Lavender, Lavandula hybrida, ext.

Endpoint summary

Description of key information

In a dermal irritation study (OECD 404) test item was not irritant to skin (GLP, Rel. K1)

In an in vitro eye irritation study (OECD 492) test item was considered irritant to the eyes (GLP, Rel K1)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin irritation study does not need to be conducted because adequate data from an in vivo skin irritation study are available

Reason / purpose for cross-reference:

data waiving: supporting information

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 October to 08 November 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 404 without any deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

16 December 2005

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: ONIPPAM / 081007
- Physical state: Green to pale yellow liquid
- Date of receipt: 15 October 2007

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at 4 °C, protected from light and under nitrogen gas
- Stability under test conditions: Test item was considered to be stable during the study period

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Grimaud frères selection S.A.S., La Corbière, Roussay, France.
- Age at study initiation: 2-4 months
- Weight at study initiation: Mean body weight: 3.0 ± 0.3 kg
- Housing: Animals were housed individually in Pajon cages (50 cm x 57 cm x 75 cm).
- Diet: 110C pelleted diet (SAFE, Villemoisson, Epinay-sur-Orge, France), ad libitum
- Water: Drinking water (filtered by a FG Millipore membrane (0.22 µm)), ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 ± 3 °C
- Humidity: 30-70 %
- Air changes: Approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod: 12 h dark/ 12 h light

IN-LIFE DATES: 23 October to 08 November 2007

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL
- Concentration (if solution): Undiluted
- pH of the test item was not measurable (non-aqueous test item)

CONTROL
- Untreated skin served as control

Duration of treatment / exposure:

4 h

Observation period:

1, 24, 48 and 72 h after removal of the dressing. Since there was a persistent irritation reaction at 72 h, the observation period was extended up to their complete reversibility (Day 15).

Number of animals:

3 males

Details on study design:

PRETREATMENT
Preparation and selection of the animals: The day before treatment, both flanks of each animal were clipped using electric clippers and just before treatment, the skin of each animal was examined in order to check the absence of any signs of skin irritation. Clipping was repeated thereafter on days 4 and 6 for the animal No. 555.

APPLICATION OF THE TEST ITEM
The test item was first evaluated on a single animal (No. 555). The durations of exposure were 3 minutes, 1 hour and 4 hours. Since the test item was not severely irritant nor corrosive on this first animal, it was then applied simultaneously for 4 hours to two other animals (Nos. 560 and 561).

TEST SITE
- Area of exposure: Test substance was applied to an area of approximately 6 cm2 of the anterior left flank (application for 3 minutes), the anterior right flank (application for 1 hour) or the posterior right flank (application for 4 hours) of the animals.
- Type of wrap if used: Test substance was placed on the gauze pad and held in contact with the skin by means of an adhesive hypoallergenic aerated semi-occlusive dressing and a restraining bandage.

REMOVAL OF TEST SUBSTANCE
- Washing: After removal of the dressing, any residual test item was wiped off by means of a dry cotton pad.
- Time after start of exposure: 4 hours

OBSERVATION TIME POINTS
1, 24, 48 and 72 h and Day 5 to 15

SCORING SYSTEM:
Draize scale, as described in OECD Guideline 404

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 8 days

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 8 days

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritant / corrosive response data:

After a 3-minute exposure (one animal)
- A very slight erythema (grade 1) was noted from day 1 until day 6.
- A dryness of the skin was observed from day 4 until day 8.
After a 1-hour exposure (one animal)
- A very slight erythema (grade 1) was noted from day 1 until day 7.
- A dryness of the skin was observed from day 4 until day 8.
After a 4-hour exposure (three animals)
- A very slight or well-defined erythema (grade 1 or 2) was noted in all animals from day 1 until day 6 (1/3 animal) or 7 (2/3 animals).
- A dryness of the skin was recorded in all animals from day 4 until day 8 (1/3 animals) or 15 (end of the observation period; 2/3 animals).
- Mean scores over 24, 48 and 72 hours for each animal were 1.0, 1.0 and 1.3 for erythema and 0.0, 0.0 and 0.0 for edema.

Other effects:

None

Table 7.3.1/1: 4-hour exposure - Individual cutaneous examinations and mean values of the scores recorded for each animal (24, 48 and 72 hours)

 

Score at time point	Erythema (Animal No. 1 / 2 / 3) Max. score 4	Oedema (Animal No. 1 / 2 / 3) Max. score 4	Other (Animal No. 1 / 2 / 3)
1 h	2/2/2	0/0/0	*/*/*
24 h	1/1/2	0/0/0	*/*/*
48 h	1/1/1	0/0/0	*/*/*
72 h	1/1/1	0/0/0	S/S/S
Day 5	1/1/1	0/0/0	S/S/S
Day 6	1/1/1	0/0/0	S/S/S
Day 7	1/1/0	0/0/0	S/S/S
Day 8	0/0/0	0/0/0	S/S/S
Day 9	0/0/0	0/0/0	*/S/S
Day 10	-/0/0	-/0/0	-/S/S
Day 11	-/0/0	-/0/0	-/S/S
Day 12	-/0/0	-/0/0	-/S/S
Day 13	-/0/0	-/0/0	-/S/S
Day 14	-/0/0	-/0/0	-/S/S
Day 15	-/0/0	-/0/0	-/S/S
Average 24, 48 and 72 h	1.0/1.0/1.3	0/0/0	-
Reversibility	Completely reversible	-	-
Average time for reversion	8 days	-	-

Animal No. 1 / 2 / 3 = Animal No. 555, 560 and 561, respectively

* = none

S = dryness of the skin

- = cutaneous examination not performed

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test substance is not classified as irritant according to Regulation (EC) No 1272/2008 (CLP) and to GHS.

Executive summary:

A dermal irritation study performed according to the OECD Guideline No. 404, and in compliance with GLP. The test substance was first applied for periods of 3 minutes, 1 hour and 4 hours to a single male New Zealand White rabbit. Since the test substance was not severely irritant nor corrosive on this first animal, it was then applied simultaneously for 4 hours to two other animals.

 

A single dose of 0.5 mL of the undiluted test substance was applied to the closely-clipped skin of one flank. The test substance was held in contact with the skin by means of a semi-occlusive dressing. Cutaneous reactions were observed approximately 1 hour, 24, 48 and 72 hours after removal of the dressing and then daily until the end of the observation period. The mean values of the scores for erythema and edema were calculated for each animal.

 

After a 3-minute exposure (one animal): A very slight erythema was noted from day 1 until day 6. A dryness of the skin was observed from day 4 until day 8.

After a 1-hour exposure (one animal): A very slight erythema was noted from day 1 until day 7. A dryness of the skin was observed from day 4 until day 8.

After a 4-hour exposure (three animals): A very slight or well-defined erythema was noted in all animals from day 1 until day 6 (1/3 animal) or 7 (2/3 animals). A dryness of the skin was recorded in all animals from day 4 until day 8 (1/3 animals) or 15 (end of the observation period; 2/3 animals).

 

Mean scores over 24, 48 and 72 hours for each animal were 1.0, 1.0 and 1.3 for erythema and 0.0, 0.0 and 0.0 for edema.

 

Under the test conditions, test substance is not classified as irritant to the skin according to Regulation (EC) No. 1272/2008 (CLP) and GHS.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

19 January to 04 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 492 without any deviations.

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)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

05 March 2015

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 150095
- Physical state: Transparent liquid
- Dates of receipt: 15 January 2016 (used for the preliminary and invalidated first main test); 09 February 2016 (used for the validated first main test); 23 May 2016 (used for the second main test); 04 July 2016 (used from the third main test)
- Expiration date of the lot/batch: 30 September 2018
- Purity test date: 11 January 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored in the refrigerator set at 5 °C, protected from light, under nitrogen atmosphere

Species:

other: Reconstructed Human cornea-like epithelium

Details on test animals or tissues and environmental conditions:

Species: Reconstructed Human cornea-like epithelium (tissues).
Supplier: MatTek, Bratislava, Slovak Republic.
Selection: At receipt, the tissues were inspected for obvious defects as they could have been rejected based on blistering, excess fluid or air bubbles below the tissue insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used.
Storage conditions: At receipt, the living EpiOcular™ tissues were stored as described in the Pre-incubation of the tissues, on their day of arrival.

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): 50 μL
- Concentration (if solution): Undiiluted

Duration of treatment / exposure:

During each main test, the test item and both negative and positive controls were applied topically on duplicate tissues and incubated at 37 °C for 30 minutes (± 2 minutes).

Duration of post- treatment incubation (in vitro):

At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 12 minutes (± 2 minutes) at room temperature to remove any remaining test item absorbed into the tissue, blotted on absorbent material, and then incubated for another 2 hours (± 15 minutes) at 37 °C, 5% CO2 in a humidified incubator.

Number of animals or in vitro replicates:

Test item, negative and positive controls were applied on duplicate tissues.

Details on study design:

- RhCE tissue construct used, including batch number: The EpiOcularTM (OCL-200, OCL-212) model consists of an airlifted, living, multilayered ocular tissue construction (surface 0.60 cm2), reconstructed from normal (non-transformed) human-derived keratinocytes. This is a non-keratinized epithelium which models the cornea epithelium with progressively stratified, but not cornified cells. The cells are cultured in proprietary serum-free culture media, which induces corneal differentiation and the formation of the organotypic 3D cornea-like model. The 3D tissue consists of highly organized cell layers similar to that found in the cornea. The model features a normal ultra-structure and is functionally equivalent to human in vivo tissue. The EpiOcular tissues were used within 72 hours of their production. Batch numbers 23700; 23711 and 23722 were used for the first, second and third main tests, respectively.

Preliminary tests: Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its coloring potential.
- Test for direct MTT reduction with the test item: 50 μL of the test item were added to 1 mL of a 1.0 mg/mL freshly prepared MTT solution; a negative control was tested concurrently by adding 50 μL of sterile deionized water to 1 mL of a 1.0 mg/mL freshly prepared MTT solution. Both mixtures were incubated in darkness at 37 °C for 3 hours (± 10 minutes) and color of the solutions obtained was evaluated.
- Test for the detection of the coloring potential of the test item: The maximum amount of test item, 50 μL was added to: (i) 1 mL of water and incubated for at least 1 hour in the dark at 37 °C, 5% CO2 and (ii) 2 mL of isopropanol, incubated in a 6-well plate and placed on an orbital plate shaker for 2 to 3 hours at room temperature. After that, the presence and intensity of the coloration were evaluated.

Main test:
- Doses of test chemical and control substances used: 50 μL

- Pre-incubation of the tissues: On the day of treatment for the first main test or on the day before treatment for the second and third main tests, tissues were equilibrated (in the 24-well shipping container) to room temperature for at least 15 minutes. The tissue inserts were transferred aseptically into the 6-well plate and pre-incubated at 37 °C, 5% CO2 in a humidified incubator for 1 hour. After the pre-incubation period, the assay medium was removed and replaced by 1 mL of fresh assay medium before incubation overnight (16-24 h) at 37 °C, 5% CO2 in a humidified incubator.

Treatment of tissues
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: During each main test, the test item and both negative and positive controls were applied topically on duplicate tissues and incubated at 37 °C for 30 minutes (± 2 minutes). At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 12 minutes (± 2 minutes) at room temperature to remove any remaining test item absorbed into the tissue, blotted on absorbent material, and then incubated for another 2 hours (± 15 minutes) at 37 °C, 5% CO2 in a humidified incubator.

- Number of tissue replicates used per test chemical and controls (positive control, negative control): One 6-well plate was used for the first test item-treated tissues. Positive and negative controls were placed on separate 6-well plates (one plate for each).Test item, negative and positive controls were applied on duplicate tissues.

- MTT viability assay: Following the post-treatment incubation, the cell viability was assessed by means of the colorimetric MTT reduction assay. Tissues were incubated with MTT solution in 24-well plates for 3 hours (± 10 minutes) at 37 °C, 5% CO2 in a humidified incubator. At the end of the 3-hour incubation period, tissues were blotted on absorbent paper and the degree of MTT staining was evaluated. For the test item, negative and positive control-treated tissues, the inserts were transferred to new wells of the 24-well plate containing 2 mL of isopropanol per well. Formazan extraction was performed overnight at 2-8 °C and protected from light.

- Optical Density measurements: At the end of the formazan extraction period, the plates were placed under orbital shaking at room temperature for at least 15 minutes before using them. Then, tissues (test item, negative and positive control treated tissues) were pierced. The extract solution was mixed and two 200 μL aliquots were transferred to the pre-labeled 96-well plate. For each 96-well plate, the average Optical Density value (OD) of 4 wells containing 200 μL of isopropanol only was used as the blank. The OD was measured at 570 nm using a plate reader.
Mean viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).

Irritation parameter:

other: relative mean viability of the tissues

Run / experiment:

First main test

Value:

64

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

mean % viability equal to 60 ± 5% is considered as a borderline result, it was agreed with the Sponsor to perform a second main test.

Irritation parameter:

other: relative mean viability of the tissues

Run / experiment:

Second main test

Value:

53

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

non concordant results were obtained in the two first main tests, it was agreed with the Sponsor to perform a third main test.

Irritation parameter:

other: relative mean viability of the tissues

Run / experiment:

Third main test

Value:

59

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

mean % viability was equal to 60 ± 5% and since viability results obtained for each individual tissue led to non-concordant classification, this was considered as a borderline result.

Other effects / acceptance of results:

PRELIMINARY TESTS
Test for direct MTT reduction with the test item: The MTT solution containing the test item did not turn blue/purple when compared with the negative control. The test item was therefore considered not to have direct MTT reducing properties. As a result, no additional controls were performed on freeze-dead tissues in parallel to the main test.
Test for the detection of the coloring potential of the test item: During this test, as both water and isopropanol solutions containing the test item did not change color, the test item was found not to have a coloring potential. As a result, no additional controls were used in parallel to the main test.

MAIN TESTS
Evaluation of the coloration of tissues at the end of the MTT incubation period: All test item-treated tissues appeared blue/white which was considered to be indicative of semi-viable tissue.
Evaluation of the MTT results:
First main test: The relative mean viability of the tissues treated with the test item was 64% with a difference of 1% between duplicate tissues. As the mean viability was > 60% after the MTT reduction, the results met the criteria for a non-irritant response. However, since a mean % viability equal to 60 ± 5% is considered as a borderline result, it was agreed with the Sponsor to perform a second main test.
Second main test: The relative mean viability of the tissues treated with the test item was 53% with a difference of 3% between duplicate tissues. As the mean viability was < 60% after the MTT reduction, the results met the criteria for an irritant response. However, since non concordant results were obtained in the two first main tests, it was agreed with the Sponsor to perform a third main test.
Third main test: The relative mean viability of the tissues treated with the test item was 59% with a difference of 13% between duplicate tissues. As the mean viability was < 60% after the MTT reduction, the results met the criteria for an irritant response. However since mean % viability was equal to 60 ± 5% and since viability results obtained for each individual tissue led to non-concordant classification, this was considered as a borderline result.
Two out of three main tests gave irritant responses (with one of them with borderline results, i.e. mean % viability equal to 60 ± 5%). When tissues (from all main tests) are considered individually: 50% gave irritant responses (i.e. below 60% viability) and 50% non-irritant response. Even if two out of three main tests gave borderline results, a fourth main test would not be necessary. When all main tests are considered together, the mean %viability tends to borderline results with positive classification (irritant), indeed a significant decrease of viability is obtained. As a result and under our experimental conditions, the test item is considered to be irritating to Reconstructed human Cornea-like Epithelium.

ACCEPTANCE OF RESULTS:
- All of the acceptance criteria for the negative and positive controls were fulfilled, therefore each main test was considered as valid.

Table 7.3.2/1: Main tests: Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls

 

Group	Exposure duration	Tissue No.	OD570 nmmeasurements		Mean blank	cOD570 nmmeasurements		Mean cOD570 nm	Viability (%)
			1st	2nd		1st	2nd
First main test
Negative control	30 min	1	1.847	1.850	0.037	1.810	1.813	1.812	96
		2	2.001	1.993		1.964	1.956	1.960	104
Positive control	30 min	1	0.686	0.695	0.037	0.649	0.658	0.654	35
		2	0.774	0.780		0.737	0.743	0.740	39
Test item	30 min	1	1.245	1.236	0.038	1.207	1.198	1.202	64
		2	1.261	1.255		1.223	1.217	1.220	65
Second main test
Negative control	30 min	1	1.992	1.936	0.041	1.952	1.896	1.924	95
		2	2.154	2.172		2.114	2.132	2.123	105
Positive control	30 min	1	0.947	0.953	0.041	0.907	0.913	0.910	45
		2	0.775	0.773		0.735	0.733	0.734	36
Test item	30 min	1	1.145	1.143	0.037	1.108	1.106	1.107	55
		2	1.091	1.084		1.054	1.047	1.050	52
Third main test
Negative control	30 min	1	1.992	1.990	0.043	1.949	1.947	1.948	102
		2	1.909	1.924		1.866	1.881	1.873	98
Positive control	30 min	1	0.679	0.677	0.043	0.636	0.634	0.635	33
		2	0.631	0.632		0.588	0.589	0.588	31
Test item	30 min	1	1.040	1.039	0.042	0.998	0.997	0.997	52
		2	1.290	1.289		1.248	1.247	1.247	65

 

Table 7.3.2/2: Main tests - Mean tissue viability and standard deviations for the test item, the negative and positive controls

 

Group	Exposure duration	cOD570 nm		Viability (%)
		Mean	SD	Mean	SD	Difference (%)
First main test
Negative control	30 min	1.886	0.105	100	6	8
Positive control	30 min	0.697	0.061	37	3	5
Test item	30 min	1.211	0.012	64	1	1
Second main test
Negative control	30 min	2.023	0.141	100	7	10
Positive control	30 min	0.822	0.124	41	6	9
Test item	30 min	1.079	0.040	53	2	3
Third main test
Negative control	30 min	1.911	0.053	100	3	4
Positive control	30 min	0.612	0.033	32	2	2
Test item	30 min	1.122	0.177	59	9	13

 

OD = optical density

cOD = blank corrected optical density

SD = Standard deviation

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Under the experimental conditions of this study and based on results of three independent and validated main tests, the test item is considered to be irritating to Reconstructed human Cornea-like Epithelium. According to the results of this study, the classification of the test item should be:category 2 (GHS 2013) and category 2 (H319) (Regulation (EC) No. 1272/2008).

Executive summary:

An in vitro eye irritation teston the EpiOcular^TM cornea epithelial model was performed according to the OECD Guideline 492 and in compliance with GLP to predict the acute eye irritation potential of the test item.

Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its coloring potential. Following the preliminary tests, the eye irritation potential of the test item was assessed in main tests. During each main test, the test item and both negative and positive controls were applied topically on duplicate tissues and incubated at 37 °C for 30 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 12 minutes at room temperature to remove any remaining test item absorbed into the tissue, blotted on absorbent material, and then incubated for another 2 hours at 37 °C, 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colorimetric MTT reduction assay. Mean viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).

 

In the preliminary tests, the test item was found not to have direct MTT reducing properties or coloring potential.

 

First main test: The relative mean viability of the tissues treated with the test item was 64% with a difference of 1% between duplicate tissues. As the mean viability was > 60% after the MTT reduction, the results met the criteria for a non-irritant response. However, since a mean % viability equal to 60 ± 5% is considered as a borderline result, it was agreed with the Sponsor to perform a second main test.

Second main test: The relative mean viability of the tissues treated with the test item was 53% with a difference of 3% between duplicate tissues. As the mean viability was < 60% after the MTT reduction, the results met the criteria for an irritant response. However, since non-concordant results were obtained in the two first main tests, it was agreed with the Sponsor to perform a third main test.

Third main test: The relative mean viability of the tissues treated with the test item was 59% with a difference of 13% between duplicate tissues. As the mean viability was < 60% after the MTT reduction, the results met the criteria for an irritant response. However since mean % viability was equal to 60 ± 5% and since viability results obtained for each individual tissues led to non-concordant classification, this was considered as a borderline result.

Two out of three main tests gave irritant responses (with one of them with borderline results,i.e.mean % viability equal to 60 ± 5%). When tissues (from all main tests) are considered individually: 50% gave irritant responses (i.e.below 60% viability) and 50% non-irritant response. Even if two out of three main tests gave borderline results, a fourth main test would not be necessary. When all main tests are considered together, the mean % viability tends to borderline results with positive classification (irritant), indeed a significant decrease of viability is obtained. As a result and under our experimental conditions, the test item is considered to be irritating to Reconstructed human Cornea-like Epithelium.

All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.

 

Under the experimental conditions of this study and based on results of three independent and validated main tests, the test item is considered to be irritating to Reconstructed human Cornea-like Epithelium. According to the results of this study, the classification of the test item should be: category 2 (GHS 2013) and category 2 (H319) (Regulation (EC) No. 1272/2008).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

A dermal irritation study performed according to the OECD Guideline No. 404, and in compliance with GLP. The test substance was first applied for periods of 3 minutes, 1 hour and 4 hours to a single male New Zealand White rabbit. Since the test substance was not severely irritant nor corrosive on this first animal, it was then applied simultaneously for 4 hours to two other animals.

 A single dose of 0.5 mL of the undiluted test substance was applied to the closely-clipped skin of one flank. The test substance was held in contact with the skin by means of a semi-occlusive dressing. Cutaneous reactions were observed approximately 1 hour, 24, 48 and 72 hours after removal of the dressing and then daily until the end of the observation period. The mean values of the scores for erythema and edema were calculated for each animal.

Mean scores over 24, 48 and 72 hours for each animal were 1.0, 1.0 and 1.3 for erythema and 0.0, 0.0 and 0.0 for edema.

Therefore the susbtance was considered not irritant to skin.

Eye irritation:

An in vitro eye irritation test on the EpiOcularTM cornea epithelial model was performed according to the OECD Guideline 492 and in compliance with GLP to predict the acute eye irritation potential of the test item.

- First main test: The relative mean viability of the tissues treated with the test item was 64% with a difference of 1% between duplicate tissues. As the mean viability was > 60% after the MTT reduction, the results met the criteria for a non-irritant response. However, since a mean % viability equal to 60 ± 5% is considered as a borderline result, it was agreed with the Sponsor to perform a second main test.

- Second main test: The relative mean viability of the tissues treated with the test item was 53% with a difference of 3% between duplicate tissues. As the mean viability was < 60% after the MTT reduction, the results met the criteria for an irritant response. However, since non-concordant results were obtained in the two first main tests, it was agreed with the Sponsor to perform a third main test.

- Third main test: The relative mean viability of the tissues treated with the test item was 59% with a difference of 13% between duplicate tissues. As the mean viability was < 60% after the MTT reduction, the results met the criteria for an irritant response. However since mean % viability was equal to 60 ± 5% and since viability results obtained for each individual tissues led to non-concordant classification, this was considered as a borderline result.

Two out of three main tests gave irritant responses (with one of them with borderline results,i.e.mean % viability equal to 60 ± 5%). When tissues (from all main tests) are considered individually: 50% gave irritant responses (i.e.below 60% viability) and 50% non-irritant response. Even if two out of three main tests gave borderline results, a fourth main test would not be necessary. When all main tests are considered together, the mean % viability tends to borderline results with positive classification (irritant), indeed a significant decrease of viability is obtained. As a result and under our experimental conditions, the test item is considered to be irritating to Reconstructed human Cornea-like Epithelium.

The substance is not expected to be classified as H318 (Causes serious eye damage), as it is not irritant to the skinand it showed results very close to criterion for the absence of classification (cell viability around 60%) in an Epiocular test (OECD guideline 492).

Justification for classification or non-classification

Self-classification:

Based on the available information (OECD 404 test), the substance is not classified for skin irritation/ corrosion according to the Regulation (EC) No. 1272/2008.

Based on the available information, the substance should be classified as eye irritant Category 2 (H319: Causes serious eye irritation) according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP). The substance is not expected to be classified as H318 (Causes serious eye damage), as it is not irritant to the skin and it showed results very close to criterion for the absence of classification (cell vialbility around 60%) in an Epiocular test (OECD guideline 492).

No data was available regarding respiratory irritation.