A mixture of Propanoic acid, -1-methyl, 2-hydroxy-, (C12-14)-alkyl ester, Propanoic acid, 2-hydroxy-, 2-(C12-14-alkyloxy)-1-methyl-2-oxoethyl ester, Propanoic acid, 2-hydroxy-, 2-(2-(C12-14-alkyloxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethyl ester, Propanoic acid, 2-hydroxy-, 2-(2-(2-(C12-14-alkyloxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethyl ester, Propanoic acid, 2-hydroxy-, 2-(2-(2-(2-(C12-14-alkyloxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethyl ester, Propanoic acid, 2-hydroxy-, 2-(2-(2-(2-(2-(C12-14-alkyloxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethyl ester, Propanoic acid, 2-hydroxy-, 2-(2-(2-(2-(2-(2-(C12-14-alkyloxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoeth oxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethyl ester, Propanoic acid, 2-hydroxy-, 2-(2-(2-(2-(2-(2-(2-(C12-14-alkyloxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoeth oxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethyl ester and Propanoic acid, 2-hydroxy-, 2-(2-(2-(2-(2-(2-(2-(2-(C12-14-alkyloxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoeth oxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethyl ester

Endpoint summary

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:

11 February - 15 July 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories S.r.l., Zona Industriale Azzida, 57, 33049 San Pietro al Natisone (UD), Italy
- Age at study initiation: 9 weeks
- Weight at study initiation: 18.5-19.8 g
- Housing: group caging in Type II polypropylene/polycarbonate cages
- Diet: ad libitum - ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice”
- Water: ad libitum tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 - 25.7°C
- Humidity (%): 31-69 %
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 16 April -21 April, 2015

Vehicle:

propylene glycol

Concentration:

25, 10 and 5% (w/v) in the main study

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: At the request of the Sponsor (based on the chemical characteristics of the test item), Propylene glycol (PG) and 1% aqueous Pluronic® PE9200 (1% Pluronic) vehicles were examined. The 50% (w/v) formulation was achievable using PG as vehicle (resulting a clear solution), while the use of 1% Pluronic resulted an inhomogeneous formulation at the same concentration (small, gel-like particles were observed in the formulation). Therefore, Propylene glycol was selected as vehicle for the study as agreed by the Sponsor. The highest achievable concentration for the test based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item (liquid) was 100% (undiluted).
- Irritation: Preliminary Irritation/Toxicity Test I. was performed on CBA/CaOlaHsd mice using two doses (2 animals/dose), at test item concentrations of 100% (undiluted) and 50% (w/v) in PG. The preliminary experiment was conducted in a similar experimental manner to the main study, but they were terminated on Day 6 with a body weight measurement.
During the Preliminary Irritation / Toxicity Test I., no mortality was observed. Hunched back, piloerection and decreased activity was recorded in the 100% dose group on Day 4; hunched back was also observed in this group on Day 5. Furthermore, minimal alopecia and minor peeling were observed in this group on Day 6. Marked body weight loss (>5%) was detected for one animal in the 100% (undiluted) dose group, but based on the result of the second animal of that group, this fact was considered to be normal animal variability.
Ear thickness of the animals was measured using by a thickness gauge on Days 1, 3 and 6, and by ear punch weight determination after the euthanasia of the experimental animals on Day 6. Increased ear thickness values were detected in several cases on Days 3 and 6; the results were clearly above the limit of acceptability in both dose groups on Day 6. The ear punch weights were within the acceptable range. Furthermore, erythema was observed in the 100% (undiluted) dose group on the ears on Day 3, and on the top of the head on Days 4-5.
The draining auricular lymph nodes of the animals were visually examined: they were larger and/or slightly larger than normal in both dose groups (subjective judgement by analogy with observations of former experiments).
Based on these results, 100% (undiluted) and 50% (w/v) doses showed strong local irritation, and were excluded from the examined concentration series of the main test. An additional preliminary experiment (Preliminary Irritation/Toxicity Test II.) was performed to provide additional data for dose selection. This experiment was started on CBA/CaOlaHsd mice using three doses (2 animals/dose) at test item concentrations of 25, 10 and 5% (w/v) in PG.
During the Preliminary Irritation / Toxicity Test II. no mortality or signs of systemic toxicity were observed. No marked body weight loss (>5%) was detected.
No erythema was observed in this experiment. Slightly increased ear thickness values were detected on Day 3 and/or Day 6 in all three dose groups; but the values were below the limit of acceptability. The observed ear punch weights were in the acceptable range.
The draining auricular lymph nodes of the animals were visually examined: they were larger than normal in the 25% (w/v) dose group, but normal in the 10 and 5% (w/v) dose groups (subjective judgement by analogy with observations of former experiments).
Based on these results of the two preliminary tests, 25% (w/v) dose was selected as top dose for the main test.

- Lymph node proliferation response: Not performed in the preliminary tests.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Proliferation assay
- Criteria used to consider a positive response:
The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
- That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.


TREATMENT PREPARATION AND ADMINISTRATION:
Formulation
To prepare the dosing formulations, the test item was used undiluted or freshly diluted with the selected vehicle to obtain appropriate concentrations in the Pharmacy of CiToxLAB Hungary Ltd. The applicable dose levels were based on the results of the Preliminary Irritation / Toxicity Test. Formulations were prepared on weight: volume basis as % (w/v), and were considered to be stable for this short period (no correction for purity of the test item was applied).
Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study, although the formulations were checked for visible homogeneity and physical stability. The test item formulation at 50-5% (w/v) concentration range were colourless clear solutions as appeared by visual inspection.
Topical application
During the study, animals were topically dosed with 25 µL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The use of the individual approach to calculate SI made the use of a statistical analysis available. The statistical analysis was performed using the SPSS/PC+ (4.0.1) software package. The heterogeneity of variance between groups was checked by Bartlett's test for the measured DPM values. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the result was positive, then Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test.

Positive control results:

SI = 9.8. Please refer to table below for individual scores

Parameter:

SI

Value:

1

Test group / Remarks:

5%

Parameter:

SI

Value:

1.2

Test group / Remarks:

10%

Parameter:

SI

Value:

2.2

Test group / Remarks:

25%

Clinical observation: No mortality or signs of systemic toxicity were observed during the study. There was no visual sign of irritation (erythema) at the site of application.

Body weight measurement: No marked body weight loss (>5%) was detected for any animals in the main test, and no treatment related effects were observed on the mean body weight in the test item treated groups.

Proliferation assay:

DPM, DPN and Stimulation Index Values for all Groups

Test Group Name	Animal Number	Measured DPM	DPM	Number of lymph nodes	DPN	Group DPN	Stimulation Index
Background (5% (w/v) TCA)	-	58 50				-	-
Negative control (PG)	1	1066	1012.0	2	506.0	378.9	1.0
	2	666	612.0	2	306.0
	3	901	847.0	2	423.5
	4	614	560.0	2	280.0
Reaction Mass of Alcohols, C12-14 (even numbered), with Dilactide 25% (w/v) in PG	5	1559	1505.0	2	752.5	818.8	2.2*
	6	2325	2271.0	2	1135.5
	7	1379	1325.0	2	662.5
	8	1503	1449.0	2	724.5
Reaction Mass of Alcohols, C12-14 (even numbered), with Dilactide 10% (w/v) in PG	9	603	549.0	2	274.5	461.8	1.2
	10	1457	1403.0	2	701.5
	11	756	702.0	2	351.0
	12	1094	1040.0	2	520.0
Reaction Mass of Alcohols, C12-14 (even numbered), with Dilactide 5% (w/v) in PG	13	597	543.0	2	271.5	379.3	1.0
	14	814	760.0	2	380.0
	15	1098	1044.0	2	522.0
	16	741	687.0	2	343.5
Positive control (25% HCA inPG)	17	9678	9624.0	2	4812.0	3719.6	9.8*
	18	4945	4891.0	2	2445.5
	19	10010	9956.0	2	4978.0
	20	5340	5286.0	2	2643.0

Note:

1.             * = Significant (p<0.05, Mann-Whitney U-test versus negative control)

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on the results of this LLNA, the substance is not a sensitiser.

Executive summary:

The skin sensitisation potential of the substance was studied in a Murine Local Lymph Node Assay performed under GLP according to OECD TG 429 and EU Method B.42. Following preliminary solubility/compatibility testing, propylene glycol was selected as the solvent. Based on irritation seen in the preliminary test, doses of 5, 10 and 25% were selected for use in the main study. In the main study groups of 4 mice were treated topically with either 5, 10 or 20% solutions of the substance in propylene glycol, a vehicle control or 25 % w/v HCA in propylene glycol as the positive control for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (³HTdR) and the values obtained were used to calculate stimulation indices (SI).

No mortality or signs of systemic toxicity were observed during the study. No treatment related effect was observed on the body weight in the test item treated groups. There were no indications of any irritancy (erythema) at the site of application.

The stimulation index values were 2.2, 1.2 and 1.0 at concentrations of 25, 10 and 5% (w/v), respectively.

The result of the positive control substancea-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A significant lymphoproliferative response (stimulation index value of 9.8) was noted for the positive control chemical, this result confirmed the validity of the assay.

Under the conditions of this assay, the substance, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitiser) in the Local Lymph Node Assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitisation potential of the substance was studied in a Murine Local Lymph Node Assay performed under GLP according to OECD TG 429 and EU Method B.42. Following preliminary solubility/compatibility testing, propylene glycol was selected as the solvent. Based on irritation seen in the preliminary test, doses of 5, 10 and 25% were selected for use in the main study. In the main study groups of 4 mice were treated topically with either 5, 10 or 20% solutions of the substance in propylene glycol, a vehicle control or 25 % w/v HCA in propylene glycol as the positive control for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6,the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (³HTdR) and the values obtained were used to calculate stimulation indices (SI).

No mortality or signs of systemic toxicity were observed during the study. No treatment related effect was observed on the body weight in the test item treated groups. There were no indications of any irritancy (erythema) at the site of application.

The stimulation index values were 2.2, 1.2 and 1.0 at concentrations of 25, 10 and 5% (w/v), respectively.

The result of the positive control substancea-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A significant lymphoproliferative response (stimulation index value of 9.8) was noted for the positive control chemical, this result confirmed the validity of the assay.

Under the conditions of this assay, the substance, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitiser) in the Local Lymph Node Assay.

Migrated from Short description of key information:
The substance is non-sensitising based on the results of a Murine Local Lymph Node Assay performed according to OECD TG 429 and EU Method B.42 under GLP. The stimulation index values were 2.2, 1.2 and 1.0 at concentrations of 25, 10 and 5% (w/v), respectively.

Justification for selection of skin sensitisation endpoint:
GLP guideline study performed on the substance itself

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance is non-sensitising based on the results of a Murine Local Lymph Node Assay performed according to OECD TG 429 and EU Method B.42 under GLP. The stimulation index values were 2.2, 1.2 and 1.0 at concentrations of 25, 10 and 5% (w/v), respectively. Since these values are below the threshold of 3, no classification is required.