Phenylacetylene

Endpoint summary

Description of key information

- Skin irritation/corrosion : Corrosive (Category 1), based on in vitro skin corrosion study (OECD 431, GLP, rel.1, K).

- Eye irritation/corrosion : Serious eye damage (Category 1) as the substance is classified for skin corrosion

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 29 October 2021 to 24 February 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

Dated to 14 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Reconstructed human epidermises (first run: epiCS, Phenion – Batch No. epiCS 21-44 with HEK cells – Batch No. 6050/ second run: epiCS, Phenion – Batch No. epiCS 22-01 with HEK cells – Batch No. 6289)

Justification for test system used:

Reconstructed human epidermis (RhE) (obtained from human derived non-transformed epidermal keratinocytes) closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

HUMAN SKIN MODEL
The 0.6 cm² reconstructed epidermises were received on 02 December 2021 (epiCS, Phenion – Batch No. epiCS 21-44 with HEK cells – Batch No. 6050) during the first run and on 25 January 2022 (epiCS, Phenion – Batch No. epiCS 22-01 with HEK cells – Batch No. 6289) during the second run. The same day, the insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The inserts were placed in a 6 wells culture plate which had been previously filled with 1 mL of culture medium (Phenion Batch No. 305-AK2250 for the first run, Phenion Batch No. 305-AL0238 for the second run). The culture plates were incubated at 37°C, 5% CO2 for 20 hours and 06 minutes before treatment.

EVALUATION OF DIRECT INTERACTION WITH MTT
The direct interaction of MTT with the test item was checked by adding 50 µL of the test item to 1mL of the solution of MTT at 1 mg/mL (same conditions as in the main test). A yellow solution was observed after 1 hour of incubation between 36.4°C and 36.6°C, 5% CO2 in the dark.
-> Therefore, there is no direct interaction between the test item and MTT.

SPECTRAL ANALYSIS OF THE TEST ITEM
- IN ISOPROPANOL:
The spectral properties at 570 nm of test item in isopropanol were checked by adding 50 μL of the test item to 2 mL of isopropanol (same conditions as in the main test). A colourless solution was obtained after 2 hours of incubation at ambient temperature with gentle shaking.
The mean of the corrected OD (blank subtracted) was 0.001 which is lower than 0.08 (value corresponding to approximately twice the OD of the extracting solvent).
-> Therefore, the test item will not interfere with the MTT assay and there is no need to add non-specific coloration controls to the study

TREATMENT
The test item was applied as supplied at the dose of 50 μL to the epidermal surface of the 2 living human skin models during 3 minutes at room temperature and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2.

In the same experimental conditions, a positive control (50 μL of 8N KOH – Fisher Scientific, Batch No. A0412420) and a negative control (50 μL of distilled water– ADL Prochilab - Batch No. 201117) were carried out.

REMOVAL OF TEST MATERIAL AND CONTROLS
3 minutes and 1 hour after the test item application the human epidermis were washed 20 times with 1 mL of DPBS (DPBS – Dutscher, Batch No. 2390821). The rinsed tissues were checked for any coloration and noted to be white, comparable coloration to that of the negative control tissues.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
The cell viability was quantified by measurement of the cell succinate dehydrogenase activity. This enzyme was responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS No. 298-93-1)] reduction into blue formazan salt. The skin sample was placed into 300 μL of MTT solution, at the concentration of 1 mg/mL, for 2h50 (during the first run) and 3 hours (during the second run) at 37°C ± 1°C, 5% CO2.
The precipitated blue formazan product was then extracted using isopropanol for 2 hours under agitation in the dark, and the concentration of formazan was measured by determining the Optical Density (OD) at 570 nm, just after dilution of the extraction in isopropanol (1:3).
The absorbance was measured in triplicate of MTT extract.
The measured absorbances were proportional to the number of living cells.

The measurement of OD was performed using the Elx800 absorbance microplate reader (controlled and calibrated every year if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.
The linearity range of optical density measured is validated for an optical density between 0 and 2.0.

VIABILITY CALCULATION
- The results were expressed as a viability percentage compared with the negative control:
Viability % = (OD test item / OD negative control) * 100
- Data from individual replicate tissues (OD values and calculated percent tissue viability data for the test item and controls), mean percent tissue viability and standard deviation for each individual test item and control were reported in Table of results

PREDICTION MODEL / DECISION CRITERIA
The OD values obtained for each test sample are used to calculate a percentage of viability relative to the negative control, which is arbitrarily set at 100%. The cut-off values for the prediction of corrosion associated with the epiCS® models are as follows:

Viability measured after exposure time points (t=3 and 60 minutes)

STEP 1 (corrosive or not corrosive)
< 50% after 3 min exposure ==> Corrosive: Corresponding hazard statement “H314: Causes severe skin burns and eye damage” and signal word “Danger”
≥ 50% after 3 min exposure AND < 15% after 60 min exposure ==> Corrosive: Corresponding hazard statement “H314: Causes severe skin burns and eye damage” and signal word “Danger”
≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure ==> Non-corrosive

STEP 2 (subcategories for items identified as corrosive in step 1)
< 15% after 3 min exposure ==> Optional Sub-category 1A*
≥ 15% after 3 min exposure ==> A combination of optional Sub-categories 1B-and-1C

* According to the data generated in view of assessing the usefulness of the RhE test methods for supporting subcategorisation, it was shown that around 33% of the Sub-category 1A results of the epiCS® test methods may actually constitute Sub-category 1B or Sub-category 1C substances/mixtures (i.e. over-classifications).
It must be noted that a limitation of this Test Guideline is that it does not allow discriminating between skin corrosive sub-categories 1B and 1C.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

Applied to the epidermal surface of the 2 living human skin models during 3 minutes at room temperature and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2.

Duration of post-treatment incubation (if applicable):

The skin sample was placed in MTT solution of 1 mg/mL concentration during 1 hour between 36.4°C and 36.6°C, 5% CO2 in the dark.

Number of replicates:

2 living human skin models for each time

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

First run - after 3 minutes exposure

Value:

61.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

First run - after 1 hour exposure

Value:

14.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Second run - after 3 minutes exposure

Value:

76.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Second run - after 1 hour exposure

Value:

6.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

MTT VIABILITY ASSAY RESULTS
- Results of the first run:
3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item was 61.2% and 14.6%, versus 0.3% and 0.6%, respectively, with the positive control item (potassium hydroxide 8N).

- Results of the second run:
3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item was 76.6% and 6.2%, versus 0.6% and 0.5%, respectively, with the positive control item (potassium hydroxide 8N).

Acceptability criteria:

- Negative control: mean OD of the tissue replicates should be ≥ 0.8 and ≤ 2.8 for epiCS® model, for every exposure time. As the extract was diluted at 1:3 just before the OD measure, the acceptability criteria should be in the range ≥ 0.3 and ≤ 0.9 for the negative control.
- Positive control: mean viability of the tissue replicates exposed for 1 hour, expressed as % of the negative control, should be < 15% for epiCS model.
- Test item: in the range 20-100% viability, and for ODs ≥ 0.3, difference of viability between the two tissue replicates should not exceed 30%.

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

According to the CLP Regulation (EC) No. 1272/2008 and to the UN GHS 7th revised edition of the Globally Harmonized System of classification and labelling of chemicals (GHS), the results of the first and the second run confirm that the test item PHENYLACETYLENE has to be classified at least in Category 1B/1C “Corrosive”. The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are required.

Executive summary:

An in vitro skin corrosion test according to the OECD Guideline OECD 431 and in compliance with GLP was performed.

The test item PHENYLACETYLENE was applied as supplied, at the dose of 50 μL, to 2 Human skin model surfaces (epiCS®, Henkel®) during 3 minutes and 1 hour. The application was followed by a rinse with 20 mL of PBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item was 61.2% and 14.6%, versus 0.3% and 0.6%, respectively, with the positive control item (potassium hydroxide 8N). However, the viability at 1 hour is close to the limit of the classification: Category 1 and Non-corrosive (14.6% while the threshold of the OECD 431 is 15%).

To confirm this result and in accordance with the Sponsor, a second run with the same conditions has been performed. 3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item was 76.6% and 6.2%, versus 0.6% and 0.5%, respectively, with the positive control item (potassium hydroxide 8N).

 

According to the CLP Regulation (EC) No. 1272/2008 and to the UN GHS 7th revised edition of the Globally Harmonized System of classification and labelling of chemicals (GHS), the results of the first and the second run confirm that the test item PHENYLACETYLENE has to be classified at least in Category 1B/1C “Corrosive”. The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are required.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion:

Skin corrosion (OECD 431, GLP, rel.1, K)

An in vitro skin corrosion test according to the OECD Guideline OECD 431 and in compliance with GLP was performed.

The test item PHENYLACETYLENE was applied as supplied, at the dose of 50 μL, to 2 Human skin model surfaces (epiCS®, Henkel®) during 3 minutes and 1 hour. The application was followed by a rinse with 20 mL of PBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item was 61.2% and 14.6%, versus 0.3% and 0.6%, respectively, with the positive control item (potassium hydroxide 8N). However, the viability at 1 hour is close to the limit of the classification: Category 1 and Non-corrosive (14.6% while the threshold of the OECD 431 is 15%).

To confirm this result and in accordance with the Sponsor, a second run with the same conditions has been performed.
3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item was 76.6% and 6.2%, versus 0.6% and 0.5%, respectively, with the positive control item (potassium hydroxide 8N).

 

According to the CLP Regulation (EC) No. 1272/2008 and to the UN GHS 7th revised edition of the Globally Harmonized System of classification and labelling of chemicals (GHS), the results of the first and the second run confirm that the test item PHENYLACETYLENE has to be classified at least in Category 1B/1C “Corrosive”. The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are required.

 

Eye irritation/corrosion:

See skin irritation/corrosion

Justification for classification or non-classification

Harmonized classification:

The registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

 

Self-classification:

According to the CLP Regulation (EC) No. 1272/2008 and to the UN GHS 7th revised edition of the Globally Harmonized System of classification and labelling of chemicals (GHS), the results of the first and the second run confirm that the test item PHENYLACETYLENE has to be classified at least in Category 1B/1C “Corrosive”. The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are required.