C-C1

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16th May 2007 to 22nd May 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

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)

Remarks:

Date of inspection: 30th August 2005 Date of signature: 21st November 2005

Type of study:

mouse local lymph node assay (LLNA)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Sponsor's identification: C-C1
Description : dark blue powder
Batch number : MB-1
Date received : 05 March 2007
Storage conditions: room temperature in the dark over silica gel

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: B & K Universal Ltd, Hull, UK.
- Age at study initiation: eight to twelve weeks old
- Weight at study initiation: 15 to 23 g
- Housing: individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet (e.g. ad libitum): (Certified Rat and Mouse Diet)
- Water (e.g. ad libitum): mains tap water
- Acclimation period: acclimatisation period of at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness

IN-LIFE DATES: From: Day 1 (dosing) To: Day 6 (termination)

Study design: in vivo (LLNA)

Vehicle:

other: ethanol/distilled water 7:3.

Concentration:

Test material at concentrations of 5%, 10% or 25% w/w in ethanol/distilled water 7:3.

No. of animals per dose:

Groups of four mice per dose.

Details on study design:

Test Material Administration
Groups of four mice were treated with the test material at concentrations of 5%, 10% or 25% w/w in ethanol/distilled water 7:3. Information available suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test material (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 µCi/ml, specific activity 2.0 Ci/mmol, GE Healthcare UK Ltd) giving a total of 20 µCi to each mouse.

Observations
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 ml of PBS was added to the pooled lymph nodes.
Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 ml of PBS into a petri dish labelled with the project number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 ml of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 ml of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 ml of 5% Trichloroacetic acid (TCA).
Determination of 3HTdR Incorporation: After approximately eighteen hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by scintillation counting. The "Poly QTM" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA).

Positive control substance(s):

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

Results and discussion

Positive control results:

Current Positive Control Study for the Local Lymph Node Assay
Introduction. A study was performed to assess the sensitivity of the strain of mouse used at these laboratories to a known sensitiser. The method was designed to meet the requirements of the following:
 OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 24 April 2002)
Study dates: 20 April 2005 to 26 April 2005
Methods. Three groups, each of four animals, were treated with 50 µl (25 µl per ear) of  Hexylcinnamaldehyde, Tech, 85% as a solution in 70% ethanol in distilled water at concentrations of 5%, 10% and 25% w/v. A further group of four animals was treated with 70% ethanol in distilled water alone.
Results. The Stimulation Index (SI) expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:
Concentration % w/v in
70% ethanol in distilled water Stimulation Index (SI) Result
5 2.64 Negative
10 8.36 Positive
25 12.94 Positive
Conclusion. -Hexylcinnamaldehyde, Tech, 85% was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 1 see below). A stimulation index of less than 3 was recorded for the three concentrations of the test material (5%, 10% and 25% w/w in ethanol/distilled water 7:3).

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 1 (see below).

Any other information on results incl. tables

Table 1               Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (% w/w) in ethanol/distilled water 7:3	dpm	dpm/Node^a	Stimulation Index^b	Result
Vehicle	3143.28	392.91	na	na
5	3690.59	461.32	1.17	Negative
10	4412.40	551.55	1.40	Negative
25	6195.24	774.41	1.97	Negative

dpm=    Disintegrations per minute

a=          Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

b=         Stimulation Index of 3.0 or greater indicates a positive result

na =      Not applicable

Clinical Observations and Mortality Data

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Blue-coloured staining on the ears and fur was noted post dose on Day 2 and for the remainder of the test in animals treated with the test material at a concentration of 25% w/w in ethanol/distilled water 7:3. Fur loss on the ears and fur was also noted in these animals on Days 5 and 6.

Bodyweight

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The test material was considered to be a non sensitiser under the conditions of the test. Classification according to Egulation (EC) No. 1272/2008 (CLP) is not required.

Executive summary:

Introduction. A study was performed to assess the skin sensitisation potential of the test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of the following:

§        OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted)

§        Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Directive 2004/73/EC

Methods. Three groups, each of four animals, were treated with 50 µl (25 µl per ear) of the test material as a solution in ethanol/distilled water 7:3 at concentrations of 5%, 10% or 25% w/w. A further group of four animals was treated with ethanol/distilled water 7:3 alone.

Results. The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (% w/w) in ethanol/distilled water 7:3	Stimulation Index	Result
5	1.17	Negative
10	1.40	Negative
25	1.97	Negative

Conclusion. The test material was considered to be a non‑sensitiser under the conditions of the test. Classification according to Egulation (EC) No. 1272/2008 (CLP) is not required.