Fatty acids, tall-oil, reaction products with pentaethylenehexamine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

In vitro eye irritation EpiOcular assay

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012/13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

MatTek Corporation, Ashland, MA 01721, USA: EpiOcularTM human cell construct: Procedure details, Version 3.1 a of February 10, 2010.

Harbell J.W. et al. (2009): COLIPA Program on Optimization of Existing ln Vitra Eye Irritation Assays for Entry into Formal Validation: Technology Transfer and Intra/Inter Labaratory Evaluation of EpiOcular Assay for Chemicals. Poster # 378, Society of Toxicology, March 2009.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, tall-oil, reaction products with pentaethylenehexamine

EC Number:

288-927-9

EC Name:

Fatty acids, tall-oil, reaction products with pentaethylenehexamine

Cas Number:

85940-40-5

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

Fatty acids, tall-oil, reaction products with pentaethylenehexamine

Test material form:

liquid: viscous

Test animals / tissue source

Species:

other: EpiOcular™ OCL-200 kit

Strain:

other: 24 OCL-200 tissues (reconstructed cornea): surface 0.6 cm2 cultured in Millicells® diameter 1 cm, MatTek Corp., Ashland MA, USA

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability : The EpiOcularTM model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinozytes used to model the human corneal epithelium. The EpiOcularTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
- Tissue for MTT reduction control: OCL-200 tissue that is killed by freezing at -20°C

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

other: MTT reduction control (KC): de-ionized water or test substance

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): undiluted test substance

Duration of treatment / exposure:

30 min at 37 °C

Duration of post- treatment incubation (in vitro):

2 h at 37 °C

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used : see above
- Tissue construct used, including batch number : see above, batch number not specified
- Doses of test chemical and control substances used : 50 µL
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable) : Exposure - 30 min at 37 °C, post incubation - 2 h at 37 °C
- Description of any modifications to the test procedure : not applicable
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable) : see above
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) : wavelength 570 nm, no further information available
- Description of the method used to quantify MTT formazan : The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model : see below
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria : see table 2
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals : Based on the historical data, a profound experience with the assay is assumed
- Positive and negative control means and acceptance ranges based on historical data : see table 2
- Acceptable variability between tissue replicates for positive and negative controls : Tissue viability is acceptable if the mean OD570 of the NC is ≥ 1.0. The mean OD570 of the NC
should not exceed 2.5. Methyl acetate used as PC usually leads to a tissue viability of approx. 25%. A viability of < 50% is acceptable.
- Acceptable variability between tissue replicates for the test chemical: A variability between the tissues is considered to be acceptable if the difference of the viability is ≤ 20%.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

Mean tissue viability

Value:

5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not reported

DEMONSTRATION OF TECHNICAL PROFICIENCY: Based on the historical data, a profound experience with the assay is assumed

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values: see table 2

Any other information on results incl. tables

Table 1: Findings

Test substance		tissue 1	tissue 2	mean KC	mean*	inter-tissue variability [%]
NC	mean OD570	2.101	2.106	0.047	2.104
	viability [% of NC]	99.9	100.1	-	100	0.3
Test substance	mean OD570	0.941	0.759	0.790	0.107
	viability [% of NC]	44.7	36.1	35.3	5	8.6
PC	mean OD570	0.593	0.494	-	0.544
	viability [% of NC]	28.2	23.5	-	26	4.7

*For the test substance the mean is given after KC-correction.

The MTT reduction control (KC) showed high values of the test substance (mean formazan production of the KC: 35%). Thus the acceptance criterion of ≤ 30% of the NC was not met. However, as all other acceptance criteria were met in the test and because the test substance would have been evaluated as irritant even without KC-correction, the test was considered valid despite this deviation.

Table 2: Historical control data

Historical Range of NC
OD570
Protocol	Historical Period	Mean OD	SD	Mean + 2 SD	Mean - 2 SD
Protocol for liquids	Apr 2010 - Aug 2012	1.532	0.213	1.96	1.11
Protocol for solids	Apr 2010 - Aug 2012	1.35	0.168	1.69	1.01

Historical Range of PC
OD570
Protocol	Historical Period	Mean OD	SD	Mean + 2 SD	Mean - 2 SD
Protocol for liquids	Apr 2010 - Aug 2012	0.390	0.151	0.69	0.09
Protocol for solids	Apr 2010 - Aug 2012	0.284	0.113	0.51	0.06

Viability (%)
Protocol	Historical Period	Mean OD	SD	Mean + 2 SD	Mean - 2 SD
Protocol for liquids	Apr 2010 - Aug 2012	24.94	6.99	38.91	10.96
Protocol for solids	Apr 2010 - Aug 2012	21.01	7.09	35.20	6.83

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Based on the observed results and applying the evaluation criteria it was concluded, that the test item shows an eye irritation potential in the EpiOcular™ eye irritation test under the test conditions chosen.