Phenyl 4-hydroxybenzoate

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:

2018-01-22 to 2018-01-22

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 21st July, 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Phenyl 4-hydroxybenzoate

EC Number:

241-698-9

EC Name:

Phenyl 4-hydroxybenzoate

Cas Number:

17696-62-7

Molecular formula:

C13H10O3

IUPAC Name:

phenyl 4-hydroxybenzoate

Specific details on test material used for the study:

TEST MATERIAL
- Name: Phenyl 4-Hydroxybenzoate
- Batch: 018964K19K
- CAS no.: 17696-62-7
- Purity: 99.1%
- Expiry date: 2018-10-01
- Appearance: white powder
- Storage conditions: room temperature (15 – 25 °C)

Method

Target gene:

Histidine locus & tryptophan locus

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

CELLS USED:
- Source of cells: MOLTOX, INC., NC 28607, USA

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

CELLS USED:
- Source of cells: MOLTOX, INC., NC 28607, USA

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction: microsome fraction prepared from Sprague Dawley rat liver homogenate

Test concentrations with justification for top dose:

1000, 500, 300, 100, 30 and 10 μg/plate

The test item concentrations to be applied in the main experiments were chosen according to the results of the preliminary cytotoxicity test in TA100.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (Sigma-41639-BCBT5423)
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

NaCl 0.15 M

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535 (5 µg/plate), TA100 (20 µg/plate), without S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Anthramine

Remarks:

TA98, TA100, TA1535, TA1537; without pre-incubation: 2 µg/plate, with pre-incubation: 1 µg/plate; with S9

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537, 50 µg/plate, without S9

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98, 2 µg/plate, without S9

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

NaCl 0.15 M

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: cis-Platinum (II)

Remarks:

E. coli, 1 µg/plate, without S9

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: dimethylbenzanthracene

Remarks:

E. coli without pre-incubation: 5 µg/plate, with pre-incubation: 2.5 µg/plate, with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation (Exp. 1), pre-incubation (Exp. 2)
without metabolic activation; with metabolic activation (with and without pre-incubation)
- Cell density at seeding (if applicable): 1-9 x10^9 bacteria/mL

EXPERIMENTAL PERFORMANCE
- I: Assay without metabolic activation
0.1 mL of the bacterial suspension containing 1-9 x10^9 bacteria/mL and 0.1 mL (in aqueous or oily vehicle) / 50 μL (in non-aqueous or non-oily vehicle) of each dilution of the original solution and 0.5 mL of sterile phosphate buffer are successively added to 2 mL of overlay agar, maintained supercooled at 45 °C
- for: S. Typhimurium strains containing 10% (v/v) of a L-Histidine-D-Biotine solution (0.5 mM).
- for E. coli strain containing 5% (v/v) of nutrient broth n° 2 to which are added 5 μL of a L-Tryptophan solution at 2 mg/mL.

Plates are incubated at 37 °C over a 48-72-hour period. The number of revertant colonies per plate is counted.

Moreover the following controls are carried out:
Negative controls:
- absolute negative control containing no test item corresponding to the spontaneous reversion rate,
- solvent used to solubilize positive controls: Acetone, DMSO, NaCl 0.15 M
Vehicle used to solubilize test item: DMSO

- II: Assay with metabolic activation
1. standard plate incorporation method: the protocol is similar to that described above, except that, 500 μL of S9-mix fraction is quickly added, before pouring the mixture onto the plates
2. pre-incubation assay: the solution of the test item solution with the test strain, and 500 μL of S9-mix fraction are preincubated with shaking for 30 min, at 37° C prior to mixing with the overlay agar and pouring onto the minimal agar plate.

DURATION
- Preincubation period (Experiment II): 30 min at 37 °C
- Exposure duration: 48 - 72 h at 37 °C

NUMBER OF REPLICATIONS: 3 plates/strain/concentration level including the controls

EVALUATION OF MUTAGENICITY
The result of the test is considered as negative if the revertant number is below three-fold the number of spontaneous reversions, for TA1535 and TA1537 strains, and below two fold the number of spontaneous reversions for TA98, TA100 and Escherichia coli WP2(uvrA-) (pKM 101) strains without and with metabolic activation.
The result of the test is considered positive if a dependent relationship concentration is obtained in one, or several of the 5 strains, without and/or with metabolic activation, a mutagenic effect being taken into account for a given dilution of test item if the number of revertant colonies is at least two-fold that of spontaneous revertant colonies for TA98, TA100 and Escherichia coli WP2(uvrA-) (pKM 101), and three-fold for TA1535 and TA1537.
All results must be confirmed in an independent experiment.

Evaluation criteria:

Ensure that the criteria of validity of the study are well respected namely:
- the bacteriostatic activity of the highest concentration tested shall be equal to or less than 75%,
- the spontaneous reversion rate of the absolute negative control shall comply with the historical values of the laboratory,
- the spontaneous reversion rate of the solvent shall not be statistically different from absolute negative control,
- the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation shall comply with the historical values of the laboratory,
- Negative and positive values should not show significant difference with the historical values of the laboratory (± 2 standard deviations).

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Key result

Species / strain:

other: TA1535, TA1537, TA98, TA100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions reported, Phenyl 4-hydroxybenzoate did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, Phenyl 4-hydroxybenzoate is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In a bacterial reverse gene mutation assay in bacteria conducted according to OECD guideline 471, strains TA98, TA100, TA 102, TA1535 and TA1537 of S. typhimurium and E. coli WP2 uvr A pKM 101 were exposed to Phenyl 4-hydroxybenzoate (99.1% purity) in DMSO, NaCl 0.15 M or acetone at concentrations of 1000, 500, 300, 100, 30 et 10 µg/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in all tester strains in both experiments (plate incorporation and pre-incubation). Based on the results, the test item is considered to be non-mutagenic in the bacterial reverse gene mutation assay.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.51001; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.