Registration Dossier

Administrative data

Description of key information

Skin sensitisation (OECD TG 429): not sensitising

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:
The study was conducted between 10 May 2016 and 25 May 2016.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
In accordance with GLP conditions
Justification for type of information:
LLNA study was performed before the REACH regulation came into force requesting in vitro information (October 2016).
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 22 July 2010.
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
Animal Information
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were 8 to 12 weeks old.

Animal Care and Husbandry
The animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70%, respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of the undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 50% or 25% v/v.
No. of animals per dose:
Three groups, each of five animals per dose. A further group of five animals was treated with acetone/olive oil 4:1 alone.
Details on study design:
Test Item Preparation and Analysis
For the purpose of the study, the test item was used undiluted and freshly prepared as a solution in acetone/olive oil 4:1.This vehicle was chosen as it produced the most suitable formulation at the required concentration. The concentrations used are given in the procedure section.The vehicle determination record is shown under 'Any other information on materials and methods incl. tables'. The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Preliminary Screening Test
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the undiluted test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the scale shown below:

Scale for Erythema (observation and score)
No erythema: 0
Very slight erythema (barely perceptible): 1
Well-defined erythema:2
Moderate to severe erythema: 3
Severe erythema (beef redness) to eschar formation preventing grading of erythema: 4

Any clinical signs of toxicity, if present, were also recorded. The body weight was recorded on Day 1 (prior to dosing) and on Day 6. The thickness of each ear was measured using a Mitutoyo 547 300S gauge (Mitutoyo Corporation), pre-dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.

Main Test

Test Item Administration
Groups of five mice were treated with the undiluted test item or the test item at concentrations of 50% or 25% (v/v) in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of five mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item or vehicle (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.

Observations
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes.
Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200 mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labeled with the study number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re suspended in 10 mL of PBS and re pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5% Trichloroacetic acid (TCA).
Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

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). 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".
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 homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one 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 U test procedures were used. Probability values (p) are presented as follows:
P<0.001: ***
P<0.01: **
P<0.05: *
P>0.05: (not significant)
Positive control results:
Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of α-Hexylcinnamaldehyde, tech., 85% as a solution in acetone/olive oil 4:1 at concentrations of 5%, 10% or 25% (v/v). A further group of five animals was treated with acetone/olive oil 4:1 alone and served as the vehicle control group. The concentration of α-Hexylcinnamaldehyde, tech., 85% expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 20%. α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.
Key result
Parameter:
SI
Remarks:
100%
Value:
1.8
Parameter:
SI
Remarks:
50%
Value:
2.53
Parameter:
SI
Remarks:
25%
Value:
0.97
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Mean DPM with standard deviation:
- Vehicle control group: 281.97 (±92.29)
- 25% (v/v): 272.97 (±143.92)
- 50% (v/v): 712.45 (±174.47)
- 100% (v/v): 507.97 (±206.79)

DETAILS ON STIMULATION INDEX CALCULATION
The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group.

EC3 CALCULATION
The test item was considered to be a non-sensitizer under the conditions of the test.

CLINICAL OBSERVATIONS
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

BODY WEIGHTS
Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.






In the preliminary screening test there were no signs of systemic toxicity or visual local skin irritation noted. No irritation was indicated by an equal to or greater than 25% increase in mean ear thickness. Based on this information the undiluted test item and the test item at concentrations of 50% and 25% (v/v) in acetone/olive oil 4:1 were selected for the main test.

Interpretation of results:
other: not sensiting
Remarks:
based on EU CLP criteria (1272/2008 and its amendments).
Conclusions:
Under the conditions of this test, the test substance did not induce a Stimulation Index (SI) above 3 when tested up to 100%. Based on these results, the substance is considered not to be a sensitiser and does not need to be classified for skin sensitisation.
Executive summary:

A study was performed to assess the skin sensitization potential of Reseda body in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear according to OECD Test Guideline 429 (LLNA). Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of100%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group were 0.97, 2.53 and 1.80 for te 25%, 50% and 100% (v/v) test groups, respectively. Under the conditions of this test, the test substance did not induce a Stimulation Index (SI) above 3. Based on these results, the substance is considered not to be a sensitiser.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A study was performed to assess the skin sensitization potential of Reseda body in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear according to OECD Test Guideline 429 (LLNA). Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of100%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group were 0.97, 2.53 and 1.80 for te 25%, 50% and 100% (v/v) test groups, respectively. Under the conditions of this test, the test substance did not induce a Stimulation Index (SI) above 3. Based on these results, the substance is considered not to be a sensitiser.

Justification for classification or non-classification

Based on the negative results found in the LLNA study, Reseda body does not need to be classified for skin sensitisation in accordance with EU CLP (1272/2008/EC and its amendments).