Fatty acids, tall-oil, reaction products with pentaethylenehexamine

Endpoint summary

Description of key information

In order to replace animal studies, tests on skin and eye irritation were performed as in vitro studies. The EpiDerm system was used to evaluate the skin corrosion and the skin irritation potential according to OECD guideline 431 and 439. A combination of the BCOP test (bovine corneal opacity, OECD guideline 437) and the EpiOcular assay was used to evaluate the eye irritation potential of the test material. The test material is not considered to be irritating to skin. The test item is considered to be irritating to eyes and is not considered to cause serious damage to eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012/13

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)

Version / remarks:

April 13, 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 22, 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

EpiDerm™ 200

Source species:

human

Cell type:

other: human-derived epidermal keratinocytes

Cell source:

other: commercially available kit (EpiDerm™ 200)

Source strain:

other: not applicable

Details on animal used as source of test system:

not applicable

Justification for test system used:

Based on the results of ECVAM (European Center for Validation of Alternative Methods) funded validation studies, it was concluded by the ECVAM Scientific Advisory Committee that the EpiDerm™ human epidermis model is suitable to be used for distinguishing between corrosive and non-corrosive chemicals (ECVAM: ESAC statement on the application of the EpidermTM human skin model for skin corrosivity testing of 14-15 Mar 2000) as well as between irritant and non-irritant chemicals (ECVAM: ESAC statement on the scientific validity of in-vitro tests for skin irritation testing of 5 Nov 2008).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ 200
- Tissue batch number(s): not specified
- Production date: not specified
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: 25 Sep 2012

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 min at room temperature or 1 h at 37 °C (corrosion test); 25 min at room temperature followed by 35 min at 37 °C (irritation test)
- Temperature of post-treatment incubation (if applicable): 42 h at 37 °C (irritation test)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 1 washing step, volume not specified
- Observable damage in the tissue due to washing: not specified
- Modifications to validated SOP: not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL assay medium
- Incubation time: 3 h
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm (OD570)
- Filter: no reference filter (not further specified)
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: not specified
- Barrier function: not specified
- Morphology: not specified
- Contamination: not specified
- Reproducibility: These historical control data demonstrate the reproducibility of results and robustness of the procedures.

NUMBER OF REPLICATE TISSUES: 2 (corrosion test); 3 (irritation test)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues (if applicable): Epi-200 tissue was killed by freezing at –20 °C
- N. of replicates: 1
- Method of calculation used: In case of direct reduction of MTT by the test substance, the OD570 values measured in the freeze-killed control tissues (KC) would be used to correct the mean OD570 of the testsubstance treated tissues (mean OD570 KC corrected). Since killed tissue might still have a residual enzyme activity that is able to produce some formazan net OD570 KC is calculated by subtracting the OD570 KC of the NC from the OD570 KC of the test substance. In case the net OD570 KC is greater than 0.1 it is subtracted from the respective mean OD570 to result in the mean OD570 KC corrected. The mean OD570 KC corrected represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 (corrosion test); 2 (irritation test)

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
- The test substance is considered to be irritating to skin if the viability is less than or equal to 50%.
- The test substance is considered to be non-irritating to skin if the viability is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

other: MTT-reduction control (De-ionized water or test substance (corrosion test); PBS or test substance (irritation test)

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL (corrosion test); 30 µL (irritation test)
- Concentration (if solution): undiluted test substance

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL (corrosion test); 30 µL (irritation test)
- Concentration (if solution): not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL (corrosion test); 30 µL (irritation test)
- Concentration (if solution): 8 n (corrosion test); 5 % (irritation test)

Duration of treatment / exposure:

Corrosion test: 3 min at room temperature or 1 h at 37 °C

Irritation test: 25 min at room temperature followed by 35 min at 37 °C

Duration of post-treatment incubation (if applicable):

Irritation test: 42 hours post-incubation period at 37 °C

Number of replicates:

see above

Vehicle:

unchanged (no vehicle)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Corrosion test, 3 min exposure

Value:

113

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Corrosion test, 1 h exposure

Value:

82

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Irritation test, 1st test run

Value:

65

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Irritation test, 2nd test run

Value:

99

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: not reported
- Direct-MTT reduction: The test substance was able to reduce MTT directly. Therefore an additional MTT reduction control (KC) was introduced (performed with the corrosion test and the 2nd test run of the irritation test). As the MTT-reduction control was used for calculation of the 1-hour exposure in the corrosion test, an influence of the test substance due to direct MTT reduction had to be excluded for the irritation test. Therefore a 2nd test run of the irritation test was performed with an additional MTT reduction control. The ability of direct MTT reduction did not impair the study result as demonstrated by the concurrently performed exposure of control tissues inactivated by freezing used during the 2nd test run.
- Colour interference with MTT: 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
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values: see tables 4 and 5

Table 1: Results of the corrosion test

		Exposure: 3 min				Exposure: 1 hour
Test substance		tissue 1	tissue 2	KC	mean	tissue 1	tissue 2	KC	mean*
NC	mean OD570	1.902	1.691	0.254	1.797	2.253	2.118	0.250	2.185
	viability [% of NC]	105.9	94.1	-	100	103.1	96.9	-	100
Test substance	mean OD570	2.041	2.010	0.335	2.025	1.943	1.950	0.412	1.785
	viability [% of NC]	113.6	111.9	-	113	88.9	89.2	-	82
PC	mean OD570	0.185	0.277	-	0.231	0.110	0.106	-	0.108
	viability [% of NC]	10.3	15.4	-	13	5.0	4.9	-	5

* The mean for the test substance after 1 hour exposure is given after KC-correction.

The result of the KC did not indicate an increased MTT reduction at the exposure period of 3 minutes (difference to KC of NC is not greater than 0.1). Thus the KC was not used for viability calculation for this exposure time.

Table 2: Results of the irritation test - 1st test run

Test substance		tissue 1	tissue 2	tissue 3	mean	SD
NC	mean OD570	2.295	2.144	2.121	2.187
	viability [% of NC]	105.0	98.1	97.0	100	4.33
Test substance	mean OD570	1.367	1.174	1.700	1.414
	viability [% of NC]	62.5	53.7	77.7	65	12.16
PC	mean OD570	0.059	0.063	0.065	0.062
	viability [% of NC]	2.7	2.9	2.9	3	0.13

 

 

Table 3: Results of the irritation test - 2nd test run

Test substance		tissue 1	tissue 2	tissue 3	KC	mean	SD
NC	mean OD570	2.404	2.415	2.401	0.057	2.407
	viability [% of NC]	99.9	100.3	99.8	-	100	0.29
Test substance	mean OD570	2.347	2.414	2.384	0.067	2.381
	viability [% of NC]	97.5	100.3	99.0	-	99	1.38
PC	mean OD570	0.096	0.090	0.105	-	0.097
	viability [% of NC]	4.0	3.7	4.4	-	4	0.32

The result of the KC did not indicate an increased MTT reduction (difference to KC of NC is not greater than 0.1). Thus the KC was not used for viability calculation.

Table 4: Historical control data - Corrosion test

Historical Range of NC
OD570
Exposure Time	Historical Period	Mean OD	SD	Mean + 2 SD	Mean - 2 SD
3 minutes	Feb 2010 - Aug 2012	1.901	0.200	2.30	1.50
60 minutes	Feb 2010 - Aug 2012	1.867	0.203	2.27	1.46
Historical Range of PC
OD570
Exposure Time	Historical Period	Mean OD	SD	Mean + 2 SD	Mean - 2 SD
3 minutes	Feb 2010 - Aug 2012	0.361	0.087	0.54	0.19
60 minutes	Feb 2010 - Aug 2012	0.155	0.048	0.25	0.06
Viability (%)
Exposure Time	Historical Period	Mean %	SD	Mean + 2 SD	Mean - 2 SD
3 minutes	Feb 2010 - Aug 2012	19.16	4.73	28.62	9.69
60 minutes	Feb 2010 - Aug 2012	8.47	2.82	14.12	2.82

Table 5: Historical control data - Irritation test

Historical Range of NC
OD570 Historical Period	Mean OD	SD	Mean + 2 SD	Mean - 2 SD
Feb 2010 - Nov 2012	2.060	0.281	2.62	1.50

 

Historical Range of PC
OD570 Historical Period	Mean OD	SD	Mean + 2 SD	Mean - 2 SD
Feb 2010 - Nov 2012	0.111	0.052	0.22	0.01

 

Viability (%)
Historical Period	Mean %	SD	Mean + 2 SD	Mean - 2 SD
Feb 2010 - Nov 2012	5.5	2.47	10.45	0.56

Interpretation of results:

GHS criteria not met

Conclusions:

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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

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)

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

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%.

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

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

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.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

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:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

September 07, 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: lsolated bovine cornea

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Schlachthof Bensheim, Am Schlachthof 7-9, 64625 Bensheim
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): age of the animals: minimum 12 months, maximum 60 months
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): not specified
- Time interval prior to initiating testing: not specified
- indication of any existing defects or lesions in ocular tissue samples: no, corneas were free of defects (opacity, scratches, pigmentation etc.)
- Indication of any antibiotics used: not specified

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL
- Concentration (if solution): undiluted test substance

Duration of treatment / exposure:

10 min at 32°C, horizontal, corneas are incubated for further 2 hours at about 32 °C (post exposure period for liquids)

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

3 corneas each for test item, positive control and negative control, respectively

Details on study design:

SELECTION AND PREPARATION OF CORNEAS : 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 anterior 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 units1 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.

QUALITY CHECK OF THE ISOLATED CORNEAS : see above ("SELECTION AND PREPARATION OF CORNEAS")

NUMBER OF REPLICATES : see above

NEGATIVE CONTROL USED : see above

POSITIVE CONTROL USED : see above

APPLICATION DOSE AND EXPOSURE TIME : 750 µL undiluted test substance, 10 min at 32 °C

TREATMENT METHOD: open chamber method

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: according to open chamber method (not further specified)
- POST-EXPOSURE INCUBATION: 2 h at 32 °C

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Final corneal opacity readings were taken for each cornea with an opacitometer.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used (see "Any other information on materials and methods incl. tables")

Irritation parameter:

in vitro irritation score

Value:

19.1

Irritation parameter:

other: Mean permeability value

Value:

0.409

Irritation parameter:

other: Mean opacity value

Value:

13.4

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

Table 1: In Vitro Irritancy score (IVIS)

Test substance	Cornea- No.	Opacity per cornea	Permeability per cornea	per cornea	IVIS per group
					mean	SD
	10	17.3	0.393	23.2
Test substance	11	15.0	0.422	21.3	19.6	4.7
	12	8.0	0.412	14.2
	1	1.6	0.001	1.6
NC	2	1.0	0.005	1.1	1.6	0.5
	3	2.0	0.006	2.1
	4	126.3	2.809	168.4
PC	5	135.9	2.689	176.2	180.2	14.2
	6	163.5	2.166	196.0

Table 2: Historical control data

Historical range of NC (protocol for liquids and surfactants)

 

Historical period   Jan 2010 - Aug 2012
Opacity	Mean 1.3	SD 1.5	Mean + 2 SD Mean - 2 SD 4.2 -1.7
Permeability	Mean 0.005	SD 0.016	Mean + 2 SD Mean - 2 SD 0.037 -0.027
Historical range of PC (1% NaOH)
Historical period   Jan 2010 - Aug 2012
Opacity	Mean 118.1	SD 20.4	Mean + 2 SD Mean - 2 SD 159.0 77.2
Permeability	Mean OD 2.814	SD 0.897	Mean + 2 SD Mean - 2 SD 4.607 1.021
In Vitro Irritation Score (IVIS)	Mean 160.3	SD 22.9	Mean + 2 SD Mean - 2 SD 206.1 114.6

Interpretation of results:

study cannot be used for classification

Remarks:

the result of the BCOP test alone is not sufficient to establish a definitive classification

Conclusions:

Based on the observed results and applying the evaluation criteria it was concluded, that the test item does not cause serious eye damage in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In order to replace animal studies, tests on skin and eye irritation were performed as in vitro studies. The EpiDerm system was used to evaluate the skin corrosion and the skin irritation potential by examination of the viability of the cells of the reconstructed skin model after 3 min and 1h or after 35 min exposure according to OECD guideline 431 and 439.

A combination of the BCOP test (bovine corneal opacity, OECD guideline 437) and the EpiOcular assay was used to evaluate the eye irritation potential of the test material.

The cell viability values obtained each for the skin corrosion and the irritation test are clearly above the threshold of 15% and 50%, respectively. The test material is therefore not considered to be irritating to skin. Measurement of the permeability and opacity of the bovine corneas after treatment with the test item gave IVIS values which were below the threshold of 55. The viability after exposure of EpiOcular tissue cells was 5%. Thus, the test item is considered to be irritating to eyes and is not considered to cause serious damage to eyes.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is considered to be classified for irritation to eyes (cat. 2) under Regulation (EC) No. 1272/2008, as amended for the 14th time in Regulation (EU) 2020/217.