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Skin Sensitisation, Gamer (2005)

Under the conditions of the study, the test material has a skin sensitising effect in the Murine Local Lymph Node Assay. The threshold concentration for sensitisation induction was > 0.3, < 1% (w/w) under the test conditions chosen.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 July 2004 to 07 October 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: About 7 - 9 weeks
- Weight at study initiation: 16.0 - 21.2 g
- Housing: individually housed in Makrolon type I cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 22 days before the first test material application

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24°C
- Humidity: 30 - 70%
- Photoperiod: 12 h light (6.00 a.m. - 6.00 p.m.), 12 h darkness (6.00 p.m. - 6.00 a.m.)
Vehicle:
other: acetone
Concentration:
0.3, 1 and 3%
No. of animals per dose:
6 animals per dose
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Acetone was used as the vehicle because good solubility of the preparation was achieved.

MAIN STUDY

TREATMENT PREPARATION AND ADMINISTRATION:
- 25 µL was applied per ear to the dorsal part of both ears. Three consecutive applications (day 0 to day 2) were performed to the same application site.
- General observations: A check for dead or moribund animals was made twice each workday and once on Saturdays, Sundays and on public holidays. No detailed clinical examination of the individual animals was performed but any obvious signs of systemic toxicity and/or local inflammation at the application sites were recorded.
- Body weight: Body weights of the individual animals were determined on study day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
- The animals were sacrificed on study day 5 by cervical dislocation.
- Ear weight: Immediately after the death of each animal a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear. The weight of the pooled punches was determined for each animal.
- Lymph node weight: Immediately after removal of the ear punches the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.
- Preparation of cell suspension and determination of cell count: After weight determination, the pooled lymph nodes of each animal were stored in phosphate buffered saline in an ice-water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing the lymph nodes through an iron mesh (mesh size 200 µm) into 6 mL of phosphate buffered saline. For determination of cell count the standard suspension was further diluted with Casy®ton in a ratio 1:500. The cell count was determined using a Casy®-Counter. Each suspension was measured in triplicate (3 dilutions of the standard suspension). The mean of the triplicate measurements was used for further evaluation.

EVALUATION OF RESULTS
- If a test material does not show a statistically significant and/or biologically relevant increase in cell count and/or lymph node weight as compared to the vehicle control in the presence of statistically significantly and/or biologically relevant increased ear weights as indication of skin irritation, it is considered not to be a sensitiser.
- If at least one concentration tested causes a concentration dependent statistically significant and/or biologically relevant increase in cell count and/or lymph node weight without being accompanied by a statistically significant and/or biologically relevant increase in ear weight, the test material is considered to be a sensitiser.
- If statistically significant and/or biologically relevant increases in ear weights are running in parallel to the increase in cell count and/or lymph node weight, it cannot be ruled out, that the lymph node response was caused by irritation and not by skin sensitisation. Then, for identification of the relevance of the statistical evaluation, a comparison of the results of the present test to appropriate historical control values may be performed . If one or a combination of the measured parameters change statistical significance, evaluation on basis of the criteria described above may be possible. If the statistical comparison with the historical control does not yield results useful for evaluation, further investigations may be necessary to differentiate between irritation and sensitisation response.
- If a test material does not elicit a statistical significant increase in lymph node weight and/or cell count but shows a clear concentration related increase in response, further investigating of the sensitisation potential at higher concentrations should be considered.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
- Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The indices of lymph node weight, cell count and ear weight were calculated as the ratio of the test group mean values for these parameters divided by those of the vehicle control group.
- Lymph node weight, cell count and ear weights were examined using the Wilcoxon test.
Positive control results:
Experimental starting date: 13 April 2004
Experimental completion date: 19 May 2004

- 3%: Lymph node weight index: 1.28*, Cell count index: 1.43* and Ear weight index: 1.10**
- 10%: Lymph node weight index: 1.81**, Cell count index: 2.28** and Ear weight index: 1.41**
- 30%: Lymph node weight index: 2.39**, Cell count index: 2.92** and Ear weight index: 1.94**
* for p ≤ 0.05, ** for p ≤ 0 .01 (Wilcoxon test)
- No signs of systemic toxicity were noticed. The positive control induced a statistically significant response in the auricular lymph nodes, when applied as 3, 10 or 30 % preparations in 1 % Pluronic® L 92 in doubly distilled water. The statistically significant increases in ear weights indicate irritation of the ear skin at all concentrations. The high ear weights in test groups 3 and 4 were accompanied by scaling and incrustations, which were observed on the day of lymph node removal.
- The increase in cell count index induced by the 3% positive control preparation was at the border of biological significance. The marked increase at 10 and 30% cannot be explained by skin irritation alone.
- Based on the results of this study, it is concluded that the test conditions and animal strain chosen are able to reveal the known skin sensitising effect of Alpha-Hexylcinnamaldehyde, techn . 85% in the Murine Local Lymph Node Assay.
- The threshold of skin sensitisation induction of the compound lies just below 3% under the test conditions used.
Key result
Parameter:
other: Threshold concentration for sensitisation
Value:
> 0.3 - < 1
Test group / Remarks:
The threshold concentration for sensitisation induction was > 0.3 % < 1 %
Parameter:
SI
Value:
1
Test group / Remarks:
Vehicle
Parameter:
SI
Value:
0.95
Test group / Remarks:
0.3 % in acetone
Parameter:
SI
Value:
1.68
Test group / Remarks:
1 % in acetone
Remarks on result:
other: Statistically significant p ≤ 0.01
Parameter:
SI
Value:
2.75
Test group / Remarks:
3 % in acetone
Remarks on result:
other: Statistically significant p ≤ 0.01
Cellular proliferation data / Observations:
LYMPH NODE WEIGHTS AND CELL COUNTS
- Treatment of the mice with 1 and 3% test material preparations induced statistically significant increase in lymph node cell counts as compared to the vehicle control group. The lymph node weights were statistically increased and therefore in congruency with the cell counts. The group mean values are presented in Table 1.

EAR WEIGHTS
- Treatment of the mice with 0.3, 1 or 3% test material preparations induced no statistically significant increase in ear weight as compared to the vehicle control group.

BODY WEIGHTS
- The expected body weight gain was generally observed in the course of the study.

DISCUSSION AND CONCLUSION
- No signs of systemic toxicity were noticed.
- The test material induced a statistically significant and biologically relevant response of the auricular lymph nodes when applied as 1 and 3% preparations in acetone to the ear of the mice.
- The ear weights did not indicate a significant irritation of the ear skin by the test material at concentrations of 0.3 - 3%.
- In conclusion, the test material has a skin sensitising effect in the Murine Local Lymph Node Assay. The threshold concentration for sensitisation induction was > 0.3% < 1% under the test conditions chosen.

Table 1: Lymph node weight and cell count: Test group mean values, standard deviations and indices

Treatment Group

Lymph Node Weight [mg]

Cell Count [Counts/Lymph Node Pair]

Mean

S.D

Index

Mean

S.D

Index*

Vehicle (acetone)

5.0

0.4

1.00

8 712 000

1 110 052

1.00

0.3 % in acetone

5.1

0.5

1.02

8 315 333

699 468

0.95

1 % in acetone

7.1

1.1

1.43##

14 597 333

2 475 710

1.68##

3 % in acetone

11.0

2.2

2.23##

23 948 667

7 014 531

2.75##

S.D. Standard deviation

* treated group/ vehicle control

## statistically significant for the value p 0.01

Interpretation of results:
other: EU Criteria: Category 1A
Conclusions:
Under the conditions of this study, the test material has a skin sensitising effect in the Murine Local Lymph Node Assay. The threshold concentration for sensitisation induction was > 0.3% < 1% under the test conditions chosen.
Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guidelines OECD 429, EU Method B.42 and OPPTS 870.2600, under GLP conditions.

The skin sensitising potential of the test material was assessed using the non-radioactive variant of the Murine Local Lymph Node Assay. The assay simulates the induction phase for skin sensitisation in mice. It determines the response of the auricular lymph nodes on repeated application of the test material to the dorsal skin of the ears.

During the study, groups of 6 female CBA/Ca mice each were treated with 0 .3, 1 and 3% (w/w) preparations of the test material in acetone or with the vehicle alone. The study used 3 test groups and 1 control group. Each test animal was applied with 25 µL per ear of the respective test material preparation to the dorsum of both ears for three consecutive days. The control group was treated with 25 µL per ear of the vehicle alone.

Three days after the last application the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring the cellular content (indicator of cell proliferation) and weight of each animal's pooled lymph nodes. A defined area with a diameter of 0.8 cm was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation.

No signs of systemic toxicity were noticed. The test material induced a statistically significant and biologically relevant response of the auricular lymph nodes when applied as 1 and 3% preparations in acetone to the ear of the mice. The ear weights did not indicate a significant irritation of the ear skin by the test material at concentrations of 0.3 – 3%.

Under the conditions of this study, the test material has a skin sensitising effect in the Murine Local Lymph Node Assay. The threshold concentration for sensitisation induction was > 0.3, < 1% (w/w) under the test conditions chosen.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Skin Sensitisation, Gamer (2005)

The skin sensitisation potential of the test material was investigated in accordance with the standardised guidelines OECD 429, EU Method B.42 and OPPTS 870.2600, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The skin sensitising potential of the test material was assessed using the non-radioactive variant of the Murine Local Lymph Node Assay. The assay simulates the induction phase for skin sensitisation in mice. It determines the response of the auricular lymph nodes on repeated application of the test material to the dorsal skin of the ears.

During the study, groups of 6 female CBA/Ca mice each were treated with 0 .3, 1 and 3% (w/w) preparations of the test material in acetone or with the vehicle alone. The study used 3 test groups and 1 control group. Each test animal was applied with 25 µL per ear of the respective test material preparation to the dorsum of both ears for three consecutive days. The control group was treated with 25 µL per ear of the vehicle alone.

Three days after the last application the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring the cellular content (indicator of cell proliferation) and weight of each animal's pooled lymph nodes. A defined area with a diameter of 0.8 cm was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation.

No signs of systemic toxicity were noticed. The test material induced a statistically significant and biologically relevant response of the auricular lymph nodes when applied as 1 and 3% preparations in acetone to the ear of the mice. The ear weights did not indicate a significant irritation of the ear skin by the test material at concentrations of 0.3 – 3%.

Under the conditions of this study, the test material has a skin sensitising effect in the Murine Local Lymph Node Assay. The threshold concentration for sensitisation induction was > 0.3, < 1% (w/w) under the test conditions chosen.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance requires classification as a skin sensitiser (Category 1A) and is assigned the hazard statement H317: May cause an alergic skin reaction.