Fatty acids, tall-oil, ethoxylated

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Jun 2017 to 22 Aug 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

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:

28 July 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, tall-oil, ethoxylated

EC Number:

500-150-1

EC Name:

Fatty acids, tall-oil, ethoxylated

Cas Number:

61791-00-2

Molecular formula:

C(18-50)H(34-98)O(3-8)

IUPAC Name:

Fatty acids, tall-oil, ethoxylated

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of the test substance (as cited in study report): Emulgane 1729
- Batch identification: 0014857532
- Purity: 100 % UVCB
- Homogeneity: The test substance was homogeneous by visual inspection.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.

OTHER SPECIFICS
- pH value: Ca. 6 (undiluted test substance determined in the lab prior to start of the GLP study)
- Physical state / color: Liquid / yellowish, clear

Test animals / tissue source

Species:

other: reconstructed human cornea model

Details on test animals or tissues and environmental conditions:

- Description of the cell system used: The EpiOcularTM model (OCL-200) is a three-dimensional, non-keratinized tissue construct composed of normal human-derived, epidermal keratinocytes used to model the human corneal epithelium. The EpiOcularTM tissues (surface 0.6 cm²) are cultured on cell culture inserts (MILLICELLs, 10 mm in diameter) and are commercially available as kits (EpiOcular™ 200) containing 24 tissues on shipping agarose.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

other: MTT reduction control (KC): Deionized water, sterile, or test substance

Amount / concentration applied:

50 µL

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

2 replicates

Details on study design:

TEST SYSTEM
- Tissue model: OCL-200
- Tissue Lot Number: 23789
- Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

MATERIALS
- Tissue for MTT reduction control: OCL-200 tissue that is killed by freezing at –20°C
- Assay medium: OCL-200-ASY assay medium
- MTT diluent: Dulbecco's modified eagle's medium (DMEM) based medium
- Pretreatment / wash buffer: Dulbecco's phosphate buffered saline (PBS), w/o Ca2+, Mg2+
- Detection agent: 3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide (MTT)
- Extracting agent: Isopropanol p.a.

EXPERIMENTAL PROCEDURE
DIRECT MTT REDUCTION
- In order to assess the ability of the test material to directly reduce MTT, a pretest (experimental conduct in accordance with GLP, but without a GLP status) was performed. The test substance was added to 0.9 mL MTT solution. The mixture was incubated in the dark at about 37°C for 3 hours. A negative control (deionized water) was tested concurrently. If the MTT solution color or in case of water-insoluble test substances the border to the water-phase turned blue / purple the test substance was presumed to directly reduce MTT.
- The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results.
- In case of direct MTT reduction, two freeze-killed control tissues were treated with the test article and the negative control each in the same way.

BASIC PROCEDURE
- Two tissues were treated with each the test substance, the positive control (PC) and the negative control (NC).
- Two killed tissues were used for each the test substance and the NC to detect direct MTT reduction.
- Pre-incubation of the tissues: On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour, the pre-incubation medium was replaced by fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours.
- Pretreatment of the tissues: After pre-incubation, the tissues were pretreated with 20 µL PBS to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.

APPLICATION OF THE TEST SUBSTANCE
- By using a pipette, fifty microliters (50 µL) undiluted liquid test substance were applied covering the whole tissue surface.
- Control tissues were concurrently applied with 50 µL sterile deionized water (NC, NC freeze-killed control tissues (KC)) or with 50 µL methyl acetate (PC) or test substance (KC).
- After application, the tissues were placed into the incubator until the total exposure time of 30 minutes was completed.

REMOVAL OF THE TEST SUBSTANCE AND POSTINCUBATION PERIOD
- In order to remove the test substance, the tissues were washed with sterile PBS. For this purpose, the tissues were immersed and swiveled three times in each of three beakers filled with PBS. Washed tissues were immediately immersed into 12-well plates pre-filled with 5 mL/well pre-warmed medium (post-soak immersion) to remove residual test substance.
- After 12 minutes of post-soak immersion, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium.
- Subsequently, the tissues were incubated at standard culture conditions for 2 hours (post-incubation period).

MTT INCUBATION
- After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours.
- After incubation, the tissues were washed with PBS to stop the MTT incubation.
- The formazan that was metabolically produced by the tissues was extracted by overnight incubation of the tissues in isopropanol at room temperature or by incubation for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was spectrophotometrically determined. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

DATA EVALUATION
- principle: The OD570 values determined for the various tissues are measures of their viability. The ratio of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether a test material was an irritant.
- Calculation of individual and mean optical densities: The OD570 value measured and corrected for each individual tissue was calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of two tissues treated in the same way was calculated.
- Application of measurements using killed control tissues: In case of direct MTT reduction by the test substance, the OD570 values measured in the freeze-killed control tissues (KC) will be used to correct the mean OD570 of the tissues treated with the test substance (mean OD570 KC-corrected). Since killed tissues might still have a residual enzyme activity that is able to produce some formazan net OD570 KC is calculated by subtracting the OD570 KC of the NC from the OD570 KC of the test substance. In case the net OD570 KC is greater than zero it is subtracted from the respective mean OD570 to result in the mean OD570 KC corrected. The mean OD570 KC-corrected represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance.
- Tissue viability: The quantification of tissue viability is presented as the ratio of the mean OD570 (or mean OD570 KC-corrected, if applicable) divided by the respective OD570 NC value in percent.

ACCEPTANCE CRITERIA
In case one of the acceptance criteria below was not met repetition of the test was considered.
- Barrier function and Quality control (QC): The supplier demonstrates that each batch of the model used meets the defined production release criteria. MatTek determines the ET50 (min) value following exposure to 100 µL of 0.3% Triton X-100 for each EpiOcular™ EIT (OCL-200) batch. The ET50 must fall within a range established based on a historical database of results. The following acceptability range (upper and lower limit) for the ET50 is established by the supplier as described in the cited OECD Guideline: Lower acceptance limit: ET50 = 12.2 min; Upper acceptance limit: ET50 = 37.5 min.
- Acceptance criteria for the NC: The absolute OD570 of the NC tissues in the MTT test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is > 0.8. The mean OD570 of the NC should not exceed 2.5.
- Acceptance criteria for the PC: Methyl acetate used as PC usually leads to a tissue viability of approx. 25%. A viability of < 50% is acceptable.
- Acceptance criteria for tissue variability: Two tissues were treated under the same conditions. A variability between the two tissues is considered to be acceptable if the relative difference of the viability is < 20%.
- Acceptance criteria for the KC: The OD570 of the killed control tissues treated as negative control should be less than or equal to 0.35. The value for direct MTT reduction of a test substance should be less than or equal to 30% of the NC.

EVALUATION OF RESULTS
- The irritation potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile water. A chemical is considered as "irritant" if the mean relative tissue viability with a test material is less than or equal to 60%.
- A single test composed of at least two tissue replicates should be sufficient for a test chemical when the result is unequivocal. However, in case of borderline results such as non-concordant replicate measurements and/or mean percent tissue viability equal to ± 5% of the cut-off value, a second test should be considered as well as a third one in case of discordant results between the first two tests. (see also table 1 in ‘Any other information on materials and methods incl. tables’)
- Combined assessment of eye irritating potential: The evaluation of the eye irritation potential of the test substance uses the results of the BCOP Test and the EpiOcular Test. If EpiOcular resulted in non-irritant in a bottom-up approach no BCOP test was conducted. In all other cases, both assays were necessary for a final assessment.

HISTORIC CONTROL DATA
Historic control values of negative and positive controls collected over an appropriate period are presented in table 2 in ‘Any other information on materials and methods incl. tables’. These data demonstrate the reproducibility of results and robustness of the procedures. They are used to derive suitable acceptance criteria for the test systems.

Results and discussion

In vitro

Results

Irritation parameter:

other: mean viability (%)

Value:

109.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Deionized wate

Positive controls validity:

valid

Remarks:

Neat methyl acetate

Other effects / acceptance of results:

- Acceptance criteria for the negative control are met.
- Acceptance criteria for the positive control are met.
- Acceptance criteria for tissue variability are met.
- Acceptance criteria for the freeze-killed control tissues are met.
- Due to the ability of the test substance to directly reduce MTT, KC tissues were applied in parallel. However, the results of the KC tissues did not indicate an increased MTT reduction. Thus, the KC was not used for viability calculation.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met