Fatty acids, tall-oil, ethoxylated

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 May 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

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008

Test guideline 4

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

6 July 2012

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 * (information provided by the sponsor)

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

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

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200
- Information test system: The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs® 10 mm Ø) and commercially available as kits (EpiDerm™ 200) containing 24 tissues on shipping agarose.
- Tissue lot number: 25812
- Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

MATERIALS
- Tissue for MTT-reduction control: EPI-200 tissue that is killed by freezing at –20°C
- Assay medium, irritation test: EPI-100-NMM assay medium
- Assay medium, MTT diluent: Dulbecco's modified eagle's medium (DMEM) based medium
- 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), 1.0 mg / mL MTT diluent
- Extracting agent: Isopropanol p.a.

EXPERIMENTAL PROCUDURE
MESH COMPATIBILITY
- For liquid test substances, a nylon mesh can be used as a spreading support. In order to exclude a reaction of the test substance with the mesh, the compatibility of the test substance with the nylon mesh was checked in a pretest.
- The test substance and the mesh are brought together on a slide and the reaction was observed after a 60-minute exposure by using a microscope. An interaction between test substance and the mesh was not noticed.
- However, it was judged that the use of a mesh was not necessary for the test substance.

DIRECT MTT REDUCTION
- 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 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 concurrently tested. If the color of the MTT solution 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.
- In case of direct MTT reduction, three freeze-killed control tissue (KC) were treated with each the test article and the negative control in the same way as the main experiment.

BASIC PROCEDURE
PREINCUBATION
- On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 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 for 18 ± 3 hours.

EXPOSURE
- Number of replicate tissues: Three tissues were treated with the test substance, the positive control (PC) and the negative control (NC), respectively. In addition, three killed control (KC) tissues were used for the test substance and the NC, respectively, to detect direct MTT reduction.
- Thirty microliters (30 µL) undiluted liquid test substance were applied by using a pipette. Control tissues were concurrently treated with 30 µL sterile PBS (NC, NC KC) or with 30 µL 5% SDS (PC) or test substance (KC). A nylon mesh was carefully placed onto the tissue surface of the NC, NC KC and PC afterwards.
- The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.

REMOVAL OF TEST MATERIAL AND CONTROLS
- The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates pre-filled with 0.9 mL fresh medium. When all tissues were rinsed the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were placed into the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours, the tissues were transferred into new 6-well plates pre-filled with 0.9 mL fresh medium and placed into the incubator for an additional 18 ± 2-hour post-incubation period.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- 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 incubation of the tissues in isopropanol. 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 quotient 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 is rritant.
- Calculation of individual and mean optical densities: The individual tissue OD570 is 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 three tissues treated in the same way is 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 tissue 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 quotient of the mean OD570 (or mean OD570 KC corrected, if applicable) divided by the respective OD570 NC value in percent for each exposure time.

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 value following exposure to Triton X-100 (1%) for each EpiDermTM 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 guidelines: Lower acceptance limit: ET50 = 4.0 hours; Upper acceptance limit: ET50 = 8.7 hours
- Acceptance criteria for the negative control (NC): The absolute OD570 of the negative control 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 2.8.
- Acceptance criteria for the positive control (PC): 5% SDS is used as PC and reflects the sensitivity of the tissues used in the test conditions. A viability of ≤ 20% is acceptable.
- Acceptance criteria for the variability of the tissues: For every treatment, three tissues are treated in parallel. The inter-tissue variability is considered to be acceptable if the SD of % viability is ≤ 18%.
- Acceptance criteria for the killed controls (KC): The OD570 of the tissues for the KC of the NC should be ≤ 0.35. The OD570 value for direct MTT reduction of a test substance should be ≤ 30% of the OD570 of the NC.

EVALUATION OF RESULTS
- The evaluation of the in vitro skin irritation potential of the test substance is based on the results of the Skin Corrosion Test (SCT) and the Skin Irritation Test (SIT).
- If a test substance is not tested in both assays or an inconclusive result is obtained in one of the studies the test strategy may still lead to an overall evaluation when the result of a single study gives a clear prediction. However, if both studies are inconclusive or contradictory results are obtained a test evaluation may not be possible.
- Irritant potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile PBS. A chemical is considered as "irritant" if the mean relative tissue viability with a test material is less than or equal to 50%. (see also table 1 in ‘Any other information on materials and methods incl. tables’)
- A single test composed of at least three 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.

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 system.

Amount/concentration applied:

30 µL

Duration of treatment / exposure:

1 hour ( 25 minutes at room temperature and for 35 minutes in the incubator)

Duration of post-treatment incubation (if applicable):

42 ± 4 hours

Number of replicates:

three replicates

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

100.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

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. The results of the KC tissues indicate an increased MTT reduction (mean viability 0.3 % of NC). Thus for the test substance, the final mean viability is given after KC correction.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met