Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate

Endpoint summary

Description of key information

Based on the available information, the test item appears to be sensitising to the skin.

Skin sensitisation (In chemico): Data waving. Study technically not feasible. The test item is not soluble in the peptide buffer. OECD 442C stipulates that the test chemical should be soluble in an appropriate solvent at a final concentration of 100  mM.

Skin sensitisation (In vitro): Weight of evidence. Test method according to the OECD 442D. Guideline with GLP. The test item showed no sensitisation potential under test conditions.

Skin sensitisation (In vitro): Data waving. The study 442E does not need to be conducted as there are more adequate options available considering the physicochemical characteristics of the test item

Skin sensitisation (In vivo): Weight of evidence. Test method according to the OECD 442B. Guideline with GLP. Under the experimental conditions the test item should be classified as a skin sensitizer using the mouse local lymph node assay (LLNA): BrdU-ELISA.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
The test item is not soluble in the peptide buffer. OECD 442C stipulates that the test chemical should be soluble in an appropriate solvent at a final concentration of 100 mM.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 8th, 2018 to October 26th, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

February 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ECVAM DB-ALM protocol 155: KeratinoSens

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

- Cell line used: KeratinoSens™ (Givaudan) cultured in maintenance medium (DMEM 1 g/l glucose, 9.1% non-heat inactivated foetal calf serum, 0.05% geneticin - stored at 5ºC ± 3ºC) at 37ºC, 5% CO2. Cells were exempt of mycoplasma.
- Passage number and level of confluence of cells: cells were used at passage 17 in repetition 1, passage 19 in repetition 2 and passage 21 in repetition 3.
- The cells were trypsinized according to the current working instruction IL 09. Cells suspension were adjusted to a density of 8.10^4 cells/ml in the seeding medium. 125 µl of the cell suspension at 8.10^4 cells/ml (i.e. 10^4 cells per well) were distributed in three white plates for the induction measurement and two transparent plates to assess the cytotoxicity. The seeded plates were incubated 24 hours ± 1 hour at 37 ºC, 5% CO2. The H12 wells were left without cells and allowed the measurement of blanks.
- Luminometer used: GloMax™ (Promega) MULTISKAN EX plate reader (Thermo life sciences) - reading range 0 - 3.5 units of Absorbance, linearity range 0 - 2.200 units of Absorbance
- Number of repetitions and replicates: the study was composed of three independent repetitions. For each repetition, the test item and the reference items were replicated on three independent plates for the measurement of induction and two plates for the measurement of cytotoxicity. Each repetition was performed on a different day with a fresh stock solution.
- Test chemical concentrations: Test item was diluted 100-fold (10X plate) in sterile water from a 200 mM stock solution and then diluted 25-fold in a new plate (4X plate). Finally, there was a further 4-fold dilution in the seeding plate
(1X plate). A positive control was diluted 100-fold in DMSO (Sigma Aldrich Batch no.W228613) from a 6.4 mM stock solution and then diluted 25-fold in a new plate in treatment medium and then further diluted 4-fold in the seeding plate. The test item was tested at 12 concentrations according to a geometric progression of ratio 2 from 0.98 µM to 2000 µM.
-Negative control: 6 wells of solvent control (1% DMSO in treatment medium) with cells and 1 well of solvent control without cell by culture plate. Positive control: 5 concentrations of cinnamaldehyde (SIGMA ALDRICH Batch no.W228613) on each culture plate. The concentration varies from 4 to 64 µM according to a geometric progression of ratio 2.

Description of evaluation and decision criteria used:
The test item is identified as potential skin sensitizer if the 4 following conditions are met in 2 of 2 or 2 of 3 repetitions. Otherwise, the keratinosens™ prediction is considered as negative:
1) The Imax is strictly 1.5 fold higher of the basal luciferase activity* statistically significantly to the value obtained for the negative control (as determined by a two-tailed, unpaired Student's t-test on the raw RLU values). If the Imax is exactly equal to 1.5, the test item is rated as negative and no EC1.5 value is calculated.
2) The EC1.5 value is strictly below 1000 μM (or <200 μg/ml for the test item with no defined molecular weight)
3) At the lowest concentration with a gene induction above 1.5, the cell viability must be strictly above 70% (i.e EC1.5 < IC70).
4) There is an apparent overall dose-response for luciferase induction, which is similar between the repetitions.
- Description of study acceptance criteria used:
1) Positive control:
- The gene induction must be statistically significant above the threshold of 1.5 in at least one dose.
- The EC1.5 value should be between IDEA Lab historical data: mean EC1.5 value ± 2 SD and the average induction, in each repetition, for cinnamaldehyde at 64 μM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of cinnamaldehyde should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
2) Negative control: for each repetition, the coefficient of variation of the solvent controls (3 x 6 wells) must be less than 20%. If for one repetition the validity criteria are not met, a third repetition should be considered.

Positive control results:

Imax = 8.46
EC1.5 = 7.06 (geometric mean)

Key result

Run / experiment:

mean

Parameter:

other: Imax

Value:

1.35

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Imax lower than 1.5, no EC1.5 is calculated

Key result

Run / experiment:

other: Geometric mean

Parameter:

other: IC50

Value:

2 000

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- The test item did not cause any precipitation or any other type of interference that might have had lead to confouding the possible sensitising effects of the test item.

DEMONSTRATION OF TECHNICAL PROFICIENCY: recommended substances for demonstrating technical proficiency with the KeratinoSens™ test method were tested to validate the method.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Average variability in yhr 3x6 control wells should be <20% (for each master plate).
- Acceptance criteria met for positive control:
1) Gene induction (luciferase activity) must be statistically significant > 1.5 in at least one dose. The luciferase activity was > in every single dose.
2) Average inductions in 3 replicates at 64 µM should be between 2-8, if this criterion is not fulfilled the test may be accepted based on dose-response: The average induction was 8.46, but there was an apparent overall dose-response for luciferase induction.
3) EC1.5 should be between 2 standard deviations of the historical mean (i.e 7-30 µM based on validation dataset: EC1.5 was 7.06 and therefore within the historical range (3-22 µM)
- Acceptance criteria met for variability between replicate measurements: yes, CV% < 20%
- Range of historical values if different from the ones specified in the test guideline: 3-22 µM

Table 1. Positive control results.

Cinnamaldehyde	4 µM	8 µM	16 µM	32 µM	64 µM	EC1.5	Imax
Rep 1	1.33	2.18	3.77	6.00	15.17	4.80	15.17
Rep 2	1.37	1.45	2.12	3.16	5.52	8.58	5.52
Rep 3	1.24	1.45	2.13	3.22	4.69	8.56	4.69
Mean	1.31	1.69	2.67	4.13	8.46	7.06*	8.46

*geometric mean

 

Table 2. Negative Control Results.

Control solvent	CV %  control solvent
Rep 1	10.5
Rep 2	8.8
Rep 3	6.8

Table 3. Test item results.

	VIABILITY		INDUCTION
ID-18/04211	IC50  µM	IC30  µM	Imax	Linear EC1.5  µM	EC1.5 Lin/Log  µM
Rep 1	53.17	44.96	1.51	30.76	30.58
Rep 2	53.85	36.30	1.11	-	-
Rep 3	71.36	53.15	1.42	-	-
Mean	-	-	1.35	-	-
Geometric mean	58.90	44.27	-	-	-

Repetition 1: Imax is slightly higher than 1.5 (1.51) at only one concentration close to the IC30. The calculated EC1.5 is lower than 1000 µM and the EC1.5 viability is higher than 70%, the repetition is therefore positive. However the corresponding positive control being higher on this repetition, a third repetition was carried out.

Repetition 2 and 3: Imax is lower than 1.5, the EC1.5 is not determined. The repetition 2 and 3 are negatives.

Interpretation of results:

GHS criteria not met

Remarks:

EU criteria

Conclusions:

The test item showed an Imax of 1.35 and an IC50 and IC30 higher than 2000 μM in KeratinoSens™. The test item showed no sensitisation potential under test conditions, therefore, the KeratinoSens prediction is considered negative.

Executive summary:

The test method KeratinoSens™ has been performed as part of an integrated approach to support the identification of the sensitization potential of the test item according to OECD 442D, under GLP conditions.

The aim of the study is to evaluate the potential of the test item to activate the gene AKR1C2 y quantifying the luciferase induction after 48h in transformed keratinocytes. 12 concentrations of the test item ranging from 0.98 to 2000 μM were prepared by serial dilution in culture medium containing 1% DMSO and added to 96-well plates of KeratinoSens cells™, in 3 separate runs of 3 replicates each. Positive and negative controls were run in parallel, as well as a cytotoxicity assay (MTT reduction). All acceptance criteria were fulfilled for the positive and negative controls in each run. The test item showed an Imax of 1.35 and an IC50 and an IC30 higher than 2000 μM in KeratinoSens™, the mean cell viability was 58.90 and 44.27 for IC50 and IC30 respectively. Under the testing conditions the test item has no sensitisation potential.

Reference 3

Endpoint:

skin sensitisation: in vitro

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
The study does not need to be conducted because there are more adequate options available considering the physicochemical characteristics of the test item. According to OECD guideline 442E for In Vitro sensitisation testing, if the test item has a log Pow> 3.5, it will tend to produce negative results. As only positive results are valid for 442E and other sensitisation tests have already been negative (442D) the most suitable test to confirm that the test item is not sensitising would be an In Vivo 442B test.

Reason / purpose for cross-reference:

data waiving: supporting information

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 24th to November 27th, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Species:

mouse

Strain:

CBA:J

Remarks:

SPF caw

Sex:

female

Details on test animals and environmental conditions:

Source: Elevage Janvier Labs (F-53941 Le Genest Saint Isle).
- Females nulliparous and non-pregnant: yes.
- Age at study initiation: 8 weeks old.
- Weight at study initiation: average weight 21.2 g (SD: 1.1)
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): Tkelad Global 18% Protein Rodent Diet (ENVIGO 2016) ad libitum.
- Water (e.g. ad libitum): Tap water from public distribution system, ad libitum. Microbiological and chemical analyses of the water were carried out once every six months by Bureau Veritas – Eurofins (FRANCE).
- Acclimation period: at least 5 days.
- Indication of any skin lesions: no.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C to 25°C
- Humidity (%): 30% to 70%
- Air changes (per hr): at least 10.
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light.

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

50%, 25% and 10%.

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
A preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the test item undiluted (100%) to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed daily from day 1 to day 6. Any signs of toxicity or excessive local irritation noted during this period were recorded. Ear thickness was recorded on day 1, day 3 and on day 6. The body weight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.
- Compound solubility: See Table 1 in 'Any other information on materials and methods incl. tables'. Due to the physical characteristics of the test item, the formulation at 100 % (w/v) using AOO (4:1 v/v) as the vehicle was suitable for the test (highest achievable concentration).
- Irritation: no.
- Systemic toxicity: no.
- Ear thickness measurements: Ear thickness was recorded on day 1, day 3 and on day 6., see 'Any other information on materials and methods incl. tables'.
- Erythema scores: no signs of erythema were observed. See 'Any other information on materials and methods incl. tables'.

MAIN STUDY
Groups of four mice were treated with the test item diluted at 25%, 50% and 100% in AOO, based on the results of the pre-screen tests. 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).
- Clinical observations: All animals were observed daily on Days 1, 2, 3, 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
- Body weight: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).
- Ear thickness: On day 1 and on day 3 (before application) as well as on day 6 (after sacrifice) of each experiment, the thickness of the right ear of each animal of the vehicle control and treated groups was measured by a micrometre. Furthermore, on day 6, punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and weighed in order to assess the irritation potential of the test item and the two lymph nodes per mouse were weighed. Furthermore, on day 6, punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and weighed in order to assess the irritation potential of the test item and the two lymph nodes per mouse were weighed.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay: BrdU - ELISA.
- Criteria used to consider a positive response: BrdU was measured by ELISA using a commercial kit (Roche Applied Science, Mannheim, Germany, Catalogue Number 11 647 229 001 – Batch No.17267000). Briefly, 100 μL of the LNC suspension was added to the wells of a flat-bottom microplate at least in triplicate. After fixation and denaturation of the LNC, anti-BrdU antibody was added to each well and allowed to react. Subsequently, the anti-BrdU antibody was removed by washing and the substrate solution was then added and allowed to produce chromogen. After 5 to 30 min, 30μL of 1 M H2SO4 was added in each well, then shaken for one minute. Absorbance at 450 nm with a reference wavelength of 690 nm was then measured. The SI value was derived as specified in the guidelines. If at least one concentration of the test item results is greater than 1.6 compared to control values, that is considered a positive response.

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of four mice were treated with the test item diluted at 25%, 50% in AOO and 100%, based on the results of the pre-screen tests. 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 four mice received the vehicle alone in the same manner.
On Day 5, 0.5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution was injected by intra-peritoneal route.
On day 6 (end of the test), the animals were anaesthetised with sodium pentobarbital and administration continued to fatal levels. The draining auricular lymph nodes from the four mice were excised.
From each mouse, a single-cell suspension of lymph node cells (LNC) excised bilaterally was prepared by gentle mechanical disaggregation through a disposable plastic pestle to crush the lymph nodes followed by passage through a #70 nylon mesh in 15 mL of DPBS (Ca2+ / Mg2+ - free) into a well of a multi-well 6. The optimised volume was based on achieving a mean absorbance of the negative control group within 0.1- 0.2. Then, BrdU was determined.

Positive control substance(s):

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

Positive control results:

The results for the last positive control (29/06/2016) were within the acceptable ranges. 5%, 10% and 25% solutions of hexyl cinnamic aldehyde in MEK generated a SI = 0.96, 1.17 and 1.61, respectively. The EC1.6 value was 24.66%.

Key result

Parameter:

SI

Value:

1.99

Variability:

0.13

Test group / Remarks:

25%

Key result

Parameter:

SI

Value:

1.57

Variability:

0.17

Test group / Remarks:

50%

Key result

Parameter:

SI

Value:

1.86

Variability:

0.15

Test group / Remarks:

100%

Parameter:

other: EC1.6

Test group / Remarks:

<25%

Remarks on result:

not determinable

Remarks:

As the SI value higher than 1.6 noted at the concentration of 100% which induced an excessive irritation, the positive response may be considered as a false positive response at this concentration. The Stimulation Index (SI) calculated by individual approach was 1.99 ± 0.13, 1.57 ± 0.17 and 1.86 ± 0.15 for the treated groups at 25%, 50% and 100%, respectively. The Stimulation Index (SI) calculated by individual approach was 1.99 ± 0.13, 1.57 ± 0.17 and 1.86 ± 0.15 for the treated groups at 25%, 50% and 100%, respectively.

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: see 'Any other information on results' below.

DETAILS ON STIMULATION INDEX CALCULATION: The SI was derived by dividing the mean BrdU labelling index/mouse within each test group by the mean BrdU labelling index for the control group. A stimulation index higher than 1.6 was recorded at the tested concentrations of 25% and 100%.

EC CALCULATION: The EC1.6 value (theoretical concentration resulting in a SI value of 1.6) was determined by linear interpolation of points on the dose-response curve, immediately above and below the 1.6-fold threshold. The equation used for calculation of EC1.6 was: EC1.6 = c + [(1.6 – d) / (b –d)] x (a – c), where a = the lowest concentration giving stimulation index > 1.6; b = the actual stimulation index caused by a; c = the highest concentration failing to produce a stimulation index of 1.6; d =the actual stimulation index caused by c.

CLINICAL OBSERVATIONS: No mortality and no signs of systemic toxicity were noted in the test and control animals during the test. Dryness of the skin was noted in all animals treated at 25%, 50% and 100% on day 6. An increase in ear thickness (+ 1.3%, + 13.6% and + 36.8%) and an increase in ear weight (+ 12.3%, + 20.9% and + 48.1%) were noted in animals treated at 25%, 50% and 100%, respectively. Therefore, the test item has to be considered as excessively irritant at a concentration of 100%.

BODY WEIGHTS: Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Table 4. Main study: Individual clinical observation and mortality data.

Groups	Test item	AnimalsNo.	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6
		Sf 1320	0	0	0	0	0	0
1	AOO	Sf 1321 Sf 1322	0	0	0	0	0	0
			0	0	0	0	0	0
		Sf 1323	0	0	0	0	0	0
		Sf 1325	0	0	0	0	0	0*
2	25%	Sf 1326 Sf 1327	0	0	0	0	0	0*
			0	0	0	0	0	0*
		Sf 1328	0	0	0	0	0	0*
		Sf 1330	0	0	0	0	0	0*
3	50%	Sf 1331 Sf 1332	0	0	0	0	0	0*
			0	0	0	0	0	0*
		Sf 1333	0	0	0	0	0	0*
		Sf 1335	0	0	0	0	0	0**
4	100%	Sf 1336 Sf 1337	0	0	0	0	0	0**
			0	0	0	0	0	0**
		Sf 1338	0	0	0	0	0	0**

0: no sign of systemic toxicity (*): slight dryness

(**): dryness

AOO: Acetone/olive oil (4:1 v/v)

Table 5. Main study: Individual body weight and body weight gain.

Groups	Test item	Animals No.	Bodyweight (g)		Body weight gain (g)
			Day 1	Day 6
1	AOO	Sf 1320	19.5	19.9	0.4
		Sf 1321	23.0	24.4	1.4
		Sf 1322	21.0	22.6	1.6
		Sf 1323	22.5	22.9	0.4
	MEAN		21.5	22.5	0.9
	Standard-deviation		1.6	1.9	0.6
2	25%	Sf 1325	21.4	20.8	-0.6
		Sf 1326	19.1	19.1	0.0
		Sf 1327	20.3	21.6	1.3
		Sf 1328	21.1	21.7	0.6
	MEAN		20.5	20.8	0.3
	Standard-deviation		1.0	1.2	0.8
3	50%	Sf 1330	21.6	23.1	1.5
		Sf 1331	19.8	20.7	0.9
		Sf 1332	21.1	22.5	1.4
		Sf 1333	20.7	21.9	1.2
	MEAN		20.8	22.1	1.3
	Standard-deviation		0.8	1.0	0.3
4	100%	Sf 1335	19.9	20.3	0.4
		Sf 1336	20.8	21.4	0.6
		Sf 1337	19.4	20.4	1.0
		Sf 1338	20.2	21.0	0.8
	MEAN		20.1	20.8	0.7
	Standard-deviation		0.6	0.5	0.3

Table 6. BrdU index & Stimulation index per group and calculation of EC1.6.

Groups	Test item	BrdU-index (mean*)	Stimulation Index SI (mean + standard deviation)	Result	EC1.6 value
1	AOO	0.472	n.a	n.a	n.a.
2	25%	0.938	1.99    ±         0.13	positive	<25%
3	50%	0.741	1.57    ±         0.17	negative#
4	100%	0.878	1.86    ±         0.15	positive

Table 7. Main study: BrdU index & Stimulation index (individual data)

Groups	Test item	Animal No.		BrdU-index (DO Indiv)	BrdU-index (DO mean)	BrdU-index mean*	Stimulation Index S.I. (indiv ± Standard deviation)
1	AOO	Sf	1320	0.529	0.503	0.472	n.a
				0.499
				0.483
		Sf	1321	0.430	0.478		n.a
				0.511
				0.494
		Sf	1322	0.470	0.448		n.a
				0.430
				0.444
		Sf	1323	0.482	0.458		n.a
				0.451
				0.442
2	25%	Sf	1325	0.955	0.928	0.938	1.97	±	0.05
				0.912
				0.918
		Sf	1326	1.088	0.996		2.11	±	0.32
				1.079
				0.823
		Sf	1327	0.807	0.854		1.81	±	0.09
				0.896
				0.860
		Sf	1328	0.954	0.974		2.06	±	0.07
				0.959
				1.011
3	50%	Sf	1330	0.781	0.768	0.741	1.63	±	0.15
				0.694
				0.831
		Sf	1331	0.686	0.670		1.42	±	0.19
				0.751
				0.574
		Sf	1332	0.711	0.684		1.45	±	0.06
				0.658
				0.683
		Sf	1333	0.927	0.842		1.78	±	0.27
				0.693
				0.907
4	100%	Sf	1335	1.013	0.904	0.878	1.92	±	0.21
				0.814
				0.885
		Sf	1336	0.917	0.887		1.88	±	0.12
				0.825
				0.921
		Sf	1337	0.973	0.939		1.99	±	0.16
				0.855
				0.989
		Sf	1338	0.716	0.780		1.65	±	0.12
				0.799
				0.826

*: mean: Σindividual value /4

AOO: Acetone/olive oil (4:1v/v)

Table 8. Individual Ear thickness and irritation level.

Groups	Test item	Animals No.	Day 1 ear thickness (mm)	Day 3 ear thickness (mm)	Day 6 ear thickness (mm)	Ear thickness increase D3/D1 (%)	Ear thickness increase D6/D1 (%)
1	AOO	Sf 1320	0.20	0.20	0.20	0.0	0.0
		Sf 1321	0.20	0.20	0.20	0.0	0.0
		Sf 1322	0.21	0.21	0.21	0.0	0.0
		Sf 1323	0.20	0.20	0.21	0.0	5.0
	MEAN		0.20	0.20	0.21	0.00	1.25
	Standard-deviation		0.00	0.00	0.01	0.00	2.50
2	25%	Sf 1325	0.21	0.21	0.21	0.0	0.0
		Sf 1326	0.21	0.21	0.21	0.0	0.0
		Sf 1327	0.20	0.20	0.20	0.0	0.0
		Sf 1328	0.20	0.21	0.21	5.0	5.0
	MEAN		0.21	0.21	0.21	1.3	1.3
	Standard-deviation		0.01	0.00	0.00	2.5	2.5
3	50%	Sf 1330	0.20	0.20	0.23	0.0	15.0
		Sf 1331	0.20	0.20	0.22	0.0	10.0
		Sf 1332	0.20	0.20	0.24	0.0	20.0
		Sf 1333	0.21	0.21	0.23	0.0	9.5
	MEAN		0.20	0.20	0.23	0.0	13.6
	Standard-deviation		0.00	0.00	0.01	0.0	4.9
4	100%	Sf 1335	0.20	0.22	0.27	10.0	35.0
		Sf 1336	0.21	0.24	0.32	14.3	52.4
		Sf 1337	0.20	0.23	0.25	15.0	25.0
		Sf 1338	0.20	0.23	0.27	15.0	35.0
	MEAN		0.20	0.23	0.28	13.6	36.8
	Standard-deviation		0.00	0.01	0.03	2.4	11.4

Table 9. Individual Ear biopsy weight and lymph node weight.

Groups	Test item	Animals No.	Ear weight Day 6 (mg)	% of ear weight increased/group1	Lymph nodes (mg)
1	AOO	Sf 1320	28.5		6.5
		Sf 1321	28.5		6.1
		Sf 1322	32.2		6.3
		Sf 1323	31.1		6.1
	MEAN		30.1		6.3
	Standard-deviation		1.9		0.2
2	25%	Sf 1325	33.0	12.3	11.6
		Sf 1326	35.5		12.8
		Sf 1327	32.2		14.9
		Sf 1328	34.4		16.0
	MEAN		33.8		13.8
	Standard-deviation		1.5		2.0
3	50%	Sf 1330	37.0	20.9	12.8
		Sf 1331	36.3		13.3
		Sf 1332	34.2		17.2
		Sf 1333	38.0		13.9
	MEAN		36.4		14.3
	Standard-deviation		1.6		2.0
4	100%	Sf 1335	42.0	48.1	13.6
		Sf 1336	52.8		19.3
		Sf 1337	43.9		18.2
		Sf 1338	39.5		18.0
	MEAN		44.6		17.3
	Standard-deviation		5.8		2.5

Table 10 . Summary of results – skin irritation.

Groups	Test item	Ear thickness increase D6/D1 (%)	Biopsy ear weight Increase (%)	Excessive irritation #
1	AOO	1.3	n.a	No
2	25%	1.3	12.3	No
3	50%	13.6	20.9	No
4	100%	36.8	48.1	Yes

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

EU

Conclusions:

The test item showed skin sensitisation potential under the tested conditions in the LLNA assay. The Stimulation Indexes were 1.99, 1.57, 1.87 at concentrations of 25, 50 and 100% test item, respectively. The results obtained, in these experimental conditions, it is possible to conclude that the test item is a sensitiser.

Executive summary:

The skin sensitisation potential of the test item was studied according to OECD 422B, under GLP conditions. Three groups of four female CBA/J mice received 25 μL/ear of 25, 50 % in Acetone/ Olive oil vehicle, and 100% (w/v) test item; and one negative control group received the vehicle (AOO). Three groups, each of four animals, were treated with 50 μL (25 μL per ear) of α-Hexylcinnamaldehyde, as a solution in acetone/olive oil (4:1 v/v) (4:1, v/v) at concentrations of 5%, 10% and 25% (v/v). A further control group of four animals was treated with acetone/olive oil (4:1, v/v) alone. The animals were treated for 3 consecutive days (D1, D2, D3), and injected with BrdU solution on Day 5. On D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by measurement of BrdU content in DNA of lymphocyte using an ELISA kit. The values obtained were used to calculate stimulation indices (SI). All acceptance criteria were fulfilled.No mortality and no signs of systemic toxicity were noted in the test and control animals during the test. Dryness of the skin was noted in all animals treated at 25%, 50% and 100% on day 6. An increase in ear thickness (+1.3%, +13.6% and +36.8%) and an increase in ear weight (+12.3%, +20.9% and +48.1%) were noted in animals treated at 25%, 50% and 100%, respectively. Therefore, the test item has to be considered as excessively irritant at the concentration of 100%. The Stimulation Indexes (SI) were 1.99, 1.57, 1.87 at concentrations of 25, 50 and 100% test item, respectively. In conclusion, the results obtained, in these experimental conditions, therefore, the test item could be considered sensitising to the skin and it is possible to conclude that the test item has to be classified as a sensitiser in Category 1 according to GHS and CLP criteria.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin Sensitisation:

Based on the available results for the 442B In vivo test, the test item should be classified as a Skin sensitiser Category I, according to CLP regulation (EC) No. 1272/2008.

Although in the in vitro test (442D) conditions the test item may be classified as not skin sensitiser, the results of the In vivo test should be accepted over those in the In vitro test, as no other in vitro or in chemico tests could be performed due to technical feasibility (442C, 442E) as part of an integrated testing approach.

In chemico skin sensitisation:

Data waving. Study technically not feasible. The test item is not soluble in the peptide buffer.  OECD 442C stipulates that the test chemical should be soluble in an appropriate solvent at a final concentration of 100  mM.

In vitro skin sensitisation:

Weight of evidence. The test method KeratinoSens™ has been performed as part of an integrated approach to support the identification of the sensitization potential of the test item according to OECD 442D, under GLP conditions. The aim of the study is to evaluate the potential of the test item to activate the gene AKR1C2 y quantifying the luciferase induction after 48h in transformed keratinocytes. 12 concentrations of the test item ranging from 0.98 to 2000 μM were prepared by serial dilution in culture medium containing 1% DMSO and added to 96-well plates of KeratinoSens cells™, in 3 separate runs of 3 replicates each. Positive and negative controls were run in parallel, as well as a cytotoxicity assay (MTT reduction). All acceptance criteria were fulfilled for the positive and negative controls in each run. The test item showed an Imax of 1.35 and an IC50 and an IC30 higher than 2000 μM in KeratinoSens™, the mean cell viability was 58.90 and 44.27 for IC50 and IC30 respectively. Under the testing conditions the test item has no sensitisation potential.

Data waving. The study does not need to be conducted because there are more adequate options available considering the physicochemical characteristics of the test item. According to OECD guideline 442E for In Vitro sensitisation testing, if the test item has a log Pow> 3.5 end to produce negative results. As only positive results are valid for 442E and other sensitisation tests have already been negative (442D) the most suitable test to confirm that the test item is not sensitising would be an In vivo 442B test.

In vivo skin sensitisation:

Weight of evidence. The skin sensitisation potential of the test item was studied according to OECD 422B, under GLP conditions. Four groups of four female CBA/J mice received 25 μL/ear of 25, 50 % in Acetone/ Olive oil vehicle, and 100% (w/v) test item ; and one negative control group received the vehicle (AOO). The values obtained were used to calculate stimulation indices (SI). No mortality and no signs of systemic toxicity were noted in the test and control animals during the test. Dryness of the skin, an increase in ear thickness (+ 1.3%, + 13.6% and + 36.8%) and an increase in ear weight (+ 12.3%, + 20.9% and + 48.1%) were noted in animals treated at 25%, 50% and 100%, respectively. Therefore, the test item has to be considered as excessively irritant at a concentration of 100%. The Stimulation Indexes (SI) were 1.99, 1.57, 1.87 at concentrations of 25, 50 and 100% test item, respectively. In these experimental conditions, enable to conclude that the test item has to be classified as a sensitiser in Category 1 according to CLP regulation (EC) No. 1272/2008

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information (negative result for OECD 442D, but positive result for OECD 442B) the substance should be classified as Sking Sensitiser Category 1, according to CLP Regulation (EC) no. 1272/2008.