2,4-dihydroxybenzoic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Remarks:

Bacterial Reverse Mutation Test

Adequacy of study:

key study

Study period:

06.03.2018 - 10.04.2018

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 21, 1997

Deviations:

yes

Remarks:

In experiments with Salmonella typhimurium TA 100 with metabolic activation higher number of revertants was observed than the upper limit of historical control for 2- aminofluorene.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,4-dihydroxybenzoic acid

EC Number:

201-946-9

EC Name:

2,4-dihydroxybenzoic acid

Cas Number:

89-86-1

Molecular formula:

C7H6O4

IUPAC Name:

2,4-dihydroxybenzoic acid

Test material form:

solid

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:

Based on the first cytotoxicity experiment (in TA 98), the first mutagenicity experiments were performed as plate incorporation test with and without metabolic activation in all the strains. The concentration of 10 µg per plate was then used as maximum in the, whereas the cytotoxicity was expected in the highest concentration only. Further doses were diluted with factor approximately 2-√10 with resulting concentration range 10.0, 3.0, 1.0, 0.3 and 0.1 µg per plate.
The second mutagenicity experiments were performed as plate incorporation test. Firstly the maximum concentration was increased by one concentration which was higher by √3 approximately i.e. 30 μg per plate. Experiments were performed in Salmonella typhimurium TA 100 and TA 98. Neither this concentration was cytotoxic for Salmonella typhimurium TA 100.
Experiments in the other indicator strains including repeated experiments in Salmonella typhimurium TA 100 (= third mutagenicity experiments) were performed with concentrations 10, 30, 75, 150 and 300 μg per plate (concentrations based on the second cytotoxicity experiment).
Some higher concentrations were cytotoxic for indicator strains Salmonella typhimurium TA 1535 and TA 1537 without metabolic activation. These experiments were repeated (third mutagenicity experiments) using the series of lower concentrations 2.5, 5.0, 10.0, 25.0 and 50.0 μg per plate.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

other: 4-nitro-o-phenylenediamine, 2-aminofluorene, 2-aminoanthracene, 9-aminoacridine hydrochloride monohydrate

Details on test system and experimental conditions:

Bacterial strains: Salmonella typhimurium TA 1535 (CCM 3814, lot. No. 2101200916917), TA 98 (CCM 3811, lot No. 0102201220053), TA 100 (CCM 3812, lot No. 0102201220054) and TA 1537 (CCM 3815, lot No. 2101200916918) as well as Escherichia coli WP2 uvrA (CCM 4751, lot No. 2104200512732).
Strains TA 98 and TA 1537 detect frame shift mutations, strains TA 100 and TA 1535 serve to detection of base-pair substitution mutations, and strain E.coli WP2 uvrA detects cross-linking mutagens.
Genotypes of strains: Genotypes of each strain were controlled (plasmid pKM 101 – ampicillin resistance, uvr mutation, rfa mutation, his/trp mutation – spontaneous reversions).
Preparation and using of S9: The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. The liver homogenate was prepared from Wistar male rats weighing approximately 200 g, previously induced with Delor 106 (mixture of PCBs). Delor 106 was diluted with olive oil to a concentration of 200 mg·mL-1, and each rat was administered a single injection of 500 mg/kg 5 days before S9 preparation. The S9 was prepared according to the methods described by Maron and Ames (1983). The liver was removed from each animal and washed in ice cold 0.15M KCl. The livers washed were mixed with another 0.15 M KCl (3 mL.g-1 wet liver) homogenized in a grinder, and the tissue suspension was centrifuged for 10 min at 9000 g. Aliquots of the supernatant (S9) were stored in plastic tubes using sterile technique at a temperature below –70 C. Cofactors (NADP and glucoso-6-phosphate) were dissolved in buffer.
Each plate in all experiments with metabolic activation contained 0.5 mL of buffer with NADP and glucoso-6-phosphate and 20, 30, 50 or 100 µL S9 (the concentration of S9 in the S9mix was 5.7, 9.1 or 16.7%). In experiments without metabolic activation only buffer was added to the top agar.
Volume of 20 µL (3.8 % of S9 in S9 mix) of S9 is used for positive controls in all strains with except of E. coli (reason – to keep constant conditions for database of historical values).
Volume of 30 µL (5.7% of S9 in S9 mix) of S9 was used for test item, solvent and negative control plates in the first experiments in all strains with except of E. coli.
Volume of 50 µL (9.1% of S9 in S9 mix) of S9 was used for test item, solvent and negative control plates in the second experiments in all strains with except of E. coli
Volume of 100 µL (16.7% of S9 in S9 mix) of S9 is used for all plates in both experiments in E. coli. The reason is small number of induced revertants in positive control at lower volumes of S9.

Statistics:

For the evaluation of results, the modified two-fold increase rule was used, which is compatible with the application of statistical methods.
Per this rule, the result is positive if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.
An increase is considered as ”biologically relevant“:
- if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion >10;
- if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤10;
A test item producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Evaluation of Results
The main criterion used for the evaluation of reversion results was a modified two-fold increase rule, which is compatible with the application of statistical methods. Per this rule, the result is positive if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.
An increase is considered as ”biologically relevant“:
- if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion >10;
- if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤10;
A test item producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
According to OECD TG 471, the biological relevance is the criterion for the interpretation of results, and a statistical evaluation of the results is not necessary.

Acceptability of Results
Minimum two experiments will be performed in each indicator strain. Only experiments complying with validity requirements will be involved in evaluation. When validity criteria are not met than concerned experiment was repeated.
The following conditions shoud be fulfilled:
1. Every strain should have appropriate properties (uvr mutation, rfa mutation, plasmids),
2. Average value of negative control (spontaneous reversion) should fall to historical limits of historical average negative control (spontaneous reversion),
3. Every test should have at least 4 acceptable doses except of negative control (acceptable dose = 2 countable dishes at least, non toxic),
4. Average value of positive control should fall to historical limits of historical average positive control,
5. The test should have most 1 unacceptable dose.

Applicant's summary and conclusion

Conclusions:

Under the above-described experimental design, the test item, 2,4-dihydroxybenzoic acid, was non mutagenic for all the used indicator strains with and without metabolic activation.

Executive summary:

The test item, 2,4-dihydroxybenzoic acid,was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The test was performed according to EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria,which is analogous to the OECD Test Guideline No. 471.

Four indicator Salmonella typhimurium strainsTA 98, TA 100, TA 1535, TA 1537and one indicatorEscherichia coli WP2 uvrA strain were used. The test item was dissolved in dimethyl sulfoxide and assayed in concentratins given further, which were applied to plates in volume of 0.02 or 0.1 mL.

The first mutagenicity experiments were performed as plate incorporation test without and with metabolic activation usinga supernatant of rat liver (S9; 30μL or 100 μL per plate) and a mixture of cofactors by the plate incorporation test with concentrations 10, 30, 100, 300 and 1000 mg per plate.

The result of first series of mutagenicity eperiments were negative. The second series of mutagenicity experiments with modified experimental conditions were performed.

The second mutagenicity experiments were performed with pre-incubation and volume of S9 was increased from 30 to 50μL per plate. Concentrations used were of 10, 50, 100, 250 and 500 mg per plate.

The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. Average revertant colony counts for the vehicle controls were within the current historical control range for the laboratory.

In the arrangement given above, the test item, 2,4-dihydroxybenzoic acid, was non-mutagenic for all the used bacterial strains.