Chlorpyrifos

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Study Type	Species	Findings	Guideline	Reliability
Bacterial Reverse Mutation Assay	S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 [bacteria] (with and without met. act.)	Negative	OECD 471, EU Method B.13/14, 5100, Japan Ministry of Agriculture, Forestry and Fisheries Testing Guidelines for Toxicology Studies, 12-Nousan-8147	1
Bacterial Reverse Mutation Assay	S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538 [bacteria] (with and without met. act.)	Negative	No guideline	2
Chromosome Aberration test	human lymphocytes	Negative	OECD 473, EU Method B.10, EPA OPP 84-2, Japan Ministry of Agriculture, Forestry and Fisheries Testing Guidelines for Toxicology Studies, 12-Nousan-8147	1
Chromosome Aberration test	Rat lymphocytes	Negative	EPA OPP 84-2, EPA OPPTS 870.5375, EU Method B.10	1
Mammalian cell gene mutation test	Chinese Hamster ovary cells	Negative	OECD Guideline 476,  EPA OPPTS 870.5300, EU Method B.17	1
Mammalian cell gene mutation test	Chinese Hamster ovary cells	Negative	No guideline	2
UDS assay	Rat hepatocytes	Negative	No guideline	2

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ISBN 92-64-12221-4

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MAFF 1988

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EK EMS 1990

Deviations:

no

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Chlorpyrifos, DURSBAN F
AGR 273801 (MM-890115-616 )
Purity: 98.6%

Species / strain / cell type:

lymphocytes: rat

Test concentrations with justification for top dose:

0 (negative control), 16.7, 50, 167, 500, 1667, and 5000 µg chlorpyrifos per ml of culture medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Key result

Species / strain:

lymphocytes: rat

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Negative in the in vitro chromosomal aberration assay utilizing rat lymphocytes

Executive summary:

The clastogenic potential of chlorpyrifos was evaluated in an in vitro chromosomal aberration assay utilizing rat lymphocytes. Approximately 48 hours after setting up of whole blood cultures, the cells were treated for 4 hours in the presence and absence of an external metabolic activation system (S-9) with 0 (negative control), 16.7, 50, 167, 500, 1667, and 5000 µg chlorpyrifos per ml of culture medium. The concentrations of the test material in the stock solutions used for treatment were verified by analytical methods. Cultures treated with 0.5 µg/ml mitomycin C and 4.2 µg/ml cyclophosphamide were used as positive controls for the non-activation and activation assays, respectively. In the first experiment, the cultures were harvested 24 h after termination of treatment. Based upon the toxicity as assessed from depression in mitotic indices, cultures treated with 0, 16.7, 50, and 167 µg/ml were selected for determining the incidence of chromosomal aberrations in the absence of S-9 and cultures treated with only 0, 16.7 and 50 µg/ml were selected for determining the incidence of chromosomal aberrations in the presence of S-9. In a second independent assay utilizing two harvest times (i.e., 24 and 48 h after termination of treatment), cultures treated with 0, 5, 16.7, and 50 µg/ml were selected for determining the incidence of chromosomal aberrations both in the presence and absence of S-9 at the first harvest time, and cultures treated with 50 µg/ml were selected for the second harvest. No significant

increase in the aberration frequency was noticed at any of the treatment levels in Assay 1 or Assay 2 when compared to the negative controls. Cultures treated with the positive control chemicals had significantly higher incidence of aberrations., It was concluded that chlorpyrifos did not exhibit clastogenic activity in cultured rat lymphocytes under the experimental conditions used.

Reference 2

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Specific details on test material used for the study:

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

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.

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.

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.

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)

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.

Reference 3

Endpoint:

in vitro gene mutation 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

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

Target gene:

hgprt locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

K1 cell line

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Japanese Collection of Research Bioresources
- Methods for maintenance in cell culture if applicable: The cells were grown as monolayer in disposable tissue culture flasks. The cell line free from mycoplasma contamination was used in the study. Cultures were free from any contamination during the conduct of the study.

MEDIA USED
α-MEM (Minimum Essential Medium, Eagle α-Modification with nucleosides) with nucleosides with 10% heat inactivated, sterile, fetal bovine serum was used as the culture medium to grow the CHO-K1 cell line. Culture medium was also supplemented with Penicillin (50 IU/mL of the medium) and Streptomycin (50 μg/mL). At the time of selection Minimum Essential Medium Eagle α-modification without nucleosides (α-MEM w/o NS) with 10% dialyzed fetal bovine serum was used.

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: 3580, 1790, 895, 447.5, 223.75, 111.875, 55.9375 and 27.96875 μg/mL
Mutagenicity test: 1.875, 3.75, 7.5, 15, 30 and 60 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was insoluble in sterile distilled water, while found soluble at 358 mg/mL in dimethyl sulfoxide (DMSO).

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

SELECTION AGENT: 2-amino-6-mercaptopurine (6-thioguanine) was used as selective agent at a concentration of 5 μg/mL α-MEM without nucleosides.

DETERMINATION OF CYTOTOXICITY
- Method: Relative cloning efficiency

Evaluation criteria:

A test substance was considered positive in the mutation assay if: Test substance causes a concentration-related biologically significant increase in mutant frequency in comparison with concurrent negative control and the test item causes a three-fold increase in the number of 6-thioguanine resistant colonies relative to concurrent negative control and such increases were statistically significant and outside of the laboratory historical negative (solvent used) control range; A net increase in mutant colonies of treated above the concurrent control was observed in at least two of the concentrations tested.
Negative results were confirmed by a repeat test (short duration), using a modification in test concentration spacing.

Statistics:

Weighted regression analysis was performed to evaluate the dose response relationship on test substance treatment groups against the negative control group (excluding positive controls).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Excessive cytotoxicity i.e., <10% cells were live at 223.75, 447.5, 895, 1790 and 3580 μg/mL both in the absence and presence of S9

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 111.875, 223.75, 447.5, 895, 1790 and 3580 μg/mL of culture medium.
- Water solubility: The test substance was insoluble in sterile distilled water at 358 mg/mL
- Precipitation: Precipitation was not observed up to the concentration of 895 μg/mL of culture medium.

RANGE-FINDING/SCREENING STUDIES: The percent relative cloning efficiency observed was 37.59, 14.22, 7.48% in the absence of metabolic activation and 31.32, 14.40, 6.96% in the presence of metabolic activation (2% v/v S9), at the tested concentrations of 27.96875, 55.9375, 111.875 μg/mL of culture medium, respectively. Reduction in the cell count observed was between 51-67% at the tested concentrations of 55.9375 and 111.875 μg/mL both in the absence and presence of metabolic activation. Excessive cytotoxicity i.e., <10% cells were live at the tested concentrations of 223.75, 447.5, 895, 1790 and 3580 μg/mL both in the absence and presence of metabolic activation. Based on the observed results and to achieve guidelines required cytotoxicity limit i.e. 10-20% relative survival, 60 μg/mL was selected as the highest concentrations both in the absence and presence of metabolic activation for the main study experiment.

Table 1: Summary of Absolute and Relative Cloning Efficiency Following Treatment

Group (μg/mL)	Absence of Metabolic Activation (Trial I)				Presence of Metabolic Activation (2% v/v S9) (Trial I)
	ACE (Mean)	ACE (%)	RCE	RCE (%)	ACE (Mean)	ACE (%)	RCE	RCE (%)
NC (DMSO)	0.7483	74.83	1.0000	100.00	0.7304	73.04	1.0000	100.00
T1 (1.875)	0.7018	70.18	0.9379	93.79	0.7215	72.15	0.9878	98.78
T2 (3.75)	0.7054	70.54	0.9427	94.27	0.7143	71.43	0.9780	97.80
T3 (7.5)	0.6839	68.39	0.9139	91.39	0.6679	66.79	0.9144	91.44
T4 (15)	0.6679	66.79	0.8926	89.26	0.6518	65.18	0.8924	89.24
T5 (30)	0.2661	26.61	0.3556	35.56	0.2233	22.33	0.3057	30.57
T6 (60)	0.1018	10.18	0.1360	13.60	0.0947	9.47	0.1297	12.97
PC	0.6464	64.64	0.8638	86.38	0.6554	65.54	0.8973	89.73
Group (μg/mL)	Absence of Metabolic Activation (Trial II)				Presence of Metabolic Activation (2% v/v S9) (Trial II)
	ACE (Mean)	ACE (%)	RCE	RCE (%)	ACE (Mean)	ACE (%)	RCE	RCE (%)
NC (DMSO)	0.7572	75.72	1.0000	100.00	0.7679	76.79	1.0000	100.00
T1 (2.5)	0.7018	70.18	0.9268	92.68	0.7233	72.33	0.9419	94.19
T2 (5)	0.7000	70.00	0.9245	92.45	0.6929	69.29	0.9023	90.23
T3 (10)	0.6929	69.29	0.9151	91.51	0.6661	66.61	0.8674	86.74
T4 (20)	0.6625	66.25	0.8749	87.49	0.6339	63.39	0.8255	82.55
T5 (40)	0.2161	21.61	0.2854	28.54	0.1929	19.29	0.2512	25.12
T6 (60)	0.0964	9.64	0.1273	12.73	0.1036	10.36	0.1349	13.49
PC	0.6286	62.86	0.8302	83.02	0.6572	65.72	0.8558	85.58

NC = Negative control, DMSO = Dimethyl sulfoxide, ACE = Absolute cloning efficiency, RCE = Relative cloning efficiency, T = Treatment concentration, PC = Positive control [0.4 μL Ethyl methanesulfonate/mL in absence of metabolic activation and 6 μg Benzo(a)pyrene/mL in presence of metabolic activation]

Conclusions:

No potential to induce gene mutations at the hgprt locus of CHO-K1 cells

Executive summary:

In a mammalian cell gene mutation assay [hgprt locus], CHO-K1 cells cultured in vitro were exposed to the test substance at different concentrations, both in the absence and presence of metabolic activation (2% v/v S9) for a period of 4 hours. The study was conducted following OECD guideline 476 and EPA OCSPP 870.5300.

Test substance was tested in two independent experiments. Cultures were exposed to the test substance at 6 dose-levels (two cultures/dose-level) between 1.875 and 60 μg/mL of culture medium, selected from a preliminary cytotoxicity test both in the absence and presence of metabolic activation (2% v/v S9) for a period of 4 hours.

A significant dose-related increase in the mutation frequency was not observed in any of the treatment concentrations between 1.875 and 60 μg/mL of culture medium both in the absence and presence of metabolic activation system (2% v/v S9) and the induced mutation frequency was comparable to that from the negative control group. All negative controls were within the historical limits and positive controls showed an increase in the mutant frequency. No relevant influence of the test item on pH value or osmolality was observed both in the absence and presence of metabolic activation during both the trials.

All criteria for a valid study were met as described in the protocol. Based on the results from this study, it was concluded that the test substance does not have potential to induce gene mutations at the hgprt locus of CHO-K1 cells both in the absence and presence of metabolic activation system under the present experimental conditions.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test substance was evaluated in the Ames with and without metabolic (S9) activation, using a pre-incubation modification of the standard assay. The studies were conducted using Salmonella typhimurium bacterial tester strains TA98, TA100, TA1535, TA1537, and TA1538. The test substance was tested at concentrations of 1.00, 3.16, 10.00, 31.62 and 100 µg/plate.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Lot Number: AGR214637
Purity: 95.7%

Target gene:

his gene

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538

Metabolic activation:

with and without

Metabolic activation system:

S-9 rat liver homogenate (from Aroclor 1254 induced male Sprague-Dawley rats)

Test concentrations with justification for top dose:

1, 3.162, 10, 31.62, and 100 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

2-nitrofluorene

other: N-methyl-N'-nitro-N-nitrosoguanine (TA100, TA1535 2 µg/plate - without S9); Quinacrine mustard.2HCl (TA1537 10 µg/plate - Without S9)

Evaluation criteria:

A test chemical is considered a bacterial mutagen if the mean number of revertant colonies observed is at least three times higher than the mean of the negative (solvent) control and it produces a dose response relationship over several concentrations. If a chemical produces reproducible reversion rates in excess of 3X over background, but no definitive dose response relationship, it is considered to be a presumptive bacterial mutagen. If a chemical produces reproducible reversion rates greater than 2X but less than 3X over controls, the results are considered to be equivocal or inconclusive.

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 100 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Non-mutagenic in the bacterial reverse mutation assay using Salmonella typhimurium

Executive summary:

The test substance was evaluated in the Ames with and without metabolic (S9) activation, using a pre-incubation modification of the standard assay. The studies were conducted using Salmonella typhimurium bacterial tester strains TA98, TA100, TA1535, TA1537, and TA1538. The test substance was tested at concentrations of 1.00, 3.16, 10.00, 31.62 and 100 µg/plate.

Bacterial toxicity was clearly noted at 100 µg/plate in TA100, TA1535, TA1537 and TA1538 in the absence of and including the S9 activation system. TA98 did not show any sign of toxicity at any concentration of the test substance tested. The highest concentration of test substance (100 µg/plate) clearly exceeded the limits of solubility as evidenced by a fine precipitate formed upon addition of the test substance. The test substance, over the concentrations tested, did not produce increased numbers of revertants, with or without S9 activation, as compared to controls. Under the conditions of the present study, the test substance did not exhibit mutagenic activity in the Ames, using Salmonella in the presence or absence of metabolic activation.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries. Testing Guidelines for Toxicology Studies, 2000. Notification No. 12-Nousan-8147. November 24, 2000. Reverse Mutation Studies 2-1-19-1

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

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

Target gene:

his gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

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: 5000, 2500, 1250, 625, 312.5, 156.25, 78.125, 39.0625, 19.53125 and 9.765625 μg/plate
Mutagenicity test: Trial I: 156.25, 312.5, 625, 1250, 2500 and 5000 μg/plate; Trial II: 51.2, 128, 320, 800, 2000 and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Solubility and precipitation tests of the test item were performed prior to the cytotoxicity test. The test item was insoluble in sterile distilled water (stock A, 50000 μg/mL), while found to be soluble in dimethyl sulfoxide (stock B, 50000 μg/mL). Therefore, dimethyl sulfoxide was selected as the vehicle for treatment.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Aminoanthracene (TA1537, TA1535, TA102 at 10 µg/plate; TA98, TA100 at 5 μg/plate) - with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)

DURATION
- Exposure duration: 48 h

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in mean revertants per concentration

Evaluation criteria:

Once criteria for a valid assay have been met, responses observed in the assay were evaluated. The conditions necessary for determining a positive result were there should be a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test article either in the absence or presence of the metabolic activation system.
Strains TA98, TA1535 and TA1537: Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 3 times the mean negative control value.
Strains TA100 and TA102: Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 2 times the mean negative control value.
A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was determined to be not mutagenic.

Key result

Species / strain:

S. typhimurium, other: TA1537, TA1535, TA98, TA100 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Normal growth was observed up to the tested concentration of 5000 μg/plate both in the absence and presence of metabolic action system. Hence, 5000 μg/plate was selected as the highest concentration to be tested in the mutagenicity experiment both in the absence and presence of metabolic activation system for the entire tester strains

NEGATIVE CONTROL: The results of the study indicate that the values for the negative control in all strains were within limits of historical ranges.

POSITIVE CONTROLS: 2-Aminoanthracene was used as the positive control in the presence of a metabolic activation for all the tester strains during both the trials. Large historical control data of this laboratory proved the efficiency and suitability of 2-aminoanthracene as a positive control in the presence of metabolic activation. The batch of S9 used in this study was characterised with benzo(a)pyrene that requires metabolic activation by microsomal enzymes.
Positive controls (both in the absence and presence of metabolic activation during both the trials) exhibited a clear increase in the number of revertants when compared with the concurrent negative control and were with the historical ranges of respective positive control. This demonstrated the efficiency of the test system and suitability of the procedures employed in the assay.
Increase in the mean number of revertants was not observed in tester strain TA100 (Trial I and II) treated with 2-aminoanthracene in the absence of metabolic activation but clear increase was observed in the presence of metabolic activation. This demonstrated the efficiency of the S9 fraction used in this assay.

Conclusions:

Non-mutagenic in the bacterial reverse mutation assay using Salmonella typhimurium

Executive summary:

The potential of the test substance to induce reverse mutations in Salmonella typhimurium (strains: TA1537, TA1535, TA98, TA100 and TA102) was evaluated in the bacterial reverse mutation test. The study was conducted according to OECD guideline 471 and OCSPP 870.5100.

The test substance was tested in two independent trials, with and without metabolic activation. Bacterial cultures were exposed to the test substance at 6 concentrations (three plates/concentration) between 51.2 and 5000 μg/plate. From a preliminary cytotoxicity test, 5000 μg/plate was selected being the recommended top dose indicated in the guidelines for testing and showing moderate cytotoxicity. After 48 hours of incubation at 37 ± 1°C, the revertant colonies were scored.

The test substance did not induce any significant increase in the number of revertants, in both trials, with and without S9 mix, in any of the five tester strains. All the values for the negative control were within historical control ranges of the laboratory and positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system.

All criteria for a valid study were met as described in the protocol. From the results of this study, under the specified experimental conditions, the test substance was concluded to be non-mutagenic in the bacterial reverse mutation assay using Salmonella typhimurium.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Chlorpyrifos was evaluated in the Chinese hamster ovary cell-hypoxanthine (guanine) phosphoribosyl transferase (CHO/HGPRT) mutagenicity assay. The studies were conducted using a Chinese hamster ovary cell line designated CHO-K1 -BH4 with and without a metabolic activation preparation. Chlorpyrifos was tested at 50, 40, 30, 25. 20, and 10 µM with metabolic activation.

GLP compliance:

no

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Chlorpyrifos
Lot: MM 820905-610
Purity: 95.7%

Target gene:

hypoxanthine (guanine) phosphoribosyl transferase (hgprt) locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Test concentrations with justification for top dose:

50, 40, 30, 25. 20, and 10 µM

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

ethylmethanesulphonate

Evaluation criteria:

For each dose level, the number of mutants per 10e6 clonable cells is calculated, and the results statistically evaluated for increasing linear or quadratic trend. Positive compounds should exhibit a significant concentration-dependent increase over at least 3 concentrations, but this depends on the concentrations chosen for the assay and the toxicity at which mutagenic activity appears. Increases at a single dose near the highest toxicity will require confirmation by a repeat assay. Test substances producing neither a significant dose-related increase nor a significant and reproducible positive response at the highest toxicity test point are considered non-mutagenic.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Chlorpyrifos was not mutagenic in the CHO/HGPRT assay, with or without metabolic activation, indicating a lack of genotoxic activity under the conditions of the present study.

Executive summary:

Chlorpyrifos was evaluated in the Chinese hamster ovary cell-hypoxanthine (guanine) phosphoribosyl transferase (CHO/HGPRT) mutagenicity assay. The studies were conducted using a Chinese hamster ovary cell line designated CHO-K1-BH4 with and without a metabolic activation preparation. Chlorpyrifos was tested at 50, 40, 30, 25. 20, and 10 µM with metabolic activation. Chlorpyrifos formed a precipitate at 50, 40, and 30 µM when the test chemical solution was added to the culture dish. Chlorpyrifos had little effect on survival of the cells in the metabolic activation assay. even at the highest concentration tested, 50 µM. Without activation, chlorpyrifos was tested at 50, 40, 30, 25, 20, and 10 µM, a concentration range which decreased the survival of the cells from approximately 100 to less than 10% of that of control cultures, and formed a precipitate at 50, 40, and 30 µM. No mutations induced by chlorpyrifos were observed. Therefore, it is concluded that chlorpyrifos was not mutagenic in the CHO/HGPRT assay, with or without metabolic activation, indicating a lack of genotoxic activity under the conditions of the present study.

Reference 7

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

The genotoxic potential of the test substance was evaluated in the rat hepatocyte unscheduled DNA synthesis (UDS) assay at concentrations of 1E-4, 3.16E-5, 1E-5, 3.16E-6, and 1E-6 M (half-log intervals).

GLP compliance:

no

Type of assay:

other: unscheduled DNA synthesis

Specific details on test material used for the study:

Lot # AGR 214637
Purity: 95.7%

Species / strain / cell type:

hepatocytes: rat

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

1E-4, 3.16E-5, 1E-5, 3.16E-6, and 1E-6 M

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Key result

Species / strain:

hepatocytes: rat

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity to the hepatocyte cultures was apparent at 1E-4 and 3.16E-5 M

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Negative in UDS assay

Executive summary:

The genotoxic potential of the test substance was evaluated in the rat hepatocyte unscheduled DNA synthesis (UDS) assay at concentrations of 1E-4, 3.16E-5, 1E-5, 3.16E-6, and 1E-6 M (half-log intervals). Toxicity to the hepatocyte cultures was apparent at 3.16E-5 and 1E-4 M test substance. The test substance failed to elicit significant UDS at any of the concentrations tested. The inability of the test substance to elicit DNA repair in primary cultures of rat hepatocytes, over the wide range of concentrations tested, suggests an apparent lack of genotoxic activity under the conditions of the present assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Study Type	Species	Findings	Guideline	Reliability
Micronucleus Test	Mouse	Negative	OECD 474, EU Method B.12, EPA OPPTS 870.5395, Japan Ministry of Agriculture, Forestry and Fisheries Testing Guidelines for Toxicology Studies, 12-Nousan-8147	1
Micronucleus Test	Mouse	Negative	No guideline	2
Micronucleus Test	Mouse	Negative	EPA OPP 84-2	1

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Specific details on test material used for the study:

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

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

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

Dose / conc.:

7.5 mg/kg bw/day

Dose / conc.:

15 mg/kg bw/day

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

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.

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.

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)

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.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Section 158.135

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Lot AGR 214637
Purity: 97.9±0.9%

Species:

mouse

Strain:

other: CD-l (ICR) BR

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

corn oil

Duration of treatment / exposure:

1 day

Frequency of treatment:

once

Post exposure period:

72 hours

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

90 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide

Tissues and cell types examined:

Bone marrow samples were obtained from both femurs. One thousand polychromatic erythrocytes (PCE) were evaluated from each animal and frequencies of micronucleated polychromatic erythrocytes were recorded.

Evaluation criteria:

The slides were coded and scored blindly. Micronuclei were identified as darkly stained bodies with sharp contours and varying shapes such as round, almond, or ring. The ratio of PCE-NCE in the bone marrow was determined by examining 1000 erythrocytes. The ratio was expressed as PCExl00/PCE+NCE.

Statistics:

The raw data on the counts of MN-PCE for each animal were first transformed by adding 1 to each count and then taking natural log of the adjusted number. The transformed MN-PCE data and the data on percent PCE were analyzed by a three-way analysis of variance (sex, dose, and time), assuming the three-way interaction to be zero. From this initial analysis, the two-way interactions were reviewed for significance. Depending upon this review, the data were analyzed by either one-; two-,or three-way analysis of variance looking only at main effects. Pairwise comparisons of treated vs. negative control groups were done, if necessary, by a t-test using Bonferroni correction for multiple comparisons. The alpha level at which all the tests were conducted was 0.01.

Key result

Sex:

male/female

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Negative in the mouse bone marrow micronucleus test

Executive summary:

Chlorpyrifos was evaluated in the mouse bone marrow micronucleus test. The micronucleus test is capable of detecting agents causing chromosomal aberrations and spindle malfunction. The test material was mixed with corn oil and administered to CD-1 (ICR) BR mice by single oral gavage at dose levels of 0 (negative control), and 90 mg/kg body weight (b.w.). Groups of animals were sacrificed at three intervals, viz., 24, 48, and 72 h after treatment. Mice treated with 120 mg/kg b.w. cyclophosphamide and sacrificed at 24 h served as positive controls. There were five animals per sex per dose level per sacrifice time. One thousand polychromatic erythrocytes (PCE) were evaluated from each animal and the frequencies of micronucleated polychromatic erythrocytes (MN-PCE) were recorded. There were no significant increases in the frequencies of MN-PCE in groups treated with the test chemical compared to negative controls. The positive control mice showed significant increases in MN-PCE. Hence, under the experimental conditions used, the test chemical was considered negative in the mouse bone marrow micronucleus test.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Chlorpyrifos was-evaluated-in the mouse bone marrow micronucleus test. The micronucleus test is capable of detecting agents causing chromosomal aberrations and spindle malfunction. Chlorpyrifos was administered to CD®-l (ICR) BR mice by single oral gavage at dose levels of a (negative control), 7, 22, and 70 mg/kg body weight (b.w.). Chlorpyrifos was dissolved in corn oil for dosing the animals. Groups of animals were sacrificed at two intervals, viz., 24 and 48 h, after treatment. Animals treated with 120 mg/kg b.w. cyclophosphamide and sacrificed at 24 h served as positive controls. There were five animals per sex per dose level per sacrifice time. One thousand polychromatic erythrocytes (PCE) were evaluated from each animal and frequencies of micronucleated polychromatic erythrocytes were recorded. The data were analyzed by contingency table methods and Chi-square statistics.

GLP compliance:

no

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

AGR214637, Lot No. MM 820905-610
Purity: 95.7%

Species:

mouse

Strain:

other: CD®-l (ICR) BR

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

corn oil

Duration of treatment / exposure:

1 Day

Frequency of treatment:

once

Post exposure period:

48 hours

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

7 mg/kg bw/day (nominal)

Dose / conc.:

22 mg/kg bw/day (nominal)

Dose / conc.:

70 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide

Tissues and cell types examined:

Bone marrow samples were obtained from both femurs. One thousand polychromatic erythrocytes (PCE) were evaluated from each animal and frequencies of micronucleated polychromatic erythrocytes were recorded.

Statistics:

The data were analyzed by contingency table methods and Chi-square statistics.

Key result

Sex:

male/female

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Negative in the mouse bone marrow micronucleus test

Executive summary:

Chlorpyrifos was-evaluated-in the mouse bone marrow micronucleus test. The micronucleus test is capable of detecting agents causing chromosomal aberrations and spindle malfunction. Chlorpyrifos was administered to CD®-l (ICR) BR mice by single oral gavage at dose levels of a (negative control), 7, 22, and 70 mg/kg body weight (b.w.). Chlorpyrifos was dissolved in corn oil for dosing the animals. Groups of animals were sacrificed at two intervals, viz., 24 and 48 h, after treatment. Animals treated with 120 mg/kg b.w. cyclophosphamide and sacrificed at 24 h served as positive controls. There were five animals per sex per dose level per sacrifice time. One thousand polychromatic erythrocytes (PCE) were evaluated from each animal and frequencies of micronucleated polychromatic erythrocytes were recorded. The data were analyzed by contingency table methods and Chi-square statistics. There were no significant increases in the frequencies of micronucleated bone marrow polychromatic erythrocytes in groups treated with chlorpyrifos compared to negative controls. The positive control mice showed significant increases in micronucleated polychromatic erythrocytes. Hence, under the experimental conditions used, chlorpyrifos was considered negative in the mouse bone marrow micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In a bacterial reverse mutation test, Salmonella typhimurium (strains: TA1537, TA1535, TA98, TA100 and TA102) were exposed with Chlorpyrifos at 6 concentrations (three plates/concentration) between 51.2 and 5000 μg/plate. From the results of this study, under the specified experimental conditions with and without S9 mix, Chlorpyrifos is concluded to be non-mutagenic.  

 

In a mammalian chromosome aberration test, human peripheral blood lymphocytes cultured in vitro were exposed to Chlorpyrifos at different concentrations both in the absence and presence of metabolic activation. Human peripheral blood lymphocyte cultures were exposed to Chlorpyrifos 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. Chlorpyrifos TGAI 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. From the results of this study, it is concluded that Chlorpyrifos 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.  

 

Chlorpyrifos was evaluated in the Chinese hamster ovary cell-hypoxanthine (guanine) phosphoribosyl transferase (CHO/HGPRT) mutagenicity assay. The studies were conducted with and without a metabolic activation. Chlorpyrifos was tested at 10, 20, 30, 40 & 50µM and was not mutagenic for Chinese hamster ovary cells indicating a lack of genotoxic activity under the conditions of the present study.  

 

Another study was performed to evaluate the micronucleus induction potential of Chlorpyrifos in mice. Chlorpyrifos was suspended in corn oil and administered orally at doses of 7.5, 15 and 30 mg for two consecutive days. 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 Chlorpyrifos up to the dose level of 30 mg/kg. From the results the study, it is concluded that Chlorpyrifos does not have micronucleus induction potential in the animals treated up to 30 mg/kg.  

 

In summary, all in vitro and in vivo studies conducted on Chlorpyrifos were negative for mutagenicity. 

Justification for classification or non-classification

The test substance was negative for mutagenicity and clastogenicity in vitro in bacterial and mammalian cells, respectively. Additionally, the test substance was negative when evaluated in vivo in laboratory animals. Based on an assessment of the robust genetic toxicity data for this substance, the substance does not need to be classified for mutagenicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.