[No public or meaningful name is available]

Endpoint summary

Description of key information

Skin corrosion in vitro (EpiDerm™, OECD 431): not corrosive

Skin irritation in vitro (EPISKIN™, OECD 439): not irritating

Eye irritation in vitro (EpiOcular™, OECD 492): not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 - 05 Nov 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted in 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.40bis (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

adopted in 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ Human Skin Model

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis
- Tissue batch number: 34102
- Delivery date: 03 November 2020
- Date of initiation of testing: 03 November 2020
- Assay medium lot number: 102920LHB

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing procedure: Rinsing was achieved by filling and emptying each tissue insert under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h (37 °C, 5% CO2)
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm™ tissue was assessed by an MTT cell viability test. The determined OD (540 - 570 nm) was 1.704 ± 0.078 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 5.75 h (acceptance criteria: 4.77-8.72 h).
- Morphology: No histological examination was reported.
- Contamination: The cells used to produce the EpiDerm™ tissue were screened for the presence of viruses, bacteria, yeast and other fungi. No contamination was detected.
- Reproducibility: No information if the results of the positive and negative controls showed reproducibility over time was reported.

NUMBER OF REPLICATE TISSUES: duplicates

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- The test item did not reduce MTT.

NUMBER OF INDEPENDENT EXPERIMENTS TO DERIVE FINAL PREDICTION: single experiment

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 min exposure is less than 50%, or if the viability after 3 min exposure is greater than or equal to 50% and the viability after 1 h exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 min exposure is greater than or equal to 50% and the viability after 1 h exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 μL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Lot/batch no.: 18L10BA1A
- Purity: 100%

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration: 8N
- Lot/batch no.: H3410

Duration of treatment / exposure:

3 and 60 min

Number of replicates:

Duplicates for each treatment and control group (3 and 60 min).

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure

Value:

91.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min exposure

Value:

85.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: Since the test substance did not directly reduce MTT, an additional test with freeze-killed or viable tissues was not performed.
- Colour interference with MTT: The test substance did not change the colour, when mixed with deionised water and thus passed the colour interference pre-test. Also its intrinsic colour was not intensive.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD of the tissue replicates treated with the negative control was ≥ 0.8 and ≤ 2.8 for every exposure time (values between 1.862 and 2.233).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control for 1 h was < 15% compared to the negative control (3.7%).
- Acceptance criteria met for variability between replicate measurements: The coefficient of variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30% (values between 1.1% and 12.8%)

Table 2: Results (summary)

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	3.9	91.7
60 minute	100*	3.7	85.7

*: The mean viability of the negative control tissues is set at 100%

Table 3: Detailed results

Tissue	Exposure Period	MeanOD570of individual tissues	Mean OD570of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	2.233	2.048	0.262	12.8	100*
		1.862
	60 Minutes	2.006	2.022	0.023	1.1
		2.038
Positive Control	3 Minutes	0.095	0.080	0.022	na	3.9
		0.064
	60 Minutes	0.096	0.076	0.029	na	3.7
		0.055
Test Item	3 Minutes	1.953	1.879	0.105	5.6	91.7
		1.805
	60 Minutes	1.755	1.733	0.032	1.8	85.7
		1.710

OD: Optical density

*: The mean percentage viability of the negative control tissue is set at 100%

na: Not applicable

Interpretation of results:

other: not corrosive

Conclusions:

There is regulatory acceptance in the EU that a substance can be considered corrosive (Skin Corrosive Cat. 1, 1A, 1B/C) based on a positive result in the Reconstructed human epidermis test method (in vitro skin corrosion). A negative in vitro corrosivity response is not conclusive with respect to non-classification or classification as a skin irritant and therefore requires further evaluation and/or data generation.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 - 25 Jan 2021

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)

Version / remarks:

adopted in 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

adopted in 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKIN™

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™
- Tissue batch number: 21-EKIN-003
- Maintenance Medium lot number: 21-MAIN3-003
- Assay Medium lot number: 21-ESSC-003
- Date of quality control: 19 Jan 2021
- Date of initiation of testing: 19 Jan 2021

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: rinsing was achieved by filling and emptying each tissue insert for approx. 40 s using a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS)
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm
- Filter bandwidth: 10 nm
- Linear OD range of spectrophotometer: yes, linearity plot provided in report

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: The barrier function was assessed by determination of the concentration of Sodium dodecyl sulphate (SDS) required to reduce tissue viability by 50% (IC50). The IC50 value was determined to be 1.8 mg/mL (acceptance criteria: 1.5 - 3.0 mg/mL).
- Morphology: The morphology of the tissues was assessed using a HES stained paraffin section. The evaluation verified a multi-layered, highly differentiated epidermis consisting of 7.5 cell layers (acceptance criteria: >= 4 cell layers).
- Contamination: The cells used to produce the EPISKIN™ tissue were screened for the presence of bacteria, fungus and mycoplasma. No contamination was detected.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Water-killed tissues
- Procedure used to prepare the killed tissues: Water-killed tissues were prepared by placing untreated EPISKIN™ tissues in sterile distilled water. The tissues were incubated at 37 °C, 5% CO2 in air for a minimum of 48 h.
- N. of replicates: triplicate s

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive or irritant to skin if the viability after 15 min exposure followed by a 42 h post-exposure incubation period is less or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15 min exposure followed by a 42 h post-exposure incubation period is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 26.3 µL/cm2

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5% (w/v) aqueous solution

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

triplicates for each treatment and control group

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 min exposure

Value:

97.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: Since a pre-test for direct MTT reduction was inconclusive due to the intrinsic colour of the test substance, an additional test with water-killed was performed.
- Colour interference with MTT: The test substance did not change the colour when mixed with sterile water and thus passed the colour interference pre-test. The use of colour correction tissues was not necessary.
- Inflammatory mediator determination: It was considered unnecessary to perform IL-1α analysis on the assay medium, retained, as the results of the MTT test were unequivocal.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD of the tissue replicates treated with the negative control was ≥ 0.6 and ≤ 1.5, and the SD was ≤ 18% for every exposure time (values obtained: OD: 0.833, SD: 6.0%). The negative control acceptance criteria were satisfied.
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control for 15 min was ≤ 40% and the SD was ≤ 18% (values obtained: viability: 3%, SD: 0.8%). The positive control acceptance criteria were satisfied.
- Acceptance criteria met for variability between replicate measurements: The SD calculated from individual percentage tissue viabilities of the three identically treated tissues was ≤ 18% (value obtained: 7.3%). The test item acceptance criterion was satisfied.

Detailed results

Table 1: Mean OD₅₇₀ Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD570 of tissues	Mean OD570 of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.853	0.833	0.050	102.4	100*	6.0
	0.870			104.4
	0.776			93.2
Positive Control Item	0.033	0.025	0.007	4.0	3.0	0.8
	0.021			2.5
	0.022			2.6
Test Item	0.858	0.812	0.061	103.0	97.5	7.3
	0.743			89.2
	0.836			100.4

 

OD: Optical Density

SD: Standard deviation

*: The mean viability of the negative control tissues is set at 100%

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.

Conclusions:

Under the conditions of the conducted test, the test substance did not possess irritating properties towards reconstructed human epidermis tissue in the EPISKIN™ model.

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:

19 - 21 Jan 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted in 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: EpiOcular™

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The EpiOcular™ EIT was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. The EpiOcular™ EIT was endorsed as an in vitro test that can be used to identify those chemicals not requiring classification and labelling for eye irritation or serious eye damage in accordance with UN GHS (No Category).
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. The EpiOcular™ tissue mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium.
- RhCE tissue or hCE cell construct used, including batch number: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT), Model; 02 October 2017, Lot No. 30692 (Assay Medium Lot No.: 011821ISA)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

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

Duration of treatment / exposure:

30 ± 2 min at 37 °C, 5% CO2 followed by 12 ± 2 min post-exposure immersion

Duration of post- treatment incubation (in vitro):

120 ±15 min at 37 °C, 5% CO2

Number of animals or in vitro replicates:

duplicates for each treatment and control group

Details on study design:

- Details of the test procedure used: The EpiOcular™ Eye Irritation Test (EIT) consists of a topical exposure of the neat test item to a human reconstructed cornea model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The ability of the test substance to directly reduce MTT and to form a blue/purple reaction product was assessed in a pre-experiment. The test substance was shown to directly reduce MTT in the direct MTT reduction test. Therefore, an additional test with freeze-killed tissues was performed.
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm, 10 nm filter band pass, linearity plot provided in report
- Description of the method used to quantify MTT formazan: The absorbance (OD570) was measured with a plate reader (Labtech LT-4500 microplate reader).
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The test substance is considered to be not irritating to eye if the tissue viability after 30 min exposure, 12 min post-exposure immersion and 120 min post-exposure incubation is > 60% relative to the negative control treated tissue.
- Acceptable variability between tissue replicates for positive and negative controls : The results are acceptable if the negative control OD is > 0.8 and < 2.5 and if the mean relative viability of the positive control is below 50% of the negative control viability.
- Acceptable variability between tissue replicates for the test chemical: The results are acceptable if the difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the killed controls (test items and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Irritation parameter:

percent tissue viability 

Run / experiment:

30 min exposure

Value:

89.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: Since a pre-test for direct MTT reduction was inconclusive due to the intrinsic colour of the test substance, an additional test with freeze-killed was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred in the main test. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results.
- Colour interference with MTT: The test substance did not change the colour when mixed with pure isopropanol and thus passed the colour interference pre-test. The use of colour correction tissues was not necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD of the tissue replicates treated with the negative control was ≥ 0.8 and ≤ 2.8 for every exposure time (value obtained: 2.220). The negative control acceptance criterion was satisfied.
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control was < 50% compared to the negative control (value obtained: 42.9%). The positive control acceptance criterion was satisfied.
- Acceptance criteria met for variability between replicate measurements: The difference in viability between the two relating tissues in each treatment group was < 20%. This acceptance criterion was satisfied.

Detailed results

Table 1: Mean OD₅₇₀ Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD570 of tissues	Mean OD570 of duplicate tissues	Individual tissue viability (%)	Relative mean viability (%)	Difference in viability (%)
Negative Control Item	2.279	2.220	102.7	100*	5.4
	2.161		97.3
Positive Control Item	0.894	0.951	40.3	42.9	5.1
	1.007		45.4
Test Item	1.901	1.993	85.6	89.8	8.3
	2.085		93.9

 

OD: Optical Density

*: The mean viability of the negative control tissues is set at 100%

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.

Conclusions:

Under the conditions of the conducted test, the test substance did not present irritating properties towards human-derived epidermal keratinocytes in the EpiOcular™ model.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro skin corrosion

The skin corrosive properties of fatty acids, C6-18 (branched and linear) alkyl and hydrocarbons, C10-18, n-alkanes, branched alkanes, cycloalkanes, aromatics (EC No. 942-654-8) have been investigated in a study using the EpiDerm™ Human Skin Model according to OECD guideline 431 under GLP conditions (Covance, 2020). Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT to formazan by viable cells in the test item treated tissues relative to the corresponding negative control.  Viable cells are able to reduce MTT to formazan whereas non-viable cells cannot. Duplicate tissues were treated with the negative control (sterile water), positive control (8 N KOH) and test item for exposure periods of 3 and 60 min. At the end of the exposure period the control items and test item were rinsed from the tissues before each tissue was taken for MTT loading. After MTT-loading the tissues were placed into 2 mL of isopropanol for formazan extraction. At the end of the formazan extraction period triplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96 well plate. The optical density (OD) was measured at 570 nm (OD₅₇₀). The mean percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues) of the test substance were 91.7% and 85.7 for the 3 min and 60 min exposure period, respectively. All criteria required for acceptance of results in the test were satisfied, incl. results of the positive controls. Under the conditions of this test, fatty acids, C6-18 (branched and linear) alkyl and hydrocarbons, C10-18, n-alkanes, branched alkanes, cycloalkanes, aromatics (EC No. 942-654-8) was considered to be non-corrosive to the skin. Since a negative in vitro corrosivity response is not conclusive with respect to non-classification or classification as a skin irritant, further evaluation and/or data generation was considered necessary.

 

In vitro skin irritation

After a skin corrosive potential of fatty acids, C6-18 (branched and linear) alkyl and hydrocarbons, C10-18, n-alkanes, branched alkanes, cycloalkanes, aromatics (EC No. 942-654-8) was excluded, the skin irritating properties were assessed using the EPISKIN™ reconstructed human epidermis model according to OECD guideline 439 under GLP conditions (Covance, 2021b). The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item in the colorimetric MTT reduction assay. Triplicate tissues were treated with the test item for 15 min. At the end of the exposure period each tissue was rinsed before incubating for 42 h. An assessment of the test item’s capability to directly reduce MTT was inconclusive due to the intrinsic color of the test item. Therefore, an additional procedure using water-killed tissues was performed. 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-labeled 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 µL samples were transferred to the appropriate wells of a pre labeled 96 well plate. The optical density was measured at 570 nm. The relative mean viability of the test item treated tissues was 97.5% after the 15 min exposure period and 42 h post-exposure incubation period. All criteria required for acceptance of results in the test were satisfied. In this study and under the experimental conditions reported, fatty acids, C6-18 (branched and linear) alkyl and hydrocarbons, C10-18, n-alkanes, branched alkanes, cycloalkanes, aromatics (EC No. 942-654-8) was evaluated to be non-irritant to the skin.

 

In vitro eye irritation

The potential of fatty acids, C6-18 (branched and linear) alkyl and hydrocarbons, C10-18, n-alkanes, branched alkanes, cycloalkanes, aromatics (EC No. 942-654-8) to induce eye irritation was assessed using the reconstructed human Cornea-like Epithelium (RhCE) test method (EpiOcular™ Eye Irritation Test (EIT)) according to the OECD guideline 492 under GLP conditions (Covance, 2021c). Duplicate tissues were treated with the test item for an exposure period of 30 min.  At the end of the exposure period each tissue was rinsed before incubating for 120 min. As the test item had been shown to directly reduce MTT additional non-viable, freeze-killed, tissues were incorporated into the testing for correction purposes. At the end of the post exposure incubation period each tissue was taken for MTT-loading. After MTT-loading the tissues was placed into 2 mL of isopropanol for formazan extraction. At the end of the formazan extraction period each well was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96 well plate.  The optical density (OD) was measured at 570 nm (OD₅₇₀). The relative mean viability of the test item treated tissues was 89.8%. All criteria required for acceptance of results in the test were satisfied. Fatty acids, C6-18 (branched and linear) alkyl and hydrocarbons, C10-18, n-alkanes, branched alkanes, cycloalkanes, aromatics (EC No. 942-654-8) was found to be non-irritant to the eye under the conditions of this test.

Justification for classification or non-classification

The available data on skin and eye irritation / corrosion of fatty acids, C6-18 (branched and linear) alkyl and hydrocarbons, C10-18, n-alkanes, branched alkanes, cycloalkanes, aromatics (EC No. 942-654-8) do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP) and are therefore conclusive but not sufficient for classification.