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Toxicological information

Genetic toxicity: in vitro

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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:
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 / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
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 resultsopen allclose all
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
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.