Docosyl acrylate

Endpoint summary

Description of key information

The test item, 2-Propenoic acid, C16-18-alkyl esters (read across) was tested in a skin irritation study and in eye irritation studies in vitro and in vivo. The test item is not irritating to skin and eye.

Additionally, Behenylacrylate (read across) was tested for skin irritation (in vitro) and eye irritation (in vitro). This test substance is also not irritating to skin and eye.
Based on these results 2-Propenoic acid, docosyl ester is also considered as not irritating to skin and eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Apr - May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

April 13, 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(from the competent authority) Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Based on the results of ECVAM funded validation studies, it was concluded by the ECVAM Scientific Advisory Committee that the EpiDerm human epidermis model is suitable to be used for distinguishing between corrosive and non-corrosive chemicals as well as between irritant and non-irritant chemicals.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200
- Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (exposure for 3 min) or 37 °C (exposure for 1 h)
- Temperature of post-treatment incubation (if applicable):37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were washed with PBS to remove residual test material 3 min or 1 h after start of the application treatment. After incubation, tissues were washed with PBS and the formazan produced by the tissues was extracted with isopropanol.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.9 mL MTT solution
- Incubation time: 55 - 65 min
- Spectrophotometer: Sunrise Absorbance Reader
- Wavelength: 570 nm (without reference filter)

NUMBER OF REPLICATE TISSUES: 2

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the mean tissue viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%
- The test substance is considered to be non-corrosive to skin if the mean tissue viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL

Duration of treatment / exposure:

3 min or 1 h

Duration of post-treatment incubation (if applicable):

3 h

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure, mean value

Value:

99

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure, mean value

Value:

98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

study cannot be used for classification

Conclusions:

No prediction can be made for skin corrosion according to GHS criteria based on the results of this in vitro study alone.
Based on the observed results and applying the evaluation criteria it was concluded, that the test substance does not show a skin corrosion potential in the EpiDerm skin corrosion test under the test conditions chosen.

Executive summary:

The potential of the test substance to cause dermal corrosion was assessed by a single topical application of 50 µL of the test substance to a reconstructed three dimensional human epidermis model (EpiDerm).

For the corrosion test two EpiDerm tissue samples were incubated with the test substance for 3 minutes and 1 hour, respectively.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure / post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The EpiDerm skin corrosion test showed the following results:

The test substance is able to reduce MTT directly. However, this ability of direct MTT reduction did not impair the study result as demonstrated by the concurrently performed exposure of control tissues inactivated by freezing.

The mean viability of the test substance treated tissues determined after an exposure period of 3 minutes was 99 %, and it was 98 % after an exposure period of 1 hour.

Based on the observed results and applying the evaluation criteria it was concluded, that the test substance does not show a skin corrosion potential in the EpiDerm skin corrosion test under the test conditions chosen.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Apr - May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

July 22, 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(from the competent authority) Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Based on the results of ECVAM funded validation studies, it was concluded by the ECVAM Scientific Advisory Committee that the EpiDerm human epidermis model is suitable to be used for distinguishing between corrosive and non-corrosive chemicals as well as between irritant and non-irritant chemicals.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200
- Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 30 °C
- Temperature of post-treatment incubation (if applicable): 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. After incubation, the tissues were washed with PBS to stop the MTT-incubation.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.9 mL MTT solution
- Incubation time: 55 - 65 min
- Spectrophotometer: Sunrise Absorbance Reader
- Wavelength: 570 nm (without reference filter)

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be an irritant to skin if the mean tissue viability is equal to or less than 50%
- The test substance is considered to be a non-irritant to skin if the mean tissue viability is greater than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5 % (w/v)

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

117

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

study cannot be used for classification

Conclusions:

No prediction can be made for skin irritation according to GHS criteria based on the results of this in vitro study alone.
Based on the observed results and applying the evaluation criteria it was concluded, that the test substance does not show a skin irritation potential in the EpiDerm skin irritation test under the test conditions chosen.

Executive summary:

The potential of the test substance to cause dermal irritation was assessed by a single topical application of 30 µL of the test substance to a reconstructed three dimensional human epidermis model (EpiDerm).

The irritation test was performed with three EpiDerm tissue samples, which were incubated with the test substance for 1 hour followed by a 42 -hours post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure / post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The EpiDerm skin irritation test showed the following results:

The test substance is able to reduce MTT directly.

The mean viability of the test substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 117 %.

Based on the observed results and applying the evaluation criteria it was concluded, that the test substance does not show a skin irritation potential in the EpiDerm skin irritation test under the test conditions chosen.

Reference 3

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2011-10-25 to 2011-11-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

July 22, 2010 (“In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Based on the results of ECVAM funded validation studies, it was concluded by the ECVAM Scientific Advisory Committee that the EpiDerm human epidermis model is suitable to be used for distinguishing between corrosive and non-corrosive chemicals as well as between irritant and non-irritant chemicals.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200
- Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature for 25 min, 37 °C for 35 min
- Temperature of post-treatment incubation (if applicable): 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. After incubation, the tissues were washed with PBS to stop the MTT-incubation.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.9 mL MTT solution
- Incubation time: 55 - 65 min
- Spectrophotometer: Sunrise Absorbance Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be an irritant to skin if the viability is less than or equal to 50%
- The test substance is considered to be a non-irritant to skin if the viability is greater than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5 % (w/v)

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

96

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

The test substance is able to reduce MTT directly. Subsequent testing of MTT reduction control was not performed, because no visible residues of the test substance remained on the tissues after washing.

The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 96%.

Based on the observed results and applying the evaluation criteria, it was concluded, that Behenylacrylate (Acrylate 22 45%) does not show a skin irritation potential in the EpiDerm™ skin irritation test under the test conditions chosen.

Test substance		Tissue 1	Tissue 2	Tissue 3	mean	SD
NC	Mean OD570	1.719	1.928	1.741	1.796
	Viability [% of NC]	95.7	107.3	96.9	100	6.39
11/0546-1	Mean OD570	1.735	1.755	1.686	1.725
	Viability [% of NC]	96.6	97.7	93.9	96	1.98
PC	Mean OD570	0.121	0.127	0.128	0.125
	Viability [% of NC]	6.7	7.0	7.1	7	0.21

Interpretation of results:

study cannot be used for classification

Conclusions:

No prediction can be made for skin irritation according to GHS criteria based on the results of this in vitro study alone.
Based on the observed results and applying the evaluation criteria it was concluded, that the test substance does not show a skin irritation potential in the EpiDerm skin irritation test under the test conditions chosen.

Executive summary:

The potential of the test substance to cause dermal irritation was assessed by a single topical application of the test substance to a reconstructed three dimensional human epidermis model (EpiDerm). Because the waxy test substance could not be applied with a pipette, a metal pin was covered with about 50 mg of the undiluted test substance.

Three EpiDerm tissue samples were incubated with the test substance for 1 hour followed by a 42 -hours post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure / post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The quotient of both values indicates the relative tissue viability. The EpiDerm skin irritation test showed the following results:

The test substance is able to reduce MTT directly. Subsequent testing of MTT reduction control was not performed, because no visible residues of the test substance remained on the tissues after washing.

The mean viability of the test substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 96 %.

Based on the observed results and applying the evaluation criteria it was concluded that the test substance does not show a skin irritation potential in the EpiDerm skin irritation test under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

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 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted May 12 th, 1981

Deviations:

no

GLP compliance:

no

Species:

rabbit

Strain:

Vienna White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Gaukler: D-6050 Offenbach/Main, FRG
- Weight at study initiation: mean weight (Male): 2.82 kg; (female): 2.88 kg
- Housing: single; cage made of stainlees steel with wire mesh walk floors, Floor area: 40 cm x 51 cm
- Diet: Kliba 341, 4mm: Firma Klingentalmuehle AG; CH-4304 Kaiseraugst, Switzerland (about 130 g per animal per day)
- Water: about 250 mL tap water per animal per day
- Acclimation period: at least 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70%
- Photoperiod (hrs dark / hrs light): 12 h /12 h

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated eye

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.1 mL bulk volume (about 32 mg of the comminuted test substance)

Duration of treatment / exposure:

single treatment

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

- 2 males
- 1 female

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: no

SCORING SYSTEM:
Scale for scoring ocular lesions:
- Chemosis (SW) and Cornea (OP) (Opacity-degree of density):
0 = None
1 = slight
2 = well-defined
3 = severe
4 = very severe

- Conjunctivae redness (RED):
0 = Normal
1 = slight
2 = well-defined
3 = severe

- Iris:
0 = Normal
1 = circum-corneal injection
2 = iritis

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Interpretation of results:

GHS criteria not met

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2011-10-18 to 2011-11-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

April 24, 2002

Deviations:

no

Principles of method if other than guideline:

no official national or international guidelines for the EpiOcularTM test:
- MatTek Corporation, Ashland, MA 01721, USA: EpiOcularTM human cell construct: Procedure details, Version 3.1a of February 10, 2010.
- Harbell J.W. et al. (2009): COLIPA Program on Optimization of Existing In Vitro Eye
Irritation Assays for Entry into Formal Validation: Technology Transfer and Intra/Inter Laboratory Evaluation of EpiOcular Assay for Chemicals. Poster # 378, Society of Toxicology, March 2009.

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE

Species:

human

Strain:

other: Three dimensional human cornea model

Details on test animals or tissues and environmental conditions:

Test system: The EpiOcularTM model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinozytes used to model the human corneal epithelium. The EpiOcularTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.

Vehicle:

unchanged (no vehicle)

Controls:

other: Negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg

Duration of treatment / exposure:

90 min

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2

Details on study design:

- Direct MTT reduction:
To assess the ability of the test material to directly reduce MTT a pretest was performed as described below. The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 55 to 65 minutes. A negative control (highly de-ionized water) was tested concurrently.
If the MTT solution color or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT. The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results.

- Basic procedure:
Two tissues were treated with the test substance, the PC and NC, respectively. In addition two killed tissues were used for the test substance and NC, respectively, in order to detect direct MTT reduction. There are two separate protocols for liquids and solids, differing in exposure time and postincubation period. Due to the physical condition of the test substance the protocol for solids was applied.

- Data evaluation
Table(s) and/or figure(s) of measured parameters presented in the report were produced using PC based tabular calculation software. The mean and individual data were not always rounded but the significant digits were produced by changing the display format. As a consequence, calculation of mean values using the individual data presented in the report will, in some instances, yield minor variations in value.
Principle : The OD570 values determined for the various tissues are measures of their viability. The quotient of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether or not a test material is an irritant.
Calculation of individual and mean optical densities: The individual tissue OD570 is calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of two tissues treated in the same way is calculated.
- Application of measurements using killed control tissues: In case of direct reduction of MTT by the test substance, the OD570 values measured in the freeze-killed control tissues (KC) will be used to correct the mean OD570 of the test substance treated tissues (mean OD570 KC corrected). Since killed tissue might still have a residual enzyme activity that is able to produce some formazan net OD570 KC is calculated by subtracting the mean OD570 KC of the NC from the mean OD570 KC of the test substance. In case the mean net OD570 KC is greater than 0.1 it is subtracted from the respective mean OD570 to result in the mean OD570 KC corrected. The mean OD570 KC corrected represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance.
- Tissue viability: The quantification of tissue viability is presented as the quotient of the mean OD570 (or mean OD570 KC corrected, if applicable) divided by the respective OD570 NC value in percent.

Irritation parameter:

other: tissue viability [%]

Run / experiment:

mean

Value:

96

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

The test substance is able to reduce MTT directly. However, this ability of direct MTT reduction did not impair the study result as demonstrated by the concurrently performed exposure of control tissues inactivated by freezing.

The mean viability of the test-substance treated tissues was 96%. Behenylacrylate (Acrylate 22 45%) does not show an eye irritation potential in the EpiOcular™ eye irritation test under the test conditions chosen.

Test substance		Tissue 1	Tissue 2	Mean KC	mean	Inter-tissue variability [%]
NC	Mean OD570	1.334	1.373	0.025	1.353
	Viability [% of NC]	98.6	101.4	-	100	2.9
11/0546-1	Mean OD570	1.299	1.299	0.029	1.299
	Viability [% of NC]	96.0	96.0	-	96	0.0
PC	Mean OD570	0.135	0.118	-	0.126
	Viability [% of NC]	10.0	8.7	-	9	1.3

Interpretation of results:

study cannot be used for classification

Conclusions:

No prediction can be made for eye irritation according to GHS criteria based on the results of this in vitro study alone.
Based on the observed results and applying the evaluation criteria it was concluded, that the test substance does not show an eye irritatio potential in the EpiOcular eye irritation test under the test conditions chosen.

Executive summary:

The potential of the test substance to cause ocular irritation was assessed by a single topical application of the test substance to a reconstructed three dimensional human cornea model (EpiOcular).

Because the waxy test substance could not be applied with a pipette, a metal pin was covered with about 50 mg of the undiluted test substance.

Two EpiOcular tissue samples were incubated with the test substance for 90 minutes followed by a 18 -hours post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure / post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The EpiOcular eye irritation test showed the following results:

The test substance is able to reduce MTT directly. However, this ability of direct MTT reduction did not impair the study result as demonstrated by the concurrently performed exposure of control tissues inactivated by freezing.

The mean viability of the test substance treated tissues was 96 %.

Based on the observed results and applying the evaluation criteria it was concluded, that the test substance does not show an eye irritation potential in the EpiOcular eye irritation test under the test conditions chosen.

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012-05-31 to 2012-03-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

September 07, 2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

Commission Regulation (EU) No. 1152/2010 of 8 December 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE

Species:

other: in vitro (bovine eyes)

Strain:

other: in vitro

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Isolated bovine cornea: The test system (target tissue) is the isolated bovine cornea. Bovine eyes are obtained as a by-product of freshly slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months).
- Source: Schlachthof Bensheim, Am Schlachthof 7-9, 64625 Bensheim
- Age at study initiation: minimum 12 months, maximum 60 months

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 µL undiluted test substance (after heating at ca. 30 °C)

Duration of treatment / exposure:

10 min

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

3 corneas

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

other: permeability value

Run / experiment:

mean

Value:

0.002

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

1.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Test Substance	Mean Opacity Value	Mean Permeability Value	In Vitro Irritation Score
12/0073-1	1.1	0.002	1.2
NC	2.8	-0.010	2.6
PC	121.7	3.204	169.7

Interpretation of results:

study cannot be used for classification

Remarks:

No predicition can be made for eye irritation according to GHS criteria.

Conclusions:

Based on the observed results and applying the evaluation criteria it was concluded, that the test substance does not cause serious eye damage in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen.
The test method according to the regulatory accepted protocol at the time of reporting does not allow for the evaluation of eye irritation. The result does not exclude an irritation potential of the test substance. For final assignment of a risk phrase at present, results from another study would be needed.

Executive summary:

The potential of the test substance to cause serious damage to the eyes was assessed by a single topical application of 750 µL of the undiluted test substance to the epithelial surface of isolated bovine corneas.

Three corneas were treated with the test substance for 10 minutes followed by a 2 -hours post-incubation period.

Corneal opacity was measured quantitatively as the amount of light transmission through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance relative to the control corneas.

The BCOP test showed the following results:

Test substance	Mean Opacity Value	Mean Permeability Value	In Vitro Irritancy Score
Test item	1.1	0.002	1.2
NC	2.8	- 0.010	2.6
PC	121.7	3.204	169.7

Based on the observed results and applying the evaluation criteria it was concluded, that the test substance does not cause serious eye damage in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen.

The test method according to the regulatory accepted protocol at the time of reporting does not allow for the evaluation of eye irritation. The result does not exclude an irritation potential of the test substance. For final assignment of a risk phrase at present, results from another study would be needed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

In the WoE study, the potential of the read across test item 2-Propenoic acid, C16-18-alkyl esters to cause dermal corrosion/irritation was assessed by a single topical application of 50 μL (corrosion test) or 30 μL (irritation test) of the test substance, 2-Propenoic acid, C16-18-alkyl esters to a reconstructed three dimensional human epidermis model (EpiDerm™). For the corrosion test two EpiDerm™ tissue samples were incubated with the test substance for 3 minutes and 1 hour, respectively. The irritation test was performed with three EpiDerm™ tissue samples, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The EpiDerm™ skin corrosion/irritation test showed the following results: The test substance is able to reduce MTT directly. However, this ability of direct MTT reduction did not impair the study result as demonstrated by the concurrently performed exposure of control tissues inactivated by freezing (performed with corrosion test, only).

- Corrosion test: The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 99%, and it was 98% after an exposure period of 1 hour.

- Irritation test:

The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 117%.

The test item does not show a skin irritation potential in the EpiDerm™ skin corrosion/irritation test under the test conditions chosen.

The potential of the Behenylacrylate to cause dermal irritation was assessed by a single topical application of the test substance to a reconstructed three dimensional human epidermis model (EpiDerm). Because the waxy test substance could not be applied with a pipette, a metal pin was covered with about 50 mg of the undiluted test substance.

Three EpiDerm tissue samples were incubated with the test substance for 1 hour followed by a 42 -hours post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure / post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The quotient of both values indicates the relative tissue viability. The EpiDerm skin irritation test showed the following results:

The test substance is able to reduce MTT directly. Subsequent testing of MTT reduction control was not performed, because no visible residues of the test substance remained on the tissues after washing.

The mean viability of the test substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 96 %.

Based on the observed results and applying the evaluation criteria it was concluded that the test substance does not show a skin irritation potential in the EpiDerm skin irritation test under the test conditions chosen.

In another supporting study, the test item Stearylacrylate was tested in a skin irritation test with three white vienna rabbits. The test concentration was 50 %. The mean erythema and edema scores were 0. The test item is not irritating to skin.

Based on these results 2-Propenoic acid, docosyl ester is also considered as not irritating to skin.

Eye irritation:

The potential of the test item, 2-Propenoic acid, C16-18-alkyl esters (read across) to cause serious damage to the eyes was assessed by a single topical application of 750 μL of the undiluted test substance to the epithelial surface of isolated bovine corneas. Three corneas were treated with the test substance for 10 minutes followed by a 2-hours post-incubation period. Corneal opacity was measured quantitatively as the amount of light transmission through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In vitro Irritancy Score of the test substance relative to the control corneas. The BCOP test showed the following results:

Test substance	Mean Opacity value	Mean Permeability value	In vitro Irritancy scrore
test item	1.1	0.002	1.2
NC	2.8	-0.010	2.6
PC	121.7	3.204	169.7

The test item does not cause serious eye damage in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen. The test method according to the regulatory accepted protocol at the time of reporting does not allow for the evaluation of eye irritation. The result does not exclude an irritation potential of the test substance. For final assignment of a risk phrase at present, results from another study would be needed.

The potential of Behenylacrylate to cause ocular irritation was assessed by a single topical application of the test substance to a reconstructed three dimensional human cornea model (EpiOcular).

Because the waxy test substance could not be applied with a pipette, a metal pin was covered with about 50 mg of the undiluted test substance.

Two EpiOcular tissue samples were incubated with the test substance for 90 minutes followed by a 18 -hours post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure / post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The EpiOcular eye irritation test showed the following results:

The test substance is able to reduce MTT directly. However, this ability of direct MTT reduction did not impair the study result as demonstrated by the concurrently performed exposure of control tissues inactivated by freezing.

The mean viability of the test substance treated tissues was 96 %.

Based on the observed results and applying the evaluation criteria it was concluded, that the test substance does not show an eye irritation potential in the EpiOcular eye irritation test under the test conditions chosen.

In another study, the test item Stearylacrylate was tested in an eye irritation test with three white vienna rabbits. The mean cornea, chemosis and iris scores were 0. The mean redness scores were 0.44. The test item is not irritating to eye.

Based on these results 2-Propenoic acid, docosyl ester is also considered as not irritating to eye.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. In the studies for both endpoints, skin irritation / corrosion and eye irritation / corrosion, the scores for the test item treated tissues were below the thresholds for classification as an irritant. As a result, the substance is not considered to be classified for skin or eye irritation under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EC) No. 2017/776.

Nevertheless, for the group of substances (monoalkyl or monoaryl or monoalkyaryl esters of acrylic acid) an entry in Table 3.1 and 3.2 of Annex VI of Regulation (EC) No 1272/2008 exists which has to be adopted for octadecyl acrylate although obtainded data show the opposite. Thus, the substance is classified as Xi (irritant), R36/37/38 (irritating to eyes, respiratory system and skin) according to Directive 67/548/EEC (DSD) and as skin irrit. cat. 2 (H315, causes skin irritation), eye irrit. cat. 2 (H319, causes serious eye irritation) and as STOT SE cat. 3 (May cause respiratory irritation) according to Regulation (EC) No 1272/2008 (CLP).