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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Hanuary - March (experimental part)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
see Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (OJ L 142, 31.5.2008, p. 1) as amended B.42
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
Species: Mouse
Strain: CBA/Ca/Ola/Hsd
Source: Harlan lnterfauna UK Limited, Blackthorne, Bicester, Oxon, UK
Sex: Female
Number used: 4 per group
Specification: Young adults (8 — 12 weeks of age)
A maximum of 4 mice was housed per cage, in cages suitable for animals of this strain and weight range. Environmental enrichment provided included tents, bases and nestlets. The animal room was designed to give the environmental conditions shown as follows.
Temperature: 22 ±3 °C
Relative humidity: 30 - 70%
Air: A minimum of 15 changes per hour
Light cycle: Artificial, giving 12 hours light, 12 hours dark
Both temperature and relative humidity were recorded daily. The recorded values were within the Specified ranges.
Diet (RM1), supplied by Special Diet Services Limited, Witham, Essex, UK, and mains water, supplied by an automatic system, were available ad libitum.
Each batch of diet is routinely analysed for composition and for contaminants. Water is also periodically analysed for contaminants. No contaminants were found in the diet or water at levels considered likely to interfere with the purpose or outcome of the study. Certificates of analyses are retained in the CTL Archives.
Acclimatisation: The animals were housed under the experimental conditions for at least 5 days, prior to the start of dosing.
Animal identification: Animals were individually identified, within each cage, by a clipped area on the side(s). The first animal in each cage had a clipped area on the left side, the second on the right side, the third on both flanks and the fourth was not clipped.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
All dose preparations were used within 24 hours of preparation. No correction was made for the purity ofthe active ingredient in the test substance. Stability
and achieved concentration were not determined.
Groups of four female mice were used for this study. Approximately 25 ul of a 2.5, 5, 10, 25 or 50 %w/v preparation of the test substance in acetone in olive oil (4:1) was applied, using a variable volume micro-pipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using acetone in olive oil (4:1) alone. The procedure was repeated daily for 3 consecutive days.
Approximately 25 µl of a 5, 10 or 25% w/v preparation of hexyl cinnamaldehyde in acetone/olive oil (4:1) was applied, and a vehicle control group was similarly treated using acetone/olive oil (4: 1) alone.
No. of animals per dose:
4 females per dose
Details on study design:
Groups of four female mice were used for this study. Approximately 25 ul of a 2.5, 5, 10, 25 or 50 %w/v preparation of the test substance in acetone in olive oil (4:1) was applied, using a variable volume micro-pipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using acetone in olive oil (4:1) alone. The procedure was repeated daily for 3 consecutive days.
Three days after the third application, all the animals were injected, via the tail vein, with approximately 250 µL of phosphate buffered saline (PBS) containing 20 µCi of a 2.0Ci/rnmol specific activity 3H-methyl thymidine. Approximately 5 hours later, the animals were humanely killed by inhalation of halothane vapour followed by cervical dislocation. The draining auricular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a container of PBS.
A single cell suspension was prepared by mechanical disaggregation of lymph nodes through a 200-mesh stainless steel gauze. The cell suspensions were then washed three times by centrifugation with approximately 10 mL of PBS. Approximately 3 mL of 5% w/v trichloroacetic acid (TCA) was added and, after overnight precipitation at 4 °C, the samples were pelleted by centrifugation and the supernatant was discarded. The cells were then resuspended in approximately 1 mL of TCA.
The lymph node suspensions were transferred to scintillation vials and 10 mL of scintillant (Optiphase) was added prior to β-scintillation counting using a Packard Tri Carb 3100TR Liquid Scintillation Counter.
Clinical observations: Animals were checked at least once daily for signs of systemic toxicity.
Bodyweights: The bodyweight of each animal was recorded prior to dosing on day 1 and prior to injection of 3H-methyl thymidine on day 6. Bodyweights were recorded.
Positive control study: The sensitisation potential of hexyl-cinnamaldehyde was assessed using the method described for the test substance.
Approximately 25 µl of a 5, 10 or 25% w/v preparation of hexyl cinnamaldehyde in acetone/olive oil (4:1) was applied, and a vehicle control group was similarly treated using acetone/olive oil (4: 1) alone.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The application of hexyl cinnamaldehyde at concentrations of 5, 10 and 25 w/v in acetone:olive oil (4: 1) resulted in a greater than 3-fold increase in isotope incorporation at the 25% concentration. Therefore, hexyl cinnamaldehyde was shown to be a skin sensitiser, confirming the validity of the protocol used for the study.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.1
Test group / Remarks:
2.5 % w/v test substance
Parameter:
SI
Value:
2.5
Test group / Remarks:
5 % w/v test substance
Parameter:
SI
Value:
4.4
Test group / Remarks:
10 % w/v test substance
Parameter:
SI
Value:
18.9
Test group / Remarks:
25 % w/v test substance
Parameter:
SI
Value:
22.9
Test group / Remarks:
50 % w/v test substance
Parameter:
EC3
Value:
6.3
Test group / Remarks:
The calculated EC3 is 6.3 % w/v for the test substance

Any other information on results incl. tables

Results of test substance

Concentration of test subst. (% w/v)

Number of lymph nodes assayed

Disintegrations per minute (dpm)

dpm per lymph node

Test:control ratio (Stimulation Index)

0 (vehicle only)

8

3716

465

N/A

2.5

8

3944

493

1.1

5

8

9408

1176

2.5

10

8

16317

2040

4.4

25

8

70392

8799

18.9

50

8

85020

10628

22.9

EC3

Calculated to be 6.3% (1575µg/cm²)

N/A = not applicable

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The estimated concentration giving rise to a 3-fold increase in lymphocyte proliferation (EC3) was 6.3% w/v (1575,µg/cm²) und thus, the substance is considered a skin-sensitiser.
Executive summary:

The application of the test substance at concentrations of 2.5, 5, 10, 25 and 50% w/v in acetone / olive oil (4:1) resulted in an increase in isotope incorporation which was greater than 3-fold at the 10, 25 and 50% w/v concentrations. Consequently, the test substance was shown to be a potential skin sensitiser. The estimated concentration giving rise to a 3-fold increase in lymphocyte proliferation (EC3) was 6.3% w/v (1575,µg/cm²).