Pepper (Piper), P. nigrum, ext.

Endpoint summary

Description of key information

- Skin irritation/corrosion: irritating, based on the rules of the CLP Regulation for classification of mixtures and on an in vitro skin corrosion test (OECD TG 431, GLP, rel.1).

- Eye irritation: not irritating based on a read-across with Olibanum oil (OECD TG 492, GLP, Rel.1) and on an in vitro eye corrosion test (OECD TG 438, GLP, rel.1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

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)

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.

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)

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

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.

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.

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.

Reference 2

Endpoint:

skin irritation / corrosion, other

Remarks:

Classification based on calculation rules for mixtures of the CLP Regulation

Type of information:

calculation (if not (Q)SAR)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

accepted calculation method

Qualifier:

no guideline required

Principles of method if other than guideline:

Classification based on calculation rules for mixtures of the CLP Regulation

Irritation parameter:

other: classification

Remarks on result:

other: skin irritant category 2

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Executive summary:

The NCS is composed of several identified constituents and in that, it can be considered as a mixture according to the definition of the CLP Regulation.

The decision logic for classification of mixtures from the ECHA Guidance on the Application of the CLP Criteria (2015) was used to determine the skin irritation/corrosion hazard of the registered substance. The decision of classification as skin irritant was based on existing data on constituents (additivity principles): the registered substance has more than 10% of its constituents classified as Skin irritant Category 2 and should be classified as a skin irritant without further testing according to the rules for classification of mixtures of Regulation (EC) No 1272/2008.

 

Constituent	Classification according to the Regulation (EC) No. 1272/2008 (CLP)		Source
	Skin irritation	Eye irritation
Dipentene (CAS#138-86-3)	SCI 2 (H315)	-	Harmonised classification
Pinene, beta- (CAS#127-91-3)	SCI 2 (H315)	-	Self-classification
Pin-2(3)-ene (CAS#80-56-8)	SCI 2 (H315)	-	Self-classification
(+)-delta-3-carene (CAS#498-15-7)	SCI 2 (H315)	-	Self-classification
Bisabolene, beta- (CAS#495-61-4)	SCI 2 (H315)	-	Self-classification
Beta-myrcene (CAS#123-35-3)	SCI 2 (H315)	EDI 2 (H319)	Self-classification

Source: ECHA disseminated dossiers or self classification

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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 July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD Guideline No.438 and under GLP compliance (GLP deviation due to the exact composition of the test item which cannot be exactly determined because is an UVCB substance. Without impact on the conclusion of the study)

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Dated to 03 September 2021

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
- Batch No.: 1005658537
- Expiration date of the lot/batch: 06 May 2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Room temperature, darkness, Kept in fridge after opening

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

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 19 July 2021 at 8:08 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 Laboratoire ICARE - Site de Martillac on 19 July 2021 at 9:41 am.

- Indication of any existing defects or lesions in ocular tissue samples: No morphological effects were noted, whatever the examination time.

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

CONTROLS
- Amount(s) applied (volume or weight with unit): 30 µL of negative control or positive control

Duration of treatment / exposure:

Test item was applied for 10 seconds to the cornea

Number of animals or in vitro replicates:

1 eye for negative control and 3 eyes for positive control and test item

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 was temperature controlled at 32 °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 61 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 to the cornea such that the entire surface of the cornea was evenly covered with the test item. The test item was applied for 10 seconds.

REMOVAL OF TEST SUBSTANCE

- After exposure, test item was rinsed twice 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:

- Mean corneal swelling (%): Corneal swelling was determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope.

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

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:

0.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Run / experiment:

mean score

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Run / experiment:

maximal mean score

Value:

11

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: 0.7, corresponding to ICE class II;

- mean score of fluorescein retention: 0.5, corresponding to ICE class I;

- maximal mean corneal swelling: 11%, corresponding to ICE class II.

The combination of the three endpoints for test item Essential oil of Piper nigrum (Piperaceae) obtained from the berries by steam distillation was 2 x II, 1 x I.

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.

- Acceptance criteria met for positive control: The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.

Interpretation of results:

GHS criteria not met

Conclusions:

In accordance with 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 berries by steam distillation does not require classification for eye irritation and serious eye damage as defined by the UN GHS (No Category).
No signal word and hazard statement are required.

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.

The test item was applied to the cornea such that the entire surface of the cornea was evenly covered with the test item. The test item was applied to 3 enucleated eyes at the dose of 30 µL, during 10 seconds and 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: 0.7, corresponding to ICE class II;

- mean score of fluorescein retention: 0.5, corresponding to ICE class I;

- maximal mean corneal swelling: 11%, corresponding to ICE class II.

 

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

The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, 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 enable to conclude that the test item Essential oil of Piper nigrum (Piperaceae) obtained from the berries by steam distillation does not require classification for eye irritation and serious eye damage as defined by the UN GHS (No Category). No signal word and hazard statement are required.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion:

The NCS is composed of several identified constituents and in that, it can be considered as a mixture according to the definition of the CLP Regulation.

The decision logic for classification of mixtures from the ECHA Guidance on the Application of the CLP Criteria (2015) was used to determine the skin irritation/corrosion hazard of the registered substance. The decision of classification as skin irritant was based on existing data on constituents (additivity principles): the registered substance has more than 10% of its constituents classified as Skin irritant Category 2 and should be classified as a skin irritant without further testing according to the rules for classification of mixtures of Regulation (EC) No 1272/2008.

 

Constituent	Classification according to the Regulation (EC) No. 1272/2008 (CLP)		Source
	Skin irritation	Eye irritation
Dipentene (CAS#138-86-3)	SCI 2 (H315)	-	Harmonised classification
Pinene, beta- (CAS#127-91-3)	SCI 2 (H315)	-	Self-classification
Pin-2(3)-ene (CAS#80-56-8)	SCI 2 (H315)	-	Self-classification
(+)-delta-3-carene (CAS#498-15-7)	SCI 2 (H315)	-	Self-classification
Bisabolene, beta- (CAS#495-61-4)	SCI 2 (H315)	-	Self-classification
Beta-myrcene (CAS#123-35-3)	SCI 2 (H315)	EDI 2 (H319)	Self-classification

Source: ECHA disseminated dossiers or self classification

 

Eye irritation:

A key study was identified on the registered substance: IDEA, 2021 (OECD 438, RS, rel.1).

 

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

The test item was applied to the cornea such that the entire surface of the cornea was evenly covered with the test item. The test item was applied to 3 enucleated eyes at the dose of 30 µL, during 10 seconds and 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: 0.7, corresponding to ICE class II;

- mean score of fluorescein retention: 0.5, corresponding to ICE class I;

- maximal mean corneal swelling: 11%, corresponding to ICE class II.

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

The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, 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 enable to conclude that the test item Essential oil of Piper nigrum (Piperaceae) obtained from the berries by steam distillation does not require classification for eye irritation and serious eye damage as defined by the UN GHS (No Category). No signal word and hazard statement are required.

Justification for classification or non-classification

Harmonized classification:

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

 

Self-classification:

Based on the available information and typical composition provided by the Lead Registrant, the registered substance is classified as skin irritant: Skin Irritant Category 2 (H315: Causes skin irritation) according to the criteria of the Regulation (EC) No. 1272/2008 (CLP).

 

Based on the available experimental data on the test substance, the registered substance is not classified for eye irritation according to the criteria of the Regulation (EC) No. 1272/2008 (CLP).

 

No information was available regarding respiratory irritation.