Disodium 4,4'-methylenebis[3-hydroxy-2-naphthoate]

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:

12 APR 2013 - 29 MAY 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4,4'-methylenebis[3-hydroxy-2-naphthoate]

EC Number:

229-653-1

EC Name:

Disodium 4,4'-methylenebis[3-hydroxy-2-naphthoate]

Cas Number:

6640-22-8

Molecular formula:

C23H16O6.2Na

IUPAC Name:

disodium 4,4'-methylenebis(3-hydroxy-2-naphthoate)

Test material form:

solid

Method

Target gene:

HIS operon (S. thyphimurium)
TRY operon (E. coli)

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Male Wistar, HSdCpb:Wu rats from Harlan®, aged 6-8 weeks, were given a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) dissolved in Miglyol 812 oil. The animals received drinking water and a standard diet ad libitum. The body weight of the animals used was 177 ± 7.68 g. About 16 hours before sacrifice, the rats remained without food. On day 5 to 7, they were sacrificed, the livers were removed and collected in ice-cooled sterilized beakers containing 0.15 M KCl. The livers were homogenized in a sterile glass potter homogenizer with a Teflon pestle containing 3 mL of 0.15 M KCl per gram of liver wet-weight. After homogenization the preparation was transferred to sterilized steel centrifuge tubes and spun at 9000 x g for 10 minutes at about 4°C and the supernatant fluid was decanted and transferred into sterilized and precooled plastic tubes. The S9 was then frozen and stored in liquid nitrogen at -196°C.
- method of preparation of S9 mix:

Quantity per ml S 9 mix
Components 1st Series 2nd Series

Liver homogenate (S9) 0.10 mL 0.30 mL
MgCQ/KCl aqueous solution (0.4 M/l .64 M) 0.02 mL 0.02 mL
Glucose-6-phosphate, disodium salt 5 µmol 5 µmol
NADP, disodium salt 4 µmol 4 µmol
Sodium phosphate buffer (0.2 M, pH 7.4) 0.50 mL 0.50 mL
Distilled water 0.38 mL 0.18 mL

- concentration or volume of S9 mix and S9 in the final culture medium : 10 % and 30 % S9 in the S9 mix were used in the 1st and 2nd series, respectively.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Every S9-batch is tested for its metabolic activity by the use of specific substrates, requiring different enzymes of the P450-isoenzyme family. The mutagenicity of 2-aminoanthracene, benzo[a]pyrene, and 3-methylcholanthrene is thus determined once for every S9-batch. Clear increases in the number of revertants for S. typhimurium TA 98, TA 100, and TA 1537 with all positive controls and for TA 1535 with 2-aminoanthracene are used as an acceptance criterion for each S9-batch. In this study 2-aminoanthracene, and benzo[a]pyrene for TA 102, are used as the concurrent positive controls for the different strains in the presence of S9 mix.

Test concentrations with justification for top dose:

1st series: 5.00, 15.8, 50.0, 158, 500, 1580, 5000 µg/plate
2nd series: 15.8, 158, 1580, 2810, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ultrapure water

- Justification for choice of solvent/vehicle: ultrapure water or DMSO are the preferred solvents

- Justification for percentage of solvent in the final culture medium: High amounts of water (added as the solvent) will dilute the top agar. Therefore, usually the maximum amount of solvent is limited to 100 µL per plate for water.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

other: Daunomycin, 2-Aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 2 - 3 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Revertant colonies were either scored automatically with the „Sorcerer" or manually with the validated „Ames Study Manager" from Perceptive Instruments, Haverhill, Suffolk, UK. Tables of individual and mean values were generated automatically.

The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies. The following criteria, based upon the historical controls of the laboratory and statistical considerations, are established:

-----------------------------------------------------------------------------------------
Mean Number of Colonies Maximal Mean Number of Colonies over the
(Solvent Control) Actual Solvent Control (Test Material)
-----------------------------------------------------------------------------------------

≤ 10 ≤ 9 ≥30
≤ 30 ≤ 19 ≥40
≤ 80 ≤ 29 ≥ 80
≤ 200 ≤ 49 ≥120
<=500 <=79 >=200

Assessment No increase Clear increase

-----------------------------------------------------------------------------------------

All further results, ranging between "no" and "clear", are assessed as "weak increases".

Interpretations:

A test material is defined as negative or non-mutagenic in this assay if
- the assay is considered valid,
and
- "no" or "weak increases" occur in the test series performed, ("weak increases" randomly occur due to experimental variation.)

A test material is defined as mutagenic in this assay if:
- the assay is considered valid,
and
- a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;
or
- "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.

There is no requirement for verification of a clear positive response.
In all further cases, a third test series with the bacterial strain in question should be performed and the results of each series should be discussed on a case by case basis.

Evaluation criteria:

see datails on test system

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: no

RANGE-FINDING/SCREENING STUDIES (if applicable):
no details given

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The negative control mutant frequencies were all in the regular range. The strain specific positive control test materials, namely daunomycin, sodium azide, 4-nitroquinolin-N-oxide and 9-aminoacridine, in the absence of S9 mix, yielded the expected mutant frequencies that were greatly in excess of the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene, which requires metabolic activation, was strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9 mix) was functioning. Thus, the study is considered valid.

Ames test:
- Signs of toxicity: No
- Mean number of revertant colonies per plate and standard deviation: please refer to file attached under 'Attached background material'

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: please refer to file attached under 'Attached background material'
- Negative (solvent/vehicle) historical control data: please refer to file attached under 'Attached background material'

Applicant's summary and conclusion

Conclusions:

With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.

Executive summary:

The investigations for the mutagenic potential of the test item were performed according to OECD Guideline 471 and GLP using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 % S9 in the S9 mix were used in the 1st and 30 % in the 2nd series, respectively. The test item was dissolved in ultrapure water and tested at concentrations ranging from 5.00 to 5000 µg/plate. No precipitation of the test material on the agar plates occurred. Toxicity to the bacteria was not observed. Daunomycin, sodium azide, 9-aminoacridine and 4-nitroquinolin-N-oxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene was used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.