7-(4-ethyl-1-methyloctyl)quinolin-8-ol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3-30 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The solubility of the test item in test specific solvents was determined in pre-tests. The test item was insoluble in all solvents indicated by the respective OECD guidelines at the required concentrations. Even after sonication, precipitates were clearly visible with the unaided eye.
Under these circumstances, in vitro skin sensitization testing in accordance with OECD 442C, OECD 442D, and OECD 442E of “7-(4-ethyl-1-methyloctyl)quinolin-8-ol” was not possible. Testing with precipitates is technically not feasible in case of DPRA and h-CLAT, and it is not recommended in case of the LuSens test, since a significant result cannot be obtained.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

22 July 2010

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:

7-(4-ethyl-1-methyloctyl)quinolin-8-ol

EC Number:

277-531-1

EC Name:

7-(4-ethyl-1-methyloctyl)quinolin-8-ol

Cas Number:

73545-11-6

Molecular formula:

C20H29NO

IUPAC Name:

7-(5-ethylnonan-2-yl)quinolin-8-ol

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor / 1702-18-01/O
- Expiration date of the lot/batch: 25/03/2021
- Purity test date: 26/03/2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature (20 ± 5° C); Keep away from light
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle:


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Velaz Prague, Czech Republic
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8-10 weeks
- Weight at study initiation:
- Housing: The animals were housed in IVC polycarbonate cages (5 animals per cage) suspended on stainless steel racks, in a room equipped with central air-conditioning.
- Diet: ssniff, ad libitum
- Water: tap water, ad libitum
- Acclimation period: The animals were acclimated in identical conditions as during the experiment for 5 days prior to the start of treatment. The acclimation was according to standard operation procedures.
- Indication of any skin lesions: The health condition of animals was examined by a veterinarian before initiation of the study. Animals were healthy, without visible signs of disease.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 - 60 %.
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 29 Dec 2018 To: 30 Dec 2019

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

The doses were selected from the concentration series 100 %, 50 %, 25 %, 10 %, 5 %, 2.5 % etc. according to OECD Guideline No. 429.
The starting concentration (25 %) was determined according to pre-screen test result.

The required amount of the test item (according to the concentration) was mixed with the vehicle shortly before the administration (i.e. 50 % concentration was obtained by mixing of 0.5mL of test item with 0.5mL of vehicle). For 100 % concentration the test item was used in undiluted form. The dose preparation data are listed in the raw data

No. of animals per dose:

5 females – negative control (vehicle)
5 females – positive control
15 females – test item
6 females - pre-screen test

Details on study design:

PRE-SCREEN TESTS:
All mice (2 mice/group) were observed daily for any clinical signs of toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to the termination (day 6). Both ears of each mouse were assessed for any signs of irritation. Ear thickness was measured by a calliper on Day 1 (pre-dose), Day 3 and Day 6. Excessive local skin irritation is indicated by an erythema score ≥ 3 and/or an increase in ear thickness of ≥ 25 %.

Pre-screen - concentration 100%
- Compound solubility: soluble
- Irritation: Both animals showed from slight to strong signs of local irritation (erythema, crusts formation) at the fourth to the sixth day of experiment. The crusts were observed between ears.
- Systemic toxicity: Reduction of body weight was observed in both animals
- Ear thickness measurements:
Mean day 1: 0.213
Mean day 3: 0.205
Mean day 6: 0.243
- Erythema scores:
Mouse No. day 1 day 2 day 3 day 4 day 5 day 6
1 0 0 0 2 3 3
2 0 0 0 2 3 3


Pre-screen - concentration 50%
7-(4-ethyl-1-methyloctyl)quinolin-8-ol administered at concentration of 50 %, caused a similar clinical signs (local irritation, crusts formation, reduction of body weight) observed after administration of 100 % concentration.

- Compound solubility:
soluble
- Irritation:
Both animals showed from slight to strong signs of local irritation (erythema, crusts formation) at the fourth to the sixth day of experiment. The crusts were observed between ears.
- Systemic toxicity:
Reduction of body weight was observed in both animals
- Ear thickness measurements:
Mean day 1: 0.200
Mean day 3: 0.200
Mean day 6: 0.203
- Erythema scores:
Mouse No. day 1 day 2 day 3 day 4 day 5 day 6
1 0 0 0 1 2 2
2
0 0 0 1 2 2




Pre-screen - concentration 25%
Administration of test item at concentration of 25 %, did not cause any changes in monitored parameters


MAIN STUDY
Day 1:
Each animal was identified and the body weight was recorded. To the dorsum of each ear, 25 µL of the appropriate dilution of the test item, or the vehicle alone was applied.
Days 2 and 3:
The application procedure carried out on day 1 was repeated.
Days 4 and 5: No treatment.
Day 6:
The body weight of each animal was recorded. 250 µL of sterile phosphate-buffered saline (PBS) containing 2 µCi (7.4 x 104 Bq) of 125I-iododeoxyuridine and 10-5M fluorodeoxyuridine was injected into all test and control mice via the tail vein.
Five hours later, the animals were sacrificed. The draining auricular lymph nodes from each ear were excised and pooled in PBS for each experimental group (pooled treatment group approach).


ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response:

TREATMENT PREPARATION AND ADMINISTRATION:

Positive control substance(s):

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

Results and discussion

Positive control results:

SI = 5.57

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

3.81

Test group / Remarks:

5% 7-(4-ethyl-1-methyloctyl)quinolin-8-ol

Results 2

Key result

Parameter:

SI

Value:

3.4

Test group / Remarks:

10% 7-(4-ethyl-1-methyloctyl)quinolin-8-ol

Results 3

Key result

Parameter:

SI

Value:

5.55

Test group / Remarks:

25% 7-(4-ethyl-1-methyloctyl)quinolin-8-ol

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
In comparison with the control group, the increase of the pooled lymph node weights was observed at all used concentration. The increase was not dose dependent. The pooled lymph node weights of treated groups were 0.0918 g for 5 % concentration, 0.0813 g for 10 % concentration and 0.1045 g for 25 % concentration of test item. The lymph node weight of control group and positive control group were 0.0464 g and 0.0734 g, respectively. The DPM values for the three treated groups were 9653.26 (5 %), 8631.16 (10 %) and 14082.08 (25 %), respectively. The SI values for the three treated groups were 3.81 (5 %), 3.40 (10 %) and 5.55 (25 %), respectively. All tested concentrations induced a S.I. greater than the threshold value of 3. The EC3 value could not be calculated correctly, because we did not observe dose dependent character of SI values. In order to correctly estimate EC value, SI indices must be dose dependent or at least decreasing in the same manner as decreasing concentration of the test item used.

DETAILS ON STIMULATION INDEX CALCULATION
Results are expressed as the Stimulation Index (SI). The SI was obtained by dividing the pooled DPM for each treatment group by the incorporation of the pooled DPM vehicle control group; this yields a mean SI. A substance is regarded as sensitizer in the LLNA test when SI≥3.
EC3 value, which induce stimulation indices, is determined by linear interpolation of points on dose-response curve, immediately above and below of SI value, according to the equation:
EC3=c+[(3-d)/(b-d)]x(a-c)
a – higher concentration, b – SI of higher concentration, c – lower concentration
d – SI of lower concentration
If all points are below the stimulation index of three, no EC3 value can be stated.
When there are no data points that fall below SI = 3, log-linear extrapolation may be applied in which the two lowest test concentrations from the dose-response curve are used, provided the lowest SI value approaches the value of 3 and that a linear dose-response exists (13). The equation for EC3 calculation:
EC3=2^{log2(c)+ (3−d)/(b−d)×[log2(a)−log2(c)]}


EC3 CALCULATION
EC 3 colud not be calculated since the SI did not show a dose-dependent increase.

CLINICAL OBSERVATIONS:

No clinical signs of toxicity were recorded

BODY WEIGHTS
see Table.

Any other information on results incl. tables

The Lymph node weight, DPM, SI, EC3 values.

			Lymph node	Number of
			weight (g)	lymph nodes	DPM	SI	EC3 (%)
Control			0.0464	10	2535.49	-	-
Positive Control			0.0734	10	8076.78	5.57*
7-(4-ethyl-1-methyloctyl)quinolin-8-ol	5 %		0.0918	10	9653.26	3.81
	10 %		0.0813	10	8631.16	3.40
	25 %		0.1045	10	14082.08	5.55

*Calculated with corresponding control value of 1449.03 DPM (Appendix 2 of test report)

Body weights

7-(4-ethyl-1-methyloctyl)quinolin-8-ol (5 %)
Mouse no.	Initial body weight	Terminal body weight
1	19.57	19.94
2	19.71	19.55
3	20.08	20.41
4	20.92	21.52
5	19.32	20.25
Mean	19.92	20.33
S.D.	0.623	0.740
7-(4-ethyl-1-methyloctyl)quinolin-8-ol(10 %)
Mouse no.	Initial body weight	Terminal body weight
1	18.19	19.83
2	17.26	17.52
3	18.10	19.92
4	18.65	18.95
5	18.03	17.77
Mean	18.05	18.80
S.D.	0.502	1.122
7-(4-ethyl-1-methyloctyl)quinolin-8-ol(25 %)
Mouse no.	Initial body weight	Terminal body weight
1	20.82	19.57
2	19.78	19.82
3	19.87	19.88
4	19.26	19.04
5	21.48	21.18
Mean	20.24	19.90
S.D.	0.892	0.790

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The sensitization potential of 7-(4-ethyl-1-methyloctyl)quinolin-8-ol was evaluated using the Local Lymph Node Assay (LLNA). The LLNA has been developed to determine the allergic contact sensitization potential of chemicals.
Based on the recommendations of the OECD Guideline 429, the test item was dissolved in DMSO (v/v). The positive control (alpha-Hexylcinnamic aldehyde) (25 %) was dissolved in the same vehicle.
The Pre-screen test was performed using the doses of 100, 50 and 25 %. Based on the observations recorded in the Pre-screen tests, the concentration of 25 % was selected as top dose for the main test.
Five female mice (CBA/Ca) per group were topically exposed (dorsum of both ears) to the test item at concentrations of 5 %, 10 % and 25 %, to the positive control and to the vehicle only. Lymphocyte proliferation was measured using incorporation of radioactive 125I-iododeoxyuridine and 10-5M fluorodeoxyuridine in the draining lymph nodes. The radioactive incorporation was expressed as disintegrations per minute (DPM)/pooled treatment group and compared with DPM value from the vehicle control group and expressed as the Stimulation Index (SI).
After application of the test item at three concentrations (5 %, 10 % and 25 % v/v) animals did not show visible clinical symptoms of neither local irritation nor systemic toxicity.
In this study the Stimulation Indices (SI) of 3.81, 3.40 and 5.55 were determined with the test item at concentrations of 5 %, 10 %, and 25 % in DMSO, respectively. The EC3 value could not be calculated correctly, because we did not record dose dependent character of SI values.
The test item 7-(4-ethyl-1-methyloctyl)quinolin-8-ol is considered a skin sensitizer under the test conditions of this study. Since the results were not sufficient to calculate EC3 values, sub-categorisation is not appropriate and therefore in accordance with Regulation (EC) No.1272/2008 (EU CLP) the substance is classified as a skin sensitizer, category 1.

Executive summary:

The sensitization potential of 7-(4-ethyl-1-methyloctyl)quinolin-8-ol was evaluated using theL ocal Lymph Node Assay (LLNA). The LLNA has been developed to determine the allergic contact sensitization potential of chemicals.

Based on the recommendations of the OECD Guideline 429, the test item was dissolved in DMSO (v/v). The positive control (a-Hexylcinnamic aldehyde) (25 %) was dissolved in the same vehicle.

The Pre-screen test was performed using the doses of 100, 50 and 25 %. Based on the observations recorded in the Pre-screen tests, the concentration of 25 % was selected as top dose for the main test. Five female mice (CBA/Ca) per group were topically exposed (dorsum of both ears) to the test item at concentrations of 5 %, 10 % and 25 %, to the positive control and to the vehicle only. Lymphocyte proliferation was measured using incorporation of radioactive125I-iododeoxyuridine and 10-5M fluorodeoxyuridine in the draining lymph nodes. The radioactive incorporation was expressed as disintegrations per minute (DPM)/pooled treatment group and compared with DPM value from the vehicle control group and expressed as the Stimulation Index (SI).

After application of the test item at three concentrations (5 %, 10 % and 25 % v/v) animals did not show visible clinical symptoms of neither local irritation nor systemic toxicity.

In this study the Stimulation Indices (SI) of 3.81, 3.40 and 5.55 were determined with the test item at concentrations of 5 %, 10 %, and 25 % in DMSO, respectively. The EC3 value could not be calculated correctly, because we did not record dose dependent character of SI values.

The test item 7-(4-ethyl-1-methyloctyl)quinolin-8-ol is considered a skin sensitizer under the test conditions of this study. Since the results were not sufficient to calculate EC3 values, subcategorization is not appropriate and therefore in accordance with Regulation (EC) No.1272/2008 (EU CLP) the substance is classified as a skin sensitizer, category 1.