[No public or meaningful name is available]

Endpoint summary

Description of key information

Skin irritation: The test material is considered not irritating to skin.
Eye irritation: The test material is considered not irritating to the eye.

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:

From April to May 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Cell type:

non-transformed keratinocytes

Justification for test system used:

In accordance with the OECD Testing Guideline 439

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™
- Delivery date: 30 April 2013

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing OPBS with Ca++ and Mg++ Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml MTT solution
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 540 nm

NUMBER OF REPLICATE TISSUES: triplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- Classification of irritation potential is based upon relative mean tissue viability following the 15-Minute exposure period followed by the 42-Hour post-exposure incubation period. Test item is classified as non-irritant if the relative mean tissue viability is > 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Approximately 1 0 mg of the test item was applied to the epidermis surface
Triplicate tissues treated with 10 µI of DPBS served as the negative controls and triplicate tissues treated with 10 µI of SDS 5% w/v served as the positive controls

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Three

Irritation / corrosion parameter:

% tissue viability

Value:

109.2

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

In an OECD 439 study, conducted according to GLP, the relative mean viability of the test material with the treated tissues (using the EPISKIN system, in vitro) was 109.2% after a 15-Minute exposure, therefore, is considered not irritating to skin.

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN™ reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed
human epidermal cultures following topical exposure to the test item by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory
mediator IL-1a in the culture medium retained following the 42-Hour post-exposure incubation period is also determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μI samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues was 109.2% after the 15-Minute exposure period.

The quality criteria required for acceptance of results in the test were satisfied.

The test item was considered to be Non-Irritant (NI).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Principles of method if other than guideline:

The physical form of the test material was a solid block. The melting point would be too high for testing in the BCOP assay and also water solubility was indicated as null suggesting that solubilising in 0.9% sodium chloride would not be possible.
Therefore in accordance with the methods described within the OECD BCOP test guideline, the test material was ground and applied neat onto the corneal surface. The cornea was exposed to the neat test item for 4 hours. The weight of the test item that adequately covered the epithelial
surface was determined. The ‘open-chamber’ method was adopted. Following application the ring & glass were replaced during the exposure period until rinsing. Rinsing was performed as the standard procedure.
These deviations were considered not to affect the purpose or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Species:

other: bovine

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System:
Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee and placed in Hanks’ Balanced Salt Solution (HBSS), supplemented with Penicillin/Streptomycin, and transported to the laboratory on ice packs. The corneas were prepared immediately on arrival.
Test Material:
For the purpose of this study the test material was used as supplied. The physical form of the test item was a solid block. The melting point would be too high for testing in the BCOP assay and also water solubility was indicated as nil suggesting that solubilising in 0.9% sodium chloride would not be possible. The test material was therefore ground and applied to the cornea for 4 hours as described in the OECD BCOP testing guideline.

Vehicle:

unchanged (no vehicle)

Remarks:

Neat substance

Controls:

not specified

Amount / concentration applied:

300 mg

Duration of treatment / exposure:

240 minutes

Details on study design:

Preparation of Corneas:
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete minimum essential medium (MEM) and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
The MEM was removed from the anterior chamber of the BCOP holder and the test item or control items applied to the corneal surface. 300mg of the ground neat test material was found to adequately cover the cornea. 750µL of each control item was applied to the appropriate corneas.
The holders were gently tilted back and forth to ensure a uniform application over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1ºC for 240 minutes.

Irritation parameter:

in vitro irritation score

Value:

1.1

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered not to be an ocular corrosive or severe irritant.

Executive summary:

A study was performed to assess the ocular irritancy potential of the test item to the isolated bovine cornea. The method was designed to be compatible with the OECD Guidelines for the Testing of Chemicals No. 437 (2009) "Bovine Corneal Opacity and Permeability Assay.

The test item was applied neat for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

A test item that induces an In Vitro Irritancy Score 55.1 is defined as an ocular corrosive or severe irritant.

The following IVIS were obtained:
Test Item 1.1
Negative Control 5.3
Positive Control 86.6

The test item was considered not to be an ocular corrosive or severe irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation
The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN™ reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed
human epidermal cultures following topical exposure to the test item by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory
mediator IL-1a in the culture medium retained following the 42-Hour post-exposure incubation period is also determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.
At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μI samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).
The relative mean viability of the test item treated tissues was 109.2% after the 15-Minute exposure period.
The quality criteria required for acceptance of results in the test were satisfied.
The test item was considered to be Non-Irritant (NI).

 

Eye irritation
A study was performed to assess the ocular irritancy potential of the test item to the isolated bovine cornea. The method was designed to be compatible with the OECD Guidelines for the Testing of Chemicals No. 437 (2009) "Bovine Corneal Opacity and Permeability Assay.
The test item was applied neat for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).
A test item that induces an In Vitro Irritancy Score 55.1 is defined as an ocular corrosive or severe irritant.
The following IVIS were obtained:
- Test Item 1.1
- Negative Control 5.3
- Positive Control 86.6
The test item was considered not to be an ocular corrosive or severe irritant.

Justification for classification or non-classification

The capacity of the registered substance to be irritating to the skin or the eye was evaluated in accordance with REACH. It was concluded that it did not meet the criteria for classification in accordance with Regulation (EC) No 1272/2008.