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

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 August 2013 - 25 November 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 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

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: Tan solid
- 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

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Animal Breeding Facility, Jai Research Foundation
- Age at study initiation: 10 to 12 weeks old
- Weight at study initiation: 23.1 - 29.6 g
- Housing: Individual solid floor polypropylene mice cages
- Diet ad libitum Teklad Certified Global High Fiber Rat/Mice feed
- Water ad libitum UV sterilised water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23
- Humidity (%): 65 to 66
- Air changes (per hr): Minimum 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle

IN-LIFE DATES: From: 29 August 2013 To: 24 September 2013

Study design: in vivo (LLNA)

Vehicle:

other: 1% Pluronic® L92

Concentration:

5%, 10%, 25% and 50% (w/v) in 1% L92 (25 μL/ear) for three consecutive days

No. of animals per dose:

5 females per dose in the main study; 2 females per dose in the preliminary irritation screen

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The test substance was not found to be soluble in any of the guideline recommended solvents, however, the test substance formed a homogenous suspension in 1% Pluronic® L92 and therefor this was used as a vehicle for the study.
- Irritation: To assess the irritant potential of the test item through ear thickness measurement and erythema, a preliminary assay was carried out. In the preliminary test, four groups of mice comprising 2 females per group were treated with X204558, applied at 5%, 10%, 25% and 50% (w/v) in 1% L92 (25 μL/ear) for three consecutive days (days 0, 1 and 2). Clinical observations were recorded daily during the experiment. Ear thickness was measured on days 0, 2 and 5 (at 72 hours post last application). Body weight was recorded on days 0 and 5. Dose concentrations of 5%, 10%, 25% and 50% (w/v) revealed no erythema and ear thickness increase was below 25%. Therefore, dose concentrations of 10%, 25% and 50% (w/v) X204558 were evaluated in the main study of LLNA.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Animal assignment was randomised using in-house developed validate computer software.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was mixed with vehicle to obtain the desired dosing concentrations. Fresh dose solutions were prepared daily prior to application. The concentrations of the dose solutions were not verified analytically.
Three groups were treated topically for three consecutive days (days 0, 1 and 2) on the dorsal surface of both ears (25 μL/ear) using a calibrated micropipette with the test substance at concentrations of 10%, 25% and 50% (w/v), respectively. Mice from vehicle control group and positive control group were handled in the same manner but received 25 μL/ear of vehicle (1% L92) or 25% α-Hexylcinnamaldehyde (v/v) in vehicle (1% L92), respectively.
On day 5 (approximately 72 h after the last treatment), all mice from the vehicle control, positive control and all the treatment groups were injected with 250 μL of sterile phosphate buffered saline (PBS) containing approximately 20 ± 1 μCi (740 KBq) of 3H-methyl thymidine via the tail vein.

Observations:
Body weights of individual mice were recorded on the first day of dosing (day 0) and prior to administration of 3H-methyl thymidine (day 5)
Individual animals were observed for clinical signs and local irritation at the site of application and systemic toxicity. the table below was used for scoring irritation
On day 5, 5 hours post-administration of 3H-methyl thymidine, all mice from the vehicle control, positive control and all the treatment groups were sacrificed and the draining auricular lymph nodes from each mouse were excised and cell suspensions were prepared.
- Lymph node proliferation response: Incorporation of 3H-methyl thymidine was measured by β-scintillation counting as disintegrations per minute (DPM) for each mouse and expressed as DPM/mouse
- Name of test method: Final results were expressed as the Stimulation Index (SI) which is calculated as a ratio of the mean DPM of test group divided by mean DPM of vehicle control group.
- Criteria used to consider a positive response: A Stimulation Index (SI) of three or more (SI value of treated group over the control) indicates potential to cause skin sensitisation.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

In addition to an assessment of the magnitude of the SI, statistical analysis was carried out for the assessment of the dose response relationship and pair-wise comparison made between the treatment and the solvent/vehicle control group. All the parameters characterised by continuous data such as body weight and radioactive disintegrations per minute (DPM) were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA). To compare vehicle and positive control data, Student's t-test was performed to calculate significance. Also Grubb’s test was also performed to check the outlier in the study.

Results and discussion

Positive control results:

The DPM value for positive control (25% α-Hexylcinnamaldehyde) was found to be 7210.80. The SI value for the 25% HCA treated positive control group was 8.66 which showed a greater than three-fold increase over the control value indicating a clear positive response for this known weak sensitiser that confirmed the reliability of this test procedure.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.91

Test group / Remarks:

10% test concentration

Results 2

Key result

Parameter:

SI

Value:

0.93

Test group / Remarks:

25% test concentration

Results 3

Parameter:

SI

Value:

0.51

Test group / Remarks:

50% test concentration

Any other information on results incl. tables

Group mean DPM values were 833.00, 755.00, 774.60 and 428.60 for the vehicle control (1% L92), 10%, 25% and 50% (w/v) dose concentrations of the test substance, respectively. Group mean body weights of treated animals were comparable with the control group and there were no indications of clinical or systemic toxicity in the test substance treated animals.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The SI obtained for the test substance at all the tested concentrations showed a less than three-fold increase over the control value. Therefore, the test substance did not demonstrate dermal sensitisation potential in the LLNA and classification is not required.

Executive summary:

A local lymph node assay (LLNA) was conducted to evaluate the skin sensitisation potential of X204558 (cloquintocet acid) in CBA/J strain mice, according to OECD 429. A preliminary assay was conducted to identify the appropriate test concentrations for the main study. Based on the results from a preliminary assay, five groups of mice (each comprising 5 females) were selected for the experiment. Three groups were treated with X204558 at concentrations of 10%, 25% and 50% (w/v) in 1% L92 applied for three consecutive days (days 0, 1 and 2) on the dorsum of both ears (25 μL per ear). One group served as vehicle control and was treated with 1% L92 and another group served as positive control and was treated with HCA (α-hexylcinnamaldehyde) at a concentration of 25% (v/v) in 1% L92.

Group mean body weights of treated animals were comparable with the control group and there were no indications of clinical or systemic toxicity in treated animals. On day 5, the uptake of 3H-methyl thymidine into the auricular (local) lymph nodes draining at the site of chemical application was measured (5 hours post-administration) to assess the lymph node proliferative response. A positive response for HCA (SI = 8.66) confirmed the reliability of the test procedure. Stimulation indices for the 10%, 25% and 50% (w/v) treated groups were 0.91, 0.93 and 0.51, respectively. The SI obtained at all tested concentrations showed a less than three-fold increase over the control value; therefore cloquintocet acid did not demonstrate dermal sensitisation potential under the conditions of the study. Based on the results of this study, cloquintocet acid is not classified as a skin sensitiser according to the CLP Regulation.