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

Skin irritation / corrosion

Administrative data

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

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

264-202-2

EC Name:

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

Cas Number:

63451-47-8

Molecular formula:

C4H6O3Zn

IUPAC Name:

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

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Hydroxy(2-methylprop-2-enoato-O)zinc
- Physical state: White powder
- Analytical purity: >85.5%
- Lot/batch No.: Y1109025
- Date of receipt: 05 July 2012
- Expiration date of the lot/batch: 15 December 2012
- Storage condition of test material: At room temperature and protected from humidity

Test animals

Species:

other: human reconstructed epidermis (tissues)

Strain:

not specified

Details on test animals or test system and environmental conditions:

Not applicable

Test system

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.

Results and discussion

In vitro

Results

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)

In vivo

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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).