Chlorpyrifos

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries, 12-Nousan-8147, Chromosomal Aberration Studies, 2-1-19-2

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration 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%

Method

Species / strain

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

CELLS USED
- Sex, age and number of blood donors if applicable: healthy, 33 year old male volunteer
- Whether whole blood or separated lymphocytes were used if applicable: Whole blood

MEDIA USED
The whole blood was cultured in RPMI-1640 (Roswell Park Memorial Institute) with L-glutamine and 25 mm HEPES (Sigma R4130) containing penicillin and streptomycin supplemented with 20% heat-inactivated (56 °C; 30 min) fetal bovine serum.

Metabolic activation:

with and without

Metabolic activation system:

rat liver (treated with Aroclor 1254) S9 fraction

Test concentrations with justification for top dose:

Cytotoxicity test: 20.48, 51.2, 128, 320, 800 and 2000 μg/mL
Main study: 25, 50, 100, 150, 200 and 250 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: As per the guideline’s requirement for highest test concentration, solubility was tested at 200000 μg/mL (100X stock in DMSO). Test item was insoluble in sterile distilled water, while found completely soluble in dimethyl sulfoxide.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

SPINDLE INHIBITOR: Colchicine

STAIN: 5% Giemsa in phosphate buffer

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Slides were prepared from each culture tube by dropping approximately about 0.5 mL of the fixed cell suspension, drop by drop on two, pre-cleaned, ice-chilled slides. The slides were dried over a slide warmer and labeled with study number, culture number and slide number. The dried slides were stained with 5% Giemsa in phosphate buffer for 8 minutes. The slides were made permanent by mounting a cover slip with DPX mountant. Out of these two slides, one was used for scoring and the other served as reserve.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 150 metaphases per replicate

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index

OTHER EXAMINATIONS:
Only cells containing 46 ± 2 chromosomes were examined for structural changes. A smaller number (e.g. 50 metaphases per slide) of metaphases were analysed in slides showing higher frequency (more than 20%) of aberrant cells. Gaps, breaks, fragments, exchanges, deletions, rings and other aberrations were recorded with their numbers and frequencies for all the treated and control cultures separately. In addition, 100 metaphases/replicate were examined for polyploidy. The number of metaphases with only gap, endoreduplication or polyploidy were recorded but not considered to calculate total aberrations and percent aberrant cells.

Evaluation criteria:

The test substance was considered to have shown clastogenic activity in this study if the following criteria were met: Increased frequency of metaphases with aberrant chromosomes observed at one or more test concentrations (concentration-related); Increases were reproducible between replicate cultures and between tests (when treatment conditions are the same); Increases in percent aberrant metaphases were statistically significant; Increases in percent aberrant metaphases were not associated with large changes in pH and osmolality of the treatment medium.

Historical control data was also considered in the evaluation.

An increase in the number of polyploid cells indicates that the test substance has the potential to inhibit mitotic processes and to induce numerical chromosome aberration. Biological relevance was considered first. The test substance was considered non-mutagenic if the results did not meet the above mentioned criteria.

Statistics:

Gaps and polyploidy were not included in the calculation of total aberration frequency. Data on mitotic index, polyploidy and percent aberrant cells were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test (Gad. and Weil, 1994). Where the data did not meet the homogeneity of variance, Student's t-test was performed to determine the level of significant difference between the negative control and three selected test concentrations (selected based on the mitotic index data) and positive controls.

Reference: Gad, S.C. and Weil, C.S. (1994). Statistics for Toxicologists. In: Principles and Methods of Toxicology, Third Edition, Hayes A.W. (Ed.). Raven Press Ltd., New York, pp. 221-274.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: Human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Significant level of cytotoxicity, i.e. >50% reduction in the mitotic index, was observed at test concentrations of 320 μg/mL both in the absence and presence of metabolic activation system

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: A significant change in pH (±1 unit) or osmolality (≥50 mOsm/kg H2O) was not observed at 0 and 4 h in any of the tested concentrations of 62.5, 125, 250, 500, 1000 and 2000 μg/mL of culture medium.
- Water solubility: Test substance was insoluble in sterile distilled water
- Precipitation: Precipitation was not observed at the tested concentrations of 62.5, 125, 250, 500 and 1000 of culture medium, while slight precipitation was observed at 2000 μg/mL of culture medium.

RANGE-FINDING/SCREENING STUDIES: The cytotoxicity was assessed based on the results of precipitation, pH and osmolality tests at the concentrations of 20.48, 51.2, 128, 320, 800 and 2000 μg/mL of culture medium. The reduction in mitotic index observed was -3.92, -1.74, -2.87, 77.91, 87.34, 98.43 % and -11.59, -2.79, 1.72, 73.79, 91.86 and 100 % at the concentrations of 20.48, 51.2, 128, 320, 800 and 2000 μg/mL of test substance, in the absence and presence of metabolic activation (2% v/v S9), respectively.
Therefore, significant level of cytotoxicity, i.e. >50% reduction in the mitotic index, was observed at test concentrations of 320 μg/mL both in the absence and presence of metabolic activation system. Hence, 250 μg/mL was selected as the highest test concentration to be tested in the main study.

Any other information on results incl. tables

Table 1: Summary of Percent Mitotic index, Percent Aberrant and Polyploid Cells

Concentration of test substance	Phase I [Absence of Metabolic Activation]
	% Mitotic Index	% Aberrant Cells	% Polyploid Cells
	Mean ± SD	Mean ± SD	Mean ± SD
NC (DMSO)	5.484 ± 0.282	2.665 ± 0.940	0.500 ± 0.707
100 μg/mL	5.172 ± 1.013	2.000 ± 0.948	0.000 ± 0.000
150 μg/mL	4.553 ± 0.682	2.335 ± 0.474	0.000 ± 0.000
200 μg/mL	2.787 ± 0.289↓	1.665 ± 0.474	0.500 ± 0.707
PC	3.282 ± 0.415↓	24.000 ± 2.828↑↑	0.000 ± 0.000
Concentration of test substance	Phase I [Presence of Metabolic Activation (1% v/v S9)]
	% Mitotic Index	% Aberrant Cells	% Polyploid Cells
	Mean ± SD	Mean ± SD	Mean ± SD
NC (DMSO)	4.672 ± 0.279	2.000 ± 0.948	0.000 ± 0.000
100 μg/mL	4.172 ± 0.547	2.000 ± 0.948	0.000 ± 0.000
150 μg/mL	3.684 ± 0.371	2.335 ± 0.474	0.000 ± 0.000
200 μg/mL	2.395 ± 0.569↓	2.000 ± 0.948	0.000 ± 0.000
PC	2.876 ± 0.441↓	24.000 ± 2.828↑↑	0.000 ± 0.000
Concentration of test substance	Phase II [Absence of Metabolic Activation
	% Mitotic Index	% Aberrant Cells	% Polyploid Cells
	Mean ± SD	Mean ± SD	Mean ± SD
NC (DMSO)	4.714 ± 0.688	2.335 ± 0.474	0.500 ± 0.707
25 μg/mL	4.315 ± 0.244	1.665 ± 0.474	0.500 ± 0.707
50 μg/mL	3.824 ± 0.332	2.335 ± 0.474	0.000 ± 0.000
100 μg/mL	2.476 ± 0.157↓	1.665 ± 0.474	0.000 ± 0.000
PC	2.836 ± 1.061	23.000 ± 1.414↑↑	0.000 ± 0.000

NC = Negative control, SD = Standard deviation, PC = Positive control (Mitomycin-C @ 0.3 μg/mL in the absence of S9 and Cyclophosphamide @ 40 μg/mL in the presence of S9), DMSO = Dimethyl sulfoxide

↑↑ = 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 potential to induce chromosomal aberrations in Human peripheral blood lymphocyte cultures; negative for clastogenicity

Executive summary:

In a mammalian chromosome aberration test, human peripheral blood lymphocytes cultured in vitro were exposed to the test substance at different concentrations both in the absence and presence of metabolic activation (1% v/v S9). The study was conducted following OECD guideline 473 and EPA OCSPP 870.5375.

The test substance was tested in two independent experiments, in the absence and presence of metabolic activation (1% v/v S9). Human peripheral blood lymphocyte cultures were exposed to the test substance at 6 dose-levels (two cultures/dose level) between 25 and 250 μg/mL of culture medium in main study experiment, selected from a preliminary cytotoxicity test.

The test substance did not induce statistically significant or biologically relevant increases in the number of cells with chromosome aberrations in the absence and presence of S9-mix in either of the two independently conducted experiments. No effects of test substance on the number of polyploid cells or number of cells with endoreduplicated chromosomes were observed either in the absence or presence of S9-mix. All negative controls were within the historical limits and positive controls showed an increase in the incidence of cells with chromosome aberrations.

All criteria for a valid study were met as described in the protocol. From the results of this study, it is concluded that the test substance did not show potential to induce chromosomal aberrations both in the absence and presence of metabolic activation system under the described experimental conditions and is negative for clastogenicity.