Methomyl

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. typhimuriumTA100, TA1535, TA97a, and TA98; E. coli WP2uvrA pKM 101	Negative	OECD 471 and 472, EU Method B.13/1, EPA OPP 84-2, Japan Ministry of Agriculture, Forestry and Fisheries Testing Guidelines for Toxicology Studies, 59 NohSan No. 4200	1
Chromosome Aberration test	human lymphocytes	Negative	OECD 473, EU Method B.10, EPA OPPTS 870.5375, MAFF Japan 59 Nousan Number 4200 Agriculture Chemicals Laws and Regulations (1985)	1
Mammalian cell gene mutation test	Chinese Hamster ovary cells	Negative	no guideline followed	2
Unscheduled DNA Synthesis (UDS) assay	rat hepatocytes	Negative	no guideline followed	2

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

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:

EPA OPP 84-2

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

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: Japan Ministry of Agriculture, Forestry and Fisheries Testing Guidelines for Toxicology Studies, 59 NohSan No. 4200

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Substance name: Methomyl Technical
- Substance ID: DPX-X1179
- Lot#: X1179-394
- Purity: 98.35%

Target gene:

his, trp

Species / strain / cell type:

S. typhimurium, other: TA100, TA1535, TA97a, and TA98

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Male SD rat liver homogenate activation system (S9 Mix)

Test concentrations with justification for top dose:

Trial I: 10, 50, 100, 250, 500, 1000, 2500, and 5000 μg/plate for TA100 and WP2 uvrA (pKM101)
10, 50, 100, 500, 1000, 2500, and 5000 μg/plate for TA97a, TA98, and TA1535
Highest concentration is the guideline limit dose for this test system
Trial II: 10, 50, 100, 500, 1000, 2500, and 5000 μg/plate for all strains

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance appeared completely soluble at 50 mg/mL producing a solution immediately upon mixing. Concentrations were calculated with the assumption that addition of the test substance to the solvent did not change the volume of the resulting solution.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene, ICR 191 Acridine

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)
- Cell density at seeding: At least 1E+8 bacteria

DURATION
- Exposure duration: 48 hours
- Expression time (cells in growth medium): Overnight culture

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in the background lawn

Evaluation criteria:

A test substance was classified as positive when: (1) the average number of revertants in any strain at any test substance concentration studied was at least two times greater than the average number of revertants in the negative control; and (2) there was a positive doseresponse relationship in that same strain.

A test substance was classified as negative when either: (1) there were no test substance concentrations with an average number of revertants which was at least two times greater than the average number of revertants in the negative control; and (2) there was no positive doseresponse relationship.

Results not meeting these criteria for positive or negative assessments were evaluated using scientific judgment.

Statistics:

Data for each tester strain were evaluated independently. For each tester strain, the average number of revertants and the standard deviation at each concentration with and without S9 activation were calculated.

Key result

Species / strain:

S. typhimurium, other: TA100, TA1535, TA97a, and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Table-1: Mutagenic activity of the test substance in strain TA100 in Trail 2

Without activation
Concentration of test substance (µg/plate)	Revertants			Average	SD
	Plate 1	Plate 2	Plate 3
0.0	99	104	107	103	4
10.0	86	96	113	98	14
50.0	108	118	113	113	5
100.0	110	107	92	103	10
500.0	94	92	99	95	4
1000.0	92	105	99	99	7
2500.0	118	91	104	104	14
5000.0	103	86	84	91	10
Sodium azide (2 µg/plate)	891	985	994	957	57
With activation
0.0	93	99	107	100	7
10.0	95	115	107	106	10
50.0	108	108	93	103	9
100.0	97	101	97	98	2
500.0	103	95	99	99	4
1000.0	107	111	93	104	9
2500.0	89	92	101	94	6
5000.0	98	80	91	90	9
2AA (1 µg/plate)	984	920	902	935	43

Table-2: Mutagenic activity of the test substance in strain TA1535 in Trail 2

Without activation
Concentration of test substance (µg/plate)	Revertants			Average	SD
	Plate 1	Plate 2	Plate 3
0.0	21	18	23	21	3
10.0	17	20	14	17	3
50.0	18	13	11	14	4
100.0	14	22	15	17	4
500.0	13	12	15	13	2
1000.0	13	19	19	17	3
2500.0	19	13	22	18	5
5000.0	17	16	17	17	1
Sodium azide (2 µg/plate)	673	799	793	755	71
With activation
0.0	17	18	17	17	1
10.0	19	12	14	15	4
50.0	14	15	15	15	1
100.0	18	14	10	14	4
500.0	12	17	13	14	3
1000.0	14	11	17	14	3
2500.0	19	14	16	16	3
5000.0	17	15	11	14	3
2AA (1 µg/plate)	314	332	328	325	9

Table-3: Mutagenic activity of the test substance in strain TA97a in Trail 2

Without activation
Concentration of test substance (µg/plate)	Revertants			Average	SD
	Plate 1	Plate 2	Plate 3
0.0	125	131	138	131	7
10.0	121	121	125	122	2
50.0	143	135	128	135	8
100.0	132	125	124	127	4
500.0	119	120	112	117	4
1000.0	116	133	137	129	11
2500.0	125	118	117	120	4
5000.0	118	114	126	119	6
ICR 191 (2 µg/plate)	1912	1745	1914	1857	97
With activation
0.0	140	124	137	134	9
10.0	121	127	125	124	3
50.0	127	123	126	125	2
100.0	133	131	130	131	2
500.0	133	136	143	137	5
1000.0	120	137	129	129	9
2500.0	132	135	126	131	5
5000.0	108	106	101	105	4
2AA (1 µg/plate)	1057	1050	1048	1052	5

Table-4: Mutagenic activity of the test substance in strain TA98 in Trail 2

Without activation
Concentration of test substance (µg/plate)	Revertants			Average	SD
	Plate 1	Plate 2	Plate 3
0.0	29	29	28	29	1
10.0	25	28	21	25	4
50.0	22	23	26	24	2
100.0	31	26	36	31	5
500.0	32	34	35	34	2
1000.0	27	21	26	25	3
2500.0	40	33	27	33	7
5000.0	29	28	38	32	6
2NF (25 µg/plate)	1327	1570	1439	1445	122
With activation
0.0	34	31	35	33	2
10.0	39	36	38	38	2
50.0	41	37	41	40	2
100.0	44	37	43	41	4
500.0	44	39	44	42	3
1000.0	34	36	31	34	3
2500.0	36	41	27	35	7
5000.0	34	28	37	33	5
2AA (2 µg/plate)	1838	1590	1789	1739	131

Table-5: Mutagenic activity of the test substance in strain WP2 uvrA (PKM101) in Trail 2

Without activation
Concentration of test substance (µg/plate)	Revertants			Average	SD
	Plate 1	Plate 2	Plate 3
0.0	192	189	178	186	7
10.0	204	182	190	192	11
50.0	182	188	187	186	3
100.0	210	187	190	196	13
500.0	218	205	190	204	14
1000.0	202	188	185	192	9
2500.0	203	213	205	207	5
5000.0	217	204	194	205	12
MMS (1000 µg/plate)	1985	1834	2183	2001	175
With activation
0.0	205	175	187	189	15
10.0	165	208	208	194	25
50.0	206	194	186	195	10
100.0	213	179	197	196	17
500.0	200	189	206	198	9
1000.0	192	196	195	194	2
2500.0	201	201	208	203	4
5000.0	196	171	194	187	14
2AA (25 µg/plate)	1878	1907	2141	1975	144

Conclusions:

Negative in Salmonella typhimurium strains and in Escherichia coli strain with and without an exogenous metabolic activation system (S9)

Executive summary:

The test substance, was evaluated for mutagenicity in Salmonella typhimurium strains TA100, TA1535, TA97a, and TA98 and in Escherichia coli strain WP2 uvrA (pKM101) with and without an exogenous metabolic activation system (S9). The study was conducted according to OECD guidelines 471 and 472, U.S. EPA 84-2.

Concentrations of 10, 50, 100, 250, 500, 1000, 2500, and 5000 μg/plate were initially evaluated using Salmonella typhimurium strain TA100 and Escherichia coli strain WP2 uvrA (pKM101) with and without an exogenous metabolic activation system (S9). In the absence of any notable bacterial toxicity or test substance precipitation, concentrations of 10, 50, 100, 500, 1000, 2500, and 5000 μg/plate were subsequently tested in Salmonella typhimurium strains TA97a, TA98, and TA1535 to complete the first trial.

In a second confirmatory trial, concentrations of 10, 50, 100, 500, 1000, 2500, and 5000 μg/plate were tested in comparison to negative (solvent) controls in all strains.

Under the conditions of this study, no evidence of mutagenic activity was detected in either of two independent trials. The test substance was negative.

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:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (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: MAFF Japan 59 Nousan Number 4200 Agriculture Chemicals Laws and Regulations (1985)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Substance name: Methomyl Technical
- Substance ID: DPX-X1179
- Lot#: X1179-512
- Purity: 98.8%

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Whole venous blood from healthy donor
- Age of blood donors: Less than 50 years old

MEDIA USED
RPMI 1640 medium supplemented with approximately 15% fetal bovine serum, 2 mM L-glutamine, 100 units penicillin/mL, 100 µg streptomycin/mL, and 1% phytohemagglutinin-M

Cytokinesis block (if used):

0.1 µg/mL Colcemid®

Metabolic activation:

with and without

Metabolic activation system:

Liver homogenate (S9) prepared from male SD rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Preliminary toxicity assay: 20.3, 40.6, 81.1, 162.2, 324.4, 648.8, 973.2, 1297.6, 1622 µg/mL (highest dose is the guideline limit dose for this test system)
Chromosome aberration assay: 9, 28, 83, 250, 300 µg/mL for the 4-hour non-activated and activated testing conditions; 9, 28, 83, 250, 500 µg/mL for the 20-hour non-activated testing condition

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was determined to be the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed an clear solution in DMSO at 500 mg/mL, the highest concentration prepared on the solubility test.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: 4 and 20 hours
- Expression time (cells in growth medium): Approximately 22 hours from the initiation of treatment
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR: Colcemid®

STAIN: Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cells were treated with 0.075 M KCl hypotonic buffer, fixed at least twice in methanol:glacial acetic acid (3:1 v/v), and stored refrigerated or frozen overnight or longer. To prepare slides, the cells were collected by centrifugation and resuspended in fresh fixative. At least two slides per culture were prepared by applying an aliquot of the fixed cells onto clean microscope slides and air-drying them. The slides were stained by Giemsa and permanently mounted.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: At least 1000 cells per concentration (at least 500 from each duplicate culture)

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index

OTHER EXAMINATIONS:
a structural aberration occurring in a polyploid cell was included in the analyses. At least 200 metaphases per concentration level (100 from each duplicate culture), when available, was analyzed for structural aberrations. Numerical aberrations were recorded as well. The number of metaphases evaluated per duplicate flask may be less if the percentage of aberrant cells reached statistical significance when 25 cells were scored. Chromatid-type aberrations included chromatid and isochromatid breaks and exchange figures. Chromosome type aberrations included chromosome breaks and exchange figures. Pulverized chromosome(s) and cells, and severely damaged cells (i.e., cells with 10 aberrations per cell) were recorded, but not included in the analyses. The XY coordinates for the microscope stage were recorded for cells with structural or numerical aberrations.

Evaluation criteria:

The following conditions were used as a guide to determine a positive response:
A statistically significant increase (p ≤0.05, Fisher's exact test) in the percentage of cells with structural aberrations was seen in one or more treatment groups relative to the vehicle control response. The observed increased frequencies were accompanied by a concentration-related increase. A statistically significant increase was observed at the highest dose only.
Statistically significant values that did not exceed the historical control range for the negative/vehicle control may be judged as not being biologically significant.

The following condition was used as a guide to determine an equivocal response:
Results observed in any of the assays resulted in statistically significant elevations in structural chromosome aberrations at more than one test concentration level, except the highest dose, without demonstrating a dose-responsive trend.

The test substance was judged negative if the following condition was met:
There was no statistically significant increase in the percentage of cells with structural aberrations in any treatment group relative to the vehicle control group.

Statistics:

Data was evaluated using scientific judgment. Statistical analysis was used as a guide to determine whether or not the test substance induced a positive response. Interpretation of the statistical analysis also relied on additional considerations including the magnitude of the observed test substance response relative to the vehicle control response and the presence of a dose responsive trend. Statistical analysis consisted of a Cochran-Armitage test for dose responsiveness and a Fisher's exact test to compare the percentage of cells with structural aberrations (or the percentage of cells with more than one aberration, if required) in the test substance treated groups with the vehicle control response.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Substantial toxicity (at least 50% reduction in mitotic index relative to the solvent control) was observed at ≥250 µg/mL in the 4-hour assay without S9, at 300 µg/mL in the 4-hour assay with S9 and at 500 µg/mL in the 20-hour assay without S9

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Based on visual inspection, the pH of the highest test substance concentration in media was not significantly different than the vehicle control (in both the assays)
- Effects of osmolality: The osmolality in treatment medium of the highest concentration tested, 1622 µg/mL, was 433 and 419 mmol/kg in the non-activated and activated testing condition, respectively. The osmolality of the vehicle (DMSO) in the treatment medium was 416 and 447 mmol/kg in the non-activated and activated testing condition, respectively. The observed increases in osmolality were 20% and therefore considered insignificant. Osmolality was not measured in the chromosome aberration assay.
- Precipitation: No visible precipitate was observed in the treatment medium at the beginning or end of the treatment periods in either the preliminary toxicity or chromosome aberration assays.
- Cytotoxicity in preliminary assay: Substantial toxicity (at least a 50% reduction in mitotic index relative to the solvent control) was observed at 324.4 and ≥973.2 µg/mL in the 4-hour non-activated testing Condition, at ≥162.2 µg/mL in the 4-hour S9 activated testing condition, and at ≥324.4 µg/mL in the 20-hour non-activated testing condition.

Table: Chromosome aberration assay summary

Treatment^a(µg/mL)	S9 activation	Treatment time	Mitotic index (%)	Cells scored		Structural aberrations per cell^b		Cells with aberrations^b
				Numerical	Structural	Mean	SD	Numerical (%)	Structural (%)
Vehicle^c	-S9	4	8.6	200	200	0.010	0.000	0.0	1.00
28	-S9	4	6.6	200	200	0.010	0.014	0.0	1.00
83	-S9	4	5.5	200	200	0.000	0.000	0.0	0.00
250^d	-S9	4	4.1	200	200	0.000	0.000	0.0	0.00
MMC 0.3	-S9	4	2.5	50	50	0.540	0.028	0.0	40.0^e
Vehicle^c	+S9	4	9.9	200	200	0.000	0.000	0.0	0.00
28	+S9	4	8.1	200	200	0.015	0.007	0.0	1.50
83	+S9	4	6.5	200	200	0.010	0.000	0.0	1.00
300^f	+S9	4	4.7	200	200	0.005	0.007	0.0	0.50
CP 10	+S9	4	2.9	50	50	0.660	0.028	0.0	44.0^e
Vehicle^c	-S9	20	10.0	200	200	0.005	0.007	0.0	0.50
83	-S9	20	6.3	200	200	0.005	0.007	0.0	0.50
250	-S9	20	5.9	200	200	0.000	0.000	0.0	0.00
500^g	-S9	20	4.6	200	200	0.015	0.007	0.0	1.00
MMC 0.3	-S9	20	3.1	50	50	0.900	0.141	0.0	54.0^e

a Human peripheral blood lymphocyte (HPBL) cells treated at 37°C

b Excluding cells with only gaps

c 1% DMSO

d Equivalent to a 1.54 mM concentration

e Statistically significant difference from control at p ≤0.05 by Fisher's exact test

f Equivalent to a 1.85 mM concentration

g Equivalent to a 3.08 mM concentration

Conclusions:

Negative for the induction of structural or numerical chromosome aberrations in the in vitro mammalian chromosome aberration assay in HPBL in the non-activated or S9 activated testing conditions

Executive summary:

The test substance was evaluated for its ability to induce structural chromosome aberrations in vitro using human peripheral blood lymphocytes (HPBL) in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9). The study was conducted following OECD guideline 473 and U.S. EPA OPPTS 870.5375. To establish a concentration range for the chromosome assay, a preliminary toxicity test was initially conducted.

The test substance was prepared in dimethyl sulfoxide (DMSO) as this vehicle was determined to be the solvent of choice based on solubility of the test substance and compatibility of the target cells. The test substance formed a clear solution in the vehicle at 500 mg/mL.

Aliquots of the vehicle control and a total of four test substance concentrations were taken to confirm dose concentrations and stability in the chromosome aberration assay. Target concentrations were verified, and the test substance was stable for the duration of the dosing period.

The maximum concentration tested in the preliminary toxicity assay was 1622 µg/mL (10 mM), the guideline limit dose for this test system. The test substance formed a clear solution in DMSO at 162.20 mg/mL, the highest stock concentration used. At concentrations ≤1622 µg/mL, no visible precipitate was observed in the treatment medium. The pH and osmolality of the highest test substance concentration in media was not significantly different from the vehicle control either in the absence or presence of S9.

In the preliminary toxicity assay, HPBL cells were treated for 4 and 20 hours in the non-activated testing conditions, and for 4 hours in the S9 activated testing condition. The cells were exposed to nine concentrations of the test substance ranging from 20.3 to 1622 µg/mL (10 mM), as well as vehicle controls. The test substance concentrations for the chromosome aberration assay were selected based on an assessment of the potential reduction in the mitotic index in the treated cultures relative to the vehicle control. Substantial toxicity (at least a reduction in mitotic index relative to the solvent control) was observed at 324.4 and ≥973.2 µg/mL in the 4-hour non-activated testing condition, at ≥162.2 µg/mL in the 4-hour S9 activated testing condition, and at ≥324.4 µg/mL in the 20-hour non-activated testing condition. Based on these findings, the concentrations chosen for the chromosome aberration assay ranged from 9 to 300 µg/mL for the 4-hour non-activated and S9 activated testing conditions, and from 9 to 500 µg/mL for the 20 -hour non-activated testing condition.

In the chromosome aberration assay, the HPBL cells were treated for 4 and 20 hours in the non-activated testing condition and for 4 hours in the S9 activated testing condition. The cells were harvested 22 hours after initiation of the treatment. The test substance was soluble in DMSO at all concentrations tested. No visible precipitate was observed in the treatment medium at the beginning or end of the treatment period at any concentration in any testing condition. Substantial toxicity (at least a 50% reduction in mitotic index relative to the solvent control) was observed at ≥250 µg/mL in the 4-hour non-activated testing condition, at 300 µg/mL in the 4-hour S9 activated testing condition, and at 500 µg/mL in the 20-hour non-activated testing condition. The selection of concentration levels for microscopic analysis was based on these data.

Cytogenetic evaluations were conducted at 28, 83, and 250 µg/mL (1.54 mM) for the 4-hour non-activated testing condition, at 28, 83, and 300 µg/mL (1.85 mM) for the 4-hour S9 activated testing condition, and at 83, 250, and 500 µg/mL (3.08 mM) for the 20-hour non-activated testing condition.

The percentage of cells with structural or numerical aberrations in the test substance-treated groups was not significantly increased above that of the solvent control at any concentration (p >0.05, Fisher's exact test).

All criteria for a valid study were met. Under the conditions of this study, the test substance did not induce structural or numerical chromosome aberrations in the in vitro mammalian chromosome aberration test in human peripheral blood lymphocytes in the non-activated or S9 activated testing conditions. The test substance was concluded as negative in this in vitro test.

Reference 3

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

The Chinese Hamster Ovary (CHO) cell line was used to detect mutation in a gene coding for the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT). The test substance was tested for mutagenic activity in both the presence and absence of metabolic activation system. The concentrations of the test substance used were 0, 10, 20, 40, 50, 55 mM (without activation) and 0, 100, 150, 200, 250, 350 mM (with activation). After the incubation period, the colonies were stained and counted. The mutant frequency was expressed as the number of mutant colonies per 1E+6 surviving cells at the time of mutant selection.

GLP compliance:

no

Type of assay:

other: in vitro mammalian cell gene mutation test using the Hprt genes

Specific details on test material used for the study:

- Substance name: Ethanimidothioic acid, N-[[(methylamino)carbonyl]oxy]-, methyl ester
- Substance ID: INX-1179-255
- Purity: ~99%

Target gene:

hgprt

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
BH4 clone of the CHO-K1 cell line

MEDIA USED
Ham's F12 medium without hypoxanthine containing 5% dialyzed heat-inactivated fetal bovine serum without antibiotics. In cytotoxicity and mutagenicity experiments, penicillin (50 units/mL) and streptomycin (50 µg/mL) were included in the culture medium. Culture dishes were incubated at 37 ± 1.5°C in an atmosphere containing 5 ± 1% CO2 and 90 ± 6% relative humidity. Prior to subculturing, the cells were removed from the dishes with 0.05% trypsin. The cell line used has been shown to be free of mycoplasma.

Metabolic activation:

with and without

Metabolic activation system:

Liver homogenate obtained from male CD rats induced with Aroclor® 1254

Test concentrations with justification for top dose:

0, 10, 20, 40, 50, 55 mM (2 trials without activation) and 0, 100, 150, 200, 250, 350 µM (2 trials with activation)

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: 9,10-dimethyl-1,2-benzanthracene (DMBA)

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity observed at ≥40.0 mM (without activation) and at ≥200 µM (wtih activation)

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test substance did not exhibit mutagenic activity in CHO/HGPRT assay with or without metabolic activation

Executive summary:

The Chinese Hamster Ovary (CHO) cell line was used to detect mutation in a gene coding for the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT). The test substance was tested for mutagenic activity in both the presence and absence of metabolic activation system. The concentrations of the test substance used were 0, 10, 20, 40, 50, 55 mM (without activation) and 0, 100, 150, 200, 250, 350 mM (with activation).

The solubility assessment for the test substance indicated that DMSO was the most appropriate solvent to use for the cytotoxicity and mutagenicity assays. The solvent, test substance and positive control solutions were added to the treatment medium in a volume of 30 µL.

Two mutagenicity trials were performed without activation and cytotoxicity was observed at concentrations of 40.0 mM and above. The statistical analysis of these trials indicated that none of the concentrations of the test substance tested caused a significant increase in the mutant frequency over that observed in the solvent control and there was not a significance positive linear dose response.

Two trials with activation were performed and cytotoxicity was evident at 200 µM and above. The statistical analysis of these trials revealed no significant increases in the mutant frequencies in the treated cultures and there was not a significant positive linear dose response.

Under the conditions of the assay, the test substance was not mutagenic in CHO cells either in the presence or absence of an S9 activation system.

Reference 4

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:

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

The purpose of the study was to determine whether the test substance induces unscheduled DNA synthesis (UDS) in freshly isolated hepatocytes from the livers of rats. The concentrations of the test substance tested ranged from 1 µM to 75 mM in the treatment medium. 20 µL of the test substance stock solution or positive control were added to each culture. The cultures were then incubated for 18 hours. Slides were prepared and the cells were scored for UDS.

GLP compliance:

no

Type of assay:

other: UDS assay

Specific details on test material used for the study:

- Substance name: Ethanimidothioic acid, N-[[(methylamino)carbonyl]oxy]-, methyl ester
- Substance ID: INX-1179-255
- Purity: 99%

Species / strain / cell type:

hepatocytes: rat

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Hepatocytes from Crl:CD® rat (Charles River, Kingston, New York)
- Sex, age: 8 week old male rats

Hepatocyte Primary Culture
Culture plates (35 mm i.d. wells, 6 wells per plate) containing 2 mL WMES and a round (25 mm dia.) coverslip were inoculated with 5E+5 viable hepatocytes per well. The cells were allowed to attach to the coverslips for 2 hours in an incubator (5 ± 1% CO2, 37 ± 1.5°C, 90+% humidity). One plate was prepared for each treatment level.

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

0, 1, 10, 100, 1000, 5000, 10000, 75000 µM

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 9,10-dimethyl-1,2-benzanthracene (DMBA)

Key result

Species / strain:

hepatocytes: rat

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Negative in UDS assay in primary rat hepatocytes

Executive summary:

The test substance was tested for its ability to induce unscheduled DNA synthesis (UDS) in primary rat hepatocyte cultures. The concentrations of the test substance tested ranged from 1 µM to 75 mM in the treatment medium. 20 µL of the test substance stock solution or positive control were added to each culture. The cultures were then incubated for 18 hours. Slides were prepared and the cells were scored for UDS.

The results do not show UDS at any concentration of the test substance and statistical analyses indicate neither a compound related effects, nor a dose-response relationship. Thus, the test substance was negative in the UDS 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 OPP 84-2, Japan Ministry of Agriculture, Forestry and Fisheries Testing Guidelines for Toxicology Studies, 59 NohSan No. 4200	1
Chromosome Aberration Test	Rat	Negative	no guideline followed	2

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:

EPA OPP 84-2

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:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japan Ministry of Agriculture, Forestry and Fisheries Testing Guidelines for Toxicology Studies, 59 NohSan No. 4200

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

- Substance name: Methomyl technical
- Substance ID: DPX-X1179
- Lot#: X1179-394
- Purity: 98.35%

Species:

mouse

Strain:

other: Crl:CD®-1(ICR)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Inc., Raleigh, North Carolina
- Age at study initiation: 62 days
- Weight at study initiation: 29.1-36.0 g (males); 21.5-27.3 g (females)
- Assigned to test groups randomly: Yes
- Housing: Standard wire mesh cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 50 ± 10
- Photoperiod: 12-hour light/dark cycle

Route of administration:

oral: gavage

Vehicle:

Sterile water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Solutions of the test substance in the vehicle were prepared at concentrations of 0.3, 0.6, and 1.2 mg/mL. Uniformity was maintained during dosing by constant stirring. Single acute doses of the appropriate solutions were administered by oral intubation at 10 mL/kg yielding treatments of 3, 6, and 12 mg/kg bw.
The negative control (vehicle) was administered by gavage in a volume of 10 mL/kg.

Duration of treatment / exposure:

Single oral dose

Frequency of treatment:

Single oral dose

Dose / conc.:

3 mg/kg bw (total dose)

Dose / conc.:

6 mg/kg bw (total dose)

Dose / conc.:

12 mg/kg bw (total dose)

No. of animals per sex per dose:

5 (solvent control; 3, 6 mg/kg; positive control) and 6 (12 mg/kg)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CP)
- Route of administration: Oral intubation
- Doses / concentrations: 40 mg/kg

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on the results from the range finding study, a dose of 12 mg/kg was selected as the maximum dose for both male and female mice

SAMPLING TIMES: 24, 48, and 72 hours

DETAILS OF SLIDE PREPARATION: Immediately after sacrifice, marrow from both femurs of each animal was aspirated and flushed into approximately 2 mL prewarmed (37°C) fetal bovine serum. The marrow was collected by centrifugation. Most of the supernatant was removed, and the cells were resuspended in the remaining 1-2 drops of serum. A Miniprep® automatic blood smearing instrument was used to prepare marrow smears. At least 3 slides per animal were prepared and fixed in absolute methanol for 8 mins. Slides were stained for 2.5 mins in 0.0125 mg/mL acridine orange in phosphate buffer (pH 7.4). Prior to scoring, a coverslip was floated on each slide using phosphate buffer.

METHOD OF ANALYSIS: Representative slides from each animal were examined blindly using incident light fluorescence microscopy. Only cells with good morphology and staining were scored. Colour was used to distinguish PCEs (reddish) from NCEs (dark green). PCEs (2000 per animal) were scored for the presence of micronuclei (round, bright yellow-green fluorescing bodies). Cellular inclusions that were irregularly shaped or stained, or out of the focal plane of the cell were considered artifacts. The unit of scoring was the micronucleated cell; PCEs with more than one micronucleus were scored as a single micronucleated PCE (MNPCE). Micronucleated NCEs seen in the optic fields scored to obtain 2000 PCEs were also counted. Additionally, the number of PCEs among 1000 erythrocytes was recorded for each animal.

Statistics:

Data for the proportion of MNPCEs among 2000 PCEs and the proportion of PCEs among 1000 erythrocytes were transformed prior to analysis using the arcsin square root function. Transformed data for PCE or MNPCE frequencies were analyzed separately for normality of distribution using the Shapiro-Wilkes test. If results indicated that the transformed values for PCE or MNPCE frequencies were normally distributed in both sexes, parametric methods (viz., Analysis of Variance and Dunnett test) were used. If there was nonnormality in either sex, nonparametric methods (viz., Krushkal-Wallis test and Mann-Whitney U tests) were used for that variable using nontransformed proportions. Positive indicator data were not included in evaluating normality of distribution. Weight gain data were assumed to be normally distributed and were analyzed by ANOVA. Data from each sex and sacrifice time were analyzed separately, and individual comparisons to the control were made using each animal as the experimental unit. All analyses were conducted at a significance level of 5%. Positive indicator data were analyzed separately.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

1 female mouse at 12 mg/kg was found dead ~24 h after dosing. Hyperactivity was displayed by 1 male at same dose. Lethargy was seen at 6 mg/kg in 3 males and at 12 mg/kg in 1 male and 1 female mice. One of these mice had half shut eyes 24 h post-dosing.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY FOR DOSE SELECTION
- Dose range: 5 to 40 mg/kg
- Clinical signs of toxicity in test animals: Deaths occurred at 15 mg/kg (1/4 males, 0/4 females), 20 mg/kg (3/5 males, 2/6 females), 25 mg/kg (2/2 females), 30 mg/kg (4/5 males, 7/7 females), and 40 mg/kg (3/3 males, 3/3 females). Animals dosed at 20, 25, 30, and 40 mg/kg exhibited tremors, convulsions, and/or gasping within 30 minutes post-exposure. These symptoms lasted up to approximately seven minutes until the animals either died or recovered. Surviving animals then became lethargic and quiet. By 3 hours post-dosing, no signs of toxicity were observed. At 15 mg/kg, the surviving mice were lethargic and had shallow respiration immediately following dosing which subsided within the following hour. No deaths or clinical signs were observed at 5 or 10 mg/kg.

Table-1: PCE Frequency

Test substance (mg/kg)	Sampling time (hrs)	Sex	N	Mean %PCE (95% conf. limits) (determined on transformed values)	Mean PCE/NCE ratio (SEM)
0	24	M	5	56.3 (44.0, 68.2)	1.38 (0.24)
0	24	F	5	56.4 (48.1, 64.6)	1.35 (0.20)
3	24	M	5	61.2 (55.9, 66.3)	1.60 (0.12)
3	24	F	5	59.4 (47.3, 70.8)	1.58 (0.33)
6	24	M	5	57.4 (43.2, 70.9)	1.51 (0.41)
6	24	F	5	52.1 (48.1, 56.2)	1.10 (0.06)
12	24	M	6	45.0 (38.3, 51.7)	0.84 (0.08)
12	24	F	5^a	53.1 (48.7, 57.6)	1.14 (0.08)
0	48	M	5	47.1 (41.6, 52.6)	0.90 (0.07)
0	48	F	5	53.0 (45.8, 60.2)	1.16 (0.14)
3	48	M	5	47.3 (39.3, 55.3)	0.92 (0.11)
3	48	F	5	50.9 (39.2, 62.6)	1.10 (0.19)
6	48	M	5	51.4 (41.6, 61.1)	1.10 (0.15)
6	48	F	5	53.7 (47.4, 59.9)	1.18 (0.11)
12	48	M	6	49.5 (40.2, 58.9)	1.03 (0.15)
12	48	F	5^b	53.2 (49.9, 56.4)	1.14 (0.05)
0	72	M	5	52.8 (43.8, 61.6)	1.16 (0.17)
0	72	F	4^b	51.2 (43.6, 58.9)	1.07 (0.09)
3	72	M	5	43.9 (35.7, 52.2)	0.80 (0.10)
3	72	F	5	54.2 (45.7, 62.6)	1.22 (0.15)
6	72	M	5	45.4 (30.6, 60.6)	0.90 (0.18)
6	72	F	5	55.1 (45.4, 64.7)	1.28 (0.18)
12	72	M	6	43.5 (40.0, 47.0)	0.77 (0.04)
12	72	F	6	56.4 (52.2, 60.6)	1.31 (0.09)
CP, 40	24	M	5	51.5 (48.6, 54.5)	1.07 (0.05)
CP, 40	24	F	5	51.5 (42.9, 60.0)	1.10 (0.15)

a Mouse died prior to scheduled sacrifice time

b Mouse was misdosed and died within 24 hours of dosing

Table-2: MNPCE Frequency

Test substance (mg/kg)	Sampling time (hrs)	Sex	N	%MNPCEs
				Mean (SEM)	Median (IQR)
0	24	M	5	0.22 (0.06)	0.30 (0.25)
0	24	F	5	0.14 (0.03)	0.15 (0.12)
3	24	M	5	0.32 (0.07)	0.30 (0.25)
3	24	F	5	0.16 (0.08)	0.15 (0.32)
6	24	M	5	0.30 (0.05)	0.30 (0.20)
6	24	F	5	0.15 (0.02)	0.15 (0.10)
12	24	M	6	0.20 (0.04)	0.22 (0.14)
12	24	F	5^a	0.21 (0.06)	0.20 (0.18)
0	48	M	5	0.14 (0.04)	0.15 (0.18)
0	48	F	5	0.17 (0.03)	0.15 (0.10)
3	48	M	5	0.18 (0.05)	0.15 (0.18)
3	48	F	5	0.15 (0.06)	0.15 (0.25)
6	48	M	5	0.18 (0.05)	0.20 (0.20)
6	48	F	5	0.14 (0.05)	0.15 (0.22)
12	48	M	6	0.17 (0.04)	0.18 (0.19)
12	48	F	5^b	0.18 (0.06)	0.10 (0.25)
0	72	M	5	0.16 (0.06)	0.15 (0.22)
0	72	F	4^b	0.14 (0.04)	0.12 (0.16)
3	72	M	5	0.11 (0.03)	0.10 (0.12)
3	72	F	5	0.15 (0.03)	0.15 (0.10)
6	72	M	5	0.10 (0.03)	0.10 (0.10)
6	72	F	5	0.16 (0.04)	0.15 (0.18)
12	72	M	6	0.13 (0.02)	0.12 (0.11)
12	72	F	6	0.19 (0.04)	0.18 (0.15)
CP, 40	24	M	5	1.97 (0.52)	1.85 (2.05)*
CP, 40	24	F	5	2.07 (0.25)	1.90 (0.92)*

a Mouse died prior to scheduled sacrifice time

b Mouse was misdosed and died within 24 hours of dosing

* p ≤0.05

Conclusions:

Negative in mouse bone marrow micronucleus assay

Executive summary:

The test substance was evaluated for its ability to induce micronuclei in bone marrow polychromatic erythrocytes (PCEs) of mice. The study was conducted following OECD guideline 474 and U.S. EPA 84-2.

A single acute dose of 0, 3, 6, or 12 mg/kg was administered by oral intubation to groups of male and female mice. In the vehicle control and all test substance treated groups, bone marrow smears were prepared approximately 24, 48, and 72 hours after dosing. 2000 PCEs per animal were scored for micronuclei.

No statistically significant increases in the frequency of micronucleated PCEs were observed in test substance treated mice at any dose level or sampling time. In addition, no statistically significant depressions in the proportion of PCEs among 1000 erythrocytes were observed. In this assay, the test substance was negative.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

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 study was designed to evaluate the clastogenic potential of the test substance as measured by increases in numerical and structural chromosomal aberrations in bone marrow cells from rats. A single dose of the test material was administered by oral gavage to three groups of 15 male and 15 female rats at levels of 2, 6, and 20 mg/kg bw. 5 males and 5 females from each group were sacrificed at 6, 24, 48 hours after the single administration of the test substance. Cytogenetic analysis for the presence of chromosomal aberrations was done in the bone marrow cells.

GLP compliance:

no

Type of assay:

mammalian bone marrow chromosome aberration test

Specific details on test material used for the study:

- Substance name: Methomyl
- Substance ID: H#15,000
- Purity: 99%

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

Distilled water

Duration of treatment / exposure:

Single oral dose

Frequency of treatment:

Single oral dose

Dose / conc.:

2 mg/kg bw (total dose)

Dose / conc.:

6 mg/kg bw (total dose)

Dose / conc.:

20 mg/kg bw (total dose)

No. of animals per sex per dose:

15 (vehicle control and all dosage groups) and 5 (positive control)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CP)
- Route of administration: Oral intubation
- Doses / concentrations: 40 mg/kg

Tissues and cell types examined:

bone marrow cells

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

One rat at 2 mg/kg was slightly depressed, rats at 20 mg/kg showed abnormal observations, including rough coat, urine stains, and slightly depressed. Two animals at 20 mg/kg were found dead prior to sacrifice.

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Negative in in vivo bone marrow chromosome study in rats

Executive summary:

The study was designed to evaluate the clastogenic potential of the test substance as measured by increases in numerical and structural chromosomal aberrations in bone marrow cells from rats. A single dose of the test material was administered by oral gavage to three groups of 15 male and 15 female rats at levels of 2, 6, and 20 mg/kg bw.

5 males and 5 females from each group were sacrificed at 6, 24, 48 hours after the single administration of the test substance. Results showed that no statistically significant increases in the frequency of chromosomal aberrations compared to control values were seen for any of the dose levels that were tested. No statistically significant differences were seen between the mean chromosome numbers and the mean mitotic indices of the test groups and the vehicle controls.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The test substance, was evaluated for mutagenicity in Salmonella typhimurium strains TA100, TA1535, TA97a, and TA98 and in Escherichia coli strain WP2uvrA (pKM101) with and without an exogenous metabolic activation system (S9). The study was conducted according to OECD guidelines 471 and 472, U.S. EPA 84-2. Concentrations of 10, 50, 100, 250, 500, 1000, 2500, and 5000 μg/plate were initially evaluated using Salmonella typhimurium strain TA100 and Escherichia coli strain WP2 uvrA (pKM101) with and without an exogenous metabolic activation system (S9). In the absence of any notable bacterial toxicity or test substance precipitation, concentrations of 10, 50, 100, 500, 1000, 2500, and 5000 μg/plate were subsequently tested in Salmonella typhimurium strains TA97a, TA98, and TA1535 to complete the first trial. In a second confirmatory trial, concentrations of 10, 50, 100, 500, 1000, 2500, and 5000 μg/plate were tested in comparison to negative (solvent) controls in all strains. Under the conditions of this study, no evidence of mutagenic activity was detected in either of two independent trials. The test substance was negative.

The test substance was evaluated for its ability to induce structural chromosome aberrations in vitro using human peripheral blood lymphocytes (HPBL) in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9). The study was conducted following OECD guideline 473 and U.S. EPA OPPTS 870.5375. Cytogenetic evaluations were conducted at 28, 83, and 250 µg/mL (1.54 mM) for the 4-hour non-activated testing condition, at 28, 83, and 300 µg/mL (1.85 mM) for the 4-hour S9 activated testing condition, and at 83, 250, and 500 µg/mL (3.08 mM) for the 20-hour non-activated testing condition. The percentage of cells with structural or numerical aberrations in the test substance-treated groups was not significantly increased above that of the solvent control at any concentration (p >0.05, Fisher's exact test). All criteria for a valid study were met. Under the conditions of this study, the test substance did not induce structural or numerical chromosome aberrations in the in vitro mammalian chromosome aberration test in human peripheral blood lymphocytes in the non-activated or S9 activated testing conditions. The test substance was concluded as negative in this in vitro test.

The Chinese Hamster Ovary (CHO) cell line was used to detect mutation in a gene coding for the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT). The test substance was tested for mutagenic activity in both the presence and absence of metabolic activation system. The concentrations of the test substance used were 0, 10, 20, 40, 50, 55 mM (without activation) and 0, 100, 150, 200, 250, 350 mM (with activation). Two mutagenicity trials were performed without activation and cytotoxicity was observed at concentrations of 40.0 mM and above. The statistical analysis of these trials indicated that none of the concentrations of the test substance tested caused a significant increase in the mutant frequency over that observed in the solvent control and there was not a significance positive linear dose response. Two trials with activation were performed and cytotoxicity was evident at 200 µM and above. The statistical analysis of these trials revealed no significant increases in the mutant frequencies in the treated cultures and there was not a significant positive linear dose response. Under the conditions of the assay, the test substance was not mutagenic in CHO cells either in the presence or absence of an S9 activation system.

The test substance was tested for its ability to induce unscheduled DNA synthesis (UDS) in primary rat hepatocyte cultures. The concentrations of the test substance tested ranged from 1 µM to 75 mM in the treatment medium. 20 µL of the test substance stock solution or positive control were added to each culture. The cultures were then incubated for 18 hours. Slides were prepared and the cells were scored for UDS. The results do not show UDS at any concentration of the test substance and statistical analyses indicate neither a compound related effects, nor a dose-response relationship. Thus, the test substance was negative in the UDS assay.

The test substance was evaluated for its ability to induce micronuclei in bone marrow polychromatic erythrocytes (PCEs) of mice. The study was conducted following OECD guideline 474 and U.S. EPA 84-2. A single acute dose of 0, 3, 6, or 12 mg/kg was administered by oral intubation to groups of male and female mice. In the vehicle control and all test substance treated groups, bone marrow smears were prepared approximately 24, 48, and 72 hours after dosing. 2000 PCEs per animal were scored for micronuclei. No statistically significant increases in the frequency of micronucleated PCEs were observed in test substance treated mice at any dose level or sampling time. In addition, no statistically significant depressions in the proportion of PCEs among 1000 erythrocytes were observed. In this assay, the test substance was negative.

The clastogenic potential of the test substance was measured by evaluating increases in numerical and structural chromosomal aberrations in bone marrow cells from rats. A single dose of the test material was administered by oral gavage to three groups of 15 male and 15 female rats at levels of 2, 6, and 20 mg/kg bw. Five males and 5 females from each group were sacrificed at 6, 24, 48 hours after the single administration of the test substance. Results show that no statistically significant increases in the frequency of chromosomal aberrations compared to control values were seen for any of the dose levels that were tested. No statistically significant differences were seen between the mean chromosome numbers and the mean mitotic indices of the test groups and the vehicle controls.

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.