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Description of key information

LLNA (OECD TG 429): EC3 = 40%, skin sensitiser Category 1B

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:
Experimental start date: 11 September 2017 Experimental completion date: 28 September 2017
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Identification: FRET 12-0492
Physical state / Appearance: very pale yellow liquid
Storage Conditions: approximately 4°C in the dark
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 ºC 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:
Undiluted test item or the test item at concentrations of 50 % or 25 % v/v in acetone/olive oil 4:1.
No. of animals per dose:
Groups of five mice were treated
Details on study design:
Preliminary Screening Test
Using available information, 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. Any clinical signs of toxicity, if present, were also recorded. The body weight of the mouse 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.
The positive control animals were similarly treated to the test animals except that 25 µL of the positive control item, α Hexylcinnamaldehyde, tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear.
Local skin irritation was scored daily. The thickness of each ear was measured and recorded as described in the preliminary screening test.

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 0.25 mL (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. Some of the lymph node cells for the suspension for animal 3-2 were lost during preparation due to a cracked centrifuge tube but after further investigation this was considered not to have affected the purpose or integrity of the study. 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 ºC, 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 B-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).
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:
The positive control item, α-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.
Key result
Parameter:
SI
Value:
2.02
Test group / Remarks:
25 % v/v
Key result
Parameter:
SI
Value:
3.68
Test group / Remarks:
50 % v/v
Key result
Parameter:
SI
Value:
4
Test group / Remarks:
100 % v/v
Key result
Parameter:
EC3
Value:
40
Cellular proliferation data / Observations:
Preliminary Screening Test
No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.
Based on this information the dose levels selected for the main test were 100%, 50% and 25% v/v in acetone/olive oil 4:1.

Clinical Observations and Mortality Data
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Body Weight
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.

Calculation of EC3 Value
EC3 = c + [[(3-d)/(b-d)] x (a-c)]
a = 50
b = 3.68
c = 25
d = 2.02

a = lowest concentration giving stimulation index >3
b = actual stimulation index caused by ‘a’
c = highest concentration failing to produce a stimulation index of 3
d = actual stimulation index caused by ‘c’


EC3 = 25 + [[(3-2.02)/(3.68-2.02)] x (50-25)] = 40
The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 40%.

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

Concentration (%v/v) in
acetone/olive oil 4:1

Stimulation Index

Result

25

2.02

Negative

50

3.68

Positive

100

4.00

Positive

Positive Control Item – 25%

8.52

Positive

 

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The test item was considered to be a sensitiser under the conditions of the test and resulted in an EC3 of 40%.
Executive summary:

The skin sensitisation potential of the test substance has been tested according to OECD TG 429: Local Lymph Node Assay" method. At 25, 50 and 100% the substance showed SI values of 2.02, 3.68 and 4.00, respectively. 100 % of the test substance caused no signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness. An EC3 has been derived resulted in an EC3 of 40 %.

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

The skin sensitisation potential of the test substance has been tested according to OECD TG 429: Local Lymph Node Assay" method. At 25, 50 and 100% the substance showed SI values of 2.02, 3.68 and 4.00, respectively. 100 % of the test substance caused no signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness.An EC3 has been derived resulted in an EC3 of 40 %.


Justification for selection of skin sensitisation endpoint:
The result of this study is reliable and adequate for covering this endpoint.

Justification for classification or non-classification

Based on the EC3 value of 40 % in the LLNA study, the substance has to be classified for skin sensitisation. The test item was also classified as a contact sensitizer (Category 1B) according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. It is reasonable to assume that the Signal Word “Warning” and the Hazard Statement “H317: May cause an allergic skin reaction” are therefore required.

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