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Description of key information

Skin corrosion (OECD TG 431): Not corrosive

Skin irritation (OECD TG 439): Skin irritant

Eye irritation (OECD 438): severely irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-05-03 till 2015-07-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. QA statement)
Remarks:
TNO Triskelion, Utrechtseweg 48, 3704 HE Zeist, The Netherlands
Test system:
human skin model
Source species:
human
Cell type:
other:
Cell source:
foreskin from a single donor
Vehicle:
unchanged (no vehicle)
Details on test system:
The EpiDerm™ (EPI-200) skin model consisted of normal human epidermal keratinocytes from one single donor, derived from neonatal-foreskin tissue. The keratinocytes were plated on chemically modified, collagen-coated, 9 mm ID cell culture inserts (surface area 0.64 cm2). The skin models are commercially available and were obtained from MatTek In Vitro Life Science Laboratories (IVLSL), Slovakia.
Amount/concentration applied:
50 ul
Duration of treatment / exposure:
Skin corrosion test: 3 min and 60 min.
Skin irritation test: 60 min and 42h of post exposure
Duration of post-treatment incubation (if applicable):
Skin corrosion test: Immediately after exposure the viability of the epidermal cells was assessed using the MTT test.
Skin irritation test: Viability of the epidermal cells was assessed using the MTT test after 42h of post exposure
Number of replicates:
2
Controls:
other: Positive controls: Skin corrosion test 8 M potassium hydroxide solution and skin irritation test: 5% aqueous sodium dodecyl sulphate (SDS). Negative controls: Skin corrosion test: Milli-Q water and skin irritation test: phosphate buffered saline (PBS).
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Corrosion: 3 minutes exposure
Value:
97
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Corrosion: 60 minutes exposure
Value:
16
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Irritation at 60 minutes exposure and 42h post exposure
Value:
5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Tissue viability (% of negative control)
Basis:
mean
Time point:
other: 3 min
Score:
97
Max. score:
100
Remarks on result:
other: Skin corrosion test (SD = 13)
Irritation parameter:
other: Tissue viability (% of negative control)
Basis:
mean
Time point:
other: 60 min
Score:
16
Max. score:
100
Remarks on result:
other: Skin corrosion test (SD = 4)
Irritation parameter:
other: Tissue viability (% of negative control)
Basis:
mean
Time point:
other: 60 min
Score:
5
Max. score:
100
Remarks on result:
other: Skin irritation test (SD = 0)
Irritant / corrosive response data:
Preliminary tests
- At the end of the incubation period of the test substance with a MTT solution, the MTT solution had neither turned blue/purple nor showed a blue/purple precipitate, indicating that the test substance did not have the potential to reduce MTT. Therefore, no additional controls were required in the in vitro skin irritation test. During the mesh compatibility test it was concluded that the test substance did not have the potential to damage the nylon mesh and therefore the nylon mesh was used in the in vitro skin corrosion test to facilitate equal distribution of the test substance over the skin membrane surface. The preliminary test to determine if the test substance has the potential to stain the skin membranes during the exposure (as required according OECD guideline no. 431) was not performed, because no tissue staining was observed during the in vitro skin irritation test.

In vitro skin corrosion test
- The mean OD of the negative control (MilliQ) and the positive control (8 M KOH) demonstrated the expected response. The CV of the test groups that showed mean viability between 20% and 100% was ≤ 30%. All acceptance criteria were met and therefore the test was considered valid.
- At the end of the 3 min and 60 exposure, the skin membranes of the positive controls were detached from the insert, most likely as a result of the corrosiveness of the positive control.
- One skin membrane of the negative control of the 3 min exposure group (i.e. replicate NC3-2) was excluded from calculations due a technical error. Prior to OD measurement at the end of the extraction with MTT extractant, it was observed that the formazan was inefficiently extracted from the skin membrane (i.e. the skin membrane was still purple stained, whereas skin membranes from other groups were not stained because the formazan was efficiently extracted from the skin membranes). The purple staining of the membrane of replicate NC3-2 confirmed viability of the skin membrane and the reason for the inefficient extraction is unknown.

In vitro skin irritation test
- The mean OD of the blank, the negative control (PBS) and the positive control (5% SDS) demonstrated the expected response. The SD calculated from individual tissue viability percentages of the three replicates was <18%. All acceptance criteria were met and therefore the test was considered valid.
Interpretation of results:
other: A skin irritant Category 2
Remarks:
according to the CLP Regulation EC 1272/2008 and its updates
Conclusions:
Under the test conditions (OECD 431, 439 and GLP) the test substance is considered to be a skin irritant.
Executive summary:

In accordance to OECD guideline 431, 439 and GLP the test substance was examined for its in vitro skin corrosion and irritation potential using EpiDerm™ reconstructed skin membranes. In the in vitro skin corrosion test, the skin membranes were topically exposed to the undiluted test substance for 3 min and 60 min. Immediately after exposure the viability of the epidermal cells was assessed using the MTT test. In the in vitro skin irritation test, the skin membranes were topically exposed to the undiluted test substance for 60 min. Viability of the epidermal cells was assessed using the MTT test after 42 h of culture. Per test group, tests were performed in duplicate. In both tests negative and positive controls were run in parallel. In the skin corrosion test the mean viability of the skin membranes was 97 ± 13% after 3 minutes and 16 ± 4% after 60 minutes. In the skin irritation test the mean viability was 5 ± 0% compared to the concurrent negative control group. Based on the results obtained in the present study the test substance is considered to be a skin irritant.

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:
2015-05-20 till 2015-06-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD 438 guideline study in compliance with GLP, available as unpublished report, no restrictions, fully adequate for assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Lot no: SM14116260
Expiry date: 24 November 2015
Physical state: Colourless liquid
Species:
other: eyes of male or female chickens (ROSS, spring chickens)
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Slaughterhouse v.d. Bor, Nijkerkerveen, The Netherlands
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: approximately 1.5 - 2.5 kg
- Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.
- The preparation and validation of the eyes prior to the ICE-test were all according to OECD guideline 438.
Vehicle:
unchanged (no vehicle)
Controls:
other: Positive controls: Benzalkonium Chloride. Negative control: Phosphate buffered saline (PBS).
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 30 µL
Duration of treatment / exposure:
10 seconds
Observation period (in vivo):
0, 30, 75, 120, 180, and 240 minutes
Number of animals or in vitro replicates:
3 eyes
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: The eyes were rinsed with 20 mL saline
- Time after start of exposure: 10 seconds

SCORING SYSTEM: According to OECD 438 guideline.

TOOL USED TO ASSESS SCORE: All examinations were carried out with the hand-slit lamp microscope. Fluorescein retention was only scored at approximately 30 minutes after treatment.

CONTROLS: A negative control (30 µL physiological saline) and 3 positive controls (30 µL Benzalkonium Chloride 5%) were included.
Irritation parameter:
percent corneal swelling
Run / experiment:
mean
Value:
5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
2.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
mean
Value:
0.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritant / corrosive response data:
Slit-lamp examination: Ylanganate caused very slight corneal swelling (5%), moderate to severe opacity (mean score of 2.5) and very slight or slight fluorescein retention (mean score of 0.8). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.

Microscopic examination: Microscopic examination of the corneas treated with Ylanganate did not reveal any abnormalities. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. The positive control BAC 5% caused severe erosion, slight vacuolation (one cornea; low region) of the epithelium, the epithelium partly detached from the basement membrane (two corneas), and endothelial necrosis (two corneas).
Interpretation of results:
other: Eye irritant Category 2 (irritant)
Remarks:
according to the CLP Regulation EC 1272/2008 and its updates
Conclusions:
Under the test conditions (OECD 438 and GLP) the test substance is considered to be an eye irritant
Executive summary:

In accordance to OECD guideline 438 and GLP the test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) Test. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL Benzalkonium Chloride (BAC)) were tested. After the exposure the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. The test substance caused very slight corneal swelling (5%), very slight or slight to moderate opacity (mean score of 2.5) and very slight fluorescein retention (mean score of 0.8). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the test substance did not reveal any abnormalities. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. The positive control BAC 5% caused severe erosion, slight vacuolation (one cornea; low region) of the epithelium, the epithelium partly detached from the basement membrane (two corneas), and endothelial necrosis (two corneas). Based on these results, the test substance is considered to be an eye irritant.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Skin corrosion: In accordance to OECD TG 431, test substance was examined for its in vitro skin corrosion potential using EpiDerm(TM) reconstructed membranes. In the in vitro corrosion test the skin membranes were topically exposed to the undiluted test substance for 3 min and 60 min. Per testgroup, tests were performed in duplicate. Immediately after exposure the viability of the epidermal cells was assessed using the MTT test. In the skin corrosion test the mean viability of the skin membranes was 97 ± 13% after 3 minutes and 16 ± 4% after 60 minutes. In both tests negative and positive controls were run in parallel and showed the required response. In view of the 16% viability being > 15% the substance is not considered corrosive.

Skin irritation: In accordance with OECD TG 439 and GLP the test substance was examined for its in vitro skin irritation potential using EpiDerm™ reconstructed skin membranes. In the in vitro skin irritation test, the skin membranes were topically exposed to the undiluted test substance for 60 min. Viability of the epidermal cells was assessed using the MTT test after 42 h of culture. Per test group, tests were performed in duplicate. In both tests negative and positive controls were run in parallel. In the skin irritation test the mean viability was 5 ± 0% compared to the concurrent negative control group. Based on the results obtained in the present study the test substance is considered to be a skin irritant.

Eye

In accordance to OECD guideline 438 and GLP the test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) Test. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL Benzalkonium Chloride (BAC)) were tested. After the exposure the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. The test substance caused very slight corneal swelling (5%), very slight or slight to moderate opacity (mean score of 2.5) and very slight fluorescein retention (mean score of 0.8). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the test substance did not reveal any abnormalities. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. The positive control BAC 5% caused severe erosion, slight vacuolation (one cornea; low region) of the epithelium, the epithelium partly detached from the basement membrane (two corneas), and endothelial necrosis (two corneas). Based on these results, the test substance is considered to be an eye irritant.

Justification for classification or non-classification

The skin irritation studies reveal that the substance is not corrosive but it is a skin irritant category 2 (H315, Causes skin irritation) in accordance withEU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and its updates.

Based on the results of the isolated chicken eye test the test substance has to be classified for eye irritation category 2 (H319 Causes serious eye irritation) in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and its updates.