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Description of key information

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
(2014)
GLP compliance:
yes (incl. certificate)
Species:
other: Assessment of corrosive potential of the test item by determination of its cytotoxic effect on an in vitro reconstructed human epidermis (exposure 3 min./rt and 60 min./37 °C, subsequently MTT).
Details on test animals and environmental conditions:
The experiment was carried out on a reconstructed human epidermis epiCS (CellSystems, Troisdorf, Germany). Inserts were of 0.6 cm² size. The model has a functional stratum corneum with an underlying layer of living cells. The barrier function of the stratum corneum is adequate, as has been shown by the supplier.
Vehicle:
unchanged (no vehicle)
Controls:
other: as negative control: physiological saline solution (0.9 % NaCl, 50 µL)
Amount / concentration applied:
50 µL per insert
Duration of treatment / exposure:
3 min./room temperature or 60 min. in an incubator (37 °C, 5 % CO2, maximum humidity)
Observation period:
post-exposure incubation: none
Details on study design:
The corrosive potential of the test item is assessed by determination of its cytotoxic effect on an in vitro reconstructed human epidermis. The test principle is based on the MTT assay reflecting the cell viability after exposure of the epidermal equivalent to topically applied test item.
All tests were performed in triplicates for each time point. The test item was applied at a 100 % concentration, i.e. 50 µL for 3 min. (room temperature) or 60 min. (37 +/- 2 °C, 5 % CO2, maximum humidity). Cell viability was measured by the amount of MTT reduction, i.e. an OD value following exposure to the negative control substance or the test item.
Irritation / corrosion parameter:
% tissue viability
Remarks:
3 min., rt
Value:
12.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
60 min., 37 °C
Value:
1.82
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
positive indication of irritation

According to OECD TG 431 a chemical should be classified as "corrosive" if the cell viability after a 3-min. treatment with a test item is decreased by more than 50 %.

 Sample No.

 Test item

 Time [min.]

 % Viability

 1 -3

  control NaCl 0.9 %

 60

 100.00

 4 -6

test item

 60

 1.82

 10 -12

  control NaCl 0.9 %

 3

 100.00

 13 -15

  test item

 3

 12.77

In this case the test item was characterized by having a significant impact on cell viability after the 3 -min. or the 60-min. period.

Interpretation of results:
other: corrosive potential
Executive summary:

An in vitro study according to OECD TG 431 for predicting a non specific, corrosive potential of a substance was conducted. In that study undiluted test item was applied topically on a reconstructed human skin (50 µL; epiCS, CellSystems, Germany). After an exposure period of 3 minutes at room temperature and 60 minutes in an incubator (37 °C, 5 % CO2, maximum humidity), the cell viability was 13 % and 2 %, respectively, as measured by a MTT conversion assay. Based on OECD TG 431 the test substance should be therefore classified as corrosive.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

Eye irritation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

No in vivo skin irritation study was performed in accordance to REACH Annex VIII, 8.1.1. Column 2 (the substance is classified as Skin Corr. 1C (H314: Causes severe skin burns and eye damage.)).

An in vitro study according to OECD TG 431 for predicting a non specific, corrosive potential of substances is available. In this study undiluted test item was applied topically on a reconstructed human skin (50 µL; epiCS, CellSystems, Germany). After an exposure period of 3 minutes at room temperature and 60 minutes in an incubator (37 °C, 5 % CO2, maximum humidity), the cell viability was 13 % and 2 %, respectively, as measured by a MTT conversion assay. Based on OECD TG 431 the test substance should be therefore classified as corrosive.

Further available is an in vitro skin irritation test according to OECD TG 439. In this study undiluted test item was applied topically on a reconstructed human epidermis (30 µL; epiCS, CellSystems, Germany). After an exposure period of 20 min. at room temperature and a post-exposure incubation of 42 hours in an incubator (37 °C, 5 % CO2, maximum humidity) the cell viability was 0.5 % as measured by a MTT conversion assay. Thus, also in this test with a 20 -min. exposure period it was demonstrated that the substance has a significant impact on cell viability.

Furthermore a structurally similar substance that is used for read across (1,6 -diisocyanatohexane, HDI; CAS 822 -06 -0) does also reveal a corrosive potential.

 

No in vivo eye irritation study was performed in accordance to REACH Annex VIII, 8.1.1. Column 2 (the substance is classified as Skin Corr. 1C (H314: Causes severe skin burns and eye damage.)).

Nevertheless, for eye irritation/corrosion three in vitro assays were performed.

At first, the test substance was investigated in the Bovine Corneal Opacity and Permeability (BCOP) test according to OECD TG 437. This in vitro test is used to identify chemicals with a potential for serious eye damage. In this study 750 µL of the unchanged test substance was applied to the extracted bovine cornea. Measurement of corneal opacity and permeability after a 10-minute exposure time and a 2-hour incubation time revealed an in vitro irritation score (IVIS) well below the cut-off value of 55, that is indicating a classification for serious eye damage according to the OECD TG 437. The positive (1 % sodium hydroxide solution) and negative (water) controls confirmed the validity of the test. Thus, under the conditions of this test no potential for serious eye damage can be concluded.

Secondly, an in vitro study for assessing ocular irritation was conducted in a human corneal epithelial (HCE) cell model. This model is recognized in the scientific community as a highly valuable model for the identification of substances that do not require classification for serious eye damage/eye irritancy (e.g. Cotovio et al., Toxicol. in Vitro, 24, 2010, 523 -537), and is routinely used by cosmetic and pharmaceutical companies. A recently conducted multi-laboratory validation study demonstrated the suitability for the assay to be used as part of a solid eye irritation risk assessment (Alépée et al., Toxicol in Vitro 31, 2016, 43–53). In this HCE-assay undiluted test item (30 µL) was applied topically to the reconstructed HCE tissue. After an exposure period of 60 minutes (room temperature), followed by a 16 hours post-treatment incubation period (37 °C, 5 % CO2, maximum humidity), the cell viability was 2.7 % as measured by a MTT conversion assay. Since the cut-off for a non-irritant to the eye is 50 %, the test substance requires classification for eye irritation or eye damage based on this assay.

Thirdly, the HET-CAM (Hen's Egg Test on the Chorioallantoic Membrane) was conducted according to a protocol of the ICCVAM Test Method Evaluation Report 2010 in order to assess a potential for severe eye damage of the substance. In this test eight days incubated fertile hen eggs were opened and 300 µL of the test substance was applied onto the chorioallantoic membrane. The effects occurring during the first 300 seconds after application were measured and used to calculate the irritation score. Based on this irritation score, which was 1 of maximal 21, the test item was identified as having a slight irritant potential.

Overall, two of three in vitro assays indicate an adverse effect to the eye. Since the substance is corrosive to the skin the risk of severe damage to the eyes is considered implicit.

With respect to respiratory irritation acute and repeated inhalation studies give evidence that the substance is a respiratory tract irritant (see endpoint summaries acute and repeated dose toxicity). Clinical observations in an acute inhalation toxicity study revealed signs reflective of respiratory tract irritation and in studies with repeated inhalation exposure irritant effect in the upper respiratory tract (epithelial alteration and inflammatory infiltrates in the nasal cavity) were histopathologically detected.

Justification for classification or non-classification

According to Regulation (EC) No 1272/2008, Annex I, classification is warranted for skin corrosion Cat. 1 (H314: Causes severe skin burns and eye damage). Based on the available in vitro assays for skin irritation/corrosion (OECD 431 and 439) no robust discrimination between Category 1A, 1B, and 1C can be made.

For substances assigned to skin corrosive Cat. 1, 1A, 1B or 1C, the risk of severe damage to eyes is considered implicit (serious eye damage Cat. 1 implicitly covered).

According to Regulation (EC) No 1272/2008, Annex I, classification is warranted for STOT SE 3 (H335: May cause respiratory irritation).