Zinc acrylate

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 22, 2018 to January 25, 2018

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)

Version / remarks:

Adopted Jul. 29, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

Dated May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

other: EpiDermTM Kit (EPI-212-SCT; Batch# 25875)

Source species:

other: Human-derived

Cell type:

other: Human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis.

Cell source:

other: Supplier: MatTek In Vitro Life Science Laboratories, Bratislava.

Source strain:

not specified

Justification for test system used:

The test system consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

Vehicle:

water

Remarks:

Demineralised water, prepared by LAUS GmbH, from an ion-exchanger, batch no: 20170815.

Details on test system:

- Environmental Condition during incubation / exposure: 37 ± 1°C and 5.0 ± 0.5% CO2
- Amount of test substance applied: 26.0 and 26.1 mg (3 minutes exposure); 26.6 mg (1 h exposure)

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

3 minutes exposure: 26.0 mg (Tissue 1); 26.1 mg (Tissue 2)
1 h exposure: 26.6 mg (Tissue 1 and 2)

Duration of treatment / exposure:

3 minutes exposure
1 h exposure

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes treatment

Value:

ca. 72.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Inconclusive, to be determined based on 1 h reading

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h treatment

Value:

ca. 10.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

All Validity criteria for the Experiment were met:
1.    The criterion for optical density of the negative control (≥ 0.8 and ≤ 2.8) was fulfilled: optical density was 2.1 (3 minutes); 2.0 (1 h).
2.    The positive control showed clear corrosive effects. The criterion for the viability of the 1 h experiment, expressed as % of the negative control (< 15%), was fulfilled, too. The mean value of relative tissue viability was 9.9%.
3.    Values for negative control and for positive control were within the range of historical data of the test facility

The mean value of relative tissue viability of the test substance was reduced to 72.5% after 3 minutes treatment. Per criteria for assessment of corrosivity, this value is above the threshold for corrosively (50%). After 1 h treatment, the mean value of relative tissue viability of the test substance was reduced to 10.7%, lying below the threshold for corrosively (15%). Therefore, the test substance is considered as corrosive to skin.

The values of the negative control met the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals, thus showing the quality of the tissues. The positive control has met the validity criterion too, thus ensuring the validity of the test system.

Absorbance values blank isopropanol (OD 570 nm)
Replicate	1	2	3	4	5	6	Mean
Absorbance	0.039	0.038	0.039	0.04	0.038	0.038	0.039
Replicate	7	8	9	10	11	12
Absorbance	0.04	0.038	0.043	0.038	0.039	0.038

Absorbance values (OD 570 nm) of negative control, test substance and positive control
Incubation	Negative Control		Test substance		Positive Control
	Tissue 1	Tissue 2	Tissue 1	Tissue 2	Tissue 1	Tissue 2
3 min	2.125	2.117	1.456	1.576	0.404	0.447
	2.115	2.09	1.527	1.587	0.427	0.446
	2.147	2.093	1.527	1.593	0.423	0.446
1 h	2	1.974	0.25	0.248	0.26	0.224
	2.093	2.009	0.259	0.25	0.244	0.221
	2.116	1.988	0.259	0.251	0.246	0.225

Mean Absorbance Values of the 3 Minutes Experiment
Designation	Negative Control	Test substance	Positive Control
Mean – blank (tissue 1)	2.09	1.464	0.379
Mean – blank (tissue 2)	2.061	1.546	0.407
Mean of the two tissues	2.076	1.505	0.393
RSD	1.00%	3.90%	5.10%

Mean Absorbance Values of the 1 h Experiment
Designation	Negative Control	Test substance	Positive Control
Mean – blank (tissue 1)	2.031	0.217	0.211
Mean – blank (tissue 2)	1.951	0.211	0.184
Mean of the two tissues	1.991	0.214	0.198
RSD	2.80%	2.10%	9.50%

Comparison of Tissue Viability
Incubation	Positive Control	Test substance
3 min	18.90%	72.50%
1 h	9.90%	10.70%

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

Under the study conditions, the test substance was considered as corrosive to skin.

Executive summary:

A study was conducted to determine skin corrosion potential of the test substance using the reconstructed human epidermis (RHE) test method according to OECD Guideline 431, in compliance with GLP. Two tissues of the human skin model EpiDermTM were treated with the test substance for 3 min and 1 h, respectively. Demineralised water was used as negative control and potassium hydroxide (8M) was used as positive control. After treatment, the respective substance was rinsed from the tissues. Then, cell viability of the tissues was evaluated by MTT, which can be reduced to a blue formazan. Formazan production was evaluated by measuring the optical density (OD) of the resulting solution. After treatment with the test substance, the mean value of relative tissue viability was reduced to 72.5% (3 min exposure) and 10.7% (1 h exposure), which qualifies as corrosive. Under the study conditions, the test substance was therefore considered as corrosive to skin (Andres, 2018).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

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 Dec. 07, 2017 to Dec. 07, 2017

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)

Version / remarks:

Oct. 09, 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals, Series on Testing and Assessment No. 160

Version / remarks:

“Guidance Document On “The Bovine Corneal Opacity and Permeability (BCOP) and Isolated Chicken Eye (Ice) Test Methods: Collection of Tissues for Histological Evaluation and Collection of Data on Non-Severe Irritants”; 25. Oct. 2011

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:

Feb. 14, 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

other: Bos primigenius Taurus (fresh bovine corneas)

Details on test animals or tissues and environmental conditions:

Test System: Bos primigenius Taurus (fresh bovine corneas). Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the Day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container. After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1°C) without phenol red was filled. The holders were then incubated for 1 h in the incubation chamber at 32 ± 1°C.

Vehicle:

Hank's balanced salt solution

Remarks:

Batch no.: 20171207, 10-fold concentrated, diluted in demineralised water (1:10)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Test substance Preparation: The test substance was a solid non-surface-active substance. It was tested as a suspension with a concentration of 20% in HBSS. The suspension was prepared freshly on the Day of the assay.

Duration of treatment / exposure:

Incubation time: 4 h

Number of animals or in vitro replicates:

For each treatment group [negative control solution (HBSS), test substance and positive control (20% imidazole solution)], three replicates were used.

Details on study design:

OPACITY TEST
After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. After removal of the pre-incubation medium (cMEM without phenol red), 750 µL negative control solution, test substance suspension and positive control solution were applied to each replicate. The test or control preparations were given directly on the epithelium in such a manner that as much as possible of the cornea was covered with test or control substances. Exposure time on the corneas was 4 h at 32 ± 1°C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, both chambers were filled with cMEM without phenol red, and the final opacity value of each cornea was recorded.

PERMEABILITY TEST
After the recording of the final opacity value, the cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution was added to the front chamber for the detection of permeability of the corneas. For the open chamber method, a sodium fluorescein solution with a concentration of 5 mg/mL was used. The chambers were then closed again and incubated for 90 min. at 32 ± 1°C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, its optical density at 492 nm was measured with the microtiter plate photometer.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of three replicates

Value:

ca. 121.95

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Relative Standard Deviation IVIS: 7.22%; The test substance induced serious eye damage on the cornea of the bovine eye.

Other effects / acceptance of results:

In the negative control, no signs of eye irritation were observed. The positive control induced serious eye damage, which would be classified as GHS category I.

Opacity and Permeability Values

 

The illuminance (unit: LUX) values measured before and after exposure

Parameter	Negative Control			Test substance			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
(I) Measured values before exposure	968	995	972	999	1022	975	994	989	979
(I) Measured values after exposure	969	938	926	250	243	267	358	390	352

Rep. = Replicate

 

Opacity Values Negative Control

Parameter	Negative Control
	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	3.85	2.67	3.67
Opacity after exposure	3.80	5.23	5.81
Opacity Difference	-0.04	2.56	2.14
Mean Opacity Difference	1.55

 

Opacity Values Test substance and Positive Control

Parameter	Test substance			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	2.51	1.56	3.54	2.72	2.93	3.36
Opacity after exposure	128.11	132.93	117.44	77.57	67.97	79.56
Opacity Difference	125.60	131.37	113.90	74.85	65.04	76.20
Opacity Difference corrected	124.05	129.82	112.35	73.30	63.49	74.65
Mean Opacity Difference corrected	122.08			70.48

 

For the permeability measurement, three replicates for each treatment group were measured three times. cMEM without phenol red was measured as blank value.

Optical density at 492 nm of Blank

Parameter	cMEM without phenol red
1. Measurement	0.041
2. Measurement	0.035
3. Measurement	0.042
Mean	0.039

 

Optical density at 492 nm of Negative Control, Test substance and Positive Control

Parameter	Negative Control			Test substance			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
1 Measurement	0.068	0.050	0.060	0.040	0.050	0.060	1.435	2.137	1.411
2. Measurement	0.064	0.047	0.054	0.036	0.048	0.057	1.463	2.140	1.413
3. Measurement	0.069	0.048	0.050	0.039	0.049	0.057	1.465	2.143	1.396
1. Measurement – blank	0.0287	0.0107	0.0207	0.0007	0.0107	0.0207	1.3957	2.0977	1.3717
2. Measurement – blank	0.0247	0.0077	0.0147	-0.0033	0.0087	0.0177	1.4237	2.1007	1.3737
3. Measurement – blank	0.0297	0.0087	0.0107	-0.0003	0.0097	0.0177	1.4257	2.1037	1.3567
Mean of each replicate	0.0277	0.0090	0.0153	-0.0010	0.0097	0.0187	1.4150	2.1007	1.3673
Mean of the 3 replicates	0.0173			--			--
Corrected	--	--	--	-0.0183	-0.0077	0.0013	1.3977	2.0833	1.3500
Corrected mean of the 3 replicates	--			-0.0082			1.6103

IVIS Value

The calculated IVIS for each replicate and the corresponding means

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control HBSS	0.37	1.81	69.47%
	2.69
	2.37
Test substance Zinc acrylate	123.78	121.95	7.22%
	129.71
	112.37
Positive Control 20% imidazole solution	94.27	94.64	0.35%
	94.74
	94.90

Validityof the Study: The validity criteria and findings

Parameter	Criterion	Found	Assessment
IVIS of negative control HBSS	≤3	1.81	ok
IVIS of positive control 20% imidazole solution	70.92 – 157.64	94.64	ok

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Under study conditions, the test substance was determined to induce serious eye damage on the cornea of the bovine eye.

Executive summary:

A study was conducted to determine the eye irritation potential of test substance using the in vitro Bovine Corneal Opacity and Permeability (BCOP) test method according to OECD Guideline 437, in compliance with GLP. The BCOP test method can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage. Fresh bovine eyes were obtained from the slaughterhouse on the day of the test. The cattle were between 12 and 60 months old. The corneas were screened for test requirements, prepared and mounted in corneal holders. Prior to treatment, the test system was previously incubated with cMEM without phenol red at 32 ± 1°C for 1 h and Corneal opacity was determined. The test substance preparation [20% suspension in Hank’s Balanced Salt Solution (HBSS)] was incubated on the cornea for 4 h at 32 ± 1°C. After removal of the test substance, opacity and permeability values were measured. For each treatment group [negative control solution (HBSS), test substance and positive control (20% imidazole solution)], three replicates were used. In the negative control, no signs of eye irritation were observed. The positive control induced serious eye damage, which would be classified as GHS category I .The negative control and the positive control met the validity criteria. The calculated IVIS (In Vitro Irritancy Score) was 121.95. Under study conditions, the test substance was therefore determined to induce serious eye damage on the cornea of the bovine eye (Geitlinger, 2018).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

A study was conducted to determine skin corrosion potential of the test substance using the reconstructed human epidermis (RHE) test method according to OECD Guideline 431, in compliance with GLP. Two tissues of the human skin model EpiDermTM were treated with the test substance for 3 min and 1 h, respectively. Demineralised water was used as negative control and potassium hydroxide (8M) was used as positive control. After treatment, the respective substance was rinsed from the tissues. Then, cell viability of the tissues was evaluated by MTT, which can be reduced to a blue formazan. Formazan production was evaluated by measuring the optical density (OD) of the resulting solution. After treatment with the test substance, the mean value of relative tissue viability was reduced to 72.5% (3 min exposure) and 10.7% (1 h exposure), which qualifies as corrosive. Under the study conditions, the test substance was therefore considered as corrosive to skin (Andres, 2018).

Eye irritation

A study was conducted to determine the eye irritation potential of test substance using the in vitro Bovine Corneal Opacity and Permeability (BCOP) test method according to OECD Guideline 437, in compliance with GLP. The BCOP test method can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage. Fresh bovine eyes were obtained from the slaughterhouse on the day of the test. The cattle were between 12 and 60 months old. The corneas were screened for test requirements, prepared and mounted in corneal holders. Prior to treatment, the test system was previously incubated with cMEM without phenol red at 32 ± 1°C for 1 h and corneal opacity was determined. The test substance preparation [20% suspension in Hank’s Balanced Salt Solution (HBSS)] was incubated on the cornea for 4 h at 32 ± 1°C. After removal of the test substance, opacity and permeability values were measured. For each treatment group [negative control solution (HBSS), test substance and positive control (20% imidazole solution)], three replicates were used. In the negative control, no signs of eye irritation were observed. The positive control induced serious eye damage, which would be classified as GHS category I .The negative control and the positive control met the validity criteria. The calculated IVIS (In Vitro Irritancy Score) was 121.95. Under study conditions, the test substance was therefore determined to induce serious eye damage on the cornea of the bovine eye (Geitlinger, 2018).

Justification for classification or non-classification

Skin irritation

In an in vitro skin irritation study (RHE), treatment with the test substance resulted in the mean value of relative tissue viability of 72.5% (3 min exposure) and 10.7% (1 h exposure). The test substance therefore warrants classification as Skin Corr. 1B - H314: Causes severe skin burns and eye damage according to EU CLP (EC 1272/2008) criteria.

Eye irritation

The calculated IVIS (In Vitro Irritancy Score) in an in vitro eye irritation study (BCOP) was 121.95. The test substance therefore warrants classification as Eye Damage 1 - H318: Causes serious eye damage according to EU CLP (EC 1272/2008) criteria.