Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-11-03 to 2010-11-05

Reliability:

1 (reliable without restriction)

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:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for the Testing of Chemicals Part 431, adopted 13. Apr. 2004 “In vitro Skin Corrosion: Human Skin Model Test”

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

hexazinc(2+) bis(2-hydroxy-3,5-bis[(1R)-1-phenylethyl]benzoate) bis(2-hydroxy-3,5-bis[(1S)-1-phenylethyl]benzoate) 2-hydroxy-3-(1-phenylethyl)-5-(1-{4-[(1R)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-3-(1-phenylethyl)-5-(1-{4-[(1S)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-3-[(1R)-1-phenylethyl]benzoate 2-hydroxy-3-[(1S)-1-phenylethyl]benzoate 2-hydroxy-5-(1-phenylethyl)-3-(1-{4-[(1R)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-5-(1-phenylethyl)-3-(1-{4-[(1S)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-5-[(1R)-1-phenylethyl]benzoate 2-hydroxy-5-[(1S)-1-phenylethyl]benzoate

EC Number:

700-321-2

Molecular formula:

C30H26O6Zn, C46H42O6Zn, C62H58O6Zn

IUPAC Name:

hexazinc(2+) bis(2-hydroxy-3,5-bis[(1R)-1-phenylethyl]benzoate) bis(2-hydroxy-3,5-bis[(1S)-1-phenylethyl]benzoate) 2-hydroxy-3-(1-phenylethyl)-5-(1-{4-[(1R)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-3-(1-phenylethyl)-5-(1-{4-[(1S)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-3-[(1R)-1-phenylethyl]benzoate 2-hydroxy-3-[(1S)-1-phenylethyl]benzoate 2-hydroxy-5-(1-phenylethyl)-3-(1-{4-[(1R)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-5-(1-phenylethyl)-3-(1-{4-[(1S)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-5-[(1R)-1-phenylethyl]benzoate 2-hydroxy-5-[(1S)-1-phenylethyl]benzoate

Test animals

Species:

other: not applicable (human skin model in vitro)

Strain:

other: not applicable (human skin model in vitro)

Details on test animals or test system and environmental conditions:

Commercially available Epi-200-Kit.
The EpiDermTM tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organised 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.
The EpiDermTM tissues are cultured on specially prepared cell cultures inserts.

Test system

Type of coverage:

other: not applicable (human skin model in vitro)

Preparation of test site:

other: not applicable (human skin model in vitro)

Vehicle:

water

Controls:

other: not applicable (human skin model in vitro)

Amount / concentration applied:

Approx. 25 mg of the wax-like test item were applied together with 25 µL H2O

Duration of treatment / exposure:

3 min and 1 h respectively

Observation period:

not applicable (human skin model in vitro)

Number of animals:

not applicable (human skin model in vitro)

Details on study design:

PERFORMANCE OF THE STUDY

Description of the Method
Four 6-well-plates were prepared with 0.9 mL assay medium in each well. The inserts containing the tissues were transferred to the wells using sterile forceps and the 6-well-plates were set into the incubator at 37°C and 5% CO2 for one hour (pre-incubation).
For each experiment (“three minutes” and “one hour”), one 24-well-plate was prepared as holding plate. 12 wells of each plate were filled with 300 µL assay medium, the other 12 with 300 µL MTT reagent. One additional plate was left empty. The plates were stored in the incubator.
For each experiment (“three minutes” and “one hour”), two 6-well-plates were used. After pre-incubation, the assay medium was replaced by fresh assay medium and the test was started, using two wells as negative control with 50 µL H2O demin., two wells as positive controls with 50 µL potassium hydroxide solution and two other wells for testing the test item.
The solid test item was ground and averagely 25.1 mg test item were applied together with 25 µL H2O.
At the start of each experiment (application of negative controls), a stop watch was started.
After the respective incubation time, the inserts were removed from the plates using sterile forceps. The inserts were thoroughly rinsed with PBS, blotted with sterile cellulose tissue and set into the respective holding plate, using the wells containing assay medium. After transfer of all inserts, they were immediately moved to the wells containing MTT reagent, blotting the bottom with cellulose tissue again before setting the insert into the MTT well. The tissues were incubated with MTT reagent for three hours. After this time, the MTT reagent was aspirated and replaced by PBSbuffer. This was then aspirated, too, and replaced several times. At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanole were pipetted, taking care to reach the upper rim of the insert. The plate was then covered with Parafilm® and left to stand over night at room temperature.
On the next day, the inserts in which formazan had been produced over night were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation.
From each well, three replicates with 200 µL solution (each) were pipetted into a 96-well-plate which was read in a plate spectral photometer at 570 nm.

Evaluation
The values of the 96-plate-reader were transferred into a spreadsheet (Microsoft Excel).
The photometric absorption of the negative controls was considered as 100%. For the mean of the three replicates of test item and positive control, formazan production was calculated as % photometric absorption compared with the negative control. 

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: % formazan production of negative control

Value:

102.5

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 3 min. Reversibility: other: not applicable. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: % formazan production of negative control

Value:

112.5

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 1 h. Reversibility: other: not applicable. (migrated information)

In vivo

Irritant / corrosive response data:

not applicable (human skin model in vitro)

Other effects:

Corrosivity of the Test Item
The relative absorbance values were increased to 102.5% after three minutes treatment. This value is above the threshold for corrosivity (50%). After one hour treatment, the relative absorbance values were reduced to 112.5%, lying above the threshold for corrosivity (15%). Therefore, the test item is considered as not corrosive.

Validity
The criterion for optical density of the negative control (> 0.8) was fulfilled: optical density was 1.772 (3 minutes) resp. 1.768 (1 hour).
The positive control showed a clear corrosive effect: the value of the three-minute-experiment was 30.5% and the value of the one-hour-experiment was 12.9%.
Values for negative control and for positive control were within the range of historical data of the test facility (see annex 2, page 15).
Therefore the experiment is considered valid.

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive

Remarks:

Criteria used for interpretation of results: other: OECD guideline 431 (2004)

Conclusions:

Based on in vitro testing, Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc is considered as not corrosive.

Executive summary:

Two tissues of the human skin model EpiDerm^TM were treated with Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc for three minutes and one hour, respectively.

In average, 25.1 mg of the solid test item were applied to each tissue and spread to match the tissue size.

Deionised water was used as negative control, 8 m KOH was used as positive control.

After treatment with the negative control the absorbance values were well above the required acceptability criterion of mean OD > 0.8 for both treatment intervals thus showing the quality of the tissues. The positive control showed clear corrosive effects for both treatment intervals.

After three minutes treatment with the test item, the relative absorbance values were increased to 102.5 %. This value is well above the threshold for corrosion potential (50%). After one hour treatment, relative absorbance values were increased to 112.5 %. This value, too, is well below the threshold for corrosion potential (15%).

Therefore, Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc is considered not corrosive in the Human Skin Model Test.