5,7-dichloro-4-(4-fluorophenoxy)quinoline;quinoxyfen (ISO)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenic effects - bacterial: Ames study. Negative. EPA OPP 84-2; Reliability = 2.

Clastogenic effects - mammalian: Chromosome aberrations in rat lymphocytes. Negative. OECD 473; Reliability = 2.

Mutagenic effects - mammalian: HGPRT in CHO cells. Negative. EPA OPP 84-2; Reliability = 1

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Substance ID: TSN 100097
- Name of substance: XDE-795
- Lot number: DECO-97-152-1
- Purity: 97.4%

Target gene:

histidine

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix prepared from Aroclor 1254-induced Sprague-Dawley rats

Test concentrations with justification for top dose:

Range finding assay: 5.0, 10, 50, 100, 500, 1000, 5000 µg/plate for both with and without S9 activation
Mutation assay and confirmatory mutation assay: 10, 50, 100, 500, 1000 µg/plate and 50, 100, 500, 1000, 5000 µg/plate with and without S9 activation, respectively

Vehicle / solvent:

Dimethyl sulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene (TA98, TA100, TA1535, TA1537 with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar
- Cell density at seeding: 5.0 x 10e7 to 1.0 x 10e8

DURATION
- Incubation temperature: 37 ± 1°C
- Incubation period: 20 minutes before inverting the plates and 68 hours after inverting the plates

NUMBER OF REPLICATIONS: Single

COLONY COUNTING: Hand counting was done for the plates where precipitate was formed. Remaining plates were counted using automatic colony counter.

NUMBER OF CELLS EVALUATED: 0.912 x 10e8

Evaluation criteria:

A response is considered negative if: all of the strains treated with test substance have mean reversion frequencies that are less than three times that of the mean reversion frequencies of the corresponding solvent control plates; and there is no evidence of a dose-dependent response.
A response is considered positive if two or more doses produce a mean reversion frequency that is three times or more greater than the mean reversion frequencies of the corresponding solvent control plates and the response is reproducible.
A response is considered equivocal if it does not fulfill the criteria of either a negative or a positive response and/or the Study Director does not consider the response to be either positive or negative.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING STUDIES: In the range finding test the test substance was evaluated at concentrations ranging from 5000 to 5.0 µg/plate in both the absence and presence of exogenous activation. The test substance concentrations of 1000 µg/plate and above produced a significant degree of toxicity in the absence of S-9 activation. The relative cloning efficiency (RCE) was 71% and 0%. The background lawn also was significantly reduced. There were not any revertants observed at the concentration of 5000 µg/plate. A moderate toxicity was observed at concentrations of 1000 µg/plate and above in the presence of S-9 activation. The toxicity was manifested only as a reduction in the number of viable colonies per plate. The relative cloning efficiency was 78% and 72%.
Based on these results, it was decided to perform the mutation assay at test substance concentrations of 1000, 500, 100, 50 and 10 µg/plate and 5000, 1000, 500, 100 and 50 µg/plate in the absence and presence of S-9, respectively.

Table-1: Initial (trial 1) and confirmatory (trial 2) mutation assays results - without metabolic activation

Compound	Conc. mg/plate	TA98		TA100		TA1535		TA1537
		Trial 1	Trial 2	Trial 1	Trial 2	Trial 1	Trial 2	Trial 1	Trial 2
Test substance	1000	22	22	81	87	23	20	11	12
	500	20	19	103	93	19	22	11	10
	100	30	29	100	92	26	26	17	16
	50	24	26	108	93	28	28	17	17
	10	28	27	101	90	28	28	17	19
Solvent control	_	30	31	109	91	30	30	18	19
Positive control	_	903	901	587	618	529	535	209	210

Table-2: Initial (trial 1) and confirmatory (trial 2) mutation assays results - with metabolic activation

Compound	Conc. mg/plate	TA98		TA100		TA1535		TA1537
		Trial 1	Trial 2	Trial 1	Trial 2	Trial 1	Trial 2	Trial 1	Trial 2
Test substance	5000	32	36	142	84	14	15	10	13
	1000	38	34	145	112	17	18	16	16
	500	37	27	129	109	22	24	22	23
	100	41	36	143	106	21	23	21	21
	50	39	41	123	106	23	22	22	22
Solvent control	_	39	41	124	98	25	23	23	23
Positive control	_	1994	1999	1775	1734	156	147	217	221

Conclusions:

Negative with and without the metabolic activation in S. typhimurium strains

Executive summary:

The test substance was evaluated for its potential to cause mutations at the histidine operon of Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537. The study was conducted following FIFRA guideline 84-2. The test substance was evaluated for toxicity to strain TA100 in the range finding test. Tester strain TA100 was exposed to the test substance in the absence of exogenous activation and in the presence of Aroclor 1254-induced rat liver S-9 plus cofactors. The toxicity was evaluated based on 1) reversion frequency, 2) viability, and 3) integrity of the background lawn. The test substance was evaluated at concentrations ranging from 5000 to 5.0 µg/plate in both the absence and presence of exogenous activation. The results of the range finding test indicated that the test substance produced a significant degree of toxicity at concentrations of 1000 µg/plate and above in the absence of S-9 activation. A moderate toxicity at concentrations of 1000 µg/plate and above was observed in the presence of S-9 activation.

The initial mutation assay was performed using concentrations of 1000, 500, 100, 50 and 10 µg/plate and 5000, 1000, 500, 100, and 50 µg/plate in the absence and presence of S-9, respectively. The same concentrations were used in the confirmatory mutation assay to confirm the results of the initial mutation assay. The results of both mutation assays indicated that the test substance did not induce any positive increase in the number of revertant colonies for any of the tester strains in the absence and presence of Aroclor 1254-induced rat liver S-9.

Under the conditions of this study, the test substance was negative in the Salmonella typhimurium preincubation mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

other: In vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Substance ID: TSN 100097
- Name of substance: XDE-795
- Purity: 97.4%

Species / strain / cell type:

lymphocytes: rat

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Outbred Crl:CD BR rat strain purchased from Charles River, Kingston, New York
- Sex, age and number of blood donors: Male rats aged approximately 18 weeks in Assay 1 and approximately 14 weeks in Assay 2. blood was collected from 2 rats in Assay 1 and 3 rats in Assay 2.
- Whether whole blood or separated lymphocytes were used: Whole blood

MEDIA USED: RPMI 1640 medium
- Properly maintained: Yes
- The medium was supplemented with 10% heat-inactivated fetal bovine serum, antibiotics and antimycotics, PHA and an additional 2 mM L-glutamine.

Metabolic activation:

with and without

Metabolic activation system:

S-9 liver homogenate prepared from Aroclor 1254 treated male Sprague-Dawley rats

Test concentrations with justification for top dose:

Assay 1: 3.1, 6.3, 12.5, 25.0, 50.0, 100 µg/mL
Assay 2: 25, 50, 100 µg/mL

Vehicle / solvent:

Dimethyl sulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: Approximately 4 hours
- Expression time (cells in growth medium): Approximately 24 and 48 hours after termination of the treatment
- Fixation time: Approximately 3 hours prior to the harvest

FIXATIVE: methanol:acetic acid (3:1)

SPINDLE INHIBITOR: Colcemid

STAIN: Giemsa

NUMBER OF REPLICATIONS: Cells plated in duplicates

NUMBER OF CELLS EVALUATED: 1000 cells/replicate

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 100 per replicate for the test substance and 50 per replicate for positive control

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index (determined as the number of cells in metaphase among 1000 cells/replicate and expressed as percentrages)

Evaluation criteria:

A test substance 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.

Key result

Species / strain:

lymphocytes: rat

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Assay-1: The average mitotic index (M.I.) of cultures treated with the highest concentration (100 µg/mL) in the absence of S-9 was reduced by 40% relative to the negative controls. However, there was no dose-related trend in the mitotic inhibition as evidenced by a lack of an effect at 50 µg/mL and the reduction in mitotic activity at the lower dose levels. This may be related to the relative insolubility of the test substance in the aqueous solution. In the presence of S-9, the M.I. of the highest dose tested (i.e., 100 µg/mL) was reduced by approximately 53% and there was no effect at the lower dose levels.
Assay-2: In the absence of S-9, at the highest concentration tested (i.e., 100 µg/mL) there was a 53% reduction in the mitotic activity in cultures harvested 24 h after treatment and a 37% reduction in the M.I. in cultures harvested at 48 h. In the activation assay, a mitotic inhibition of 39% was observed in the 24 h harvest and a 46% inhibition in the 48 h harvest time at the highest concentration, i.e., 100 µg/mL of test substance.

Table-1: Results of the chromosomal aberration assay 24 hours after treatment in the absence of S-9 (Assay-1)

	Negative control (1% DMSO)	25 µg/mL	50 µg/mL	100 µg/mL	Positive control
	Replicate A+B	Replicate A+B	Replicate A+B	Replicate A+B	Replicate A+B
Chromatid gaps	14	28	13	9	15
Chromosome gaps	2	8	2	2	5
Chromatid breaks	13	23	2	2	7
Chromatid exchanges	0	5	0	0	37
Chromosome breaks	0	4	0	0	10
Chromosome exchanges	0	0	0	0	0
Total aberrations (excluding gaps)^a	13 (6.5)	33 (16.5)	2 (1.0)	2 (1.0)	54 (54.0)
No. of cells with Aberr. (excluding gaps)^a	13 (6.5)	25 (12.5)	2 (1.0)	2 (1.0)	48^b (48.0)
Miscellaneous Aberr.	0	1	0	0	0
Cells with multiple Aberr. (5 or more aberr.)	1	0	0	0	14

a = Values in parentheses are percentages; b = Significantly (alpha <0.01) different from the negative control

Table-2: Results of the chromosomal aberration assay 24 hours after treatment in the presence of S-9 (Assay-1)

	Negative control (1% DMSO)	25 µg/mL	50 µg/mL	100 µg/mL	Positive control
	Replicate A+B	Replicate A+B	Replicate A+B	Replicate A+B	Replicate A+B
Chromatid gaps	6	7	7	12	10
Chromosome gaps	0	2	3	2	3
Chromatid breaks	1	3	1	7	19
Chromatid exchanges	0	0	0	0	7
Chromosome breaks	0	1	1	1	12
Chromosome exchanges	0	0	0	0	0
Total aberrations (excluding gaps)^a	1 (0.5)	4 (2.0)	2 (1.0)	8 (4.0)	38 (38.0)
No. of cells with Aberr. (excluding gaps)^a	1 (0.5)	3 (1.5)	2 (1.0)	8 (4.0)	28^b (28.0)
Miscellaneous Aberr.	0	0	0	0	0
Cells with multiple Aberr. (5 or more aberr.)	0	0	0	0	1

a = Values in parentheses are percentages; b = Significantly (alpha <0.01) different from the negative control

Table-3: Results of the chromosomal aberration assay 24 hours after treatment in the absence of S-9 (Assay-2)

	Negative control (1% DMSO)	25 µg/mL	50 µg/mL	100 µg/mL	Positive control
	Replicate A+B	Replicate A+B	Replicate A+B	Replicate A+B	Replicate A+B
Chromatid gaps	7	2	6	6	7
Chromosome gaps	1	1	1	1	3
Chromatid breaks	5	2	1	4	29
Chromatid exchanges	0	0	0	0	42
Chromosome breaks	0	0	0	0	14
Chromosome exchanges	0	0	0	0	0
Total aberrations (excluding gaps)^a	5 (2.5)	2 (1.0)	1 (0.5)	4 (2.0)	85 (85.0)
No. of cells with Aberr. (excluding gaps)^a	5 (2.5)	2 (1.0)	1 (0.5)	3 (1.5)	53^b (53.0)
Miscellaneous Aberr.	0	0	0	0	0
Cells with multiple Aberr. (5 or more aberr.)	0	0	0	0	4

a = Values in parentheses are percentages; b = Significantly (alpha <0.01) different from the negative control

Table-4: Results of the chromosomal aberration assay 48 hours after treatment in the absence of S-9 (Assay-2)

	Negative control (1% DMSO)	100 µg/mL
	Replicate A+B	Replicate A+B
Chromatid gaps	1	6
Chromosome gaps	1	3
Chromatid breaks	0	2
Chromatid exchanges	0	1
Chromosome breaks	0	2
Chromosome exchanges	0	0
Total aberrations (excluding gaps)^a	0 (0.0)	6 (3.0)
No. of cells with Aberr. (excluding gaps)^a	0 (0.0)	6 (3.0)
Miscellaneous Aberr.	0	0
Cells with multiple Aberr. (5 or more aberr.)	0	0

a = Values in parentheses are percentages

Table-5: Results of the chromosomal aberration assay 24 hours after treatment in the Presence of S-9 (Assay-2)

	Negative control (1% DMSO)	25 µg/mL	50 µg/mL	100 µg/mL	Positive control
	Replicate A+B	Replicate A+B	Replicate A+B	Replicate A+B	Replicate A+B
Chromatid gaps	2	2	3	8	4
Chromosome gaps	0	0	2	0	5
Chromatid breaks	3	0	2	2	10
Chromatid exchanges	0	0	0	0	17
Chromosome breaks	2	1	0	0	10
Chromosome exchanges	0	0	0	0	1
Total aberrations (excluding gaps)^a	5 (2.5)	1 (0.5)	2 (1.0)	2 (1.0)	38 (38.0)
No. of cells with Aberr. (excluding gaps)^a	5 (2.5)	1 (0.5)	2 (1.0)	2 (1.0)	27^b (27.0)
Miscellaneous Aberr.	0	0	0	0	0
Cells with multiple Aberr. (5 or more aberr.)	0	0	0	0	2

a = Values in parentheses are percentages; b = Significantly (alpha <0.01) different from the negative control

Table-6: Results of the chromosomal aberration assay 48 hours after treatment in the presence of S-9 (Assay-2)

	Negative control (1% DMSO)	100 µg/mL
	Replicate A+B	Replicate A+B
Chromatid gaps	2	6
Chromosome gaps	2	3
Chromatid breaks	1	3
Chromatid exchanges	0	0
Chromosome breaks	0	1
Chromosome exchanges	0	0
Total aberrations (excluding gaps)^a	1 (0.5)	4 (2.0)
No. of cells with Aberr. (excluding gaps)^a	1 (0.5)	4 (2.0)
Miscellaneous Aberr.	0	0
Cells with multiple Aberr. (5 or more aberr.)	0	0

a = Values in parentheses are percentages

Conclusions:

The test substance was not clastogenic to rat lymphocytes in culture

Executive summary:

The clastogenic potential of the test substance was evaluated in an in vitro chromosomal aberration assay utilizing rat lymphocytes following OECD guideline 473 and EPA OPP 84-2. Approximately 48 hours after the initiation of whole blood cultures, cells were treated for 4 hours in the presence and absence of an external metabolic activation system (S-9) with 0 (negative control), 3.1, 6.3, 12.5, 25, 50, and 100 µg test substance per ml of culture medium. Cultures treated with 0.5 µg/ml mitomycin C or 6 µg/ml cyclophosphamide were used as positive controls for the non-activation and activation assays, respectively. In the first experiment, the cultures were harvested 24 h after termination of treatment. Based upon the mitotic indices, cultures treated. with 0, 25, 50 and 100 µg/ml in the absence and presence of S-9 were selected for determining the incidence of chromosomal aberrations. In a confirmatory assay utilizing 2 harvest times (i.e., 24 and 48 h after termination of treatment), the incidence of chromosomal abnormalities was determined from cultures treated. with 0, 25, 50, and 100 µg/ml both in the presence and absence of S-9 at the first harvest time, and from cultures treated with 0 and 100 µg/ml at the second harvest. No significant increase in the incidence of abnormal cells was noticed at any of the treatment levels in Assay 1 or Assay 2 when compared to the negative controls. The positive control chemicals had significantly higher incidences of abnormal cells. Hence, the test substance was considered to be negative in the in vitro chromosomal aberration assay utilizing rat lymphocytes.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

GLP compliance:

yes

Type of assay:

other: gene mutation test at HGPRT locus

Specific details on test material used for the study:

Substance ID: TSN 100097
Name of substance: XDE-795
Purity: 96.2%

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HGPRT)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Subcultured cells used which were originally obtained from Dr. Patrick O'Neill, University of Vermont
- Suitability of cells: Cells exhibited a high cloning efficiency and a short doubling time of 12-14 hours

MEDIA USED
- Type and identity of media including CO2 concentration: The CHO cells were cultured in hypoxanthine-free (Hx-free) nutrient medium supplemented with 10% heat-inactivated fetal bovine serum (HIFBS), 2mM of L-glutamine, 50 units/ml of penicillin and 50 µg/ml of streptomycin (complete culture medium) in T-75 cm2 plastic tissue culture flasks at 37 ± 1°C in a humidified incubator set at 5% CO2 and 95% air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver homogenate (S-9 fraction) and the cofactor pool

Test concentrations with justification for top dose:

2.5, 5.0, 10, 15 and 20 µg/ml without activation and 10, 20, 40, 60 and 80 µg/ml with activation. The dose were selected based on the range finding study

Vehicle / solvent:

- Solvent used: DMSO (for test substance and positive control EMS); Acetone (for positive control DMBA)

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium
- Cell density at seeding: Duplicate cultures seeded with 3x10e6 cells/flask were used at each dose level

DURATION
- Exposure duration: 4 h
- Expression time: The cells from each of the duplicate culture flasks were subcultured in hypoxanthine-free Ham's F-12 nutrient medium supplemented with 5% dialyzed HIFBS, 2mM L-glutamine, 50 units/ml penicillin and 50 µg/ml streptomycin {cloning medium} at a density of 2x10e6 cells/T-150 cm² flask. The cells were subcultured at 2- to 3- day intervals for 8 or 9 days, prior to selecting the mutant phenotypes.
- Selection time: To determine the cloning efficiency of the cells at the time of selection, 200 cells/60 mm dish were plated in triplicate in the cloning medium. All clonable test doses and appropriate positive and solvent controls were cloned for mutant selection. The cultures were then incubated for 7 days without disturbing the plates to minimize the formation of satellite colonies.

STAIN: Giemsa stain

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The colonies were washed with phosphate buffered saline (PBS), fixed with methanol, stained with Giemsa stain, and counted.

NUMBER OF CELLS EVALUATED: 200 cells per plate

DETERMINATION OF CYTOTOXICITY
- Method: The cells from each flask were trypsinized, and then seeded in triplicate at a density of 200 cells/60 mm dish. The cells were allowed to grow for a period of 7 days without any disturbance to minimize the formation of satellite colonies. The colonies were then washed with phosphate buffered saline (PBS), fixed with methanol, stained with Giemsa stain, and counted. A cluster of more than 50 cells growing within a confined area was considered a colony. The average number of colonies per plate was calculated, and the Relative Cloning Efficiency (RCE) was determined

Evaluation criteria:

For an assay to be acceptable, the mutation frequency in the positive controls should be significantly higher than the background frequency, and the mutation frequency in the negative controls should be within reasonable limits of the historical control values of the test facility and the literature values. The test substance was considered positive in the assay if it induced a statistically significant and reproducible increase in mutation frequency at more than one of the dose levels tested. The final interpretation of the data also took into consideration such factors as the mutation frequency and cloning efficiencies in the negative controls and dose-response relationships.

Statistics:

The frequency of mutants 1x10e6 clonable cells was analyzed by the Cochran-Armitage test for trend (≤0.025, one-sided, increasing) and the Fisher-Irwin exact test for group comparisons for proportions (≤0.01, one-sided).
Within-group comparisons were made by the Fisher-Irwin exact test (≤0.01).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No change
- Effects of osmolality: Within limit
- Precipitation: The concentration of 80 µg/ml formed a precipitate in the culture medium at the time of treatment, indicating that the solubility limit of the test article in the culture medium had been exceeded.

Parallel Toxicity Test of Definitive Mutation Assay: In the non-activated system, the concentrations of 5.0, 10, 15, and 20 µg/ml had average RCEs of 78%, 62%, 53% and 32%, respectively. The concentration of 2.5 µg/ml was relatively nontoxic.
In the activated system, the concentrations of 10, 20, 40, 60 and 80 µg/ml had average RCEs of 71%, 41%, 24% 12% and 3%, respectively.

RANGE-FINDING STUDY: In the range finding test, the test article was tested at dose levels of 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16, 31, 63 and 125 µg/ml. In the non-activated system, the test substance concentrations of 63 and 125 µg/ml were completely toxic. The concentrations of 4.0, 8.0, 16 and 31 µg/ml had RCEs of 74%, 73%, 20%, and 2%, respectively. The lower concentrations were relatively nontoxic. In the activated system, the top two concentrations of 63 and 125 µg/ml had RCEs of 36% and 5%, respectively. The lower concentrations were nontoxic.

Assay results:

Conc. µg/mL	Definitive assay -Mutants per 1 x 10e^6surviving cell without activation	Definitive assay -Mutants per 1 x 10e^6surviving cell with activation	Confirmatory assay - Mutants per 1 x 10e^6surviving cell without activation	Confirmatory assay - Mutants per 1 x 10e^6surviving cell with activation
Solvent control	11.5	13.5	6	3.5
2.5	4	7.5	2.5	3
5.0	11.5	8	3.5	2
10.0	1	13.5	4.5	3.5
15.0	2	8	0	0
20.0	3.5	6	0	0
Positive control	139	238.5	175.5	182.5

Conclusions:

Under the conditions of the study, the test substance did not induce gene mutation in Chinese hamster ovary cells.

Executive summary:

The test substance was evaluated for its potential to cause gene mutation at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in cultured Chinese hamster ovary cells (CHO) with and without exogenous metabolic activation per the FIFRA Guideline No. 84-2.

A range finding test was performed to assess the toxicity to CHO cells within this test system. Ten concentrations at doses ranging from 0.25 to 125 µg/ml were tested with and without metabolic activation. Toxicity was assessed by measuring the reduction in Relative Cloning Efficiency (RCE). Doses for the Mutation Assays were then selected based on 0-100% cytotoxicity.

The Mutation Assay was performed in two independent trials: A definitive and a confirmatory Assay. Doses were selected based on data from the Range Finding Test. The doses in the definitive mutation assay were: 2.5, 5.0, 10, 15 and 20 µg/ml without activation and 10, 20, 40, 60 and 80 µg/ml with activation. The same concentrations were tested in the confirmatory mutation assay, also. After treatment, the cells underwent an 8-9 day expression period. At the conclusion of the expression period, the cultures were cloned in medium containing 6-thioguanine to select HGPRT enzyme-deficient mutants. These cultures were then incubated for 7 days. After 7 days, the cultures were fixed, stained, and the cloning efficiency and mutant frequencies were calculated for each culture by counting the number of colonies.

The test substance did not cause a statistically significant reproducible increase in the mutant frequency at the HGPRT locus among the test article-treated cultures either in the absence or presence of exogenous metabolic activation. The mutant frequencies of the solvent controls were within the test facility historical negative control values. The positive controls caused a significant increase in the mutant frequencies. The test substance did not induce gene mutations in Chinese hamster ovary cells when tested under the conditions of the study and, therefore, was negative in this study.

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. OECD 474; Reliability = 1

Link to relevant study records

Reference

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:

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:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: bone marrow micronucleus test

Specific details on test material used for the study:

- Substance ID: TSN 100097
- Name of substance: XDE-795
- Purity: 97.4%

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, Michigan
- Assigned to test groups randomly: Yes
- Housing: Individually housed
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least 7 days

Route of administration:

oral: gavage

Vehicle:

Corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was mixed with corn oil for dosing. Cyclophosphamide (CP) was dissolved in distilled water. Freshly prepared solutions were used for dosing the animals. The concentrations of the test substance in the dosing solutions were verified by high pressure liquid chromatography (HPLC).

Duration of treatment / exposure:

24, 48, or 72 h after treatment

Frequency of treatment:

single

Dose / conc.:

1 250 mg/kg bw/day

Dose / conc.:

2 500 mg/kg bw/day

Dose / conc.:

5 000 mg/kg bw/day

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control: Cyclophosphamide was selected to induce an unequivocal positive response
- Route of administration: Oral
- Doses / concentrations: 120 mg/kg bw (10 mL/kg)

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The highest dose level of 5000 mg/kg bw was based upon the results of a range-finding test and it is also the generally accepted limit dose for relatively non-toxic test substances.

BONE MARROW SAMPLING: At the end of the specified intervals following dosing, the animals were sacrificed by cervical dislocation. Bone marrow samples were obtained from both femurs in the following way. After separating the bone from the adjoining muscles, the distal end of the femur was severed to expose the marrow cavity. A 25-gauge needle was used to aspirate the bone marrow into a 3 ml disposable plastic syringe containing approximately 0.5 ml of fetal bovine serum. After aspiration, the contents of the syringe were transferred into a 1.5 ml centrifuge tube containing 0.5 ml of serum. The cells were resuspended in the serum by gentle aspiration using the syringe and needle. The tubes were centrifuged at 1000 rpm (approximately 80 g) for approximately 5 minutes in a table-top centrifuge. The supernatant was discarded leaving a small amount of serum covering the pellet. The cell pellet was resuspended using a disposable transfer pipette. Wedge smears were prepared on microscope slides using small portions of the cell suspension. The slides were allowed to air dry and stained with Wright-Giemsa using a Hematek automatic slide stainer.

SLIDE SCORING: The slides were coded and scored blindly. One thousand polychromatic erythrocytes were examined from each animal and the number of micronucleated polychromatic erythrocytes (MN-PCE) was recorded. Micronuclei were identified as darkly stained bodies with smooth contours and varying shapes such as round, almond, or ring. The ratio of PCE-NCE in the bone marrow was determined by examining 1000 erythrocytes. The ratio was expressed as PCEx100/PCE+NCE.

Statistics:

The raw data on the counts of MN-PCE for each animal were first transformed by adding 1 to each count and then taking natural log of the adjusted number. The transformed MN-PCE data and the data on percent PCE were analyzed by a three-way analysis of variance (sex, dose, and time), assuming the three-way interaction to be zero. From this initial analysis, the two-way interactions were reviewed for significance. Depending upon this review, the data were analyzed by either one, two, or three-way analysis of variance looking only at main effects. Pairwise comparisons of treated vs. negative control groups were done, if necessary, by Dunnett's t-tests, one-sided (upper) for MN-PCE and two-sided for percent PCE. The alpha level at which all the tests were conducted was 0.01.

The final interpretation of biological significance of the responses was based on both statistical outcome and scientific judgment.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2500 and 5000 mg/kg bw
- Mortality: 10 of 10 animals treated with 5000 mg/kg bw of the test substance survived. At the 2500 mg/kg bw dose level, one female died on day two of observation. Necropsy of the dead animal indicated no evidence of gavage error. The cause of death was undetermined.
- Clinical signs of toxicity in test animals: All animals appeared normal immediately after dosing. At three hours post-dosing, all animals were exhibiting signs of lethargy. Six hours after dosing, one female and three males in the 2500 mg/kg bw group and two females and all males in the 5000 mg/kg bw group appeared to be lethargic. On day two of observation, one female in the 2500 mg/kg bw dose group was recorded as jumping violently and displaying erratic behavior. This animal died the same day. Two males in the 2500 mg/kg bw group remained lethargic on day 2 of observation. On day three of observations, two males were recorded as extremely lethargic and not eating as evidence of lack of fecal droppings under their cages. One of these males also had perineal staining.

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity: Lethargy was observed in most of the male mice treated with the test material two hours after dosing. At four hours post dosing, males in the 1250 mg/kg bw dose group appeared somewhat lethargic. Those in the 2500 mg/kg bw dose group were expressing varying degrees of lethargy and 7 of the 10 males in the 5000 mg/kg bw dose group appeared lethargic. At the 24-hour sacrifice, all males treated with the test materials had varying degrees of perianal staining. Lethargy was also observed in the 5000 mg/kg bw male group. The remaining males appeared to return to normal on day three of observation. At the 72 h sacrifice, a 1250mg/kg bw male group could not open its eyes due to ocular discharge.
- Induction of micronuclei: There were no significant differences in MN-PCE frequencies between the groups treated with the test substance and the negative controls. The positive control chemical, CP, induced a significant increase in the frequencies of micronucleated polychromatic erythrocytes. The percent PCE values observed in the test substance-treated animals were not significantly different from the negative control values at any dose level tested. However, the %PCE values in the test substance treated males tended to be lower than the negative controls especially in the groups harvested at 48 and 72 h after treatment. The biological significance of this trend, nevertheless, is uncertain. The positive control chemical (CP), on the other hand, had a significant effect on the ratio of PCE to NCE.

Conclusions:

Negative mouse micronucleus test

Executive summary:

The test substance was evaluated in the mouse bone marrow micronucleus test following OECD guideline 474 and US EPA 798.5395. The micronucleus test is capable of detecting agents causing chromosomal aberrations and spindle malfunction. The test substance was administered to CD-1 mice by single oral gavage at dose levels of 0 (negative control), 1250, 2500, and 5000 mg/kg body weight. The highest dose level of 5000 mg/kg bw was based. upon the results of a range-finding test and it is also the generally accepted limit dose for relatively non-toxic test substances. The concentrations of the test substance in the dosing solutions were verified by analytical methods. Groups of animals were sacrificed at 24, 48, or 72 h after treatment. Mice treated with 120 mg/kg bw cyclophosphamide and sacrificed at 24 h served as positive controls. There were five animals per sex per dose level per sacrifice time. One thousand polychromatic erythrocytes (PCE) were evaluated from each surviving animal and the frequencies of micronucleated polychromatic erythrocytes (MN-PCE) were recorded. There were no statistically significant increases in the frequencies of MN-PCE in groups treated with the test substance as compared to negative controls. The positive control mice showed significant increases in MN-PCE. Hence, under the experimental conditions used, the test substance was considered to be negative in the mouse bone marrow micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

A bacterial reverse mutation assay was conducted using 4 strains of S. typhimurium. The initial assay involved the standard plate incorporation protocol using 6 test concentrations over a dose range of 5 to 5000 µg per plate. The experiment was conducted both in the presence and absence of an auxiliary metabolic activation system (rat liver S-9 mix). The results of the range finding test indicated that the test substance produced a significant degree of toxicity at concentrations of 1000 µg/plate and above in the absence of S-9 activation. A moderate toxicity at concentrations of 1000 µg/plate and above was observed in the presence of S-9 activation. The initial mutation assay was performed using concentrations of 1000, 500, 100, 50 and 10 µg/plate and 5000, 1000, 500, 100, and 50 µg/plate in the absence and presence of S-9, respectively. The same concentrations were used in the confirmatory mutation assay to confirm the results of the initial mutation assay. The results of both mutation assays indicated that the test substance did not induce any positive increase in the number of revertant colonies for any of the tester strains in the absence and presence of Aroclor 1254-induced rat liver S-9. The test substance was negative in the Salmonella typhimurium preincubation mutation assay. 

 

The clastogenic potential of the test substance was evaluated in an in vitro chromosomal aberration assay utilizing rat lymphocytes. Approximately 48 hours after the initiation of whole blood cultures, cells were treated for 4 hours in the presence and absence of an external metabolic activation system (S-9) with 0 (negative control), 3.1, 6.3, 12.5, 25, 50, and 100 µg test substance per ml of culture medium. Cultures treated with 0.5 µg/ml mitomycin C or 6 µg/ml cyclophosphamide were used as positive controls for the non-activation and activation assays, respectively. In the first experiment, the cultures were harvested 24 h after termination of treatment. Based upon the mitotic indices, cultures treated. with 0, 25, 50 and 100 µg/ml in the absence and presence of S-9 were selected for determining the incidence of chromosomal aberrations. In a confirmatory assay utilizing 2 harvest times (i.e., 24 and 48 h after termination of treatment), the incidence of chromosomal abnormalities was determined from cultures treated. with 0, 25, 50, and 100 µg/ml both in the presence and absence of S-9 at the first harvest time, and from cultures treated with 0 and 100 µg/ml at the second harvest. No significant increase in the incidence of abnormal cells was noticed at any of the treatment levels in Assay 1 or Assay 2 when compared to the negative controls. The positive control chemicals had significantly higher incidences of abnormal cells. Hence, the test substance was considered to be negative in the in vitro chromosomal aberration assay utilizing rat lymphocytes.

 

The test substance was evaluated for its potential to cause gene mutation at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in cultured Chinese hamster ovary cells (CHO) with and without exogenous metabolic activation. A range finding test was performed to assess the toxicity to CHO cells within this test system. Ten concentrations at doses ranging from 0.25 to 125 µg/ml were tested with and without metabolic activation. Toxicity was assessed by measuring the reduction in Relative Cloning Efficiency (RCE). Doses for the mutation assays were then selected based on 0-100% cytotoxicity. The mutation assay was performed in two independent trials: A definitive and a confirmatory assay. Doses were selected based on data from the rangefinding test. The doses in the definitive mutation assay were: 2.5, 5.0, 10, 15 and 20 µg/ml without activation and 10, 20, 40, 60 and 80 µg/ml with activation. The same concentrations were tested in the confirmatory mutation assay, also. The test substance did not cause a statistically significant reproducible increase in the mutant frequency at the HGPRT locus among the test article-treated cultures either in the absence or presence of exogenous metabolic activation. The mutant frequencies of the solvent controls were within the test facility historical negative control values. The positive controls caused a significant increase in the mutant frequencies. The test substance did not induce gene mutations in Chinese hamster ovary cells when tested under the conditions of the study and, therefore, was negative in this study.

 

In the mouse bone marrow micronucleus test, the test substance was administered to CD-1 mice by single oral gavage at dose levels of 0 (negative control), 1250, 2500, and 5000 mg/kg body weight. The highest dose level of 5000 mg/kg bw was based upon the results of a range-finding test and it is also the generally accepted limit dose for relatively non-toxic test substances. Groups of animals were sacrificed at 24, 48, or 72 h after treatment. Mice treated with 120 mg/kg bw cyclophosphamide and sacrificed at 24 h served as positive controls. There were five animals per sex per dose level per sacrifice time. There were no statistically significant increases in the frequencies of MN-PCE in groups treated with the test substance as compared to negative controls. The positive control mice showed significant increases in MN-PCE. Hence, under the experimental conditions used, the test substance was considered to be negative in the mouse bone marrow micronucleus test.

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.