Tri-2-propyn-1-yl phosphate

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:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 26. Jun. 2020

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 30. May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

tris(prop-2-yn-1-yl) phosphate

EC Number:

853-966-8

Cas Number:

1779-34-6

Molecular formula:

C9H9O4P

IUPAC Name:

tris(prop-2-yn-1-yl) phosphate

Specific details on test material used for the study:

Test Item: Phosphoric acid,tri-2-propargyl ester
Batch No.: H219121101
CAS No.: 1779-34-6
EINECS-No.: 853-966-8
Molecular Formula: C9H9O4P
Molecular Weight: 212 g/mol
Purity: >= 99 % LDY196
Appearance: Yellow liquid
Homogeneity: Homogenous

Method

Target gene:

hisC3076, hisD3052, hisG46, hisG428, uvrB, rfa, pKM101, pAQ1

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: S9 was obtained by Trinova Biochem GmbH, Gießen
- Batch nos.: 4272 (exp. 1, 1b, 1c, 2, 2b, repetition exp. 1 / 2nd approach, exp. 2c), 4088 (exp. 1), 4209 (exp. 1b, 1c, 2, 2b, repetition exp. 1), 4335 (repetition exp. 1), 4100 (repetition exp. 1), 4318 (repeti-tion exp. 1 / 2nd approach, exp. 2c)
- Specification: produced from the livers of male Sprague-Dawley rats which were treated with Phenobarbital/5,6-Benzoflavone.
- method of preparation of S9 mix: Phosphate buffer(22.5 mL), 0.1M NADP-solution(1.0 mL), 1M G6P-solution(0.125 mL), Salt solution(0.5 mL), Rat liver S9(1.0 mL)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): metabolic activation by microsomal enzymes (e.g., benzo(a)pyrene).

Test concentrations with justification for top dose:

Experiment 1: 5, 1.5, 0.5, 0.15, 0.05 µL/plate
Experiment 1b: 5, 1.5, 0.5, 0.15, 0.05, 0.015, 0.005 µL/plate
Experiment 2: 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08 µL/plate
Experiment 2b: 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08 µL/plate
Repetition Experiment 1: 5, 1.5, 0.5, 0.15, 0.05 µL/plate
Repetition Experiment 1b: 5, 1.5, 0.5, 0.15, 0.05 µL/plate
Experiment 2c: 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08 µL/plate

Vehicle / solvent:

Vehicle: Dimethylsulfoxide (DMSO)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

mitomycin C

other: 4-Nitro-1,2-phenylene diamine, C6H7N3O2, CAS-No.: 99-56-9; 2-Amino-anthracene, C1 4H11N, CAS-No.: 613-13-8;

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation; in suspension
DURATION
- Preincubation period: 20min at 37 ± 1 °C
- Exposure duration: 48 hours at 37 ±1 °C
SELECTION AGENT (mutation assays): mutagenic substances: 4-Nitro-1,2-phenylene diamine (CAS#99-56-9), Sodium azide (CAS#26628-22-8), 2-Amino-anthracene(CAS#613-13-8), Benzo-apyrene(CAS#50-32-8) and Mitomycin C(CAS#50-07-7)
NUMBER OF REPLICATIONS: 3

Rationale for test conditions:

All negative and nearly all strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Evaluation criteria:

Acceptance criteria:
- The number of revertant colonies in the negative controls is within the range of the historical control data
- The sterility controls revealed no indication of bacterial contamination
- The positive controls induced a significant increase in the number of revertant colonies within the range of the historical control data
- The determination of titre should give a number of at least 10^9 cells/mL, correlating to 100 colonies/plate after dilution.
All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and with-out metabolic activation and nearly all were within the historical control data ranges.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

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

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

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

Test results 3

Key result

Species / strain:

S. typhimurium TA 102

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

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

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

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

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:

Based on the results of this study it is concluded that testing of Phosphoric acid,tri-2-propargyl ester (LDY196) in the Salmonella typhimurium strains under the experi-mental conditions in this study led to an inconclusive outcome.

Executive summary:

The study procedures described in this report were based on the most recent Guideline OECD 471 (2020) and EU Method B.13/14 (2008).

The test item Phosphoric acid,tri-2-propargyl ester (LDY196) was tested in the Bacterial reverse mutation assay with five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537).

The test was performed in seven experiments in the presence and absence of metabolic activation, with +S9 standing for the presence of a metabolic activation, and -S9 standing for absence of metabolic activation.

After performance of Exp. 1, 1b, 1c, 2 and 2b, a transciption error in the evaluation sheet of Exp. 1 was noticed. As the corrected results showed an increase factor f(I) > 3 for TA1535, but the number of spontanous revertants was at the lower bond of the historical control data, a repetition of Exp. 1 was performed.

The invalid experiments or experimental parts are not reported in this report, but the raw data are kept in the test facility in the GLP-archive.

 

An overview over the experiments is given in the following table.

Table: Overview over the experiments performed

Experiment	Tested strains	Validity	Additional information
1 (plate incorporation)	TA98, TA100, TA102, TA1535, TA1537	valid for TA98, TA100, TA1535, TA1537   invalid for TA102	results evaluated for TA98, TA100, TA1535, TA1537   Invalid positive controls for TA102 (+/-S9)
1b (plate incorporation)	TA102	valid for TA102 (+S9)   invalid for TA102 (-S9)	results evaluated for TA102 (+S9)   Invalid positive control for TA102 (-S9)
1c (plate incorporation)	TA102 (-S9)	invalid for TA102 (-S9)	New positive control MMC implemented for TA102 (-S9) Solvent control DMSO for TA102 (-S9) not in historical range (mean ± 3 sd)
2 (pre-incubation)	TA98, TA100, TA102, TA1535, TA1537	valid for TA98, TA100, TA102 (+S9), TA1535, TA1537   invalid for TA102 (-S9)	results evaluated for TA98, TA100, TA102 (+S9), TA1535, TA1537   Invalid positive control for TA102 (-S9)
2b (pre-incubation)	TA102 (-S9)	invalid for TA102 (-S9)	Solvent control DMSO for TA102 (-S9) not in historical range (also not inside mean ± 3 sd)
2c (pre-incubation)	TA102 (-S9)	valid	New positive control MMC implemented for TA102 (-S9)
Repetition Exp. 1 (plate incorporation)	TA98, TA100, TA102, TA1535, TA1537	valid for TA98, TA100, TA1535, TA1537   invalid for TA100	results evaluated for TA98, TA102, TA1535, TA1537   Solvent controls for TA100 not in historical range (also not inside mean ± 3 sd)
Repetition Exp. 1 / 2^ndapproach	TA100	valid	results evaluated for TA100 (+/-S9)

 

Experiment 1:

In the first experiment,the test item (dissolved in Dimethyl sulfoxide, DMSO) was tested up to concentrations of 5 µL/plate in the absence and presence of S9 mix in the strains TA98, TA100, TA102, TA1535 and TA1537 using the plate incorporation method.

The test item showed no precipitates and no signs of toxicity on the plates at any of the concentrationsin boththe presence and the absence of metabolic activation.

At the highest test item concentration (5 µL/plate), a significant increase (f(I) > 3) in the number of revertants was found for TA1535 (+S9). Additionally, a dose-dependency over the tested range could be observed for TA100 (+/-S9) and TA1535 (+S9).

As the positive controls for TA102 (+/-S9) showed no positive response, exp. 1 was invalid for TA102 and was repeated in exp. 1b

 

Experiment 1b:

Based on the results of exp.1, the experiment was repeated for TA102 with and without metabolic activation.

For the approach with S9, a valid result could be obtained.The test item showed no precipitates and no signs of toxicity on the plates at any of the concentration.

As the positive control for TA102 (-S9) showed no positive response, exp. 1b was invalid for TA102 (-S9) and was repeated in exp. 1c.

 

Experiment 1c:

Based on the results of exp.1 and 1b, the experiment was repeated for TA102 without metabolic activation.

As the number of spontaneous revertants of DMSO for TA102 (-S9) was not within the historical control data, exp. 1c was invalid for TA102 (-S9). A separate repetition was not performed, but a valid result could be obtained in the repetition exp. 1.

 

Experiment 2:

Based on the results of the first experiment,the test item was tested up to concentrations of 5 µL/plate in the presence and absence of S9 mix in all bacteria strains using the pre-incubation method.

The test item showed no precipitates and no signs of toxicity on the plates at any of the concentrationsin boththe presence and the absence of metabolic activation.

A dose-response over the tested range could be observed for TA100 (+/-S9) and TA1535 (-S9) and in tendency also for TA1535 (+S9).

As the positive controls for TA102 (-S9) showed no positive response, exp. 2 was invalid for TA102 (-S9) and was repeated in exp. 2b.

 

Experiment 2b:

Based on the results of exp.2, the experiment was repeated for TA102 without metabolic activation.

As the number of spontaneous revertants of DMSO for TA102 (-S9) was not within the historical control data range, exp. 2b was invalid for TA102 (-S9).

 

Repetition Experiment 1:

The test item was tested up to concentrations of 5 µL/plate in the absence and presence of S9 mix in the strains TA98, TA100, TA102, TA1535 and TA1537 using the plate incorporation method.

The test item showed no precipitates and no signs of toxicity on the plates at any of the concentrationsin boththe presence and the absence of metabolic activation.

A tendency of a dose-response over the tested range could be observed for TA1535 (-S9).

As the number of spontaneous revertants of DMSO and demin. water was not within the historical control data range, the repetition exp. 1 was not valid for TA100 (+/-S9) and was repeated.

 

Repetition Experiment 1 / 2^ndapproach:

Thetest item was tested up to concentrations of 5 µL/plate in the absence and presence of S9 mix in the strains TA100 using the plate incorporation method.

The test item showed no precipitates and no signs of toxicity on the plates at any of the concentrationsin boththe presence and the absence of metabolic activation.

A dose-response over the tested range could be observed for TA100 (+/-S9).

 

Experiment 2c:

Based on the results of exp. 2 and 2b, the experiment was repeated for TA102 without metabolic activation.

A valid result could be obtained. The test item showed no precipitates and no signs of toxicity on the plates at any of the concentration.

No relevant or dose-related increase in the number of revertants could be observed.