Phosphoric acid, mixed esters with biphenyl-4,4'-diol and phenol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization. Study is adequate for assessment with acceptable restrictions. Stability of the test material was the responsibility of the sponsor and therefore an expiry date was not reported for the batch of test material used.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

of 2000

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

biphenyl-4,4'-diyl tetraphenyl bis(phosphate)

EC Number:

700-627-6

Cas Number:

17270-01-8

Molecular formula:

C36H28O8P2

IUPAC Name:

biphenyl-4,4'-diyl tetraphenyl bis(phosphate)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: essential amino acid requiring strains

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from male Sprague Dawley rats treated by oral gavage on 3 consecutive days with phenobarbitone and β-naphthoflavone (80 and 100 mg/kg/day, respectively) for enzyme induction. (S9 mix = 10% liver S9 in standard cofactors)

Test concentrations with justification for top dose:

Preliminary Toxicity Study (TA100, WP2 uvrA without or with metabolic activation (S9))
Doses: 0 (vehicle control); 0.15; 0.5; 1.5; 5; 15; 50; 150; 500; 1500 and 5000 μg/plate

Experiments 1 and 2,
(direct plate incorporation method, TA 98, TA100, TA1535, TA1537, WP2 uvrA, each without and with metabolic activation (S9) tested in triplicate)
Doses: 0 (vehicle control); 50; 150; 500; 1500 and 5000 μg/plate.

Vehicle / solvent:

Dimethyl sulphoxide (DMSO) (solvent was dried using molecular sieves, sodium alumino-silicate i.e. 2 mm pellets, nominal pore diameter 4 A)

Justification for choice of solvent/vehicle:
Test material was insoluble in water according to information of the sponsor, but it was soluble in dimethyl sulphoxide (DMSO) at the required concentration of 50 mg/mI in solubility checks performed in this laboratory. Vehicle (DMSO) control plates produced counts of revertant colonies within the normal range.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

In addition, sterility controls were included to assess sterility of the test material

Negative solvent / vehicle controls:

yes

Remarks:

without and with S9 mix

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Positive control substances for tests without metabolic activation (S9 mix). All of them are well established reference mutagens.

Controls 2

Untreated negative controls:

yes

Remarks:

In addition, sterility controls were included to assess sterility of the test material and S9 mix

Negative solvent / vehicle controls:

yes

Remarks:

without and with S9 mix

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene for TA 100, TA 1535, TA 1537 & WP2 uvrA

Remarks:

Positive control substance for tests with metabolic activation (S9 mix). Both are well established reference mutagen.

Details on test system and experimental conditions:

Direct Plate Incorporation Tests, both without and with metabolic activation, were performed in both experiments (Experiments 1 and 2).

Prior to use the master strains were checked for characteristics, viability and spontaneous reversion rate and were all found to be satisfactory.

The following positive controls were used to check mutability of the bacteria and activity of the S9 mix:

Without metabolic activation (S9 mix):

N-ethyl-N'-nitro-N-nitrosoguanidine:
- 2 μg/plate: - strain WP2 uvrA,
- 3 μg/plate: - strain TA 100,
- 5 μg/plate: - strain TA 1535

9-Aminoacridine:
- 80 μg/plate: - strain TA 1537

4-Nitroquinoline-1-oxide:
- 0.2 μg/plate: - strain TA 98

With metabolic activation (S9 mix):

2-Aminoanthracene:
- 1 μg/plate: - strain TA 100,
- 2 μg/plate: - strains TA 1535 and TA 1537,
- 10 μg/plate: - strain WP2 uvrA,

Benzo(a)pyrene:
- 5 μg/plate: - strain TA 98

Evaluation criteria:

The test chemical was considered to exhibit mutagenic activity in this assay if the following criteria were met:
A dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain in the presence and/or absence of the S9 microsomal enzymes.

The test chemical was considered to be non-mutagenic (negative) if the above criteria were not met.

Positive controls were considered to be valid if their mean revertant count (per strain without or with metabolic acitvation) was at least two times the respective vehicle control value.

Statistics:

The study report does not clearly indicate whether or not the data were analysed for statistically significant differences of revertant counts although reference is given to:
Kirkland DJ (Ed) 1989: Statistical Evaluation of Mutagenicity Test Data.
UKEMS Sub-committee on Guidelines for Mutagenicity Testing, Report - Part Ill, Cambridge University Press.

Comment:
However, the study report states that biological relevance of the results would be considered first and the study result was unequivocal. Statistical analysis of the data was not required to determine the result of the test.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Preliminary Toxicity study

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Preliminary Toxicity study

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Preliminary Toxicity study

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Preliminary Toxicity study

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Key result

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:

valid

Positive controls validity:

valid

Additional information on results:

S9 mix used in each experiment and the test material formulations tested for sterility in the preliminary toxicity test showed no evidence of contamination.
An opaque film was noted from 1500 µg/plate and an associated oily precipitate observed at 5000 µg/plate. Neither of these observations interfered with the scoring of revertant colonies.

Applicant's summary and conclusion

Conclusions:

negative without and with metabolic activation (S9 mix)