Pirimiphos-methyl

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:

14 Jan 2015 to 30 Jan 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Pirimiphos-methyl

EC Number:

249-528-5

EC Name:

Pirimiphos-methyl

Cas Number:

29232-93-7

Molecular formula:

C11H20N3O3PS

IUPAC Name:

O-2-(diethylamino)-6-methylpyrimidin-4-yl O,O-dimethyl phosphorothioate

Method

Target gene:

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

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain 3

Species / strain / cell type:

E. coli, other: WP2 pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Phenobarbital/ β-naphthoflavone induced rat liver S9
- Method of preparation of S9 mix: The S9 was prepared from 8 – 12 weeks old male Wistar rats (RjHan:WI; weight approx. 220 – 320 g, induced by peroral administration of 80 mg/kg b.w. phenobarbital and by peroral administrations of β-naphthoflavone each, on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at –80 °C.
- Concentration or volume of S9 mix and S9 in the final culture medium: S9 supernatant was 10 % v/v in the S9 mix. S9 mix contains: 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
- Quality controls of S9: Each batch of S9 mix was routinely tested with 2-aminoanthracene as well as benzo[a]pyrene. The protein concentration in the S9 preparation was 39.3 mg/mL in both experiments.

Test concentrations with justification for top dose:

Experiment I (-S9 and +S9): 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II (-S9 and +S9): 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: The solvent was chosen because of its solubilisation properties and its relative non-toxicity to the bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-aminoanthracene, 2-AA

Details on test system and experimental conditions:

PRE-EXPERIMENT FOR CYTOTOXICITY
To evaluate the cytotoxicity of the test substance a pre-experiment was performed with all strains. Eight concentrations were tested for cytotoxicity and mutation induction each with three replicate plates. The experimental conditions in this pre-experiment were the same as described below for experiment I (plate incorporation test). Cytotoxicity of the test substance results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn. The pre-experiment is reported as the main experiment I since the criteria mentioned under Acceptability of the assay were met.

EXPERIMENTAL PERFOMANCE
For each strain and concentration including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 μL Test solution at each concentration, solvent (negative control) or reference mutagen solution (positive control),
- 500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer* (for test without metabolic activation),
- 100 μL Bacteria suspension (cf. test system, pre-culture of the strains; OD = 0.9 - 1.2, wavelength = 500 nm; approx. 8x108 cells/mL)),
- 2000 μL Overlay agar
For the pre-incubation method 100 μL test solution (solvent or reference mutagen solution (positive control)), 500 μL S9 mix / S9 mix substitution buffer* and 100 μL bacteria suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for 72 hours at 37 °C in the dark, plates were then stored at 4 °C until counted.
* Substitution buffer: 7 parts of the 100 mM sodium-ortho-phosphate-buffer pH 7.4 with 3 parts of KCl solution 0.15 M

DATA RECORDING
The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB9 7BN, UK) with the software program Ames Study Manager. The counter was connected to an IBM AT compatible PC with printer to print out the individual values and the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates (see tables of results). Due to precipitation of the test item the colonies were partly counted manually.

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- Regular background growth in the negative and solvent control
- The spontaneous reversion rates in the negative and solvent control are in the range of the historical data
- The positive control substances should produce a significant increase in mutant colony frequencies
- A minimum of five analysable concentrations should be present with at least four showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

EVALUATION OF RESULTS
A test substance is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed.
A concentration dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A concentration dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls, such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA98, TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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:

E. coli, other: WP2 pKM101, WP2 uvrA pKM101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate in experiment I and in experiment II from 2500 to 5000 µg/plate in all strains with S9 mix and at 5000 µg/plate in all strains without S9 mix. The undissolved particles had no influence on the data recording.

STUDY RESULTS
- Signs of toxicity: Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (μg/plate) shown in table 1 in 'Any other information on results incl. tables'
- Mean number of revertant colonies per plate and standard deviation: No increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test substance at any concentration, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations and all mutation rates were within the range of normal biological variability. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

Any other information on results incl. tables

Table 1. Toxic effects, evident as a reduction in the number of revertants

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	/	/	/	/
TA 1537	5000	/	/	/
TA 98	2500 – 5000	5000	/	/
TA 100	/	/	/	/
WP2 pKM101	/	5000	/	/
WP2uvrApKM101	/	/	/	/

/= no toxic effects

Applicant's summary and conclusion

Conclusions:

The test substance is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This AMES test was performed under GLP following the OECD 471 guideline to investigate the potential of the test substance to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli strains WP2 uvrA pKM101 and WP2 pKM101.

Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate in experiment I and in experiment II from 2500 to 5000 µg/plate in all strains with S9 mix and at 5000 µg/plate in all strains without S9 mix. The undissolved particles had no influence on the data recording. The plates incubated with the test substance showed normal background growth in all strains used up to the highest concentration. Cytotoxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in experiment I in strain TA 1537 without S9 mix, strain TA 98 with and without S9 mix and strain WP2 pKM101with S9 mix. No increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations and all mutation rates were within the range of normal biological variability. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.