Registration Dossier

Skin irritation:

Key: EpiDerm (2018):not irritating to skin

Read-across substance:

- OECD 404 (Bioassay, 2013): not irritating to the skin
- Epiderm (2012): not irritating

Eye irritation:

Key: EpiOcular (2018): not irritating to the eye

Read-across substance:

- OECD 405 (Bioassay, 2013): not irritating to the eye

- EpiOcular (2012): not irritating

- BCOP (2012): does not cause serious eye damage

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 17/0241-1, Batch 294-296

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The stability under storage conditions over the study period was guaranteed.
- Stability under test conditions: room temperature

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

not specified

Details on animal used as source of test system:

- Tissue model: EPI-200
- Tissue Lot Number: 25844
- Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Justification for test system used:

The test system is part of an integrated approach on testing and assessment (IATA) for skin corrosion and irritation and is one of the accepted guideline studies.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm model
- Tissue batch number(s): 25844

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant if the viability is less than 45% compared to negative control.
- The test substance is considered to be non-irritant if the viability is higher than 55% compared to control.
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 45%, or if the viability after 3 minutes exposure is greater than or equal to 45 % and the viability after 1 hour exposure is less than 10%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than 55% and the viability after 1 hour exposure is greater than 20%.
- The borderline evaluation was determined statistically using historic data and hence considers the variance of the test method.This evaluation is an amendment to the evaluation provided in OECD Guideline 431.

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µl undiluted liquid test substance

NEGATIVE CONTROL
- PBS, sterile

POSITIVE CONTROL
- 5% (w/v) sodium dodecyl sulfate (SDS) in water

Duration of treatment / exposure:

1h

Duration of post-treatment incubation (if applicable):

32 - 36 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 1

Value:

76.1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 2

Value:

79

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 3

Value:

66

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Individual and mean OD570 values, individual and mean viability values and standard deviations

Test substance		Tissue 1	Tissue 2	Tissue 3	Mean	SD
NC	Mean OD570	1.370	1.417	1.298	1.362
NC	Viability (% of NC)	100.6	104.1	95.3	100.0	4.4
Test substance	Mean OD570	1.037	1.076	0.899	1.004
Test substance	Viability (% of NC)	76.1	79.0	66.0	73.7	6.8
PC	Mean OD570	0.046	0.054	0.049	0.050
PC	Viability (% of NC)	3.4	3.9	3.6	3.7	0.3

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 item does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

Executive summary:

The objective was to assess the skin irritation and corrosion potential of the test item. Using the methods currently available, a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT).

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

The potential of the test substance to cause dermal irritation was assessed by a single topical application of 30 μL of the 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 test substance is not able to reduce MTT directly. The 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 73.7%. Slight compound residues remained on the tissues treated with the test substance after the washing procedure.

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

Endpoint conclusion:

no adverse effect observed (not irritating)

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 17/0241-1, Batch 294-296

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

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- Source: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Tissue model: OCL-200
- Tissue Lot Number: 27004 and 27009
- Incubation conditions: 37°C +/- 1°C, 5% +/- 1% CO2, 90% +/- 5% relative humidity
- Detection agent: MTT
- Ectracting agent: Isopropanol
- Wash buffer: PBS

Vehicle:

unchanged (no vehicle)

Controls:

yes

Details on study design:

- Negative control: Deionized water, sterile
- Positive control: Neat methyl acetate

Irritation parameter:

other: tissue viability (% of NC)

Run / experiment:

Mean 1st Run

Value:

130.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

other: tissue viability (% of NC)

Run / experiment:

Mean of 2nd Run

Value:

95

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

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

Test substance		Tissue 1	Tissue 2	Mean	Inter-tissue variability (%)
NC	Mean OD570	0.814	0.788	0.801
NC	Viability (% of NC)	101.6	98.4	100.0	3.2
Test item	Mean OD570	0.842	1.249	1.045
Test item	Viability (% of NC)	105.1	155.9	130.5	50.8
PC	Mean OD570	0.255	0.215	0.235
PC	Viability (% of NC)	31.8	26.8	29.3	5.1

2nd Run: Individual and mean OD570 values, individual and mean viability values and inter-tissue variability

Test substance		Tissue 1	Tissue 2	Mean	Inter-tissue variability (%)
NC	Mean OD570	1.594	1.727	1.660
NC	Viability (% of NC)	96.0	104.0	100.0	8.0
Test item	Mean OD570	1.540	1.616	1.578
Test item	Viability (% of NC)	92.7	97.3	95.0	4.6
PC	Mean OD570	0.445	0.325	0.385
PC	Viability (% of NC)	26.8	19.6	23.2	7.2

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results observed for the EpiOcular Test alone and by applying the evaluation criteria it was concluded that the test item does not show 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 item. 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. However, in the current case the results derived with EpiOcular Eye Irritation Test alone were sufficient for a final assessment. Therefore, further testing in BCOPwas waived.

EpiOcular Eye Irritation Test

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 test runs were performed. Two EpiOcular™ tissues per test run 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 test substance is not able to directly reduce MTT.

As the acceptance criterion for inter-tissue variability of the test substance was not met (values for single tissues: 105.1% and 155.9%) and the mean OD570 of the NC was exceptionally low (0.801), a 2nd experiment was performed to clarify the result. In the 2nd test run, the mean viability of the tissues treated with the test substance was 95.0%. All acceptance criteria were met. Minimal compound residues remained on the tissues treated with the test substance after the washing procedure of both test runs.

Based on the results observed in the EpiOcular Test alone and by applying the evaluation, it was concluded that the test substance does not show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

Endpoint conclusion:

no adverse effect observed (not irritating)

Endpoint conclusion:

no study available

Skin irritation:

The potential of the test substance to cause dermal irritation was assessed by a single topical application of 30 μL of the 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 test substance is not able to reduce MTT directly. The 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 73.7%. Slight compound residues remained on the tissues treated with the test substance after the washing procedure.

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

The potential of the read-across substance to cause acute dermal irritation or corrosion was assessed by a single topical application of an amount of 0.5 mL of the test item for 4 hours to the intact skin of three White New Zealand rabbits (stepwise procedure starting with one animal and supplementing two additional animals), using a patch of 2.5 cm x 2.5 cm, covered with semi-occlusive dressing. After removal of the patch the application area was washed off. The study was conducted according to OECD 404 guideline and GLP (Bioassay, 2013).

The cutaneous reactions were assessed immediately after removal of the patch, approximately 1, 24, 48 and 72 hours after removal of the patch and in weekly intervals until day 14 at the latest.

The following test item-related clinical observations were recorded during the course of the study:

- Very slight erythema (grade 1)

- Scaling

The cutaneous reactions, with the exception of scaling, were reversible in one animal within 14 days after removal of the patch. In one animal cutaneous reactions were reversible within 72 hours and in one animal within 7 days after removal of the patch. Mean scores over 24, 48 and 72 hours for each animal were 0.7, 0.7 and 0.7 for erythema and 0.0, 0.0 and 0.0 for edema.

Considering the described cutaneous reactions as well as the average score for irritation, the test item does not show a skin irritating potential under the test conditions chosen.

The potential of the read-across substance to induce skin irritation was assessed according to OECD431/439 and GLP in an in vitro human epidermis skin model. The corrosion test involved application of 50 µl of the test item for 3 min or 1 h, respectively to two skin samples. Immediately after incubation, the viability of the human epidermis was assessed by MTT assay; the negative control was set at 100% cell viability. Since the viability was 93% after 3 min and 95% after 1 h, the test substance is not corrosive. To identify a irritating potential of the test item, 30 µl of the undiluted test item were applied to three epidermis samples and incubated for 1 hr at 37°C. Following removal of the test item, the tissue samples were incubated for 48 h at 37°C, before cell viability was measured by MTT assay. Cell viability was 77% in the treated samples, indicating that the test item does not possess a skin irritating potential. Based on the observed results and applying the evaluation criteria cited above 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.

Eye irritation:

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 test runs were performed. Two EpiOcular™ tissues per test run 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 test substance is not able to directly reduce MTT.

As the acceptance criterion for inter-tissue variability of the test substance was not met (values for single tissues: 105.1% and 155.9%) and the mean OD570 of the NC was exceptionally low (0.801), a 2nd experiment was performed to clarify the result. In the 2nd test run, the mean viability of the tissues treated with the test substance was 95.0%. All acceptance criteria were met. Minimal compound residues remained on the tissues treated with the test substance after the washing procedure of both test runs.

Based on the results observed in the EpiOcular Test alone and by applying the evaluation, it was concluded that the test substance does not show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

The potential of the read-across substance to cause damage to the conjunctiva, iris or cornea was assessed by a single ocular application of 0.1 mL of the test item to one eye of three White New Zealand rabbits (stepwise procedure starting with one animal and supplementing two additional animals). The study was conducted according to OECD 405 guideline and GLP (Bioassay, 2013). About 24 hours after application the eye was rinsed with tap water. The ocular reactions were assessed approximately 1, 24, 48 and 72 hours after application.

The following test item-related clinical observations were recorded during the course of the study:

- Slight and obvious conjunctival redness (grade 1-2)

- Slight conjunctival chemosis (grade 1)

- Slight or obvious discharge (grade 1-2).

- Additional findings like injected scleral vessels in a circumscribed area at hour 1 or hair loss from hour 24 until hour 72.

The ocular reactions were reversible in all animals within 48 or 72 hours after application, respectively.

Mean scores calculated for each animal over 24, 48 and 72 hours were 0.0, 0.0 and 0.0 for corneal opacity, 0.0, 0.0 and 0.0 for iris lesions, 0.7, 0.7 and 0.3 for redness of the conjunctiva and 0.0, 0.0 and 0.0 for chemosis.

Considering the described ocular reactions as well as the average score for irritation, the read-across substance does not show an eye irritating potential under the test conditions chosen.

The potential of the the read-across substance to induce eye irritation was assessed by means of an in vitro EpiOcular assay. 50 μL of the test substance was applied topically to a reconstructed three dimensional human cornea model (EpiOcular™). Two EpiOcular™ tissue samples were incubated with the test substance for 30 minutes followed by a 2-hours post-incubation period. Water was used as negative control and methyl acetate as positive control.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. Using this assay, the mean viability of the test-substance treated tissues was 96%, indicating that the test item does not have the potential to irritate the eyes.

The potential of the the read-across susbstance to cause serious damage to the eyes was assessed in an in vitro bovine cornea opacity and permeability (BCOP) test according to OECD guideline 437 and GLP. Three isolated cornea from freshly slaughtered cattle were prepared per treatment group (positive control (PC), negative control (NC) and test item); 750 μL of the undiluted liquid test substance was applied directly to the epithelial surface of the cornea using a pipette (open chamber method). Control tissues were concurrently applied into the anterior chamber with 750 μL of de-ionized water (NC) or with 750 μL of 1% (w/v) solution of sodium hydroxide in deionized water (PC) using a pipette. The corneas were incubated in a horizontal position at about 32 °C for approximately 10 minutes (liquids and surfactants) and the test item or controls removed thereafter. The corneas were incubated for further 2 hours at about 32 °C. After the incubation period the medium was removed and both chambers were then refilled with fresh Eagle’s MEM.

Corneal opacity was subsequently measured with an opacitometer; for determination of permeability the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (4 mg/mL for liquid test substances and surfactants) and incubated for 90 ± 5 min in a horizontal position at about 32 °C. The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was measured spectrophotometrically. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined.

The mean corneal opacity and permeability values of each treatment group were used to calculate an In Vitro Irritancy Score (IVIS). The positive and negative controls were within the hisorical control, indicating reproducibility and robustness of the test method. The calculated IVIS value is -1.5 and therefore below the threshold value of 55, it can be concluded that the test item does not cause any serious damage to the eyes.

Based on the available data, the test substance is not classified with regard to skin and eye irritation according to Directive 67/548/EEC (DSD) and Regulation (EC) No 1272/2008 (CLP), respectively.