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

Skin irritation / corrosion

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

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 material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

951-779-7

Molecular formula:

Not relevant, UVCB substance

IUPAC Name:

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

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: room temperature, darkness (Optimal temperature at 20°C)

In vitro test system

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

Results and discussion

In vitro

Results

Results 1

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

Results 2

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.