Benzoic acid, 2-hydroxy-, reaction products with formaldehyde, coupled with diazotized 5-amino-8-[[4-[(4-nitro-2-sulfophenyl)amino]phenyl]azo]-2-naphthalenesulfonic acid disodium salt

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from 21 Jun. 2018 to 02 Jul. 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzoic acid, 2-hydroxy-, reaction products with formaldehyde, coupled with diazotized 5-amino-8-[[4-[(4-nitro-2-sulfophenyl)amino]phenyl]azo]-2-naphthalenesulfonic acid disodium salt

EC Number:

276-521-4

EC Name:

Benzoic acid, 2-hydroxy-, reaction products with formaldehyde, coupled with diazotized 5-amino-8-[[4-[(4-nitro-2-sulfophenyl)amino]phenyl]azo]-2-naphthalenesulfonic acid disodium salt

Cas Number:

72245-24-0

Molecular formula:

not applicable

IUPAC Name:

Benzoic acid, 2-hydroxy-, reaction products with formaldehyde, coupled with diazotized 5-amino-8-[[4-[(4-nitro-2-sulfophenyl)amino]phenyl]azo]-2-naphthalenesulfonic acid disodium salt

Test material form:

solid: particulate/powder

Method

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:

S9

Test concentrations with justification for top dose:

-Toxicity test: 5000, 1580, 500, 158 and 50.0 μg/plate
- Main test: 5000, 2500, 1250, 625 and 313 µg/plate.
On the basis of the results obtained in the preliminary toxicity test, 5000 µg/plate was selected as the top concentration for the Main Assay.

Vehicle / solvent:

Sterile water for injection and Dimethylsulfoxide (DMSO)

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

Remarks:

without metabolic activation (S9)

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation system (S9)

Details on test system and experimental conditions:

FORMULATION PROCEDURE
Solutions of the test item, as received, were prepared immediately before use in sterile water for injection. Concentrations were expressed in terms of test material material as received. All test item solutions were used within 51 minutes from the initial formulation.

METHOD OF APPLICATION: in agar (plate incorporation)
-Nutrient Broth: Oxoid Nutrient Broth No. 2 was prepared at a concentration of 2.5 % in distilled water and autoclaved prior to use. This was used for the preparation of liquid cultures of the tester strains.
-Nutrient Agar: Oxoid Nutrient Broth No. 2 (25 g) and Difco Bacto-agar (15 g) were added to distilled water (1 litre) and autoclaved. The solutions were then poured into 9 cm plastic Petri dishes and allowed to solidify and dry before use. These plates were used for the non-selective growth of the tester strains.
-Minimal Agar: Minimal medium agar was prepared as 1.5 % Difco Bacto-agar in Vogel-Bonner Medium E, with 2% Glucose, autoclaved and poured into 9 cm plastic Petri dishes.
-Top Agar: "Top Agar" (overlay agar) was prepared as 0.6% Difco Bacto-agar + 0.5% NaCl in distilled water and autoclaved. Prior to use, 10 mL of a sterile solution of 0.5 mM Biotin + 0.5 mM Histidine (or 0.5 mM tryptophan) was added to the top agar (100 mL).

DETERMINATION OF CYTOTOXICITY
A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the Main Assay. In this test a wide range of dose levels of the test item, set at half-log intervals, were used. Treatment was performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included. Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

SOLUBILITY
Solubility of the test item was evaluated in a preliminary trial using distilled water. This solvent was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity.

INCUBATION AND SCORING
The prepared plates were inverted and incubated for approximately 72 hours at 37 °C. After this period of incubation, plates were held at 4°C for 24 hours before scoring of colonies.

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only.
In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

The regression analysis fits a regression line to the data by the least squares method, after square root transformation of the plate counts to satisfy normal distribution and homoscedasticity assumptions.
The regression equation is expressed as: y = a +bx
where:
y = transformed revertant numbers
a = intercept
b = slope value
x = dose level (in the units given).
Regression lines are calculated using a minimum of the three lowest dose levels, and then including the further dose levels in turn. The correlation co-efficient (r), the value of students "t" statistic, and the p-value for the regression lines are also given.

Results and discussion

Test results

Species / strain:

other: TA1537, TA98 and TA100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

SOLUBILITY
The test item was found to be soluble at 50.0 mg/mL. This result permitted a maximum concentration of 5000 µg/plate to be used in the toxicity test.

TOXICITY TEST
Neither toxicity, nor precipitation of the test item was observed at the end of the incubation period at any concentration tested, in the absence or presence of S9 metabolic activation.
Plates treated with the test item showed a dose dependent orange colour of the agar, which did not interfere with the scoring of revertant colonies and evaluation of the background lawn.
Dose-related increases in revertant numbers were observed both in the absence and presence of S9 metabolism with all tester strains with the exception of TA1535 and WP2 uvrA tester strains in the presence of S9 metabolism.

MAIN TEST
Neither toxicity, nor precipitation of the test item was observed at the end of the incubation period with any tester strain, at any dose level, in the absence or presence of S9 metabolic activation. Plates treated with the test item showed a dose dependent orange colour of the agar, which did not interfere with the scoring of revertant colonies and evaluation of the background lawn.
Dose-related increases in revertant numbers were observed both in the absence and presence of S9 metabolic activation, with all tester strains with the exception of TA1535 and WP2 uvrA.
The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.

VALIDITY CRITERIA:
The assay was considered valid if the following criteria were met:
- Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.
- The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 millions for each strain.
- No more than 5% of the plates should be lost through contamination or other unforeseen event.
The study was accepted as valid:
- Results show that mean plate counts for untreated and positive control plates fell within the normal range based on historical control data.
- The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain.
- No plates were lost through contamination or cracking.

EVALUATION
Test item treatments of TA1537 and TA98 tester strains yielded biologically relevant, statistically significant (based on the doubling rule) and dose related increases in the number of revertant colonies, both in the absence and presence of S9 metabolism, that can be considered as clear evidence of mutation induction. In addition, biologically relevant and dose related increases in revertant numbers were also observed with TA100 tester strain and were considered to be a further evidence of this mutagenic activity.
Since a clear positive response was observed, no further experiment was undertaken.

Applicant's summary and conclusion

Conclusions:

The test item induces reverse mutation in bacteria both in the absence and presence of S9 metabolism, under the reported experimental conditions.

Executive summary:

The test item was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The test was performed according to the OECD Guideline 471 (1997) and the EU method B.13/14 of EC 440/2008. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone.

The test item was used as a solution in sterile water for injection.

The test item was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals.

On the basis of toxicity test results, in the Main Assay using the plate incorporation method, the test item was assayed at five dose levels from 313 to 5000 µg/plate. Neither precipitation of the test item, nor toxicity was observed at the end of the incubation period, with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism.

Test item treatment yielded dose-related increases in revertant numbers, both in the absence and presence of S9 metabolic activation, with all tester strains with the exception of TA1535 and WP2 uvrA.

These increases were greater than twice the concurrent negative control values with TA1537 and TA98 tester strains and so can be considered a clear evidence of mutation induction.

Since a clear positive response was observed, no further experiment was undertaken.

It is concluded that the test item induces reverse mutation in bacteria both in the absence and presence of S9 metabolism, under the reported experimental conditions.