heptan-2-yl [(5-chloroquinolin-8-yl)oxy]acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation: not mutagenic with and without metabolic activation (OECD 471, Sokolowski 2010)

Mutation in mammalian cells: not mutagenic with and without metabolic activation (OECD 476, Dollenmeier 1987)

Chromosomal aberration, clastogenicity: negative with and without metabolic activation (OECD 473, Ogorek 1998)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 June 1998 to 03 September1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Chinese hamster ovary cells (ATCC CCL61)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

Cytotoxicity test: 0, 0.39, 0.78, 1.56, 3.13, 6.25, 12.50, 25 and 50 µg/mL
Experiments without metabolic activation: 0, 6.25, 12.50 and 25.00 µg/mL
Experiments with metabolic activation: 0, 12.50, 25.00 and 50.00 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 Migrated to IUCLID6: 0.2 µg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 Migrated to IUCLID6: 20.0 µg/mL

Details on test system and experimental conditions:

DURATION
- Preincubation period: approximately 24 hours
- Exposure duration: 21 or 45 hours (without S9); 3 hours treatment followed by 18 hours recovery or 3 hours treatment followed by 18 hours recovery (with S9)

SPINDLE INHIBITOR
- Two hours prior to harvesting, the cultures were treated with 0.4 µg/mL Colcemide to arrest cells in metaphase. After harvesting the cells were spread on glass slides and air dried.

NUMBER OF REPLICATIONS: 4

NUMBER OF CELLS EVALUATED:
- The percentage of mitotic suppression was determined by evaluating at least 2000 cells from each slide. Whenever possible two hundred well spread metaphase figures with 19 to 21 centromeres from two cultures (100 metaphases per replicate culture) in the vehicle control and in the treated groups were scored. At least fifty metaphases were scored in the positive controls (25 per replicate culture).
- The slides were examined for specific and unspecific structural aberrations.
- The percentages of mitotic suppression, in comparison with the controls, were evaluated by counting at least 2000 cells per slide for each treatment group.

DETERMINATION OF CYTOTOXICITY
- Determination of cloning efficiency: Cell cultures were exposed to the test substance for 3 hours in the presence of a metabolic activation system. Four concentrations of the test substance and the vehicle (DMSO) control were tested. The treatment was terminated by washing the cultures with phosphate buffered saline (PBS). The cells were then suspended by trypsinisation, pelleted, resuspended in fresh growth medium and counted with a haemocytometer. The cultures were diluted so that 100 cells were seeded per 9.6 cm² in 3 ml of growth medium. After seven to eight days of growth at 37°C the cultures were fixed and stained with Giemsa and the surviving colonies (cloning efficiency) were determined by eye.

Evaluation criteria:

Criterion for positive response based on an increased incidence of specific chromosomal aberrations.

Criteria for a positive test: the percentage of metaphases containing specific aberrations in a treatment group is higher than 6.0, and differs statistically from the respective negative control, with a demonstrable concentration-related response.

Criteria for a negative response: percentage of metaphases containing specific aberrations in a treatment group is less than or equal to 6.0, and does not differ statistically from the respective negative control.

Statistics:

Not applicable

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Analysis for test article content of the lowest, by serial dilution prepared stock solutions used in the mutagenicity test revealed that the test article was stable in the vehicle used and that the cells were exposed to the intended concentrations. The actual concentrations ranged between 94.4 –95.6% of the nominal concentrations.

Cytotoxicity test - Concentrations higher than 50 µg/mL could not be tested, due to solubility limitations of the test substance in the vehicle DMSO. In the experiments with and without metabolic activation, marked cytotoxicity was observed at the highest concentrations.

In all experiments performed with or without metabolic activation no statistically significant increase in the number of metaphases containing specific chromosomal aberrations was observed.

Remarks on result:

other: strain/cell type: CHO cells ATCC CCL61

Remarks:

Migrated from field 'Test system'.

Table 1: CGA185072: Mitotic Index (% of control) of CHO cells

 

	Original study			Confirmatory study
Experiment	1	2	3	4	5	6
Metabolic Activation	-	+	-	+	-	+
Treatment (h)	21	3	21	3	45	3
Recovery (h)	-	18	-	18	-	42
CGA185072
50.00 µg/mL	0	95	0	138	1	79
25.00 µg/mL	20	107	19	106	50	95
12.50 µg/mL	103	96	89	93	89	99
6.25 µg/mL	96		71		128
3.13 µg/mL	109		94		87
1.56 µg/mL
0.78 µg/mL
0.39 µg/mL		103		101		94

 

Table 2: Percent incidence of specific chromosomal aberrations in CHO cells treated with CGA185072

	Original study			Confirmatory study
Experiment	1	2	3	4	5	6
Metabolic Activation	-	+	-	+	-	+
Treatment (h)	21	3	21	3	45	3
Recovery (h)	-	18	-	18	-	42
Negative control	2.0	2.0	2.5	2.0	3.0	3.5
CGA185072
50.00 µg/mL
25.00 µg/mL		3.0		4.0		4.0
12.50 µg/mL	3.0	2.0	4.5	4.5	2.5	1.0
6.25 µg/mL	2.0	2.5	0.5	3.5	3.0	3.0
3.13 µg/mL	0.5		2.0		2.0
1.56 µg/mL
0.78 µg/mL
Positive control^a,b	80.0***	60.0***	80.0***	70.0***

200 cells with well spread metaphase figures were scored, except for positive controls where 50 cells were scored.

-  no positive control

^aCyclophosphamide, 20 µg/mL             ^bMitomycin-C, 0.2 µg/mL

* p ≥ 0.05; *** p ≤ 0.001 (Chi-square test)

Conclusions:

negative with and without metabolic activation

Executive summary:

The clastogenicity of the test substance was tested under GLP to OECD TG 473 in an in vitro study. Chinese hamster ovary cells (ATCC CCL61) were incubated with several concentrations of cloquintocet-mexyl. Mitomycin C (0.2 µg/mL, without metabolic activation), and cyclophosphamide (20.0 µg/mL, with metabolic activation), were used as positive controls. Experiments without metabolic activation were conducted at 0, 6.25, 12.50 and 25.00 µg/mL in the main study, experiments with metabolic activation were conducted at 0, 12.50, 25.00 and 50.00 µg/mL in the main study.

In all experiments performed with or without metabolic activation, no statistically significant increase in the number of metaphases containing specific chromosomal aberrations was observed.

Cloquintocet-mexyl caused no clastogenic effects in Chinese hamster ovary cells in vitro.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 February 1987 to 24 June 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

No analytical confirmation of dosing solution

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

6-GT resistance in hgprt gene

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Chinese hamster cells V79

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal fraction S9 derived from Arochlor 1254 induction

Test concentrations with justification for top dose:

Toxicity test:
0-250 µg/mL (without metabolic activation),
0-500 µg/mL (with metabolic activation)
Original mutagenicity test:
0-250 µg/L (without metabolic activation),
0-500 µg/mL (with metabolic activation)
Confirmatory mutagenicity test:
0-250 µg/mL (without metabolic activation),
0-500 µg/mL (with metabolic activation)

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: Ethylmethane sulfonate 300 nmol/mL (+S9); N-Nitrosodimethylamine 1µl/mL (-S9)

Details on test system and experimental conditions:

Duration: 5 hours (+S9) , 21 hours (-S9)
Expression period: 5 days
Mutant selection: 5 days at 37°C in the presence of 8 µl/mL 6-TG

Selection agent: 6-TG

Number of replications: 2
Number of cells evaluated: 18 x 10^5

Determination of cytotoxicity: Cloning efficiency

Evaluation criteria:

Comparison in number of mutant clones

Statistics:

Not conducted

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cytotoxicity:
100% cytotoxicity at 500 µg/mL cloquintocet-mexyl with metabolic activation
100% cytotoxicity at 250 µg/mL cloquintocet-mexyl without metabolic activation

Mutagenicity tests:
Evaluated at concentrations from 25 to 500 µg/mL cloquintocet-mexyl with metabolic activation and 12.5 to 250 µg/mL cloquintocet-mexyl without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

In the experiments performed with and without microsomal activation comparison of the number of mutant clones revealed no significant deviations between cultures treated with cloquintocet-mexyl and untreated controls. The positive control substances showed clear mutagenic effects with and without metabolic activation.

Table 1: V79 assay with cloquintocet-mexyl: Summary of the results of the mutagenicity experiments with metabolic activation (Incidence of mutant clones (mutant frequency x10^-6, normalised to plating efficiency of 100%).

Concentration [µg/mL]	Mutant frequency Original experiment	viable clones per dish	Mutant frequency Confirm. experiment	viable clones per dish
Solvent control	4.0	46.00	5.1	54.83
Positive control	164.1	16.00	75.3	45.00
CGA185072 mg/mL
25	4.0	53.83	4.0	74.83
50	8.0	34.67	4.0	64.67
100	4.0	47.50	4.0	77.33
200	4.0	50.00	4.0	55.00
300	14.6	34.17	4.0	66.00
400	10.7	26.00	4.0	61.83
500	4.0	24.50	5.1	54.67

 

Table 2: V79 assay with cloquintocet-mexyl: Summary of the results of the mutagenicity experiments without metabolic activation (Incidence of mutant clones (mutant frequency x 10^-6, normalised to plating efficiency of 100%).

Concentrations [µg/mL]	Mutant frequency Original experiment	viable clones per dish	Mutant frequency Confirm. experiment	viable clones per dish
Solvent control	4.0	35.33	4.0	42.67
Positive control	1973.3	15.0	1388.2	19.33
CGA185072 mg/mL
12.5	4.0	31.67	4.0	49.33
25	4.0	42.50	4.0	50.17
50	6.9	32.00	4.0	49.00
100	11.6	33.67	4.0	46.83
150	4.0	36.17	4.0	47.17
200	4.0	36.33	4.0	43.33
250	4.0	33.67	4.0	54.33

Conclusions:

negative with metabolic activation
negative without metabolic activation

Executive summary:

Cloquintocet mexyl was tested under GLP to OECD TG 476 for mutagenic activity in Chinese hamster V79 cells with and without metabolic activation. A range of concentrations was tested. 100% cytoxicity at 500 µg/mL cloquintocet-mexyl with metabolic activation and at 250 µg/mL cloquintocet-mexyl without metabolic activation was observed. The mutation tests were evaluated at concentrations from 25 to 500 µg/mL cloquintocet-mexyl with metabolic activation and 12.5 to 250 µg/mL cloquintocet-mexyl without metabolic activation. There were no increases in mutant clones at any dose level tested. Cloquintocet-mexyl is not mutagenic to mammalian cells in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiated 11 August 2009. Experimental phase 17 to 28 August 2009

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 OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain / cell type:

E. coli, other: WP2 pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 [phenobarbital (80 mg/kg) i.p. and β-naphthoflavone p.o. induced]

Test concentrations with justification for top dose:

3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate (active ingredient)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (purity > 99 %)
- Justification for choice of solvent/vehicle: because of its solubility properties and its relative non-toxicity to the bacteria

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9, 10 µg/plate, TA1535 and TA100

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

without S9, 10 µg/plate, TA1537 and TA98

Positive control substance:

methylmethanesulfonate

Remarks:

without S9, 3 µL/plate, WP2 uvrA pKM101 and WP2 pKM101

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9, 2.5 µg/plate (TA 1535, TA 1537, TA 98, TA 100) and 10 µg/plate (WP2 uvrA pKM 101, WP2 pKM 101)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar; plate incorporation (experiment 1) and preincubation (experiment II)

DURATION
- Preincubation: 60 minutes
- Incubation period: at least 48 hours (at 37°C in the dark)

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

Not required

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative 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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli, other: WP2 pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at higher concentrations in strain TA 1535 without metabolic activation and in strains TA 1535, TA 98, and TA 100 with metabolic activation in experiment I. In experiment II, toxic effects were observed in strain TA 1537 without metabolic activation and in strains TA 100, WP2 uvrA pKM101, and WP2 pKM101 with metabolic activation.

Table 1: Toxic effects observed at the following concentrations (µg/plate)

Strains	Experiment I		Experiment II
	With S9 mix	Without S9 mix	With S9 mix	Without S9 mix
TA 1535	5000	2500, 5000	/	/
TA 1537	/	/	1000	/
TA 98	/	5000	/	/
TA 100	/	2500, 5000	/	5000
WP2 uvrA pKM101	/	/	/	2500, 5000
WP2 pKM101	/	/	/	2500, 5000

/ = no toxic effects observed

Table 2: Summary of Results Experiment I

Test Group	Dose Level µg/ plate	Revertant Colony Counts (Mean ± SD)
Without Activation		TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA pkm 101	WP2pKm 101
DMSO		13 ± 4	14 ± 2	29 ± 8	138 ± 14	569 ± 26	238 ± 15
Untreated		15 ± 5	11 ± 2	34 ± 3	152 ± 18	539 ± 36	226 ± 3
Cloquintocet- mexyl	3	16 ± 4	11 ± 1	32 ± 9	135 ± 12	600 ± 25	215 ± 2
	10	14 ± 2	13 ± 6	28 ± 5	144 ± 12	630 ± 37	206 ± 11
	33	14 ± 1	14 ± 6	29 ± 2	135 ± 13	589 ± 39	207 ± 15
	100	14 ± 4	15 ± 1	28 ± 6	132 ± 9	604 ± 18	211 ± 14
	333	13 ± 4^P	17 ± 2^P	26 ± 2^P	111 ± 6^P	542 ± 35^P	223 ± 14^P
	1000	11 ± 3^P M	15 ± 1^P	28 ± 5^P	122 ± 8^P	564 ± 23^P	197 ± 7^P
	2500	8 ± 1^P M	13 ± 1^P M	27 ± 3^P M	72 ± 4^P M	525 ± 22^P M	153 ± 13^P M
	5000	4 ± 2^P M	14 ± 4^P M	20 ± 2^P M	71 ± 7^P M	502 ± 17^P M	129 ± 8^P M
NaN3	10	1343 ± 733			1564 ± 417
4-NOPD	10			329 ± 13
4-NOPD	50		89 ± 2
MMS	3.0 µL					2871 ± 453	2797 ± 455
With Activation
DMSO		17 ± 2	20 ± 4	36 ± 7	154 ± 8	627 ± 34	224 ± 15
Untreated		19 ± 2	23 ± 4	39 ± 9	181 ± 7	645 ± 46	224 ± 20
Cloquintocet- mexyl	3	16 ± 4	20 ± 3	35 ± 9	159 ± 9	580 ± 51	218 ± 7
	10	17 ± 5	20 ± 8	35 ± 8	154 ± 5	624 ± 17	212 ± 8
	33	16 ± 3	21 ± 10	39 ± 4	147 ± 21	640 ± 69	181 ± 22
	100	17 ± 3	24 ± 5	39 ± 10	141 ± 16	595 ± 45	207 ± 6
	333	17 ± 2	21 ± 8	36 ± 6	155 ± 11	473 ± 42	226 ± 22
	1000	13 ± 3^P	19 ± 6^P	29 ± 8^P	145 ± 7^P	426 ± 8^P	199 ± 2^P
	2500	6 ± 3^P M	12 ± 2^P M	28 ± 4^P M	65 ± 4^P M	434 ± 20^P M	188 ± 19^P M
	5000	3 ± 2^P M	18 ± 3^P M	15 ± 3^P M	51 ± 4^P M	400 ± 12^P M	181 ± 11^P M
2-AA	2.5	324 ± 17	259 ± 12	1578 ± 109	2744 ± 155
2-AA	10.0					1828 ± 75	2428 ± 145
P = Precipitate M = Manual count

 

Table 3: Summary of Results Experiment II

Test Group	Dose Level µg/plate	Revertant Colony Counts (Mean ±SD)
Without Activation		TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA pkm 101	WP2pKm 101
DMSO		22 ± 6	11 ± 2	35 ± 6	115 ± 15	410 ± 29	198 ± 13
Untreated		20 ± 6	11 ± 1	40 ± 2	146 ± 5	431 ± 7	221 ± 6
Cloquintocet- mexyl	3	20 ± 6	12 ± 2	36 ± 12	122 ± 12	397 ± 19	180 ± 6
	10	16 ± 1	13 ± 4	37 ± 5	117 ± 2	413 ± 32	198 ± 15
	33	23 ± 1	15 ± 4	34 ± 3	126 ± 8	400 ± 23	158 ± 4
	100	18 ± 2	11 ± 4	25 ± 3	101 ± 4	427 ± 41	174 ± 4
	333	19 ± 4^P	8 ± 3^P	34 ± 11^P	90 ± 12^P	406 ± 8^P	171 ± 7^P
	1000	16 ± 5^P	5 ± 3^P	29 ± 1^P	93 ± 8^P	409 ± 42^P	177 ± 1^P
	2500	20 ± 6^P	9 ± 2^P	24 ± 10^P	97 ± 14^P	388 ± 10^P	156 ± 7^P
	5000	19 ± 5^P M	10 ± 3^P M	25 ± 7^P M	73 ± 11^P M	361 ± 14^P M	131 ± 15^P M
NaN3	10	1551 ± 119			1570 ± 44
4-NOPD	10			420 ± 5
4-NOPD	50		99 ± 8
MMS	3.0 µL					2036 ± 48	2437 ± 98
With Activation
DMSO		22 ± 4	19 ± 4	52 ± 7	137 ± 9	423 ± 21	203 ± 24
Untreated		23 ± 11	19 ± 3	50 ± 12	175 ± 16	513 ± 16	230 ± 5
Cloquintocet- mexyl	3	24 ± 3	15 ± 5	50 ± 8	148 ± 34	437 ± 5	186 ± 14
	10	22 ± 6	20 ± 4	60 ± 7	144 ± 5	451 ± 14	207 ± 15
	33	20 ± 8	23 ± 1	43 ± 7	146 ± 4	459 ± 20	211 ± 18
	100	29 ± 4	19 ± 8	49 ± 2	160 ± 11	423 ± 36	193 ± 15
	333	20 ± 3	23 ± 7	43 ± 2	147 ± 14	364 ± 7	197 ± 13
	1000	20 ± 1^P	21 ± 2^P	43 ± 4^P	123 ± 2^P	272 ± 13^P	170 ± 13^P
	2500	12 ± 8^P	14 ± 4^P	31 ± 4^P	63 ± 11^P	91 ± 18^P	76 ± 38^P
	5000	19 ± 9^P M	20 ± 5^P M	27 ± 4^P M	38 ± 8^P M	19 ± 6^P M	20 ± 11^P M
2-AA	2.5	334 ± 43	216 ± 22	1054 ± 24	1378 ± 222
2-AA	10.0					1633 ± 47	2136 ± 515
P = Precipitate M = Manual count

 

Conclusions:

negative with metabolic activation
negative without metabolic activation

Executive summary:

The potential of cloquintocet-mexyl to induce gene mutations was investigated under GLP to OECD TG 471, using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strains WP2 uvrA pKM 101 and WP2 pKM 101.

A range of doses was tested up to the maximum dose of 5000 µg/ plate. No substantial increase in revertant colony numbers of any of the six tester strains was observed following treatment with cloquintocet-mexyl at any dose level, in the presence or absence of metabolic activation (S9 mix). There was no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Positive control chemicals showed appropriate responses in the relevant strains.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Negative, no clastogenic or aneugenic effects in vivo, Chinese hamster (OECD 474, Strasser 1987)

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

Study period:

22 December 1986 to 03 September 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

a top dose level of 2500 mg/kg was used, 1000 PCE were examined per animal

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

hamster, Chinese

Strain:

other: random outbred strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 4-9 weeks (males), 6-10 weeks (females)
- Weight at study initiation: 22 to 34 g
- Assigned to test groups randomly: yes, by computer generated random numbers
- Housing: individual caging
- Diet: standard diet ad libitum
- Water: ad libitum
- Acclimation period: 4-5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21°C
- Humidity: 52-66%
- Air changes: air conditioned room
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 22 December 1986 To: 03 September 1987

Route of administration:

oral: gavage

Vehicle:

0.5% CMC

Details on exposure:

Tolerability test: 2500 mg/kg bw, administered to 2 males + 2 females.
Main study: 625,1250, 2500 mg/kg bw (no mortality occurred in the toxicity test at 2500 mg/kg)
Control groups were treated with 0.5% CMC and the positive controls cyclophosphamide, 64 mg/kg bw.

The mutagenicity study was conducted in 8 male and 8 female hamsters per dose group and sampling time. In addition to the treated groups, negative and positive control groups were used. They received the vehicle (0.5% CMC) or the mutagen cyclophosphamide (CPA, 64 mg/kg).

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Once

Post exposure period:

16, 24 or 48 hours

Dose / conc.:

625 mg/kg bw/day (nominal)

Dose / conc.:

1 250 mg/kg bw/day (nominal)

Dose / conc.:

2 500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

8 males and 8 females

Control animals:

yes, concurrent vehicle

Positive control(s):

- cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 64 mg/kg bw

Tissues and cell types examined:

Bone marrow smears were prepared from the shafts of both femurs. After staining, coded slides of 5 animals/sex/dose were scored. 1000 polychromatic erythrocytes per animal were scored for the incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was determined for each animal.

Details of tissue and slide preparation:

- Bone marrow smears were prepared from the shafts of both femurs.
- After staining, coded slides of 5 animals/sex/dose were scored.

Evaluation criteria:

The test substance is considered active if a statistically significant increase in the number of polychromatic erythrocytes with micronuclei in comparison with the negative control occurs at any dose and sampling time.

Statistics:

The significance of differences was assessed by the chi square test.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- No mortality occurred
- Micronucleus test: There was no significant increase of micronuclei. The positive control substance yielded clearly enhanced incidences of polynucleated erythrocytes.

Table 1: Percentage of micronucleated PCE's at different preparation times

Compound and concentration	Sex	16 hours	24 hours	48 hours
Test article CGA185072 (625 mg/kg)	males females		0.04 0.10
Test article CGA185072 (1250 mg/kg)	males females		0.02 0.06
Test article CGA185072 (2500 mg/kg)	males females	0.06 0.08	0.04 0.12	0.04 0.16
Negative control Vehicle (0.5% CMC)	males females	0.06 0.08	0.02 0.08	0.06 0.04
Positive control Cyclophosphamide (64 mg/kg)	males females		4.54 4.22

 

Conclusions:

negative

Executive summary:

The genotoxicity of cloquintocet-mexyl was studied under GLP to OECD TG 474 in an in vivo micronucleus assay using Chinese hamsters. Dose levels of 625, 1250 and 2500 mg/kg were orally administered. Animals were killed 16, 24 or 48 hours after dosing and bone marrow smears prepared from each femur. After staining the smears, slides from 5 animals/sex/dose were scored for 1000 polychromatic erythrocytes per animal. The number of micronuclei were assessed. Under the conditions of this test there were no clastogenic or aneugenic effects.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Cloquintocet-mexyl technical has been evaluated for possible mutagenic activity in a range of assays including both in vitro and in vivo studies and covering a range of core endpoints (gene mutation, clastogenicity, DNA repair). The key studies are considered to be those for gene mutation in bacteria (Sokolowski 2010) and in mammalian cells (Dollenmeier 1987), and for clastogenicity both in vitro (Ogorek 1998) and in vivo (Strasser 1987).

In vitro evaluation

The test substance was examined in four strains of S typhimurium (TA 1535, TA 1537, TA98 and TA100) and two strains of E coli (E. coli WP2 pKM 101 and E. coli WP2 uvr A pKM 101), in both the absence and presence of auxiliary metabolic activation (S9) (Sokolowski 2010). Cloquintocet-mexyl was negative in this assay. A negative result was also reported in an earlier supporting study on the same four Salmonella strains and E coli strain WP2 uvr A (Deparade 1990). The test substance was examined for gene mutation in mammalian cells using the HPRT locus in V79 cells, and at dose levels limited by toxicity in both the absence and presence of S9 (Dollenmeier 1987). Cloquintocet-mexyl was negative in this assay. The test substance was examined for clastogenic activity in CHO cells, again at dose levels limited by toxicity and solubility in both the absence and presence of S9 (Ogorek 1998). Cloquintocet-mexyl was negative in this assay. This finding confirmed an earlier supporting cytogenetic study using human lymphocytes (Strasser 1987a). Negative results for Cloquintocet-mexyl were also obtained in supporting studies examining the DNA repair endpoint in both rat hepatocytes (Hertner 1987) and human fibroblasts (Meyer 1987).

In vivo evaluation

Cloquintocet-mexyl has been examined in the Chinese hamster bone marrow micronucleus assay, which can detect both clastogenic and aneugenic activity, in both male and female animals at doses up to 2500 mg/kg (Strasser 1987). Cloquintocet-mexyl was negative in this assay.

Justification for classification or non-classification

Cloquintocet-mexyl showed no evidence of genotoxicity in vitro or in vivo and does not warrant classification for this end point under Regulation (EC) 1272/2008, Annex I, Part 3, 3.5.2.