Acetic acid, 2-[(5-chloro-8-quinolinyl)oxy]-, 1-methylhexyl ester

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

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:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

619-447-3

Cas Number:

99607-70-2

Molecular formula:

C18H22ClNO3

IUPAC Name:

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

Details on test material:

- Name of test material (as cited in study report): cloquintocet-mexyl
- Physical state: beige solidified melt
- Analytical purity: 95.4% w/w
- Purity test date: 09 July 2009
- Reanalysis date: end of July 2011
- Storage condition of test material: <30°C

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain 3

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

Controls

Controls 1

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

Controls 2

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

without S9, 10 µg/plate, TA1537 and TA98

Controls 3

Positive control substance:

methylmethanesulfonate

Remarks:

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

Controls 4

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

Results and discussion

Test results

Test results 1

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

Remarks:

see table 1

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see table 1

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli, other: WP2 pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see table 1

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Cloquintocet-mexyl did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

The potential of cloquintocet-mexyl to induce gene mutations 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 was investigated.

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.