Chlorpyrifos

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

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 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Agriculture, Forestry and Fisheries, 12-Nousan-8147, Micronucleus Studies 2-1-19-3

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Chlorpyrifos

EC Number:

220-864-4

EC Name:

Chlorpyrifos

Cas Number:

2921-88-2

Molecular formula:

C9H11Cl3NO3PS

IUPAC Name:

O,O-diethyl O-3,5,6-trichloropyridin-2-yl phosphorothioate

Test material form:

solid

Specific details on test material used for the study:

Substance ID: TSN308540
Lot Number: 2K04161692
Purity: 98.0 wt%

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Breeding Facility, Jai Research Foundation
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 30-38 g
- Assigned to test groups randomly: Yes (censored randomisation method)
- Fasting period before study: 2-3 h before dosing and 1 h after dosing
- Housing: 3 to 4 male animals per cage
- Diet: ad libitum, except during fasting
- Water: ad libitum
- Acclimation period: Five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 65-67
- Air changes (per hr): Minimum 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil
- Justification for choice of solvent/vehicle: Test substance was insoluble in distilled water and formed a suspension in corn oil hence corn oil was selected as the vehicle for the study.
- Concentration of test material in vehicle: 200 mg/mL

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: On day 1 and day 2, 18.76 and 18.77 mg of test substance were sampled respectively and made up to 25 mL for group II, to attain a final concentration of 0.75 mg/mL. For group III and IV simultaneously 15 and 30 mg of test substance were sampled and made up to 10 ml by suspending in to corn oil to attain final concentrations of 1.5 and 3.0 mg/mL, respectively. The test substance was administered orally to mice using a metal cannula attached to a 1 mL disposable syringe. Mice from the vehicle control group (Group I) received only corn oil orally on both the days.

Duration of treatment / exposure:

Two days

Frequency of treatment:

Once/day

Post exposure period:

23-24 hours after the final treatment

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

7.5 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

30 mg/kg bw/day

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin-C
- Route of administration: Intraperitonial
- Doses / concentrations: 1.0 mg/kg body

Examinations

Tissues and cell types examined:

Femur boone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:

TREATMENT AND SAMPLING TIMES: All the animals from the vehicle, treatment and positive control groups were sacrificed by CO2 asphyxiation approximately between 23-24 hours after the final treatment. Femur bones from the sacrificed animals were excised and the epicondyle tips were removed. The bone marrow content was expelled by flushing and aspirating approximately 3 mL of foetal bovine serum using a 1 mL syringe and 24 gauge needle into centrifuge tubes. The aspirated bone marrow content was mixed using the syringe to dissociate the cells in order to avoid cell clump formation.

DETAILS OF SLIDE PREPARATION: The tubes were centrifuged at around 2000 rpm for 10 minutes and the supernatant was discarded leaving about 0.2 - 0.3 mL of medium with the cell pellet. The cell pellet was dissociated thoroughly using a pasteur pipette and a drop of suspension was placed on a clean slide. A smear was prepared and allowed to air dry. The slides were marked with study number, animal number and slide number. Two slides were prepared per animal and the cells were fixed with methanol and allowed to air dry for 20 minutes. Slides were stained using 5% Giemsa in phosphate buffer for 25 minutes. Subsequently the slides were rinsed in distilled water, air-dried and mounted.

METHOD OF ANALYSIS: In order to prevent bias in the scoring, the slide numbers were masked with code numbers. The slides were examined for the presence of micronuclei in polychromatic and normochromatic erythrocytes under microscope (Eclipse E600, Nikon, Eclipse 80i, Nikon, OPTIPHOT-2, Nikon, Eclipse Ni-U (Fluoroscence), Nikon). A minimum of 4000 polychromatic erythrocytes were screened per animal to evaluate the incidence of micronuclei. A minimum of 500 normochromatic erythrocytes to its corresponding polychromatic erythrocytes were recorded to determine the P/E ratio. The masked labels were removed and all the slides were decoded after scoring.

Evaluation criteria:

The conditions necessary for determining a positive result were,
Dose related increase in the incidence of micronuclei or increase in a single dose group; biological relevance of the results was considered first; statistical methods were used to support evaluation of the results; a response was considered negative in the absence of increases in the numbers of MNPCEs relative to the concurrent and established historical control frequencies for MNPCEs induction.

Statistics:

The data of percent micronucleated polychromatic erythrocytes (% MNPCE) and P/E ratio was statistically analysed using Bartlett’s test and Analysis of Variance (ANOVA) followed by the Dunnett’s t-test to determine the level of significant differences between the vehicle control and the treatment group. Where data did not meet the homogeneity of variance, a Student's t-test was performed to determine the level of significant difference between the vehicle control, treatment group and the positive control group.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Clinical symptoms like lethargy and abnormal gait were observed in animals treated at dose level of 15 and 30 mg/kg body weight, respectively.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 40-180 mg/kg
- Solubility: The test item was found insoluble in distilled water (Stock A, 200 mg/mL), while formed suspension in corn oil (Stock B, 200 mg/mL)
- Clinical signs of toxicity in test animals: 100% mortality was observed in animals treated at 180 mg/kg body weight. Mortality in two male and female animals was observed at the treated dose of 80 mg/kg body weight, while mortality in one male animal was observed at the treated dose of 40 mg/kg body weight. Lacrimation, clonic convulsion, abdominal breathing, hunched back posture, salivation were observed in animals treated at 180 mg/kg body weight. Mortality was not observed in any of the animals of vehicle group. Clinical symptoms of clonic convulsion, tail erection, hunched back posture, lacrimation, spastic locomotion, salivation, abdominal breathing, rough hair coat were observed in animals treated at the dose level of 80 mg/kg body weight. Clinical symptoms viz., spastic locomotion, clonic convulsion, tail erection, hunched back posture, lacrimation were observed in animals treated at the dose level of 40 mg/kg body weight. Significant decrease in body temperature i.e. > 3 °C was observed after day 1 and day 2 of dosing in animals treated at the dose level of 80 and 180 mg/kg body weight, while decrease of 2°C in body temperature was observed in male and female animals treated at the dose level of 40 mg/kg body weight, when compared with the concurrent vehicle control group.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: The mean percent micronucleated polychromatic erythrocytes (% MNPCE) observed in male animals was 0.027, 0.023, 0.023 and 0.020 at the dose levels of 0.0 (vehicle control group), 7.5, 15 and 30 mg of test substance/kg body weight, respectively. The mean percent micronucleated polychromatic erythrocytes (% MNPCE) observed in animals treated with Mitomycin-C (1.0 mg/kg body weight) was 1.157.
- Ratio of PCE/NCE: The ratio of polychromatic erythrocytes (PCE) to total erythrocytes (P/E ratio) in treated groups, treated at the dose levels of 7.5 and 15 mg/kg body weight was comparable to the vehicle control group, while statistically significant decrease in P/E ratio was observed in male animals treated at the dose level of 30 mg/kg body weight and in animals from positive control group. Although statistically significant decrease in P/E ratio was observed in male animals of positive control group, it was within the historical data range.
- Statistical evaluation: Statistical analysis of the results did not reveal any significant difference in percent micronucleated polychromatic erythrocytes (% MNPCE) in animals belonging to treatment groups treated at the dose levels of 7.5, 15 and 30 mg/kg body weight when compared with the vehicle control group.

Any other information on results incl. tables

Table 1: Summary of Micronucleated Polychromatic Erythrocytes in Bone Marrow Cells (number of animals = 6 males/group)

Group and Dose of the test substance	Male				Mean P/E Ratio
	Total PCE	MNPCE
		Total	Mean	Mean % MNPCE
G I Vehicle control (Corn oil)	27053	8	1.333	0.027	0.521
G II 7.5 mg/kg bw	27040	7	1.167	0.023	0.506
G III 15 mg/kg bw	27027	7	1.167	0.023	0.522
G IV 30 mg/kg bw	27158	6	1.000	0.020	0.461↓
G V Positive control Mitomycin C 1.0 mg/kg bw	27068	313	52.167↑↑	1.157↑↑	0.468↓

% MNPCE = (MNPCE x 100)/Total PCE

PCE = Polychromatic Erythrocytes

MNPCE = Micronucleated Polychromatic Erythrocytes

P/E = Polychromatic Erythrocytes corresponding to Normochromatic Erythrocytes/Total Erythrocyte

↑↑ = Significantly higher than the control at 1% level (p ≤0.01)

↓ = Significantly lower than the control at 5% level (p ≤0.05)

Applicant's summary and conclusion

Conclusions:

No micronucleus induction potential in mice

Executive summary:

The study was performed to evaluate the micronucleus induction potential of test substance in mice following the guidelines OECD 474 and OCSPP 870.5395. 30 healthy CD1 mice (males) were divided into 5 groups, each group comprising 6 male animals. The test substance was suspended in corn oil and administered orally at doses of 7.5, 15 and 30 mg/kg body weight (Group II, III and IV respectively) for two consecutive days. Animals were sacrificed approximately between 23-24 hours after the final treatment. A concurrent positive control group was treated with a single intraperitoneal injection of mitomycin C at the dose level of 1.0 mg/kg body weight.

All animals were normal in the vehicle control group (Group I) and treatment group II (7.5 mg/kg body weight) and positive control group (Group V) both post-treatment and pre-sacrifice. Clinical symptoms like lethargy and abnormal gait were observed in animals treated at the dose levels of 15 and 30 mg/kg body weight, respectively. Mortality was not observed in any of the animals from the control or treatment groups. Body weights were comparable among the groups during the experimental period.

Number and percentage of micronucleated polychromatic erythrocyte (MNPCE) were not increased in male animals treated with the test substance up to the dose level of 30 mg/kg body weight when compared with the vehicle control group.

The positive control group treated with mitomycin-C yielded a statistically significant increase in the number of micronucleated polychromatic erythrocytes (MNPCE) in comparison to the vehicle control group, both groups giving results in the range of the historical controls.

From the results of the present study, it is concluded that the test substance does not have micronucleus induction potential in the animals treated up to 30 mg/kg body weight following oral administration for two consecutive days.