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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
From May 13 to 21, 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Source study has reliability 1. Details on the read across are attached in section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
male mice instead of female mice were used; group housing instead of single housing; no body weight measurement
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Interfauna UK Limited, Blackthorne, Bicester, Oxon, UK
- Sex: male
- Age at study initiation: young adults
- Weight at study initiation:
- Housing: 4/cage
- Diet (e.g. ad libitum): RM1 ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 d
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30-70 %
- Air changes: minimum 15 per hr
- Photoperiod: 12/12 hrs dark / hrs light
- IN-LIFE DATES: the main study was initiated on 13 May 2002. The experimental phase started on 14 May 2002 and was completed on 21 May 2002.
For the positive control study, the experimental phase started on 18 September 2001 and was completed on 25 September 2001.

Study design: in vivo (LLNA)

Vehicle:
other: acetone
Concentration:
3 %, 10 % and 50 % w/v
No. of animals per dose:
groups of 4 males
Details on study design:
Groups of four male mice were used for this study. Approximately 25 µl of a 3 %, 10 % or 30 % w/v preparation of test substance in acetone was applied, using a variable volume micro-pipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using acetone alone. The procedure was repeated daily for 3 consecutive days.
Three days after the third application, all animals were injected, via the tail, with approximately 250 µl of PBS containing about 20 µCi of a 2.0Ci/mmol specific activity 3H-methyl thymidine (for beta-scintillation counting using a Packard Tri-Carb 2500TR Liquid Scintillation Counter). After 5 d, the animals were sacrificed. 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.

Results are expressed as a counts per minute (cpm) value per lymph node for each group. The stimulation index for each test group is then calculated by dividing the cpm value per lymph node by the equivalent value for the control (vehicle only) group.
The criterion for a positive response is that one or more concentrations of the test substance should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group. The assay is able to identify those materials that elicit responses in standard guinea pig tests for skin sensitisation. Consequently, a test substance which does not fulfil the above criterion is designated as unlikely to be a sensitiser.


Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The application of hexylcinnamaldehyde at concentrations of 1, 3 and 10 % w/v in acetone resulted in a greater than 3-fold increase in isotope incorporation at all 3 concentrations.
The validity of the protocol was confirmed.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
0.83
Test group / Remarks:
3% of test substance
Parameter:
SI
Value:
1.19
Test group / Remarks:
10% of test substance
Parameter:
SI
Value:
1.06
Test group / Remarks:
30% of test substance
Cellular proliferation data / Observations:
The application of test substance at concentrations of 3 %, 10 % and 30 % w/v in acetone resulted in an increase in isotope incorporation which was less than 3-fold at all three concentrations.

Any other information on results incl. tables

concentration of
test substance
(% w/v)

number of
lymph nodes
assayed

counts per
minute

(cpm)

cpm per
lymph node
(×10-2)

test control
ratio

0 (vehicle only)

8

1092

1.37

N/A

3

8

913

1.14

0.83

10

8

1303

1.63

1.19

30

8

1160

1.45

1.06

Applicant's summary and conclusion

Interpretation of results:
other: not classified according to the CLP Regulation (EC 1272/2008)
Conclusions:
Test substance was considered to be not sensitizing to mouse skin (LLNA).
Executive summary:

Method

Skin sensitisation potential of the test substance was assessed based on OECD guideline 429 (local lymph node assay). Male CBA/Ca mice were exposed to test substance at concentrations of 0, 3, 10 and 30 % in acetone. Isotope (3H-methyl thymidine) incorporation was measured in lymph nodes. The negative (vehicle alone) and positive (hexylcinnamaldehyde at concentrations of 1, 3, and 10 % w/v in acetone) controls were tested.

Results

Positive and negative controls were valid. The isotope incorporation was increased by less than a threefold at all concentrations of test substance. Under test conditions, test substance was considered to be not sensitizing to mouse skin.