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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:
26 February 2016 to 15 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Test material form:
other: yellow semi-solid
Specific details on test material used for the study:
- Physical state: Yellow semi-solid
- Analytical purity: 100 %
- Expiration date of the lot/batch 23 November 2017
- Storage condition of test material: Room temperature in the dark

In vitro test system

Details on study design:
Not applicable

In chemico test system

Details on study design:
Not applicable

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands.
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: Animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: Food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Ltd., Oxon, UK), ad libitum
- Water: Mains tap water, ad libitum
- Acclimation period: at least 5 days
-Randomly allocated to cages.

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: at least 15 changes per hour
- Photoperiod: 12 h dark / 12 h light

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Preliminary test: 50% w/w in acetone/olive oil 4:1
Main test: 1.5, 15 and 50% w/w in acetone/olive oil 4:1
No. of animals per dose:
Preliminary screening test: 1 female/dose
Main test: 5 females/dose
Details on study design:
PRELIMINARY SCREENING TEST:
- The mouse was treated by daily application of 25 µL of test item at concentrations of 50% w/w in acetone/olive 4:1 to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3).

MAIN TEST
- Mice were treated by daily application of 25 µL of test item at concentrations of 1.5, 15 and 50% w/w in acetone/olive 4:1 to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3) using an automatic micropipette.
- A further group of 5 mice received the vehicle alone in the same manner.
- The positive control animals were similarly treated except that alpha-Hexylcinnamaldehyde, tech., 85% was used at a concentration of 25% v/v in acetone/olive oil 4:1.

3H-METHYL THYMIDINE ADMINISTRATION AND TERMINAL PROCEDURES
- Following first topical application of test item, vehicle control or positive control item, on 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, ARC UK Ltd) giving a total of 20 μCi to each mouse.
- Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation.
- The draining auricular lymph nodes were excised and pooled for each individual animal of each group. For each group 1 mL of PBS was added to the pooled lymph nodes.
- A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze.
- Lymph node cells were rinsed with 4 mL PBS into a petri dish, and cells suspension was transferred to a centrifuge tube. Petri dish was washed with 5 mL PBS and cells added to the centrifuge tube.
- Lymph node cells were pelleted at 1400 pm for 10 mins and re-suspended in 10 mL PBS and re-pelleted.
- The pellet was re-suspended in 3 mL of 5 % (w/v) trichloroacetic acid (TCA) to precipitate radioactive material.
- After 18 hours incubation at 4 °C, precipitate were recovered by centrifugation at 2100 rpm for 10 mins and re-suspended in 1 mL TCA and transferred to 10 mL of scintillation fluid.
- 3HTdR incorporation was measured by β –scintillation counting.

DATA EVALUATION
- The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
- Criteria used to consider a positive response: The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a “non-sensitizer.”
- The EC3 value was also calculated (EC3 is the concentration of test item expected to cause a 3 fold in 3HTdR incorporation.
- The equation used for the calculation of EC3 is EC3= c + [[(3-d)/(b-d)] x (a-c)].
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate.
Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeinity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric once way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non-parametric Kruskal-Wallis Rank Sum and Mann-Whitney test procedures were used.

Results and discussion

Positive control results:
Stimulation index for α-hexylcinnamaldehyde at 25 % v/v in acetone/olive oil 4:1 was 5.30 and classified as a sensitizer.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
EC3
Remarks:
(%)
Value:
9
Key result
Parameter:
SI
Value:
0.76
Test group / Remarks:
Stimulation index for 1.5% test item.
Remarks on result:
other: Negative
Key result
Parameter:
SI
Value:
4.81
Test group / Remarks:
Stimulation index for 15% test item.
Remarks on result:
other: Positive
Key result
Parameter:
SI
Value:
8.18
Test group / Remarks:
Stimulation index for 50% test item.
Remarks on result:
other:
Remarks:
positive
Cellular proliferation data / Observations:
PRELIMINARY SCREENING TEST
- Clinical observations, body weight and mortality data are given in Appendix 1 (attached) and local skin irritation is given in Appendix 2 (attached).
- The ear thickness measurements and mean ear thickness changes are given in Appendix 3 (attached).
- No signs of systemic toxicity, visual skin irritation or irritation were observed.
-Based on this information, dose levels of 1.5, 15 and 50% were selected for the main test.

MAIN TEST
- The radioactive disintegrations per minute per lymph node and the stimulation index are given in Appendix 4 (attached).
- The Stimulation Index for 1.5, 15 and 50% were 0.76, 4.81 and 8.18, respectively. Thus, the test item was found negative at 1.5% and positive at 15 in 50%.
- Individual clinical observations and mortality data for test and control animals are given in Appendix 5 (attached) and local irritation is given in Appendix 6 (attached).
- The ear thickness measurements and mean ear thickness are given in Appendix 7 (attached).
- No death occurred and no signs of systemic toxicity were observed during the test.
- Individual body weights and body weight change are given in Appendix 8 (attached).
- Body weight change of test animals between Day 1 and 6 was comparable to that observed in the corresponding control group animals.

Any other information on results incl. tables

The Stimulation Index for the test tiem was 0.76 (negative), 4.81 (positive), 8.18 (positive) at concentration of 1.5, 15 and 50% test item in acetone/olive oil 4:1, respectively.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test item was considered to be a sensitizer under the conditions of the test.
The positive control alpha-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 25% v/v in acetone/olive oil 4:1, thus, demonstrating the sensitivity and reliability of the test system.
Executive summary:

INTRODUCTION

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.


METHOD

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50% w/w, this concentration was selected as the highest dose level to be investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in acetone/olive oil 4:1 at concentration of 1.5, 15 and 50% w/w. A further group of five animals was treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using alpha-hexyclinnamaldehyde tech., 85% at a concentration of 25% v/v in acetone/olive oil 4:1.


RESULTS

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

The Stimulation Index for the test tiem was 0.76 (negative), 4.81 (positive), 8.18 (positive) at concentration of 1.5, 15 and 50% test item in acetone/olive oil 4:1, respectively.

The Stimulation Index for the positive control item was 5.30 (positive) at a concentration of 25% positive control in acetone/olive oil 4:1.

The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 9%.

 

CONCLUSION

The test item was considered to be a sensitizer under the conditions of the test.

The positive control alpha-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 25% v/v in acetone/olive oil 4:1, thus, demonstrating the sensitivity and reliability of the test system.