Pepper (Piper), P. nigrum, ext.

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 July to 08 July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD Guideline No.431 and under GLP compliance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Dated to 18 June 2019.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Pepper (Piper), P. nigrum, ext.

EC Number:

284-524-7

EC Name:

Pepper (Piper), P. nigrum, ext.

Cas Number:

84929-41-9

Molecular formula:

Not available

IUPAC Name:

Essential oil of Piper nigrum (Piperaceae) obtained from the berries by steam distillation

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): ESSENTIAL OIL OF PIPER NIGRUM (PIPERACEAE) OBTAINED FROM THE BERRIERS BY STEAM DISTILLATION code ID-21/11768
- CAS number: 84929-41-9
- Analytical purity: 100%
- Lot/batch No.: 1005658537
- Expiration date of the lot/batch: 06/05/2022
- Appearance: pale yellow to green liquid
- Storage condition of test material: Room temperature (20°C ± 5°C) and darkness

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test substance: ESSENTIAL OIL OF PIPER NIGRUM (PIPERACEAE) OBTAINED FROM THE BERRIERS BY STEAM DISTILLATION code ID-21/11768
- Batch No.: 1005658537
- Expiration date of the lot/batch: 06 May 2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage: Room temperature (20°C ± 5°C) and darkness

FORM AS APPLIED IN THE TEST
The test item was used as supplied in the study.

In vitro test system

Test system:

human skin model

Source species:

other: reconstituted epidermis (epiCS, Cell Systems)

Cell type:

other: epiCS, Cell Systems - Batch No.21-23 supplied by PHENION

Vehicle:

unchanged (no vehicle)

Details on test system:

HUMAN SKIN MODEL
The 0.6 cm² reconstituted epidermis (epiCS, Cell Systems - Batch No.21-23) were received on 07 July 2021. The insert (filter + epidermis) was gently detached from the agar avoiding leaving agar pieces onto the polycarbonate membrane and remove any remaining agarose by gentle blotting onto a sterile tissue paper.
The inserts were placed immediately in wells (6 wells culture plate) previously filled with 1 ml of warmed culture medium epiCS supplied by PHENION - batch 305-AK1402 (the absence of bubbles was verified).
Cultures were incubated for 2 to 3 hours at 37°C, 5% CO2.

EVALUATION OF DIRECT INTERACTION WITH MTT:
The direct interaction of MTT with the test item was checked by adding 50 µL ± 2 µl of the test item to 1mL of the staining solution, mixed and incubated 1 hour ± 2 minutes at 37°C, 5% CO2. A blue coloration indicates a non-specific reduction induced by the test item (untreated MTT medium was used as control). The colour was assessed visually at the end of the exposure time.
> No colour was observed. No additional control for the direct MTT reduction by the test item is required.

EVALUATION OF TEST ITEM COLOURING POTENTIAL:
The spectral properties at 570 nm of test item in isopropanol were checked by adding 50 µL ± 2 µl of the test item to 0.3 mL of distilled water and incubated 1 hour ± 2 minutes at 37°C, 5% CO2 and 50 µl ± 2 µl of the test item were added to 2 ml of isopropanol and incubated 2 hours ± 2 minutes at 37°C, 5% CO2. The colour was assessed by absorbance reading at 540 nm against water or isopropanol as blanks.
> The absorbances read are not significative. No additional control for coloured and/or colouring test item is required.

EVALUATION OF THE COMPATIBILITY OF THE NYLON MESHES WITH THE TEST ITEM:
50 µl ± 2 µl of the test item were deposited on a nylon meshes and incubated 1 hour ± 2 minutes at room temperature. The integrity of the nylon meshes was observed with a phase-contrast microscope.
> No reaction was observed. A nylon mesh was used for the contact of the test item with the epidermis.

TREATMENT
Before the contact with the test or reference items, the epiCS culture medium was replaced by 1 ml of fresh warmed culture medium at 37°C as well as, two 24-well plates were prepared with 300 µl of culture medium epiCS and two 24-well plates were prepared with 300 µl of staining solution, maintained at 37°C, 5% CO2 until their use.

50 µl ± 2 µl of the test item or reference item were applied on the tissues surface with a micropipette and a nylon mesh was deposited gently on the surface of the epidermis with pliers.
The epidermises were incubated 1 hour ± 2 minutes at 37°C, 5% CO2 and 3 minutes exactly at room temperature.

REMOVAL OF TEST MATERIAL AND CONTROLS
At the end of the periods of treatment, the epidermises were rinsed with 1 ml of PBS 20 times (from a dispenser) according to the time of departure interval.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
They were stored in the previously prepared 24-well plate containing 300 µl of culture medium epiCS and other tissues were treated in the same way. The epidermises were then transferred in the 24-well plate with 300 µl of staining solution per well, except the epidermises that should not be colored, which were incubated in 300 µl of epiCS Assay medium MTT epiCS without MTT.
After 3 hours ± 10 minutes incubation at 37°C, 5% CO2, MTT was aspirated and the wells were washed three times with PBS.
The epidermis were transferred in a 24-well plate and immersed with 2 ml of isopropanol. MTT extraction is performed overnight without shaking at room temperature.
At the end of extraction, epidermises were pierced and removed from the wells and plates are shaken again 3 minutes until the extraction solution is homogeneous. The absorbance was measured in triplicate on 200 µl of MTT extract at 540 nm using isopropanol as blank.


The measurement of OD was performed using MULTISKAN EX plate reader (Thermo life sciences), reading range 0 - 3.5 units of Absorbance and linearity range 0 - 2.200 units of Absorbance at 540 nm.


VIABILITY CALCULATION:
- The results were expressed as a viability percentage compared with the negative control: viability % = OD test item / OD negative control * 100
- Data from individual replicate tissues (OD values and calculated percent tissue viability data for the test item and controls), mean percent tissue viability and standard deviation for each individual test item and control were reported in Table 7.3.1/1.

PREDICTION MODEL / DECISION CRITERIA:

The OD values obtained for each test sample are used to calculate a percentage of viability relative to the negative control, which is arbitrarily set at 100%. The cut-off values for the prediction of corrosion associated with the epiCS® models are as follows:

Viability measured after exposure time points (t=3 and 60 minutes)

STEP 1 (corrosive or not corrosive)
< 50% after 3 min exposure ==> Corrosive: Corresponding hazard statement “H314: Causes severe skin burns and eye damage” and signal word “Danger”
≥ 50% after 3 min exposure AND < 15% after 60 min exposure ==> Corrosive: Corresponding hazard statement “H314: Causes severe skin burns and eye damage” and signal word “Danger”
≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure ==> Non-corrosive

STEP 2 (subcategories for items identified as corrosive in step 1)
< 15% after 3 min exposure ==> Optional Sub-category 1A*
≥ 15% after 3 min exposure ==> A combination of optional Sub-categories 1B-and-1C

* According to the data generated in view of assessing the usefulness of the RhE test methods for supporting subcategorisation, it was shown that around 33% of the Sub-category 1A results of the epiCS® test methods may actually constitute Sub-category 1B or Sub-category 1C substances/mixtures (i.e. over-classifications).
It must be noted that a limitation of this Test Guideline is that it does not allow discriminating between skin corrosive sub-categories 1B and 1C.

ACCEPTABILITY CRITERIA:
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
-The positive control viability percentage must be less than 15% after 1 hour of exposure.
- The negative control mean absorbance must be higher than or equal to 0.8 and less than 2.8.
- The difference of viability between two tissue replicates should not exceed 30%.

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

Duration of treatment / exposure:

During 3 minutes at room temperature and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2.

Duration of post-treatment incubation (if applicable):

Cultures were incubated for 2 to 3 hours at 37°C, 5% CO2.

Number of replicates:

2 living human skin models (two by exposition time 3 minutes and 1 hour)

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

at 3 minutes

Value:

99

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

At 1 hour

Value:

113

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

MTT VIABILITY ASSAY RESULTS
The viability percentage of the test item is 99% after 3 minutes of exposure and 113% after 1 hour versus 7.9% and 0.0%, respectively, with the positive control item (potassium hydroxide 8N).

These results are in accordance with the acceptability criteria:

- The positive control shows a viability percentage of 7.9% and 0%, less than 15% after 1 hour of exposure.
- The negative control mean absorbance is higher than or equal to 0.8 and less than 2.8.
- The difference of viability between the two tissue replicates is less than or equal to 30%.

These results allow to validate the test.

Any other information on results incl. tables

Table 7.3.1/1: Individual and average values for the test item, the negative and positive controls

INDIVIDUAL AND AVERAGE VALUES AFTER 3 MINUTES EXPOSURE

 

	Skin	OD	Mean OD / disc (#)	Viability %	Mean viability %	Viability difference between replicates %
Negative control	-	-	-	-	-	-
Positive control	Ep.1	0.091 0.091 0.091	0.091	6.0	7.9	3.8
	Ep.2	0.142 0.160 0.146	0.150	9.8
Test item	Ep.1	1.403 1.476 1.476	1.452	94.6	99.0	8.8
	Ep.2	1.474 1.806 1.479	1.587	103.4

 

Acceptability criteria:

- The positive control shows a viability percentage of 7.9% and 0%, less than 15% after 1 hour of exposure.
- The negative control mean absorbance is higher than or equal to 0.8 and less than 2.8.
- The difference of viability between the two tissue replicates is less than or equal to 30%. These results allow to validate the test.

 

INDIVIDUAL AND AVERAGE VALUES AFTER 1 HOUR EXPOSURE

	Skin	OD	Mean OD / disc (#)	Viability %	Mean viability %	Viability difference between replicates %
Negative control	Ep.1	1.526 1.556 1.532	1.538	100.2	100.00	0.4
	Ep.2	1.671 1.429 1.495	1.532	99.8
Positive control	Ep.1	0.000 0.000 0.000	0.000	0.0	0.0	0.0
	Ep.2	0.000 0.000 0.000	0.000	0.0
Test item	Ep.1	1.635 1.595 1.651	1.627	106.0	113.0	14.0
	Ep.2	1.846 1.831 1.850	1.843	120.0

Note #: mean of 3 values

OD: optical density

SPL: sample

 

Acceptability criteria:

- The positive control shows a viability percentage of 7.9% and 0%, less than 15% after 1 hour of exposure.
- The negative control mean absorbance is higher than or equal to 0.8 and less than 2.8.
- The difference of viability between the two tissue replicates is less than or equal to 30%. These results allow to validate the test.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the retained experimental conditions and in accordance with the Regulation (EC) No. 1272/2008, the test item ESSENTIAL OIL OF PIPER NIGRUM (PIPERACEAE) OBTAINED FROM THE BERRIERS BY STEAM DISTILLATION doesn’t show any corrosive effect and, therefore, does not have to be classified in Category 1 “Corrosive”. The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are not required.

Executive summary:

An in vitro skin corrosion test according to the OECD Guideline OECD 431 and in compliance with GLP was performed.

The aim of the study was to assess the capability of the test item, ESSENTIAL OIL OF PIPER NIGRUM (PIPERACEAE) OBTAINED FROM THE BERRIERS BY STEAM DISTILLATION, to induce a skin corrosive effect on in vitro reconstructed epidermis after the test item application at the dose of 50 μL, undiluted, on the epiCS® reconstructed epidermises model, for 3 minutes and 1 hour.
The application was followed by a rinse with 1 ml of PBS 20 times. Cell viability was then measured by the measurement of the succinate deshydrogenase mitochondrial activity of the alive cells. This enzyme is involved in the transformation of MTT (3(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide) into blue formazan salt. A spectrophotometric measurement is performed after the crystal dissolution. The measured absorbances are proportional to the number of living cells (Mosmann, J. Immunol. Meth., 1983, 55 63). This study is carried out according to the protocol provided by the O.E.C.D. Test Guideline No. 431 (dated 18 June 2019) and the epidermises supplier’s Standard Operating Protocol.

 

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 99% and 113%, versus 7.9% and 0.0%, respectively, with the positive control item (potassium hydroxide 8N).

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item, ESSENTIAL OIL OF PIPER NIGRUM (PIPERACEAE) OBTAINED FROM THE BERRIERS BY STEAM DISTILLATION, does not have to be classified in Category 1 “Corrosive”. The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are not required.