Amines, rosin

Endpoint summary

Description of key information

Skin corrosion:

In one key in vitro skin corrosion test using a human skin model, undiluted Rosin Amine 90 was topically applied on a human three-dimensional epidermal model for 3 minutes and 1 hour. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with Rosin Amine 90 compared to the negative control tissues was 72% and 48%, respectively. Because the mean relative tissue viability for Rosin Amine 90 was not below 50% after 3 minutes treatment and not below 15% after 1-hour treatment, Rosin Amine 90 is considered to be not corrosive.

 

Skin irritation:

In one key in vitro skin irritation test using the EPISKINTM reconstructed human epidermis model, Rosin Amine 90 was applied topically for a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The relative mean viability of the test item treated tissues was 3.9% The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 3.3%. 

Under the conditions of the study, the test item, Rosin amine 90 demonstrated the ability to cause a positive response in the EPISKIN™ Reconstructed Human Epidermis Model. However, this n vitro study does not allow the conclusion on whether the test item is EU CLP/UN GHS Category 1 or Category 2.

 

Eye irritation:

In one key study evaluating the eye hazard potential of Rosin Amine 90 using the Bovine Corneal Opacity and Permeability test (BCOP test), undiluted sample of Rosin Amine 90 was applied on the epithelium of the bovine cornea for 10 minutes After exposure the cornea was thoroughly rinsed to remove the test item and incubated for 2 hours with fresh medium followed by opacity measurement and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein.

The results demonstrated that Rosin Amine 90 induced serious eye damage through both endpoints, resulting in a mean in vitro irritancy score of 55 after 10 minutes of treatment and therefore can be included that it induced serious eye damage under the conditions of the test.

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:

21-25 September 2015

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)

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

GLP compliance:

yes

Remarks:

Except for the following: - Information about purity/composition of the test item was not available prior to completion of the study. - The quality environment in which the characterisation of the test item was performed was not known.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Rosin, Batch: 542700
- Appearance: Transparent pale amber viscous liquid
- Expiration date of the lot/batch: 31 March 2016
- Purity test date: Not indicated

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

Test system:

human skin model

Source species:

human

Cell type:

other: EPI-200, normal, human-derived epidermal keratinocytes

Cell source:

other: MatTek Corporation, Ashland MA, U.S.A.

Details on animal used as source of test system:

Not applicable

Justification for test system used:

Recommended test system in international guidelines (OECD and EC)

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION:
EpiDerm Skin Model (EPI-200, Lot no.: 22676 kit K and kit J). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to
form a multilayered, 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) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Environmental conditions:
- Prior to the assay, skin tissues were kept refrigerated from the day they were received.
- All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 58 - 90%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C
(actual range 36.3 - 37.3°C).
- Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. - Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door.
- Based on laboratory historical data these deviations are considered not to affect the study integrity.

APPLICATION OF TEST SUBSTANCE
- 1 hour before the start of the assay, tissues were transferred to 6-well plates containing 0.9 mL DMEM medium per well. The plates were incubated for approximately 1.5 hours at 37 ± 1°C
- The medium was replaced with fresh DMEM medium just before Rosin Amine 90 was applied. The test was performed on a total of 4 tissues per test item together with a negative control and positive control.
- Two tissues were used for a 3-minute exposure to Rosin Amine 90 and two for a 1-hour exposure. Fifty µl of the undiluted test item was added into the 6-well plates on top of the skin tissues.
- The remaining tissues were treated with 50 µl Milli-Q water (negative control) and with 50 µl 8N KOH (positive control), respectively.
- Following the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. Rinsed tissues were kept in 24 well plates on 300 µl DMEM medium until 6 tissues (= one application time) were dosed and rinsed.
 
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporatio.

CELL VIABILITY MEASURMENT / NUMBER OF INDEPENDENT TESTING RUNS
The DMEM medium was replaced by 300 µl MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
 
Test for the interference of the test item with the MTT endpoint:
A test item may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.

PREDICTION MODEL / DECISION CRITERIA:
- Justification for the selection of the cut-off point(s) if different than recommended in TG 430:
Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test item was classified according to remaining cell viability following exposure of the test item with either of the two exposure times.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- 50 µl of the undiluted test item was added into the 6-well plates on top of the skin tissues

NEGATIVE CONTROL
- 50 µl Milli-Q water

POSITIVE CONTROL
- th 50 µl 8N KOH

After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item. Rinsed tissues were kept in 24 well plates on
300 µl DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

Duration of treatment / exposure:

3-minute and 1-hour exposure

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 72

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

15%

Remarks on result:

other: not corrosive

Remarks:

3-minute application viability (percentage of control)

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 48

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

11%

Remarks on result:

other:

Remarks:

1-hr application viability (percentage of control)

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Rosin Amine 90 was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that Rosin Amine 90 did not interfere with the MTT endpoint.

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3-minute exposure to the positive control was 15%. The maximum inter-tissue variability in viability between two tissues treated identically was less than 23% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 13%. It was therefore concluded that the test system functioned properly.

ACCEPTANCE OF RESULTS:
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 3-minute exposure to the positive control should be ≤ 30%.
c) In the range of 20 – 100% viability, the maximum inter-tissue variability (in viability) is ≤ 30% between two tissues treated identically.
d) In the range of 20 – 100% viability, the maximum difference in percentage between the mean
viability of two tissues and one of the two tissues is ≤ 15%.

Interpretation of results:

other: Rosin Amine 90 is considered to be not corrosive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

It is concluded that this test is valid and that Rosin Amine 90 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in the report.

Executive summary:

The objective of this study was to evaluate the ability of the test substance, Rosin Amine 90, to induce skin corrosion when applied topically on a human three dimensional epidermal model. The study was conducted in accordance with OECG Guideline 431 and EC Commission Regulation No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: 'In vitro Skin Corrosion: Human Skin Model Test'.

The possible corrosive potential of Rosin Amine 90 was tested through topical application for 3 minutes and 1 hour. The test substance was applied undiluted (50 µL) on top of the skin tissue and then following the exposure period, the tissues were washed with phosphate buffered saline. Rinsed tissues were kept in 24 -well plates on 300 µl DMEM medium until 6 tissues were dosed and rinsed (6 tissues = 1 application time).

Rosin Amine 90 was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Since the solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that Rosin Amine 90 did not interfere with the MTT endpoint.

The positive control had a mean relative tissue viability of 15% after 3 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The acceptability criteria for the maximum inter-tissue variability in viability between two tissues treated identically and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues were met, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3 -minute and 1 -hour treatments with Rosin Amine 90 compared to the negative control tissues was 72% and 48% respectively. Because the mean relative tissue viability for Rosin Amine 90 was not below 50% after the 3 -minute treatment and not below 15% after the 1 -hour treatment Rosin Amine 90 is considered to be not corrosive.

Finally, it is concluded that this test is valid since the acceptability criteria were met and that Rosin Amine 90 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 17 January 2018 - Experimental completion date: 29 January 2018

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:

updated 28 July 2015

Deviations:

yes

Remarks:

Due to a technical issue, the Labtech LT-4500 microplate reader and LT-com analysis software were unavailable to perform the OD measurement . The study was repeated. This deviation did not affect the integrity or validity of the study.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Rosin amine 90, 20170314028
- Manufacturing Date: 17 March 2017
- Expiration date of the batch: 01 March 2019
- Purity test date: 100% (UVCB)
- Physical state/Appearance: amber viscous liquid


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark

Test Item Preparation: the test item was used undiluted as supplied

Test system:

human skin model

Remarks:

3.3.1 EPISKIN™ Reconstructed Human Epidermis Model Kit

Source species:

human

Cell type:

other: reconstructed human epidermis tissues

Cell source:

other: EpiSkin Laboratories, Lyon, France

Justification for test system used:

Following a successfully completed ECVAM validation study, the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.
The procedure followed is based on the recommended EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: 3.3.1 EPISKIN™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): EpiSkinTM Tissues (0.38cm2) lot number: 18-EKIN-004
- EpiSkinTM Tissues expiry date: 29 January 2018
- Date received: 23 January 2018
- Date of initiation of testing: 24 January 2018

The EPISKINTM Kit consisted of one EPISKIN™ plate containing 12 reconstructed human epidermis tissues (0.38 cm2), maintenance medium (used for tissue pre-conditioning and testing purposes) and assay medium (used for MTT assay).

The maintenance medium and assay medium were refrigerated at 1 to 10 ºC.
Immediately upon receipt of the EPISKIN™ at Envigo Research Limited, Shardlow, UK, the kit was inspected for the color of the agar medium used for transport and for acceptable pH.


TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
Pre-incubation (Day 0: Tissue Arrival) - 37 °C, 5% CO2 in air overnight
Main Test - 37 °C, 5% CO2 in air
- Temperature of post-treatment incubation (if applicable): 37 °C, 5% CO2 in air

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: at the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: none


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL MTT
- Incubation time: 3 hours at 37 °C, 5% CO2 in air
- Spectrophotometer: Labtech LT 4500 microplate reader
- Wavelength: 570 nm (without a reference filter)


NUMBER OF REPLICATE TISSUES: Triplicate tissues

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- the determination of skin irritation potential was performed in parallel on viable and water killed tissues
- Procedure used to prepare the killed tissues (if applicable): Water-killed tissues were prepared prior to the study by placing untreated EPISKINTM tissues (0.38 cm2) in a 12 well plate containing 2.0 mL of sterile distilled water in each well. The tissues were incubated at 37 C, 5% CO2 in air for a minimum of 48 hours. At the end of the incubation the water was discarded. Once killed the tissues were stored in a freezer (14 to 30 °C) for up to 6 months. Before use each tissue was thawed by placing in 2.0 mL of maintenance medium, warmed to approximately 37 oC, for approximately 1 hour at room temperature.
- N. of replicates : triplicates
- Method of calculation used: As the test item, Rosin amine 90 was shown to directly reduce MTT and water-killed tissues were employed, the results of the MTT assay were therefore corrected as given in section

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
1. Assessment of Direct Test Item Reduction of MTT
1A. Test for Direct MTT Reduction (Step 1)
1B. Determination of skin irritation potential on viable and water killed tissues (Step 2)
2. Assessment of Color Interference with the MTT endpoint
3. Pre-incubation (Day 0: Tissue Arrival)
4. Main Test
4A. Application of Test Item and Rinsing (Day 1)
4B. Post- exposure incubation
5. MTT Loading/Formazan Extraction (Day 3)
6. Absorbance/Optical Density Measurements (Day 6)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
-Classification of irritation potential is based upon relative mean tissue viability following the 15 Minute exposure period followed by the 42 Hour post exposure incubation period:
Relative mean tissue viability is ≤50% - Irritant (Category 1 or 2)
Relative mean tissue viability is >50% - Non-irritant (Not classified for irritation)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL (26.3 µL/cm2) of the test item was applied to the epidermis surface
- Concentration (if solution): undiluted Rosin amine 90

VEHICLE - not applicable

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL of Dulbecco’s Phosphate Buffered Saline (DPBS), was used.


POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL of Sodium dodecyl sulphate (SDS) T
- Concentration (if solution): the positive control item, SDS, was prepared as a 5% w/v aqueous solution. The positive control was formulated within 2 hours of being applied to the test system.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

triplicates

Irritation / corrosion parameter:

% tissue viability

Remarks:

Relative mean viability

Run / experiment:

3

Value:

ca. 3.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: positive response in the EPISKIN™ Reconstructed Human Epidermis Model.

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: not applicable
- Direct-MTT reduction: The MTT solution containing the test item turned purple which indicated that the test item directly reduced MTT. Therefore, the determination of skin irritation potential was performed in parallel on viable and water killed tissues.
- Colour interference with MTT: The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

ACCEPTANCE OF RESULTS:

Positive Control:
The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%.
The relative mean tissue viability for the positive control treated tissues was 4.6% relative to the negative control treated tissues and the standard deviation value of the viability was 0.6%. The positive control acceptance criteria were therefore satisfied.

Negative Control:
The assay establishes the acceptance criterion for an acceptable test if the mean OD570 for the negative control treated tissues is 0.6 and ≤1.5, and the SD value of the percentage viability is ≤18%. The mean OD570 for the negative control treated tissues was 0.772 and the standard deviation value of the viability was 3.3%. The negative control acceptance criteria were therefore satisfied.

Test Item:
The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%.
The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 3.3%. The test item acceptance criterion was therefore satisfied.

All values obtained for the negative and positive control groups were within the historical range of the testing laboratory. Historical control data is provided in Appendix 2, and comprises OD570 values for both control groups as well as relative mean viability values for positive control groups (please see appendix 2) .

The individual and mean OD570values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Appendix 1. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in Appendix 1. For Appendix 1, please see attached background material.

 

Classification of irritation potential is based upon relative mean tissue viability following the 15‑Minute exposure period followed by the 42‑Hour post‑exposure incubation period according to the following table:

Criteria forin vitro Interpretation	Prediction	EU CLP (Regulation (EC) No 1272/2008)	UN GHS
Relative mean tissue viability is ≤50%	Irritant	H314 or H315 Category 1 or 2	H314 or H315 Category 1 or 2
Relative mean tissue viability is >50%	Non-irritant	Not classified for irritation	Not classified or UN GHS Category 3 can not be determined

Interpretation of results:

other: positive response in the EPISKIN™ Reconstructed Human Epidermis Model

Conclusions:

Under the conditions of the study, the test item, Rosin amine 90 demonstrated the ability to cause a positive response in the EPISKIN™ Reconstructed Human Epidermis Model.
However, thisin vitro study does not allow the conclusion on whether the test item is EU CLP/UN GHS Category 1 or Category 2.

Executive summary:

The purpose of this in vitro study was to evaluate the skin irritation potential of the test item, Rosin amine 90(CAS number: 61790-47-4) using the EPISKIN^TMreconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours.

The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. 

To identify possible interference of the test item with MTT, the test item was checked for the ability to directly reduce MTT. The MTT solution containing the test item turned purple which indicated that the test item directly reduced MTT. Therefore, the determination of skin irritation potential was performed in parallel on viable and water killed tissues.

As the test item, Rosin amine 90 was shown to directly reduce MTT and water-killed tissues were employed, the results of the MTT assay were therefore

corrected.

Next, the assessment of color interference with the MTT endpoint was performed.

10 µL of test item, Rosin amine 90 was added to 90 µL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made. The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

For the main test, Triplicate tissues (0.38 cm²) were treated with the test item for an exposure period of 15 minutes. The relative mean viability of the test item treated tissues was 3.9% after a 15‑Minute exposure period and 42‑Hourpost‑exposure incubation period.

The test item, Rosin amine 90 is a viscous amber liquid. It was noted that the test item treated tissue culture surfaces remained sticky after the rinsing step at the end of the 15-Minute exposure period. Therefore, it was reasonable to assume that residual test item remained on the tissue culture surface throughout the 42-Hour post exposure incubation period.

It was considered unnecessary to perform IL-1aanalysis as the results of the MTT test were unequivocal.

Under the conditions of the study, the test item, Rosin amine 90 demonstrated the ability to cause a positive response in the EPISKIN™ Reconstructed Human Epidermis Model.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

25 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006

Version / remarks:

The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM).

Qualifier:

according to guideline

Guideline:

other: In Vitro Techniques in Toxicology Database (INVITTOX) study plan 127. Bovine Opacity and Permeability (BCOP) Assay, 2006

Qualifier:

according to guideline

Guideline:

other: Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Rosin amine 90
- Expiration date of the lot/batch: 31 March 2016
- Purity test date: No correction factor required
- pH (1% in water, indicative range) 7.2 – 8.5 (determined by WIL Research Europe)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
No correction was made for the purity/composition of the test item. The test item was tested neat.

OTHER SPECIFICS:
Negative control:
A negative control, physiological saline (Eurovet Animal Health, Bladel, The Netherlands) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.

Positive control:
10% (w/v) Benzalkonium Chloride (Merck KGaA, Darmstadt, Germany) [CAS Number 63449-41-2] solution prepared in physiological saline.

Species:

other: Bovine eyes

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the ey
- Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

PREPARATION OF CORNEAS
The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

CORNEA SELECTION AND OPACITY READING
- Following the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations will be performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany).
- The opacity of each cornea will be read against a cMEM filled chamber, and the initial opacity reading thus determined will be recorded.
- Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.
 

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µl of either the test substance, negative or positive control was introduced onto the epithelium of the cornea.

Duration of treatment / exposure:

10 ± 1 minutes at 32 ± 1°C

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

Not applicable

Number of animals or in vitro replicates:

Three corneas were selected at random for each treatment group.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C. The holders were slightly rotated, with the corneas maintained in a horizontal position, to
ensure uniform distribution of the control or the test item over the entire cornea.
- After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations was performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.
- After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Eagle’s Minimum Essential
Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM.
- Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was
performed. Each cornea was inspected visually for dissimilar opacity patterns.
- Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck Darmstadt, Germany) was evaluated.

SCORING SYSTEM:
- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
 - In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
 - Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.

TOOL USED TO ASSESS SCORE: opacitometer

Irritation parameter:

cornea opacity score

Value:

29.9

Negative controls validity:

valid

Remarks:

-0.6

Positive controls validity:

valid

Remarks:

97.1

Remarks on result:

positive indication of irritation

Irritation parameter:

in vitro irritation score

Remarks:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Value:

55.3

Negative controls validity:

valid

Remarks:

-0.6

Positive controls validity:

valid

Remarks:

144.1

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 144 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

ACCEPTANCE OF RESULTS: Yes
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean = Yes: the mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 144 and was within two standard deviations of the current historical positive control mean
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range = Yes: the negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas

- Historical control data can be found in the 'Attached background information' below.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Rosin Amine 90 induced serious eye damage through both endpoints: Corneal Opacity and Permeability, resulting in a mean in vitro irritancy score of 55 after 10 minutes of treatment.

Executive summary:

The eye hazard potential of the test substance, Rosin Amine 90, was evaluated using the Bovine Corneal Opacity and Permeability test (BCOP test), in a procedure compatible with OECD 437, EC Commission Regulation No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.47 'Bovine corneal opacity and permeability method for identifying ocular corrosives and severe irritants, OTWG: The Bovine Corneal Opacity and Permeability (BCOP) Test Method (March 2006), In Vitro Techniques in Toxicology Database study plan 127: Bovine Opacity and Permeability (BCOP) Assay (2006) and Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy (Gautheron, P. et al., 1992).

 

The test substance's potential to cause eye damage was tested through topical application of 750 µl directly to the top of bovine corneas for 10 minutes. The same volume of positive and negative controls were also tested. The negative control was physiological saline, employed in order to detect non-specific change in the test system and to provide a baseline for the assay endpoints. The positive control used was 10% (w/v) Benzalkonium Chloride solution, prepared in physiological saline.

 

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 144 and was within two standard deviations of the current historical positive control mean. These results met the acceptability criteria, and therefore can be concluded that the test conditions were adequate and that the test system functioned properly.

 

Rosin Amine 90 induced serious eye damage through both endpoints, resulting in a mean in vitro irritancy score of 55 after 10 minutes of treatment. Therefore, it can be concluded that Rosin Amine 90 induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report and should be classified category 1 according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008. 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin corrosion and Skin irritation

Based on the available data, it is concluded, that Rosin Amine 90 is not corrosive in the in vitro skin corrosion test. Rosin Amine 90 demonstrated the ability to cause a positive response in the EPISKIN™ Reconstructed Human Epidermis Model. Category 2 classification according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations is therefore in place.

 

Eye irritation

Rosin Amine 90 induced a serious eye damage in the Bovine Corneal Opacity and Permeability test (in vitro irritancy score (IVIS) ≥ 55).

Therefore, Rosin amine 90 should be classified category 1 according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations.