Reaction mass of biphenyl-2-yl diphenyl phosphate and triphenyl phosphate and bis(biphenyl-2-yl) phenyl phosphate

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:

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

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of biphenyl-2-yl diphenyl phosphate and triphenyl phosphate and bis(biphenyl-2-yl) phenyl phosphate

EC Number:

700-393-5

IUPAC Name:

Reaction mass of biphenyl-2-yl diphenyl phosphate and triphenyl phosphate and bis(biphenyl-2-yl) phenyl phosphate

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.