Hydroxy(2-methylprop-2-enoato-O)zinc

Endpoint summary

Description of key information

In a GLP and OECD guideline 439 -compliant study, hydroxy(2 methylprop-2-enoato-O)Zinc was not irritating.
In a GLP and OECD guideline 405 -compliant study, hydroxy(2 methylprop-2-enoato-O)Zinc was irritating to eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 September - 09 October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in compliance with OECD Guideline 439 without any deviation

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Species:

other: human reconstructed epidermis (tissues)

Strain:

not specified

Details on test animals or test system and environmental conditions:

Not applicable

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

other: negative control: D-PBS; positive control: 5 % (w/v) SDS solution

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ± 2 mg
- Concentration: Test material: undiluted; negative control: Dulbecco’s Phosphate-Buffered Saline (D-PBS); positive control: Sodium Dodecyl Sulphate (SDS) at a 5% (w/v) aqueous solution

Duration of treatment / exposure:

15 ± 1 min

Observation period:

Post-treatment incubation period: 42 ± 1 h

Number of animals:

Three epidermis units were used per test material, positive and negative controls

Details on study design:

TEST SYSTEM:
- Cell system used: Episkin™ model
- Source: SkinEthic Laboratories (Lyon, France)


METHODOLOGY
Preliminary tests:
- Test for direct MTT reduction with the test item: 10 mg of the test item was added to 2 mL of a 0.3 mg/mL freshly prepared MTT solution. The mixture was incubated in darkness at 37 °C for 3 h ± 5 minutes and then the colour of the solutions obtained was evaluated. A negative control was tested concurrently by adding 10 μL of water for injections to 2 mL of a 0.3 mg/mL freshly prepared MTT solution.
- Test for the detection of the colouring potential of the test item: 10 mg of test item was added to 90 μL of water for injection in a transparent recipient and after 15 minutes of mixing, the colouration was checked.

Main tests:
- MTT conversion assay: The epidermis units were transferred from the kit into maintenance medium filled wells and pre-incubated at 37 °C, 5 % CO2 in a humidified incubator overnight. After pre-incubation, the test materials (10 mg) were applied topically to the epidermal model (three epidermis units per test material, positive and negative controls) for 15 ± 1 min at room temperature. After rinsing with D-PBS, the epidermises were then incubated at 37 °C, 5 % CO2 in a humidified incubator for 42 ± 1 h. Aliquots of culture media were kept frozen (-20 °C) for cytokine (IL-1α) further measurements. The viability was assessed by incubating the tissues for 3 h ± 5 min with MTT solution in a 12 well plate (0.3 mg/mL; 2 mL/well). The formazan precipitated was then extracted using acidified isopropanol (0.5 mL) and quantified spectrophotometrically at 540-595 nm using 96 well plates (200 μL/well).
- IL-1α assay: Culture media samples retained from the three negative controls and the three test item-treated tissues were analysed by ELISA according to standard laboratory procedures. The data are presented as the mean of IL-1α concentration in pg/mL.

Irritation / corrosion parameter:

other: other: relative viability %

Value:

99

Remarks on result:

other:

Remarks:

Basis: mean. Time point: exposure: 15 min. Max. score: 100.0. Reversibility: other: not applicable. (migrated information)

Irritant / corrosive response data:

Negative control:
- Mean optical density: 0.912 ± 0.082
Positive control:
- Mean viability %: 14 ± 8
Test material:
- Mean relative viability %: 99 ± 7
- IL-1 α mean concentration: 11.8 pg/mL

- For more data, see tables in the attached PDF document

Other effects:

None

Preliminary test:

- In the preliminary test, the test item was found not to have direct MTT reducing properties as the MTT solution containing the test item did not turn blue/purple when compared with the negative control. As a result, no additional controls were performed on water-killed tissues in parallel to the main test.

- The test item was found not to have a colouring potential in the preliminary test as the water solution containing the test item did not change colour. As a result, no additional controls were used in parallel to the main test.

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the test conditions, hydroxy(2 methylprop-2-enoato-O)zinc, has cell viability > 50 % on reconstructed human epidermis model and IL-1 α concentrations < 60 pg/mL therefore it is considered as non-irritant according to the criteria of Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272-2008).

Executive summary:

In a GLP-compliant in vitro skin irritation study performed according to the OECD Guideline 439, 10 ± 2 mg of the test item, hydroxy(2 methylprop-2-enoato-O)zinc, was applied topically on the surface of human reconstructed epidermis for 15 ± 1 min at room temperature. The test item and both the negative (Dulbecco’s Phosphate-Buffered Saline) and positive controls (5 % (w/v) SDS solution) were applied topically on triplicate tissues. At the end of the contact period, each tissue was rinsed with D-PBS and incubated for 42 ± 1 h at 37 °C, 5 % CO2 in a humidified incubator. Cell viability was then assessed through the colorimetric MTT reduction assay. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100 % (reference viability). Also, the concentration of the inflammatory mediator IL-1α was evaluated in the culture medium retained following the 42-h recovery period.

Mean relative viability for the test item was 99 ± 7 %. Mean viability for the positive control was 14 ± 8% and optical density value for the negative control epidermis was 0.912 ± 0.082. Acceptance criteria were therefore met and the study was considered valid. Mean IL-1 α concentrations in the culture medium of test item-treated tissues was 11.8 pg/mL.

Under the test conditions, as hydroxy(2 methylprop-2-enoato-O)zinc produced > 50 % cell viability on a reconstructed human epidermis model with IL-1 α culture medium concentrations < 60 pg/mL, it is considered as non-irritant according to the criteria of Annex VI to the Directive 67/548/EEC and the CLP Regulation (EC) N° (1272-2008).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 March - 09 April 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in compliance with OECD Guideline 405 without deviation

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Elevage de Gérome, Linxe, France
- Age at study initiation: 12 or 13 weeks
- Weight at study initiation: 2.37-2.96 kg
- Housing: Animals were kept in individual boxes installed in conventional air conditioned animal husbanding.
- Diet: Food (SAFE – 112), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 17-23 °C
- Humidity: 30-70 %
- Air changes: Approximately 10-15 changes per hour
- Photoperiod: 12 h dark / 12 h light

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g

Duration of treatment / exposure:

1 h (physiological rinse was done at 1 h post-installation)

Observation period (in vivo):

21 days
- Examination of the eyes was made after 1, 24, 48 and 72 h and Days 7, 14 and 21 after treatment.

Number of animals or in vitro replicates:

3 females

Details on study design:

TREATMENT
- Initially, a single animal was treated. After consideration of the responses produced on Day 7 in the first treated animal, two additional animals were treated.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Physiological rinse was done on Day 0 (at 1 h post-installation) to remove the remaining test item.
- Time after start of exposure: 1 h

SCORING SYSTEM: According to OECD Guideline 405

Irritation parameter:

cornea opacity score

Basis:

animal #1

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

0.7

Max. score:

2

Reversibility:

fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Basis:

animal #1

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

2.3

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 21 days

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

0.7

Max. score:

2

Reversibility:

fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Basis:

animal #2

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

2.7

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

2.7

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

cornea opacity score

Basis:

animal #3

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

iris score

Basis:

animal #3

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

0.3

Max. score:

2

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Basis:

animal #3

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

2.7

Max. score:

3

Reversibility:

fully reversible within: 14 days

Irritation parameter:

chemosis score

Basis:

animal #3

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

2.3

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

The ocular reactions observed during the study have been moderate to important and totally reversible:
- at the conjunctivae level: an important redness noted 24 h after the test item instillation and totally reversible between Days 7 and 14, associated with a moderate to important chemosis noted 24 h after the test item instillation and totally reversible on Day 7;
- at the iris level: a congestion, noted 24 h after the test item instillation, and totally reversible between Days 2 and 3;
- at the corneal level: a moderate corneal opacity, noted 24 h after the test item instillation, and totally reversible between Days 7 and 21.
A corneal neovascularization was noted on Day 7 in one animal and on Day 14 in a second one. This reaction was totally reversible between Days 14 and 21.

Other effects:

None

See the attached document for information on tables of results

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the test conditions, Zinc monomethacrylate / Dymalink 709 is classified as “R36 irritating to eyes”, according to the criteria of Annex VI to the Directive 67/548/EEC and "Category 2A - irritating to eyes" according to CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an eye irritation study conducted according to the OECD Guideline 405 and in compliance with GLP, 3 healthy female rabbits of the New Zealand White strain were exposed to 0.1 g of test item, Zinc monomethacrylate / Dymalink 709 in one eye while the another eye remained untreated and served as control. The eyelids were then gently held together for about one second to avoid any loss of test item. Physiological rinse was done on Day 0 (at 1 h post-installation) to remove the remaining test item. The eyes were examined and the changes were observed at 1, 24, 48 and 72 h and Days 7, 14 and 21 after treatment and graded according to the method of OECD Guideline 405. Initially, a single animal was treated and after consideration of the responses produced on Day 7 in the first animal, two additional animals were treated.

The ocular reactions observed during the study have been moderate to important and totally reversible. At the conjunctivae level, an important redness noted 24 h after the test item instillation and totally reversible between Days 7 and 14, associated with a moderate to important chemosis noted 24 h after the test item instillation and totally reversible on Day 7; at the iris level, a congestion, noted 24 h after the test item instillation, and totally reversible between Days 2 and 3; at the corneal level, a moderate corneal opacity, noted 24 h after the test item instillation, and totally reversible between Days 7 and 21. A corneal neovascularization was noted on Day 7 in one animal and on Day 14 in a second one. This reaction was totally reversible between Days 14 and 21. Mean individual scores at 24, 48 and 72 h after exposure for the 3 animals were 2.0, 2.0, 2.0 for cornea score; 0.7, 0.7, 0.3 for iris score; 2.3, 2.7, 2.7 for conjunctivae score and 1.3, 2.7, 2.3 for chemosis score. In this study, test item is an eye irritant on female rabbits.

 

Under the test conditions, Zinc monomethacrylate / Dymalink 709 is classified as “R36 irritating to eyes”, according to the criteria of Annex VI to the Directive 67/548/EEC and "Category 2A - irritating to eyes" according to CLP Regulation (EC) N° (1272-2008).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a GLP-compliant in vitro skin irritation study performed according to the OECD Guideline 439, 10 ± 2 mg of Hydroxy(2 methylprop-2-enoato-O)zinc applied topically on the surface of human reconstructed epidermis for 15 ± 1 min at room temperature followed by a 42 ± 1 h recovery incubation produced 99 ± 7 % mean cell viability through a colorimetric MTT reduction assay and mean IL-1 α concentrations of 11.8 pg/mL in the culture medium. The positive control (5 % (w/v) SDS solution) applied under the same conditions produced 14 ± 8% mean cell viability and the negative control (Dulbecco’s Phosphate-Buffered Saline) produced an optical density value of 0.912 ± 0.082. As it produced > 50 % cell viability on a reconstructed human epidermis model with IL-1 α culture medium concentrations < 60 pg/mL, Hydroxy(2 methylprop-2-enoato-O)Zinc is considered as non-irritant according to the criteria of Annex VI to the Directive 67/548/EEC and the CLP Regulation (EC) N° (1272-2008).

In an eye irritation study conducted according to the OECD Guideline 405 and in compliance with GLP, 3 healthy female rabbits of the New Zealand White strain were exposed to 0.1 g of test item, Zinc monomethacrylate / Dymalink 709 in one eye while the another eye remained untreated and served as control. The ocular reactions observed during the study have been moderate to important and totally reversible. At the conjunctivae level, an important redness noted 24 h after the test item instillation and totally reversible between Days 7 and 14, associated with a moderate to important chemosis noted 24 h after the test item instillation and totally reversible on Day 7; at the iris level, a congestion, noted 24 h after the test item instillation, and totally reversible between Days 2 and 3; at the corneal level, a moderate corneal opacity, noted 24 h after the test item instillation, and totally reversible between Days 7 and 21. A corneal neovascularization was noted on Day 7 in one animal and on Day 14 in a second one. This reaction was totally reversible between Days 14 and 21. Mean individual scores at 24, 48 and 72 h after exposure for the 3 animals were 2.0, 2.0, 2.0 for cornea score; 0.7, 0.7, 0.3 for iris score; 2.3, 2.7, 2.7 for conjunctivae score and 1.3, 2.7, 2.3 for chemosis score. In this study, test item is an eye irritant on female rabbits.

Justification for selection of skin irritation / corrosion endpoint:
Only one study available

Justification for selection of eye irritation endpoint:
Only one in vivo study available

Effects on eye irritation: irritating

Justification for classification or non-classification

As it produced > 50 % cell viability on a reconstructed human epidermis model with IL-1 α culture medium concentrations < 60 pg/mL, Hydroxy(2 methylprop-2-enoato-O)Zinc is considered as non-irritant to skin and it should therefore not be classified for skin irritation/corrosion according to the Annex VI to the Directive 67/548/EEC and to the CLP Regulation (EC) N° (1272-2008).

In a GLP and OECD guideline 405 -compliant study, Hydroxy(2 methylprop-2-enoato-O)Zinc showed reversible signs of irritation to eye therefore it should be classified as “R36 irritating to eyes”, according to the criteria of Annex VI to the Directive 67/548/EEC and "Category 2A - irritating to eyes" according to CLP Regulation (EC) No 1272-2008.