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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:
2008-10-08 to 2009-01-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): #5 Biphenyl diphenyl phosphate (mixture of the phosphates of ortho-Phenyl Phenol and Phenol and containing 25 % of Triphenyl Phosphate)
- Substance type: organic
- Physical state:Colorless to light yellowish clear liquid
- Analytical purity: > 99 %
- Impurities (identity and concentrations): no data
- Lot/batch No.: F01003
- Expiration date of the lot/batch: 2009-03-15
- Stability under test conditions: no data
- Storage condition of test material: room temperature; store in a cool, dry, well-ventilated area; keep containers tightly closed when not in use.

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
5, 2.5, 1.25, 0.625 and 0.3125 µL/plate
Controls
Untreated negative controls:
yes
Remarks:
sterile water
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: different positive control substances were used - please see Table 2.1 and 2.2 in "Any other information on materials and methods incl. tables"

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

1) Evaluation of test results

- The raw data of spontaneous revertants values were represented with Mean ± S.D.

- The raw data were analyzed by ANOVA to evaluate the difference between negative control group and test article groups, and p <0.05 indicates the significant difference.

- A increase in revertants over the negative control was as the cut-off between a mutagenic and non-mutagenic response:

A. TA97a, TA98, TA100 and TA102: had more than two-fold increase in revertants over the negative control, then the test articles were considered a mutagen.

B. TA1535: had more than three-fold increase in revertants over the negative control, then the test articles were considered a mutagen. 

2) Results

a) Result of genotyping of Salmonella typhimurium strains

The genotype of five Salmonella typhimurium strains (TA97a, TA98, TA100, TA102 and TA1535) for this study were identified and met the standard as described in Table 1:

Table 1. Result of Genotyping of Salmonella typhimurium strains

Salmonella typhimurium strains Histidine requirement UV sensitivity Crystal violet sensitivity Ampicillin resistance Tetracycline resistance Spontaneous revertants
His+
Bio-
 Plate
His-
Bio-
 Plate
His+ Bio- Plate Zone of growth inhibition* Ampicillin plate Ampicillin Tetracycline plate without S9
TA97a + + + 143.0 ± 6.1
TA98 + + + 12.7 ± 2.1
TA100 + + + 95.0 ± 5.0
TA102 + + + + + 127.0 ± 9.5
TA1535 + + 16.0 ± 1.0

* +: a clear zone of inhibition appeared around the disc

b) Result of dose range finding of test article in TA100 and bacterial reverse mutation test

- The C.V. of 3 plates/group for the negative control group, positive control group and testing groups were met the criterion.

- Based on the result of dose range finding, the concentrations of 5, 2.5, 1.25, 0.625 and 0.3125 uL/plate of "#5 Biphenyl diphenyl phosphate" were used for bacterial reverse mutation test in five Salmonella typhimurium strains. The positive control articles were caused more than twofold increase in revertant colonies on TA97a, TA98, TA100 and TA102; more than three-fold increase in revertants colonies on TA1535 (Table 3).

- In all five Salmonella typhimurium strains, there were no significant increase in the numbers of revertant colonies in the presence and absence of a S9 mixture metabolic activation system (Table 3).

Table 2. Result of Dose Range Finding of Test Article in Salmonella typhimurium TA100

Dose (µL /plate) Number of revertants / plate (Mean ± S.D., n=3)
TA100 without S9 mixture
Negative control (Sterile water) 111.0 ± 13.1
Positive control 523.3 ± 74.6
#5 Biphenyl diphenyl phosphate (CAS number: 132-29-6)  
5 110.0 ± 10.5
2.5 110.0 ± 11.4
1.25 111.0 ± 10.6
0.625 106.3 ± 9.3
0.3125 115.3 ± 9.6

Table 3. Result of Bacterial Reverse Mutation Test

Dose (µL /plate) Number of revertants /plate (without S9 mixture, Mean ± S.D., n=3)
TA97a TA98 TA100 TA102 TA1535
Negative control (sterile water) 146.7 ± 0.6 13.0 ± 3.5 108.3 ± 16.9 145.3 ± 13.6 13.3 ± 0.6
Positive control 570.7 ± 78.9 159.3 ± 19.0 437.3 ± 28.1 640.0 ± 36.7 298.7 ± 20.1
#5 Biphenyl diphenyl phosphate (CAS number: 132-29-6)          
5 182.3 ± 14.4 12.0 ± 1.7 129.7 ± 5.8 139.3 ± 9.0 12.0 ± 3.0
2.5 171.3 ± 6.7 13.0 ± 1.0 123.3 ± 15.3 136.0 ± 13.9 15.7 ± 3.1
1.25 178.3 ± 2.5 10.7 ± 0.6 107.3 ± 5.7 150.0 ± 4.0 15.3 ± 3.2
0.625 163.0 ± 7.9 11.7 ± 1.5 115.7 ± 13.7 154.7 ± 4.6 14.0 ± 2.0
0.3125 174.7 ± 4.9 11.7 ±2.1 122.3 ± 10.7 154.0 ± 12.5 12.7 ±3.2
Dose (µL /plate) Number of revertants /plate (with S9 mixture, Mean ± S.D., n=3)
Negative control (sterile water) 186.3 ± 13.0 14.3 ± 3.5 95.7 ± 4.0 123.3 ± 3.1 13.7 ± 3.5
Positive control 1920.0 ± 80.0 430.7 ± 48.1 480.0 ± 40.0 608.0 ± 21.2 180.0 ± 12.5
#5 Biphenyl diphenyl phosphate (CAS number: 132-29-6)          
5 192.0 * 7.9 16.7 ± 2.5 105.3 ± 11.9 130.0 ± 7.2 12.0 ± 1.7
2.5 198.3 ± 4.2 19.7 ± 2.9 119.0 ± 9.5 126.0 ± 7.2 12.3 ± 4.0
1.25 193.0 ± 1.0 16.7 ± 3.5 95.7 ± 9.8 142.0 ± 5.3 9.7 ± 0.6
0.625 197.3 ± 6.5 21.0 ± 0.0 94.0 ± 4.4 158.0 ± 7.2 9.3 ± 0.6
0.3125 206.7 ± 11.6 18.7 ± 2.1 104.0 ± 9.5 153.3 ± 11.7 12.7 ± 2.1

Table 4. Concentration of positive control

Strain S9 mixture Positive control Concentration (µg /plate)
TA97a without 9-Aminoacridine (9-AA) 50
TA98 2-nitrofluorene (2-NF) 1
TA100 sodium azide (SA) 1
TA102 mitomycin C (MMC) 0.2
TA1535 sodium azide (SA) 1
TA97a with 2-aminoanthracene (2-AA) 5
TA98 2-aminoanthracene (2-AA) 1
TAIOO benzo(a)pyrene (B(a)P) 1
TA102 2-aminoanthracene (2-AA) 5
TA1535 2-aminoanthracene (2-AA) 5

Applicant's summary and conclusion

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

Based on the results of bacterial reverse mutation test, for five Salmonella typhimurium strains, no significant increase in the numbers of revertant colonies were observed in the presence and absence of a S9 mixture metabolic activation system.
In conclusion, the test item did not cause mutagenicity in this study.
Executive summary:

"Bacterial Reverse Mutation Test" was used in this study to evaluate the genotoxicity of "#5 Biphenyl diphenyl phosphate". The test was performed in accordance with OECD Guideline 471.

Five doses (5, 2.5, 1.25, 0.625 and 0.3125 µL /plate) were used for dose range finding test in Salmonella typhimurium TA100 to determinate the testing dosage of bacterial reverse mutation test. The results showed that the test article was non-cytotoxicity and five doses (5, 2.5, 1.25, 0.625 and 0.3125 µL/plate) were used for bacterial reverse mutation test in Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535. Plate incorporation method was used in the presence and absence of a S9 mixture metabolic activation system.

Based on the results of bacterial reverse mutation test, for -fiveSalmonella typhimuriumstrains, no significant increase in the numbers of revertant colonies were observed in the presence and absence of a S9 mixture metabolic activation system. In conclusion, "#5 Biphenyl diphenyl phosphate" did not cause mutagenicity in this study.