Prop-2-yn-1-ol

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:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: modern guideline study conduceted according to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Prop-2-yn-1-ol

EC Number:

203-471-2

EC Name:

Prop-2-yn-1-ol

Cas Number:

107-19-7

Molecular formula:

C3H4O

IUPAC Name:

prop-2-yn-1-ol

Details on test material:

Name of test substance: 2-Propyn-1-ol
Test substance No.: 00/0529-4
Batch identification: 93006936W0
Purity/composition: 99.621 area-%.
Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
Storage stability: The stability of the test substance under storage conditions is guaranteed until 04 Nov 2015 as indicated by the sponsor, and the sponsor holds this responsibility.

ADDITIONAL TEST SUBSTANCE INFORMATION
Date of production: 04 Nov 2014
Physical state, appearance: Liquid, colorless, clear
Storage conditions: Room temperature

Method

Target gene:

his- (S. typhimurium), trp- (E. coli)

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction

Test concentrations with justification for top dose:

Experiment 1: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate (with and without S9 mix, all strains) Standard Plate Test
Experiment 2: 0; 3.3; 10; 33; 100, 333 and 1000 μg/plate (with and without S9 mix, all strains) Standard Plate Test
Experiment 3: 0; 3.3; 10; 33; 100, 333 and 1000 μg/plate (with and without S9 mix, all strains) Preincubation Test

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, water was used as vehicle.

Controls

Untreated negative controls:

yes

Remarks:

Additional plates were treated with soft agar, S9 mix, buffer, vehicle or the test substance but without the addition of tester strains

Negative solvent / vehicle controls:

yes

Remarks:

The vehicle control with and without S9 mix only contains the vehicle used for the test substance at the same concentration and volume for all tester strains

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene, N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylenediamine

Details on test system and experimental conditions:

Standard plate test
The experimental procedure of the standard plate test (plate incorporation method) was based on the method of Ames et al. (1, 2).
• Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were be poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

• Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were be poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

Preincubation Test
The experimental procedure was based on the method described by Yahagi et al. (7) and Matsushima et al. (8).
0.1mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perseptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Positive controls
With S9 mix
• 2-aminoanthracene (2-AA): 2.5 μg/plate, TA 1535, TA 100, TA 1537, TA 98; 60 μg/plate, Escherichia coli WP2 uvrA
Without S9 mix
• N-methyl-N'-nitro-N-nitrosoguanidine (MNNG): 5 μg/plate, TA 1535, TA 100
• 4-nitro-o-phenylenediamine (NOPD): 10 μg/plate, TA 98
• 9-aminoacridine (AAC): 100 μg/plate, TA 1537
• 4-nitroquinoline-N-oxide (4-NQO): 5 μg/plate, E. coli WP2 uvrA

Three test plates per dose or control were used.

Evaluation criteria:

The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control data range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

SPT: from about 333 µg/plate onward; PIT:from about 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TOXICITY
A bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test depending on the strain and test conditions from about 333 μg/plate onward.
In the preincubation assay bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ revertants) was observed depending on the strain and test conditions from about 1000 μg/plate.
SOLUBILITY
No test substance precipitation was found with and without S9 mix.
MUTAGENICITY
A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

For detailed result tables see attached document.

Applicant's summary and conclusion

Conclusions:

The test substance 2-Propyn-1-ol is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Executive summary:

According to the results of the study, the test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in three experiments carried out independently of each other (standard plate test and preincubation assay). The results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within or nearby the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data.