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Administrative data

Description of key information

Skin: Guinea pig Primary Irritation, No irritation. Reliability = 2

Eye: OECD 492. Relative viability of 92.2% (not irritating) Reliability = 1

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reference:
Composition 0
Qualifier:
no guideline followed
Principles of method if other than guideline:
The primary dermal irritation of the test substance was examined in guinea pigs.
GLP compliance:
not specified
Test material information:
Composition 1
Vehicle:
physiological saline
Species:
guinea pig
Strain:
Hartley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Japan SLC, Inc. (3371-8 Koto-cho, Harnamatsu-shi, Shizuoka, Japan)
- Age at study initiation: 6 weeks
- Weight at study initiation: not reported
- Housing: Aluminum cages (W 350 mm x D 400 mm x H 230 mm). Animals were kept in groups of 5 or 6 per cage during the quarantine period and in groups of 5 per cage during the study period.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±2°C
- Humidity (%): 50±10%
- Air changes (per hr): 17 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours/dark
Type of coverage:
occlusive
Preparation of test site:
shaved
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g

VEHICLE
- Amount(s) applied (volume or weight with unit): physiological saline

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): physiological saline

POSITIVE CONTROL: No
Duration of treatment / exposure:
24 hours
Observation period:
3, 24, and 48 hours after patch removal
Number of animals:
5
Details on study design:
TEST SITE
- Area of exposure: lateral abdomen (normal and abraded skin)
- % coverage: not reported
- Type of wrap if used: non-permeable adhesive plaster and elastic adhesive plaster

REMOVAL OF TEST SUBSTANCE
- Washing (if done): not reported
OBSERVATION TIME POINTS: 3, 24, and 48 hours

SCORING SYSTEM:
- Method of calculation: Draize scale
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Time point:
48 h
Score:
0
Max. score:
8
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
No skin reaction was observed at any observation time in either the normal skin or abraded skin similarly to the control group.
Other effects:
No abnormality was observed in general conditions dnring the stndy period.
Interpretation of results:
GHS criteria not met
Conclusions:
No irritation was observed.
Executive summary:

Skin irritability of the test substance was examined using five albino Hartley guinea pigs. Normal and abraded skin regions were provided on the shaven lateral abdomen of each animal. The pure test substance as a white powder was used in the study with dropping physiological saline on it in order to improve contact with the skin. For administration, 0.1 g of the test substance was placed on the cloth portion of the adhesive plaster for patch test with a drop of physiological saline on it and applied as a 24-hour closed patch using an adhesive sponge plaster and elastic plaster. Irritation reactions were observed at 3, 24 and 48 hours after removal of the patch in accordance with the Draize’s assessment criteria. As a result, no abnormality was observed in general conditions during the study period. No skin reaction was observed at any observation time in either the normal skin or abraded skin similarly to the control group, and the primary irritation index (P.I.I.) was 0.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
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)
Deviations:
yes
Remarks:
Stability of test or controls was not determined by testing facility. Test and controls not characterized by testing facility (performed under non-GLP conditions by sponsor prior to initiation of study).
GLP compliance:
yes
Test material information:
Composition 1
Species:
other: In vitro EpiOcular™ tissues containing stratified human keratinocytes
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability: Test was performed according to OECD Guideline 492 using EpiOcular™ Eye Irritation Test (EIT)
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: commercially available RhCE tissue constructs
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
50 mg
Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used: After an overnight incubation for 16-24 hours, 6-well plates containing the EpiOcular™ tissues were removed from the incubator. Prior to test or control substance applications, each tissue surface was moistened with 20 μL of Ca++Mg++-free D-PBS and incubated at standard culture conditions for 30 minutes. After incubation, the tissues were tested in duplicate with 50 μL of the positive control or negative control, or approximately 50 mg of test substance and incubated at standard culture conditions for 6 hours. At the end of the 6-hour treatment time, the test substance, or control articles were removed by extensively rinsing the tissues followed by transference to 5 mL of Assay Medium, in a prelabeled 12 well plate for 25 minutes of immersion incubation (Post-Soak) at room temperature to remove any test substance absorbed into the tissue. At the end of the Post-Soak immersion, each insert was removed, the medium decanted off the tissue, the insert blotted on absorbent material, and transferred to the appropriate well of the prelabeled 6-well plate containing 1 mL of warm Assay Medium. The tissues were incubated for 18 hours at Standard Culture Conditions (Posttreatment Incubation). At the end of the Post-treatment Incubation, the constructs were removed, gently blotted on absorbent material, and transferred to appropriate wells containing 300 μL of MTT solution. The trays were incubated for 180 minutes at standard culture conditions, after which they were transferred to a prelabeled 24-well plate containing 2.0 mL of isopropanol in a like manner. The plates were sealed with parafilm and stored in the refrigerator (2-8ºC) until the last exposure time was harvested. To extract the MTT, the plates were then placed on an orbital plate shaker and shaken for 2-3 hours at room temperature. At the end of the extraction period, the liquid within the cell culture inserts was decanted into the well from which the cell culture insert was taken. The extract solution was mixed, and two aliquots of 200 μL were transferred to the appropriate wells of a 96-well plate. Two hundred microliters of isopropanol were added to the two wells designated as the blanks. The absorbance at 550 nm (OD550) of each well was measured with a Molecular Devices Vmax plate reader.

- RhCE tissue construct used, including batch number: stratified human keratinocytes
- Doses of test chemical and control substances used: 50 mg test substance; 50 µL of positive or negative control
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): 6-hour exposure and 18-hour post treatment incubation at 37°C
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: negative control of sterile deionized water (50 µL) for MTT and isopropanol blank for colorant control test
- Number of tissue replicates used per test chemical and controls: duplicate for test substance, positive and negative controls
- Wavelength used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 550 nm
- Description of the method used to quantify MTT formazan: If the MTT solution colour turned blue/purple, the test substance was presumed to have reduced the MTT
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: Based on OECD Guideline 492, if the test article-treated tissue viability is >60% relative to negative control-treated tissue viability, the test article is identified as not requiring classification and labelling according to UN GHS (No Category). If the test article-treated tissue viability is ≤60% relative to negative control-treated tissue viability, the test article is identified as potentially requiring classification and labelling according to UN GHS (Category 1 or 2)
- Positive and negative control means and acceptance ranges based on historical data: no
- Acceptable variability between tissue replicates for positive and negative controls: yes; the corrected mean OD550 value of the negative control was >0.8 and <2.5, and the mean relative viability of the positive control was ≤50%
- Acceptable variability between tissue replicates for the test chemical: yes
Irritation parameter:
other: % tissue viability
Value:
92.2
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes; OD550 was 1.473
- Acceptance criteria met for positive control: yes; viability was 17.5%

The test substance was not observed to reduce MTT directly in the absence of viable cells; therefore, a killed control experiment was not performed. The test substance was not observed to be a colorant in isopropanol; therefore, a colorant control was not performed.
Interpretation of results:
GHS criteria not met
Conclusions:
The test substance was predicted to be a non-irritant (mean viability was 92.2%).
Executive summary:

The EpiOcular™ Eye Irritation Test (EIT) was used to evaluate the ocular irritation potential of the test substance in the context of classification of ocular irritation according to the UN GHS classification system. The ocular irritation potential was evaluated based upon measuring the relative conversion of MTT (3-[4, 5 - dimethylthiazol-2-yl] - 2,5 - diphenyltetrazolium bromide)in the test substance-treated tissues after exposure to the test substance for 6 hours, followed by an 18 hour post-exposure expression period. Ocular irritation potential of the test article was predicted if the relative viability was less than or equal to 60%. If the relative viability was greater than 60%, the test article was predicted to not require classification or labelling for ocular irritation (GHS No Category). The protocol met the requirements of the OECD test guideline “Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labeling for eye irritation or serious eye damage: (TG 492).

The corrected mean OD550value for the negative control was 1.473, and the viability of the positive control, Methyl Acetate, was less than 50%; therefore, the assay results were considered valid. Based upon the results of this assay the test substance resulted in a relative viability of 92.2%, and is predicted to not require classification or labelling for ocular irritation (GHS No Category).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritability of the test substance was examined in guinea pigs (normal and abraded skin). 0.1 g of the test substance, as received, was used. Physiological saline was dropped on it in order to improve contact with the skin. Application was for 24 hours under semi-occlusive conditions. No skin reaction was observed at any observation time in either the normal skin or abraded skin similarly to the control group, and the primary irritation index (P.I.I.) was 0.

An in vitro EpiOcular™ eye irritation test was performed with the test substance. The test substance had a relative viability of 92.2%, indicating that it is not predicted to be an eye irritant.

Justification for classification or non-classification

No dermal irritation was observed in a primary irritation test in guinea pigs. In an in vitro Epiocular™ eye irritation test, the test substance was not irritating based on a relative viability of 92.2%. Therefore, the substance does not need to be classified for skin or eye irritation according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.