Methyl (R)-2-(4-(3-chloro-5-trifluoromethyl-2-pyridyloxy)phenoxy)propionate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenic effects - bacterial: Ames study. Negative. OECD 471; Reliability = 1.

Clastogenic effects - mammalian: Chromosome aberrations in rat lymphocytes. Negative. OECD 473; Reliability = 1.

 

Gene mutation in mammalian cells:HGPRT locus in the CHO-K1 Chinese hamster cell line. Negative; Reliability = 2

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

Additional documentation provided in IUCLID assesment reports (Chapter 13) supports the read across approach

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

no guideline followed

Principles of method if other than guideline:

The objective of this study was to evaluate the test item for its ability to induce forward mutation at the HGPRT locus in the CHO-K1 Chinese hamster cell line, as assessed by colony growth in the presence of 6-thioguanine. The test item, was evaluated for mutagenic activity over a concentration range of 25 µg/ml to 500 µg/ml.

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:

Substance ID: XR00453
AGR-187381
Purity: Not specified

Target gene:

hypoxanthine guanine phosphoribosyl transferase (hgprt) locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

(CHO-K1)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: S9 from Arochlor 1254 induced SD rat
- method of preparation of S9 mix: A 9000 X g supernatant in 0.25 M sucrose buffered with phosphate at pH 7.4 was prepared by the test facility

Test concentrations with justification for top dose:

25, 100, 250, 500, 1000, 1500 & 2000 µg/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

other: 5-Bromodeoxyuridine

Details on test system and experimental conditions:

- The cells were quantitatively seeded at 200 cells/dish, allowed to attach for 16 to 19 hours, then exposed to approximately 10 dilutions of the test article for 4 hours. The cells were then washed and incubated in F12 culture medium for 7 days to allow colony development. The number of colonies compared to the negative control was used to select 6 to 8 doses for the mutation assay covering the range from approximately 0% to 90% reduction in colony-forming ability. This dose selection procedure was performed both with and without S9 metabolic activation.
- Non activation assay: The assay was initiated by exposing about 4E6 cells in a 250 nm flask to each concentration of the test article for 4 hours. Concurrently, three 50 mm dishes containing 200 cells each were exposed in the same manner as each flask. After treatment, the cell monolayers were washed and fresh culture medium was added. The high density cell monolayers were then trypsinized and replated at about 1.5E6 cells into each of two 150 mm dishes per dose level. The small dishes were incubated for 7-8 days to permit colony development and the determination of the toxicities of the treatments. The large dishes were incubated to permit growth and expression of induced mutations and were subcultured on day 3 and again on day 5 to allow for additional growth and expression time. At each subculture the two cultures for each dose level were combined and reseeded at 1.5E6 cells into each of two 150 mm dishes.
At the end of the expression period (6 to 7 days), each culture was reseeded at 2E6 cells per 100 mm dish (12 dishes total) in mutant selection medium. Also, three 60 mm dishes were seeded at 200 cells each in culture medium to determine the cloning efficiency of each cu1ture. After incubation for 7 to 9 days, the ratio of TG-resistant colonies in the mutant selection dishes to the number of colonies in the cloning efficiency dishes was used to calculate the mutant frequency.
- Activation assay: The activation assay was performed independently with its own set of negative and positive controls. The procedure was identical to the nonactivation assay except for the addition of the S9 fraction of rat liver homogenate and necessary cofactors during the 4-nour treatment period. The fetal bovine serum content of the medium was reduced to 5% by volume. The cofactors consisted of nicotinamide adenine dinucleotide phosphate (NADP, sodium salt), glucose- 6-phosphate, calcium chloride, potassium chloride and magnesium chloride all in a pH 7.8 sodium phosphate buffer.

Evaluation criteria:

The observation of a mutant frequency that meets the minimum criterion for a single treated culture within a range of assayed concentrations is not sufficient evidence to evaluate a test article as a mutagen. The following test results must be obtained to reach this conclusion for either activation or nonactivation conditions:
A dose-related or toxicity-related increase in mutant frequency should be observed. It is desirable to obtain this relation for at least three doses, but this depends on the concentration steps chosen for the assay and the toxicity at which mutagenic activity appears.
An increase in mutant frequency may be followed by only small or no further increases at higher concentrations or toxicities. However, a decrease in mutant frequency to values below the minimum criterion in a single assay is not acceptable for classifying the test article as a mutagen. If the mutagenic activity at the lower concentrations or toxicities is large, a repeat assay is performed to confirm the mutagenic response pattern. An increase at a single dose near the highest testable toxicity requires confirmation by a repeat assay.
Either parameter, applied concentration or toxicity (percent survival), can be used to establish whether the mutagenic activity is related to an increase in effective treatment. A negative correlation with dose is acceptable only if a positive correlation with toxicity exists. An apparent increase in mutagenic activity as a function of decreasing toxicity is not acceptable evidence for mutagenicity.
A test article is evaluated as non-mutagenic in a single assay only if the minimum increase in mutant frequency is not observed for a range of applied concentrations that extends to toxicity causing about 10% to 15% survival.
If a repeat assay does not confirm an earlier, minimal response as discussed above, the test article is evaluated as nonmutagenic in this assay system.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

73% survival at 500 µg/ml

Vehicle controls validity:

valid

Positive controls validity:

valid

Table 1:Mean absolute cloning efficiency (%) and mean mutant frequency

	- S9			+ S9
	Mean ACE	Mean MF		Mean ACE	Mean MF
NC (DMSO)	81.7	2.0	NC (DMSO)	76.0	2.7
25 µg/ml	65.4	1.3	25 µg/ml	67.4	1.2
100 µg/ml	59.9	2.8	100 µg/ml	57.4	8.7*
250 µg/ml	74.9	0.0	250 µg/ml	67.2	8.1*
500 µg/ml	56.2	1.5	500 µg/ml	63.0	0.0
1000 µg/ml	61.0	2.0	1000 µg/ml	-	-
1500 µg/ml	31.7	0.0	1500 µg/ml	-	-
PC	51.7	84.6**	PC	53.9	205.66**

**Significant increase, p≤0.01

*Significant increase, p≤0.05

Conclusions:

The test material is considered inactive in the CHO/HGPRT forward mutation assay under the conditions of the present study.

Executive summary:

The objective of this study was to evaluate the test item for its ability to induce forward mutation at the HGPRT locus in the CHO-K1 Chinese hamster cell line, as assessed by colony growth in the presence of 6-thioguanine. The test item, was evaluated for mutagenic activity over a concentration range of 25 µg/ml to 500 µg/ml. The latter treatment was near the solubility limit and caused 73% survival. Treatments with 1000 µg/ml and higher concentrations caused excessive arrest of cell division or lethality. No mutants were induced by the 500 µg/ml treatment and small increases in mutant frequency at lower concentrations were evaluated as representing assay variability. The test material was therefore considered to be inactive in the CHO/HGPRT Forward Mutation Assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Additional documentation provided in IUCLID assesment reports (Chapter 13) supports the read across approach

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

73% survival at 500 µg/ml

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test material is considered inactive in the CHO/HGPRT forward mutation assay under the conditions of the present study.

Executive summary:

The objective of this study was to evaluate the test item for its ability to induce forward mutation at the HGPRT locus in the CHO-K1 Chinese hamster cell line, as assessed by colony growth in the presence of 6-thioguanine. The test item, was evaluated for mutagenic activity over a concentration range of 25 µg/ml to 500 µg/ml. The latter treatment was near the solubility limit and caused 73% survival. Treatments with 1000 µg/ml and higher concentrations caused excessive arrest of cell division or lethality. No mutants were induced by the 500 µg/ml treatment and small increases in mutant frequency at lower concentrations were evaluated as representing assay variability. The test material was therefore considered to be inactive in the CHO/HGPRT Forward Mutation Assay.

Reference 3

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:

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:

other: EEC: Directive 97/69/EEC, of July 31, 1992 Adapting to Technical Progress for the Seventeenth Time Council Directive 67/548/EEC, EEC Publication No. L383A, 1992

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Haloxyfop-R Methyl Ester Technical
Lot #: NB10150101 (TSN101748)
Purity: 98.4%

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system
- Source of S9 : Molecular Toxicology, Inc.
- Method of preparation of S9 mix: The homogenate was prepared from male Sprague-Dawley rats that had been injected (i.p.) with Aroclor™ 1254 (200 mg per ml in com oil) at 500 mg/kg.
- Concentration or volume of S9 mix: 1.00 mL

Test concentrations with justification for top dose:

Dose range finding study: 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3330, 5000 µg/plate
Mutagenicity study: 33.3, 100, 333, 1000, 3330, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: At 100 mg per mL, which was the most concentrated stock dilution prepared, the test article formed a transparent, beige solution. The test article remained a solution in all succeeding dilutions prepared for the mutagenicity assay.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene: TA100, TA1535, TA1537 (2.5 µg; with S9); WP2uvrA (25.0 µg; with S9), ICR-191: TA1537 (2.0 µg; without S9)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: Two (initial mutagenicity assay and confirmatory assay)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium: Preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 ± 2 minutes
- Exposure duration/duration of treatment: 52 ± 4 hour


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Background growth inhibition

Evaluation criteria:

For a test substance to be considered positive, it had to produce at least a 3-fold (TA98, TA1535, TA1537, and WP2uvrA) or 2-fold (TA100) dose related and reproducible increase in the mean revertants per plate of at least one tester strain over the mean revertants per plate of the appropriate vehicle control. An observed response which did not meet all three of the above criteria (magnitude, dose-responsiveness, reproducibility) was not evaluated as positive.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Indications of cytotoxicity were observed with tester strain TA100 with S9 mix at 3330 µg/plate and above as evidenced by a slight reduction of the bacterial background lawn.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: A dose range finding study was conducted on the test article using tester strains TA100 and WP2uvrA in both the presence and absence of S9 mix (one plate per dose). Ten doses of test substance, from 5000 to 6.67 µg per plate, were tested. Indications of cytotoxicity were observed with tester strain TA100 in the presence of S9 mix at 3330 ug per plate and above as evidenced by a slight reduction of the bacterial background lawn. No cytotoxicity was observed with tester strain TA100 in the absence of S9 mix or with WP2uvrA in either the presence or absence of S9 mix as evidenced by a normal background lawn and no decrease in the number of revertants per plate. Slight article precipitate was observed at 3330 µg per plate and above in the presence of S9 mix and at 1000 and 3330 µg per plate in the absence of S9 mix. Moderate test article precipitate was observed at 5000 µg per plate in the absence of S9 mix.

Table-1: Mutagenicity assay results (mean revertants per plate)

Conc. (µg/plate)	TA1537		TA1535		TA98		TA100		WP2uvrA
	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9
DMSO	6	12	7	9	9	24	96	93	17	13
33.3	3	8	8	10	15	30	79	94	16	14
100	4	9	9	13	9	20	100	98	20	12
333	8	9	12	12	13	26	106	101	14	14
1000	8	9	11	12	9	20	109	80	14	15
3330	9	11	8	9	9	16	96	70	13	12
5000	9	8	7	12	11	20	98	71	9	15
Positive cotnrol	1678	168	588	107	178	500	623	787	451	205

Table-2: Confirmatory assay results (mean revertants per plate)

Conc. (µg/plate)	TA1537		TA1535		TA98		TA100		WP2uvrA
	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9
DMSO	7	7	10	11	14	26	92	96	17	15
33.3	8	12	10	10	11	28	87	101	14	16
100	4	10	17	13	16	21	90	96	14	16
333	7	10	11	10	13	25	91	86	17	12
1000	9	5	10	8	11	22	84	103	15	14
3330	7	8	10	11	12	19	89	85	14	15
5000	7	6	15	10	9	26	102	74	18	13
Positive cotnrol	1898	153	710	108	243	423	673	979	547	258

Conclusions:

Negative in reverse bacterial mutation test

Executive summary:

The test substance was evaluated in the Salmonella-Escherichia coli/mammalian-microsome reverse mutation assay following OECD guieline 471 and U S EPA OPPTS 870.5100. The tester strains used in this study were Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537, and Escherichia coli tester strain WP2uvrA. The assay was conducted using 6 doses of test substance in both the presence and absence of an externally supplied metabolic activation system (S9) along with concurrent vehicle and positive controls. The concentration of the test material ranged from 33.3 to 5000 µg/plate in the presence and absence of metabolic activation. The results of the initial mutagenicity assay were confirmed in an independent experiment. The test material did not induce a positive increase in the number of revertants per plate of any of the tester strains either in the presence or absence of microsomal enzymes (S9). Hence the test substance was classified as negative in this bacterial mutagenicity test under the experimental conditions used.

Reference 4

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 Chromosomal Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Haloxyfop-R methyl ester
Lot #: NB10150101 (TSN101748)
Purity: 98.4%

Species / strain / cell type:

lymphocytes: rat

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Lymphocytes from rats

For lymphocytes:
- Whether whole blood or separated lymphocytes were used: Whole blood
- Whether blood from different donors were pooled or not: The blood samples from individual rats were pooled
- Mitogen used for lymphocytes: PHA (20 µg/mL)

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI 1640 medium supplemented with 10% heat inactivated fetal bovine serum, antibiotics and antimycotics (Fungizone 0.25 µg/mL; penicillin G, 100 µ/ml; and streptomycin sulfate, 0.1 mg/ml), 20 µg/mL PHA, and an additional 2 mM L-glutamine. Cultures were initiated by inoculating approximately 0.5 mL of whole blood/5 ml of culture medium. Cultures were set up in duplicate at each dose level in T-25 plastic tissue culture flasks or in glass medicine bottles (35 mL) (without S9 activation) and incubated at 37°C.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system
- Source of S9 : Molecular Toxicology, Inc., Boone, North Carolina
- Method of preparation of S9 mix: The mix consists of 10 mM MgCl2·6H2O, 5 mM glucose-6-phosphate, 4mM nicotinamide adenine dinucleotide phosphate, 10 mM CaCl2, 30 mM KCI, and 50 mM sodium phosphate (pH 8.0).
- Concentration or volume of S9 mix and S9 in the final culture medium: 10% (v/v) in a mix and 2% v/v is used as final concentration

Test concentrations with justification for top dose:

Assay 1: 1.0, 3.33, 10, 33.3, 100, 333.3, and 1000 µg/mL (The highest concentration evaluated was based upon solubility limitations)
Assay 2: 24 h without S9: 1.0, 3.33, 10, 33.3, 100, 333.3, 666.6, and 1000 ug/mL; 4 h with S9: 33.3, 100, 333.3, 666.6, and 1000 µg/mL

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

FOR CHROMOSOME ABERRATION:
- Spindle inhibitor (cytogenetic assays): Colcemid was added approximately 3 hr prior to harvest at a final concentration of 0.2 µg/mL
- Methods of slide preparation and staining technique used including the stain used: The cells were swollen by hypotonic treatment (0.075 M KCI), fixed with methanol:acetic acid (3:1), dropped on microscope slides, and stained in Giemsa.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): Mitotic indices were determined as the number of cells in metaphase among 1000 cells/replicate and expressed as percentages. 100 metaphases/replicate were examined, where possible, from coded slides at each selected concentration of the test substance and the negative controls (a total of 200 cells/treatment) for structural abnormalities. In addition, 100 metaphase/replicate were examined for the incidence of polyploidy.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Only those metaphases that contained 42 ± 2 centromeres were scored with the exception of cells with multiple aberrations, in which case accurate counts of the chromosomes were not always possible. Those cells having 5 or more aberrations/cell were classified as cells with multiple aberrations. Gaps were not included in calculations of total cytogenetic aberrations.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Mitotic index (MI)

Evaluation criteria:

A test chemical is considered positive in this assay if it induces a significant dose-related and reproducible increase in the frequency of cells with aberrations.

Statistics:

The frequencies of cells with aberrations (excluding gaps) were compared by the following statistical methods. At each dose level, data from the replicates were pooled. A two way contingency table was constructed to analyze the frequencies of cytogenetic abnormalities. An overall Chi-square statistic, based on the table, was partitioned into components of interest. Specifically, statistics were generated to test the two global hypotheses of (1) no differences in average number of cells with aberrations among the dose groups, and (2) no linear trend of increasing number of cells with aberrations with increasing dose. An ordinal metric (0, 1, 2, etc.) was used for the doses in the statistical evaluation. If either statistic was found to be significant at alpha=0.01 versus a one-sided increasing alternative, pairwise tests (i.e., control vs. treatment) were performed at each dose level and evaluated at alpha=0.01 again versus a one-sided alternative.

Polyploid cells were analyzed by the Fisher Exact probability test. The number of polyploid cells were pooled across replicates for the analysis and evaluated at alpha=0.05. The data was analyzed separately based on the presence or absence of S-9 and based on the exposure time.

Key result

Species / strain:

lymphocytes: rat

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Assay 1: Reduction in mitotic activity at ≥ 1000 µg/mL without S9, at 1000 µg/mL with S9; Assay 2: With S9 at ≥333.3 µg/mL

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- Concurrent vehicle negative and positive control data :
Assay 1: Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemicals. Aberrant cell frequencies in MMC (without S-9) and CP (with S-9) treated cultures were 27.0 and 27.7%, respectively.
Assay 2: Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemicals. Aberrant cell frequencies in MMC (without S-9) and CP (with S-9) treated cultures were 24 and 46%, respectively.

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For lymphocytes in primary cultures: Assay 1: Without metabolic activation, cultures treated with 1000 µg/mL had a 69% reduction in mitotic activity. The next highest dose (333.3 µg/mL) had a 43% reduction in mitotic activity and the remaining cultures had 13 to 26% reductions in mitotic activity. In the presence of S-9, the only noticeable reduction in the mitotic index (40%) occurred in cultures treated with 1000 µg/mL.
Assay 2: The higher concentrations tested in the absence of S-9, i.e., 333.3, 666.6, and 1000 µg/mL, showed excessive toxicity while the remaining cultures had no toxicity. In the presence of S-9 activation, concentrations of 33.3-333.3 µg/mL, showed no toxicity. Cultures treated with 666.6 and 1000 µg/mL had reductions in mitotic activity of 31.7 and 23.5%, respectively.

Table-1: Incidence of Polyploidy (Assay 1)

Dose (µg/mL)	%Polyploidy	Dose (µg/mL)	%Polyploidy
	Without S9		With S9
1% DMSO	1.0	1% DMSO	0.5
33.3	1.0	100.0	1.0
100.0	0.0	333.3	0.0
333.3	1.5	1000.0	0.5
MMC (0.5)	0.5	CP (4.0)	0.0

Table-2: Incidence of Polyploidy (Assay 2)

Dose (µg/mL)	%Polyploidy	Dose (µg/mL)	%Polyploidy
	Without S9		With S9
1% DMSO	0.5	1% DMSO	0.5
10.0	0.0	100.0	0.0
33.3	0.0	333.3	0.5
100.0	0.0	1000.0	0.5
MMC (0.075)	0.0	CP (4.0)	0.0

Table-3: Assay-1: Results of the Chromosomal Aberration Assay 4 Hours After Treatment in the Absence of S-9

	Negative control	33.3 µg/mL	100 µg/mL	333.3 µg/mL	Positive control
No. of cells scored	200	200	200	200	100
Chromatid Gaps	1	1	3	0	4
Chromosome Gaps	0	0	0	0	0
Chromatid Breaks	1	2	1	1	9
Chromatid Exchanges	0	0	0	0	17
Chromosome Breaks	1	0	0	0	4
Chromosome Exchanges	0	0	0	0	0
Total Aberrations (excluding gaps)	2 (1.0)	2 (1.0)	1 (0.5)	1 (0.5)	30 (30.0)
No. of cells with Aberr. (excluding gaps)	2 (1.0)	2 (1.0)	1 (0.5)	1 (0.5)	27* (27.0)
Miscellaneous Aberr.	0	0	0	0	0
Cells with Multiple Aberr. (5 or more aberr.)	0	0	0	0	4

Values in parentheses are percentages

*Significantly (alpha <0.01) different from negative control.

Table-4: Assay-1: Results of the Chromosomal Aberration Assay 4 Hours After Treatment in the Presence of S-9

	Negative control	100 µg/mL	333.3 µg/mL	1000 µg/mL	Positive control
No. of cells scored	200	200	200	200	100
Chromatid Gaps	4	1	2	3	3
Chromosome Gaps	0	0	0	0	0
Chromatid Breaks	1	0	1	5	11
Chromatid Exchanges	0	0	0	0	19
Chromosome Breaks	0	0	0	0	6
Chromosome Exchanges	0	0	0	0	0
Total Aberrations (excluding gaps)	1 (0.5)	0 (0.0)	1 (0.5)	5 (2.5)	36 (35.6)
No. of cells with Aberr. (excluding gaps)	1 (0.5)	0 (0.0)	1 (0.5)	4 (2.0)	28* (28.0)
Miscellaneous Aberr.	0	0	0	0	0
Cells with Multiple Aberr. (5 or more aberr.)	0	0	0	0	5

Values in parentheses are percentages

*Significantly (alpha <0.01) different from negative control.

Table-5: Assay-2: Results of the Chromosomal Aberration Assay 24 Hours After Treatment in the Absence of S-9

	Negative control	10.0 µg/mL	33.3 µg/mL	100 µg/mL	Positive control
No. of cells scored	200	200	200	200	100
Chromatid Gaps	6	3	1	2	12
Chromosome Gaps	0	0	0	0	0
Chromatid Breaks	4	3	2	1	13
Chromatid Exchanges	0	0	0	0	15
Chromosome Breaks	0	1	0	1	2
Chromosome Exchanges	0	0	0	0	0
Total Aberrations (excluding gaps)	4 (2.0)	4 (2.0)	2 (1.0)	2 (1.0)	30 (30.0)
No. of cells with Aberr. (excluding gaps)	4 (2.0)	4 (2.0)	2 (1.0)	2 (1.0)	24* (24.0)
Miscellaneous Aberr.	0	0	0	0	0
Cells with Multiple Aberr. (5 or more aberr.)	0	0	0	0	3

Values in parentheses are percentages

*Significantly (alpha <0.01) different from negative control.

Table-6: Assay-2: Results of the Chromosomal Aberration Assay 4 Hours After Treatment in the Presence of S-9

	Negative control	10.0 µg/mL	33.3 µg/mL	100 µg/mL	Positive control
No. of cells scored	200	200	200	200	100
Chromatid Gaps	1	2	4	2	3
Chromosome Gaps	0	0	0	0	0
Chromatid Breaks	1	1	6	0	21
Chromatid Exchanges	0	0	1	0	31
Chromosome Breaks	1	1	0	1	4
Chromosome Exchanges	1	0	0	0	1
Total Aberrations (excluding gaps)	3 (1.5)	2 (1.0)	7 (3.5)	1 (0.5)	57 (57.0)
No. of cells with Aberr. (excluding gaps)	3 (1.5)	4 (2.0)	6 (3.0)	1 (0.5)	46* (46.0)
Miscellaneous Aberr.	0	0	1	0	0
Cells with Multiple Aberr. (5 or more aberr.)	0	2	1	0	18

Values in parentheses are percentages

*Significantly (alpha <0.01) different from negative control.

Conclusions:

Negative in the in vitro chromosomal aberration assay utilizing rat lymphocytes

Executive summary:

The test substance was evaluated in an in vitro chromosomal aberration assay utilizing rat lymphocytes. The study was conducted following OECD guideline 473 and EPA OPPTS 870.5375. Approximately 48 hr after the initiation of whole blood cultures, cells in the absence and presence of S-9 activation were treated for 4 hours with targeted concentrations of 0 (negative control) to 1000 µg test substance per mL of culture medium and harvested at the end of treatment (Assay 1). Based upon the mitotic indices, cultures treated with targeted doses of 0, 33.3, 100, and 333.3 µg/ml in the absence of S-9 activation and cultures treated with targeted doses of 0, 100, 333.3, and 1000 µg/ml in the presence of S-9 activation were selected for determining the incidence of chromosomal aberrations. In a confirmatory assay (Assay 2), cultures were treated as above except that in the absence of S-9, they were treated continuously for 24 hours until the time of their harvest. The incidence of chromosomal abnormalities was determined from cultures treated with 0, 10, 33.3 and 100 µg/ml in the absence of S-9 and from cultures treated with 0, 100, 333.3, and 1000 µg/ml in the presence of S-9 activation. No significant increase in the incidence of aberrant cells 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 (i.e., mitomycin C without S-9 and cyclophosphamide with S-9) had significantly higher incidences of abnormal cells in both the assays. Hence, the test substance was considered to be negative in the in vitro chromosomal aberration assay utilizing rat lymphocytes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Clastogenic effects - mammalian: in vivo mouse micronucleus study; Negative. GLP study; Reliability = 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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

Additional documentation provided in IUCLID assesment reports (Chapter 13) supports the read across approach

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test substance was administered to male and female SD rats by single oral gavage at dose levels of 0, 30, 100 and 300 mg/kg. The animals were sacrificed at 30 and 42 h after treatment. 1000 PCE were examined from the bone marrow smears of each animal and the number of micronucleated normochromatic erythrocytes, the number of micronucleated normochromatic erythrocytes and the number of normochromatic erythrocytes were recorded.

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

DOWCO 453
Lot #: AGR-187381
Purity: 99.55%

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Timco, Inc., Houston, Texas
- Age at arival: Approximately 8 weeks
- Assigned to test groups randomly: Yes
- Housing: Housed singly in suspended wire-bottom cages
- Diet: Purina Certified Rodent Chow #5002
- Water: Well water
- Acclimation period: 7-8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72 ± 5
- Humidity (%): 40-60%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 h dark/light

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: The test substance was solubilized in 100 mL of 1N NaOH. To this, 70 mL of deionized distilled water was added.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: As test substance was not soluble in water, aqueous solutions of this chemical were prepared in the following way: To prepare a 7.0% stock solution, 17.5 g of test substance was solubilized in 100 mL of 1 N NaOH. To this, 70 ml of deionized distilled (d.d) water was added and the pH was adjusted to about 6.0 by adding (approximately 35 mL) 1.2 N HCl. The contents were brought up to 250 ml by adding d.d water. The stock solution was diluted with d.d. water to the desired concentration. All the solutions used for dosing were less than 60 hours old. The test substance solutions prepared by solubilization in 1 N NaOH were found to be stable for at least 42 days.

Duration of treatment / exposure:

Single oral administration

Frequency of treatment:

Single oral administration

Dose / conc.:

30 mg/kg bw (total dose)

Dose / conc.:

100 mg/kg bw (total dose)

Dose / conc.:

300 mg/kg bw (total dose)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control used: Cyclophosphamide
- Route of administration: Oral gavage
- Doses / concentrations: 20 mg/kg

Tissues and cell types examined:

Bone marrow cells from femurs

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The dose levels of the test substance used in the study were selected on the basis of LD50 studies conducted in the test facility, which allowed to assume that the single-dose oral LD50 is 500 mg/kg body weight for both sexes. The top dose level of 300 mg/kg used in the study was 60% of the LD50.

TREATMENT AND SAMPLING TIMES: Single oral dose and the treated animals were sacrificed at 30 and 42 h after treatment

DETAILS OF SLIDE PREPARATION: Cell smears were prepared on clean microscope slides using small portions of the cell suspension. The slides were allowed to air dry at least over night. For staining, the slides were fixed for 5 minutes in absolute methanol, rinsed thoroughly in buffer (pH 6.8), stained in Giemsa (1:6 Gurr's R66 Giemsa:buffer, pH 6.8) for 10 minutes, and rinsed thoroughly in buffer (pH 6.8). The slides were blotted with filter paper to remove excess water and allowed to air dry before being cover slipped.

METHOD OF ANALYSIS: The slides were divided into two groups according to sex, coded, and scored blindly. One thousand polychromatic erythrocytes were examined from each animal and the following information was recorded: (1) the number of micronucleated polychromatic erythrocytes (MN-PCE); (2) the number of micronuc1eated normochromatic erythrocytes (MN-NCE) in the optical field containing 1000 PCE; and (3) the number of normochromatic erythrocytes in the optical field containing 1000 PCE. The frequencies of micronucleated cells were expressed as the number of micronucleated erythrocytes in the optical field containing 1000 PCE.

Statistics:

The frequencies of total micronucleated erythrocytes were analyzed by two-way analysis of variance (sex and dose) followed by linear dose-response tests. The frequencies were not transformed prior to analysis. The level of confidence chose was 95% or greater. The data on the proportion of PCE were no evaluated statistically.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

There was a noticeable decrease in the proportions of PCE in females treated with 100 and 300 mg/kg at both sacrifice times. In males, such an effect was observed only in the 300 mg/kg group sacrificed at 42 h. Nevertheless, the decreases in the proportion of PCE noticed in the treatment groups were not sufficient to interfere with detection of any induced micronucleated erythrocytes. It is possible that at least part of the decreases in the proportion of PCE may be due to the nonspecific toxicological stress (in the present situation, decreased food consumption) encountered by the animals.

Table-1: Summary of the data on the incidence of micronucleated erythrocytes and the % of PCE in the bone marrows of male and female rats in the various treatment groups

			30 h sacrifice		42 h sacrifice
Treatment	Sex	No. of animals^A	Total MN^B± SD	% PCE^C± SD	Total MN^B± SD	% PCE^C± SD
Negative control	M	5	3.2 ± 1.9	72.9 ± 2.7	4.0 ± 2.3	79.6 ± 7.3
	F	5	3.2 ± 0.4	71.5 ± 5.6	2.6 ± 2.1	67.6 ± 3.2
30 mg/kg	M	5	2.4 ± 1.5	76.2 ± 3.5	4.2 ± 3.3	74.2 ± 4.4
	F	5	4.2 ± 1.9	68.8 ± 5.2	3.8 ± 3.6	62.1 ± 5.5
100 mg/kg	M	5	2.4 ± 1.5	69.8 ± 3.4	5.0 ± 1.2	73.4 ± 4.5
	F	5	2.8 ± 1.5	60.7 ± 5.0	3.2 ± 2.9	51.9 ± 15.5
300 mg/kg	M	5	3.0 ± 2.3	67.2 ± 4.3	2.6 ± 2.1	65.1 ± 6.0
	F	5	2.6 ± 0.5	55.8 ± 6.6	1.6 ± 1.3	54.9 ± 6.3
20 mg/kg (cyclophosphamide)	M	5			37.2^D± 7.2	46.2 ± 6.4
	F	5			37.8^D± 8.1	30.1 ± 3.2

A 1000 PCE were examined from each animal

B Total number of micronucleated erythrocytes per 1000 PCE per animal

C PCE x 100 / PCE + NCE

D Analysis of variance showed significant difference between negative control and CP treatment, p <0.0001

Conclusions:

Negative in rat bone marrow micronucleus erythrocytes assay

Executive summary:

The test substance was evaluated in the rat bone marrow micronucleus test for genotoxic activity. The micronucleus test is capable of detecting agents that cause chromosomal aberrations and spindle malfunction. The test substance was administered to male and female SD rats by single oral gavage at dose levels of 0 (negative control), 30, 100 and 300 mg/kg. The positive control rats received a single dose oral gavage of 20 mg/kg cyclophosphamide, a well-known clastogen. The animals were sacrificed at 30 and 42 h after treatment. 1000 PCE were examined from the bone marrow smears of each animal and the number of micronucleated normochromatic erythrocytes, the  number of micronucleated normochromatic erythrocytes and the number of normochromatic erythrocytes were recorded. The frequency of micronucleated bone marrow erythrocytes was unaffected by test substance treatment. Rats treated with cyclophosphamide, on the other hand, had significant numbers of micronucleated bone marrow erythrocytes. The results of the present study indicate a lack of genotoxic activity for test substance within the test system employed.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Additional documentation provided in IUCLID assesment reports (Chapter 13) supports the read across approach

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Negative in rat bone marrow micronucleus erythrocytes assay

Executive summary:

The test substance was evaluated in the rat bone marrow micronucleus test for genotoxic activity. The micronucleus test is capable of detecting agents that cause chromosomal aberrations and spindle malfunction. The test substance was administered to male and female SD rats by single oral gavage at dose levels of 0 (negative control), 30, 100 and 300 mg/kg. The positive control rats received a single dose oral gavage of 20 mg/kg cyclophosphamide, a well-known clastogen. The animals were sacrificed at 30 and 42 h after treatment. 1000 PCE were examined from the bone marrow smears of each animal and the number of micronucleated normochromatic erythrocytes, the  number of micronucleated normochromatic erythrocytes and the number of normochromatic erythrocytes were recorded. The frequency of micronucleated bone marrow erythrocytes was unaffected by test substance treatment. Rats treated with cyclophosphamide, on the other hand, had significant numbers of micronucleated bone marrow erythrocytes. The results of the present study indicate a lack of genotoxic activity for test substance within the test system employed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The test substance was evaluated in the Salmonella-Escherichia coli/mammalian-microsome reverse mutation assay following OECD guieline 471 and U S EPA OPPTS 870.5100. The tester strains used in this study were Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537, and Escherichia coli tester strain WP2uvrA. The assay was conducted using 6 doses of test substance in both the presence and absence of an externally supplied metabolic activation system (S9) along with concurrent vehicle and positive controls. The concentration of the test material ranged from 33.3 to 5000 µg/plate in the presence and absence of metabolic activation. The results of the initial mutagenicity assay were confirmed in an independent experiment. The test material did not induce a positive increase in the number of revertants per plate of any of the tester strains either in the presence or absence of microsomal enzymes (S9). Hence the test substance was classified as negative in this bacterial mutagenicity test under the experimental conditions used.

The test substance was evaluated in an in vitro chromosomal aberration assay utilizing rat lymphocytes. The study was conducted following OECD guideline 473 and EPA OPPTS 870.5375. Approximately 48 hr after the initiation of whole blood cultures, cells in the absence and presence of S-9 activation were treated for 4 hours with targeted concentrations of 0 (negative control) to 1000 µg test substance per mL of culture medium and harvested at the end of treatment. The test substance was considered to be negative in the in vitro chromosomal aberration assay utilizing rat lymphocytes.

The test substance was evaluated in the rat bone marrow micronucleus test for genotoxic activity. The micronucleus test is capable of detecting agents that cause chromosomal aberrations and spindle malfunction. The test substance was administered to male and female SD rats by single oral gavage at dose levels of 0 (negative control), 30, 100 and 300 mg/kg. The results of the present study indicate a lack of genotoxic activity for test substance within the test system employed.

The test item was evaluated for mutagenic activity in CHO-K1 Chinese hamster cell line over a concentration range of 25 µg/ml to 500 µg/ml. The latter treatment was near the solubility limit and caused 73% survival. Treatments with 1000 µg/ml and higher concentrations caused excessive arrest of cell division or lethality. No mutants were induced by the 500 µg/ml treatment and small increases in mutant frequency at lower concentrations were evaluated as representing assay variability. The test material was therefore considered to be inactive in the CHO/HGPRT Forward Mutation Assay.

Justification for classification or non-classification

The test substance was negative for mutagenicity and clastogenicity in in vitro studies. 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.