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Endpoint summary

Description of key information

LLNA (OECD TG 429): EC3 = 31%.

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 26 September 2016 and 23 November 2016

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:

22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

EC No. 440/2008

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:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor Batch No. RB390-61
- Expiration date of the lot/batch: 01 June 2018
- Purity test date: 16 June 2016
- Purity: 99.4%
- Appearance: Clear colorless liquid
- Storage conditions: approximately 4”C in the dark

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

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 acclimatisation period of at least five 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 eight to twelve weeks old.

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) Ltd, Oxon, UK) was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 19 to 25°C and 30 to 70 %, respectively. The rate of air exchange was approximately 15 changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light and twelve 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:

Preliminary Test
The test item undiluted or at a concentration of 50% v/v in vehicle e.g.: 4:1

Main Test
The test item at concentrations of 50% or 25% v/v in vehicle e.g.: 4:1

No. of animals per dose:

Preliminary Test
One per concentration

Main Test
Groups of five mice were treated per concnetration

Details on study design:

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 two mice, one mouse per test item concentration. The mice were treated by daily application of 25 µL of the undiluted test item or the test item at a concentration of 50% v/v in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice were 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 erythema (see below). Any clinical signs of toxicity, if present, were also recorded. The body weight of each 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. Due to a technician error, the undiluted test item was used as the maximum concentration rather than the 50%, as the preliminary screening test suggested that the undiluted test item had the potential to cause excessive local skin irritation. 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, 97.3%, at a concentration of 25% v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item, vehicle control or positive control item (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR:80µCi/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.

Bodyweights: The bodyweight 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 labelled with the project 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 ten minutes. The pellet was resuspended in 10 ml of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 ml of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation: After approximately eighteen hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by beta scintillation counting. The "Poly QTM" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty 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 twenty 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:

Dissiminated (delete when editing)
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:

EC3

Value:

31

Parameter:

SI

Value:

2.42

Test group / Remarks:

25%

Parameter:

SI

Value:

4.74

Test group / Remarks:

50%

Parameter:

SI

Value:

4.93

Test group / Remarks:

100%

Preliminary Screening Test

No signs of systemic toxicity or visual local skin irritation were noted.  A greater than 25% increase in mean ear thickness was noted in the animal treated with the undiluted test item.  No irritation indicated by an equal to or greater than 25% increase in mean ear thickness was noted in the animal treated with the test item at a concentration of 50% v/v in acetone/olive oil 4:1.

Based on this information, the dose levels selected for the main test were 50%, 25% and 10% v/v in acetone/olive oil 4:1, but due to a technician error, the actual concentrations used were 100%, 50% and 25% v/v in acetone/olive oil 4:1.

 Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute per lymph node and the stimulation index are given in Appendix 4.

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:

Treatment Group	Concentration	Stimulation Index	Result
Test Item	25% v/v in acetone/olive oil 4:1	2.42	Negative
	50% v/v in acetone/olive oil 4:1	4.74	Positive
	100%	4.93	Positive
Positive Control Item	25% v/v in acetone/olive oil 4:1	7.42	Positive

                    

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       =       4.74

c       =       25

d       =       2.42

EC3 = 25 + [[(3-2.42)/(4.74-2.42)] x (50-25)] = 31

The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 31%

   

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 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.
The test item was classified as a contact sensitizer (Category 1B) according to the Globally Harmonized System of Classification and Labelling of Chemicals.
The positive control α Hexylcinnamaldehyde, 97.3% gave a Stimulation Index of greater than 3 (7.42) 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.
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Executive summary:

Dissiminated; Only study results should be added, additional reasoning when applicable should be done in Endpoint summary (deleted when editing)

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 2.42, 4.74 and 4.93 respectively. At these concentrations also ear thickness was increased with 60% at the 100% test substance application indicating slight irritation of the test substance but unlikely to have influenced the result of the study. An EC3 has been derived resulted in an EC3 of 31%.

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 2.42, 4.74 and 4.93, respectively.

At these concentrations also ear thickness was increased with 60% at the 100% test substance application indicating slight irritation of the test substance but unlikely to have influenced the result of the study. An EC3 has been derived resulted in an EC3 of 31%.

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

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Justification for classification or non-classification

Based on the EC3 value of 31% in the LLNA study, the substance has to be classified for skin sensitisation. According to EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, it has to be classified for skin sensitisation,Category 1B, H317: May cause an allergic skin reaction.