Soybean oil, epoxidized, reaction products with methanol

Endpoint summary

Description of key information

In-vitro and in-vivo studies on skin and eye irritation are available for the substance itself.

Skin irritation:
The substance does not show skin irritating potential in the acute dermal irritation/corrosion study in rabbits and in the EpiDerm™ skin corrosion/irritation test.

Eye-irritation:
The substance does not show eye irritation potential in the EpiOcular™ eye irritation test and does not cause serious eye damage in the Bovine Corneal Opacity and Permeability Test (BCOP Test). Furthermore, the substance does not show eye irritation properties in a non-guideline in vivo study in rabbits.

Information on the category member CAS 188831-96-1 support this conclusion.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation / corrosion, other

Remarks:

In vitro EpiDerm skin corrosion/irritation test

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006; In Vitro Skin Corrosion: Human Skin Model Test; Official Journal of the European Union, No. L 142.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Species:

other: EpiDerm™ 200 kit

Details on test animals or test system and environmental conditions:

TEST SYSTEM:
- Source: MatTek In Vitro Life Sciences Laboratories, Bratislava, Slovakia (corrosion test)

Three dimensional human epidermis model
The EpiDerm™ 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 EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37
- Humidity: 92-95%
- CO2: 5%

Vehicle:

unchanged (no vehicle)

Controls:

other: historical control values

Amount / concentration applied:

Single topical application of 50 μL (corrosion test) or 30 μL (irritation test)

Duration of treatment / exposure:

For the corrosion test two EpiDerm™ tissue samples were incubated with the test substance for 3 minutes and 1 hour, respectively. The irritation test was performed with three EpiDerm™ tissue samples, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period.

Details on study design:

EXPERIMENTAL PROCEDURE
Mesh compatibility: For liquid test substances a nylon mesh can be used as a spreading support. However, it was judged that this was not necessary for the test substance. Thus no pretest for mesh compatibility was performed.

Direct MTT reduction
To assess the ability of the test material to directly reduce MTT a pretest was performed as described below. The test substance was added to 0.9 mL
of the MTT solution. The mixture was incubated in the dark at about 37 °C for 55 to 65 minutes. A negative control (de-ionized 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 that direct MTT reduction occurred, one freeze-killed control tissue per exposure time was treated with, each, the test article and the negative control, in the same way as described in section “Basic procedure”, additionally.

Basic procedure
- Corrosion test: From the day of arrival in the laboratory, tissues were kept in the refrigerator. At least 1 hour but not more than 1.5 hours before test-substance application, tissues were transferred to 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. The preincubation medium was replaced with fresh medium immediately before application. Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator, as a rule) and test group (test material, negative control and positive control; 12 tissues per test) were used. Fifty microliter (50 μL) of the undiluted liquid test substance was applied using a pipette. Control tissues were concurrently applied with 50 μL of de-ionized water (negative control, NC) or with 50 μL of 8 n potassium hydroxide (positive control, PC). The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment. Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time were dosed and rinsed. The assay medium was then replaced by MTT solution and tissues were incubated for 3 hours. After incubation, tissues were washed with PBS and the formazan produced by the tissues was extracted with isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

- Irritation test: 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 preincubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours. Three tissues were treated with the test substance, the PC and NC, respectively. Thirty microliter (30 μL) of the undiluted liquid test substance was applied using a pipette. Control tissues were concurrently applied with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC). A nylon mesh was placed carefully onto the tissue surface afterwards. The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator. 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 incubated in 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 of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period. 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 determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

- Data evaluation: 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 or not a test material is corrosive or irritant. 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 two tissues (corrosion test) or three tissues (irritation test) treated in the same way is calculated. The quantification of tissue viability is presented as the quotient of the mean OD570 divided by the respective OD570 NC value in percent for each exposure time.

ACCEPTANCE CRITERIA
- Assay acceptance criterion 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 ≥ 1.0. The mean OD570 of the NC should not exceed 2.5.
- Acceptance criteria for the positive control (PC): Corrosion test: Potassium hydroxide as 8.0 normal ready made solution is used as positive reference. A 3-minute treatment with 8.0 n KOH usually reveals a mean relative tissue viability of ~20%. An assay is acceptable if mean relative tissue viability of the 3 min positive control is ≤ 30%. Irritation test: 5% SDS is used as PC and reflects the sensitivity of the tissues used in the test conditions. A viability of ≤ 20% is acceptable.
- Assay acceptance criterion for tissue variability: For every treatment, 2 tissues (corrosion test) or 3 tissues (irritation test) are treated in parallel. The inter-tissue variability is considered to be acceptable if the difference of the OD570 values of the two tissues is ≤ 0.3 (corrosion test) or if the SD of %-viability is ≤ 20 (irritation test).

EVALUATION OF RESULTS
Corrosive potential of the test materials is predicted from the mean relative tissue viabilities obtained after 3 min treatment compared to the negative control tissues concurrently treated with de-ionized water. A chemical is considered as "corrosive", if the mean relative tissue viability after 3 min treatment with a test material is decreased below 50%. In addition, those materials with a viability of ≥ 50% after 3 min treatment are considered as "corrosive" if the mean relative tissue viability after 1 hour treatment with a test material is decreased below 15%.
Mean tissue viability (% of negative control); Prediction
3 min: < 50; corrosive
3 min: ≥ 50 and 1 hour: < 15; corrosive
3 min: ≥ 50 and 1 hour: ≥ 15; non-corrosive
Although the method is not finally validated for categorizing the severity of corrosivity according to certain classification and labeling systems, it is suggested to use the most stringent category for test substances leading to viabilities below 50% after 3 min treatment.
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%.
Mean tissue viability (% of negative control); Prediction
≤ 50; irritant
> 50; non-irritant

Irritation parameter:

other: [% viability of NC]

Basis:

mean

Score:

94

Remarks on result:

other: corrosion, exposure: 3 min, mean OD570: 1.807

Irritation parameter:

other: viability [% of NC]

Basis:

mean

Score:

101

Remarks on result:

other: corrosion, exposure: 1 hour, mean OD570: 1.863

Irritation parameter:

other: viability [% of NC]

Basis:

mean

Score:

108

Remarks on result:

other: irritation, mean OD570: 2.161

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the observed results and applying the evaluation criteria it was
concluded, that the test substance does not show a skin irritation potential in the EpiDerm™ skin corrosion/irritation test under the test conditions chosen.

Executive summary:

The potential of the test substance to cause dermal corrosion/irritation was assessed by a single topical application of 50 μL (corrosion test) or 30 μL (irritation test) of the test substance to a reconstructed three dimensional
human epidermis model (EpiDerm™).
For the corrosion test two EpiDerm™ tissue samples were incubated with the test substance for 3 minutes and 1 hour,respectively. The irritation test was performed with three EpiDerm™ tissue samples, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period.
Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The EpiDerm™ skin corrosion/irritation test showed the following results:

The test substance is not able to reduce MTT directly.

Corrosion test:
The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 94%, and it was 101% after an exposure period of 1 hour.

Irritation test:
The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 108%.

Compound residues remained on the tissues after the washing procedure. However, this did not interfere with the MTT assay, as the test substance was not able to reduce MTT directly, which was demonstrated in a pretest.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Qualifier:

according to guideline

Guideline:

other: Japan MAFF Testing Guideline of 12 Nousan No. 8147, November 24, 2000

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

GLP compliance:

yes (incl. QA statement)

Remarks:

Bioassay Labor für biologische Analytik GmbH, 69120 Heidelberg

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: Approx. 8 - 9 months
- Weight at study initiation: 4.41 kg – 4.82 kg
- Housing: One rabbit per cage in stainless steel wire mesh cages with grating with shallow cage body; floor area: 4225 cm2. Wooden gnawing blocks
(Type KNH E-041); Abedd ® Lab. and Vet. Service GmbH Vienna, Austria were added as enrichment.
- Diet: STANRAB (P) SQC; SDS Special Diets Services, 67122 Altrip, Germany
- Water: Tap water ad libitum.
- Acclimation period: Acclimatization for at least 5 days before application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23
- Humidity (%): 30-70
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

other: Negative control: Untreated skin sites of the same animal.

Amount / concentration applied:

0.5 mL

Duration of treatment / exposure:

4 h

Observation period:

14 days

Number of animals:

3 males

Details on study design:

TEST SITE
- Area of exposure: 2.5 cm x 2.5 cm
- Type of patch: test patch (Idealbinde, Pfälzische Verbandstoff-Fabrik, Kaiserslautern) and Fixomull® stretch (adhesive fleece), Beiersdorf AG

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Lutrol® E 400 = Polyethylenglycol, BASF SE and Lutrol® / water (1 : 1).
- Time after start of exposure: end of the exposure period

READINGS:
- Immediately after removal of the patch, approx. 1, 24, 48 and 72 h after removal of the patch and then in weekly intervals maximally up to day 14.
- Illumination used for reading: Daylight tubes "Lumilux" (L 58W/860 PLUS ECO 25X1, Osram, Germany)

MORTALITY:
A check for any dead or moribund animal was made at least once each workday.

SCORING SYSTEM:
The evaluation of skin reactions was performed according to the quoted guidelines.
Erythema and eschar formation. Grading:

0) No erythema
1) Very slight erythema (barely perceptible)
2) Well- defined erythema
3) Moderate to severe erythema
4) Severe erythema (beet redness) to eschar formation preventing grading of erythema

Edema formation. Grading:

0) No edema
1) Very slight edema (barely perceptible)
2) Slight edema (edges of area well- defined by definite raising)
3) Moderate edema (raised approx. 1 mm)
4) Severe edema (raised more than 1 mm and extending beyond area of exposure)

Descriptions of any dermal findings not covered by this scale were recorded.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.7

Max. score:

4

Reversibility:

fully reversible within: 72h

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: No edema was observed at any of the readings

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: No edema was observed at any of the readings

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: No edema was observed at any of the readings

Irritant / corrosive response data:

In two animals slight erythema (grade 1), observed immediately and 1 hour after removal of the patch, increased to well-defined (grade 2) after 24 hours and persisted in one animal until the 72-hour reading. In this animal slight erythema was noted at study day 7 again, while in the other animal this grade was observed at hour 48 and 72. In one animal very slight erythema was observed immediately after removal of the patch and persisted until hour 48. The cutaneous reactions were reversible in all animals within 72 hours, 7 days or 14 days after removal of the patch. Mean scores over 24, 48 and 72 hours for each animal were 1.3, 2.0 and 0.7 for erythema and 0.0, 0.0 and 0.0 for edema.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non GLP, non-guideline, animal experimental study. Restrictions in reporting but otherwise adequate for assessment.

Qualifier:

no guideline followed

Principles of method if other than guideline:

A quantity of 100 µL of Cetiol R/animal was instilled once into the conjunctival sac of the right eyes of a group of 6 male New Zealand rabbits. The contact of the test substance with the outer contact areas of the eye was permanent. The assessment of the inflammatory symptoms on the cornea, iris and the conjunctivas was made 1, 6, 24, 48 and 72 hours after the instillation by means of the point scheme by Draize (J.W. Draize, Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. Ass. of Food and Drug Officials of the U.S., pp. 48-52 (1960)).

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

100 µL

Duration of treatment / exposure:

72 hours

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

6 males

Details on study design:

SCORING SYSTEM:
The assessment of the inflammatory symptoms on the cornea, iris and the conjunctivas was made 1, 6, 24, 48 and 72 hours after the instillation by means of the point scheme by Draize (J.W. Draize, Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. Ass. of Food and Drug Officials of the U.S., pp. 48-52 (1960)).

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

other: 1 and 6 hours

Score:

1.2

Reversibility:

fully reversible within: 24 hours

Remarks on result:

other: Score is a percentage of the maximum reaction possible.

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

other: 24 and 48 hours

Score:

0

Remarks on result:

other: Score is a percentage of the maximum reaction possible.

Irritant / corrosive response data:

While at the points in time mentioned no inflammatory symptoms could be observed on the cornea and iris, inflammatory symptoms of low degree in the form of slight reddening were observed on the conjunctivas of 3 animals, which had, however, entirely disappeared 24 hours after the treatment.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant non-guideline study, available as unpublished report, fully adequate for assessment.

Qualifier:

no guideline available

Principles of method if other than guideline:

There are no official national or international guidelines for the EpiOcularTM test yet; however, the study was performed according to the methods described in the following publications:
- MatTek Corporation, Ashland, MA 01721, USA: EpiOcularTM human cell construct: Procedure details, Version 3.1a of February 10, 2010.
- Harbell J.W. et al. (2009): COLIPA Program on Optimization of Existing In Vitro Eye Irritation Assays for Entry into Formal Validation: Technology Transfer and Intra/Inter Laboratory Evaluation of EpiOcular Assay for Chemicals. Poster # 378, Society of Toxicology, March 2009.
In addition the study follows the testing strategy for determination of eye irritation/corrosion as given in the following OECD guideline:
- OECD Guideline for Testing of Chemicals No. 405, April 24, 2002 (“Acute Eye Irritation/Corrosion”).

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Species:

other: EpiOcular™ OCL-200 kit

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- Source: MatTek Corp., Ashland MA, USA

Three dimensional human cornea model
The EpiOcular™ 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 EpiOcular™ 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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37

Vehicle:

unchanged (no vehicle)

Controls:

other: historical control values

Amount / concentration applied:

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

Duration of treatment / exposure:

Samples were incubated with the test substance for 30 minutes followed by a 2-hours post-incubation period.

Details on study design:

EXPERIMENTAL PROCEDURE
- Direct MTT reduction: To assess the ability of the test material to directly reduce MTT a pretest was performed as described below.
The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 55 to 65 minutes. A negative control (sterile 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 that direct MTT reduction occurred, two freeze-killed control tissues were treated with, each, the test article and the negative control, in the same way as described in section “Basic procedure”, additionally.
- Basic procedure: Two tissues were treated with the test substance, the PC and NC, respectively. There are two separate protocols for liquids and solids, differing in exposure time and postincubation period. Due to the physical condition of the test substance the protocol for solids was applied.
- 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 standard culture conditions for 16 – 24 hours (pre-incubation).
- Pretreatment of the tissues: After the pre-incubation the tissues were pre-treated with 20 μL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.
- Application of the test substance: Using a pipette, fifty microliter (50 μL) of the undiluted liquid test substance was applied covering the whole tissue surface. Control tissues were concurrently applied with 50 μL of sterile de-ionized water (NC) or with 50 μL of methyl acetate (PC). After application, the tissues were placed into the incubator until the total exposure time of 90 minutes (solids) was completed.
- Removal of the test substance and postincubation period: 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 prewarmed medium (post-soak immersion) in order 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 (postincubation 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 incubation of the tissues in isopropanol at room temperature overnight or for at least 2 hours on a plate shaker. 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.

- Data evaluation: 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 or not a test material is an irritant. 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 two tissues treated in the same way is calculated. The quantification of tissue viability is presented as the quotient of the mean OD570 divided by the respective OD570 NC value in percent.

ACCEPTANCE CRITERIA:
- Assay acceptance criterion 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 ≥ 1.0. 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.
- Assay acceptance criterion for tissue variability: Two tissues were treated under the same conditions. A variability between the tissues is considered to be acceptable if the difference of the viability ≤ 20%.

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 50%. At present no prediction is performed if the mean relative tissue viability with a test material is > 50 ≤ 60% as the cut off value is currently being evaluated to lie in this range.

Mean tissue viability
(% of negative control); Prediction
≤ 50; irritant
> 50 ≤ 60; no prediction
> 60; non-irritant

Irritation parameter:

other: Viability [% of NC]

Run / experiment:

Mean

Value:

100

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

Commission Regulation (EU) No. 1152/2010

Principles of method if other than guideline:

The study follows the testing strategy for determination of eye irritation/corrosion as given in the following OECD guideline:
- OECD Guideline for Testing of Chemicals No. 405, April 24, 2002 (“Acute Eye Irritation/Corrosion”)

In addition, the study was conducted according to the methods described in the following documents/publications:
• Gautheron P, Dukic M, Alix D and Sina JF (1992): Bovine Corneal Opacity and Permeability Test: An In Vitro Assay of Ocular Irritancy. Fundamental and Applied
Toxicology 18, 442-449.
• Sina JF, Galer DM, Sussman RS, Gautheron PD, Sargent EV, Leong B, Shah PV, Curren RD, and Miller K (1995): A collaborative evaluation of seven alternatives to the Draize eye irritation test using pharmaceutical intermediates. Fundam Appl Toxicol 26:20-31.
• INVITTOX (1999). Protocol 124: Bovine Corneal Opacity and Permeability Assay – SOP of Microbiological Associates Ltd. Ispra, Italy: European Centre for the Validation of Alternative Methods (ECVAM).
• ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods) Test Method Evaluation Report “In Vitro Ocular Toxicity Test Methods for Identifying Severe Irritants and Corrosives”, November 2006 (NIH Publication No.: 07-4517).
• Schrage A, Kolle SN, Rey Moreno MC, Norman K, Raabe H, Curren R, van Ravenzwaay B, and Landsiedel R (2011). The Bovine Corneal Opacity and Permeability Test in Routine Ocular Irritation Testing and its Improvement within the Limits of OECD Test Guideline 437. ATLA 39: 37–53.

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Species:

other: Isolated bovine cornea

Details on test animals or tissues and environmental conditions:

TEST SYSTEM:
- Source: The test system (target tissue) is the isolated bovine cornea. Bovine eyes are obtained as a by-product of freshly slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months).
- Supplier: Schlachthof Bensheim, Am Schlachthof 7-9, 64625 Bensheim

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

750 μL of the undiluted liquid test substance

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

- 2 hours post-exposure measurements
- 90 ± 5 min determination of permeability

Number of animals or in vitro replicates:

3 corneas per treatment group

Details on study design:

- Preparation of the bovine corneas and measurement of initial corneal opacity: Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in specially designed corneal holders that consists of interior and posterior chambers. Both chambers were filled to excess with prewarmed Eagles’s MEM (without phenol red) and then equilibrated in a vertical position at about 32°C for at least 1 hour. After the equilibration period the medium in both chambers was replaced with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that show macroscopic tissue damage or an opacity value < 512 opacity units were discarded. Corneas with opacity values close to the median value of all corneas were selected as negative control (NC). The remaining corneas were then distributed into treatment and positive control groups. Each corneal holder was uniquely identified with a number on the chambers.
- Application of the test substance and washing: Each treatment group (test substance, NC and PC) consisted of 3 corneas. Before application the medium in the anterior chamber was removed using a syringe. 750 μL of the undiluted liquid test substance was applied into the anterior chamber using a pipette. Control tissues were concurrently applied into the anterior chamber with 750 μL of de-ionized water (negative control, NC) or with 750 μL of 1% (w/v) solution of sodium hydroxide in deionized water (positive control, PC) using a pipette. The corneas were incubated in a horizontal position at about 32°C for approximately 10 minutes (liquids and surfactants). The NC and PC were then removed from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red). Because the test substance could not be removed using a syringe, the epithelium was rinsed with the open chamber method.
- Post-exposure incubation for liquid test substances and surfactants: The corneas were incubated for further 2 hours at about 32°C. After the incubation period the medium was removed and both chambers were then refilled with fresh Eagle’s MEM.
- Measurement of final corneal opacity: Before measurement, each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.
- Determination of permeability: For determination of permeability the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (4 mg/mL for liquid test substances and surfactants) and incubated for 90 ± 5 min in a horizontal position at about 32°C. The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was measured spectrophotometrically. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined.

DATA EVALUATION:
The mean corneal opacity and permeability values of each treatment group were used to calculate an In Vitro Irritancy Score (IVIS). The measured data of the opacitometer and spectrophotometer were copied electronically into EXCEL data spreadsheets which carried out the necessary calculations.
- Calculation of the corneal opacity value: First, the opacity was calculated using the opacitometer specific algorithm, with a and b device specific and with Io being the illuminance (lux) through the empty corneal holder with windows and liquid and I being the illuminance (lux) through the holder with the cornea; opacity value = a * Io/I + b. Then the opacity change per cornea was calculated by subtracting the initial from the final opacity; opacity change per cornea = final opacity - initial opacity. Subsequently, the corrected opacity change was calculated by subtracting the mean opacity change of the negative control; corrected opacity change = opacity change - mean opacity change of NC. Finally, the mean opacity value for each test substance could be determined as the mean of all corrected opacity changes per treatment group; mean opacity value = mean of all corrected opacity changes per group.
- Calculation of permeability value: First, the OD490 value was calculated by subtracting the mean blank OD490 (blank = Eagle´s MEM w/o phenol red) from the OD490 of each cornea; OD490 value = OD490 - mean blank OD490. If the OD490 value of the treated cornea was above 1.5, the OD490 of a 1:5 dilution was used to calculate the OD490 value; OD490 value = 5 * (OD490 of a 1:5 dilution - mean blank OD490). Subsequently, the corrected OD490 value was calculated by subtracting the mean OD490 value of the negative control; corrected OD490 value = OD490 value - mean OD490 value of NC. Finally, the mean OD490 value for each test substance could be determined as the mean of all corrected OD490 values per treatment group; mean OD490 value = mean of all corrected OD490 values per group.
- Calculation of the In Vitro Irritancy Score (IVIS): The IVIS could be calculated per treated cornea and finally the mean IVIS per treatment group ± standard deviation was determined: IVIS per cornea = corrected opacity change + 15 * corrected permeability OD change; IVIS per treatment group = mean opacity value + 15 * mean permeability OD value.

ACCEPTANCE CRITERIA:
In case one of the following acceptance criteria is not covered, repetition of the test was considered. A study is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean. The negative control responses should result in opacity and permeability values that are less than the established upper limits. Since the IVIS per treatment group is determined from the mean of three single corneas, the variability between the corneas treated per test substance should be acceptably low. If no clear prediction is possible, e.g. different predictions are obtained for single corneas, the test will be repeated.

EVALUATION OF RESULTS:
The following rules of assessment apply:
IVIS; Prediction
> 55; risk of serious damage to the eyes
≤ 55; no risk of serious damage to the eyes

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

1.1

Vehicle controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Permeability score

Run / experiment:

Mean

Value:

0.001

Vehicle controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

1.1

Vehicle controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

In a GLP compliant guideline study (OECD 404), 3 male New Zealand white rabbits were exposed to 0.5 mL of the undiluted test substance (Bioassay, 2013). The rabbits were exposed for 4h. After exposure, the effects of this exposure on the rabbit skin were checked after 1, 24, 48 and 72 hours. In two animals slight erythema (grade 1), observed immediately and 1 hour after removal of the patch, increased to well-defined (grade 2) after 24 hours and persisted in one animal until the 72-hour reading. In this animal slight erythema was noted at study day 7 again, while in the other animal this grade was observed at hour 48 and 72. In one animal very slight erythema was observed immediately after removal of the patch and persisted until hour 48. The cutaneous reactions were reversible in all animals within 72 hours, 7 days or 14 days after removal of the patch. Mean scores over 24, 48 and 72 hours for each animal were 1.3, 2.0 and 0.7 for erythema and 0.0, 0.0 and 0.0 for edema. Therefore, it was concluded that, based on this study, the test substance is not corrosive or irritating to the rabbit skin.

In addition, in a GLP compliant in vitro EpiDerm™ test (BASF SE, 2012) according to OECD guidelines 431 and 439, the potential of Soybean oil, epoxidized, reaction products with methanol to cause dermal corrosion/irritation was assessed by a single topical application of 50 μL (corrosion test) or 30 μL (irritation test) of the test substance to a reconstructed three dimensional human epidermis model. For the corrosion test two EpiDerm™ tissue samples were incubated with the test substance for 3 minutes and 1 hour, respectively. The irritation test was performed with three EpiDerm™ tissue samples, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The test substance is not able to reduce MTT directly. In the corrosion test, the mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 94%, and it was 101% after an exposure period of 1 hour. In the irritation test, the mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 108 %. Based on the observed results and applying the evaluation criteria it was concluded, that Soybean oil, epoxidized, reaction products with methanol does not show a skin irritation potential in the EpiDerm™ skin corrosion/irritation test under the test conditions chosen.

Eye irritation:

In a GLP compliant in vitro EpiOcular™ test (BASF SE, 2012), the potential of Soybean oil, epoxidized, reaction products with methanol to cause ocular irritation was assessed by a single topical application of 50 μL of the test substance to a reconstructed three dimensional human cornea model. Two EpiOcular™ tissue samples were incubated with the test substance for 30 minutes followed by a 2-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues was 100%. Based on the observed results and applying the evaluation criteria it was concluded, that Soybean oil, epoxidized, reaction products with methanol does not show an eye irritation potential in the EpiOcular™ eye irritation test under the test conditions chosen.

In a GLP compliant in vitro Bovine Corneal Opacity and Permeability Test (BASF SE, 2012) according to OECD guideline 437, the potential of Soybean oil, epoxidized, reaction products with methanol to cause serious damage to the eyes was assessed by a single topical application of 750 μL of a undiluted test-substance preparation to the epithelial surface of isolated bovine corneas. Three corneas were treated with the test substance for an exposure period of 10 minutes followed by a 2-hours post-incubation period. Corneal opacity was measured quantitatively as the amount of light transmission through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance relative to the control corneas. The mean opacity value was 1.1, the mean permeability value was 0.001 and the In Vitro Irritancy Score was 1.1. The negative control values were 2.0, 0.000 and 2.0 and the positive control values were 120.2, 3.963 and 179.7, respectively.

Based on the observed results and applying the evaluation criteria it was concluded, that Soybean oil, epoxidized, reaction products with methanol does not cause serious eye damage in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen. The test method according to the regulatory accepted protocol at the time of reporting does not allow for the evaluation of eye irritation. The result does not exclude an irritation potential of the test substance. For final assignment of a risk phrase at present, results from another study would be needed.

 

In addition, in a non-GLP, non guideline study (Henkel AG, 1983) 6 male New Zealand white rabbits were exposed to Cetiol R. A quantity of 100 µL of Cetiol R/animal was instilled once into the conjunctival sac of the right eye of a group of 6 male New Zealand rabbits. The contact of the test substance with the outer contact areas of the eye was permanent. The assessment of the inflammatory symptoms on the cornea, iris and the conjunctivas was made 1, 6, 24, 48 and 72 hours after the instillation by means of the point scheme by Draize (1960). While at the points in time mentioned no inflammatory symptoms could be observed on the cornea and iris, inflammatory symptoms of low degree in the form of slight reddening were observed on the conjunctivas of 3 animals, which had, however, entirely disappeared 24 hours after the treatment. Therefore, it was concluded that, based on this study, the test substance is not irritating to the rabbit eye.

Justification for classification or non-classification

The test substance does not have to be classified according to the classification and labelling of Directive 67/548/EEC and according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.