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Description of key information

The test substance was determined to be skin irritating in an in vivo study using rabbits.

A weight of evidence approach determined irritating properties of the test substance to the eye in vitro. 

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:
2003-07-21 to 2003-08-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
2002
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
other: Chbb:HM(SPF) - Littlerussian
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 17 to 20 months
- Weight at study initiation: 2.7 — 3.1 kg b. w.
- Housing: individually in PPO cages (floor area: 2576 sq. cm) with perforated floor
- Diet: ad libitum, Altromin 2123" from Altromin, D-32791 Lage, Lippe
- Water: ad libitum, domestic quality drinking water acidified with hydrochloric acid to pH 2.5
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 55 ± 15
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.5 mL
- Concentration: undiluted (100 %)
Duration of treatment / exposure:
3 min, 1 (for 1 animal) and 4 h (for all 3 animals)
Observation period:
immediately and after 1, 24, 48, 72 h and 7, 14, 21 days
Number of animals:
1 animal (3min, 1 and 4 h), 2 animals (4 h)
Details on study design:
TEST SITE
- Area of exposure: back skin, 2.5 x 2.5 cm
- Type of wrap if used: 16-layer gauze patch secured with adhesive Gothaplast tape and Gothaplast tape wound around the trunk of the animal

REMOVAL OF TEST SUBSTANCE
- Washing: mild soap and lukewarm water
- Time after start of exposure: after exporsure time was over test field was marked and directly washed

SCORING SYSTEM:
- Erythema and Eschar Formation: 1-4
- Oedema Formation: 1-4
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Score:
2.9
Max. score:
4
Reversibility:
fully reversible within: 21 days
Remarks on result:
other: exposure time 4 h
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Score:
2.3
Max. score:
4
Reversibility:
fully reversible within: 21 days
Remarks on result:
other: exposure time 4 h
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
3
Max. score:
4
Reversibility:
fully reversible within: 21 days
Remarks on result:
other: exposure time 4 h
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
3
Max. score:
4
Reversibility:
fully reversible within: 21 days
Remarks on result:
other: exposure time 4 h
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
2.67
Max. score:
4
Reversibility:
fully reversible within: 21 days
Remarks on result:
other: exposure time 4 h
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
3
Max. score:
4
Reversibility:
fully reversible within: 21 days
Remarks on result:
other: exposure time 4 h
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 21 days
Remarks on result:
other: exposure time 4 h
Irritation parameter:
edema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 21 days
Remarks on result:
other: exposure time 4 h

Results of Animal 1 after 3 min and 1 h exposure:

Exposure time  Timepoint of observation [h] Erythema score (of max. 4) Edema score (of max. 4)
3 min 0 2 0
1 1 0
24 2 0
48 1 0
72 0 0
1 h 0 2 0
1 1 0
24 2 2
48 2 2
72 2 2
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
The test substance was determined to cause skin irritation in rabbits.
Executive summary:

The skin irritant effect of the test substance was investigated according to OECD Guideline 404. During an initial test using one female albino rabbit a sequential application (exposure time 3 min, 1 h and 4 h) of the test substance was carried out on one site of the left back area. The skin was examined for signs of irritation directly after application as well as 1 h, 24 h, 48 h and 72 h after the termination of exposure as well as on day 7, 14 and 21. Since the animal did show very well defined but reversible signs of skin irritation within the observation period of 21 days it was decided to carry out a confirmatory test with two additional animals in order to allow a categorization of the test item based on the classification criteria. Two female albino rabbits were exposed to the test item at one skin site on the backs during the confirmatory test. After 4 hour exposure time the test item was removed and the skin was examined 1 h, 24 h, 48 h and 72 h after the termination of exposure as well as on day 7, 14 and 21. Very well defined skin reactions were observed in these animals, too. All effects were fully reversible within 21 days. Under the experimental conditions described, the mean score (24/48/72h) for erythema was 2.9 and for edema 2.3. For individual animals the erythema score for 24/48/72h was 3 for animals 1 and 2 and 2.67 for animal 3. The edema score for individual animals at 24/48/72h was 3 for animal 1 and 2 for animals 2 and 3. Considering these results the test substance was determined to be irritating to the skin.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
weight of evidence
Study period:
2015-11-16 to 2015-11-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline for Testing of Chemicals 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage (July, 2015).
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Envigo CRS GmbH, 64380 Rossdorf, Germany
Species:
human
Strain:
other: Reconstructed human Cornea-like Epithelium (RhCE)
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability
In a prevalidation study performed by Avon Products Inc. and MatTek Corporation, the in vitro eye test using the human cornea model EpiOcular™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for eye irritancy potential. The EpiOcular ™ Eye Irritation Test (EIT) was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
RhCE tissue was used.
Cells used for the tissue production were screened for biological contamination and determined to be free of any contamination.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 μL
Duration of treatment / exposure:
30 min for test item and controls
Duration of post- treatment incubation (in vitro):
122 minutes
Number of animals or in vitro replicates:
2
Details on study design:
- RhCE tissue construct used, including batch number
- EpiOcular Kit (Lot number: 21581) Kits MatTek Corporation (Ashland, MA 01721, USA)
- Doses of test chemical and control substances used: 50 μL
- Duration and temperature of exposure (30 min, 37 ± 1.5 °C), post-exposure immersion (12 ± 2 minutes, room temperature) and post-exposure incubation (122 minutes, 37 ± 1.5 °C) periods
- Description of any modifications to the test procedure: No modifications were done.
- Number of tissue replicates used per test chemical and controls: 2
- Description of the method used to quantify MTT formazan: OD measurement, no data on wavelength etc provided
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
The mean OD value of the blank control wells (ODBlk) for each experiment were calculated. The ODBlk from each OD value of the same experiment (blank corrected values) were subtracted. The mean value of the two aliquots for each tissue (= corrected test item OD) were calculated. The mean value of the two relating tissues for each control and test item (= corrected mean OD) were calculated. For further calculations only the corrected mean negative control OD value was needed. The corrected OD value of the negative control corresponds to 100% viability. Corrected negative control OD = Negative Control OD - ODBfk = 100% Viability
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant. If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labeled irritant.
- Positive and negative control means and acceptance ranges based on historical data:
negative control OD: > 0.8 and < 2.5; mean relative viability of the positive control: below 60% of the negative control viability
- Acceptable variability between tissue replicates for positive and negative controls: < 20%
- Acceptable variability between tissue replicates for the test chemical: < 20%
Irritation parameter:
other: tissue viability [%]
Run / experiment:
mean
Value:
39.3
Vehicle controls validity:
not examined
Negative controls validity:
valid
Remarks:
100 %
Positive controls validity:
valid
Remarks:
32.0 %
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No damages described.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Results:

 

Dose Group

Mean Absorbance* Tissue 1 and 2 minus Mean Blank

Mean Absorbance of 2 Tissues*

Rel. Absorbance [%] Tissue 1 and 2**

Absolute Value of the Difference of the Rel. Absorbances [%] Tissue 1 and 2

Rel. Absorbance [% of Negative Control]**

Negative control

1.339

1.307

102.5

5.0

100.0

1.274

97.5

Positive control

0.442

0.418

33.8

3.6

32.0

0.395

30.2

Test item

0.531

0.514

40.6

2.6

39.3

0.497

38.0

* Mean of three replicate wells after blank correction

** relative absorbance [rounded values]: (100x(absorbance test item/positive control))/ absorbance negative control

 

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol did not led to a change in colour.

Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue colour.

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 39.3 % (threshold for irritancy:60 %), consequently the test item was irritant to eye.

 

Concerning acceptance criteria:

-The negative control OD is >0.8 and < 2.5 (1.274 and 1.339).

-The mean relative viability of the positive control is below 60 % of the negative control viability (32.0 %).

-The difference of viability between the two relating tissues of a single item is < 20 % (values between 2.6 % to 5.0 %) in the same run (for positive and negative control tissues and tissues of single test items).

Interpretation of results:
other: Category 1 or 2 based on GHS criteria
Conclusions:
The test substance was determined to be an eye irritant in the in vitro Reconstructed human Cornea-like Epithelium.
Executive summary:

The in vitro Reconstructed human Cornea-like Epithelium study according to OECD 492 was performed to assess the irritation potential of the test substance. Independent duplicate tissues of EpiOcular were exposed to either the test item, the negative control (deionised water) or the positive control (Methyl acetate) for 30 min. 50 μL of the test item were dispensed directly onto duplicate EpiOcular tissue surface. After exposure of the tissues to the test substance the mean tissue viability decreased to 39.3 %. This value is below the threshold for irritation, which is defined to be ≤ 60 %. Therefore, the test substance was considered to be eye irritating or cause serious damage to the eye.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2016-03-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Envigo CRS GmbH, 64380 Rossdorf, Germany
Species:
chicken
Strain:
other: Gallus Gallus
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Number of animals: multiple
- Characteristics of donor animals: 3 kg, approx. 56 days old
- Storage, temperature and transport conditions of ocular tissue: Following slaughter, the intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline. The heads were transported to the test facility at ambient temperature.
- Time interval prior to initiating testing: used during the same day as sacrifice
- indication of any existing defects or lesions in ocular tissue samples: Eyes that had a high baseline fluorescein staining (>0.5) or corneal opacity score (>0.5) after the enucleation process were rejected.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.03 mL
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
240 min
Number of animals or in vitro replicates:
3 for test substance and positive control, 3 for negative control
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
Eyes that had a high baseline fluorescein staining (>0.5) or corneal opacity score (>0.5) after the enucleation process were rejected. Acceptable eyes were dissected from the skull and pulled from the orbit by holding the nictitating membrane firmly with surgical forceps. The tissue behind the eye was carefully removed with bent, blunt-tipped scissors. Once the eye was removed from the orbit a portion of the optic nerve remained. Other connective tissue was removed from the eye on an absorbent tray liner.

EQUILIBRATION AND BASELINE RECORDINGS
After the approval process the eyes were incubated for 45 minutes for equilibrium purposes. Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.

NUMBER OF REPLICATES
3 for test substance and positive control, 2 for negative control

NEGATIVE CONTROL USED
Sodium chloride 0.9 % w/v

POSITIVE CONTROL USED
Benzalkonium chloride 5 % in distilled water

APPLICATION DOSE AND EXPOSURE TIME
The test substance was used undiluted and the concentrations of the controls were 0.9 % for negative and 5 % for positive control. All eyes were exposed for 10 seconds.

OBSERVATION PERIOD
Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (± 5 minutes) after the eyes had been decontaminated with the isotonic saline.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL of isotonic saline, rinsing

METHODS FOR MEASURED ENDPOINTS:
The Haag-Streit BQ 900 (Switzerland) microscope was used for examinations. Corneal opacity, damage to epithelium based on fluorescein retention (only at 30 minutes), swelling, and macroscopic morphological damage to the surface (pitting, sloughing, roughening of the corneal surface, and adhering of test item) were evaluated.

SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

DECISION CRITERIA: The decision criteria of UN GHS was applied. A test was considered acceptable if the concurrent negative or vehicle/solvent items and the concurrent positive controls were identified as GHS Non-Classified and GHS Category 1, respectively.
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
0.5
Positive controls validity:
valid
Remarks:
4.0
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Run / experiment:
mean
Value:
1.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
0.5
Positive controls validity:
valid
Remarks:
3.0
Remarks on result:
no indication of irritation
Irritation parameter:
percent corneal swelling
Run / experiment:
mean
Value:
13.53
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
1.47
Positive controls validity:
valid
Remarks:
34.27
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Corneal Opacity Scores
Very faint to easily discernible translucent area of opacity of the cornea was noted in all test item treated eyes. Complete corneal opacity was noted in all positive control treated eyes. Very faint opacity was noted in the negative control treated eyes. No morphological effects were noted in the test item or control item treated eyes. Sloughing was noted in all positive control treated eyes.

Fluorescein Retention Scores
Very minor single cell staining to focal or confluent dense single cell staining of the cornea was noted in all test item treated eyes. Confluent large areas of the cornea retaining fluorescein were noted in all positive control treated eyes. Very minor single cell staining was noted in both negative control treated eyes.
Interpretation of results:
other: not Category 1 based on GHS criteria
Conclusions:
The test substance was determined to not cause serious eye damage in an Isolated Chicken Eye Test.
Executive summary:

An Isolated Chicken Eye Test (ICE) according to OECD 438 was conducted to evaluate the irritation potential of the test substance to the eye. The undiluted test substance was applied to an isolated chicken eye for 10 seconds. The experiment was done in triplicate. Sodium chloride was used as a negative control and Benzalkonium chloride as positive control. The eyes were evaluated 30, 75, 120, 180 and 240 minutes after exposure. The mean values for cornea opacity, fluorescein retention and corneal swelling were calculated. For the test substance a cornea opacity score of max. 2.0, a fluorescein retention score of max. 1.2 and a corneal swelling of max. 13.53 % were detected. The values for the positive control were 4.0, 3.0 and 34.27 % respectively, while for negative control 0.5, 0.5 and 1.47 % were established. Due to these observations it can be concluded that the test substance does not cause serious damage to the eyes.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion

A semi-occlusive skin irritation test was performed using 3 rabbits. One rabbit was exposed for 3 min, 1 and 4 h while the other 2 were exposed for 4 h only. The animals were observed for 21 days. After 4 h exposure to the test substance erythema and edema development was observed that was reversible within 21 days. The mean erythema score for 24/48/72h was 2.9 and the mean edema score for the same timeframe was 2.3. For individual animals the erythema score for 24/48/72h was 3 for animals 1 and 2 and 2.67 for animal 3. The edema score for individual animals at 24/48/72h was 3 for animal 1 and 2 for animals 2 and 3. Due to these results the test substance was classified as being skin irritating.

 

Eye irritation

A weight of evidence approach was conducted to evaluate the eye irritation potential of the test substance. Therefore two in vitro studies were conducted.

The in vitro Reconstructed human Cornea-like Epithelium study according to OECD 492 was performed to assess the potential to cause irritation or serious eye damage of the test substance. Independent duplicate tissues of EpiOcular were exposed to either the test item, the negative control or the positive control for 30 min. After exposure of the tissues to the test substance the mean tissue viability decreased to 39.3 %. Therefore, the test substance was considered to be eye irritating or cause serious damage to the eye.

An Isolated Chicken Eye Test (ICE) according to OECD 438 was conducted to evaluate the potential of the test substance to cause serious damage to the eye. The undiluted test substance was applied to three isolated chicken eyes for 10 seconds. The eyes were evaluated 30, 75, 120, 180 and 240 minutes after exposure. For the test substance a cornea opacity score of max. 2.0, a fluorescein retention score of max. 1.2 and a corneal swelling of max. 13.53 % were detected. The values for the positive control were 4.0, 3.0 and 34.27 % respectively, while for negative control 0.5, 0.5 and 1.47 % were established. Due to these observations it can be concluded that the test substance does not cause serious damage to the eyes.

 

Conclusion

The study with Reconstructed human Cornea-like Epithelium determined the test substance causes an adverse effect. Whether this effect is eye irritation or serious damage to the eye could not be concluded from this study. Therefore the ICE study was conducted to determine if the substance causes serious eye damage. This study concluded that the test substance does not cause serious damage to the eyes. In a weight of evidence approach the results of both studies combined showed that the test substance was irritating (Category 2) to eye according to EU CLP and UN GHS.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

Based on available data on skin irritation/corrosion, the test item is classified into category 2 (H315: Causes skin irritation) according to Regulation (EC) No 1272/2008 (CLP), as amended for the ninth time in Regulation (EU) No 2016/1179.

Based on available data on eye irritation/serious eye damage, the test item is classified into category 2 (H319: Causes serious eye irritation) according to Regulation (EC) No 1272/2008 (CLP), as amended for the ninth time in Regulation (EU) No 2016/1179.