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Administrative data

Description of key information

The test substance is neither corrosive nor irritating to skin in two in vitro tests (LPT, 2014). The test substance shows no irritant effects on eyes in one in vitro test (LPT, 2014).

Key value for chemical safety assessment

Skin irritation / corrosion

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:
2014-02-26 to 2014-03-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
other: In Vitro Testing Strategy for Ocular Hazard Classification and Labeling of Antimicrobial Cleaning Products, Appendix B: ICCVAM-Recommended EpiOcular™ (EO) Test Method Protocol, published 2010.
GLP compliance:
yes
Species:
other: in vitro EpiOcular-EIT Model
Details on test animals or tissues and environmental conditions:
The following EpiOcular™-EIT Model was used:
Lot. No. 19146; OCL-200-EIT, MatTek In Vitro Life Science Laboratories, s.r.o Mlynské Nivy 73, 821 05 Bratislava II, Slovak Republic.
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
Test item was applied as solid.
50 mg of test item and 50 μL of the negative and positive controls were administered neat by topical application onto the tissue surface, so that the
surface of the tissue was completely covered by the test item.







Duration of treatment / exposure:
6-hour treatment period
Details on study design:
Assessment of coloured or staining materials
- To identify the potential of the test item to interfere with the MTT assay, the following checks were performed:
50 mg test item were added to 1 ml sterile deionised water and incubated in the dark at 37°C in a humidified atomosphere of 5% CO2 for 60 minutes. Furthermore 50 mg test item were added to 2 ml isopropanol, incubated for 2 hours. No discoloration of the test item either in water or
isopropanol was noted. Hence, no possible interacting with the MTT measurement had to be considered and no additional test had to be performed.

ADMINISTRATION
- Wetted tissues were incubated at standard culture conditions for 30 +/- 2 minutes
- 50 mg of test item was applied as solid to the tissue, forming a relatively smooth even layer on the surface
- Three replicate tissues were employed
- Positive control item was metyl acetate
- Negative control was sterile deionised water
- 50 μL of negative and positive controls were used
- Tissues were placed back into the culture medium after dosing and incubated at standard culture conditions for 6 hours ± 15 minutes
- Test item and controls were removed by extensively rinsing the tissues with Ca++Mg++free D-PBS
- After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously-warmed assay medium in a pre-labeled 12-well
plate for a 25 ± 2 minute immersion incubation at room temperature
- Each insert was removed from the assay medium, the medium was decanted off the tissues, the inserts were blotted on absorbent material and
transferred to the appropriate wells of the pre-labeled 6-well plate containing 1 mL of warm assay medium
- Tissues were incubated for 18 ± 0.25 hours at standard culture conditions
- After post-treatment incubation of 18 hours, the MTT assay was performed
- 300 μL of the MTT solution was added to each designated well of a pre-labeled 24-well plate, each insert was removed from the 6-well plate and
gently blotted on absorbent material
- Tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Three construct tissues were used for each treatment and concurrent control groups
- All tissues were placed into the 24-well plate, the plate was incubated for 180 ± 10 minutes at standard culture conditions
-Each insert was removed from the 24-well plate after 180 ± 10 minutes, bottom of each insert was blotted on absorbent material and transferred
to a pre-labeled 24-well plate containing 2.0 mL of propanol-2
- Plates were sealed with parafilm and stored overnight at 2-8°C in the dark for extraction
- Subsequently plates were placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature
- Extract solution was mixed and 200 μL aliquots were transferred to the appropriate wells of pre-labeled 96-well plates
- Optical density (OD) was measured at a wavelength of 570 nm in a spectrophotometer (Tecan Sunrise Magellan Version 6.47)
- Measurements were made for each of the three tissues in duplicate


Remarks on result:
other: The test item was considered to be non-cytotoxic and predicted to be non-irritant (see Executive summary below).

Acceptance criteria for test results

The results are acceptable if:

- the negative control OD > 1.0 and < 2.3

- the mean relative viability of the positive control methyl acetate is below 60% of negative control viability

- the difference of viability between the tissues of a single chemical is < 20% in the same run (for positive and negative control

tissues and tissues of single chemicals).

Data evaluation and interpretation

The individual % of control OD570 values were averaged to calculate the mean percent of control. The relative tissue viability was determined against the negative control. If the test item-treated tissue viability is > 60% relative to negative controltreated tissue viability, the test item is predicted to be non-irritant. If the test item-treated tissue viability is ≤ 60% relative to negative controltreated tissue viability, the test article is predicted to be irritant.

Conclusions:
The test item was considered to be non-cytotoxic and predicted to be non-irritant.
Executive summary:

The purpose of the experiment was to evaluate the potential ocular irritation of the test item by measuring

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) dye conversion by the EpiOcular™ tissue construct after topical exposure to test item.

The EpiOcular (OCL-200) was used. Three construct tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined by photometrical measurement of formazan production as a result of enzymatic reduction of the vital dye MTT (3-[4,5 - dimethylthiazol-2-yl] - 2,5 - diphenyltetrazolium bromide) and expressed as relative percentage of viability of the negative control-treated constructs.

50 mg of test item and 50 μL of the concurrent negative (sterile deionised water) or positive control (methyl acetate) were administered by topical application onto the construct on cultures for 6 hours, followed by a post-incubation for 18 hours.

The mean viability of the cells exposed to the test item was 93.3% of the mean negative control value. Hence, the OD570 values were above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 60%.

Result: The test item was considered to be non-cytotoxic and predicted to be non-irritant.

The mean optical density (OD) of the negative control of 3 tissues was 1.498 and was well within the acceptable range of ≥ 1.0 to ≤ 2.3. The viability of cells treated with the positive reference item, methyl acetate, was 19.5% of the negative controls and below the 60% cut-off value of the negative controls. The standard deviation of all triplicates determined was below the limit of acceptance of 20%. Hence, all acceptance criteria required were fulfilled.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The substance is considered to be lipohilic (log Kow=4.88) and has a very low water solubility. Water solubility (Lange, 2014) was determined to be lower than 2 µg/l and the measured pH was 7. In general a neutral pH is expected for alcohols. Furthermore an oral acute toxicity study was conducted with the substance (dose: 2000 mg/kg bw) and a necropsy 14 days after ingestion showed no pathological changes. Hence there is no indication for severe corrosion/irritation effects in the examined organs after oral uptake of the substance in this study. Furthermore two in vitro studies showed that the substance has no corrosive and no irritation potential with regard to exposed skin.

In summary based on available information there are no indications regarding potential corrosion or irritation effects of the substance to eyes. Hence the substance is expected to be a no or low eye irritant. As described by Scott et al. (Toxicology in vitro, 24, 1-9, 2010) in such case a bottom up approach was chosen and a testing strategy for in vitro examination of irritation effects on eyes was developed. According to Scott et al. epithelial models such as the chosen EpiOcular-Test should have the greatest potential to distinguish non-classified substances from irritants.

Based on the results of the conducted in vitro eye irritation study (EpiOcular-Test) the test item was considered to be non-cytotoxic and predicted to be non-irritant.

Taking the above mentioned considerations into account and in addition to the information described above the conducted in vitro eye irritation study is considered to be sufficient to assess the substance regarding the irritation potential to eyes.


Justification for selection of skin irritation / corrosion endpoint:
No study was selected, since both in vitro studies, the in vitro skin corrosion test (LPT, 2014) and the in vitro skin irritation test (LPT, 2014), showed neither corrosive nor irritant effects on the skin.

Justification for selection of eye irritation endpoint:
Only one study available.

Justification for classification or non-classification

Based on the assessment of three in vitro studies (skin corrosion / skin irritation and eye irritation) and according to criteria of EC Directive 67/548/EEC and EC Regulation 1272/2008 the test item is neither corrosive nor irritating to skin and not irritating to eyes. Therefore, the test item must not be classified.