Absolute of Nicotiana tabacum (Solanaceae) obtained from leaves by organic solvent treatment and subsequent ethanol extraction

Endpoint summary

Description of key information

Skin :

- Skin irritation (OECD 439, K, Rel.1): “Irritating to skin” or in Category 1 “Corrosive”.

- Skin corrosion (OECD 431, K, Rel.1): not classified for skin corrosion,

<=> The test substance is therefore classified as Skin irritant (Category 2)

 

Eye:

- Eye irritation (OECD 492, K, Rel.1): potentially requiring classification and labeling according to UN GHS Category 2 or Category 1.

- Eye corrosion (OECD 438, K, Rel.1): not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method.

<=> The test item can be classified as eye irritant (Category 2).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 14 February and 29 April 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 431 without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

18 June 2019

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

14 October 2019

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: foreskin

Justification for test system used:

Following the REACH bottom-up strategy described in the ECHA R.7a guidance, after the positive result obtained in the SkinEthic RHE® model method for skin irritation, the EpiCS™ RHE Model method was used to assess skin corrosion.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: 0.6 cm² reconstituted epidermis and four additional killed control tissues (EpiCS™, supplied by CellSystems)
- Tissue batch number(s): 100-AJ0806-1 (living tissues) / 100-AI1214-1 (killed control tissues)
- Expiration date: no data
- Date of received: 28 April 2020 for the living tissues and defrozen on 29 April 2020 for the killed control tissues

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minutes (room temperature) and 1 hour (37°C +/- 1°C)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1 mL DPBS, 20 times
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL concentration for the skin sample and MTT assay medium for the 4 tissues used for the non-specific control
- Incubation time: 3 hours at 37 +/- 1°C and 5% CO2.
- Spectrophotometer: ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: viability % = OD test item/OD negative control x 100. For each tissue, OD values and calculated percentage cell viability data for the test item, positive and negative controls, were calculated.
- As the test item was identified as producing direct MTT reduction, the true tissue viability is then calculated as follows: True viability % = [(OD of living tissues exposed to test item - OD of killed tissues exposed to test item) / OD of living tissues exposed to negative control] x 100

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The direct interaction of MTT with the test item was checked by adding 50 µL of the test item to 1 mL of solution of MTT at 1 mg/mL. A purple suspension was observed after 1 hour of incubation between 37.1°C and 37.4°C, 5% CO2.
> Therefore, the test item was identified as a direct MTT reducer. Four additional killed control tissues were added to the study. These four control tissues (two for 3-minute treatment and two for 1-hour treatment) were incubated with culture medium instead of MTT solution during the MTT incubation step to generate a non-specific colour control (NSC control).

SPECTRAL ANALYSIS OF THE TEST ITEM IN ISOPROPANOL
The spectral properties at 570 nm of test item in isopropanol were checked by adding 50 µL of the test item to 2 mL of isopropanol. A yellow solution was obtained after 3 hours of incubation at ambient temperature with gentle shaking. The mean of the corrected OD was 0.067 which is lower than 0.08.
> Therefore, the test item will not interfere with the MTT assay and there is no need to add non-specific coloration controls to the study.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

PREDICTION MODEL / DECISION CRITERIA
- Step 1: The test substance is considered to be classified to skin as follows if the viability is:
< 50% after 3 min exposure = Corrosive
≥ 50% after 3 min exposure AND < 15% after 60 min exposure = Corrosive
≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure = Non-corrosive
- Step 2: The test substance is subcategorised for items identified as corrosive in step 1:
1A if viability is < 15% after 3 min exposure
1B/1C if viability is ≥ 15% after 3 min exposure

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Remarks:

corrected with 4 killed human skin model surfaces to generate non-specific MTT reduction

Run / experiment:

3 minutes

Value:

84.47

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

58.06%

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

corrected with 4 killed human skin model surfaces to generate non-specific MTT reduction

Run / experiment:

1 hour

Value:

86.98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

0.5 %

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

MTT VIABILITY ASSAY RESULTS
- The mean percent viability of the epidermis skins treated with the test item were 84.47% and 86.98%, versus 58.06% and 0.5%, respectively, with the positive control item (potassium hydroxide 8N).

- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: Yes, four additional killed control tissues were used to generate non-specific MTT reduction
- Colour interference with MTT: none.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Proficiency chemicals were tested according to the OECD TG 431.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the negative control OD of the 2 replicates is >= 0.8 and <= 2.8 for every exposure time. As the extract was diluted at 1:3 just before the OD mesure, the acceptability criteria should be in the range >= 0.3 and <=0.9 for the negative control (values 0.667 for 3-minute exposure and 0.599 for 1-hour exposure).
- Acceptance criteria met for positive control: yes, the mean tissue viability of the 2 replicates exposed for 1 hour are < 20% (0.50 %)
- Acceptance criteria met for variability between replicate measurements: yes, in the range 20-100% viability, and for ODs ≥ 0.3, difference of viability between the two tissue replicates was < 30%.
- Acceptance criteria of range of historical values: The mean viability of epidermises treated with positive control during 3 minutes was 58.06%, which is in the range of historical data (0.40% and 85.12%) for 3-minute exposure and the mean viability of epidermises treated with positive control during 1-hour was 0.50%, which is in the range of historical data (0.00% and 1.32%) for 1-hour exposure.

Table 7.3.1/1: Main test - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls

 

Tissue	Exposure Period	OD	Mean OD/ disc (#)	Mean OD / Product	Viability (%)	Mean viability (%)	Viability difference between replicates (%)
Negative Control	3 Minutes	0.685 0.697 0.713	0.698	0.667	104.73	100.00	4 9.5
		0.644 0.635 0.627	0.635		95.27
	60 Minutes	0.595 0.656 0.643	0.631	0.599	105.34	100.00	10.7
		0.554 0.557 0.590	0.567		94.66
Positive Control	3 Minutes	0.319 0.347 0.345	0.337	0.387	50.56	58.06	15.0
		0.450 0.435 0.428	0.437		65.57
	60 Minutes	0.003 0.002 0.004	0.003	0.003	0.50	0.50	0.0
		0.002 0.003 0.003	0.003		0.50
Test Item	3 Minutes	0.625 0.624 0.620	0.623	0.576	93.47	86.42	14.1
		0.521 0.542 0.526	0.529		79.37
	60 Minutes	0.578 0.611 0.602	0.597	0.532	99.67	88.81	21.7
		0.459 0.477 0.465	0.467		77.96
Test item killed tissues	3 minutes	0.017 0.017 0.016	0.016	0.013	2.40	1.95	0.9
		0.010 0.011 0.011	0.010		1.50
	60 minutes	0.012 0.013 0.010	0.012	0.011	2.00	1.84	0.3
		0.009 0.009 0.011	0.010		1.67
Test item corrected	3 minutes					84.47
	60 minutes					86.98

 

OD: optical density

#: mean of 3 values

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the experimental conditions of this study, the test substance is not classified for skin corrosion according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS. Based on the result of the in vitro skin irritation study, the test substance is therefore classified as Skin irritant Category 2 (H315: Causes skin irritation) according to CLP and GHS

Executive summary:

An in vitro skin corrosion study was performed according to the OECD Guideline 431 and in compliance with GLP, using the EpiCS reconstructed human epidermis model.

The test item TOBACCO ABSOLUTE was applied as supplied, at the dose of 50 µL, to 2 living Human skin model surfaces (epiCS®, CellSystems®) during 3 minutes and 1 hour, followed by a rinse with 20 mL of DPBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. Additionally, 4 killed Human skin model surfaces were treated under the same experimental conditions in order to generate non-specific MTT reduction. 

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 84.47% and 86.98%, versus 58.06% and 0.5%, respectively, with the positive control item (potassium hydroxide 8N).

All the acceptability criteria were met and the quality criteria required for acceptance of results in the test were satisfied:

- the negative control OD of the 2 replicates is >= 0.8 and <= 2.8 for every exposure time. As the extract was diluted at 1:3 just before the OD mesure, the acceptability criteria should be in the range >= 0.3 and <=0.9 for the negative control (values 0.667 for 3-minute exposure and 0.599 for 1-hour exposure).
- the mean tissue viability of the 2 replicates exposed for 1 hour are < 20% (0.50 %)
- Variability between replicate measurements in the range 20-100% viability, and for ODs ≥ 0.3, difference of viability between the two tissue replicates was < 30%.
- The mean viability of epidermises treated with positive control during 3 minutes was 58.06%, which is in the range of historical data (0.40% and 85.12%) for 3-minute exposure and the mean viability of epidermises treated with positive control during 1-hour was 0.50%, which is in the range of historical data (0.00% and 1.32%) for 1-hour exposure.

Under the experimental conditions of this study, the test substance is not classified for skin corrosion according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS. Based on the result of the in vitro skin irritation study, the test substance is therefore classified as Skin irritant Category 2 (H315: Causes skin irritation) according to CLP and GHS.

This study is considered as acceptable and satisfies the requirement for skin corrosion endpoint.

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:

key study

Study period:

15 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 438 without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

adopted 25 June 2018

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

Signed 14 October 2019

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES

- Source: The eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption have been used for this assay.

- Characteristics of donor animals (e.g. age, sex, weight): The age and weight of the chickens used in this test method are that of spring chickens traditionally processed by a poultry slaughterhouse (i.e., approximately 7 weeks old, 1.5 - 2.5 kg).

- Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. The heads have been collected on 15 June 2020 at 8:20 am.

- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline. The eyes were enucleated at Phycher on 15 June 2020 at 10:00 am.

- Indication of any existing defects or lesions in ocular tissue samples: None

- Indication of any antibiotics used: None

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): 30 µL of the pure test substance.

- Concentration (if solution): Test item was used as supplied

Duration of treatment / exposure:

Test item was applied for 10 seconds to the cornea

Number of animals or in vitro replicates:

1, 3 and 3 eyes for negative & positive control and test item, respectively.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
- The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.

- The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus were at a controlled temperature of 32.0 °C.

- After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.

- Once all eyes had been examined and approved, the eyes were incubated between 45 and 65 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

APPLICATION DOSE AND EXPOSURE TIME
- Immediately following the zero reference measurements, the eye (in its holder) was removed from the superfusion apparatus, placed in a horizontal position, and 30 µL of the test item was applied for 10 seconds to the cornea such that the entire surface of the cornea was evenly covered with the test item.

REMOVAL OF TEST SUBSTANCE
- After exposure, test item was rinsed from the eye with 10 mL of physiological saline at ambient temperature. The eye (in its holder) was subsequently returned to the superfusion apparatus in the original upright position.

OBSERVATION PERIOD
- Treated corneas were evaluated before the pre-treatment and at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

METHODS FOR MEASURED ENDPOINTS:
- All observations of the cornea and measurement of corneal thickness were performed using a Haag-Streit BP900 slit-lamp microscope with depth-measuring device no. I. For the measurement of corneal thickness, the slit-width was set at 9½, equalling 0.095 mm.

- The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which was determined only at pretreatment and 30 minutes after exposure to the test item) were determined at each of the above time points.

SCORING SYSTEM:
- The Corneal swelling was determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope. It was expressed as a % and was calculated from corneal thickness measurements according to the following formula:
Corneal swelling (%) = ((corneal thickness at time t - corneal thickness at time = 0) / (corneal thickness at time = 0)) x 100

The mean percentage of corneal swelling for all tested eyes was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was then given for the test item.

- Mean maximum opacity score: Corneal opacity was calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all tested eyes was calculated for all observation time points. Based on the highest mean score for corneal opacity, as observed at any time point, an overall category score was then given for each test or control item.

0: No opacity

0.5: Very faint opacity

1: Scattered or diffuse areas; details of the iris clearly visible

2: Easily discernible translucent area; details of the iris are slightly obscured

3: Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible

4: Complete corneal opacity; iris invisible

- Mean fluorescein retention score at 30 minutes post-treatment: The mean fluorescein retention value for all tested eyes was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item.

0: No fluorescein retention

0.5: Very minor single cell staining

1: Single cell staining scattered throughout the treated area of the cornea

2: Focal or confluent dense single cell staining

3: Confluent large areas of the cornea retaining fluorescein

- Morphological effects include "pitting" of corneal epithelial cells, "loosening" of epithelium, "roughening" of the corneal surface and "sticking" of the test item to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the the interpretation of the investigator.

DECISION CRITERIA:

- Results from corneal opacity, swelling, and fluorescein retention were evaluated separately to generate an ICE class for each endpoint. The ICE classes for each endpoint were then combined to generate an Irritancy Classification for the test item.

- Once each endpoint was evaluated, ICE classes were assigned based on a predetermined range. Interpretation of corneal thickness, opacity, and fluorescein retention using four ICE classes was done according to the table 7.3.2/1, 7.3.2/2, 7.3.2/3.

Irritation parameter:

cornea opacity score

Run / experiment:

maximal mean score

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Run / experiment:

mean score

Value:

1.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Run / experiment:

maximal mean score

Value:

7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OCULAR REACTIONS:

- maximal mean score of corneal opacity: 1.0, corresponding to ICE class II;

- mean score of fluorescein retention: 1.7, corresponding to ICE class III;

- maximal mean corneal swelling: 7%, corresponding to ICE class II.

The combination of the three endpoints for test item was 2 x II, 1 x III.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected. It is also within the results obtained in the historical negative control data.

- Acceptance criteria met for positive control: The combination of the three endpoints for the positive control, sodium hydroxide was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe Irritant", as expected. It is also similar to the results obtained in the historical control data.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

In accordance with Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category "no prediction can be made", as defined by the OECD guideline No.438. Therefore, the test item TOBACCO ABSOLUTE is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method. Based on the results from the previous epiOcular study OECD 492, the test item can be classified as eye irritant (Category 2).

Executive summary:

An ex vivo eye irritation study was performed according to the OECD Guideline 438 and in compliance with GLP to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

Test item TOBACCO ABSOLUTE was applied as supplied , at the dose of 30 µL, to 3 enucleated chicken eyes during 10 seconds.

Then the eyes were rinsed twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose.

 

The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 1.0, corresponding to ICE class II;

- mean score of fluorescein retention: 1.7, corresponding to ICE class III;

- maximal mean corneal swelling: 7%, corresponding to ICE class II.

 

The combination of the three endpoints for test item was 2 x II, 1 x III.

The combination of the three endpoints for the positive control, sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.

The combination of the three endpoints for the negative control, physiological saline, was 3 x I.

Therefore, the negative control is classified as “No Category”, as expected.

In accordance with Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category "no prediction can be made", as defined by the OECD guideline No.438. Therefore, the test item TOBACCO ABSOLUTE is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method. 

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 2019 to April 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 492 without any deviation.

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:

Adopted 25 June 2018

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Species:

other: Reconstructed human Cornea-like Epithelia

Details on test animals or tissues and environmental conditions:

Description of the cell system used: 0.60 cm² Reconstructed human Cornea-like Epithelia [EpiOcular(TM) OCL-212-ver2.0, supplied by MatTek Corporation]

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Concentration: Undiluted

Duration of treatment / exposure:

30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions)

Duration of post- treatment incubation (in vitro):

- Post-exposure immersion period: 12 minutes at room temperature - Post-exposure incubation period: 2 hours at standard culture conditions

Number of animals or in vitro replicates:

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

Details on study design:

TISSUES CONSTRUCTS
- RhCE tissue construct used, including batch number: 0.60 cm² Reconstructed human Cornea-like Epithelia [EpiOcular(TM) OCL-212-ver2.0, supplied by MatTek Corporation, batch No. 30640] were received on 15 January 2020. The 2 additional killed Human skin models [EpiOcular(TM) OCL-212-ver2.0, supplied by MatTek Corporation, batch No. 30614] had been frozen on 02 July 2019 and defrozen on 13 November 2019 (1st freeze-thaw cycle), refrozen on 14 November 2019 to be defrozen the day of the treatment, on 16 January 2020 (2nd freeze-thaw cycle).

- Pre-incubation of the tissues:
The same day, the tissues in their well shipping container were equilibrated to room temperature for 15 minutes. Then the inserts (filter + epithelium) were gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. They were placed in 6 wells culture plate which had been previously filled with 1 mL of 37°C pre-warmed culture medium and incubated during 20 hours and 40 minutes, at standard culture conditions.

- Indication of controls used for direct MTT-reducers:
The direct interaction of MTT with the test item was checked by adding 50 µL of the test item to 1 mL of solution of MTT at 1 mg/mL. A purple suspension was observed after 3 hours of incubation between 36.6°C and 37.5°C, 5% CO2.
> Therefore, the test item was identified as a direct MTT reducer. Two additional killed control tissues were added to the study which underwent the entire testing procedure to generate a non-specific colour control (NSC control).

- Indication of controls used for colouring test chemicals:
The spectral properties at 570 nm of test item in isopropanol were checked by adding 50 µL of the test item to 2 mL of isopropanol. A yellow solution was obtained after 2 hours and 1 minute of incubation at ambient temperature with gentle shaking. The mean of the corrected OD was 0.071 which is lower than 0.08.
> Therefore, the test item will not interfere with the MTT assay and there is no need to add non-specific coloration controls to the study.

MAIN TEST
- Pre-treatment:
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca2+Mg2+Free-DPBS. The tissues were incubated at standard culture conditions for 30 minutes.

- Treatment and post-treatment incubation of the tissues:
The test item was applied as supplied, at the dose of 50 µL, to the entire surface of 2 living RhCE tissue replicates and 2 killed RhCE (EpiOcular(TM) tissue model) (NSC control) during 30 minutes at standard culture conditions.

In the same experimental conditions, a positive control (Methyl acetate - Sigma-Aldrich, batch No. BCBX8836) and a negative control (distilled water - ADL Prochilab - Batch No. 190521) were carried out. The controls were applied, as supplied, at the dose of 50 µL, to the surface of 2 RhCE tissue replicates during 30 minutes at standard culture conditions.

After the treatment, the test item and control substances were carefully washed from the RhCE tissues by extensive rinsing with Ca2+Mg2+Free-DPBS (Dutscher - Batch No. 2190919). The rinsed tissues were checked for any coloration and noted to be yellowish.
This rinsing step was followed by a 12-minute post-exposure immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue.
The RhCE constructs were then incubated for a 2 hours post-exposure incubation at standard culture conditions in 1 mL of fresh medium at 37°C, 5% CO2.

- MTT viability assay:
Following the exposure to the test item, viability measurements are performed immediately after the post-exposure incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects.
The RhCE tissue viability was measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1] by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.
The RhCE constructs were placed in 300 µL of a MTT solution at 1.0 mg/mL for 2 hours and 50 minutes at standard culture conditions.

The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol during 19 hours and 03 minutes at 7+/-3°C in the dark.
The concentration of formazan was measured by determining the OD at 570 nm, just after dilution of the extractions in isopropanol (1:2).

The OD at 570 nm was measured in triplicate samples of formazan extracts.
The measured OD are proportional to the number of living cells.

The measurement of OD was performed using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

Irritation parameter:

other: % mean corrected percent tissue viability

Value:

21.58

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

MTT VIABILITY ASSAY RESULTS
- The mean corrected percent tissue viability of the RhCE replicates treated with the test item was 21.58% versus 29.59% in the positive control (Methyl acetate).

- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: Yes, two additional killed control tissues were used to generate non-specific MTT reduction
- Colour interference with MTT: none.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Proficiency chemicals were tested according to the OECD TG 492.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for mean tissue viability: yes, tissues treated with the positive control substance showed a mean tissue viability < 50% (value of 29.59%).
- Acceptance criteria met for negative control: yes, the negative control OD of the 2 replicates is > 0.8 and < 2.5 for every exposure time. As the extract was diluted at 50% just before the OD measurement, the acceptability criteria should be in the range > 0.8 and <2.5 for the negative control.
- Acceptance criteria met for variability between replicate measurements: yes, less than 20% between two tissue replicates (value of 11.69%).

Table 7.3.2/1: Main test - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls after 30 minutes exposure

 

Tissue	OD	Mean OD/ disc (#)	Mean OD / Product	Viability (%)	Mean viability (%)	Viability difference between replicates (%)	Conclusion
Negative Control	1.154 1.127 1.117	1.133	1.087	104.28	100.00	8.56	-
	1.051 1.047 1.021	1.040		95.72
Positive Control	0.279 0.274 0.262	0.272	0.322	25.03	29.59	9.11	UN GHS Category 2 or 1
	0.375 0.377 0.362	0.371		34.15
Test Item	0.372 0.406 0.388	0.389	0.326	35.80	29.96	11.69
	0.264 0.269 0.252	0.262		24.11
Test item NSMTT	0.072 0.072 0.071	0.072	0.091	6.63	8.38	3.50
	0.112 0.110 0.107	0.110		10.12
Test item corrected					21.58		UN GHS Category 2 or 1

#: mean of 3 values

OD: optical density

SPL: sample

Interpretation of results:

other: to be identified as potentially requiring classification and labeling according to UN GHS Category 2 or Category 1

Conclusions:

The mean corrected percent tissue viability of the RhCE replicates treated with the test item TOBACCO ABSOLUTE was 21.58% versus 29.59% in the positive control (Methyl acetate). In conclusion, under the experimental conditions adopted and in accordance with the Regulation EC No. 1272/2008, the test item TOBACCO ABSOLUTE has to be identified as potentially requiring classification and labeling according to UN GHS Category 2 or Category 1.

Executive summary:

An OECD 492 study was performed to evaluate the eye hazard potential of test item after topical administration on in vitro reconstructed human cornea-like epithelium tissues (EpiOcular^TM tissue model).

 

Test item was coated on a nylon mesh then applied , at the approximate dose of 19 mg to 2 DPBS pre-treated RhCE (EpiOcular^TM tissue model) during 6 hours at 37°C, 5% CO₂, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 25 minutes post-exposure immersion period at room temperature and 18 hours post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay.

 

The mean corrected percent tissue viability of the RhCE replicates treated with the test item TOBACCO ABSOLUTE was 21.58% versus 29.59% in the positive control (Methyl acetate).

  

In conclusion, under the experimental conditions adopted and in accordance with the Regulation EC No. 1272/2008, the test item TOBACCO ABSOLUTE has to be identified as potentially requiring classification and labeling according to UN GHS Category 2 or Category 1.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

One key study was identified (Phycher 2019). In this study, the Reconstructed Human Epidermis (SkinEthic RHE® model) was performed according to the OECD Guideline 439 and in compliance with GLP to predict the acute skin irritation potential of the test item.

Test item was coated on a nylon mesh then applied, at the approximate dose of 16 mg, to 3 living Reconstructed Human epidermis (SkinEthic RHE®model) during 42 minutes. The application was followed by a rinse with 25 mL of DPBS and a 41 hours and 40 minutes post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

The mean percent viability of the treated tissues is 6.3%, versus 1.6% in the positive control (5% Sodium Dodecyl Sulfate). In accordance with the Regulation EC No. 1272/2008 and in absence of information on skin corrosion, the test item TOBACCO ABSOLUTE has to be classified in Category 2 “Irritating to skin” or in Category 1 “Corrosive”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” or “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are required.

Skin corrosion:

One key study was identified (Phycher 2020). In this study, the in vitro skin corrosion study was performed according to the OECD Guideline 431 and in compliance with GLP, using the EpiCS reconstructed human epidermis model.

The test item TOBACCO ABSOLUTE was applied as supplied, at the dose of 50 µL, to 2 living Human skin model surfaces (epiCS®, CellSystems®) during 3 minutes and 1 hour, followed by a rinse with 20 mL of DPBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. Additionally, 4 killed Human skin model surfaces were treated under the same experimental conditions in order to generate non-specific MTT reduction. 

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 84.47% and 86.98%, versus 58.06% and 0.5%, respectively, with the positive control item (potassium hydroxide 8N).

All the acceptability criteria were met and the quality criteria required for acceptance of results in the test were satisfied.

Under the experimental conditions of this study, the test substance is not classified for skin corrosion according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS. Based on the result of the in vitro skin irritation study, the test substance is therefore classified as Skin irritant Category 2 according to CLP and GHS. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” is required.

This study is considered as acceptable and satisfies the requirement for skin corrosion endpoint.

Eye irritation:

One key study was identified (Phycher, 2019). An OECD 439 study was performed to evaluate the eye hazard potential of test itemafter topical administration on in vitro reconstructed human cornea-like epithelium tissues (EpiOcularTMtissue model). Test item was coated on a nylon mesh then applied , at the approximate dose of 19 mg to 2 DPBS pre-treated RhCE (EpiOcularTMtissue model) during 6 hours at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 25 minutes post-exposure immersion period at room temperature and 18 hours post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay.

The mean corrected percent tissue viability of the RhCE replicates treated with the test item TOBACCO ABSOLUTE was 21.58% versus 29.59% in the positive control (Methyl acetate). Therefore, nunder the experimental conditions adopted and in accordance with the Regulation EC No. 1272/2008, the test item TOBACCO ABSOLUTE has to be identified as potentially requiring classification and labeling according to UN GHS Category 2 or Category 1. The hazard statement “H319: Causes serious eye irritation” with the signal word “Warning” or “H318: Causes serious eye damage” with the signal word “Danger” are required.

Eye corrosion:

One key study was identified (Phycher, 2020). An ex vivo eye irritation study was performed according to the OECD Guideline 438 and in compliance with GLP to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

Test item TOBACCO ABSOLUTE was applied as supplied , at the dose of 30 µL, to 3 enucleated chicken eyes during 10 seconds.Then the eyes were rinsed twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose.

The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 1.0, corresponding to ICE class II;

- mean score of fluorescein retention: 1.7, corresponding to ICE class III;

- maximal mean corneal swelling: 7%, corresponding to ICE class II.

The combination of the three endpoints for test item was 2 x II, 1 x III.

The combination of the three endpoints for the positive control, sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.

The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected.

In accordance with Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category "no prediction can be made", as defined by the OECD guideline No.438. Therefore, the test item TOBACCO ABSOLUTE is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method. Based on the results from the previous epiOcular study OECD 492, the test item can be classified as eye irritant (Category 2). The hazard statement “H319: Causes serious eye irritation” with the signal word “Warning” is required.

Respiratory irritation:

No data was available.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self classification:

Skin irritation/Corrosion

1/ Skin irritation (OECD 439, K, Rel.1): The mean percent viability of the treated tissues is 6.3%, versus 1.6% in the positive control (5% Sodium Dodecyl Sulfate).

Therefore, the test item TOBACCO ABSOLUTE require classification and labelling according to CLP and GHS as Category 2 or Category 1.

2/ Skin corrosion (OECD 431, K, Rel.1): 3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 84.47% and 86.98%, versus 58.06% and 0.5%, respectively, with the positive control item (potassium hydroxide 8N).

=> Based on the results from both in vitro studies, the test item can be classified as skin irritant (Category 2 CLP/GHS). The hazard statement “H315: Causes skin irritation” with the signal word “Warning” is required.

Eye irritation/Corrosion:

1/ Eye irritation (OECD 492, K, Rel.1): The mean corrected percent tissue viability of the RhCE replicates treated with the test item TOBACCO ABSOLUTE was 21.58% versus 29.59% in the positive control (Methyl acetate).

Therefore, the test item TOBACCO ABSOLUTE require classification and labelling according to CLP and GHS as Category 2 or Category 1.

2/ Eye corrosion (OECD 438, K, Rel.1): The combination of the three endpoints for test item was 2 x II, 1 x III. Therefore, "No prediction can be made", as defined by the OECD guideline No.438.

=> Based on the results from both in vitro studies, the test item can be classified as eye irritant (Category 2 CLP and GHS as based on current data it is not possible to subcategorize). The hazard statement “H319: Causes serious eye irritation” with the signal word “Warning” is required.

 Respiratory irritation:

No data was available regarding respiratory irritation.