6-chlorohexan-2-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 13th to March 1st 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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:

6-chlorohexan-2-one

EC Number:

233-546-5

EC Name:

6-chlorohexan-2-one

Cas Number:

10226-30-9

Molecular formula:

C6H11ClO

IUPAC Name:

6-chlorohexan-2-one

Test material form:

liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd mice

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo, San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks old
- Weight at study initiation: 19.9 – 21.9 grams. The weight variation in animals in the study did not exceed ± 20 % of the mean weight.
- Housing: Group caging / mice were provided with glass tunnel-tubes. Type II. polypropylene / polycarbo nate cages
- Diet (e.g. ad libitum): ad libitum. Ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice (Batch number: 575 4308, Expiry date: 31 March 2016 and Batch number: 540 5117, Expiry date: 31 July 2016)
- Water (e.g. ad libitum): ad libitum. Municipal supply from 500 mL bottle.
- Acclimation period: 14 days
- Indication of any skin lesions: Only healthy animals were used for the study. Health status was certified by the veterinarian.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0 – 24.8°C
- Humidity (%): 22- 77%
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

10, 25, 50 and 100 % w/v in vehicle

No. of animals per dose:

4 animals per dose

Details on study design:

PRE-SCREEN TESTS: 3 Preliminary tests were performed: The preliminary experiments were conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed:
1) The Preliminary Irritation/Toxicity Test I: Two doses (2 animals/dose) at test item concentrations of 100 % (undiluted) and 50 % (w/v) in AOO. Based on the high variability of the observed body weight loss, an additional test was performed.
2) The Preliminary Irritation/Toxicity Test II: Three doses (2 animals/dose) at test item concentrations of 50%, 25% and 10 % (w/v) in AOO. Based on the equivocal results of body weight loss an additional test was performed.
3) The Preliminary Irritation/Toxicity Test III: One dose (2 animals/dose) at test item concentration of 100% (undiluted).

- Compound solubility: The following standard OECD vehicle was assessed: AOO (acetone:olive oil 4:1 (v:v) mixture). The highest achievable concentration based on the regulatory requirements and the physical characteristics of the test item was 100 % (undiluted). The formulation at 50 % (w/v) using AOO as vehicle was suitable for the test.
- Irritation/Erithema scores: In the Preliminary Test I slight erythema (score 1) was observed for the animals of the 100% (undiluted) dose group on Day 3. In the Preliminary Test II slight erythema (score 1) was observed for the animals of the 50 % (w/v) dose group on Days 2-3.
- Systemic toxicity: During the Preliminary Irritation / Toxicity Tests, no mortality or signs of systemic toxicity were observed. In the Preliminary Irritation / Toxicity Test III minimal amount of test item precipitate was observed on the ears of the animals in 100% (undiluted) dose group on Days 1-3.
- Ear thickness measurements: The ear thickness values and ear punch weights were within the acceptable range.
- Draining auricular lymph nodes: They were normal in all examined dose groups.
- Body weight: Marked body weight loss (>5%) were observed on several animals in the Preliminary Experiment I and III. There was no dose relationship found and these changes were considered as individual variability.

MAIN STUDY
Based on the results of pre-screen tests, 100% (w/v) dose group is selected as top dose for the main test. However based on the equivocal results of the observed body weight loss, an additional fourth dose group was also included to ensure that there will be three analysable concentrations in the main test.
The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
a. That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
b. The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
In the main study, four doses of the test item and one negative and one positive control were tested. Animals were topically dosed with 25 μL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

Proliferation assay: On Day 6, each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS containing approximately 20 μCi of 3HTdR. Once injected, the mice were left for 5 hours (± 30 minutes). After this period mice were euthanized by asphyxiation with ascending doses of carbon dioxide. The draining auricular lymph nodes were removed and single cells suspensions of pooled lymph node cells were prepared and after overnight (approximately 18 hours) incubation at 2-8 °C, the 3HTdR incorporation was measured (10-minute measurement). The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Back ground level was also measured in duplicates.

Observations:
Clinical Observations: During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.
Measurement of body weight: Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

Positive control substance(s):

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

Results and discussion

Positive control results:

The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at a concentration of 25 % in the relevant vehicle (AOO) using CBA/CaOlaHsd mice.
No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historic positive control data (stimulation index value of 7.2) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay. Furthermore, the DPN values observed for the positive control substance in this experiment were within the historical control range.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

ca. 1.4

Test group / Remarks:

10 % (w/v) in AOO

Results 2

Key result

Parameter:

SI

Value:

ca. 0.9

Test group / Remarks:

25 % (w/v) in AOO

Results 3

Key result

Parameter:

SI

Value:

ca. 1.2

Test group / Remarks:

50 % (w/v) in AOO

Results 4

Key result

Parameter:

SI

Value:

ca. 1.1

Test group / Remarks:

100 % (w/v) in AOO

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Test item 100% (w/v) in AOO: 5451 DPM
Test item 50% (w/v) in AOO: 5859 DPM
Test item 25% (w/v) in AOO: 4311 DPM
Test item 10% (w/v) in AOO: 6733 DPM

DETAILS ON STIMULATION INDEX CALCULATION
The stimulation index values were 1.1, 1.2, 0.9 and 1.4 at a concentrations of 100 (undiluted), 50, 25 and 10% (w/v), respectively

CLINICAL OBSERVATIONS:
No mortality or signs of systemic toxicity were observed during the study. Minimal amount of test item precipitate was observed on the ears of the experimental animals in the 100 % (undiluted) dose group on Days 1 3. There were no indications of any irritancy at the site of application.

BODY WEIGHTS
No treatment related effects were observed on the mean body weight of the groups. However, marked body weight loss (>5%) was detected for one animal of the negative control group and for one animal of the 25% (w/v) dose group, these changes are considered as animal variability.

OTHER: The appearance of the lymph nodes was normal in the negative control group and in all the test item treated dose groups. Larger than normal lymph nodes were observed in the positive control group.

Any other information on results incl. tables

Table 1: Main test: DPM, DPN and Stimulation Index Values for all Groups

Test Group Name	Measured DPM / group	DPM	Number of lymph nodes	DPN	Stimulation Index
Background	32	-	-	-	-
(5 % (w/v) TCA)	36
Negative control	4873	4839.0	8	604.9	1.0
(AOO)
6-Chlorohexan-2-one 100% (undiluted)	5485	5451.0	8	681.4	1.1
6-Chlorohexan-2-one 50 % (w/v) inAOO	5893	5859.0	8	732.4	1.2
6-Chlorohexan-2-one 25 % (w/v) inAOO	4345	4311.0	8	538.9	0.9
6-Chlorohexan-2-one 10 % (w/v) inAOO	6767	6733.0	8	841.6	1.4
Positive control	35008	34974.0	8	4371.8	7.2
(25 % (w/v) HCA inAOO)

Notes:

1. DPM (Disintegrations Per Minute)

2. DPN (Disintegrations Per Node) = DPM divided by the number of lymph nodes.

3. Stimulation Index = DPN of a treated group divided by DPN of the negative control group.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under these test conditions, the test item showed to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

Executive summary:

The skin sensitisation potential of the test item was evaluated in accordance with the OECD 429 Guideline with GLP. 3 Preliminary Irritation/Toxicity Tests were performed in CBA/CaOlaHsd mice (2 animals/dose) at different doses in AOO (acetone:olive oil 4:1 (v:v) mixture). Based on their results, the 100% (w/v) was selected as top dose for the main test. In the main assay, 24 female CBA/CaOlaHsd mice were used (n=4/group): four groups test item at 100%, 50%, 25% and 10% (in AOO), a negative control group (vehicle, AOO) and a positive control group (25 % HCA in AOO). The test item solutions were applied on the dorsal surface of ears of experimental animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI). No mortality or systemic clinical signs were observed during the study. Minimal amount of test item precipitate was observed on the ears of the experimental animals in the 100% (undiluted) dose group on Days 1-3. There were no indications of any irritancy at the site of application. No treatment related effects were observed on the mean body weight changes in the study. The stimulation index values were 1.1, 1.2, 0.9 and 1.4 at concentrations of 100% (undiluted), 50%, 25% and 10%(w/v), respectively. Under these test conditions, the test item showed to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.