Reaction mass of 2-dodecylhexadecyl methacrylate and 2-Tetradecyloctadecyl methacrylate

Endpoint summary

Description of key information

The skin corrosion potential of Reaction mass of 2-Propenoic acid, 2-methyl-, 2-dodecylhexadecyl ester and 2-Propenoic acid, 2-methyl-, 2-tetradecyloctadecyl ester was evaluated using OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method). The test was GLP compliant.

Triplicate EpiDerm skin model tissues were treated with a single topical application of 50µl of Reaction mass of 2-Propenoic acid, 2-methyl-, 2-dodecylhexadecyl ester and 2-Propenoic acid, 2-methyl-, 2-tetradecyloctadecyl ester. Additional triplicate tissues were treated with 50µl of Sterile Water (Negative control) and 50µl of Potassium Hydroxide (Positive control). All tissues were exposed for 30 minutes and 60 minutes at 37°C, 5% CO2, ≥95% Relative Humidity.

All controls were valid and demonstrated the reliability of the test system. The test substance did not interfere with MTT.

Mean tissue viability (as a percentage of the negative control), was 104.23% and 101.94 % after 3 and 60 minutes of exposure, respectively.

The substance is not corrosive to the skin.

The in vitro skin irritation of Reaction mass of 2-Propenoic acid, 2-methyl-, 2-dodecylhexadecyl ester and 2-Propenoic acid, 2-methyl-, 2-tetradecyloctadecyl ester was determined in accordance with the OECD Guideline for Testing of Chemicals 439 during a GLP-compliant study. This in vitro risk assessment assay predicts the Skin irritation potential of a chemical by measurement of its cytotoxic effect on the EpiDerm™ tissue model.

Prior to testing, the test substance was checked for interference with water and/or MTT. No interference was identified.

Skin irritation of the test substance and controls was evaluated in triplicate. After 60±1 minutes exposure on the surface of the EpiDerm™ reconstructed human epidermis and a 42±4h post-exposure incubation time, viability of the tissues was assessed and compared to the negative control.

The percentage of viability obtained with Reaction mass of 2-Propenoic acid, 2-methyl-, 2-dodecylhexadecyl ester and 2-Propenoic acid, 2-methyl-, 2-tetradecyloctadecyl ester was 100.052%, therefore it was considered as non-irritant to the skin according to the criteria laid down in the OECD Guideline for Testing of Chemicals 439.

An in vitro Bovine Corneal Opacity and Permeability (BCOP) assay was performed in line with OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The IVIS determinated for Reaction mass of 2-Propenoic acid, 2-methyl-, 2-dodecylhexadecyl ester and 2-Propenoic acid, 2-methyl-, 2-tetradecyloctadecyl ester was 1.1.

The substance is not irritant or damaging to the 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:

key study

Study period:

05 February 2019 - 06 February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

EpiDerm™ reconstructed human epidermis

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: MatTek Corporation EpidermTM reconstructed tissue model: EPI-200
- Tissue batch number(s): Lot 28682
- Delivery date: 05 February 2019
- Date of initiation of testing: 05 February 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C 5% CO2 - Relative Humidity (%): ≥95%

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Spectrophotometer: BMG LabTech FluoStar Optima
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: PASS
- Barrier function: PASS
- Morphology: PASS
Tissue thickness: PASS
- Contamination: No contamination reported = PASS

NUMBER OF REPLICATE TISSUES : 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE :
Prior to the assay, the test item was checked for interference (water colouration or MTT interference) and found not to interfere.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3 tissues per condition

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the relative tissue viability after 3 minutes treatment 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 relative tissue viability after 3 minutes treatment exposure is more and after 1 hour is less than or equal to 15%.
- Justification for the selection of the cut-off point(s) : Justification in accordance with Table 5 of testing guideline OECD 431

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Single topical administration of 50ul of neat test item

NEGATIVE CONTROL : Sterile water (tissue grade)
Lot number: RNBG8380
- Amount(s) applied : 50µl
- Concentration (if solution): Neat

POSITIVE CONTROL : Potassium Hydroxide
Lot number: SLBD3295V
- Amount(s) applied : 50µl
- Concentration (if solution): 8N

Duration of treatment / exposure:

3 minutes and 60 minutes

Duration of post-treatment incubation (if applicable):

N/A

Number of replicates:

three tissues per condition (n=3).

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 Minutes

Value:

104.23

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes

Value:

101.94

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None reported
- Direct-MTT reduction: No direct reduction
- Colour interference with MTT: No colour interference

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 of the negative control tissues must be ≥0.8. results for 3 minute = 1.94 and for 1 hour = 2.07 therefore passed.
- Acceptance criteria met for positive control: The mean of the positive control relative percentage viability, after 1hour exposure must be < 15% of the mean of the negative control. The result was a pass at 5.51
- Acceptance criteria met for variability between replicate measurements:should not exceed 0.3 (30%) results= Pass.

3 Minute Endpoint		NC	PC	TA1
Tissue 1	Aliquot 1 OD	1.74	0.29	2.10
	Aliquot 2 OD	1.79	0.28	2.07
Tissue 2	Aliquot 1 OD	1.90	0.34	1.85
	Aliquot 2 OD	1.88	0.33	1.85
Tissue 3	Aliquot 1 OD	2.15	0.32	2.12
	Aliquot 2 OD	2.17	0.31	2.12
All tissues	Average OD	1.94	0.31	2.02
	Average % Viability	100.00	15.96	104.23
	Average % SD	8.37	1.09	6.25
	Average % CV	8.37	6.83	5.99

1 Hour Endpoint		NC	PC	TA1
Tissue 1	Aliquot 1 OD	2.08	0.11	2.23
	Aliquot 2 OD	2.09	0.09	2.25
Tissue 2	Aliquot 1 OD	2.12	0.12	2.05
	Aliquot 2 OD	2.08	0.12	2.09
Tissue 3	Aliquot 1 OD	2.01	0.12	2.01
	Aliquot 2 OD	2.03	0.13	2.01
All tissues	Average OD	2.07	0.11	2.11
	Average % Viability	100.00	5.51	101.94
	Average % SD	1.69	0.54	4.74
	Average % CV	1.69	9.90	4.65

Interpretation of results:

GHS criteria not met

Conclusions:

The skin corrosion potential of Reaction mass of 2-Propenoic acid, 2-methyl-, 2-dodecylhexadecyl ester and 2-Propenoic acid, 2-methyl-, 2-tetradecyloctadecyl ester was evaluated. The tissue viability of the test item treated tissues were assessed and compared to a negative control. The percentage viability obtained after 3 minutes was 104.23% and after 60 minutes was 101.94%.The substance did not meet the criteria for classification as a corrosive in accordance with Regulation (EC) No. 1272/2008.

Executive summary:

The skin corrosion potential of Reaction mass of 2-Propenoic acid, 2-methyl-, 2-dodecylhexadecyl ester and 2-Propenoic acid, 2-methyl-, 2-tetradecyloctadecyl ester was evaluated using OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method). The test was GLP compliant.

Triplicate EpiDerm skin model tissues were treated with a single topical application of 50µl of Reaction mass of 2-Propenoic acid, 2-methyl-, 2-dodecylhexadecyl ester and 2-Propenoic acid, 2-methyl-, 2-tetradecyloctadecyl ester. Additional triplicate tissues were treated with 50µl of Sterile Water (Negative control) and 50µl of Potassium Hydroxide (Positive control). All tissues were exposed for 30 minutes and 60 minutes at 37°C, 5% CO2, ≥95% Relative Humidity.

All controls were valid and demonstrated the reliability of the test system. The test substance did not interfere with MTT.

Mean tissue viability (as a percentage of the negative control), was 104.23% and 101.94 % after 3 and 60 minutes of exposure, respectively. Therefore, 2-Propenoic acid, 2-methyl-, 2-dodecylhexadecyl ester and 2-Propenoic acid, 2-methyl-, 2-tetradecyloctadecyl ester does not meet the criteria for classification according to CLP Regulation (EC) No. 1272/2008.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Model used: EpiDerm (EPI-200) Reconstituted Human Epidermis
- Tissue batch number(s): 30827
- Production date:
- Shipping date:
- Delivery date: 24SEP19
- Date of initiation of testing: 30SEP19

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Optical Density (O.D.) values (the mean and SD of MTT value of 3 tissues exposed to sterile water) should be within the Test Guideline acceptance range of 1.0 – 3.0. QC result = 1.914 ± 0.076 = PASS
- Barrier function: The ET50 of tissues exposed to 100 µL Triton X-100 1%, n=3 should be within the Test Guideline acceptance range of 4.77 hours - 8.72 hours. QC result = 6.1 hours = PASS
- Morphology: Histological examination should demonstrate human epidermis-like structure: including multiple layers (at least 4) of viable epithelial cells (basal layer, stratum spinosum, stratum granulosum) which are present under multilayered stratum corneum. QC result = 11 layers are present = PASS. Tissue thickness should be within the acceptance range of >70 µm and <130 µm. QC result = 92.4 µm = PASS
- Contamination: There should be no evidence of contamination during long term antibiotic and antimycotic free culture. QC result = No contamination reported = PASS

NUMBER OF REPLICATE TISSUES:
Triplicate

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

Duration of treatment / exposure:

60 ± 1 minute (25 minutes at room temperature and 35 minutes at 37°C, 5% CO2, ≥95% RH)

Duration of post-treatment incubation (if applicable):

42 ± 4h post-treatment incubation (at 37°C, 5% CO2, ≥ 95% RH)

Number of replicates:

Triplicate

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean value

Value:

100.052

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None observed
- Direct-MTT reduction: None observed
- Colour interference with MTT: None observed

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean OD570 = 1.605. PASS.
- Acceptance criteria met for positive control: mean relative percentage viability = 6.118. PASS.
- Acceptance criteria met for variability between replicate measurements: standard deviation of viability percentages for triplicate skin models in each experimental condition = 4.948 (NC), 0.708 (PC) and 6.636 (TA1). PASS.

Viability measurements after 60±1 minutes of application and 42±4 hours post-incubation of test and reference items and controls.

Condition	Tissue #	Raw data		Blank corrected data		Mean OD	% of Viability
		Aliquot 1	Aliquot 2	Aliquot 1	Aliquot 2
NC	Tissue 1	1.757	1.831	1.651	1.725	1.688	105.204
	Tissue 2	1.744	1.659	1.638	1.553	1.596	99.439
	Tissue 3	1.622	1.650	1.516	1.544	1.530	95.357
PC	Tissue 1	0.217	0.212	0.111	0.106	0.109	6.762
	Tissue 2	0.204	0.208	0.098	0.102	0.100	6.232
	Tissue 3	0.187	0.197	0.081	0.091	0.086	5.360
TA1	Tissue 1	1.990	1.993	1.884	1.887	1.886	117.513
	Tissue 2	1.669	1.696	1.563	1.590	1.577	98.255
	Tissue 3	1.459	1.461	1.353	1.355	1.354	84.388

Interpretation of results:

GHS criteria not met

Conclusions:

Reaction mass of 2-dodecylhexadecyl methacrylate and 2-Tetradecyloctadecyl methacrylate did not reduce the tissue viability. The test substance does not meet the criteria for classification as irritant to the skin according to Regulation (EC) No.1272/2008.

Executive summary:

The in vitro skin irritation of Reaction mass of 2-Propenoic acid, 2-methyl-, 2-dodecylhexadecyl ester and 2-Propenoic acid, 2-methyl-, 2-tetradecyloctadecyl ester was determined in accordance with the OECD Guideline for Testing of Chemicals 439 during a GLP-compliant study. This in vitro risk assessment assay predicts the Skin irritation potential of a chemical by measurement of its cytotoxic effect on the EpiDerm™ tissue model.

Prior to testing, the test substance was checked for interference with water and/or MTT. No interference was identified.

Skin irritation of the test substance and controls was evaluated in triplicate. After 60±1 minutes exposure on the surface of the EpiDerm™ reconstructed human epidermis and a 42±4h post-exposure incubation time, viability of the tissues was assessed and compared to the negative control.

The percentage of viability obtained with Reaction mass of 2-Propenoic acid, 2-methyl-, 2-dodecylhexadecyl ester and 2-Propenoic acid, 2-methyl-, 2-tetradecyloctadecyl ester was 100.052%, therefore it was considered as non-irritant to the skin according to the criteria laid down in the OECD Guideline for Testing of Chemicals 439.

The test item did not meet the criteria for classification as irritant to the skin according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 04 July 2019 to 11 July2019

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Three corneas (triplicate)

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

QUALITY CHECK OF THE ISOLATED CORNEAS
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer.

NUMBER OF REPLICATES
Three corneas (triplicate)

NEGATIVE CONTROL USED
Sodium chloride 0.9% w/v

POSITIVE CONTROL USED
Ethanol

APPLICATION DOSE AND EXPOSURE TIME
The test article was not diluted prior to administration. A volume of 0.75 mL of the test article was a pplied to each of three corneas followed by a 10 minute incubation at 32±1°C

POST-INCUBATION PERIOD: yes at 32±1 ºC for 120 minutes

REMOVAL OF TEST SUBSTANCE
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer
- Corneal permeability: optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

Irritation parameter:

in vitro irritation score

Value:

1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The corneas treated with the test item were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

The positive control In Vitro Irritancy Score was within the acceptance range. The positive control acceptance criterion was therefore satisfied.

The negative control gave opacity and permeability values below the established upper limits. The negative control acceptance criteria were therefore satisfied.

Interpretation of results:

GHS criteria not met

Conclusions:

The IVIS determinated for Reaction mass of 2-Propenoic acid, 2-methyl-, 2-dodecylhexadecyl ester and 2-Propenoic acid, 2-methyl-, 2-tetradecyloctadecyl ester was 1.1. The test substance does not meet the criteria for classification as irritant or damaging to the eye according to Regulation (EC) No.1272/2008.

Executive summary:

An in vitro Bovine Corneal Opacity and Permeability (BCOP) assay was performed in line with OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The IVIS determinated for Reaction mass of 2-Propenoic acid, 2-methyl-, 2-dodecylhexadecyl ester and 2-Propenoic acid, 2-methyl-, 2-tetradecyloctadecyl ester was 1.1. The test substance does not meet the criteria for classification as irritant or damaging to the eye according to Regulation (EC) No.1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

The tissue viability was 104.23% and 101.94 % after 3 and 60 minutes of exposure during the in vitro skin corrosion test. The tissue viability was 100.052% duuring the in vitro skin irritation test. The test substance does not meet the criteria for classification as irritant or corrosive to the skin according to Regulation (EC) No.1272/2008.

The IVIS determinated for Reaction mass of 2-Propenoic acid, 2-methyl-, 2-dodecylhexadecyl ester and 2-Propenoic acid, 2-methyl-, 2-tetradecyloctadecyl ester was 1.1. The test substance does not meet the criteria for classification as irritant or damaging to the eye according to Regulation (EC) No.1272/2008.