2-ethylhexyl acrylate

Endpoint summary

Description of key information

2-EHA demonstrated weak dermal sensitization potential in the local Lymph node assay in mice (EC3 = 18.96 %; 4740 µg/cm²). In various former tests involving guinea pigs 2-EHA proved sensitising, too, with and without adjuvants. 2-EHA was also predicted to be a skin sensitizer in a battery of in vitro assays addressing key steps of the adverse outcome pathway (AOP) for skin sensitization as defined by OECD (DPRA, LuSens, MUSST and h-CLAT).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

25 Oct 2005 - 22 Nov 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD guideline study conducted in compliance with GLP regulations

Remarks:

the lymph nodes were pooled instead to be counted individually, no reliable data on skin irritation potential are documented

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Strain: CBA/Ca/Ola/Hsd
- Source: Harlan Interfauna UK Limited, Blackthorne, Bicester, Oxon, UK
- Age at study initiation: Young adults (8-12 weeks of age)
- Mean body weight at study initiation: 19.2 g
- Housing: maximum of 4 mice per cage
- Diet (ad libitum): Diet (RM1), supplied by Special Diet Services Limited, Witham, Essex, UK
- Water (ad libitum): mains water
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%): 30-70%
- Air changes (per hr): A minimum of 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Vehicle:

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

Concentration:

0.5, 1, 2.5, 5 and 10 % w/v

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS
Female CBA/Ca strain mice (n = 1) were exposed topically on the dorsum of both ears to 25 µl of 2.5, 10 and 50 % w/v concentrations daily for 3 consecutive days. Control animais were untreated (naïve). 5 days after the initiation of exposure, mice were terminated and auricular lymph nodes were excised and pooled per animal. A single cell suspension of draining lymph node cells (LNC) was prepared under aseptic conditions by mechanical disaggregation through sterile 200-mesh stainless steel gauze and resuspended in phosphate buffered saline (PBS). Viable cell counts were performed by exclusion of trypan blue dye, and the cellularity per lymph node was recorded.
Topical exposure to EHA (50% and 10%) resulted in marked increases in cellularity (approximately ninefold to fourfold changes compared with naïve controls). At lower doses, changes in cellularity were more modest, or were absent.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The criterion for a positive response is that one or more concentrations of the test substance
should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group.

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of four female mice were used for the main LLNA study. Approximately 25µl of a 0.5, 1, 2.5, 5 or 10 % w/v preparation of the test substance in acetone in olive oil (4:1) was applied, using a variable volume micro-pipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using acetone in olive oil (4:1) alone. The procedure was repeated daily for 3 consecutive days.

Three days after the third application, all the animals were injected, via the tail vein, with approximately 250µl of phosphate buffered saline (PBS) containing 20µCi of a 2.0Ci/mmol specific activity 3H-methyl thymidine. Approximately 5 hours later, the animals were humanely killed by inhalation of halothane vapour followed by cervical dislocation. The draining auricular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a container of PBS.

A single cell suspension was prepared by mechanical disaggregation of lymph nodes through a 200-mesh stainless steel gauze. The cell suspensions were then washed three times by centrifugation with approximately 10mL of PBS. Approximately 3mL of 5 % w/v trichloroacetic acid (TCA) was added and, after overnight precipitation at 4°C, the samples were pelleted by centrifugation and the supernatant was discarded. The cells were then resuspended in approximately 1 mL of TCA.

The lymph node suspensions were transferred to scintillation vials and 10 mL of scintillant (Optiphase) was added prior to ß-scintillation counting using a Packard Tri-Carb 3100TR Liquid Scintillation Counter.

Animals were checked at least once daily for signs of systemic toxicity.

The bodyweight of each animal was recorded prior to dosing on day 1 and prior to injection of 3H-methyl thymidine on day 6 for LLNA study mice or prior to termination for sighting study mice.

Positive control substance(s):

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

Statistics:

The EC3 value was derived by interpolating between two points on the Stimulation Index (SI) axis, one immediately above and the other immediately below the SI value of 3 (vehicle-treated control values [SI=1] not being used for the latter). Where the data points lying immediately above and below the SI value of three have the co-ordinates a (the concentration giving the SI immediately above 3), b (the SI of a), c (the concentration giving the SI immediately below 3) and d (the SI of c), the EC3 value was calculated using the following equation:
EC3 = [(3-d)/(b-d)] x (a-c) + c

Positive control results:

The application of hexylcinnamaldehyde at concentrations of 5 %, 10 % and 25 % w/v in acetone : olive oil (4:1) resulted in a greater than 3-fold increase in isotope incorporation at the 10 and 25 % w/v concentrations. Therefore, hexylcinnamaldehyde was shown to be a skin sensitiser, confirming the validity of the protocol used for the study.

Parameter:

EC3

Remarks:

%

Value:

9.7

Parameter:

SI

Value:

1.1

Test group / Remarks:

0.5%

Parameter:

SI

Value:

1.2

Test group / Remarks:

1%

Parameter:

SI

Value:

1

Test group / Remarks:

2.5

Parameter:

SI

Value:

1.2

Test group / Remarks:

5%

Parameter:

SI

Value:

3.1

Test group / Remarks:

10%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The application of the test substance at concentrations of 0.5, 1, 2.5, 5 and 10 %w/v in acetone in olive oil (4:1) resulted in an increase in isotope incorporation which was greater than 3-fold at the 10 %w/v concentration. Consequently, the test substance was shown to be a potential skin
sensitiser. The concentration giving rise to a 3 fold increase in lymphocyte proliferation (EC3) was calculated to be 9.7 %w/v (2425µg/cm2), indicative of a sensitiser of moderate potency.

The application of hexylcinnamaldehyde at concentrations of 5%, 10% and 25% w/v in acetone in olive oil (4:1) resulted in a greater than 3-fold increase in isotope incorporation at the 10 and 25 %w/v concentrations. Therefore, hexylcinnamaldehyde was shown to be a skin sensitiser, confirming the validity of the protocol used for the study.

Concentration of test substance (% w/v)	Number of lymph nodes assayed	Disintegrations per minute (dpm)	dpm per lymph node	Test : control ratio (SI)
0 (vehicle only)	8	5822	728	N/A
0.5	8	6172	772	1.1
1	8	7026	878	1.2
2.5	8	5503	688	1.0
5	8	7012	877	1.2
10	8	17989	2249	3.1
EC3	Calculated to be 9.7 % (2425µg/cm2)

N/A - not applicable

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Ethylhexyl acrylate is a skin sensitiser under the conditions of the test with an EC3 value of 9.7 %w/v (2425µg/ml).

Executive summary:

In a Local Lymph Node Assay (OECD TG # 429), 2-ethylhexyl acrylate was applied as 0.5, 1, 2.5, 5 or 10 %w/v preparations in acetone in olive oil (4:1). A vehicle control group was similarly treated using acetone in olive oil (4:1) alone. Dose levels were set according to sighting study results which indicated safe application levels and the dose range most likely to result in the ability to calculate a potency value. The estimated concentration giving rise to a 3 fold increase in lymphocyte proliferation (EC3) was calculated as percentage dose and µg/cm².

The test substance had the capacity to cause skin sensitisation when applied as a dose of 10 %w/v preparation in acetone in olive oil (4:1). The EC3 value giving rise to a 3 fold increase in lymphocyte proliferation was calculated to be 9.7 %w/v (2425µg/cm2), indicative of a sensitizer of moderate potency.

In a positive control study, hexylcinnamaldehyde was shown to have the capacity to cause skin sensitisation when applied as 10 and 25% w/v preparations in acetone in olive oil (4:1), confirming the validity of the protocol used for this study.

Ethylhexyl acrylate is a skin sensitiser under the conditions of the test with an EC3 value of 9.7 %w/v (2425µg/ml).

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Apr 2017 - Jan 2018 (Pre-study and Main study)

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:

July 2010

Deviations:

yes

Remarks:

Animals were kept for at least 16 days (cLLNA), a challenge was performed

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2012

Deviations:

yes

Remarks:

Animals were kept for at least 16 days (cLLNA), a challenge was performed

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Remarks:

CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS BV Inc, Postbus 6174, Netherlands
- Females nulliparous and non-pregnant: not specified
- Microbiological status of animals, when known: not specified
- Age at study initiation: 8 weeks
- Weight at study initiation: 15.8 g - 22.0 g (main study); 17.5 g - 20.2 g (pre-study)
- Housing: single
- Diet: ad libitum:
- Water: ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12; 6 am to 6 pm illumination period

IN-LIFE DATES (From - To)
- 06 Jun 2017 - 29 Jun 2017 (main study);
- 28 Mar - 10 Apr (Test groups 1 and 2) , 28 Mar - 25 Apr (Test group 7), 28 Mar - 27 Apr (Test groups 3 and 6) (Pre-study)

Vehicle:

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

Concentration:

PRE-STUDY
50% (induction phase)
50%, 10% (challenge phase)
The highest dose (50%) was chosen in accordance with previously performed experiments.

MAIN STUDY
50% (induction phase)
50%, 10%, 2.5% and 1% (challenge phase)
The highest dose (50%) was chosen in accordance with previously performed experiments.

No. of animals per dose:

5 (Main study); 4 (Pre-study)

Details on study design:

PRE-STUDY:
- Treatment scheme: 4 test groups (2, 5, 6 and 7) and 3 control groups (test groups 1, 3 and 4)
- Test groups 1 and 2 were used to quantify the primary immune response (induction phase). Test group 3 served as control group throught the challenge phase. Control groups 1 and 3 were used to rule out any influence of the vehicle. Test group 4 was used to identify the levels of "unspecific" induced lymphoproliferation after a single application in the absence of a memory response. Test group 7 was used to confirm that responses induced during induction phase have diminished by the time when animals were challenged.
- Selection of doses: Two mice were treated topically with 10%, 25%, 50%, 75%, and 100% of the test substance each on three consecutive days. Clinical signs were recorded after each application and on day 6. No signs of systemic toxicity were observed. In a second pre-test, the potential to induce unspecific lymphoproliferation was investigated. Mice were treated once with 25%, 50%, 75%, and 100% of the test substance. Two days after treatment, lymphoproliferation was quantified by means of 3H-thymidine incorporation. Unspecific proliferation after a single application of = 75% test item was reported. Thus, 50% test item was the highest dose, which could be used.

TREATMENT PREPARATION AND ADMINISTRATION:
- The study comprised four treatment groups and three control groups. Each group consisted of 4 mice
- Form of application: epicutaneous
- Application volume: 25 µl
- Application site: Dorsal surface of both ears
- Frequency of application: 3 consecutive applications (study day 1 – study day 3) to the same application site. Where applicable, single challenge treatment on study day 21.
- On study day 6 and, where applicable, on day 21 and day 23, mice were injected intravenously (i.v.) with 20 µCi of 3H-thymidine1 in 250 µL of sterile saline into a tail vein.

TERMINAL PROCEDURES:
- The animals were sacrificed on study day 6 (test groups 1 and 2) or day 21 (test group 7) or day 23 (test groups 3, 4, 5 and 6) about 5 hours after 3H-thymidine injection by cervical
dislocation under Isoflurane anesthesia.
- Determination of ear and lymph node weight, determination of cell count, and measurement of 3H-thymidine incorporation was performed

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Challenge local lymph node assay
- Criteria used to consider a positive response: The stimulation index (SI) should be greater or equal to 3 before classification as a potential skin sensitizer is warranted.
- Body weight: Individual body weights were taken on day 1 (prior to administration) and day 23
- Clinical signs/mortality: Obvious signs of systemic toxicity and/or local inflammation as well as a check for moribund animals was performed

TREATMENT PREPARATION AND ADMINISTRATION:
- The study comprised four treatment groups, one vehicle control group and one challenge control group. Each group consisted of 5 mice
- Form of application: epicutaneous
- Application volume: 25 µl
- Application site: Dorsal surface of both ears
- Frequency of application: 3 consecutive applications (study day 1 – study day 3) to the same application site. Single challenge treatment on study day 21.
- On study day 23, mice were injected intraveneously (i.v) with 20 µCi 3H-thymidine1 in 250 µL sterile saline into the tail vein

TERMINAL PROCEDURES:
- The animals were sacrificed on study day 23 about 5 hours after 3H-thymidine injection by cervical dislocation under Isoflurane anesthesia.
- Determination of ear and lymph node weight, determination of cell count, and measurement of 3H-thymidine incorporation was performed

Positive control substance(s):

other: no positive control included

Statistics:

The stimulation indices of 3H-thymidine incorporation, cell count, lymph node weight and ear weight measurements were calculated as the ratio of the test group mean values for these parameters divided by that of the vehicle control group. In addition, WILCOXON-test was used. Mean values and standard deviations of the measured parameters were determined.

Positive control results:

No positive control was included in the study.

Parameter:

other: Elicitation threshold

Remarks:

%

Value:

> 2.5

Remarks on result:

other: Main study

Parameter:

SI

Value:

1.14

Test group / Remarks:

induction 50%/challenge 1%

Remarks on result:

other: Main study

Parameter:

SI

Value:

1.33

Test group / Remarks:

induction 50%/challenge 2.5%

Remarks on result:

other: Main study

Parameter:

SI

Value:

13.03

Test group / Remarks:

induction 50%/challenge 10%

Remarks on result:

other: Main study

Parameter:

SI

Value:

26.16

Test group / Remarks:

induction 50%/challenge 50%

Remarks on result:

other: Main study

Cellular proliferation data / Observations:

PRE-STUDY

CLINICAL OBSERVATIONS:
No signs of systemic toxicity were noticed in all animals during general observation. Very slight erythema on the ear skin was observed in all animals treated with the test substance
(test groups 2, 4, 5, 6 and 7) during the observation period. In addition, slight swelling was noted in test group 4 on study day 23.

BODY WEIGHTS:
The expected body weight gain was generally observed during the study.

MAIN STUDY

CLINICAL OBSERVATIONS:
No signs of systemic toxicity were noticed in all animals during general observation. Slight swelling of the ear skin was observed in all animals at all concentrations, induced with
the 50% concentration on study day 2 and 3.

BODY WEIGHTS:
The expected body weight gain was generally observed during the study.

Table 1: Summary of stimulation indices (Pre-study)

TestGroup	Induction Treatment day 1, 2, 3	Challenge Treatment day 21	Lymph node preparation on day	³H-thymidine incorporation Stimulation Index^1,2	Cell Count Stimulation Index^1,2	Lymph Node Weight Stimulation Index^1,2	Ear Weight Stimulation Index^1,2
1	vehicle AOO		6	1.00	1.00	1.00	1.00
2	50% in AOO		6	20.41	3.30	3.15	1.10
3	vehicle AOO	vehicle AOO	23	1.00	1.00	1.00	1.00
4	vehicle AOO	50% in AOO	23	1.63	1.47	1.39	0.94
5	50% in AOO	50% in AOO	23	20.66	4.38	3.69	1.01
6	50% in AOO	10% in AOO	23	8.02	2.78	2.40	1.00
7	50% in AOO		21	0.18	0.71	0.96	1.03

1test group 2 and 7 / test group 1 (vehicle control)

2test group 4, 5 and 6 / test group 3 (vehicle control)

The statistical evaluations were performed using the WILCOXON-test ( # for p=0.05, ##for p=0.01 )

Table 2: Summary of stimulation indices (Main study)

 

Test Group	Induction Treatment day 1, 2, 3	Challenge Treatment day 21	³H-thymidine incorporation Stimulation Index^1		Cell Count Stimulation Index^1		Lymph Node Weight Stimulation Index^1		Ear Weight Stimulation Index^1
12	vehicle AOO	vehicle AOO	1.00		1.00		1.00		1.00
23	vehicle AOO	50% in AOO	1.82	##	1.45	##	1.32	##	1.06
3	50% in AOO	50% in AOO	26.16	##	3.49	##	3.36	##	1.03
4	50% in AOO	10% in AOO	13.03	##	2.90	##	2.44	##	1.09	##
5	50% in AOO	2.5% in AOO	1.33		1.20		1.12		1.08	#
6	50% in AOO	1% in AOO	1.14		1.15		1.29	#	1.11	##

1test group x / test group 1 (vehicle control)

2vehicle control group

3challenge control group

The statistical evaluations were performed using the WILCOXON-test ( # for p=0.05, ## for p=0.01 )

The determination of the elicitation threshold in the challenge phase was achieved by treating different groups of primed animals with decreasing doses of the test item. Appropriate control groups are required for determination of the stimulation indices and exclusion of irritation effects (challenge control group).

Interpretation of results:

study cannot be used for classification

Conclusions:

By results obtained, the test substance was found to have a skin sensitizing potential and is able to induce a secondary immune response (Pre-study). It was concluded that the threshold concentration for inducing a secondary immune response (elicitation threshold) of the test substance was > 2.5% in the Challenge Murine Local Lymph Node Assay (cLLNA) under the test conditions chosen (Main study).

Executive summary:

This supporting LLNA assay aimed at addressing the level of 2-EHA required for the induction of a secondary immune response (elicitation threshold) in already sensitized animals. In a previously performed study (Pre-study), the potential of 2-EHA to induce a secondary immune response was examined using four test groups and three control groups of four female mice each. The experiment was also devided into two parts, namely induction (50%) and challenge phase (10%, 50%). It was concluded that 2-EHA exhibits a skin sensitizing potential and is able to induce a secondary immune response (Pre-study).

In the main study, during the induction treatment groups of 5 female CBA/CaOlaHsd mice each were treated three times with a 50% (w/w) preparation of 2-EHA in AOO (4:1) or with the vehicle alone. During the challenge treatment, the animals were treated once with 1%, 2.5%, 10% and 50% (w/w) preparations of 2-EHA in AOO (4:1) or with the vehicle alone.

The study used 4 test groups, a vehicle control group and a challenge control group. Each animal was treated with 25 µL per ear of the appropriate test-substance preparation or the vehicle applied to the dorsal surfaces of both ears on three consecutive days (day 1, 2 and 3; induction treatment) or on day 21 (challenge treatment). 20 µCi 3H-thymidine in 250 µL sterile saline were injected into the tail vein of the mice on day 23. About 5 hours after the 3H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring 3H-thymidine incorporation (indicator of cell proliferation). Cell counts and weights of each animal’s pooled lymph nodes were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined to obtain an indication of possible skin irritation.

No signs of systemic toxicity were noticed in all animals during general observation.

The 50% concentration applied to the animals during the challenge treatment, which were induced with the vehicle alone (challenge control group), induced statistically significant increases of 3H-thymidine incorporation into the cells and in the auricular lymph node cell counts. However, both failed to reach the cutoff (SI 3 for 3H-thymidine incorporation and SI 1.5 for the auricular lymph node cell counts). In addition the increase in lymph node weight was statistically significant.

When applied as 50% preparation in AOO (induction treatment) and as 10% and 50% in AOO (challenge treatment), 2-EHA induced a biologically relevant (increase above the cutoff Stimulation Index of 3), statistically significant and concentration-dependent increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes.

Concomitantly, a biologically relevant, statistically significant and concentration-dependent response (increase to 1.5-fold or above of control value = stimulation index (SI) = 1.5) in the auricular lymph node cell counts was observed at 10% and 50%. In addition, statistically significant increases in lymph node weights were noted at the 1%, 10% and 50% concentration.

2-EHA concentrations did not cause relevant increases (SI = 1.25) in ear weights demonstrating the absence of significant ear skin irritation. The increases were statistically significant at 1%, 2.5% and 10%. However, slight swelling of the ear skin was observed in all animals at all concentrations, induced with the 50% concentration on study day 2 and 3.

Thus, it is concluded that the threshold concentration for inducing a secondary immune response (elicitation threshold) of 2-EHA was > 2.5% in the Challenge Murine Local Lymph Node Assay (cLLNA) under the test conditions chosen.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

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:

July 22, 2010

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 06, 2012

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Jai Research Foundation
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: no data
- Age at study initiation: 9 to 10 weeks old
- Weight at study initiation: 18.0 - 23.9 g
- Housing: individually in solid floor polypropylene
- Diet: Teklad Certified Global High Fiber Rat/Mice feed manufactured by Envigo, USA, was provided ad libitum
- Water: UV sterilised water (Reverse Osmosis water filtration system) was provided ad libitum in polypropylene water bottle
- Acclimation period: 6 days
- Indication of any skin lesions: none

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23
- Humidity (%): 57 to 66
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

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

Concentration:

screening study: 2.5%, 10%, 25%, 50%, 75% (v/v) in AOO and 100% (undiluted).
main study: 2%, 10%, 30% (v/v) in AOO and 100% (undiluted)

No. of animals per dose:

12 (2 mice/group) for screening study
30 (5 mice/group) for main study

Details on study design:

PRE-SCREEN TESTS:
six groups of mice (2 mice per group) that were treated with 2-Ethylhexyl Acrylate at concentrations of 2.5%, 10%, 25%, 50%, 75% (v/v) in AOO and 100% (undiluted, as received) (25 ìL/ear) for three consecutive days (days 1, 2 and 3). The dose was gently spread evenly over the dorsal surface of the ear using the tip of the pipette. No treatment was made on days 4 and 5.
- Compound solubility: the test item was found soluble in guideline recommended LLNA vehicle, acetone/olive oil (4:1 v/v) at 75%
- Irritation: The ears were evaluated daily for erythema. Ear swelling and erythema were used to identify concentrations of the test material that produced irritation to the ears of mice.
- Erythema scores:
Observation 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
- Systemic toxicity: Clinical observations were recorded daily during the experiment. Body weight was recorded on days 1 and 6 (prior to termination).
- Ear thickness measurements: Ear thickness of each animal was measured (apex of the pinna) using a micrometer (digital micrometer; serial N° 293.821) on days 1 (pre-dose), 3 and 6. Increases in ear swelling on days 3 and 6 were calculated for each animal relative to the thickness measurement taken on day 1.

MAIN STUDY
- The Local Lymph Node Assay
Prior to treatment, the animals were weighed and the ears were checked for any abnormalities or clinical signs of diseases or injury. Thirty healthy naive female mice without pre-existing ear irritation were selected and distributed into treatment groups (5 mice per group). Four treatment groups (G17 to G20) were treated topically once daily for three consecutive days (days 1, 2 and 3) on the dorsal surface of both ears (25 µL/ear) using a calibrated micropipette with 2-Ethylhexyl Acrylate at concentrations of 2%, 10%, 30% (v/v) in AOO and 100% (undiluted, as received.), respectively. The dose was gently spread evenly over the dorsal surface of the ear using the tip of the pipette.
Mice from the vehicle control group (G16) and positive control group (G21) were handled in the same manner but received 25 µL/ear of vehicle (AOO) and 25% a-Hexylcinnamaldehyde (v/v) in vehicle (AOO), respectively. No treatment was applied on days 4 and 5 for any group. All dosage preparations were freshly prepared on the day of application.
- Administration of 3H-methyl thymidine
On day 6 (approximately 72 h after the last treatment), all mice from the vehicle control, positive control and all treatment groups were intravenously injected via the tail vein with 250 µL of sterile phosphate buffered saline (PBS) containing approximately 20 ± 1 µCi (740 KBq) of 3H-methyl thymidine (Lot N° 09/16).
- Body Weight
Body weights of individual mice were recorded on the first day of dosing (day 1) and prior to administration of 3H-methyl thymidine (day 6). Group mean body weights were calculated.
- Observations
Individual animals were observed carefully daily for clinical signs, local irritation at the site of application and systemic toxicity. All the observations were systematically recorded for individual mice. Local irritation responses were made as per criteria given in OECD 429, 2010;
- Collection of Lymph Nodes
On day 6, 5 hours post-administration of 3H-methyl thymidine, all mice from the vehicle control, positive control and all treatment groups were euthanised by CO2 asphyxiation. The draining auricular lymph nodes from each individual mouse were excised and pooled in phosphate buffered saline.
- Preparation of Cell Suspension
The draining auricular lymph nodes of individual mice were collected in separate petri dishes containing phosphate buffered saline (PBS). A single cell suspension of lymph node cells was prepared by gentle mechanical disaggregation through 200 to 210 µm-mesh stainless steel gauze with the plunger of the syringe and collected in a petri dish. The gauze was washed with PBS into the petri dish and a single cell suspension was transferred into a 15 mL graduated centrifuge tube. The single cell suspension was finally made up to 10 mL with PBS used to rinse the petri dish. The cell suspension was centrifuged approximately at 190 to 200 g for 10 minutes in a centrifuge at 4 °C.
After centrifugation, the supernatant was removed by aspiration using a micropipette leaving 1 – 2 mL of supernatant above each pellet. Each pellet was gently agitated before making up to 10 mL with PBS; this procedure was repeated twice. After the final wash, the supernatant was removed leaving a minimal volume (approximate 0.5 mL) of supernatant above each pellet.
Each pellet was agitated before re-suspending with 3 mL of 5% trichloroacetic acid (TCA) and kept for precipitation of macromolecules in the refrigerator for approximately 18 hours. After incubation with 5% TCA at 4 ¿¿1 °C, each precipitate was recovered by centrifugation (190 to 200 g) for 10 minutes and the supernatant was removed.
The precipitate was re-suspended in 1 mL of 5% TCA. Each precipitate was transferred to a scintillation vial with 10 mL of scintillation fluid (Hionic flour) and thoroughly mixed. The vials were loaded into a ß–scintillation counter and after minimum 30 minutes, 3H-TdR incorporation was measured. Background 3H-TdR level was measured into 1 mL aliquots of 5% TCA.
- Determination of Cellular Proliferation
Incorporation of 3H-methyl thymidine was measured by ß-scintillation counting as DPM for each mouse and expressed as DPM/mouse. The total radioactivity of each sample was counted using LSA (Liquid Scintillation Analyser) after 30 minutes of mixing with scintillation fluid and required quench corrections were made.
The quench curve was established using extended quench standards (PerkinElmer) of DPM ß source. Computer constructed quench curve was derived from the above commercially available series of scaled standards which automatically converts Counts Per Minute (CPM) to DPM.
- Liquid Scintillation Analyzer Parameters
LSA : PerkinElmer (TRI-CARB®3100 TR)
Detector : High Performance Photo Multiplier Tube (PMT) Method : Conventional DPM
Counting Time: 10 minutes (2 sigma value of counting reaction 0.5%) Counting Region: 0 – 18.6 keV
SNC DPM (standard) : 267700 DPM (3H)
- Evaluation of Results
The proliferate response of lymph nodes from each mouse was expressed as the number of radioactive DPM per mouse, calculated by subtracting out background DPM (measured in 1 mL of 5% TCA aliquot).

SI = mean DPM of test group divided by mean DPM of solvent/vehicle control group

The DPM/mouse, along with an appropriate measure of inter-animal variability (i.e., mean ¿ standard deviation), were calculated for each test group and vehicle and positive control groups. Final results were expressed as the (SI) which is calculated as a ratio of the mean DPM of test group divided by mean DPM of vehicle control group. Any test item that produces a SI > 3 in the LLNA is considered “positive” for dermal sensitization potential (Kimber et al., 1994).

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test item is regarded as a skin sensitizer when the SI for a dose group is ¿¿3 together with consideration of a dose-response relationship.

Positive control substance(s):

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

Statistics:

In addition to an assessment of the magnitude of the SI, statistical analysis was carried out for the assessment of the dose response relationship and pair-wise comparison made between the treatment and the solvent/vehicle control group. All the parameters characterised by continuous data such as body weight and radioactive disintegrations per minute (DPM) were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA). To compare vehicle and positive control data, Student's t-test was performed to calculate significance.

Parameter:

SI

Value:

1.19

Test group / Remarks:

2%

Parameter:

SI

Value:

2.57

Test group / Remarks:

10%

Parameter:

SI

Value:

3.53

Test group / Remarks:

30%

Parameter:

SI

Value:

5.5

Test group / Remarks:

100%

Parameter:

SI

Value:

4.2

Test group / Remarks:

Positive control 25% HCA

Parameter:

EC3

Remarks:

%

Value:

18.96

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
See Table 1
The SI of 4.20 obtained for the concurrent positive control, ¿-Hexylcinnamaldehyde, showed greater than a three-fold increase over the control value indicating a clear positive response for this known weak sensitizer that confirmed the reliability of this test procedure.
The SI obtained for 2-Ethylhexyl Acrylate at 2% and 10% showed less than three-fold increase and at 30% and 100% showed greater over the control value. The EC3 value obtained for 2-Ethylhexyl Acrylate is 18.96.

CLINICAL OBSERVATIONS:
No clinical signs were observed in any mice from the vehicle control, positive control and treated groups at 2%, 10%, 30% (v/v) 2-Ethylhexyl Acrylate in AOO and 100% (undiluted, as received). All mice appeared active and healthy.

No dermal irritation was observed at any site in the mice treated at concentrations of 2%, 10%, 30% (v/v) 2-Ethylhexyl Acrylate in AOO and 100% (undiluted, as received) on day 1 to day 6. Very slight erythema (score of 1, barely perceptible) was noted on days 2 to 5 in all mice of the positive control group (25% HCA).

BODY WEIGHTS
The mean body weight of positive control as well as 2-Ethylhexyl Acrylate treated mice was comparable to that of the control group.

TABLE1: Summaryof DPM and SI Value                                                                       Sex:Female

 

Group N°	Dose Concentration(%)	N°of Mice Used	Group Mean DPM	Standard Deviation	Stimulation Index (SI)
G16	VehiclecontrolAOO	5	1446.60	820.33	1
G17	2%2-EthylhexylAcrylate	5	1718.00	1440.54	1.19
G18	10%2-EthylhexylAcrylate	5	3711.00	1244.23	2.57
G19	30%2-EthylhexylAcrylate	5	5105.40*	1887.66	3.53
G20	100% 2-EthylhexylAcrylate	5	7954.40**	3411.62	5.50
G21	Positivecontrol25%HCA	5	6069.40**	626.36	4.20

 

Note    :    Values are mean with standard deviation.

Key     :    HCA = a-Hexylcinnamaldehyde

DPM = Disintegrations per minute

AOO = Acetone:olive oil

Stimulation Index = Mean DPM of test group divided by mean DPM of solvent/vehicle control group

* = Significantly higher than control (p <= 0.05)

 

** = Significantly higher than control (p <= 0.01)

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Based on the results of this study, 2-Ethylhexyl Acrylate is considered weak sensitiser for dermal sensitisation potential in the LLNA.
Proper conduct of the LLNA was confirmed via a positive response with 25% ¿-Hexylcinnamaldehyde (HCA), a weak contact sensitizer.

Executive summary:

The dermal sensitisation potential of 2- Ethylhexyl Acrylate was evaluated in the mouse local lymph node assay (LLNA) (OECD TG# 429). A preliminary assay was conducted to identify the appropriate test concentrations for the main study. Based on the results from a preliminary assay, six groups of mice (each comprising 5 females) were selected for the experiment. Four groups were treated with 2-Ethylhexyl Acrylate at concentrations of 2%, 10%, 30% (v/v) in acetone: olive oil (AOO) and 100% for three consecutive days (days 1, 2 and 3) on the dorsum of both ears (25mL per ear). One group served as a vehicle control and was treated with AOO, and another group served as a positive control and was treated witha-hexylcinnamaldehyde (HCA) at a concentration of 25% (v/v) in AOO. Group mean body weights of treated animals were comparable with the control group. One animal at 100% lost 0.5 grams of bodyweight and three animals in the positive control lost 0.9, 2.0 and 1.0 grams of body weight by the end of the study. There were no indications of clinical or systemic toxicity in 2-Ethylhexyl Acrylate treated animals. On day 6, the uptake of 3H-methyl thymidine into the auricular (local) lymph nodes draining at the site of chemical application was measured (5 hours post 20 ± 1 µCi intravenous (i.v.) injection) to assess the lymph node proliferative response. All animals were euthanized via CO2 asphyxiation and the lymph nodes were harvested and prepared for analysis in a scintillation counter. The results are presented in Disintegrations Per Minute per mouse (DPM/mouse). Each animal's ears were also evaluated for erythema and edema prior to each application and ·again on days 4 and 5 and on day 6, prior to the i.v. injection. A positive response for HCA (Stimulation Index; SI = 4.20) confirmed the reliability of the test procedure. Mean stimulation indices for the 2%, 10%, 30% (v/v) and 100% 2-Ethylhexyl Acrylate treated groups were 1.19, 2.57, 3.53 and 5.50, respectively. The EC3 value obtained .for 2- Ethylhexyl Acrylate is 18.96. Therefore, 2-Ethylhexyl Acrylate demonstrate weak dermal sensitization potential in the local Lymph node assay. All criteria for a valid study were met as described in the protocol. The vehicle control and positive control in the definitive LLNA were within the acceptable ranges and fulfilled the requirements for a valid assay.

Reference 4

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

Feb 2015

GLP compliance:

not specified

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

PEPTIDES
Synthetic peptides: Cysteine- (C-) containing peptide and Lysine- (K-) containing peptide
The peptides are custom material containing phenylalanine to aid in UV-detection and either cysteine or lysine as the reactive center.
The C-containing peptide was incubated with the test substance in a ratio of 1:10 and the K-containing peptide in a ratio of 1:50.

CONTROLS
Negative control (NC): vehicle control = acetonitrile
Positive control (PC): p-Benzoquinone, puriss. (CAS 106-51-4)

TEST SUBSTANCE PREPARATIONS
The test-substance was solved at a 100 mM concentration in acetonitrile.

EXPERIMENTAL PROCEDURE
The test substance is incubated with synthetic peptides for 24 hours at room temperature and the remaining non-depleted peptide concentration is determined thereafter by high performance liquid chromatography with gradient elution and UV-detection at 220 nm.
The test substance was solved at a 100 mM concentration in acetonitrile. Per test run three samples of the test substance were incubated. Additionally triplicates of the concurrent vehicle control (=NC) were incubated with the peptides. Two test runs were performed with the C-peptide; the 2nd test run was performed with 6 samples per treatment group.

DATA EVALUATION
The peptide depletion of test-substance incubated samples was compared to the peptide depletion of the NC samples and expressed as relative peptide depletion.
For the test substance the mean peptide depletion as average of C- and K-peptide depletion is calculated and used for evaluation of the chemical reactivity.
According to the classification tree model described by Gerberick et al. (2007) highly reactive test substance (mean peptide depletion > 42.47 %) is predicted to be a strong sensitizer, a moderately reactive test substance (22.62 % < mean peptide depletion < 42.47 %) a moderate sensitizer, a test substance of low reactivity (6.38 % < mean peptide depletion < 22.62 %) a weak sensitizer, and a test substance of minimal reactivity (mean peptide depletion < 6.38 %) a non-sensitizer.

Run / experiment:

other: Test substance

Parameter:

other: mean depletion of C-peptide

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Test substance

Parameter:

other: mean depletion of K-peptide

Value:

60.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Visual observation after the 24-hour incubation time revealed precipitates in all samples of the test substance with the C-peptide. Thus the samples were centrifuged prior to HPLC analysis. No precipitates were observed in all samples of the test substance with the K-containing peptide after the incubation time.

Table 1: Results of reaction with cysteine-peptide

Reaction with cysteine-peptide		peptide depletion [%]
		sample1 sample4*	sample2 sample5*	sample3 sample6*	mean	SD
NC: acetonitrile	Run 1	-0.2	0.3	-0.1	0.0	0.3
	Run 2	-3.5	1.9	1.6	0.0	3.0
		**	-0.1	0.1	0.0	0.2
PC: p-Benzoquinone - 10/0214-1	Run 1	100	100	100	100	-
	Run 2	100	100	100	100	-
		100	100	100	100	-
2-Ethylhexylacrylat - 00/0903-4	Run 1	100	100	100	100	-
	Run 2	100	100	100	100	-
		100	100	100	100	-

*In test run 2 six samples were analyzed for each treatment group.

Table 2: Results of reaction with lysine-peptide

Reaction with lysine-peptide	peptide depletion [%]
	sample 1	sample 2	sample 3	mean	SD
NC 1: acetonitrile	0.6	-0.3	-0.3	0.0	0.5
PC: p-Benzoquinone - 10/0214-1	100	100	100	100	-
2-Ethylhexylacrylat - 00/0903-4	20.0	21.1	21.7	20.9	0.8

Table 3: Mean peptide depletion

	Cysteine-Peptide   mean depletion [%] SD		Lysine-Peptide   mean depletion [%] SD		mean of both depletions [%]
PC: p-Benzoquinone - 10/0214-1	100.0	-	100.0	-	100.0
2-Ethylhexylacrylat - 00/0903-4	100.0	-	20.9	0.8	60.5

Chemical reactivity was determined by mean peptide depletion and was rated as high, moderate, low, or minimal:

Mean peptide depletion [%] Reactivity

> 42.47: high reactivity

> 22.62 < 42.47: moderate reactivity

> 6.38 < 22.62: low reactivity

< 6.38: minimal reactivity

Interpretation of results:

other: positive indication of skin sensitisation

Conclusions:

Based on the observed results and applying the prediction model proposed in Gerberick et al. (2007) it was concluded that the test substance shows a high chemical reactivity in the DPRA under the test conditions chosen.
Based on the results and applying the evaluation criteria the test substance is predicted to be a skin sensitizer. A single in vitro assay is not sufficient to adequately assess skin sensitization endpoint. This study is part of a test battery strategy.

Executive summary:

Chemical reactivity has been shown to be well associated with allergenic potency. Within this context measuring the amount of proteins with nucleophilic side chains such as cysteine or lysine residues after incubation with putative allergens may serve as surrogate markers. For the test substance, the mean peptide depletion as average of cysteine- and lysine-peptide depletions was calculated to be 60.5% and thus, showing a high chemical reactivity in the DPRA.

This study is part of a test battery combining several in vitro methods addressing key steps of the adverse outcome pathway (AOP) for skin sensitization as defined by OECD. The following tests have been conducted to assess the skin sensitizing potential of the test substance.

• protein reactivity (DPRA),

• activation of keratinocytes (LuSens), and

• activation of dendritic cells (MUSST).

ln a separate report, the results of the individual studies were summarized and evaluated to predict the presence or absence of skin sensitizing potential of the test substance.

The combination of test methods and the evaluation of their results has been evaluated and published by Bauch et al., 2012. Based on the performance standards of the OECD test guideline no. 429 (Local Lymph Node Assay, LLNA, OECD 2010) the evaluation based on the DPRA, LuSens and MUSST methods yields an overall accuracy of 95% compared to results in humans (for comparison: for the same data set the LLNA yielded an overall accuracy of 86%).

A skin sensitizing (quantitative) potency assessment using the reported results was not validated at the time of writing of this report.

Based on the results and applying the evaluation criteria the test substance is predicted to be a skin sensitizer.

Reference 5

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 442E (In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome pathway for Skin Sensitisation)

Version / remarks:

Oct 2017

GLP compliance:

not specified

Type of study:

activation of dendritic cells

Details on the study design:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Test substance preparation: Emulsion 1000 µg/ml onward
- Vehicle: DMSO
- Precipitation in medium: no precipitation

TEST SYSTEM:
- Type of cells: human pro-monocytic cell line U937

CONCENTRATIONS
- 125, 250, 500, 1000, and 2000 µg/ml

NEGATIVE AND POSITIVE CONTROLS
- Positive control: Ethylene diamine (70 µg/ml)
- Negative control: Lactic acid (LA, 200 µg/ml)

EXPERIMENTAL PROCEDURE:
- Number of experiments/replicates: 3 experiments
- Exposure period: 48 h
- Quantitative measuremtn of changes of CD86: flow cytometric analysis

Positive control results:

CD 86 expression was not induced after 48 hour treatment with lactic acid and was induced after 48 hours exposure to EDA. The level of expression was within the range of the historical negative and positive control data.

Run / experiment:

other: 1

Parameter:

other: CD 86 induction

Value:

1.86

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: CD 86 induction

Value:

1.25

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 3

Parameter:

other: CD 86 induction

Value:

1.37

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

A test substance was predicted to have a dendritic cell activating potential when the marker expression exceeded the threshold of 1.2 with respect to vehicle treated cells (VC) at any tested sufficiently non-cytotoxic (cell viability =70%) concentration in two experiments. Three independent experiments were performed.
The test substance was tested in a concentration range of 125 to 2000 µg/mL. Cell viability was decreased below 70% at 2000 µg/mL. In all experiments, an induction of the expression of CD 86 was observed at sufficiently non-cytotoxic (cell viability =70%) concentration.

Table 1: Results of main experiment

Concentration (µg/ml)	First	experiment		2nd	experiment		3rd	experiment
	CD 86 induction		rel. viability	CD 86induction		rel.viability	CD 86induction		rel. viability
vc	1.00		100.00	1.00		100.0	1.00		100.0
125.0	1.86		92.20	1.29		98.3	1.37		92.0
250.0	2.25		85.02	1.18		99.0	1.67		85.9
500.0	2.05		91.76	1.25		98.1	1.43		89.1
1000.0	1.96		75.45	2.16		79.3	1.25		66.1
2000.0	2.47		37.46	1.37		0.0	1.10		7.9

Values in bold represent valid CD 86 inductions at non-cytotoxic concentrations exceeding the threshold of 1.2.

Interpretation of results:

other: positive indication of skin sensitisation

Conclusions:

In summary, after 48 hours of exposure to test substance, CD 86 expression was induced in U937 cells at concentration 125 µg/mL affording at least 70% viability. From this it has to be concluded that test substance does induce dendritic cell activation. Based on the outcome of the test battery strategy, the test substance is predicted to be a skin sensitizer. A single in vitro assay is not sufficient to adequately assess skin sensitization endpoint. This study is part of a test battery strategy.

Executive summary:

The myeloid U937 skin sensitization test is a dendritic cell activation test to predict skin sensitizing potential. The test is performed using the human pro-monocytic cell line U937 as surrogate tor dendritic cells. As readout, the change in the expression of the cell membrane marker CD 86 measured by flow cytometry after 48 hours of test substance exposure is determined. A test substance is predicted to activate dendritic cells when CD86 cell surface expression exceeds the threshold of 1.2 in relation to vehicle control in at least two independent experiments.

This study is part of a test battery combining several in vitro methods addressing key steps of the adverse outcome pathway (AOP) for skin sensitization as defined by OECD. The following tests have been conducted to assess the skin sensitizing potential of the test substance.

• protein reactivity (DPRA),

• activation of keratinocytes (LuSens), and

• activation of dendritic cells (MUSST).

ln a separate report, the results of the individual studies were summarized and evaluated to predict the presence or absence of skin sensitizing potential of the test substance.

The combination of test methods and the evaluation of their results has been evaluated and published by Bauch et al., 2012. Based on the performance standards of the OECD test guideline no. 429 (Local Lymph Node Assay, LLNA, OECD 2010) the evaluation based on the DPRA, LuSens and MUSST methods yields an overall accuracy of 95% compared to results in humans (for comparison: for the same data set the LLNA yielded an overall accuracy of 86%).

A skin sensitizing (quantitative) potency assessment using the reported results was not validated at the time of writing of this report.

Based on the results and applying the evaluation criteria the test substance is predicted to be a skin sensitizer.

Reference 6

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

other:

Version / remarks:

OECD Guideline 442E, version october 2017

Deviations:

not specified

Principles of method if other than guideline:

h-CLAT assay

GLP compliance:

not specified

Type of study:

activation of dendritic cells

Details on the study design:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator
- Vehicle: DMSO (0.2% final concentration)
- Precipitation in culture medium after 24 h incubation: No precipitation

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Weighed 0.1 OOOg of test substance in 1 ml volumetric flask, added saline up to 1 ml, mixed by vortexing, sonicated 5 min, test substance did not dissolve. Weighed 0.500 g of test substance in the 1 ml volumetric flask, added DMSO up to 1 ml, mixed by vortexing, sonicated 5 min, test substance as disperse phase formed an unstable emulsion with DMSO. Through constant swirling and sonication, this emulsion was stabilized. The highest miscible dose in the plate is 1000.0 µg/ml. In the main test, test substance was used at the maximal dose at 1000.0 µg/ml.

POSITIVE CONTROLS
- The strong sensitizer DNCB (4.0 µg/ml) was used as positive control.

CYTOTOXICITY ASSESSMENT:
- The cytotoxicity of the test substance was evaluated by flow cytometry using propidium iodide staining after 24 hours exposure.
- In the main test, test substance was used at eight final concentrations determined with regard to the maximal soluble dose:
max, max/1.2, max/1.2^2, max/1.2^3, max/1.2^4, max/1.2^5, max/1.2^6 und max/1.2^7.

SURFACE MARKER EXPRESSION:
After 24 hours of exposure, THP-1 cells were stained with FITC labeled anti-human-CD 861 anti-human-CD 54 antibody and propidium iodide and the fluorescence intensity was analyzed using flow cytometry. A test substance was predicted to have a dendritic cell activating potential when the marker expression exceeded the threshold of 150% with respect to vehicle treated cells for CD86 and for a threshold of 200% for CD54 at any tested sufficiently non-cytotoxic (cell viability = 50%) concentration in at least two independent experiments. Three independent experiments were performed and the expression of CD 86 and CD 54 was analyzed

CONCENTRATION
The Test substance was tested in a concentration range of 279.1 to 1000.0 µg/ml.

EVALUATION OF THE RESULTS
As readout, the change in the expression of the cell membrane markers CD 54 and CD 86 measured by flow cytometry after 24 hours of test substance exposure is used. A test substance is predicted to be a skin sensitizer when marker expression exceeds the threshold in relation to vehicle control.

Positive control results:

Positive control: DNCB 4 µg/mL:
While the tested concentration of the positive control was in the acceptable range for viability (>50% ), for THP-1 cells according to SOP, CD 54 and CD 86 expression were induced after 24 hours
exposure to DNCB. The level of expression were within specification with >150% for CD 86 and >200% for CD 54 as stated in the SOP.

Run / experiment:

other: 1

Parameter:

other: Induction of CD86

Value:

154

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: Induction of CD86

Value:

162

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 3

Parameter:

other: Induction of CD86

Value:

153

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 1-3

Parameter:

other: no CD54 induction

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

RESULTS OF CYTOTOXICITY ASSESSMENT:
The highest miscible dose at 1000.0 µg/ml was used as the maximal test concentration instead of CV75-based dose without affecting cytotoxicity at all tested doses (data of preliminary cytotoxicity range finding experiment is shown in Table 1).

RESULTS OF SURFACE MARKER EXPRESSION
Cell viability was decreased below 90% at the highest dose of 1000.0 µg/ml in all three experiments performed (experiment 1-3). No induction above the threshold of the expression of CD 54 was observed in all three experiments. An induction above the threshold of the expression of CD 86 was observed in all three experiments at sufficiently non-cytotoxic (cell viability=50%) concentrations from 401 .9 µg/ml to 1000.0 µg/ml (experiment 1) and at 1000.0 µg/ml in experiments 2 and 3.

Table 1: h-CLAT. Results of preliminary cytotoxicity assessment.

Date	Test conc. (µg/mL)	7.8	15.6	31.3	62.5	125	250	500	1000
10. Oct. 2013	Viability (%)	98.5	98.3	98.2	98.0	97.8	97.7	97.9	94.0
	Test conc. (µg/mL)	7.8	15.6	31.3	62.5	125	250	500	1000
26. Sep. 2013	Viability (%)	97.9	97.6	97.4	95.9	95.5	96.2	95.0	92.0

Table 2: Results of the main experiments 1 -3

Experiment 1:

sample	conc. (µg/mL)	MFI (Geo Mean)			corrected MFI		RFI (CD86)	RFI (CD54)	viability
		CD86	CD54	isotype	CD86	CD54	vs DMSO control	vsDMSOcontro1	lgG	CD86	CD54
control		0.38	0.31	0.23	0.15	0.08	91	104	97.9	97.4	98.0
DMSO	0.20%	0.40	0.31	0.24	0.166	0.074	100	100	97.9	97.9	98.1
DNCB	4.0	0.85	0.51	0.25	0.594	0.26	358	349	89.3	90.7	90.1
Test substance (DMSO)	279.1	0.46	0.31	0.24	0.224	0.074	135	100	96.1	96.6	96.2
	334.9	0.46	0.31	0.24	0.219	0.071	132	96	95.5	96.3	95.7
	401.9	0.52	0.33	0.25	0.263	0.076	158	103	89.8	91.7	91.6
	482.3	0.46	0.32	0.24	0.224	0.079	135	107	93.4	94.4	94.3
	578.7	0.56	0.36	0.26	0.3	0.1	181	135	84.9	86.7	86.l
	694.4	0.52	0.33	0.25	0.265	0.081	160	109	89.9	90.4	89.2
	833.3	0.51	0.34	0.25	0.256	0.084	154	114	90.6	91.6	91.2
	1000.0	0.59	0.37	0.29	0.298	0.079	180	107	60.1	62.3	62.9

Experiment 2:

sample	conc. (µg/mL)	MFI (Geo Mean)			corrected MFI		RFI (CD86)	RFI (CD54)	viability
		CD86	CD54	isotype	CD86	CD54	vs DMSO control	vsDMSOcontro1	CD86	CD54	isotype
control		0.58	0.39	0.30	0.29	0.10	83	87	98.2	97.8	98.1
DMSO	0.20%	0.64	0.41	0.30	0.3'15	0.109	100	100	97.8	97.9	97.9
DNCB	4.0	2.05	0.95	0.34	1.711	0.61	496	563	85.3	87.5	86.9
Test substance (DMSO)	279.1	0.69	0.41	0.30	0.385	0.109	112	100	97.8	97.5	97.7
	334.9	0.68	0.41	0.30	0.382	0.112	111	103	97.1	97.4	97.2
	401.9	0.65	0.42	0.30	0.346	0.119	100	109	97.6	97.1	97.2
	482.3	0.67	0.41	0.31	0.355	0.095	103	87	96.9	97.0	97.1
	578.7	0.64	0.41	0.30	0.34	0.112	99	103	97.2	97.2	97.1
	694.4	0.64	0.41	0.30	0.335	0.112	97	103	97.2	97.0	96.9
	833.3	0.72	0.43	0.30	0.411	0.124	119	11'1	95.3	94.4	94.5
	1000.0	0.92	0.51	0.36	0.558	0.147	162	135	73.8	7'1.2	7'1.5

Experiment 3:

sample	conc. (µg/mL)	MFI (Geo Mean)			corrected MFI		RFI (CD86)	RFI (CD54)	viabilitv
		CD86	CD54	isotype	CD86	CD54	vs DMSO control	vsDMSOcontro1	CD86	CD54	isotype
control		0.80	0.60	0.42	0.38	0.18	96	10)	97.6	97.3	97.2
DMSO	0.20%	0.82	0.59	0.42	0.401	0.175	100	100	97.4	97.3	97.0
DNCB	4.0	1.89	1.11	0.44	1.45	0.67	362	383	86.3	86.9	87.1
Test substance (DMSO)	279.1	0.80	0.57	0.42	0.383	0.146	96	83	96.1	96.6	96.7
	334.9	0.81	0.58	0.42	0.386	0.152	96	87	95.2	95.1	95.0
	401.9	0.79	0.57	0.42	0.372	0.154	93	88	96.6	96.7	97.0
	482.3	0.76	0.57	0.42	0.337	0.147	84	84	95.9	96.1	96.1
	578.7	0.78	0.56	0.43	0.347	0.127	87	73	94.8	94.6	95.2
	694.4	0.80	0.56	0.43	0.364	0.129	91	74	95.1	95.4	95.6
	833.3	1.03	0.64	0.46	0.57	0.)81	142	103	73.1	74.7	72.7
	1000.0	1.10	0.65	0.49	0.615	0.168	153	96	57.8	59.3	59.1

Interpretation of results:

other: positive indication of skin sensitisation

Conclusions:

No prediction can be made for skin sensitisation according to GHS criteria based on the results of this in vitro study alone.
According to the results of the present study, the test substance induces dendritic cell activation, which is indicative of skin sensitisation.

Executive summary:

The human Cell Line Activation Test (h-CLAT) is an in vitro skin sensitization test based on the enhancement by sensitizers of CD86 and/or CD54 expression on THP-1 cells. After 24 hours of exposure to the test substance, CD86 expression was induced in THP-1 cells at concentrations between 279.1 and 1000.0 µg/ml affording at least 50% viability. CD 54 expression was not induced in THP-1 cells at maximal test concentration and at all tested doses. From this, it has to be concluded that the test substance does induce dendritic cell activation.

Reference 7

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

Feb 2015

GLP compliance:

not specified

Type of study:

activation of keratinocytes

Details on the study design:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Test substance preparation: Solution in DMSO (100 x stock preparations), Solution in 1% DMSO in culture medium 3 (final concentrations)
- Vehicle: 1% DMSO in culture medium 3
- Precipitation in culture medium 3 after 48 h incubation: no precipitation

TEST SYSTEM:
- Type of cells: cell line LuSens

SELECTIION OF CONCENTRATIONS:
The test concentrations used for the main experiment have been established in a preliminary cytotoxicity test. For the main experiment concentrations of 5.36 to 27.67 µg/ml were used.
- Experiment 1: 7.72, 9.27, 11.12, 13.34, 16.01, 19.22, 23.06, and 27.67 µg/ml
- Experiment 2: 5.36, 6.44, 7.72, 9.27, 11.12, 13.34, 16.01, and 19.22 µg/ml

CONTROLS.
- Negative control (NC): DL-lactic acid (LA, 45 µg/ml)
- Positive control (PC): ethylene glycol dimethacrylate (EGDMA), 18 µg/ml
- Vehicle control (VC): 1% DMSO in culture medium

EXPERIMENTAL PROCEDURE:
- Number of experiments/replicates: 2 experiments were performed consisting of three replicates of each test substance concentration
- Exposure period: 48 h
- Determination of cell viability: MTT assay
- Absorbance: measured at 570 nm with reference wavelength at 690 nm (Sunrise Absorbance Reader)
- Quantitative measuremtn of luciferase gene induction: luciferase assay

Positive control results:

An upregulation of the luciferase activity after 48 hours treatment occurred with EGDMA but not with LA. The Ievei was within the range of the historical negative and positive control data.

Run / experiment:

other: 1

Parameter:

other: Fold induction

Value:

2.04

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: Fold induction

Value:

2.17

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

The CV75 value was derived from the concentration response curve. The CV75 is the estimated concentration that affords 75% cell viability and it was determined to be 16.01 µg/ml (corresponding to a final active ingredient concentration of approximately 15.97 µg/mL taking the purity of 99.75% into account) for the test substance und er the chosen exposure conditions on LuSens cells. ln the first main test the test substance was used at eight final concentrations determined with regard to the CV75 value: CV75 x 1.2³, CV75 x 1.2², CV75 x 1.2, CV75, CV75/1.2, CV75/1.2², CV75/1.2³ and CV75/1.2^4. Due to the observed toxicity lower concentrations were tested in the second experiment.

Luciferase activity after test substance treatment exceeded 1.5 fold induction with respect to the vehicle control at concentrations that did not reduce cell viability below 70% in two independent experiments.

Table 1: Results experiment 1

	First experiment
Concantration µg/mL	Fold induction		Rel. viability [%]
	mean	SD	mean	SD
vc	1.00	0.21	100.0	4.1
7.72	3.10	0.58	98.9	3.4
9.27	2.78	0.25	90.7	0.6
11.12	3.40	0.48	92.1	3.3
13.34	3.05	0.21	93.1	9.4
16.01	2.04	1.06	68.5	0.5
19.22	1.34	0.90	23.2	23.0
23.06	0.84	0.20	14.2	14.1
27.67	0.80	1.09	4.5	3.3
EGDMA (18µg/mL)	6.85	0.44	112.5	3.9
LA(450µg/mL)	1.01	0.26	106.2	1.9

Table 2: Results experiment 2

	Second experiment
Concentration µg/ml	Fold induction		rel. viability[%]
	mean	SD	mean	SD
vc	1.00	0.27	100.0	3.2
5.36	2.24	0.71	99.5	3.1
6.44	2.17	0.71	97.6	3.9
7.72	2.38	0.04	102.1	2.7
9.27	2.50	0.24	100.2	1.6
11.12	2.74	0.22	104.5	3.0
13.34	3.39	0.33	100.9	3.4
16.01	3.02	0.78	95.4	21.6
19.22	3.25	0.51	72.0	17.8
EGDMA (18 µg/ml)	5.82	0.30	113.5	2.2
LA (450 µg/ml)	1.14	0.31	110.3	2.3

Values in bold demonstrate valid results exceeding the threshold of 1.5 and viability of >70%.

Interpretation of results:

other: positive indication of skin sensitisation

Conclusions:

ln summary, after 48 hours of exposure to test substance luciferase activity in LuSens cells was induced at concentrations affording at least 70% viability in two independent experiments. From this it has to be concluded that test substance has a keratinocyte activating potential. Based on the outcome of the test battery strategy, the test substance is predicted to be a skin sensitizer. A single in vitro assay is not sufficient to adequately assess skin sensitization endpoint. This study is part of a test battery strategy.

Executive summary:

The LuSens assay is an in vitro method for the identification of keratinocyte activating substances using the genetically modified keratinocytes. The cell line Lu Sens was treated with 10 test substance concentrations for 48 hours in at least two independent experiments with each 3 replicates. Cells were lysed and luciferase induction was evaluated by measuring luminescence signal after substrate addition. In parallel, a MTT assay was performed to assess cytotoxicity. A test substance was considered to have an antioxidant response element induction potential if the fold induction of luciferase activity was >1 .5 and viability determined in the MTT assay was >70% at any test concentration. Luciferase activity after test substance treatment exceeded 1.5 fold induction with respect to the vehicle control at concentrations that did not reduce cell viability below 70% in two independent experiments.

This study is part of a test battery combining several in vitro methods addressing key steps of the adverse outcome pathway (AOP) for skin sensitization as defined by OECD. The following tests have been conducted to assess the skin sensitizing potential of the test substance.

• protein reactivity (DPRA),

• activation of keratinocytes (LuSens), and

• activation of dendritic cells (MUSST).

ln a separate report, the results of the individual studies were summarized and evaluated to predict the presence or absence of skin sensitizing potential of the test substance.

The combination of test methods and the evaluation of their results has been evaluated and published by Bauch et al., 2012. Based on the performance standards of the OECD test guideline no. 429 (Local Lymph Node Assay, LLNA, OECD 2010) the evaluation based on the DPRA, LuSens and MUSST methods yields an overall accuracy of 95% compared to results in humans (for comparison: for the same data set the LLNA yielded an overall accuracy of 86%).

A skin sensitizing (quantitative) potency assessment using the reported results was not validated at the time of writing of this report.

Based on the results and applying the evaluation criteria the test substance is predicted to be a skin sensitizer.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In vivo studies

The dermal sensitisation potential of 2-EHA was evaluated in the mouse local lymph node assay (LLNA) (OECD TG# 429) (Ritu Chhimwal, 2017). A preliminary assay was conducted to identify the appropriate test concentrations for the main study. Based on the results from a preliminary assay, six groups of mice (each comprising 5 females) were selected for the experiment. Four groups were treated with 2-EHA at concentrations of 2%, 10%, 30% (v/v) in acetone: olive oil (AOO) and 100% for three consecutive days (days 1, 2 and 3) on the dorsum of both ears (25mL per ear). One group served as a vehicle control and was treated with AOO, and another group served as a positive control and was treated witha-hexylcinnamaldehyde (HCA) at a concentration of 25% (v/v) in AOO. Group mean body weights of treated animals were comparable with the control group. One animal at 100% lost 0.5 grams of bodyweight and three animals in the positive control lost 0.9, 2.0 and 1.0 grams of body weight by the end of the study. There were no indications of clinical or systemic toxicity in 2-EHA treated animals.On day 6, the uptake of 3H-methyl thymidine into the auricular (local) lymph nodes draining at the site of chemical application was measured (5 hours post 20 ± 1 µCi intravenous (i.v.) injection) to assess the lymph node proliferative response. All animals were euthanized via CO2 asphyxiation and the lymph nodes were harvested and prepared for analysis in a scintillation counter. The results are presented in Disintegrations Per Minute per mouse (DPM/mouse). Each animal’s ears were also evaluated for erythema and edema prior to each application and ·again on days 4 and 5 and on day 6, prior to the i.v. injection. A positive response for HCA (Stimulation Index; SI = 4.20) confirmed the reliability of the test procedure. Mean stimulation indices for the 2%, 10%, 30% (v/v) and 100% 2-EHA treated groups were 1.19, 2.57, 3.53 and 5.50, respectively. The EC3 value obtained for 2-EHA is 18.96% (4740 µg/cm²). Therefore, 2-EHA demonstrate weak dermal sensitization potential in the local Lymph node assay. All criteria for a valid study were met as described in the protocol. The vehicle control and positive control in the definitive LLNA were within the acceptable ranges and fulfilled the requirements for a valid assay.

 

A further LLNA assay (BASF SE, 2019) aimed at addressing the level of 2-EHA required for the induction of a secondary immune response (elicitation threshold) in already sensitized animals. In a previously performed study (Pre-study, BASF SE, 2018), the potential of 2-EHA to induce a secondary immune response was examined using four test groups and three control groups of four female mice each. The experiment was also devided into two parts, namely induction (50%) and challenge phase (10%, 50%). It was concluded that 2-EHA exhibits a skin sensitizing potential and is able to induce a secondary immune response (Pre-study).

In the main study, during the induction treatment groups of 5 female CBA/CaOlaHsd mice each were treated three times with a 50% (w/w) preparation of 2-EHA in AOO (4:1) or with the vehicle alone. During the challenge treatment, the animals were treated once with 1%, 2.5%, 10% and 50% (w/w) preparations of 2-EHA in AOO (4:1) or with the vehicle alone.

The study used 4 test groups, a vehicle control group and a challenge control group. Each animal was treated with 25 µL per ear of the appropriate test-substance preparation or the vehicle applied to the dorsal surfaces of both ears on three consecutive days (day 1, 2 and 3; induction treatment) or on day 21 (challenge treatment). 20 µCi 3H-thymidine in 250 µL sterile saline were injected into the tail vein of the mice on day 23. About 5 hours after the 3H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring 3H-thymidine incorporation (indicator of cell proliferation). Cell counts and weights of each animal’s pooled lymph nodes were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined to obtain an indication of possible skin irritation.

No signs of systemic toxicity were noticed in all animals during general observation.

The 50% concentration applied to the animals during the challenge treatment, which were induced with the vehicle alone (challenge control group), induced statistically significant increases of 3H-thymidine incorporation into the cells and in the auricular lymph node cell counts. However, both failed to reach the cutoff (SI 3 for 3H-thymidine incorporation and SI 1.5 for the auricular lymph node cell counts). In addition the increase in lymph node weight was statistically significant.

When applied as 50% preparation in AOO (induction treatment) and as 10% and 50% in AOO (challenge treatment), 2-EHA induced a biologically relevant (increase above the cutoff Stimulation Index of 3), statistically significant and concentration-dependent increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes.

Concomitantly, a biologically relevant, statistically significant and concentration-dependent response (increase to 1.5-fold or above of control value = stimulation index (SI) = 1.5) in the auricular lymph node cell counts was observed at 10% and 50%. In addition, statistically significant increases in lymph node weights were noted at the 1%, 10% and 50% concentration.

2-EHA concentrations did not cause relevant increases (SI = 1.25) in ear weights demonstrating the absence of significant ear skin irritation. The increases were statistically significant at 1%, 2.5% and 10%. However, slight swelling of the ear skin was observed in all animals at all concentrations, induced with the 50% concentration on study day 2 and 3.

Thus, it is concluded in this study that the threshold concentration for inducing a secondary immune response (elicitation threshold) of 2-EHA was > 2.5% in the Challenge Murine Local Lymph Node Assay (cLLNA) under the test conditions chosen.

In an older study, the dermal sensitisation potential of 2-EHA was evaluated in the mouse local lymph node assay (LLNA) (OECD TG # 429) (Betts, 2006). 2-EHA was applied as 0.5, 1, 2.5, 5 or 10 %w/v preparations in acetone in olive oil (4:1). A vehicle control group was similarly treated using acetone in olive oil (4:1) alone. Dose levels were set according to sighting study results which indicated safe application levels and the dose range most likely to result in the ability to calculate a potency value. The estimated concentration giving rise to a 3 fold increase in lymphocyte proliferation (EC3) was calculated as percentage dose and µg/cm². 2-EHA had the capacity to cause skin sensitisation when applied as a dose of 10 %w/v preparation in acetone in olive oil (4:1). The EC3 value giving rise to a 3 fold increase in lymphocyte proliferation was calculated to be 9.7 %w/v (2425µg/cm²), indicative of a sensitizer of moderate potency. In a positive control study, hexylcinnamaldehyde was shown to have the capacity to cause skin sensitisation when applied as 10 and 25% w/v preparations in acetone in olive oil (4:1), confirming the validity of the protocol used for this study.

 

In vitro studies

The following in vitro studies are part of a test battery combining several methods addressing key steps of the adverse outcome pathway (AOP) for skin sensitization as defined by OECD. The following tests have been conducted to assess the skin sensitizing potential of 2-EHA.

• protein reactivity (DPRA),

• activation of keratinocytes (LuSens),

• activation of dendritic cells (MUSST),

• human Cell Line Activation Test (h-CLAT).

The combination of test methods and the evaluation of their results has been evaluated and published by Bauch et al., 2012. Based on the performance standards of the OECD test guideline no. 429 (Local Lymph Node Assay, LLNA, OECD 2010) the evaluation based on the DPRA, LuSens and MUSST methods yields an overall accuracy of 95% compared to results in humans (for comparison: for the same data set the LLNA yielded an overall accuracy of 86%).

Based on the results and applying the evaluation criteria the test substance is predicted to be a skin sensitizer.

 

Chemical reactivity has been shown to be well associated with allergenic potency. Within this context measuring the amount of proteins with nucleophilic side chains such as cysteine or lysine residues after incubation with putative allergens may serve as surrogate markers. For 2-EHA, the mean peptide depletion as average of cysteine- and lysine-peptide depletions was calculated to be 60.5% and thus, showing a high chemical reactivity in the DPRA (BASF SE, 2011, 2013).

The LuSens assay is an in vitro method for the identification of keratinocyte activating substances using the genetically modified keratinocytes. The cell line Lu Sens was treated with 10 test substance concentrations for 48 hours in at least two independent experiments with each 3 replicates. Cells were lysed and luciferase induction was evaluated by measuring luminescence signal after substrate addition. In parallel, a MTT assay was performed to assess cytotoxicity. A test substance was considered to have an antioxidant response element induction potential if the fold induction of luciferase activity was >1 .5 and viability determined in the MTT assay was >70% at any test concentration. Luciferase activity after 2-EHA treatment exceeded 1.5 fold induction with respect to the vehicle control at concentrations that did not reduce cell viability below 70% in two independent experiments (BASF SE, 2013).

 

The myeloid U937 skin sensitization test is a dendritic cell activation test (MUSST) to predict skin sensitizing potential. The test is performed using the human pro-monocytic cell line U937 as surrogate tor dendritic cells. As readout, the change in the expression of the cell membrane marker CD 86 measured by flow cytometry after 48 hours of test substance exposure is determined. A test substance is predicted to activate dendritic cells when CD86 cell surface expression exceeds the threshold of 1.2 in relation to vehicle control in at least two independent experiments.After 48 hours of exposure to 2-EHA, CD 86 expression was induced in U937 cells at concentration 125 µg/mL affording at least 70% viability. From this it has to be concluded that 2-EHA does induce dendritic cell activation (BASF SE, 2011, 2013).

 

The human Cell Line Activation Test (h-CLAT) is an in vitro skin sensitization test based on the enhancement by sensitizers of CD86 and/or CD54 expression on THP-1 cells. After 24 hours of exposure to 2-EHA, CD86 expression was induced in THP-1 cells at concentrations between 279.1 and 1000.0 µg/ml affording at least 50% viability. CD 54 expression was not induced in THP-1 cells at maximal test concentration and at all tested doses. From this, it has to be concluded that the test substance does induce dendritic cell activation (BASF SE, 2013).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification according to:

GHS classification (GHS UN rev. 7, 2017): Skin Sensitization: Category 1B

Annex VI of Regulation (EC) No 1272/2008 (CLP Regulation): Category 1.