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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 June 2013 - 24 October 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

November 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

[(5-chloroquinolin-8-yl)oxy]acetic acid

Cas Number:

88349-88-6

Molecular formula:

C11H8ClNO3

IUPAC Name:

[(5-chloroquinolin-8-yl)oxy]acetic acid

Test material form:

solid: particulate/powder

Remarks:

powder

Specific details on test material used for the study:

- Name of test material (as cited in study report): X204558
- Physical state: Colourless powder
- Analytical purity: 98.3% ± 0.03% wt/wt
- Purity test date: 14 September 2014
- Lot/batch No.: 2GHB0002
- Storage condition of test material: in its original container at ambient condition

Method

Target gene:

Specific histidine loci in the tester strains of Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

The S9 fraction was buffered and supplemented with the essential co-factors β-NADP and Glucose- 6-phosphate to form the “S9 mix”. This mix was added to the top agar in this activated assay. The S9 fraction was procured from Molecular Toxicology Inc.

Test concentrations with justification for top dose:

156.25, 312.5, 625, 1250, 2500 and 5000 μg/plate both in the absence and presence of metabolic activation (5% v/v S9 mix) for Trial I (three plates/dose level)
51.2, 128, 320, 800, 2000 and 5000 μg/plate both in the absence and presence (10% v/v S9 mix) of metabolic activation for Trial II (three plates/dose level)

The selected top dose was the recommended maximum test concentration as specified in OECD 471.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in distilled water, and was found to be soluble in dimethyl sulfoxide.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-aminoanthracene

Remarks:

In the Absence of Metabolic Activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
Stock solutions were prepared for testing at six concentrations 156.25, 312.5, 625, 1250, 2500 and 5000 μg/plate both in the absence and presence of metabolic activation for Trial I.
In Trial II, the concentration spacing was modified using a factor of 2.5 and the concentration of S9 mix was increased from 5% v/v to 10% v/v. The tester strains were exposed at the concentrations of 51.2, 128, 320, 800, 2000 and 5000 μg/plate both in the absence and presence of metabolic activation.

DURATION
- The numbers of revertant colonies were recorded after the 48 h incubation period in both trials.

NUMBER OF REPLICATIONS:
- Triplicate plates were maintained for each test concentration, negative and positive controls in both trials.

DETERMINATION OF CYTOTOXICITY
- Before commencing the mutagenicity study, X204558 was tested for cytotoxicity to Salmonella typhimurium tester strain TA100, both in the absence and presence of metabolic activation
- Method: assessment of the state of background bacterial growth inhibition and reduction in number of colonies.
Stock solutions were prepared for testing at ten concentrations, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/plate. Volumes of 100 μL of relevant stock solutions were used to obtain the required test concentrations for treatment both in the absence and presence of the metabolic activation system. Tubes containing 2 mL of molten top agar with 0.5 mM histidine/biotin were maintained at 45.0 ± 2 °C. A volume of 500 μL of 5% v/v S9 mix was added in the presence of metabolic activation and 500 μL of 0.2 M phosphate buffer was added in the absence of metabolic activation. Volume of 100 μL of the relevant stock solution of test item, dimethyl sulfoxide and relevant positive control were used for treatment, as a negative control and as a positive control, respectively. Finally, 100 μL of bacterial culture was added to the tubes and mixed. This treatment mixture was poured on MGA plates and allowed to solidify. Triplicate sets were maintained for each concentration of X204558 and negative control. The petri plates were incubated at 37.0 ± 1 °C for 48 hours and then examined to assess the state of background bacterial growth inhibition and reduction in number of colonies.

OTHER:
Prior to the cytotoxicity test, a solubility and precipitation test was conducted and based on this, 5000 μg/plate was selected as the highest concentration for the cytotoxicity test.
Prior to use the cultures were tested for cell viability, genotype confirmation, histidine requirement and/or biotin requirement, rfa mutation, uvrB mutation, ampicillin resistance and tetracycline resistance. An operating system sterility check was performed including the top agar, S9 mix, solvent, test system, minimum glucose agar plate oxoid nutrient broth solution, and the sodium phosphate buffer.

Rationale for test conditions:

As recommended by the guideline

Evaluation criteria:

A positive result was considered when there was a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test article either in the absence or presence of the metabolic activation system.
Strains TA98, TA1535, and TA1537: Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean negative control value.
Strains TA100 and TA102:
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean negative control value.
A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was determined to be not mutagenic. Negative results obtained in the first trial were confirmed by a second trial, using the same method as specified above, with an alteration in concentration spacing and metabolic activation.

Statistics:

Not required; group means and standard deviation were calculated

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

partial inhibition of background lawn at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

partial inhibition of background lawn at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

partial inhibition of background lawn at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: None
- Evaporation from medium: None
- Water solubility: The test item was insoluble in distilled water, and was found to be soluble in dimethyl sulfoxide, therefore, dimethyl sulfoxide was used as the solvent
- Precipitation: None
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
Before commencing the mutagenicity study, X204558 was tested for cytotoxicity to Salmonella typhimurium tester strain TA100. The experiment was conducted both in the absence and presence of metabolic activation. Normal growth was observed up to the test concentration of 2500 μg/plate both in the absence and presence of metabolic action system, while partial inhibition was observed at the concentration of 5000 μg/plate both in the absence and presence of metabolic action system. Hence, 5000 μg/plate was selected as the highest concentration to be tested in the mutagenicity experiment in the absence and presence of metabolic activation system.

COMPARISON WITH HISTORICAL CONTROL DATA:
The results of the study indicate that the values for the negative control in all strains were within limits of historical ranges. Positive controls (both in the absence and presence of metabolic activation during both the trials) exhibited a clear increase in the number of revertants when compared with the concurrent negative control and were with the historical ranges

Any other information on results incl. tables

Mean Count of His+ Revertant Colonies in Negative Control, Positive Controls and Treatment Plates in the Absence of Metabolic Activation (Trial I)

Concentration of X204558 (μg/plate)	His+ Revertant Colonies/Plate (Absence of Metabolic Activation)
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DMSO)	7.67	3.21	21.00	4.00	26.33	2.08	135.67	3.79	240.00	2.65
156.25	7.33	2.31	21.33	3.21	25.33	1.53	143.00	10.82	243.67	5.03
312.5	7.00	2.00	20.33	2.08	28.00	2.65	138.67	4.16	237.33	12.74
625	7.67	3.06	19.67	1.15	27.67	0.58	136.67	7.02	236.33	8.02
1250	11.00	0.00	20.00	4.00	24.33	4.04	142.67	3.51	235.67	2.31
2500	8.33	4.62	21.33	4.04	21.00	2.00	138.33	5.51	238.00	10.44
5000	13.67	0.58	20.00	2.00	23.33	1.53	141.00	8.00	230.33	7.77
PC	187.33	12.74	396.67	20.43	434.00	123.01	932.00	63.59	998.33	19.73
PC-2Aa	-	-	-	-	-	-	131.33	4.16	-	-

 

Mean Count of His+ Revertant Colonies in Negative Control, Positive Control and Treatment Plates in the Presence of Metabolic Activation (Trial I)

Concentration of X204558 (μg/plate)	His+ Revertant Colonies/Plate [Presence of Metabolic Activation (5% v/v S9 mix)]
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DMSO)	9.33	2.89	22.33	1.53	24.00	2.00	128.00	6.24	241.33	5.69
156.25	5.67	2.89	19.67	1.15	23.67	2.31	137.33	10.02	244.67	6.03
312.5	8.33	2.31	24.33	2.08	27.00	2.00	134.67	18.18	236.00	11.14
625	7.33	6.66	22.67	1.15	24.33	1.53	144.33	15.53	243.67	6.43
1250	9.67	2.08	21.00	2.65	24.00	2.00	138.33	5.51	240.67	6.66
2500	9.00	2.65	23.00	1.73	25.00	2.00	134.67	3.79	237.00	6.24
5000	14.33	2.08	19.67	0.58	22.33	2.08	129.67	4.16	238.67	9.61
PC-2Aa	298.00	23.58	603.00	22.61	567.00	38.00	880.33	101.52	1140.33	30.66

 

Mean Count of His+ Revertant Colonies in Negative Control, Positive Controls and Treatment Plates in the Absence of Metabolic Activation (Trial II)

Concentration of X204558 (μg/plate)	His+ Revertant Colonies/Plate [Absence of Metabolic Activation]
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DMSO)	9.67	2.08	20.67	3.51	25.67	4.51	146.33	8.62	234.67	7.09
51.2	6.33	2.31	20.67	4.16	19.67	2.52	143.33	3.21	239.00	15.52
128	7.00	1.00	18.67	1.53	22.67	1.53	147.33	9.45	231.00	5.00
320	5.33	3.21	18.00	2.00	24.33	2.08	142.00	6.00	238.00	11.14
800	6.33	1.53	21.00	2.00	20.67	2.52	144.33	3.79	237.33	7.09
2000	5.00	2.65	19.00	4.58	25.33	1.53	141.33	9.02	235.33	11.68
5000	6.00	2.65	19.67	3.21	26.00	2.00	138.33	7.02	242.33	5.51
PC	238.00	13.75	423.33	58.01	594.67	58.73	882.33	60.53	935.67	37.82
PC-2Aa	-	-	-	-	-	-	144.33	13.50	-	-

 

Mean Count of His+ Revertant Colonies in Negative Control, Positive Control and Treatment Plates in the Presence of Metabolic Activation (Trial II)

Concentration of X204558 (μg/plate)	His+ Revertant Colonies/Plate [Presence of Metabolic Activation (10% v/v S9 mix)]
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DMSO)	9.67	3.51	22.00	4.58	24.67	3.21	136.33	7.77	235.67	15.57
51.2	7.33	2.89	20.67	5.86	21.67	2.52	143.00	11.36	234.33	4.16
128	6.67	1.53	19.00	3.00	22.67	2.52	133.00	3.00	239.67	11.06
320	7.00	3.61	19.00	3.61	27.33	4.04	149.67	6.66	250.67	4.73
800	6.00	2.65	23.67	2.52	21.00	2.65	147.67	7.77	231.00	6.24
2000	7.67	1.53	19.67	3.06	23.67	2.08	142.33	10.69	240.33	9.07
5000	8.00	2.65	23.00	3.00	22.33	2.52	142.00	10.15	229.67	5.51
PC-2Aa	294.33	13.58	524.33	48.00	630.33	33.56	963.00	37.47	1114.33	35.22

 

Applicant's summary and conclusion

Conclusions:

Under the specified experimental conditions, X204558 is considered to be non-mutagenic in the Bacterial Reverse Mutation Assay using Salmonella typhimurium.

Executive summary:

The potential of X204558 (cloquintocet acid) to induce reverse mutations in Salmonella typhimurium (Tester strains: TA1537, TA1535, TA98, TA100 and TA102) was evaluated in the bacterial reverse mutation test according to OECD 471. The substance was tested with and without metabolic activation in two independent trials. Bacteria were exposed to 6 dose levels (three plates/dose level) between 51.2 and 5000 μg/plate. From a preliminary toxicity test, 5000 μg/plate was selected being the recommended top dose indicated in the guidelines for testing. After 48 hours of incubation at 37 ± 1°C, the revertant colonies were scored.

X204558 (cloquintocet acid) did not induce any increase in the number of revertants in both the trials, with and without S9 mix, in any of the five strains. All the values for the negative control were within the historical control ranges of the laboratory and positive controls showed an increase in the number of revertant colonies demonstrating the efficiency of the test system. All criteria for a valid study were considered met. Under the conditions of the study, X204558 (cloquintocet acid) is concluded to be non-mutagenic in the Bacterial Reverse Mutation Assay.