p-menth-1-en-4-ol

Endpoint summary

Description of key information

In vitro skin irritation/corrosion (OECD 431 and OECD 439): irritating

In vitro eye irritation/corrosion (OECD 437 and OECD 492): irritating

 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Draize skin irritation test as described in Draize et al. (1944) Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes; Journal of Pharmacology and Experimental Therapeutics, Nov 1944, 82 (3) 377-390
- Parameters analysed / observed: erythema and oedema using a scoring system by Draize et al. (1944)

GLP compliance:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Sigma Aldrich, Milan, Italy
- Purity: analytical-grade

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BioLASCO, Taiwan
- Age at study initiation: 8-10 weeks
- Females non-pregnant and nulliparous: yes
- Weight at study initiation: 200-220
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 +/- 1
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

not specified

Preparation of test site:

shaved

Vehicle:

not specified

Controls:

not specified

Amount / concentration applied:

Amount applied: 50 µl
Concentrations: 0.094%, 0.188%, 0.375%, 0.750%, 1.500%

Duration of treatment / exposure:

4 h

Observation period:

24, 48 h

Number of animals:

Each group consisted of five female rats

Details on study design:

TEST SITE
- Area of exposure: 2.5 x 2.5 cm at each site of the back
- Site preparation: 24 h before the test, the backs of the animals were shaved and checked for any abnormalities the next day.

REMOVAL OF TEST SUBSTANCE
- Washing: with distilled water
- Time after start of exposure: 4 h

OBSERVATION TIME POINTS
- 24 and 48 h

SCORING SYSTEM:
- Method of calculation: Scoring system by Draize et al. (1944)

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48 h

Score:

0

Max. score:

4

Reversibility:

other: no evidence of any skin reaction

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48 h

Score:

0

Max. score:

4

Reversibility:

other: no evidence of any skin reaction

Remarks on result:

no indication of irritation

The prepared tea tree oil from leaves and from twigs contained 47.31% and 39.09% terpinen-4 -ol, respectively.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions chosen, the test substance did not induce skin irritancy.

Executive summary:

Five female Wistar rats per concentration group were treated with 0.094%, 0.188%, 0.375%, 0.750%, 1.500% of terpinen-4 -ol. Exposure duration was 4 hours and the test substance was removed with distilled water after the exposure period. Twenty-four and 48 h after application, skin irritation was observed and quantified through the scoring system by Draize et al. (1944). No evidence of erythema, oedema, or any other skin reaction was observed at 24 h and 48 h.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Oct 2017 - Jan 2018

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:

Jul 2016

Deviations:

yes

Remarks:

Decision criteria based on company data

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

yes

Remarks:

Decision criteria based on company data

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: BASF SE, Ludwigshafen, Germany
- Batch No.of test material: 01767-1010
- Identitiy: Confirmed
- Purity: 97.3%
- Expiry date: 17 Mar 2019
- Physical state / color: liquid / colorless, clear
- pH value: ca. 5

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, under light exclusion
- Stability under test conditions: Stability was guaranteed
- Homogeneity: Homogeneous by visual inspection

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm model (EPI-200)

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200
- Tissue lot number: 25857, test run 1; 25863, test run 2
- Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation: Room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
-Tissues were washed with PBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/ml MTT diluent
- Incubation time: 3 h
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORIC DATA
- Viability: Tissue viability is presented as quotient of the mean OD570 divided by the respective OD570 NC value in percent for each exposure time.
- Barrier function: Lower acceptance limit: ET50 = 4.0 hours; Upper acceptance limit: ET50 = 8.7 hours
- Contamination: no contamination
- Reproducibility: Variability between the tissues was considered to be acceptable if the coefficient of variation (CV) of % vaibility was below or equal to 30%.

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Results of pre-test: The application of killed control tissues was not necessary

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 3 minutes exposure is greater than or equal to 50 % and 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%
- Justification for the selection of the cut-off points if different from OECD TG 431: The “borderline“ evaluation (50 ± 5%, 25 ± 5% and 15 ± 5%) was determined statistically using historic company data and hence considers the variance of the test method. This evaluation is an amendment to the evaluation provided in OECD Guideline 431.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µl undiluted test substance

Duration of treatment / exposure:

3 min and 1 h

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test run 1, tissue 1, 3 min

Value:

102.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication for corrosion

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test run 1, tissue 2, 3 min

Value:

99.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication for corrosion

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test run 1, tissue 1, 1 h

Value:

10.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: positive indication of corrosion

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test run 1, tissue 2, 1 h

Value:

52.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication for corrosion

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test run 2, tissue 1, 1 h

Value:

23.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication for corrosion

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test run 2, tissue 2, 1 h

Value:

25.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication for corrosion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results observed and by applying the evaluation criteria, it was concluded that the test substance shows a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy including transport classification under the test conditions chosen.

Executive summary:

The objective was to assess the skin corrosion and irritation potential of the test substance. Using the methods currently available, a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential including transport classification. Therefore, three in vitro assays were proposed for this in vitro skin irritation and corrosion test strategy including transport classification: The Skin Corrosion Test (SCT), Skin Irritation Test (SIT) and In vitro Membrane Barrier Test (Corrositex®).

However, in the current case, the results derived with SIT and SCT were sufficient for a final assessment. Therefore, further testing in Corrositex® was waived.

The potential of the test substance to cause dermal corrosion was assessed by a sinlge topical application of 50 µl undiluted test substance to a reconstructed three-dimensional, human epidermis model (EpiDerm™).

For the corrosion test, two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour each.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by 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 epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The following results were obtained in the EpiDerm™ skin corrosion/irritation test:

The test substance is not able to reduce MTT directly.

Two test runs (1-hour exposure) of the EpiDermTM skin corrosion test were performed.

Results of the 1st test run:

The relative mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 101%. However, non-concordant replicate measurements of the test-substance treated tissues were obtained at the 1-hour exposure period (individual values: 10.4% and 52.2%) and the acceptance criteria for the variability of the tissues was not met. Thus, a 2nd test run (only the 1-hour exposure) was performed.

Results of the 2nd test run (1-hour exposure):

The relative mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour was 24.7% (individual values: 23.8% and 25.7%). All acceptance criteria were met.

Thus, the test substance is concluded not to have a skin corrosion potential.

Reference 3

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Oct 2017 - Jan 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:

Jul 2015

Deviations:

yes

Remarks:

Decision criteria based on company data

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Jul 2012

Deviations:

yes

Remarks:

Decision criteria based on company data

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: BASF SE, Ludwigshafen, Germany
- Batch No.of test material: 01767-1010
- Identitiy: Confirmed
- Purity: 97.3%
- Expiry date: 17 Mar 2019
- Physical state / color: liquid / colorless, clear
- pH value: ca. 5

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, under light exclusion
- Stability under test conditions: Stability was guaranteed
- Homogeneity: Homogeneous by visual inspection

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm model (EPI-200)

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200
- Tissue lot number: 25857
- Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature (25 min), 37°C (35 min)
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Tissues were washed with PBS 1 h after application

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/ml MTT diluent
- Incubation time: 3 h
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Tissue viability is presented as quotient of the mean OD570 divided by the respective OD570 NC value in percent for each exposure time.
- Barrier function: Lower acceptance limit: ET50 = 4.0 hours; Upper acceptance limit: ET50 = 8.7 hours
- Contamination: no contamination
- Reproducibility: The inter-tissue variability is considered to be acceptable if the SD of % viability is ≤ 18%.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Results of pre-test: The application of killed control tissues was not necessary

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered as "irritant" if the mean relative tissue viability with a test material is less than or equal to 50%.
- - Justification for the selection of the cut-off point(s) if different than recommended in TG 439: The “borderline“ evaluation (50 ± 5%) was determined statistically using historic BASF data and hence considers the variance of the test method. This evaluation is confirming the borderline range provided in OECD Guideline 439.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

30 µl undiluted test substance

Duration of treatment / exposure:

1 h

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Vehicle:

unchanged (no vehicle)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 1

Value:

2.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 2

Value:

2.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 3

Value:

2.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Based on the results observed and by applying the evaluation criteria, it was concluded that the test substance shows a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy including transport classification under the test conditions chosen.

Executive summary:

The objective was to assess the skin corrosion and irritation potential of the test substance. Using the methods currently available, a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential including transport classification. Therefore, three in vitro assays were proposed for this in vitro skin irritation and corrosion test strategy including transport classification: The Skin Corrosion Test (SCT), Skin Irritation Test (SIT) and In vitro Membrane Barrier Test (Corrositex®).

However, in the current case, the results derived with SIT and SCT were sufficient for a final assessment. Therefore, further testing in Corrositex® was waived.

The potential of the test substance to cause dermal irritation was assessed by a sinlge topical application of 30 µl undiluted test substance to a reconstructed three-dimensional, human epidermis model (EpiDerm™).

The irritation test was performed with three EpiDerm™ tissues, which were incubated with the test substance for 1 hour followed by a 42-hour post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by 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 epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The following results were obtained in the EpiDerm™ skin corrosion/irritation test:

The test substance is not able to reduce MTT directly.

The relative mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42-hour post-incubation was 2.9%. Thus, the test substance is concluded to have a skin irritation potential.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Nov 2017 - Jan 2018

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:

Jul 2013

Deviations:

yes

Remarks:

decision criteria

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:

Dec 2010

Deviations:

yes

Remarks:

decision criteria

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: BASF SE, Ludwigshafen, Germany
- Batch No.of test material: 01767-1010
- Identity: Confirmed
- Purity: 97.3%
- Expiry date: 17 Mar 2019
- Physical state / color: Liquid / colorless, clear
- pH value: ca. 5 (undiluted test substance)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature; under light exclusion
- Stability under test conditions: The stability was guaranteed
- Homogeneity: The test substanc was homogeneous by visual inspection

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was applied undiluted, thus, no preparation was necessary.

Species:

other: Isolated bovine cornea

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Schlachthof Alzey, Emil Färber GmbH & Co. KG, Robert-Bosch-Strasse 23, 55232 Alzey, Germany
- Characteristics of donor animals (age): minimum 12 months, maximum 60 months
- Detailed information of ocular tissue: Bovine eyes are obtained as a by-product of freshly slaughtered cattle

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

yes, concurrent positive control

Amount / concentration applied:

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

VEHICLE
- not applicable

CONTROLS
- Amount(s) applied (volume or weight with unit): 750 µl

Details on study design:

SELECTION AND PREPARATION OF CORNEAS

QUALITY CHECK OF THE ISOLATED CORNEAS: yes

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: yes

POSITIVE CONTROL USED: yes

APPLICATION DOSE AND EXPOSURE TIME: 750 µl, 10 min

TREATMENT METHOD: not specified

POST-INCUBATION PERIOD: yes/no. If YES please specify duration

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3
- POST-EXPOSURE INCUBATION: 2 hours

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: see "any other information on materials and methods"

Irritation parameter:

in vitro irritation score

Run / experiment:

cornea number 19

Value:

28.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of corrosive potential

Irritation parameter:

in vitro irritation score

Run / experiment:

cornea number 20

Value:

26.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of corrosive potential

Irritation parameter:

in vitro irritation score

Run / experiment:

cornea number 21

Value:

24.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of corrosive potential

Table 1: Opacity score of the test substance, the negative control and the positive control

Test substance identification	Cornea- No.	Initial opacity	Final opacity	Opacity Change	Corrected Opacity Change	Mean	SD
	19	4.5	21.3	16.8	10.6
17/0495-1	20	3.7	17.0	13.3	7.2	9.0	1.7
	21	3.8	18.9	15.2	9.0
	10	3.9	11.0	7.1	NA
NC	11	2.8	7.6	4.8	NA	6.1	1.2
	12	4.0	10.5	6.5	NA
	13	4.7	32.7	27.9	21.8
PC1	14	2.8	34.3	31.6	25.4	21.9	3.5
	15	4.6	29.0	24.5	18.3
	16	5.3	109.4	104.1	98.0
PC2	17	5.2	97.5	92.3	86.2	93.7	6.5
	18	3.8	106.8	103.0	96.8

Table 2: Permeability score of the test substance, the negative and the positive control

Test substanceidentification	Cornea-No.	MeanOD490	DilutionFactor	Mean CorrectedOD490	Mean	SD
	19	1.170	1	1.165
17/0495-1	20	1.270	1	1.266	1.161	0.107
	21	1.057	1	1.053
	10	0.002	1	NA
NC	11	0.004	1	NA	0.004	0.003
	12	0.007	1	NA
	13	0.986	1	0.982
PC1	14	0.552	1	0.548	0.734	0.223
	15	0.677	1	0.672
	16	0.217	1	0.212
PC2	17	0.559	1	0.555	0.388	0.171
	18	0.401	1	0.397

Table 3: In Vitro Irritancy Score (IVIS) of the test substance, the negative and the positive control

Test substanceidentification	Cornea- No.	Opacity per cornea	Permeability per cornea	per cornea	IVIS per group
					mean	SD
	19	10.6	1.165	28.1
17/0495-1	20	7.2	1.266	26.2	26.4	1.7
	21	9.0	1.053	24.8
	10	7.1	0.002	7.2
NC	11	4.8	0.004	4.8	6.2	1.2
	12	6.5	0.007	6.6
	13	21.8	0.982	36.5
PC1	14	25.4	0.548	33.6	32.9	4.1
	15	18.3	0.672	28.4
	16	98.0	0.212	101.1
PC2	17	86.2	0.555	94.5	99.5	4.4
	18	96.8	0.397	102.8

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Based on the results of the BCOP and EpiOcular Tests and by applying the evaluation criteria, it was concluded that the test substance shows an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

Executive summary:

The objective was to assess the eye irritating potential of the test substance. By using the methods currently available a single in vitro assay is not sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test.

BCOP

The potential of the test substance to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 μL undiluted test substance to the epithelial surface of isolated bovine corneas. Three corneas were treated with the test substance for 10 minutes followed by a 2-hour postincubation period.

In addition to the test substance, a negative control (NC; deionized water) and two positive controls (PC1 / PC2; 100% ethanol / 100% dimethylformamide) were applied to three corneas each.

Corneal opacity was quantitatively measured as the amount of light transmitted through the cornea. Permeability was quantitatively measured 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.

The following results were obtained in the BCOP Test:

Test substance identification	MeanOpacityValue	MeanPermeabilityValue	Mean In Vitro Irritancy Score
Test substance	9.0	1.161	26.4
NC	6.1	0.004	6.2
PC1	21.9	0.734	32.9
PC2	93.7	0.388	99.5

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Nov 2017 - Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

Jul 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: BASF SE, Ludwigshafen, Germany
- Batch No.of test material: 01767-1010
- Identity: Confirmed
- Purity: 97.3%
- Expiry date: 17 Mar 2019
- Physical state / color: Liquid / colorless, clear
- pH value: ca. 5 (undiluted test substance)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature; under light exclusion
- Stability under test conditions: The stability was guaranteed
- Homogeneity: The test substanc was homogeneous by visual inspection

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was applied undiluted, thus, no preparation was necessary.

Species:

human

Strain:

other: epidermal keratinocytes

Details on test animals or tissues and environmental conditions:

Description of the cell system used: Reconstructed human cornea-like epithelium
- Model used: EpiOcular™ model (OCL-200)
- Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Tissue lot number: 27015
- Certificate of authenticity: provided by the supplier
- Verification of tissue functionality and quality (performed by the supplier):
- Tissue viability: acceptance criteria met
- Barrier function: acceptance criteria met
- Sterility: no contamination

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

yes, concurrent positive control

Details on study design:

- Details of the test procedure used: Protocol for liquids
- RhCE tissue construct used: OCL-200 (reconstructed cornea, surface 0.6 cm² cultured in Millicells with a diameter of 1 cm)
- Tissue lot number: 27015
- Doses of test chemical and control substances used: undiluted test chemical, deionized water and neat methyl acetate
- Pre-incubation: 16-24 h
- Pretreatment with 20 µl PBS for 30 minutes
- Application of the test substance: 50 µl
- Duration and temperature of exposure: 30 minutes at 37 +/- 1°C
- Washing post-exposure: Three times
- Post-exposure immersion: 12 minutes of post-soak immersion
- Post-exposure incubation period: 2 hours
- Indication of controls used for direct MTT-reducers: A pre-test was performed in order to assess the potential of the test substance to directly reduce MTT. No potential of directly inducing MTT was observed.
- Number of tissue replicates used per test chemical and controls: 2
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan: The assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated for 3 hours. After incubation, the tissues were washed with PBS. The formazan that was metabolically produced by the tissues was extracted by overnight incubation of the tissues in isopropanol at room temperature or by incubation for at least 2 hours on a plate shaker. The optical density of the extracts was spectrophotometrically determined. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: If applicable, two freeze-killed control tissues were treated with the test article and the negative control each in the same way as the "main experiment". Based on the result of the pretest, it was judged that application of killed control tissues was not necessary.
- Description of evaluation criteria used: The OD570 values determined for the various tissues are measures of their viability. The ratio of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether a test material was an irritant.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: yes

- Positive and negative control means and acceptance ranges based on historical data: yes
- Acceptable variability between tissue replicates for positive and negative controls: yes
- Acceptable variability between tissue replicates for the test chemical: yes
- The "borderline" evaluation (60 +/- 5%) was statistically determined by using historic BASF data and hence considers the variance of the test method. This evaluation is an amendment to the evaluation provided in OECD Guideline 492.

Irritation parameter:

other: viability

Run / experiment:

tissue 1

Value:

6.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

other: viability

Run / experiment:

tissue 2

Value:

5.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Table 1: Individual and mean OD570 values, individual and mean viability values and inter-tissue variability

Testsubstance identification		tissue 1	tissue 2	mean	Inter-tissue variability [%]
NC	mean OD570	1.927	1.903	1.915
	viability [% of NC]	100.6	99.4	100.0	1.3
17/0495-1	mean OD570	0.120	0.110	0.115
	viability [% of NC]	6.3	5.7	6.0	0.5
PC	mean OD570	0.435	0.575	0.505
	viability [% of NC]	22.7	30.0	26.4	7.3

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Based on the results of the BCOP and EpiOcular Tests and by applying the evaluation criteria, it was concluded that the test substance shows an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

Executive summary:

The objective was to assess the eye irritating potential of the test substance. By using the methods currently available a single in vitro assay is not sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test.

EpiOcular

The potential of the test substance to cause ocular irritation was assessed by a single topical application of 50 μL undiluted test substance to a reconstructed three-dimensional, human cornea model (EpiOcular™).

Two EpiOcular™ tissues were incubated with the test substance for 30 minutes followed by a 2-hour post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after

exposure/post-incubation by 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 epidermal tissues treated with the test substance is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability.

The following results were obtained in the EpiOcular™ eye irritation assay: The test substance is not able to directly reduce MTT.

The relative mean viability of the tissues treated with the test substance was 6%.

Summary of the individual test results of the in vitro eye irritation turnkey testing strategy:

Test Method	Test Result	Test Evaluation	Evaluation Test Strategy
BCOP Test	The mean IVIS of the corneas treated with the test substance was 26.4.	Not identified as corrosive or severe irritant	Ocular irritant
EpiOcular	The mean relative viability of the tissues treated with the test substance was 6%.	Irritant

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin corrosion:

The skin corrosion and irritation potential of the test substance was assessed using a testing strategy including three in vitro assays: The Skin Corrosion Test (SCT)(OECD 431), Skin Irritation Test (SIT) (OECD 439) and In vitro Membrane Barrier Test (Corrositex®)(OECD 435).

In the current case, the results derived with SIT and SCT were sufficient for a final assessment. Therefore, further testing in Corrositex^® was waived.

The potential of the test substance to cause dermal corrosion was assessed by a sinlge topical application of 50 µl undiluted test substance to a reconstructed three-dimensional, human epidermis model (EpiDerm™). Two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour each.

The extent of tissue destruction due to the test substance was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test (MTT) and compared to the solvent control. The following results were obtained in the EpiDerm™ skin corrosion/irritation test:

The test substance is not able to reduce MTT directly.

Two test runs (1-hour exposure) of the EpiDermTM skin corrosion test were performed.

The results of the 1st test run did not meet the acceptance criteria for the variability of the tissues, thus a 2nd test run for the 1-hour exposure was performed.

Results of the 2nd test run (1-hour exposure):

The relative mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour was 24.7% (individual values: 23.8% and 25.7%). All acceptance criteria were met.

Thus, the test substance is concluded not to have a skin corrosion potential.

Skin irritation:

The potential of the test substance to cause dermal irritation was assessed by a sinlge topical application of 30 µl undiluted test substance to a reconstructed three-dimensional, human epidermis model (EpiDerm™).

The irritation test was performed with three EpiDerm™ tissues, which were incubated with the test substance for 1 hour followed by a 42-hour post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test (MTT).  The quotient of the values of the substance treated tissues versus the control treated tissues, indicates the relative tissue viability. The following results were obtained in the EpiDerm™ skin corrosion/irritation test:

The test substance is not able to reduce MTT directly.

The relative mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42-hour post-incubation was 2.9%.

Thus, the test substance is concluded to have a skin irritation potential.

Skin irritation in vivo:

As a supporting study, data from a non-guideline in vivo study relying on the principles of the Draize skin irritation test (Draize et al., 1944) is considered  (Lee et al., 2013). Five female Wistar rats per concentration group were treated with 0.094%, 0.188%, 0.375%, 0.750%, 1.500% of the test substance. Exposure duration was 4 hours and the test substance was removed with distilled water after the exposure period. Twenty-four and 48 h after application, skin irritation was observed and quantified through the scoring system by Draize et al. (1944). No evidence of erythema, oedema, or any other skin reaction was observed at 24 h and 48 h.

As the in vitro studies have a higher reliability as they were conducted accoding to GLP and OECD guidelines, the test substance is considered a skin irritant (Cat. 2).

 

Eye irritation/corrosion:

In line with the eye irritation/corrosion turnkey testing strategy, two in vitro assays were applied: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test.

BCOP

The BCOP assay was performed to evaluate the corrosive potential of the test substance. A single topical application of 750 μL undiluted test substance to the epithelial surface of isolated bovine corneas was performed. Three corneas were treated with the test substance for 10 minutes followed by a 2-hour postincubation period.

In addition to the test substance, a negative control (NC; deionized water) and two positive controls (PC1 / PC2; 100% ethanol / 100% dimethylformamide) were applied to three corneas each.

Corneal opacity was quantitatively measured as the amount of light transmitted through the cornea. Permeability was quantitatively measured 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.

EpiOcular^TM

The potential of the test substance to cause ocular irritation was assessed by a single topical application of 50 μL undiluted test substance to a reconstructed three-dimensional, human cornea model (EpiOcular™).

Two EpiOcular™ tissues were incubated with the test substance for 30 minutes followed by a 2-hour post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test (MTT). The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability.

Summary of the individual test results of the in vitro eye irritation turnkey testing strategy:

 

Test Method	Test Result	Test Evaluation	Evaluation Test Strategy
BCOP Test	The mean IVIS of the corneas treated with the test substance was 26.4.	Not identified as corrosive or severe irritant	Ocular irritant
EpiOcular	The mean relative viability of the tissues treated with the test substance was 6%.	Irritant

 

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes  under Regulation No. 1272/2008.

Skin irritation/corrosion:

Based on the results, the test substance is classified as irritating to the skin (Skin Irrit. Cat. 2).

Eye irritation/corrosion:

Based on the results, the test substance is classified as irritating to the eyes (Eye Irrit. Cat. 2).