Phenylacetylene

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:

From October 28, 2021 to January 19, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 471 and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 21st, 1997 and corrected June 26th, 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

QA statement signed on 2022-01-19

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Phenylacetylene

EC Number:

208-645-1

EC Name:

Phenylacetylene

Cas Number:

536-74-3

Molecular formula:

C8H6

IUPAC Name:

ethynylbenzene

Test material form:

liquid

Method

Target gene:

Histidine and tryptophan

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Strains of Salmonella typhimurium and Escherichia coli were purchased from MOLTOX (TM) and maintained in the laboratory according to internal SOP. The genotype of bacterial strains was checked for histidine and tryptophane requirements, loss of cell wall LPS (rfa mutation), ampicillin resistance, UVB and UVA sensitivity, spontaneous revertant rate and sensitivity to reference mutagens.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: S9 fraction, microsome fraction prepared from Sprague Dawley rat liver homogenate, was provided by MOLTOX (TM).
- method of preparation of S9 mix:
The final concentration of co-factors and salts was as follows:
S9 fraction: 10%
MgCl2-6H2O: 8 mM
KCl: 33 mM
Glucose-6-phosphate Na2: 5 mM
NADP Na2: 4 mM
Phosphate buffer (pH 7.4): 0.1 M

Test concentrations with justification for top dose:

- Assay 1 (+/- S9): 50, 150, 500, 1500 and 5000 µg/plate (up to cytotoxic concentration) for plate incorporation method.
- Assay 2 (+/- S9): 50, 150, 500, 1500 and 5000 µg/plate (up to cytotoxic concentration) for pre-incubation method.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item is not soluble in water at 50 mg/mL but soluble in DMSO. A stock solution for each assay was prepared extemporaneously at 100 mg/mL in DMSO. The aspect of solution for assay n°1 and n°2 was a light-yellow solution for stock solution colorless for dilutions. The highest dose tested was 5 000 μg/plate.

Sterility tests:
Test item and the corresponding dilutions were added to 2 mL of top agar maintained at 45°C, and poured after homogenization on the bottom agar (20 ml) onto a Petri plate (90 mm in diameter) (n = 3). Plates were incubated for 48 - 72 hours at 37°C and then examined. There should be no bacterial growth on any plate. S9-mix sterility was checked using the same protocol.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO or Acetone

True negative controls:

no

Positive controls:

yes

Remarks:

In the presence of S9-mix

Positive control substance:

other: 2-Anthramine 2 µg/plate for TA98, TA100, TA1535 and TA 1537 without pre-incubation 1 µg/plate for TA98, TA100, TA1535 and TA 1537 with pre-incubation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO or NaCl 0.15 M

True negative controls:

no

Positive controls:

yes

Remarks:

In the absence of S9-mix

Positive control substance:

9-aminoacridine

2-nitrofluorene

other: Sodium azide 20 µg/plate for TA 100 and 5 µg/plate for TA 1535

Details on test system and experimental conditions:

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

PRELIMINARY CYTOTOXICITY TESTING (Strain TA 100)
In a test tube, 0.1 mL of the bacterial suspension (1-9 x 10^3 bacteria/mL) and 0.1 mL (in -aqueous or -oily vehicle / 50 μL (in non-aqueous or non-oily vehicle as ethanol ….) of the stock solution and dilutions, were successively added to 2 mL of top agar at 45°C, containing 10 % (v/v) of a solution of L-Histidine-D-Biotine (2.5 mM). After homogenization, the content of the tube was poured onto a Petri plate (90 mm in diameter) containing minimal agar (20 mL). 3 plates per concentration were incubated for 24-72 hours at 37°C, and the colonies counted. A negative control containing the blank alone was run in parallel.

In case bacteriostatic activity is detected, the highest concentration to be retained is that exhibiting a bacteriostatic activity of 75 % or less. The precipitate, if present, should not interfere with the scoring. The following four dilutions studied are distributed according to a semi-logarithmic progression.

METHOD OF TREATMENT/ EXPOSURE
- Assay without S9:
For each Salmonella typhimurium strains, 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 as ethanol ….) of each dilution of the original solution and 0.5 mL of sterile phosphate buffer were successively added to 2 mL of overlay agar, maintained supercooled at 45° C, containing 10 % (v/v) of a L-Histidine-D-Biotine solution (0.5 mM).
For Escherichia coli strain, in a test tube 0.1 mL of the bacterial suspension containing 1-9 x 10^9 bacteria/mL and 0.1 mL (in -aqueous or -oily vehicle / 50 μL (in non-aqueous or non-oily vehicle as ethanol ….) of each dilution of the original solution and 0.5 mL of phosphate buffer were successively added to 2 mL of overlay agar maintained super cooled in 45° C containing 5% (v/v) of nutrient broth n° 2 to which are added 5 μL of a L-Tryptophane solution at 2 mg/mL.

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

- Assay with S9:
Two methodologies were used:
* either a standard plate incorporation method where 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;
* or the pre-incubation assay where 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.

This method is known to increase the detection sensitivity of a number of promutagens like alkaloïds, aliphatic N-Nitroso compounds (OECD n° 471).
For the first assay, direct incorporation method was used.

- Assay repetition:
If the first assay is positive, the second one is performed in the same manner.
If the first assay, in presence of test item, is negative, the pre-incubation test is performed for the second assay.

Moreover the following controls were 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
- Positive control

TREATMENT AND HARVEST SCHEDULE
- Preincubation period, if applicable: not reported
- Exposure duration/duration of treatment: 48-72 hours at 37°C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Rationale for test conditions:

Tested up to cytoxicity concentration (5000 µg/plate).

Evaluation criteria:

EVALUATION CRITERIA:
The following validity criteria were checked to validate each experiment:
- 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,
- In presence of the solvent the spontaneous reversion rate shall comply with the historical values of the absolute negative control of the laboratory.
- 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).


CRITERIA CONCLUSION:
The result of the test is considered as negative if the revertant number is below three fold the number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of spontaneous reversions for TA 98, TA 100 and Escherichia coli WP2 (uvrA-) (pKM 101) strains without and with metabolic activation.

The result of the test is considered positive if a dose-reponse 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 TA 98, TA 100 and Escherichia coli WP2 (uvrA-) (pKM 101), and three fold for TA 1535 and TA 1537.

All results must be confirmed in an independent experiment.

Statistics:

Not reported

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

up to 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

up to 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

up to 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

up to 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

up to 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

STERILITY CONTROLS
- Results show the absence of any bacterial growth in the presence of the various concentrations of the test item.
- Results show the absence of any bacterial growth in the presence of "S9-mix".

BACTERIOSTATIC ACTIVITY CONTROL
Results show a high toxicity in presence of the highest dose 5 000 μg/plate and close to the maximum tolerated 75%. Therefore, the test item was tested at the following doses: 5 000, 1 500, 500, 150 and 50 μg/plate.

MUTATION ASSAY INTERPRETATION
- There is no difference between the number of spontaneous reversions, the number of reversions obtained for the positive controls (without and with metabolic activation), and the mean of corresponding experimental historical values obtained in the laboratory.
- There is no evidence of any increase in the number of revertant colonies in the presence of the test item without and with metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 strains and Escherichia coli WP2 (uvr A¯) (pKM101) strain.
- Results are confirmed in an independent experiment.
- Evidence of toxicity demonstrated by :
* a thinning of the bacterial lawn in all strains was measured without and with metabolic activation.
* an absence of the bacterial lawn in Salmonella typhimurium TA 1535, TA 1537, TA 100 strains with metabolic activation and preincubation in presence of the highest dose tested 5 000 μg/plate.

HISTORICAL CONTROL DATA
- Positive historical control data: cf. attached document
- Negative (solvent/vehicle): cf. attached document

Any other information on results incl. tables

Tables of results were attached in the section "Attached background material".

Applicant's summary and conclusion

Conclusions:

In the assay conditions, in presence of doses (range from 50 to 5000 µg/plate) prepared from the test item PHENYLACETYLENE BATCH: LTWK2021-02, the numbers of revertant colonies in all cases did not exceed the criteria for a positive response and did not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 strains and Escherichia coli WP2 (uvrA-) (pKM101) strain without, or with metabolic activation, according to the OECD Guideline n°471.

Executive summary:

In a reverse gene mutation assay in bacteria, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2 (uvrA-) were treated with the test item PHENYLACETYLENE BATCH: LTWK2021-02. This study was performed in the absence and presence of metabolic activation. Two independent assays were carried out.

 

To determine the dose-range concentrations, a preliminary cytotoxicity testing was performed (strain TA 100). A high toxicity in presence of the highest dose 5 000 μg/plate and close to the maximum tolerated 75%. Therefore, the test item was tested at the following doses: 5 000, 1 500, 500, 150 and 50 μg/plate.

 

For assay n° 1, various concentrations were put in contact with the strains in the absence and presence of a metabolic activation system (S9-mix 10% (v/v)). For assay n° 2, various concentrations were put in contact with the strains in the absence of metabolic activation and with pre-incubation in the presence of metabolic activation system (S9-mix 10% (v/v)).

For the two assays, negative and positive controls were carried out in parallel. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. There is no difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental “historical” values obtained in the laboratory.

 

Evidence of toxicity demonstrated by a thinning of the bacterial lawn of non-revertant in all strains was shown. There is no evidence of any increase in the number of revertant colonies in the presence of the various concentrations of the test item (5 000, 1 500, 500, 150 and 50 μg/plate), without and with metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2 (uvrA¯) (pKM 101). These results validate the two tests.

 

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test, the test item was considered to be non-mutagenic.

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