or without metabolic activation.

Positive control substances showed increases in revertant colony countmore than twice of the solvent control values with bacterial strains in various tests, and these suggested that susceptibilities of test bacterial strains were adequately maintained and the test was adequately carried out.

Based on the above results, the present test substance was assessed to be negative.