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Description of key information

OECD guideline, GLP-compliant in vivo skin corrosion/irritation study. No skin corrosion/irritation was recorded during the observation period up to 72h after the exposure period.


 


2 OECD guideline, GLP compliant studies assessed eye corrosion/irritation using in vitro methods. Based on a weight-of evidence assessment of the two studies, they indicate that the substance is predicted to be irritating to the eye but not corrosive.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Jul - 16 Aug 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
adopted in Apr 2002
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
other: Japanese white species (Kbs:JW)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Ina Breeding Center, Kitayama Labes, Co., Ltd., Nagano, Japan
- Age at study initiation: 10 - 11 weeks
- Weight at study initiation: 2.71 - 2.38 kg
- Housing: The animals were individually housed in aluminium cages (W350 x D550 x H350 mm) in stainless-steel automatic-washing racks.
- Diet: pelleted diet RC4 (Lot. No. 160330, 160414 from Oriental Yeast Co., Ltd.), 150 g/day
- Water: municipal tap water, ad libitum
- Acclimation period: 8 - 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5 - 25.9
- Humidity (%): 48.2 - 65.4
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 Jul 2016 To: 23 Jul 2016
Type of coverage:
semiocclusive
Preparation of test site:
other: clipped and thereafter shaved
Vehicle:
other: olive oil
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.5 g

VEHICLE
- Amount(s) applied: 0.5 mL olive oil was dropped and blended well with the test substance.
Duration of treatment / exposure:
4 h
Observation period:
72 h
Reading time points: 1, 24, 48 and 72 h
Number of animals:
3
Details on study design:
TEST SITE
- Area of exposure: 2.5 cm x 2.5 cm on the dorsal area of the drunk

REMOVAL OF TEST SUBSTANCE
- After patch removal, the test substance remaining on the skin was removed as much as possible (not further specified)

SCORING SYSTEM:
- Method of calculation: Draize scoring system
Irritation parameter:
erythema score
Basis:
mean
Remarks:
of all 3 animals
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
edema score
Basis:
mean
Remarks:
of all 3 animals
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritant / corrosive response data:
No skin reaction was observed on the skin in any rabbit after 24, 48 and 72 h of observation.
Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008
Conclusions:
A reliable study conducted in accordance with OECD 404 and GLP, found the test material to be non-irritating to the skin of rabbits.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 - 17 Sep 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
adopted in Oct 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Government of India, Department of Science and Technology, India
Vehicle:
other: corn oil
Remarks:
The test substance was insoluble in saline, therefore a homogenous suspension of the test substance in corn oil was prepared.
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration: 20% suspension (w/v) in corn oil

VEHICLE
- Amount(s) applied: 750 µL

POSITIVE CONTROL: Imidazole
- Amount(s) applied: 750 μL
- Concentration: 20% (w/v) in 0.9% sodium chloride solution
Duration of treatment / exposure:
4 h ± 5 min at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
90 ± 5 min at 32 ± 1 °C
Number of animals or in vitro replicates:
Triplicates for each treatment and control group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The eyes were dissected with a 2 to 3 mm rim and were transferred to container containing Hank`s Blanced Salt Solution. Each cornea was mounted in a specially designed cornea holder.

QUALITY CHECK OF THE ISOLATED CORNEAS
Only corneas which were free from defects were used. The baseline opacity was determined for each cornea. Any corneas with opacity values >7 opacity units were discarded.

TREATMENT METHOD
Each cornea was mounted in a cornea holder with the endothelial side against the O-ring of the posterior part of the holder. Afterwards, the anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium, ensuring that no bubbles were present within the holders. For equilibration, the corneas in the holder were incubated in at 32 ± 1 °C for at least 1 h.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: washing was performed until no visual evidence of the test substance was observed using medium with phenol red and thereafter using medium without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The light transmission through the corneas, given as lux value, was recorded and thereafter converted into an opacity value (baseline opacity values).
- Corneal permeability: The amount of fluorescein that crossed the cornea was measured spectrophotometrically (OD490) in the medium from the posterior chamber.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA
Test substance with an IVIS > 55 was regarded as Category 1.
Test substance with an IVIS ≤ 3 was regarded as No Category.
Test substance with an IVIS > 3; ≤ 55: no prediction can be made.
Irritation parameter:
in vitro irritation score
Run / experiment:
mean value of 3 corneae
Value:
3.54
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the vehicle control (corn oil) the calculated IVIS was 0.48, and therefore within two standard deviations of the reported historical mean of the negative control (IVIS: 0.09 - 1.48).
- Acceptance criteria met for positive control: After treatment with the positive control (20% imidazole) the calculated IVIS was 120.52 and, therefore within two standard deviations of the reported historical mean of the positive control (IVIS 63.28 - 257.23).
Interpretation of results:
other: non-corrosive (Eye Irrit. 2 or not classified according to Regulation (EC) No 1272/2008)
Conclusions:
Under the conditions of the test, the test substance was shown to have no corrosive potential in the Bovine corneal opacity and permeability (BCOP) test prediction model. The result does not allow for the non-classification or classification as irritant of the test substance and therefore further evaluation and/or data generation is required.
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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 in Jul 2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz, Germany
Species:
human
Strain:
other: EpiOcular™
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 51.3 mg (tissue 1) and 50.1 mg (tissue 2)

POSITIVE CONTROL:
- Amount applied: 50 µL

NEGATIVE CONTROL:
- Amount applied: 50 µL
Duration of treatment / exposure:
6 h
Duration of post- treatment incubation (in vitro):
25 min
Number of animals or in vitro replicates:
Duplicate tissues for each treatment and control group
Details on study design:
- RhCE tissue construct used, including batch number: EpiOcular™ tissue (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia), Batch number: 27006, Day of delivery: 26 Sep 2017

- Viability: The quality of the final tissue was assessed by undertaking a MTT cell viability test. A MTT-solution with a concentration of 1 mg/mL was used. The plates with the tissue inserts were incubated for 180 min at 37 ± 1 °C, 5 ± 1% CO2. The OD (540 – 570 nm) was determined to be 1.534 ± 0.049.

- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 0.3% Triton X-100. The ET-50 value was determined to be 17.77 min.

- Contamination: The cells used to produce the EpiOcular™ tissue were screened for the presence of HIV-1 virus, Hepatitis B and C virus, bacteria, yeast and other fungi.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
6 h exposure, 25 min post-exposure immersion and 18 h post-exposure incubation at 37 °C.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The ability of the test substance to directly reduce MTT and to form a blue/purple reaction product was assessed in a pre-experiment.
The MTT solution did not change its colour; therefore, direct MTT reduction had not taken place, and no data correction was necessary. No colour development was visible. Therefore, the main test was performed without colourant controls.

- Number of tissue replicates used per test chemical and controls: 2

- Wavelength: 570 nm

- Acceptance criteria: The results are acceptable if the negative control OD is >0.8 and <2.5 and if the mean relative viability of the positive control is below 50% of the negative control viability.

- Reference to historical data of the RhCE tissue construct: Historical control data was used to assess the validity of the test.

- Acceptable variability between tissue replicates for the test chemical, positive and negative controls: The difference of viability between the two relating tissues of a single test substance is < 20% in the same run.
Irritation parameter:
other: % tissue viability mean value of 2 tissues
Run / experiment:
6 h exposure
Value:
4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The OD of the negative control was 1.620 and 1.624 for tissue 1 and tissue 2, respectively. Both values fulfill the validity criteria (> 0.8 and < 2.5) and fell within the range of the historical control data (range: 1.047 - 2.340).
- Acceptance criteria met for positive control: The OD of the positive control was 0.713 and 0.657 for tissue 1 and tissue 2, respectively. The tissue viability mean value of 2 tissues was 42.2% and fell within the range of the historical control data (range: 21.1 - 53.9%).
- The variation within replicates was < 20% in the same run for positive (3.5%) and negative (0.2%) control tissues and tissues treated with the test substance (0.2%).

Table 1: Absorbance values of the negative and positive control and of the test substance (OD at 570 nm)

  Negative Control Positive control Test substance
Tissue 1 1.596 0.754 0.101
1.719 0.748 0.101
Tissue 2 1.672 0.697 0.105
1.651 0.692 0.105

Table 2: Viability of Tissue 1 and 2

  Positive control Test substance
% Viability (Tissue 1) 44.0 3.9
% Viability (Tissue 1) 40.5 4.1
% Viability Mean 42.2 4.0
Interpretation of results:
other: irritating potential (Eye Irrit. 2 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 EpiOcular™ EIT prediction model. The result allows for the classification of the test substance as eye irritant, since a Bovine corneal opacity and permeability (BCOP) test method according to OECD 437 is available, showing non-corrosive properties of the test substance.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

The substance is not judged to be classified as corrosive or irritating under CLP regulation (EC 1272/2008, as amended) to skin due the lack of irritation or corrosive effects in the in vivo skin corrosion/irritation study conducted on rabbit.


 


The substance is judged to be classified as Eye Irritant 2 (H319) under the CLP regulation (EC 1272/2008, as amended) based on the combination of results obtained from 2 in vitro assays.