Registration Dossier

Administrative data

Description of key information

Skin irritation (similar to OECD TG 404): not irritating

Eye irritation (OECD TG 438): not 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:
09 May 1988 to 24 May 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
GLP compliance:
not specified
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: commercial supplier
- Age: adults
- Diet: ad libitum
- Water: ad libitum
- housing: caged individually
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.5 mL
- Concentration: undiluted


Duration of treatment / exposure:
4 hours
Observation period:
24,48, and 72 hours after exposure
Number of animals:
3
Details on study design:
TEST SITE
- Area of exposure: Dorsum
- Type of wrap if used: 25mm x 25mm gauze pad, backed by a 20mm x 30mm strip of thin polythene film stuck to a 25mm x 75 mm strip of zinc oxide sticking plaster.

REMOVAL OF TEST SUBSTANCE
- Washing: excess substance was wiped from the test site with a damp tissue.
- Time after start of exposure: 4 hours

OBSERVATION TIME POINTS
one hour after removal of the test patch thereafter at 24, 48, and 72 hours.

SCORING SYSTEM:
- Method of calculation: Draize scoring system
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no edema observed
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no edema observed
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no edema observed
Remarks on result:
no indication of irritation
Irritation parameter:
erythema score
Basis:
animal #1
Remarks:
1 hour after patch removal
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
erythema score
Basis:
animal #2
Remarks:
1 hour after patch removal
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
erythema score
Basis:
animal #3
Remarks:
1 hour after patch removal
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritant / corrosive response data:
One hour after patch removal, all three animals showed slight erythema, without oedema. These effects reduced in intensity and all three animals appeared normal at 72 hours after patch removal.
Interpretation of results:
other: not irritating
Remarks:
in accordance with EU CLP (EC 1272/2008 and its updates)
Conclusions:
Benzoin Hypersoluble showed very slight irritancy to rabbit skin. These very slight effects were reversible within 72 hours. Under the conditions of the test, the test substance is not considered to be a skin irritant.
Executive summary:

In a study equivalent to OECD TG 404 the irritating potential of the test substance was investigated. 3 New Zeeland white rabbits were exposed to 0.5mL undiluted test substance for 4 hours under semiocclusive conditions. Observations were made one hour after removal of the test patch thereafter at 24, 48, and 72 hours. Irritation was scored according to the Draize method. One hour after patch removal, all three animals showed slight erythema, without oedema. These effects reduced in intensity and all three animals appeared normal at 72 hours after patch removal. Under the conditions of the test, the test substance is not considered to be a skin irritant.

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
Study period:
17 Nov 2017 to 04 Dec 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)
Version / remarks:
26 July 2013
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)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
30 mg test substance
Duration of treatment / exposure:
10 seconds followed by a 20 mL saline rinse
Number of animals or in vitro replicates:
Test group and positive control: triplicates
Negative control: Singlo
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. Corneal thickness was measured using the Depth Measuring Attachment No. I for the Haag-Streit slit-lamp microscope, set at 0.095 mm. Corneal thickness was expressed in instrument units. 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.

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:
1
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
Remarks on result:
other: maximum mean values
Other effects / acceptance of results:
- Slit-lamp examination: The test substance only caused very slight corneal swelling (mean of 1%). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control NaOH caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.
- Microscopic examination: 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 NaOH revealed severe erosion (3/3 corneas), severe necrosis (3/3 corneas) of the epithelium, stromal (3/3 corneas) and endothelial (3/3 corneas) necrosis.
Interpretation of results:
other: not classified
Remarks:
in accordance with the criteria outlined in Annex I of the CLP regulations (1272/2008/EC).
Conclusions:
Under the test conditions (OECD 438 and GLP) the test substance is not considered eye-irritant.
Executive summary:

The test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) test, in accordance with OECD guideline 438 and GLP. In the ICE test, chicken eyes were exposed to 30 mg test substance for 10 seconds. In addition, the test included a negative control eye (30 µL saline) and a positive control eye (30 mg NaOH). 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. Mean fluorescein retention score was determined at 30 minutes post-treatment. The test substance caused very slight corneal swelling (mean score 1 %). Microscopic examination of the cornea did not reveal any abnormalities. The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control NaOH caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of these corneas generally revealed severe erosion, severe necrosis of the epithelium, stromal and endothelial necrosis. Based on these results, the test substance is not considered irritating to the eyes.

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

Additional information

Skin irritation:

In a study equivalent to OECD TG 404 the irritating potential of the test substance was investigated. Three New Zeeland white rabbits were exposed to 0.5mL undiluted test substance for 4 hours under semiocclusive conditions. Observations were made one hour after removal of the test patch thereafter at 24, 48, and 72 hours. Irritation was scored according to the Draize method. One hour after patch removal, all three animals showed slight erythema, without oedema. These effects reduced in intensity and all three animals appeared normal at 72 hours after patch removal. Under the conditions of the test, the test substance is not considered to be a skin irritant.

Eye irritation

The test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) test, in accordance with OECD guideline 438 and GLP. In the ICE test, chicken eyes were exposed to 30 mg test substance for 10 seconds. In addition, the test included a negative control eye (30 µL saline) and a positive control eye (30 mg NaOH). 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. Mean fluorescein retention score was determined at 30 minutes post-treatment. The test substance caused very slight corneal swelling (mean score 1 %). Microscopic examination of the cornea did not reveal any abnormalities. The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control NaOH caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of these corneas generally revealed severe erosion, severe necrosis of the epithelium, stromal and endothelial necrosis. Based on these results, the substance was not considered irritating to the eyes.

 

Justification for classification or non-classification

Based on the available information the test substance does not need to be classified for skin and eye irritation in accordance with the criteria outlined in EU CLP (EC no. 1272/2008 and its amendments).