4-hydroxybutyl acrylate

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Jul 2012 to 09 Aug 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

other: In Vitro Skin Sensitisation: U937 Skin Sensitisation Test

Version / remarks:

20 July 2016

Principles of method if other than guideline:

There are no official national or international guidelines for the MUSST Assay; however, the study is performed according to the methods described in the following publications:

Python F, Goebel C, Aeby P. (2007) Assessment of the U937 cell line for the detection of contact allergens. Toxicol Appl. Pharmacol. 220(2), 113-24.

Bauch C, Kolle SN, Fabian E, Pachel C, Ramirez T, Wiench B, Wruck CJ, van Ravenzwaay B, Landsiedel R. (2011) Intralaboratory validation of four in vitro assays for the prediction of the skin sensitizing potential of chemicals. Toxicology in Vitro 25, 1162 – 1168.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

- Name of test material: 4-hydroxybutyl acrylate
- Lot/batch No.: 010232EDA0
- Expiration date of the lot/batch: 30 Nov 2012
- Stability under test conditions: at least until 30 Nov 2012

Details on the study design:

Concentrations: 3.26, 6.53, 13.05, 26.10, 52.20 µg/mL

PRE-TEST
In order to determine the concentrations suitable for the main experiment a pre-test (experimental conduct in accordance with GLP but without a GLP status) was performed. Cells were exposed to 9 concentrations of the test substance (0.5 μg/mL up to 2000 μg/mL) and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA.

MAIN-TEST
Test substance preparation
The test-substance preparation was performed on a weight per volume basis shortly before application by stirring.

Test-substance preparation: The test substance was dissolved in medium as a 2x stock solution of the highest concentration. Further concentrations were prepared as 2x concentrations by serial dilution.
Vehicle: Culture medium
Reason for the vehicle: Culture medium was used according to the physiological conditions.
Form of application: Solutions in culture medium

The test substance preparations were prepared within 4 hours of performing the assay (preparation of test-substance samples).

CONTROLS
Negative control (NC): Lactic acid (LA – 200 μg/mL), CAS no.: 50-21-5
Positive control (PC): Ethylene diamine (EDA – 70 μg/mL), CAS no.: 97-90-5
Vehicle control: Culture medium Isotype control: In order to help distinguish non-specific (“background”) staining from specific antibody staining each test-substance concentration and control is additionally incubated with IgG1 FITC (CD86)

PREPARATION OF CELLS
U937 cells from the working cell bank were thawed and cultured in suspension using complete RPMI 1640 medium with 25 mM HEPES buffer and 2 mM L-glutamine supplemented with 10% fetal bovine serum and 100 U/mL penicillin and 100 μg/mL streptomycin under standard culture conditions (37°C, ca. 5% CO2, ≥ 90% humidity) until for 5 passages but not longer than passage 13 prior to testing. For substance incubation, cells seeded in 96-well microtiter plates (100 μL of 0.5x10^6 cells/mL). As a rule, two independent experiments were performed. In each experiment, duplicates of each treatment were tested.

TEST SUBSTANCE APPLICATION
Treatment was performed by adding 100 μL of test-substance preparation to the cells, thus diluting the 2x concentrated test-substance preparations to their final concentration and the cells to 0.25x10^6 cells/mL. After application the plates were sealed with semi-permeable plate sealers in order to prevent evaporation of the test substance. The plates were placed into the incubator under standard culture conditions for the exposure period of 48 hours.

VISUAL INSPECTIONS
A visual inspection for test-substance precipitates was performed for each test-substance concentration prior to application. In addition, each well was inspected under a microscope after the exposure period of 48 hours in order to detect irregularities in cell morphology or test-substance precipitates.

CELL STAINING AND FLOW CYTOMETRIC ANALYSIS
After visual inspection the cells were transferred into V-shaped 96-well microtiter plates and centrifuged. Supernatants were discarded. The cells were washed once with PBS with 5% FBS at 4°C. Cells were resuspended in 100 μL PBS (with 5% FBS) and labeled for 30 min at 4°C in the dark with 5 μL IgG-FITC (isotype control) or 5 μL anti-CD86-FITC antibody. Following incubation, cells were washed twice with PBS (with 5% FBS) and once with PBS and were then resuspended in 200 μL PBS. For cell viability analysis, cells were stained with PI (1.25 μg/mL final concentration in PBS) for 5 min at 4°C in darkness. Fluorescence intensity was analyzed using flow cytometry.

DATA EVALUATION
Analysis of the membrane markers was performed in 10,000 viable cells, determined by PI staining. Concentrations affording viability less than 70% were not considered for further assessment of dendritic cell activation. For data analysis, the CXP software (Beckman Coulter) was used. Data evaluation was performed with percentage of CD86 positive cells among the viable cells. An isotype control was used to quantify and remove non-specific antibody binding. The CD86 result was expressed as fold induction of CD86 expression compared to the respective vehicle control.

ACCEPTANCE CRITERIA
A tested concentration is not to be further evaluated when relative viability is less than 70%. A study is considered acceptable if the positive and negative and vehicle control data lie within the range of the historical data. The cell viability of untreated cells must yield at least 90%. The expression marker CD86 of the vehicle control cells should lie between 8% – 20%.

EVALUATION OF RESULTS
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.

Key result

Run / experiment:

other: 2 experiments (concentration in μg/mL)

Parameter:

other: CD86

Value:

3.26 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Raw data main experiments

Concentration µg/mL	1st Experiment		2nd Experiment
	CD 86 induction	Rel. viability	CD 86 induction	Rel. viability
VC	1.00	100.0	1.00	100.0
3.26	1.29	99.9	1.45	99.6
6.53	1.54	99.4	1.97	99.0
13.05	1.59	95.5	2.13	88.6
26.10	0.65	73.3	0.74	53.3
52.20	0.33	59.9	0.44	21.0

Control data

Concentration µg/mL	1st Experiment		2nd Experiment
	CD 86 induction	Rel. viability	CD 86 induction	Rel. viability
VC	1.00	100.0	1.00	100.0
LA 200 µg/mL	1.09	100.0	1.02	99.9
EDA 70 µg/mL	2.09	92.0	2.21	80.6

Pre-test

The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve to be 26.10 μg/mL.

Main tests

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 noncytotoxic (cell viability ≥ 70%) concentration in at least two independent experiments.

The MUSST showed the following results:

The test substance was soluble in culture medium.

The dilutions of the test substance were solutions.

In summary, after 48 hours of exposure to test substance 4 -hydroxybutyl acrylate CD86 expression was induced in U937 cells at concentration between 3.26 and 13.05 μg/mL affording at least 70% viability. From this it has to be concluded that test substance 4 -hydroxybutyl acrylate does induce dendritic cell activation.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

in combination with DPRA and LuSens in chemico/vitro tests

Reference 2

Endpoint:

skin sensitisation: in chemico

Remarks:

Direct Peptide Reactivity Assay (DPRA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

DPRA Assay for in vitro sensitisation with dendritic cells

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

- Gerberick GF, Vassallo JD, Bailey RE, Chaney JG, Morrall SW, Lepoittevin JP. Development of a Peptide Reactivity Assay for Screening Contact Allergens. Toxicological Sciences 81,332-343, 2004.
- Gerberick GF, Vassallo JD, Foertsch LM, Price BB, Chaney JG, Lepoittenvin JP. Quantificationn of Chemical Peptide Reactivity for Screening Contact Allergens: A Classification Tree Model Approach. Toxicological Sciences 97(2), 417-427, 2007.
- Bauch C, Kolle SN, Fabian E, Pachel C, Ramirez T, Wiench B, Wruck CJ, van Ravenzwaay B, Landsiedel R. Intralaboratory validation of four in vitro assays for the prediction of the skin sensitizing potential of chemicals. Toxicology in Vitro 25, 1162 – 1168, 2011.

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Direct Peptide Reactivity Assay (DPRA)

Specific details on test material used for the study:

- Name of test material (as cited in study report): 4-Hydroxybutyl Acrylate
- Analytical purity: >98 %
- Lot/batch No.: 010232EDA0

Details on the study design:

The test substance was dissolved at a 100 mM concentration in acetonitrile.
Further, a co-elution control was performed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover the samples were analyzed by measuring UV absorbance at 258 nm and the area ratio 220 / 258 was calculated as a measure of peak purity.

The samples were prepared in triplicates for each peptide.
The test substance was incubated with the C-containing peptide in a ratio of 1:10 (0.5 mM peptide, 5 mM test substance) and with the K-containing peptide in a ratio of 1:50 (0.5 mM peptide, 25 mM test substance).
The samples were prepared in suitable tubes, capped tightly and incubated at room temperature in the dark for 24 +/- 2 hours. Prior to HPLC analysis the samples were visually investigated for any precipitate that may have formed during the exposure period. The HLPC analysis of the batch of samples started about 24 hours after sample preparation and the analysis time itself did not exceed 30 hours.

Synthetic peptides:
Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol)
Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)
The peptides are custom material (Supplier: RS Synthesis, Louisville KY, USA) containing phenylalanine to aid in detection and
either cysteine or lysine as the reactive center.

Negative control (NC): vehicle control = acetonitrile
Positive control (PC): Ethylene glycol dimethacrylate (EGDMA; CAS-no. 97-90-5) (prepared as a 50 mM solution in acetonitrile).

Parameter:

other: mean C-peptide depletion (%)

Value:

100 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Parameter:

other: mean K-peptide depletion (%)

Value:

95.7 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Parameter:

other: mean peptide depletion (%)

Value:

97.8 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

The test substance was solved in acetonitrile. The samples of the test substance with the peptides were solutions. Visual observation after the 24-hour incubation time did not reveal precipitates in all samples of the test substance with both peptides.

The mean C-peptide depletion, caused by the test substance was determined to be 100.0%.

The mean K-peptide depletion, caused by the test substance was determined to be 95.7%.

Thus, the mean peptide depletion was calculated to be 97.8%.

No co-elution of test substance and peptides was noticed.

Based on the observed results and applying the prediction model proposed in Gerberick et. al (2007) and cited in chapter 3.10 it was concluded that 4-Hydroxybutyl Acrylate shows a high chemical reactivity in the DPRA under the test conditions chosen.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

in combination with LuSens and MUSST in vitro tests

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

There are no official national or international guidelines for the LuSens Assay; however, the study is performed according to the methods described in the following publication:
Bauch C, Kolle SN, Ramirez T, Eltze T, Fabian E, Mehling A, Teubner W, van Ravenzwaay B, Landsiedel R, (2012), Putting the parts together: Combining in vitro methods to test for skin sensitizing potentials, Regul Toxicol Pharmacol, 63(3):489-504.

GLP compliance:

yes (incl. QA statement)

Type of study:

other: LuSens Assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): 4-Hydroxybutyl Acrylate
- Physical state:
- Analytical purity: 98.8 corr. area% was detemined
- Lot/batch No.: 010232EDA0

Details on the study design:

In order to determine the concentrations suitable for the main experiment a pre-test (experimental conduct in accordance with GLP but without a GLP status) was performed. Cells were exposed to 9 concentrations of the test substance (1.95 μg/mL up to 500 μg/mL) and cytotoxicity was determined thereafter by MTT assay. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve to be 9.53 μg/mL.

In the main test, test substance was used at six final concentrations determined with regard to the CV75 value: CV75 x 1.22, CV75 x 1.2, CV75, CV75/1.2, CV75/1.22 and CV75/23. After 48 hour exposure luciferase activity was measured in a luminometer. A test substance was concluded to exhibit a keratinocyte activating potential when the luciferase activity exceeds 1.5 fold induction with respect to the vehicle control and at concentrations that did not reduce a viability below 70%. In parallel a MTT assay was performed to assess cytotoxicity of the test substance.

Two independent experiments were performed. In each experiment, three replicates of each treatment were tested

CONTROLS
Negative control (NC): 450 μg/mL DL-Lactic acid (LA, Sigma, Cat.No. 69785)
Positive control (PC): 15 μg/mL Ethylene glycol dimethacrylate (EGDMA, Sigma, Cat.No 335681)
Vehicle control: 1% DMSO in culture medium 3

Key result

Remarks on result:

other: see "Other effects / acceptance results"

Other effects / acceptance of results:

The test substance was soluble in 1% DMSO. The dilutions of the test substance were solutions.
In summary, after 48 hours of exposure to test substance 4-Hydroxybutyl Acrylate luciferase activity in LuSens cells was induced affording at least 70% viability viability in two independent experiments. From this it has to be concluded that test substance 4-Hydroxybutyl Acrylate has a keratinocyte activating potential.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

in combination with DPRA and MUSST in chemico/vitro tests

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A combination of several in vitro methods addressing key steps of the adverse outcome pathway (AOP) for skin sensitization [1] as defined by OECD, has been conducted to assess the skin sensitizing potential of the test substance.

The reactivity of test substance towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA) (BASF SE, 2013).

For this purpose the test substance was incubated with synthetic peptides for ca. 24 hours at room temperature and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm.

The test substance was dissolved at a 100 mM concentration in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide). Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides.

Further, a co-elution control was performed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover the samples were analyzed by measuring UV absorbance at 258 nm and the area ratio 220 / 258 was calculated as a measure of peak purity.

The following results were obtained in the DPRA:

The test substance was solved in acetonitrile. The samples of the test substance with the peptides were solutions. Visual observation after the 24-hour incubation time did not reveal precipitates in all samples of the test substance with both peptides.

The mean C-peptide depletion, caused by the test substance was determined to be 100.0%.

The mean K-peptide depletion, caused by the test substance was determined to be 95.7%.

Thus, the mean peptide depletion was calculated to be 97.8%. No co-elution of test substance and peptides was noticed. Based on the observed results and applying the prediction model proposed in Gerberick et. Al (2007) and cited in chapter 3.10 it was concluded thattest substance shows ahigh chemical reactivityin the DPRA under the test conditions chosen.

The potential of test substance to induced the cell membrane marker CD86 expression was evaluated in the Myeloid U937 Skin Sensitization Test (MUSST Assay) (BASF SE, 2012). For this purpose the test substance was incubated with human pro-monocytic cell line U937 for ca. 48 hours at 37°C and membrane marker expression measured by flow cytometry.

A solubility experiment was performed. The test substance was soluble in culture medium at a concentration of 2000μg/mL. In order to determine the concentrations suitable for the main experiment a pre-test (experimental conduct in accordance with GLP but without a GLP status) was performed. Cells were exposed to 9 concentrations of the test substance (0.5μg/mL up to 2000μg/mL) and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA.

The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve to be 26.10μg/mL. In the main test, test substance was used at five final concentrations determined with regard to the CV75 value: CV75 x 2, CV75, CV75/2, CV75/4, CV75/8. After 48 hour exposure U937 cells were stained with FITC labeled anti-human-CD86 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 1.2 with respect to vehicle treated cells (VC) at any tested sufficiently noncytotoxic (cell viability ≥ 70%) concentration in at least two independent experiments.

The MUSST showed the following results:

The test substance was soluble in culture medium. The dilutions of the test substance were solutions.

After 48 hours of exposure to test substance, CD86 expression was induced in U937 cells at concentration between 3.26 and 13.05μg/mL affording at least 70% viability. From this it has to be concluded that test substancedoes induce dendritic cell activation.

The keratinocyte activating potential of test substance was evaluated also in the LuSens assay (BAS SE, 2013). For this purpose the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and incubation was measured in a luminometer. The test substance was soluble in 1% DMSO at a concentration of 500μg/mL (50 mg/mL 100x stock solution).

In order to determine the concentrations suitable for the main experiment a pre-test (experimental conduct in accordance with GLP but without a GLP status) was performed. Cells were exposed to 9 concentrations of the test substance (1.95μg/mL up to 500μg/mL) and cytotoxicity was determined thereafter by MTT assay. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve to be 9.53μg/mL.

In the main test, test substance was used at six final concentrations determined with regard to the CV75 value: CV75 x 1.22, CV75 x 1.2, CV75, CV75/1.2, CV75/1.22 and CV75/23. After 48 hour exposure luciferase activity was measured in a luminometer. A test substance was concluded to exhibit a keratinocyte activating potential when the luciferase activity exceeds 1.5 fold induction with respect to the vehicle control and at concentrations that did not reduce a viability below 70%. In parallel a MTT assay was performed to assess cytotoxicity of the test substance.

The LuSens showed the following results:

The test substance was soluble in 1% DMSO. The dilutions of the test substance were solutions.After 48 hours of exposure to test substance luciferase activity in LuSens cells was induced affording at least 70% viability viability in two independent experiments. From this it has to be concluded that test substance has a keratinocyte activating potential.

The results of the individual studies are evaluated to predict the presence or absence of skin sensitizing potential of 4-Hydroxybutyl Acrylate.

 

The combination of test methods and the evaluation of their results has been evaluated and published by Bauch et al., 2012[2]. Based on the performance standards of the OECD test guideline no. 429 (Local Lymph Node Assay, LLNA, OECD 2010[3]), 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.

Evaluation criteria for individual tests are:

Test method	Endpoint	Evaluation criteria
Direct Peptide Reactivity Assay (DPRA)	Peptide depletion	Positive if ≥6.38% mean peptide depletion Negative if <6.38% mean peptide depletion
Keratinocyte Activation Assay - LuSens	ARE-dependent luciferase activity	Positive if ≥1.5-fold luciferase activity when viability is >70% of the vehicle control Negative if <1.5-fold luciferase activity
Dendritic Cell Line Activation Assay Myeloid U937 Skin Sensitization Test (MUSST)	CD86 expression	Positive if ≥1.2-fold of CD86 when viability is >70% of the control Negative if <1.2-fold of CD86

Direct Peptide Reactivity Assay: Positive

97.8% mean peptide depletion(100.0% cysteine peptide depletion; 95.7% Lysine peptide depletion)

 

Keratinocyte Activation Assay (LuSens): Positive

In at least two independent experiments a luciferase activity induction above 1.5-fold at test substance concentrations that did not reduce cell viability below 70% was observed.

 

Dendritic Cell Line Activation Assay Myeloid U937 Skin Sensitization Test(MUSST):Positive

ln at least two independent experiments induction of the expression of CD 86 above 1.2-fold was observed at sufficiently non-cytotoxic (cellviability≥70%) concentration.

 

Based on the results of the in vitro tests applying the evaluation criteria 4-Hydroxybutyl Acrylate predicted to be a skin sensitizer.

 

[1]OECD 2012:The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins (2012) OECD ENVIRONMENT, HEALTH AND SAFETY PUBLICATIONS: No.168; ENV/JM/MONO(2012)10

http://search.oecd.org/officialdocuments/displaydocumentpdf/?cote=env/jm/mono(2012)10/part1&doclanguage=en last accessed 05 Feb 2013

[2]Bauch C Kolle SN, Ramirez T, Eltze T, Fabian E, Mehling A, Teubner W, van Ravenzwaay B, Landsiedel R., 2012.Putting the parts together: combining in vitro methods to test for skin sensitizing potentials. Regul Toxicol Pharmacol. 63: 489-504.

^[3]OECD 2010: OECD GUIDELINE FOR THE TESTING OF CHEMICALS 429 Skin Sensitization: Local Lymph Node Assay (adopted July 2010)

http://www.oecd-ilibrary.org/docserver/download/9742901e.pdf?expires=1361285265&id=id&accname=guest&checksum=0F8779438DAD60D05DF9CD1DDABC2555

 

In vivo:

4-Hydroxybutyl Acrylate

In contrast to these results, another study conducted by BASF showed no sensitising potential of 4 -hydroxybutyl acrylate although the molecular structure and the positive results with structurally very similar compounds indicate a sensitising potential. Sixteen female white guinea pigs were assigned to one test group of 10 animals and two control groups of 3 animals each. During the induction phase, repeated painting with the test substance (20 % and 2 % in ether) produced erythema in all animals, followed by scaling and superficial crust formation (slight necrosis). Following challenge 13 days after last induction application, no skin reaction was observed in test or control animals that were exposed to 0.2% of the test substance for 12 h. Re-challenge with 1% test substance 24 h later induced slight to distinct, in part patchy erythema in 6/10 (=60%) test animals and in 1/3 (33%)control animals. Skin reactions in test and control animals were comparable concerning the degree of redness observed. Due to these findings in the controls, it is difficult to judge whether the substance induced sensitisation or simply irritation.

  

Supportingly, in vitro and in vivo data confirming skin sensitization were identified for 4-hydroxybutyl acrylate (4-HBA).Furthermore,the chemically related compounds 2-Hydroxyethyl acrylate (CAS: 818 -61 -1; 2-HEA) and Hydroxypropyl acrylate (CAS: 25584 -83 -2, HPA) were shown to be skin sensitizers in several animal studies:

Read across with 2 -HEA:

The skin sensitising potential of the read across substance 2-HEA was investigated in an interlaboratory study in four independent laboratories (Scholes et al., 1992). The Local Lymphnode Assay was conducted according to the method described by Kimber et al. (1989, 1991) and Basketter et al. (1991) with one small modification. In the study described here the animals received topical applications of test chemical on three consecutive days and the assay was terminated after 5 days. The application of the test substance at concentrations of 5, 10, 25, and 50 % in acetone:olive oil, 4:1 v/v and dimethylformamide (DMF) respectively resulted in an increase in isotope incorporation which was greater than 3-fold at the 5 % w/v concentration. 2 -Hydroxyethyl acrylate elicited positive LLNA responses in all four contributing laboratories. Consequently, the test substance was shown to be a potential skin sensitizer. The available data do not show a clear dose-response relationship, except for the data from one laboratory. Thus, derivation of an EC3 value and quantification of the substance's sensitizing potency was not possible. Based on the available data, it can be assumed that 2 -HEA is a sensitizer of moderate to strong potency according to the criteria proposed by the EU Expert Group (ECHA, R.8, December 2010).

In addition, 2-HEA was tested in the Guinea Pig Maximization test in the same Interlaboratory study (Scholes et al., 1992). Induction concentrations were 0.25 % (intradermal) and 5.0 % (topical); the challenge concentration was 1.0 %. Positive results were noted in 70% of the test animals at 24 and 48 hr after challenge. Thus, 2 -hydroxyethyl acrylate was assessed by the authors to be a strong sensitizer in the GPMT under the test conditions.

This result was confirmed by a GPMT conducted with 2-HEA by Bio Dynamics Inc. for Union Carbide Corporation (1982). 10/10 animals treated with the test material (5% concentration at intradermal induction) exhibited dermal responses at the first reading after challenge. No significant dermal responses were seen in the six irritation control animals, thus confirming that the concentration used was non-irritating (Union Carbide Corporation, 1982).

Read across with HPA:

This assessment is supported by data from the read across substance hydroxypropyl acrylate. The substance was tested in the Guinea Pig Maximization Test according to the method described by Magnusson and Kligman (1970). In this study cross reaction patterns of selected acrylate and methacrylate esters were investigated (Clemmensen 1984). The hydroxyalkylesters sensitized between 25 and 100 % of the treated animals (HPA 100 %) and exhibited extensive cross-reactions. The intensity of reaction was most pronounced with acrylates and occurred at much lower concentrations than with methacrylates.

Justification for classification or non-classification

Based on the available data, the substance has to be classified as Skin Sens. 1, H317 (May cause an allergic skin reaction) according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.