Registration Dossier

Administrative data

Description of key information

Skin irritation (OECD 439 and 431): irritating

Eye irritation (OECD 405): irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 June - 15 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
/ no application of correction factor on positive control, no demonstration of technical proficiency, 3 min incubation at room temperature instead of at 37°C (OECD 431)
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 July 2016
Deviations:
yes
Remarks:
/ no application of correction factor on positive control, no demonstration of technical proficiency, 3 min incubation at room temperature instead of at 37°C (OECD 431)
Qualifier:
according to
Guideline:
other: EU method B.40 BIS. (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
30 May 2008
Deviations:
yes
Remarks:
/ no demonstration of technical proficiency
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbrauchersch utz, Wiesbaden, Germany
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Source strain:
other: EpiDermTM; reconstructed three-dimensional human epidermis (EPI-200); 00267
Justification for test system used:
The normal, human-derived epidermal keratinocytes (NHEK) attain levels of differentiation at the cutting edge of in vitro skin technology. Ultrastructurally, the skin models closely parallel human skin.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Kit (Epi-200, MatTek Corporation (Bratislava, Slovakia)
- Tissue lot number: 23345
- Delivery date: 12 June 2016
- Date of initiation of testing: 27 June 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature for the 3 ± 0.5 min exposure periods, 37 ± 1.5 °C for the 60 ± 5 min exposure period

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: rinsed 20 times with DPBS
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader (Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1))
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD = 1.783 ± 0.027 (Acceptance criteria: 1.0-3.0)
- Barrier function: ET-50 = 6.16 h (Acceptance criteria: 4.77 - 8.72)
- Morphology: functional stratum corneum, a viable basal cell layer and intermediate spinous and granular layers
- Contamination: no contamination

NUMBER OF REPLICATE TISSUES: duplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues: freezing
- N. of replicates : 2
- Method of calculation used: True viability = Viability of treated tissue – Interference from test chemical = ODtvt – ODkt; ODkt = (mean ODtkt – mean ODukt)
tvt = treated viable tissue
kt = killed tissues
tkt = treated killed tissue
ukt = untreated killed tissue (NC treated tissue)
Since the interference by the test item extract is < 30% of the negative control value, the net OD of the test item extract treated killed control was subtracted from the mean OD of the test item extract treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50% (optional Sub-category 1A), or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15% (optional Sub-category 1B and 1C).
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 50 µl
- Concentration: 79.4 μL/cm2

NEGATIVE CONTROL
- Amount(s) applied: 50 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration: 8 N
Duration of treatment / exposure:
3 ± 0.5 min and 60 ± 5 min
Number of replicates:
2
Type of coverage:
other: in vitro system
Preparation of test site:
other: intact reconstructed human epidermis
Vehicle:
unchanged (no vehicle)
Irritation / corrosion parameter:
% tissue viability
Remarks:
of negative control
Run / experiment:
mean value of the test item (100%), 3 min exposure
Value:
114
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
of negative control
Run / experiment:
mean value of the test item (100%), 60 min exposure
Value:
56.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: yes
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, as the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time (values between 1.693 and 1.924)
- Acceptance criteria met for positive control: yes, as the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control (7.9%)
- Acceptance criteria met for variability between replicate measurements: yes, as the Coefficient of Variation (CV) is in the range of 20 – 100% viability between tissue replicates is ≤ 30% (values between 3.8% and 16.6%)

Table 1: MTT assay after 3 min exposure

Negative control

Positive control

Test item

Tissue sample

1

2

1

2

1

2

OD570

1.795

1.775

0.594

0.647

2.276

2.062

1.820

1.717

0.603

0.685

2.019

1.944

1.850

1.693

0.619

0.658

2.002

1.949

OD570(mean)

1.821

1.728

0.606

0.663

2.099

1.985

OD570(mean values* of replicates)

1.737

0.597

2.005

CV (%)

3.8

6.8

4.0

rel. viability (%)

100.0

34.4

114.0**

Blanc (mean) = 0.038

* after blanc correction of single values

** after data correction because of MTT interference

CV = Coefficient of Variation

Table 2: MTT assays after 60 min exposure

Negative control

Positive control

Test item

Tissue sample

1

2

1

2

1

2

OD570

1.924

1.845

0.198

0.158

1.232

0.968

1.895

1.715

0.184

0.166

1.181

0.949

1.768

1.728

0.184

0.167

1.190

0.951

OD570(mean)

1.862

1.763

0.189

0.164

1.201

0.956

OD570(mean values* of replicates)

1.776

0.140

1.042

CV (%)

4.0

12.6

16.6

rel. viability (%)

100.0

7.9

56.7**

Blanc (mean) = 0.038

* after blanc correction of single values

** after data correction because of MTT interference

CV = Coefficient of Variation

Interpretation of results:
other: not corrosive
Conclusions:
The test item PIPERITONE was non corrosive to skin according to EU CLP and UN GHS
Executive summary:

After exposure of the tissues to the test item the relative absorbance value did not decrease (114.0%) after 3 minutes exposure. After the 1 hour exposure the viability was reduced to 56.7%. The correction factor derived from the additional test with freeze-killed tissues was considered. Both values did not affect the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item was not considered to be corrosive.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 May - 06 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Commission Regulation 440/2008, 1st ATP 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Source strain:
other: EpiDerm™; Reconstructed Human Epidermal Model (EPI-200), Strain 00267, Lot Nr 23339
Details on animal used as source of test system:
SOURCE ANIMAL
- Source: MatTek Corporation, Bratislava, Slovakia

The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.
Justification for test system used:
Systemic reactions play a minor role in modulating local skin toxicity potential of chemicals, so skin irritation potential may be predicted by in vitro systems, provided they are sufficiently complex to mimic human skin barrier and cell reactivity.
Vehicle:
unchanged (no vehicle)
Details on test system:
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C
- Temperature of post-treatment incubation (if applicable): room temperature, later again 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: at least 15

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: microplate reader (Versamax® Molecular Devices, Softmax Pro, version 4.7.1)
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: One experiment was carried out in triplicate plating.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the mean relative tissue viability of three individual tissues is reduced to ≤ 50% of the negative control.
- The test substance is considered to be non-irritant to skin if the mean relative tissue viability of three individual tissues is > 50% of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 30 μL
- Concentration (if solution): 47 μL/cm²

NEGATIVE CONTROL
- Amount(s) applied: 30 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 5% SLS
Duration of treatment / exposure:
1 h
Duration of post-treatment incubation (if applicable):
43 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value of test item (47 μL/cm²)
Value:
4.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: No. The test item did not reduce MTT (test for direct MTT reduction).
- Colour interference with MTT: No. The test item did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control the absorbance values (1.525, 1.957, 1.950) were in the range of the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 min treatment.
- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.3% thus confirming the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: yes, but value (19%) is slightly above the threshold of the OECD Guideline 439 ( < 18%)

Table 2: MTT assay after 60 min exposure

Dose
Group

Tissue No.

OD570

OD570(mean
absorbance of replicates)

Mean absorbance of replicates blank corrected

Mean absorbance of replicates after blank correction

Rel. Absorbance [%]
Tissue 1, 2 + 3*

Relative Standard Deviation
[%]

Viability (Mean Rel. Absorbance
[% of Negative Control]***)

Well 1

Well 2

Well 3

Blank

0.036

0.037

0.036

0.036

Negative
Control

1

1.578

1.549

1.558

1.562

1.525

1.811

84.2

13.7

100.0

2

2.026

1.984

1.971

1.994

1.957

108.1

3

2.024

1.971

1.965

1.986

1.950

107.7

Positive
Control

1

0.098

0.097

0.097

0.097

0.061

0.078

3.4

19.0

4.3

2

0.118

0.121

0.118

0.119

0.082

4.6

3

0.125

0.126

0.126

0.126

0.089

4.9

Test
Item

1

0.135

0.137

0.133

0.135

0.098

0.087

5.4

11.6

4.8

2

0.117

0.116

0.116

0.116

0.080

4.4

3

0.119

0.119

0.118

0.119

0.082

4.5

* = relative absorbance per tissue [rounded values]: 100 x (absorbance tissue)/(mean adsorbance negative control)

** = relative absorbance per treatment group [rounded values]: 100 x (mean absorbance test item/ positive control)/(mean adsorbance negative control)

Interpretation of results:
other: irritating potential (Skin Irrit. 2 or Skin Corr. 1 according to Regulation (EC) No 1272/2008)
Conclusions:
Under the conditions of the test, the test substance was shown to have an irritating potential towards reconstructed human epidermis tissue in the EpiDerm™ model.
Executive summary:

The skin irritation potential of the test substance was determined by an in vitro skin irritation test using a reconstructed human skin model according to OECD Guideline 439 and in compliance with GLP (2016).

After treatment with the test substance for 60 min the tissue viability decreased to 4.8% compared to the negative control (threshold for irritancy ≤ 50%). Therefore, the test substance is considered to possess an irritant potential.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 June - 04 July 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
/ GLP guideline study with minor deviations (no information about animal age, body weight determined only once)
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
02 Oct 2012
Deviations:
yes
Remarks:
/ GLP guideline study with minor deviations (no information about animal age, body weight determined only once)
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
30 May 2008
Deviations:
yes
Remarks:
/ GLP guideline study with minor deviations (no information about animal age, body weight determined only once)
GLP compliance:
yes
Species:
rabbit
Strain:
other: SPF albino rabbits, Chbb:HM(SPF) - Littlerussian
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: BI Pharma KG, Biberach, Germany
- Weight at study initiation: 1.5 - 1.7 kg (females)
- Housing: individual in PPO cages with perforated floor
- Diet : pelleted diet Altromin 2123 (Altromin, Lage, Lippe Germany), ad libitum
- Water: water (acidified with hydrochloric acid, pH 2,5), ad libitum (analysis was performed)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 55 ± 15
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/ 12

IN-LIFE DATES: From: 26 June To: 04 July 2000
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL of the test article were applied into one eye of the rabbits.
Duration of treatment / exposure:
single application without washing
Observation period (in vivo):
7 days
Reading time points: 1, 24, 48, 72 h and 7 days after the treatment
Number of animals or in vitro replicates:
4 females
Details on study design:
SCORING SYSTEM: Draize scoring system

TOOL USED TO ASSESS SCORE: fluorescein
Irritation parameter:
cornea opacity score
Remarks:
before fluorescein application
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
cornea opacity score
Remarks:
before fluorescein application
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.7
Max. score:
4
Reversibility:
fully reversible within: 3 days
Irritation parameter:
cornea opacity score
Remarks:
before fluorescein application
Basis:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
cornea opacity score
Basis:
animal #4
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.7
Max. score:
2
Reversibility:
fully reversible within: 3 days
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.7
Max. score:
2
Reversibility:
fully reversible within: 3 days
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 7 days
Irritation parameter:
iris score
Basis:
animal #4
Time point:
24/48/72 h
Score:
0.7
Max. score:
2
Reversibility:
fully reversible within: 3 days
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1.7
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.3
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Basis:
animal #4
Time point:
24/48/72 h
Score:
1.3
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1.3
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1.3
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #4
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritant / corrosive response data:
Based on the readings before fluorescein application, all 4 animals showed a cornea opacity (scored with 1) after 1h , which was reversible within 72 h in 1/4 animals. All animals showed an Iris score of 1 after 1 h, which was reversible within 72h in 2/4 animals. Conjunctivae was observed in all animals after 1 h (scored with 3 in 1 animal and with 2 in the other 3 animals). After 24 h all animals showed a conjunctivae scored with 2. After 48 h or 72 h the scoring decreased to 1 (in 2 or 1 animal, respectively). The fourth animal showed a consistent scoring of 2 within the 72 h. Chemosis was observed in all animals after 1 h (scored with 2). 2/2 animals showed a decrease in scoring to 1 after 24 h, the other 2 animals after 48 h.
7 days after application of the test article all four animals were free of any signs of eye irritation.

Table 1: Individual scores of eye irritation

Rabbit #

Time [h]

conjunctivae

iris

cornea

cornea (after Fluorescein)

redness

chemosis (swelling)

1

1

3

2

1

1

-

24

2

2

1

1

1

48

2

1

1

1

1

72

1

1

0

1

1

mean

1.7

1.3

0.7

1.0

1.0

2

1

2

2

1

1

-

24

2

1

1

1

1

48

1

1

1

1

1

72

1

1

0

0

1

mean

1.3

1.0

0.7

0.7

1.0

3

1

2

2

1

1

-

24

2

2

1

1

1

48

2

1

1

1

1

72

2

1

1

1

0

mean

2.0

1.3

1.0

1.0

0.7

4

1

2

2

1

1

-

24

2

1

1

1

1

48

1

1

1

1

1

72

1

1

0

1

1

mean

1.3

1.0

0.7

1.0

1.0

Iris: 0 - 2 numerical scores, Redness: 0 - 3 numerical scores, Chemosis and Cornea opacity score : 0 - 4 numerical scores

 

Only the scores from the readings after 24, 48 and 72 hours are included in the calculation of the individual mean scores.

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
CLP: Eye irrit 2, H319
Executive summary:

The eye irritation potential of the test substance was determined by an in vivo eye irritation test according to OECD Guideline 405 and in compliance with GLP (2016).Application of the test substance to the rabbit eye resulted in a mean corneal opacity score of 1 in 3/4 animals, which was reversible within maximum 7 days. Thus, the test substance is considered to be irritant to the eye.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin

The skin irritation potential of the test substance was determined by an in vitro skin irritation test using a reconstructed human skin model according to OECD Guideline 439 and in compliance with GLP (2016).

After treatment with the test substance for 60 min the tissue viability decreased to 4.8% compared to the negative control (threshold for irritancy ≤ 50%). Therefore, the test substance is considered to possess an irritant potential.

The skin corrosive potential of the test substance was determined by an in vitro skin irritation test using a reconstructed human skin model according to OECD Guideline 431 and in compliance with GLP.

After exposure of the tissues to the test item the relative absorbance value did not decrease (114.0%) after 3 minutes exposure. After the 1 hour exposure the viability was reduced to 56.7%. The correction factor derived from the additional test with freeze-killed tissues was considered. Both values did not affect the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item was not considered to be corrosive.

In conclusion, based on available studies, the test substance is considered to be irritating to the skin and therefore is classified as Skin Irrit. 2, H315 according to CLP.

Eye

The eye irritation potential of the test substance was determined by an in vivo eye irritation test according to OECD Guideline 405 and in compliance with GLP (2016). Application of the test substance to the rabbit eye resulted in a mean corneal opacity score of 1 in 3/4 animals, which was reversible within maximum 7 days. Thus, the test substance is considered to be irritant to the eye.

Justification for classification or non-classification

The available data on skin irritation meet the criteria for classification as Skin Irrit. Cat. 2 (H315) according to Regulation (EC) 1272/2008.

The available data on eye irritation meet the criteria for classification as Eye Irrit. Cat. 2 (H319) according to Regulation (EC) 1272/2008.