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Description of key information

Skin corrosion (OECDTG 404): Not corrosive

Skin irritation (OECD TG 404): Skin irritant
Eye severe damage (OECD TG 438): Not eye irritating


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:
24 April 2001 - 16 May 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The in vivo test was performed before the REACH regulation came into force, requesting in vitro studies.
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
17 July 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Version / remarks:
31 July 1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Elevage Scientifique des Dombes, F-01400 Chatillon sur Chalaronne, France
- Age at study initiation: 11 weeks
- Weight at study initiation: 2.1 - 2.2 kg
- Housing: Individually in stainless steel cages.
- Diet: Free access to pelleted standard Provimi Kliba 3418 rabbit maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst).
- Water: Free access to community tap water from Füllinsdorf
- Acclimation period: no data.

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 20 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 14
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
other: Adjacent areas of the untreated skin of each animal served as controls.
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.5 mL
Duration of treatment / exposure:
4 hours
Observation period:
14 days
Number of animals:
1 male and 2 females
Details on study design:
TEST SITE:
Four days before treatment, the left flank was was clipped with an electric clipper, exposing an area of approximately 100 square centimeters (10x10 cm). The skin of the animals was examined one day before treatment, and regrown fur of all animals was again clipped.

TREATMENT
On the day of treatment, 0.5 mL of the test substance was places on a surgical gauze patch (ca. 2.5 cm x 2.5 cm). This gauze patch was applied to approx. 6 square centimeters of the intact skin of the clipped area. The patch was covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and enchored with tape.

REMOVAL OF TEST SUBSTANCE
After 4 hours the dressing was removed and the skin was flushed with lukewarm tap water to clean the application site.

OBSERVATIONS
- Mortality/Viability and Clinical signs: Daily from delivery of the animals to the termination of the test.
- Body Weight: At the start of acclimatization, on the day of application and at termination of observation.
- Irritation: The skin reactions were assessed at approximately 1, 24, 48 and 72 hours and at 7, 10 and 14 days after the removal of the dressings and test substance. The irritation scores and a description of all other (local) effects were recorded.

SCORING SYSTEM:
The irritation was assessed according to the numerical scoring system listed in the EEC Commission Directive 92/69/EEC, July 31, 1992.
If evident, corrosive or staining properties of the test substance were described and recorded.
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 14 days
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.67
Max. score:
4
Reversibility:
fully reversible within: 14 days
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
2.33
Max. score:
4
Reversibility:
fully reversible within: 14 days
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2.67
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
edema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1.67
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritant / corrosive response data:
At 1 hour, in one animal moderate erythema was seen; at the same time in 2/3 severe swelling was seen. At 24/48/72 hours well-defined erythema were seen in all animals with maximum score 2; at this time point 2/3 animals showed moderate to severe swelling with average score of 2.33 and one animal scored 1.33. All skin reactions were clear 14 days after treatment.
Other effects:
No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred.
Scaling was evident at the test site of all animals from 7 to 10 days after treatment. In addition, the skin of one female was noted to be dry and inelastic at these readings.
No staining by the test substance of the treated skin was observed. No irreversible alterations of the treated skin were observed nor were corrosive effects evident on the skin.
The body weights of all animals were considered to be within the normal range of variability.
Interpretation of results:
other: Skin irritant Cat 2
Remarks:
According to Regulation (EC) No. 1272/2008 and its updates.
Conclusions:
In this skin irritation study with rabbits, performed according to OECD 404 guideline and GLP principles, moderate erythema (score 2) were seen and moderate to severe edema (score 1-3) were seen at 24/48 and 72hours. Two out of three animals have an average edema score of = 2.33, while the other animal scored an average of 1.33. The irritant effects were reversible within 14 days. This means that the substance is a skin irritant.
Executive summary:

The substance was tested for 4 hours in a skin irritation test in 3 rabbits according to OECD TG 404 test guideline and GLP principles.

No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred. In this study moderate erythema (score 2) were seen and moderate to severe edema (score 1 -3) were seen at 24/48 and 72hours. Two out of three animals have an average edema score of = 2.33, while the other animal scored average of 1.33 for edema. The irritant effects were reversible within 14 days. This means that the substance is 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:
23-12-2016 to 08-02-2017
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)
GLP compliance:
yes (incl. QA statement)
Remarks:
Triskelion B.V., Utrechtseweg 48, 3700 AV, Zeist
Species:
other: Eyes of male or female chickens (ROSS, spring chickens)
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Slaughterhouse v.d. Bor, Nijkerkerveen, The Netherlands
- Characteristics of donor animals: Approximately 7 weeks old, male or female chickens, body weight range approximately 1.5-2.5 kg, were used as eye donors.
- Storage, temperature and transport conditions of ocular tissue: 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.
- Time interval prior to initiating testing: Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus.
- Indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: No
Vehicle:
unchanged (no vehicle)
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using the following procedure: First the eye-lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium 2.0% w/v was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. Next, the head with the fluorescein-treated cornea was examined with a slit-lamp microscope (Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland) to ensure that the cornea was not damaged. If undamaged (e.g., fluorescein retention and corneal opacity scores of ≤ 0.5), the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye-ball from the orbit without cutting off the optical nerve too short. The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a target rate of 0.10-0.15 mL/min. The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32 °C (water pump set at 36.4 °C). After placing in the superfusion apparatus, the eyes were examined again with the slit-lamp microscope to ensure that they were not damaged. An accurate measurement was taken at the corneal apex of each eye. Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes showing opacity (score higher than 0.5), or were unacceptably stained with fluorescein (score higher than 0.5) indicating the cornea to be permeable, or eyes that showed any other signs of damage, were rejected as test eyes and replaced.

EQUILIBRATION AND BASELINE RECORDINGS
Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. For that purpose, after an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured again to determine the zero reference value for corneal swelling calculations.

NUMBER OF REPLICATES
Negative control: 1
Positive control: 3
Test group: 3

NEGATIVE CONTROL USED
Physiological saline

POSITIVE CONTROL USED
Benzalkonium Chloride 5%

APPLICATION DOSE AND EXPOSURE TIME
30 μL for 10 seconds

OBSERVATION PERIOD
240 minutes

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL saline. After rinsing, each eye in the holder was returned to its chamber.
- Indicate any deviation from test procedure in the Guideline: none

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Slit-lamp microscope examination
- Damage to epithelium based on fluorescein retention: Slit-lamp microscope examination
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: set at 0.095 mm
- Others: After the final examination, the test substance treated eyes, the negative and positive control eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at ca 4 μm and stained with PAS (Periodic Acid-Schiff). The microscopic slides were subjected to histopathological examination.

SCORING SYSTEM:
Defined scoring scales were used for each parameter to define the severity of effects into four categories (I-IV).
- Mean corneal swelling (%): According to OECD 438 guideline. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes
- Mean maximum opacity score: According to OECD 438 guideline. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes
- Mean fluorescein retention score at 30 minutes post-treatment: According to OECD 438 guideline.

DECISION CRITERIA: According to OECD 438 guideline
Irritation parameter:
percent corneal swelling
Run / experiment:
slit-lamp examination
Value:
3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: maximum mean values
Irritation parameter:
cornea opacity score
Run / experiment:
slit-lamp examination
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: maximum mean values
Irritation parameter:
fluorescein retention score
Run / experiment:
slit-lamp examination
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Slit-lamp examination: The test substance caused no or very slight corneal swelling (mean of 3%). 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. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight, moderate or severe erosion and very slight or slight vacuolation of the epithelium and the epithelium partly detached from the basement membrane.
Interpretation of results:
other: Not an eye irritant.
Remarks:
According to Regulation (EC) No. 1272/2008 and its updates.
Conclusions:
Under the test conditions (OECD 438 and GLP) the test substance is not 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 no or very slight corneal swelling (mean of 3%). 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. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight, moderate or severe erosion and very slight or slight vacuolation of the epithelium and the epithelium partly detached from the basement membrane. Based on these results, the test substance is considered to be not eye irritating.

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

Additional information

In vivo skin corrosion and irritation:

The substance was tested for 4 hours in a skin corrosion/irritation test in 3 rabbits according to OECD TG 404 test guideline and GLP principles.

No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred. In this study moderate erythema (score 2) were seen and moderate to severe edema (score 1 -3) were seen at 24/48 and 72hours. Two out of three animals have an average edema score of = 2.33, while the other animal scored average of 1.33 for edema. The irritant effects were reversible within 14 days. This means that the substance is not corrosive for skin but it is a skin irritant.

In vitro eye irritation:

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 no or very slight corneal swelling (mean of 3%). 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. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight, moderate or severe erosion and very slight or slight vacuolation of the epithelium and the epithelium partly detached from the basement membrane. Based on these results, the test substance is considered to be not eye irritating.

Justification for classification or non-classification

Based on the absence of skin corrosion the substance does not need to be classified for skin corrosion according to Regulation (EC) No. 1272/2008 and its updates.

Based on the skni irritation results, the substance needs to be classified for skin irritation, Cat 2 according to Regulation (EC) No. 1272/2008 and the phrase: H315 causes skin irritation.

Based on the eye irritation results, the test substance is considered to be not eye irritating according to Regulation (EC) No. 1272/2008 and its updates.