4-hydroxybenzoic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-12-03 to 2009-02-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to OECD guideline 471

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

and EU B.13/14

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-hydroxybenzoic acid

EC Number:

202-804-9

EC Name:

4-hydroxybenzoic acid

Cas Number:

99-96-7

Molecular formula:

C7H6O3

IUPAC Name:

4-hydroxybenzoic acid

Constituent 2

Reference substance name:

p-Hydroxybenzoesäure

IUPAC Name:

p-Hydroxybenzoesäure

Details on test material:

- Name of test material (as cited in study report): p-Hydroxybenzoesäure
-Composition: p-Hydroxybenzoic acid, Batch No.: 08/1000/10, 1-20
-date of production: 2008-10-20
-purity 99.94%, white powder
-storage: 15°C-25 °C, dry and dark

Method

Target gene:

uvr: UV sensitivity pKM101/ pAQ1: Ampicilline/ Tetracycline resistance His: Histidine requirements rfa: crystal violet sensitivity

Species / strain

Species / strain / cell type:

S. typhimurium, other: LT2: Strains: TA1535, TA97a, TA98, TA100, TA102

Metabolic activation:

with and without

Metabolic activation system:

S9 (Trinova Biochem, Giessen) from liver of male Sprague-Dawley rats (treated with 500 mg Aroclor 1254/ kg b.w. i.p.)

Test concentrations with justification for top dose:

1st experiment: 50, 150, 500, 1500, 4999 µg/plate
2nd experiment: 1251, 2501, 5001 µg/plate

Vehicle / solvent:

DMSO,
A stock solution of the test substance in DMSO was prepared containing 50g/l p-Hydroxybenzoic acid

Controls

Untreated negative controls:

yes

Remarks:

Water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

all control experiments were valid

Migrated to IUCLID6: and: 4-Nitro-1,2-phenylenediamine , Sodium azide , 2-Amino-anthracene

Details on test system and experimental conditions:

-Origin of strains: from Prof. Ames (2001 to 2002)
-Storage: -80°C
-overnight culture of bacteria: 10 h before the start: an aliquot of stock culture was thawed and incubated in 70 mL nutrient broth for 10 h at 37°C
-S9 mix: freshly prepared and stored at 0°C.
METHOD OF APPLICATION: Two different incubation methods were used subsequently to confirm results of the plate incorporation method:
1st experiment - Plate incorporation method: 0.1 ml of the appropriate test item solution was mixed with overnight culture of the
respective strain, 0.5 ml phosphate buffer (for treatments without S9), or 0.5 ml S9 mix, 2 ml Top-Agar were added. The mixture was vortexed and
poured on a minimal glucose agar and distrubuted evenly.
- Exposure duration: 48 hours
- Incubation: in the dark at 37°C
2nd Experiment - Pre-incubation method: The components of the mixture were prepared as described above. Mixtures were incubated at 37°C for 30 min and aerated through careful shaking. Then 2 ml of Top Agar were added, the mixture vortexed and poured onto minimal glucose agar and distributed evenly.
- Exposure duration: 48 hours
- Incubation: in the dark at 37°C

Evaluation criteria:

A test item is consiered to have mutagenic potential, if a significant increase in revertant colonies per plate (increase factor >= 2 compared to
control) is recorded in at least one strain. A concentration related increase over time can also be taken as a sign of mutagenic activity.

Statistics:

Each test was performed in four replicates and mean values were calculated

Results and discussion

Test results

Key result

Species / strain:

bacteria, other: Salmonella typhimurium LT2 strains: TA1535, TA 97a, TA98, TA100, TA102

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:

valid

Positive controls validity:

valid

Additional information on results:

The results were confirmed in a 2nd experiment using the pre-incubation method

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Mean revertant values (colonies per plate) (first experiment) of the four replicates (table 8.1 -a)

Strain	97a	97a	98	98	100	100	102	102	1535	1535	-	-	-
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Water	112	152	12	13	148	134	239	242	11	11
DMSO	131	118	11	12	116	120	214	265	15	11
positive Control	>1000	>1000	>1000	>1000	>1000	>1000	>1000	>1000	>1000	>1000
4999 ul/pl	133	122	12	6	114	172	232	225	9	9
1500 ul/pl	110	113	9	8	123	150	233	170	6	8
500 ul/pl	106	109	9	9	169	119	212	227	6	9
150 ul/pl	165	123	9	10	124	140	245	249	12	11
50 ul/pl	141	136	10	12	126	115	232	195	14	8

Results are mean of four replicate experiments

Standard deviations ranged from 50% to 0% (positive control)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
With and without metabolic activation no signs of mutagenicity were detected in the Ames test (OECD471).

Executive summary:

Mutagenicity of p-Hydroxybenzoesäure was tested according to OECD guideline 471 (Ames test, Bacterial Reverse Mutation Assay) using five different genetically manipulated strains of Salmonella typhimurium bacteria both with and without a metabolic activation system (S9) for 48 hours.

As the test item was not cytotoxic in a pretest, it was applied in concentrations ranging from 50 to 4999 µg/plate to the test strains (1st experiment) and in a 2nd experiment ranging from 1251 µg/plate to 5001 µg/plate.

For Experiment 1 and 2:

The test item did not cause an increase in revertant mutants compared to plates treated with water or the vehicle DMSO only, both with and without a metabolic activation system (S9).

p-Hydroxybenzoesäure is considered as not bacterial mutagen under the conditions of the test.