Pirimiphos-methyl

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 Mar 2017 to 16 Jun 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

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

Test animals

Species:

mouse

Strain:

other: Hsd:ICR (CD-1)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Six to ten weeks old
- Weight at study initiation: 22.1 to 30.9 g
- Housing: The animals were housed in groups of up to seven in solid-floor polypropylene cages with wood-flake bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: a minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 1 Mar 2017 to 16 Jun 2017

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Carboxymethyl-cellulose (CMC)
- Justification for choice of vehicle: 0.5% CMC was used as the vehicle as it gave an emulsion suitable for dosing.
- Amount of vehicle: 10 mL/kg
- Concentration of test material in vehicle: 17.5, 35 and 70 mg/mL

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was freshly prepared as required as an emulsion at the appropriate concentration in 0.5% CMC.

Frequency of treatment:

Single dose

Post exposure period:

24 hours and 48 hours (highest dose group only)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

175 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

350 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

700 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control: Cyclophosphamide is a positive control item known to produce micronuclei under the conditions of the test.
- Route of administration: Single oral administration
- Actual dose: 50 mg/kg
- Dose volume: 10 mL/kg

Examinations

Tissues and cell types examined:

Bone marrow, Polychromatic erythrocytes (PCEs)

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION: Immediately following termination (i.e. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal bovine serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grünwald/Giemsa, allowed to air-dry and a cover slip applied using mounting medium.

SLIDE EVALUATION: Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 4000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei.
The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

METHOD OF ANALYSIS: A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the corresponding vehicle control group.

CRITERIA FOR DOSE SELECTION:
A range-finding toxicity test was performed to determine a suitable dose level for the main test. The dose level selected should ideally be the maximum tolerated dose level or a maximum recommended dose of 2000 mg/kg if no clinical signs are observed. The range- finding toxicity test was also used to determine if the main test was to be performed using both sexes or males only. Bone marrow samples were taken from the range-finding animals 48 hours after dosing and slides were prepared and qualitatively assessed to ensure that any bone marrow toxicity observed was within acceptable limits for the main test. In addition, three satellite groups of three male mice were dosed with the maximum proposed dose level for the main test alongside the confirmatory range-finding dose level. These animals were terminated at 1, 4, and 24 hours after dosing and blood samples were taken to provide plasma and blood cell samples for proof of exposure analysis. The main study would only be performed if the Study Director and Sponsor agreed that the analysis from the satellite groups indicated that exposure to the bone marrow had been achieved.

Evaluation criteria:

Acceptability criteria
The test is considered acceptable if the following criteria are met:
- The concurrent negative control data are considered acceptable for addition to the laboratory historical control database
- The concurrent positive controls or scoring controls should induce responses that are compatible with those generated in the historical control database and produce a statistically significant increase compared with the concurrent negative control
-The appropriate number of doses and cells has been analysed

Statistics:

All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Range-Finding Toxicity Test
In animals dosed with the test item at 2000 mg/kg, premature death was observed in the male animal and the following clinical signs were observed: hunched posture, ptosis, tiptoe gait, and emaciation. At 1000 mg/kg, the following clinical signs were observed: hunched posture, ptosis, lethargy, ataxia, splayed gait, tonic convulsions, and emaciation. The male animal was euthanized due to the severity of the clinical signs. Clinical signs of hunched posture and ptosis were observed in animals dosed at 300 and 600 mg/kg. In animals dosed at 800 mg/kg the following clinical signs were observed: hunched posture, ptosis, tonic convulsions, splayed gait, and emaciation. In animals dosed at 700 mg/kg the clinical signs included hunched posture, ptosis and splayed gait. Emaciation was also observed in one animal at this dose level. However, following discussions with the animal technician, the emaciation was considered to be due to the other animals not allowing this animal to feed. Qualitative analysis of the bone marrow slides indicated that there was no evidence of any significant toxicity to the bone marrow in any of the range-finding animals. The micronucleus test was therefore conducted, following agreement from the Sponsor and the Study Director, using the oral route in groups of seven mice (males) at the considered maximum tolerated dose of 700 mg/kg, with 350 and 175 mg/kg as the two lower dose levels. There was no marked difference in toxicity of the test item between the sexes; therefore the main test was performed using only male mice.

Proof of exposure
Analysis of the plasma and blood cell samples indicated that the test item was present in the satellite group animals at all timepoints and that bone marrow exposure would most likely be achieved.

Micronucleus test
There were no premature deaths observed in any of the dose groups in the main test. Clinical signs of hunched posture and ptosis were observed in the 350 mg/kg dose group. In the 700 mg/kg dose groups, the clinical signs were more severe than those observed at the same dose in the range-finding test and included: hunched posture, ptosis, lethargy, ataxia, splayed gait, hypothermia, elevated tail, decreased respiratory rate, laboured respiration, increased salivation, occasional body tremors, and increased respiratory rate. However, the more severe clinical signs were only observed at the 24 or 48-hour observations and were only observed in 2 of the animals in the 24-hour dose groups, and 2 of the animals in the 48-hour dose group. The reason for this was not known however it may be considered to be due to variable sensitivity of the animals to the test item.

Evaluation of bone marrow slides
There were no marked decreases in the PCE/NCE ratio observed in the 24 or 48-hour test item dose groups when compared to the vehicle control group.
There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test item dose groups when compared to the vehicle control group and were well within the negative control historical control data. Since none of the values for micronucleus frequency in pirimiphos-methyl treated groups were statistically significant, and all were within the HCD, a trend test was not conducted.
The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

See Table 1 in ‘Any other information on results incl. tables'.

Any other information on results incl. tables

Table 1. Micronucleus test -summary of group mean data

Treatment Group	Number of PCE with Micronuclei per 4000 PCE		PCE/NCE Ratio
	Group Mean	SD	Group Mean	SD
Vehicle Control (0.5% CMC) 10 mL/kg 24-hour Sampling Time	1.6	2.1	0.56	0.28
Positive Control (Cyclophosphamide) 50 mg/kg 24-hour Sampling Time	85.8***	42.0	0.84	0.14
Test substance 700 mg/kg 48-hour Sampling Time	2.4 (P = 0.262)	1.1	0.41 (P=0.295)	0.16
Test substance 700 mg/kg 24-hour Sampling Time	3.4 (P = 0.206)	2.5	0.73	0.46
Test substance 350 mg/kg 24-hour Sampling Time	2.3 (P = 0.464)	1.6	0.67	0.32
Test substance 175 mg/kg 24-hour Sampling Time	2.3 (P = 0.390)	1.3	0.66	0.21

Applicant's summary and conclusion

Conclusions:

The test substance was considered to be non-genotoxic under the conditions of the test.

Executive summary:

The micronucleus test was conducted under GLP following the OECD 474 guideline, using the oral route in groups of 7 mice (males) at the maximum tolerated dose of 700 mg/kg, with 350 and 175 mg/kg as the two lower dose levels using 0.5% carboxymethyl-cellulose (0.5% CMC) as the vehicle. A range-finding test was performed to find suitable dose levels of the test item, and to investigate if there was a marked difference in toxic response between the sexes. In addition, three satellite groups of three male mice were dosed with the maximum proposed dose level for the main test alongside the confirmatory range-finding dose level. These animals were terminated at 1, 4, and 24 hours after dosing and blood samples were taken to provide plasma and blood cell samples for proof of exposure analysis. The analysis samples indicated that the test item was present in the plasma and blood cells of the satellite group animals and that bone marrow exposure would most likely be achieved. Validation of the analytical method was performed by spiking known amounts of the test item into control plasma and blood cells and determining accuracy and precision data to meet SANCO/3029/99 guideline. There was no marked difference in toxicity of the test item between the sexes; therefore the main test was performed using only male mice. Animals were killed after 24 hours and 48 hours (highest dose level only), the bone marrow extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Additional groups of 5 mice were given a single oral dose of 0.5% CMC or dosed orally with cyclophosphamide (50 mg/kg bw), to serve as vehicle and positive controls respectively. Vehicle and positive control animals were terminated after 24 hours.

There were no premature deaths observed in any of the dose groups in the main test. The clinical signs of hunched posture and ptosis were observed in the 350 mg/kg dose group. The clinical signs observed in the 700 mg/kg dose groups were more severe than those observed at 700 mg/kg in the range-finding test and the following were observed: hunched posture, ptosis, lethargy, ataxia, splayed gait, hypothermia, elevated tail, decreased respiratory rate, laboured respiration, increased salivation, occasional body tremors, loss of righting reflex, and increased respiratory rate. However, the more severe clinical signs were only observed at the 24 or 48-hour observations and were only observed in 2 of the animals in the 24-hour dose group, and 2 of the animals in the 48-hour dose group. The reason for this was not known however it may be considered to be due to variable sensitivity of the animals to the test item. There were no marked decreases in the PCE/NCE ratio observed in the 24 or 48-hour test item dose groups when compared to the vehicle control group. There was no evidence of any significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

In conclusion, the test substance was considered to be non-genotoxic under the conditions of the test.