2,2''-dihydroxy-4,4''-(2-hydroxy-propane-1,3-diyldioxy)dibenzophenone

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996-08-07 to 1996-08-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

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:

-

EC Number:

424-210-0

EC Name:

-

Cas Number:

23911-85-5

Molecular formula:

C29 H24 O7

IUPAC Name:

2-benzoyl-5-[3-(4-benzoyl-3-hydroxyphenoxy)-2-hydroxypropoxy]phenol

Method

Target gene:

S. typhimurium: His;
E.coli: Trp

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: uvrB deletion and rfa mutation (S. typhimurium)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Aroclor 1254-induced rat liver S9
- source of S9: livers of male Sprague-Dawley rats which received a single i.p. injection of Aroclor 1254 at 500 mg/kg, five days before S9 preparation
- method of preparation of S9 mix: not specified
- volume of S9 mix in the final culture medium: 0.5 mL of S9-mix in 2 mL of molten, trace histidine/tryptophan supplemented top agar
- quality controls of S9: yes, using a recognised mutagenic compound (2AA) which requires metabolic activation

Test concentrations with justification for top dose:

Concentration range in the main test with metabolic activation: 0, 50, 150, 500, 1500 and 5000 µg/plate;
Concentration range in the main test without metabolic activation: 0, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

Solvent: Dimethyl sulphoxide

Controls

Untreated negative controls:

yes

Remarks:

spontaneous reversion rates examined

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10^9/mL
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration: approx. 48 h, at 37°C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
The plates were assessed for revertant colonies using a Domino colony counter and examined for a thinning of the background lawn. A manual count was performed at 5000 µg/plate because of test material precipitation.

METHODS FOR MEASUREMENTS OF GENOTOXICITY
The frequency of revertant colonies assessed using a Domino colony counter. A manual count was performed at 5000 µg/plate because of test material precipitation. Area and miscount-correction was used as correction method.

Evaluation criteria:

For a substance to be considered positive in this test system, it should have induced a doserelated and statistically significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. ln the event of the two experiments giving conflicting or equivocal results, then a third experiment may be performed to confirm the correct response, To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between two and five fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate. ln this case the limiting factor was the maximum recommended dose.

Statistics:

according to:
Kirkland D J (Ed) (1989) Statistical Evaluation of Mutagenicity Test Data.
UKEMS Sub-committee on Guidelines for Mutagenicity Testing, Report - Part
lll, Cambridge University Press.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING STUDIES:
Five concentrations of the test material were assayed in triplicate against each tester strain, using the direct plate incorporation method in accordance with the standard methods for mutagenicity tests using bacteria.
The dose range of the test material used in the preliminary toxicity study was 0, 50, 150, 500, 1500 and 5000 µg/plate. The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-).The mean number of revertant colonies for the toxicity assay were:

Strain Dose µg/plate
0 50 150 500 1500 5000
TA 100 82 87 91 96 103P 94P
WP2uvrA- 17 18 14 21 18P 14P
P = precipitate

STUDY RESULTS:
- Concurrent vehicle negative and positive control data:
Solvent control plates gave counts of revertant colonies within the normal range. All positive control chemicals gave increases in revertants, either with or without the metabolising system as appropriate, within expected ranges.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible: yes

Ames test:
- Signs of toxicity: No toxicity was exhibited to any of the strains of bacteria used.

HISTORICAL CONTROL DATA:
No histroical contral data were indicated in the study report. However, spontaneous mutation rates of untreated controls were presented (please refer to “Evaluation Criteria") which is sufficient according to the OECD TG 471 version which was in place when the test was perfomed (OECD TG 471 from 1997).

Any other information on results incl. tables

Prior to use the master strains were checked for characteristics, viability and spontaneous reversion rate and were all found to be satisfactory. Results for the negative controls are presented in the Attachment (refer to "Attached background material"). The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in in the Attachment (refer to "Attached background material").
No toxicity was exhibited to any of the strains of bacteria used. A precipitate was observed at and above 1500 µg/plate, this did not interfere with the scoring of revertant colonies. No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material either with or without metabolic activation.
All of the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

 

Notes to attached material shown in "Attached background material":

1. When bacterial growth inhibition is found, the applicable value is marked with an asterisk.
2. The average number of colonies for each concentration is recorded in parentheses.
3. Figures immediately below the average values refer to standard deviation.
4 "Number of revertants" - The observed values and average value are shown in order, beginning with the lowest concentration of the test substance.
5. The following postfixes are used when required:
C : contaminated
P : precipitate
X : plate unscorable

Applicant's summary and conclusion

Conclusions:

The test material, FADEX He 1819 PK, was considered to be negative with and wthout metabolic activation in the in vitro gene mutation assay in bacteria and therefore non-mutagenic under the conditions of this test.

Executive summary:

Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2uvrA- were treated with the test material, FADEX He 1819 PK, using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard cofactors).

This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. lt also meets the requirements of the OECD, EC and USA, EPA (TSCA) guidelines. The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. The vehicle (dimethyl sulphoxide) control plates produced counts of revertant colonies within the normal range. All of the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies, both with and without the metabolising system. The test material caused no visible reduction in the growth of the bacterial lawn at any dose level either with or without metabolic activation, it was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A precipitate was observed at and above 1500 µg/plate, this did not interfere with the scoring of revertant colonies.

No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.