Registration Dossier

Administrative data

Description of key information

Skin-irritation:

The test substance was assessed for skin irritation/corrosion potential according to an in-vivo study conducted to OECD 404.

Three rabbits were tested.

The test substance was liquid and was used without dilution.

The exposure time was for 4 hours. The gauze patch was removed after the exposure, and the application site was cleaned with tissue paper moistened with warm water to remove the test article. Immediately before and after the application of the test article, and at 30 minutes, 60 minutes, 24 hours, 48 hours, 72 hours, 4 days and 5 days after removing the gauze patch, the test animals were individually observed and scored for signs for erythema and edema based on the skin reaction evaluation criteria

The application site had extremely mild erythema or clear erythema as skin reaction to the SiMAA2 application in all cases from the observation at 30 minutes after removing the gauze patch. Erythema was observed at observation 60 minutes, 24 hours, and 48 hours after on the application site in all the cases; however, it decreased over time and disappeared at 72 hours after in one case and by 5 days after in the other two cases.

There was no crust or edema formation at any application site in any animal throughout the observation period, and corrosivity of SiMAA2 to the skin was not detected in rabbits.

Eye-irritation:

The purpose of the study was to determine the eye irritation potential of the test materials using the SkinEthic Reconstituted Human Corneal model after a treatment period of 10 minutes.  The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.

The experimental design of the study consists of a test for Direct Reduction of MTT by the test materials, followed by the main test.

For the main test, triplicate SkinEthic tissues were treated with 30 ml of the test material for 10 minutes.  Triplicate tissues treated with 30 µl of Solution A served as the negative control and triplicate tissues treated with 30 µl of Sodium Dodecyl Sulphate at concentrations of 1% w/v and 0.5% w/v served as the positive control.

At the end of the exposure period each SkinEthic tissue was rinsed.  The rinsed tissues (two per group) were taken for MTT loading.  The remaining tissues were retained for possible histopathology.  Following MTT loading the reduced MTT was extracted from the tissues.

After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured (OD540).  Data is presented in the form of % viability (MTT conversion relative to negative controls).

The test material was classified according to the following criteria:

i)                   If the % relative viability was ≥ 60% the test material was considered to not likely to be a severe ocular irritant.

ii)                  If the % relative viability was 60% the test material was considered to be irritant.

The relative mean viability of the test material treated tissues after a 10 minute exposure was 104.8%.

The quality criteria required for acceptance of results in the test were satisfied.

According to the protocol followed the test material was considered not likely to be a severe ocular irritant (NI).

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:
September 9, 2002 to September 23, 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of relevant results.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
yes
Remarks:
Elizabethan collars were placed in order to protect the application site after the application of the test article. There were no other deviations from the protocol that might have negatively influenced the reliability of the study.
GLP compliance:
not specified
Species:
rabbit
Strain:
other: rabbits bred at Japan SLC, Inc. (Std:JW/CSK)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Japan SLC
- Age at study initiation: 17 weeks
- Weight at study initiation: 2.66-2.88 kg
- Housing: Aluminium cage, W40 x D50 x H33 cm ,1 animal per cage.
- Diet : Throughout the quarantine, acclimation, and study periods, 150 g of radiation-sterilized pellet diet (Labo R Stock, Nosan Corporation) per day was fed chlorinated well water ad libitum.
- Water chlorinated well water ad libitum.
- Acclimation period: 7 days
-Floor shavings: not used


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0-27.0 deg C
- Humidity : 30.0-70.0 % RH
- Air changes (per hr): ~13 times/hour
- Photoperiod (hrs dark / hrs light):12 hours/day ( 7am-7pm)


IN-LIFE DATES: From:9th September 2002 To: 23rd September 2002
Type of coverage:
occlusive
Preparation of test site:
shaved
Vehicle:
unchanged (no vehicle)
Controls:
other: The clipped area to the right of the dorsal midline was for the test article and the left of the dorsal midline was for the control,which was treated in the same way as the application of the test article using the gauze patch without the test article
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL




Duration of treatment / exposure:
4 hours
Observation period:
5 Days.
Number of animals:
3
Details on study design:
TEST SITE
- Area of exposure: Ca 6 cm2
- Type of wrap if used: a gauze patch
(approximately 6 cm2) with 0.5 mL of the test article applied to the skin and adhered with approximately 5 cm × 5 cm non-irritating tape (Blenderm™, 3M). In addition, it was wrapped with adhesive bandage (Silkytex, Alcare Co., Ltd.)


REMOVAL OF TEST SUBSTANCE
- Washing (if done): tissue paper moistened with warm water to remove the test article.
- Time after start of exposure: 4 hours.


SCORING SYSTEM: Draize scale

OBSERVATION OF GENERAL CONDITIONS AND BODYWEIGHT MEASUREMENT:
Animals were observed for general conditions once daily throughout quarantine, acclimation, and test periods. Body weight was measured at receipt, on the day of the application of the test article (just prior to application) and the last day of observation.

SKIN CONDITION:
Immediately before and after the application of the test article, and at 30 minutes, 60 minutes, 24 hours, 48 hours, 72 hours, 4 days and 5 days after removing the gauze patch, test animals were individually observed and scored for signs for erythema and edema based on the skin reaction evaluation criteria described below (see evaluation of skin reaction).

Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
not fully reversible within: 5 days
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.66
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1.33
Max. score:
4
Reversibility:
not fully reversible within: 5 days
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: No edema recorded ay any observation
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: No edema recorded ay any observation
Irritation parameter:
edema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: No edema recorded ay any observation
Irritant / corrosive response data:
The application site had extremely mild erythema (Score 1 in No. 2102) or clear erythema (Score 2 in Nos. 2101 and 2103) as skin reaction to the SiMAA2 application in all cases from the observation at 30 minutes after removing the gauze patch. Erythema was observed at observation 60 minutes, 24 hours, and 48 hours after on the application site in all the cases; however, it decreased over time and disappeared at 72 hours after in one case (No. 2102) and by 5 days after in the other two cases (Nos. 2101 and 2103). There was no crust or edema formation at any application site in any animal throughout the observation period, and
corrosivity of SiMAA2 to the skin was not detected in rabbits.
Other effects:
Although the test animal No. 2103 had laceration and bleeding on the lips, which was probably caused by the Elizabethan collar, there were no other abnormalities in general condition seen in any of the test animals throughout the study period.

None

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
There was no crust or edema formation at any application site in any animal throughout the observation period, and corrosivity of SiMAA2 to the skin was not detected in rabbits.
Executive summary:

The test article was liquid; therefore, it was used as it was without dilution.

Animals were restrained with a restraint system with collar, and then a gauze patch (approximately 6 cm2) with 0.5 mL of the test article applied to the skin and adhered with approximately 5 cm × 5 cm non-irritating tape (Blenderm™, 3M). In addition, it was wrapped with adhesive bandage (Silkytex, Alcare Co., Ltd.), and a Elizabethan collar (Natsume Seisakusho Co., Ltd.) was placed on the neck before returning to the animals to the cages. The exposure time was for 4 hours. The gauze patch was removed after the exposure, and the application site was cleaned with tissue paper moistened with warm water to remove the test article. The clipped area to the right of the dorsal midline was for the test article and the left of the dorsal midline was for the control, which was treated in the same way as the application of the test article using the gauze patch without the test article.

Immediately before and after the application of the test article, and at 30 minutes, 60 minutes, 24 hours, 48 hours, 72 hours, 4 days and 5 days after removing the gauze patch, - 3 - test animals were individually observed and scored for signs for erythema and edema based on the skin reaction evaluation criteria

Although the test animal No. 2103 had laceration and bleeding on the lips, which was probably caused by the Elizabethan collar, there were no other abnormalities in general condition seen in any of the test animals throughout the study period.

The application site had extremely mild erythema (Score 1 in No. 2102) or clear erythema (Score 2 in Nos. 2101 and 2103) as skin reaction to the SiMAA2 application in all cases from the observation at 30 minutes after removing the gauze patch. Erythema was observed at observation 60 minutes, 24 hours, and 48 hours after on the application site in all the cases; however, it decreased over time and disappeared at 72 hours after in one case (No. 2102) and by 5 days after in the other two cases (Nos. 2101 and 2103). There was no crust or edema formation at any application site in any animal throughout the observation period, and corrosivity of SiMAA2 to the skin was not detected in rabbits.

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:
The study was performed between 03 June 2008 and 05 June 2008.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
other: SkinEthic HCE model
Deviations:
no
Qualifier:
equivalent or similar to guideline
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:
no
Principles of method if other than guideline:
The test is based on the hypothesis that irritant chemicals are able to penetrate the stratum corneum of the SkinEthic HCE model and are sufficiently cytotoxic to cause cell death in the underlying cell layers.
GLP compliance:
yes (incl. QA statement)
Species:
other: SkinEthic Reconstituted Human Corneal model
Strain:
other: Reconstituted HCE model
Details on test animals or tissues and environmental conditions:
Tissue: Consists of the transformed human kerotinocytes of the cell line HCE that formed a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye.
- Source: HCE, SkinEthic Laboratories, Nice, France



ENVIRONMENTAL CONDITIONS
- Temperature (°C): Room temperature
- Storage: wells containing 1ml of maintenance medium
-Incubation: Incubated over night at 37 deg C, 5% CO2 in air

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µl
- Concentration (if solution): NA


Duration of treatment / exposure:
10 minutes
Observation period (in vivo):
None, at the end of the expsoure period, each skinEthic tissue was rinsed. The rinsed tissues (two per group) were taken for MTT loading. The remaining tissues were retained for possible histopathology. Following MTT loading the reduced MTT was extracted from the tissues.
Number of animals or in vitro replicates:
Not applicable as tissues were used. Triplicate SkinEthic tissues were used.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the relevant exposure period, each tissue insert was removed from the well using forceps and rinsed using a wash bottle containing DPBS.
- Time after start of exposure: 10 minutes

SCORING SYSTEM: After exposure of tissues to the test material, the tissues are removed and rinsed. The rinsed tissues are taken for MTT loading, were the reduced MTT is extracted from the tissues. After extraction, the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured (OD50). Data is presnted in the form of % viability (MTT conversion relative to negative controls).


TOOL USED TO ASSESS SCORE: The test material was classified according to the following criteria:
* If the % relative viability was greater than or equal to 60% the test material was considered to not likely to be a severe ocular irritant.
* If the % relative viability was less than 60% the test material was considered to be irritant.
Irritation parameter:
other: Relative mean viability (%)
Run / experiment:
10 minute exposure
Value:
104.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Not likely to be a severe ocular irritant

Assessment of Direct test Material Reduction of MTT

The test material was not able to directly reduce MTT.

Assessment of Eye Irritation Potential:

The mean OD540 values and mean viabilities for each treatment group are given in Table 1.

The relative mean viability of the test material treated tissues after a 10 minute exposure was 104.8%.

It was considered unnecessary to proceed with tissue histopathology.

Table1:

Material

Mean OD540

% Viability

Negative Control

0.888

100*

Positive Control
0.5% w/v

0.566

63.7

Positive Control
1% w/v

0.376

42.3

Test Material

0.931

104.8

* =          The mean viability of the negative tissues is set at 100%

Quantitative Evaluation of Tissue Viability (MTT Uptake Visaul Assessment)

The quantitative evaluation of tissue viability is presented in table 2.

The negative control and test material treated tissues appeared blue. This was considered to be indicative of viable tissue.  The positive control material at each concentration appeared blue/white and was considered to be indicative of semi‑viable tissue.

Table 2:

Material

Score

Tissue 1

Tissue 2

Negative Control

-

-

Positive Control
0.5% w/v

+

+

Positive Control
1% w/v

+

+

Test Material

-

-


Interpretation of results:
other: not irritating
Conclusions:
According to the protocol followed the test material was considered not likely to be a severe ocular irritant (NI).
Executive summary:

Introduction.  The purpose of this study was to determine the eye irritation potential of the test materials using the SkinEthic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories, , ) after a treatment period of 10 minutes.  The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.

Methods.  The experimental design of the study consists of a test for Direct Reduction of MTT by the test materials, followed by the main test.

For the main test, triplicate SkinEthic tissues were treated with 30 ml of the test material for 10 minutes.  Triplicate tissues treated with 30 µl of Solution A served as the negative control and triplicate tissues treated with 30 µl of Sodium Dodecyl Sulphate at concentrations of 1% w/v and 0.5% w/v served as the positive control.

At the end of the exposure period each SkinEthic tissue was rinsed.  The rinsed tissues (two per group) were taken for MTT loading.  The remaining tissues were retained for possible histopathology.  Following MTT loading the reduced MTT was extracted from the tissues.

After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured (OD540).  Data is presented in the form of % viability (MTT conversion relative to negative controls).

The test material was classified according to the following criteria:

i)                    If the % relative viability was ≥ 60% the test material was considered to not likely to be a severe ocular irritant.

ii)                   If the % relative viability was 60% the test material was considered to be irritant.

Results.  The relative mean viability of the test material treated tissues after a 10 minute exposure was 104.8%.

It was considered unnecessary to proceed with tissue histopathology.

Quality criteria.  The quality criteria required for acceptance of results in the test were satisfied.

Conclusion. According to the protocol followed the test material was considered not likely to be a severe ocular irritant (NI).

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The test substance is not an skin or eye irritant.

Justification for classification or non-classification

Skin corrosion / irritation

Skin corrosion is defined as the production of irreversible damage to the skin following application of the test substance. Skin irritation is the production of reversible damage to the skin following application of the test substance. 

There was no evidence of skin corrosion (no visivble necrosis) from an in-vivo study so the substance is not classified as a skin irritant.

 

During in vivo testing, substances are classified as Category 2 irritants where a mean value of 2.3 - ≤4 is observed for erythema or for edema in at least two of the three animals tested from observations at 24, 48 and 72 hours after patch removal. Substances may be classified for skin irritation if inflammation persists to the end of the observation period in at least two animals.

The test substance was assessed for skin irritation potential and all three animals tested had scores for erythema ≤2.3 and no edema was observed. All effects observed were fully reversible within 5 days.

The test substance is therefore not classified as a skin irritant.

 

Eye irritation

Serious eye damage is defined as the production of tissue damage in the eye, or serious physical decay or vision following application of the test substance to the anterior surface of the eye which is not fully reversible. Eye irritation means the production of changes in the eye following application of the test substance which is fully reversible. 

Eye irritation was assessed using an in-vitro test method.

According to the protocol followed the test material was considered not likely to be a severe ocular irritant (NI) and the test substance is therefore not classified as an eye irritant. Further testing is not considered necessary.