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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
Remarks:
source of read across
Adequacy of study:
key study
Study period:
From May 13th to 21st, 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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; groupe 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

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
- Housing: 4 per cage
- Diet: RM1, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: minimum 15 per hr
- Photoperiod: 12 hrs dark / 12 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 the 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.

The 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 (Kimber et al 1994). Consequently, a test substance which does not fulfil the above criterion is designated as unlikely to be a sensitiser.

Animals were checked at least once daily for signs of systemic toxicity.
The results were expressed as a counts per minute (cpm) value per lymph node for each group. The stimulation index for each test group was then calculated by dividing cpm value per lymph node by the equivalent value for the control (vehicle only) group.

The criterion for a positive response (sensitiser) was 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.
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 three 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 the 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 (x10-2) Test control ratio
0 (vehicle control) 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:
Non skin sensitizer
Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance according to OECD Guideline 429 (local lymph node assay), in compliance with GLP. Male CBA/Ca mice were exposed to the 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 valid. The isotope incorporation was increased by less than a threefold at all concentrations of the test substance. Under the study conditions, the test substance was considered to be not sensitizing to mouse skin.

Conclusion

Non skin sensitizer