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Diss Factsheets

Administrative data

Description of key information

Skin irritation: not irritating to the skin based on an in vivo key study (OECD 404, K, rel.1) and an in vitro supporting study (eq. to OECD 439, S, rel.2).


Eye irritation: not irritating to the eyes based on an in vivo key study (OECD 405, K, rel.1) and an in vitro supporting study (eq. to OECD 492, S, rel.2).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 June 2008 to 23 June 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
According to the OECD Guideline 404 and in compliance with GLP practices.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2500 (Acute Dermal Irritation)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
Adopted April 24, 2002
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Version / remarks:
April 29, 2004
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Swiss legislation on Good Laboratory Practice (signed in September 04, 2008)
Species:
rabbit
Strain:
New Zealand White
Remarks:
Young Adult New Zealand White Rabbit, SPF
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands BV, Kreuzelweg 53, NL-5961 NM Horst / The Netherlands Postbus 6174
- Age at study initiation: 12 weeks (male); 16-20 weeks (females)
- Weight at study initiation: 2.3, 2,4 and 2,8 kg
- Housing: Individually in stainless steel cages equipped with feed hoppers, drinking water bowls, with autoclaved wood (RCC Ltd, Füllinsdorf) and haysticks 4642 for gnawing.
- Diet (e.g. ad libitum): Pelleted standard Provimi Kliba 3418 rabbit maintenance diet ad libitum (batch nos. 03/08 and 19/08) provided by Provimi Kliba AG, CH-4303 Kaiseraugst.
- Water (e.g. ad libitum): Community tap water from Füllinsdorf, ad libitum.
- Acclimation period: 7 days under laboratory conditions after health examination.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23°C
- Humidity (%): 30-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
- Music was played during the light cycle.
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL measured with a syringe and applied undiluted as it was delivered by the sponsor.
Duration of treatment / exposure:
4 hours
Observation period:
Until 7 days after the removal of the dressing, gauze patch and test article.
Number of animals:
1 male and 2 females
Details on study design:
TEST SITE
- Area of exposure: left flank, approximately 100 cm² (10 cm x 10 cm)
- Type of wrap if used: test substance was applied to the intact skin using a surgical gauze patch (ca. 2.5 x 2.5 cm) covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and anchored with tape.

REMOVAL OF TEST SUBSTANCE
- Washing: The dressing was removed and the skin was flushed with lukewarm tap water to clean the application site so that any reactions (erythema) were clearly visible at that time.
- Time after start of exposure: 4h

OBSERVATION TIME POINTS
- Viability/Mortality and clinical signs: Dai!y from delivery of the animals to the termination of test.
- Body weights: At start of acclimatization, on the day of application and at termination of observation.
- Clinical signs: Daily from acclimatization of the animals to the termination of test.
- Skin reaction: approx 1, 24, 48, 72 hours, as well as 7 days after exposure (removal of the dressing, gauze patch and test item).

SCORING SYSTEM:
Erythema and eschar formation
No erythema: 0
Very slight erythem: 1
Well-defined erythema:2
Moderate to severe erythema: 3
Severe erythema to slight eschar formation: 4

Oedema formation
No oedema:0
Very slight oedema: 1
Slight oedema:2
Moderate oedema:3
Severe oedema:4

The mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) for each animal for erythema/eschar gardes and for oedema grades, separately. An animal is positive when the mean score is 2 or greater. The test is positive for irritation when at least 2 animals are positive for the same endpoint (erythema/eschar or oedema).

All rabits were sacrificed by an intravenous injection of Pentobarbitone into the ear vein at a dose of at least 1 mL/kg bw and discarded.
No necrospy was performed on the animals sacrificed at termination of observation.
Irritation parameter:
edema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Remarks on result:
probability of weak irritation
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Remarks on result:
probability of mild irritation
Irritant / corrosive response data:
The mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) for each animal for erythema/eschar grades and for edema grades, separately. The mean erythema/eschar score of the three animals was 1.00, 0.33 and 0.33, respectively and the mean edema score was 0.00 for each of the three animals.

A very slight erythema was observed in all animals 1 hour after test item exposure and persisted up to the 24- and 72-hour reading, respectively.
Other effects:
No abnormal findings were observed on the treated skin of any animal 48 hours and 7 days after treatment, respectively.
No staining produced by the test item of the treated skin was observed.
Neither alterations of the treated skin were observed nor were corrosive effects evident on the skin.
The body weights of all rabbits were considered to be within the normal range of variability.

Individual findings:

Evaluation interval* Rabbit No. Sex Erythema Oedema
1 hour 66 M 1 0
67   1 0
68 F 1 0
24h 66 M 1 0
67 F 1 0
68 F 1 0
48h 66 M 1 0
67 F 0 0
68 F 0 0
72h 66 M 1 0
67 F 0 0
68 F 0 0
7 days 66 M 0 0
67 F 0 0
68 F 0 0

*Examinations were performed at the specified times after removal of the dressing.

 

Mean score at 24, 48 and 72h after a 4 hours exposure

 

Animal number Sex Erythema N Oedema N
66 M 1.00 3 0.00 3
67 F 0.33 3 0.00 3
68 F 0.33 3 0.00 3

N= number of available data points.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, test substance is not classified as irritating to skin according to the criteria of the Annex I of the Regulation EC No. 1272/2008 (CLP) and to the GHS.
Executive summary:

In a dermal irritation study performed according to the OECD Guideline No. 404, and in compliance with GLP, 0.5 mL of test material was applied on the clipped skin of the dorsal flank area of 1 male and 2 females New Zealand rabbits. Test sites were covered with a semi-occlusive dressing for 4 hours, followed by several observations at approximately 1, 24, 48 and 72 hours and 7 days after removal of dressing, gauze patch and test substance.

The observed skin irritation consisted of a very slight erythema observed in all animals 1 hour after test item exposure and persisted up to the 24- and 72-hour reading, respectively. All skin reactions were clear within 7 days after treatment.

No abnormal findings were observed on the treated skin of any animal 48 hours and 7 days after treatment, respectively. No staining produced by the test item of the treated skin was observed. Neither alterations of the treated skin were observed nor were corrosive effects evident on the skin. The body weights of all rabbits were considered to be within the normal range of variability.

The mean erythema/eschar score of the three animals was 1.00, 0.33 and 0.33, respectively and the mean oedema score was 0.00 for each of the three animals.

Under the test conditions, test substance is not classified as irritating to skin according to the criteria of the Annex I of the Regulation EC No. 1272/2008 (CLP) and to the GHS. This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From June 18, 2008 to June 23, 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
GLP study performed in 2008 in accordance with the ECVAM international validation study on in vitro tests which followed the current OECD Guideline 439 originally adopted in 2010 with the following deviations: - no check of colour interference was mentioned to identify potential interference by coloured test chemicals or test chemicals that become coloured when in contact with water or isopropanol and allowing to decide on the need for additional controls. - Application temperature for 15 ± 1 min at 37 ± 1°C, 5 ± 0.5% CO2 (into the incubator) instead of room temperature. - 15 µL of test item and controls substances were applied instead of 10 µL.
Qualifier:
according to guideline
Guideline:
other: ECVAM International validation study on in vitro tests for acute skin irritation: Report on the validity of the EPISKIN and EpiDerm assays and on the Skin Integrity Function Test (Altern Lab Anim. 2007 Dec; 35 (6): 559-601)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
See the section "Rationale for reliability incl. deficiencies"
GLP compliance:
yes (incl. QA statement)
Remarks:
German Statement of GLP compliance/Date of inspection: 02.09.2006
Specific details on test material used for the study:
- Storage conditions: In the refrigerator at +2 to +8°C, protected from light
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: foreskin
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
EPISKIN Standard Model TM (EPISKIN-SM(TM), purchased from Skinethic Laboratories, 0.38 cm², Lot no.: 08-EKIN-023):
This tissue model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EPISKIN-SM (TM) tissues are cultured on specially prepared cell culture inserts.
Tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate and reached RCC-CCR GmbH on June 17, 2008. On day of receipt, tissues were transfered to 12-well plates with maintenance medium.

EVALUATION OF DIRECT INTERACTION WITH MTT
- For correct interpretation of results it was necessary to assess the ability of the test item to directly reduce MTT. The direct interaction of MTT with the test item was checked by adding 15 µL of the test item to 1mL of the solution of MTT. No colour change could be observed in the present study. Therefore, there is no direct interaction between the test item and MTT.

TREATMENT
- After approximately 24 hours incubation of the tissues, they were treated with the test item. Under sterile condition using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1°C) maintenance medium).
- The negative and positive control and the test item were added into the insert atop the concerning triplicate tissues, as supplied, at the approximate dose of 15 µL. The 12-well plates were placed into the incubator for 15 ± 1 min at 37 ± 1°C, 5 ± 0.5% CO2.

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the end of the treatment interval the inserts were removed immediately from the 12-well plate. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. Tissues were incubated for 42 hours 15 minutes at 37 ± 1°C, 5 ± 0.5% CO2.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE

- The MTT concentrate was prepared freshly and diluted with the MTT diluent. A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well.
- After the treatment procedure was completed for all tissues of each time point cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1°C, 5 ± 0.5% CO2) MTT solution was aspirated from the wells and wells were rinsed three times with PBS. Tissues were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissues were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol) into each vial. The level rised above the tissues. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for 69 hours room temperature.
- Per each tissue 2 x 200 µL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax (R) Molecular Devices, D-85737 Ismaning) at 570 nm without reference filter. Mean values were calculated from the 2 wells per tissue.

VIABILITY CALCULATION:

- The mean OD of the three negative control tissue was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control, the individual relative tissue viability is calculated according to the following formulas: relative viability % = (OD test item / OD negative control) * 100

- Data from individual replicate tissues (OD values and calculated percent tissue viability data for the test item and controls) for each individual test item and control were reported in Table 7.3.1/1.

PREDICTION MODEL / DECISION CRITERIA

For the test item and the positive control the mean relative viability ± standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model :
- For the current test, an irritation potential of a test item according to EU classification R38 is predicted if the mean relative tissue viability of three individual tissues is reduced below 50% of the negative control.
- For the current test, a non classification of a test item according to EU non-irritant (NI) classification is predicted if the mean relative tissue viability of three individual tissues is increased above 50% of the negative control.

ACCEPTABILITY OF THE ASSAY
- The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the two tissues is ≥ 0.6.
An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 40%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 15 µL of the neat test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 15 µL Deionised water (Lot no. 18.6.08)

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 15 µL
- Concentration (if solution): 5% (aq) Sodium lauryl sulphate (Sigma 82024 Taufkirchen, Lot No. 043K0015) solution in deionised water, prepared freshly prior to the performance of the experiment
Duration of treatment / exposure:
Exposure: 15 minutes
Post incubation period: 42 hours
Duration of post-treatment incubation (if applicable):
The skin sample was placed in MTT solution of 1 mg/mL concentration for 3 hours at 37°C ± 1°C, 5 ± 0.5 % CO2.
Number of replicates:
Triplicate
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 minutes
Value:
98.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
After treatment with the negative control the absorbance values were well above the required acceptability criterion of mean OD ≥ 0.6 for the 15 minutes treatment interval thus showing the quality of the tissues.
Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 28.1% thus ensuring the validity of the test system.
After treatment with the test item, the relative absorbance values were irrelevantly decreased to 98.6%. This value is well above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Table 7.3.1/1: Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls



 










































Dose group 



Treatment interval



Absorbance 570 nm Tissue 1*



Absorbance 570 nm Tissue 2*



Absorbance 570 nm Tissue 3*



Mean absorbance of 3 tissues



Rel. Absorbance [% of negative control]**



Negative control



15 minutes



1.3294



1.1599



1.2799



1.2564



100.0



Positive control



15 minutes



0.3653



0.3502



0.3437



0.3530



28.1



Test item



15 minutes



1.2158



1.1091



1.3901



1.2383




98.6




 




* Mean of three replicate wells after blank correction


** Relative absorbance (rounded values): (100 x ABSORBANCEtest item)/(ABSORBANCEnegative control)




 


Acceptability criteria:


- Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.


- The mean OD of negative control tissues for the treatment of 15 minutes was 1.2564. The acceptability criteria should be ≥ 0.6 for the negative control.



- The mean viability of positive control tissues for the treatment of 15 minutes was 28.1 %.It should be < 40 % for Episkin model.



 


Interpretation of results:
GHS criteria not met
Conclusions:
The mean percent viability of the treated tissues is 98.6%, versus 28.1% in the positive control (5% Sodium Lauryl Sulfate). Under the test conditions, test substance is not classified as irritating to skin.
Executive summary:

An in vitro skin irritation test using the Reconstructed human Epidermis (Episkin Standard model) was performed in accordance with the ECVAM international validation study on in vitro tests which followed the current OECD Guideline 439 originally adopted in 2010. The study was compliant with GLP to predict the acute skin irritation potential of the test substance.


 


Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues was 98.6%. Since the mean relative tissue viability for the test item was above 50% after 15 minutes treatment, the test item is considered to be non-irritant.


The positive control had a mean cell viability after 15 minutes exposure of 28.1%. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean viability of positive control tissues was within the laboratory historical control data range.


 


Finally, it is concluded that this test is valid and that the test item is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 25 June 2008 to 03 July 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
According to the OECD Guideline 405 and in compliance with GLP practices.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
Adopted April 24, 2002
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
April 29, 2004
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Swiss legislation on Good Laboratory Practice (signed in September 04, 2008)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands BV, Kreuzelweg 53, NL-5961 NM Horst / The Netherlands Postbus 6174
- Age at study initiation: 16-17 weeks (male); 14 weeks (females)
- Weight at study initiation: 2.6, 2.4 and 2.4 kg
- Housing: Individually in stainless steel cages equipped with feed hoppers, drinking water bowls, with autoclaved wood (RCC Ltd, Füllinsdorf) and haysticks 4642 for gnawing.
- Diet (e.g. ad libitum): Pelleted standard Provimi Kliba 3418 rabbit maintenance diet ad libitum (batch nos. 03/08 and 19/08) provided by Provimi Kliba AG, CH-4303 Kaiseraugst.
- Water (e.g. ad libitum): Community tap water from Füllinsdorf, ad libitum.
- Acclimation period: 7 days under laboratory conditions after health examination.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23°C
- Humidity (%): 30-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
- Music was played during the light cycle.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
Duration of treatment / exposure:
Single exposure
Observation period (in vivo):
1, 24, 48 and 72 hours after instillation of test material.
Number of animals or in vitro replicates:
1 male and 2 females
Details on study design:
On the day of treatment, the undiluted test substance was placed in the conjunctival sac of the left eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of test article. The right eye remained untreated and served as the reference control.

REMOVAL OF TEST SUBSTANCE
- Washing: not reported

OBSERVATION TIME POINTS
- Viability/Mortality and clinical signs: Daily from acclimatization of the animals to the termination of test.
- Body weights: At start of acclimatization, on the day of application and at termination of observation.
- Clinical signs: Daily from acclimatization of the animals to the termination of test.
- Ocular reaction: approx 1, 24, 48 and 72 hours after the instillation.

SCORING SYSTEM: The eye reactions were assessed according to the following Draize score. Scleral reddening and ocular discharge were also assessed.

GRADING OF OCULAR LESIONS
CORNEA

Opacity: degree of density (area most dense taken for reading):
No ulceration or opacity = 0
Scattered or diffuse areas of opacity (other than slight dulling of normal luster), details of iris clearly visible = 1
Easily discernible translucent area, details of iris slightly obscured = 2
Nacreous area, no details of iris visible, size of pupil barely discernible = 3
Opaque cornea, iris not discernible through the opacity = 4

Area of cornea involved:
Zero = 0
One quarter (or less) but not zero = 1
Greater than one quarter, but less than half = 2
Greater than half, but less than three quarters = 3
Greater than three quarters, up to whole area = 4

IRIS
Normal = 0
Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperemia, or injection, any of these or combination of any thereof, iris still reacting to light (sluggish reaction is positive) = 1
No reaction to light, hemorrhage, gross destruction (any or all of these) = 2

CONJUNCTIVAE
Redness (refers to most severe reading of palpebral and bulbar conjunctivae when compared with control eye)
Blood vessels normal = 0
Some blood vessels, definitely hyperemic (injected) = 1
Diffuse, crimson color, individual vessels not easily discernible = 2
Diffuse beefy red = 3

Chemosis: lids and/or nictitating membranes
No swelling = 0
Any swelling above normal (including nictitating membranes) = 1
Obvious swelling with partial eversion of lids = 2
Swelling with lids about half-closed = 3
Swelling with lids more than half-closed = 4
Irritation parameter:
conjunctivae score
Basis:
animal: #2 and #3
Time point:
24/48/72 h
Score:
0.67
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Remarks on result:
probability of mild irritation
Irritation parameter:
cornea opacity score
Basis:
animal: #1, #2 and #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal: #1, #2 and #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal: #1, #2 and #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately. The individual mean scores for corneal opacity and iris were 0.00 for all three animals. The individual mean scores for the conjunctivae were 0.00, 0.67 and 0.67 for reddening and 0.00 for chemosis for all three animals.

No abnormal findings were observed in the cornea of iris of any animal at any of the measurement intervals.

A slight to moderate reddening of the conjunctivae was noted at the 1-hour reading and persisted as slight in two animals up to 48 hours after treatment. A slight swelling (chemosis) of the conjunctivae was observed in one animal 1 hour after treatement.

No abnormal findings were observed in the treated eye of any animal 72 hours after treatment, the end of the observation period for all animals.
Other effects:
No staining produced by the test item of the treated eye was observed.
No corrosion of the cornea was observed at any of the reading times.
The body weight of all rabbits were considered to be within the normal range of variability.

Individual findings:


































































































































Time after exposureRabbit No.SexCorneal opacityIrisConjunctivaeSclera
RednessChemosis
1 hour69M00100
70F00100
71F00210
24h69M00000
70F00100
71F00100
48h69M00000
70F00100
71F00100
72h69M00000
70F00000
71F00000

 


Mean score at 24, 48 and 72h after a single exposure of 0.1 mL


 

























































Animal numberSexCorneal opacityNIrisNConjunctivae 
RednessNChemosisN
69M0.0030.0030.00303
70F0.0030.0030.67303
71F0.0030.0030.67303

N=number of available data points.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, test substance is not classified as irritating to eyes according to the criteria of the Annex I of the Regulation EC No. 1272/2008 (CLP) and to the GHS.
Executive summary:

In an eye irritation study performed according to the OECD Guideline No. 405, and in compliance with GLP, 0.1 mL of undiluted test material was instilled into one eye of one male and two females New Zealand White rabbits. The other eye remained untreated and served as control. The upper and lower eyelids were held together for about one second immediately after application, to prevent loss of the test material, and then released. The eyes were examined and the changes were observed at 1, 24, 48 and 72 h after treatment and graded according to the Draize method.


The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately. The individual mean scores for corneal opacity and iris were 0.00 for all three animals. The individual mean scores for the conjunctivae were 0.00, 0.67 and 0.67 for reddening and 0.00 for chemosis for all three animals.


No abnormal findings were observed in the cornea of iris of any animal at any of the measurement intervals. A slight to moderate reddening of the conjunctivae was noted at the 1-hour reading and persisted as slight in two animals up to 48 hours after treatment. A slight swelling (chemosis) of the conjunctivae was observed in one animal 1 hour after treatement.


No abnormal findings were observed in the treated eye of any animal 72 hours after treatment, the end of the observation period for all animals.


No staining produced by the test item of the treated eye was observed.
No corrosion of the cornea was observed at any of the reading times.
The body weight of all rabbits were considered to be within the normal range of variability.


Mean individual scores at 24, 48 and 72 h after exposure for the 3 animals were 0 / 0 / 0 for cornea score; 0 / 0 / 0 for iris score; 0 / 0.67 / 0.67 for conjunctivae score and 0 / 0 / 0 for chemosis score.


Under the test conditions, test substance is not classified as irritating to eyes according to the criteria of the Annex I of the Regulation EC No. 1272/2008 (CLP) and to the GHS. This study is considered as acceptable and satisfies the requirement for eye irritation endpoint.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From June 12, 2008 to June 13, 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
GLP study performed before the adoption of the OECD Guideline on in vitro eye irritation and comparable with the OECD Guideline 492 with some deviations: - no check of colour interference was mentioned to identify potential interference by test chemicals absorbing light in the same range as formazan dye (naturally or after treatment) and allowing to decide on the need for additional controls. - 100 µL of test item and controls substances were applied instead of 50 µL. - 0.3% Triton X100 used as positive control instead of the recommended positive control, Methyl acetate. - The formazan salt was extracted for 18 hours without shaking at room temperature instead of 2-3 h with shaking. - Different treatment intervals instead of only 30 minutes of exposure time.
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:
yes
Remarks:
See the section "Rationale for reliability incl. deficiencies".
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. QA statement)
Remarks:
German Statement of GLP compliance/Date of inspection: 02.09.2006.
Specific details on test material used for the study:
- Storage conditions: In the refrigerator at +2 to +8°C, protected from light
Species:
other: Reconstructed human Cornea-like Epithelia
Details on test animals or tissues and environmental conditions:
Description of the cell system used:
EpiOcular kits and MTT-100 assays were pruchased from MatTek Corporation (Ashland, MA 01721, USA). The EpiOcular (TM) tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamos epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular(TM) tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts (MILLICELL (R), 10 mm of diameter).
EpiOcular(TM) tissues were shipped at 4°C on medium-supplemented agarose gels in a 24-well plate. Including time in transit, tissues may be stored at 4°C for up to 6 days prior to use. On day of receipt, the tissues were kept in the refrigerator. Next day, at least one hour before starting the assay, tissues were transferred to 6-well plates with assay medium, which is immediately replaced before the test is started.
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): 100 µL of the undiluted test item applied to each of duplicate tissues.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 100 µL deionised water (Lot no. 10.04.08) applied to each of duplicate tissues for 60 minutes.

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 100 µL
- Concentration (if solution): 0.3 % Triton X-100 (Lot. No. 050108 TTA) solution in deionised water, prepared freshly prior to the performance of the experiment and applied to each of duplicate tissues for 15 as well as for 45 min.
Duration of treatment / exposure:
1, 30 and 60 minutes for the test item, 15 and 45 minutes for the positive control and 60 minutes for the negative control in culture medium at 37 ± 1°C, 5 ± 0.5% CO2.
Duration of post- treatment incubation (in vitro):
- Post-exposure immersion period: The inserts were placed in 6-well plates containing 5 mL medium per well and remained there for 10 to 20 minutes.
- Post-exposure incubation period: After the 10 to 20 minutes incubation was completed the cell culture insert were transferred from the holding plates to the MTT-plates for a 3 hours incubation period at 37 ± 1°C, 5 ± 0.5% CO2.
Number of animals or in vitro replicates:
Test item, negative and positive controls were applied on duplicate tissues.
Details on study design:
MAIN TEST
- PRE-INCUBATION OF THE TISSUES:
At least 1 hour before dosing, EpiDerm(TM) tissues were removed from the refrigerator. Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1°C) maintenance medium.

EVALUATION OF DIRECT INTERACTION WITH MTT
- For correct interpretation of results it was necessary to assess the ability of the test item to directly reduce MTT. The direct interaction of MTT with the test item was checked by adding 50 µL of the test item to 1mL of the solution of MTT and the mixture was incuabted in the dark at room temperature for 60 minutes. No colour change could be observed in the present study. Therefore, there is no direct interaction between the test item and MTT.

- TREATMENT AND POST-TREATMENT INCUBATION OF THE TISSUES:
Each duplicate EpiOcular(TM) tissues as for the test item and duplicate for the positive and negative control were treated with the test item, positive and negative controls for different treatment times. The negative control was tested for 60 min, the positive control was tested for 15 and 45 minutes, the test item was tested for 3, 30 and 60 minutes.
After pre-incubation tissues was completed, medium was replaced by 1 mL freshly assay medium in both 6-well plates. The negative and positive control, and the test item was added into the insert atop the concerning EpiOcular duplicate tissues. The 6-well plates were placed into the incubator for the different intervals at 37 ± 1°C, 5 ± 0.5 % CO2.
After the end of the treatment intervals the inserts were removed immediately from the 6-well plates. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material. The inserts were placed in a 6-well plates containing 5 mL medium per well and remained there for 10 to 20 min. Afterwards the cell viability was determined with the MTT assay.

- MTT VIABILITY ASSAY:
The MTT concentrate was prepared on the day of testing and diluted with the MTT diluent. The remaining MTT solution was stored in the dark at 4°C for later use on the same day (not until next day). 24-well plates were prepared before end of the tissue pre-warming period. 300 µL of the MTT solution were added to each well and the plates were kept in an incubator (37 ± 1°C, 5 ± 0.5 % CO2) until use.
After the 10 to 20 min incubation was completed the cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1°C, 5 ± 0.5 % CO2) MTT solution was aspirated from the wells and wells was rinced three times with PBS. Inserts were transferred into new 24 well plates. The inserts were immersed into extractant solution by gently pipetting 2 mL extractant solution (isopopranol) into each insert. The level rised above the upper edge of the insert, thus completely covering the tissue from both sides. The 24 well plate was sealed to inhibit isopropanol evaporation. The formazan salt was extracted for 18 hours without shaking at room temperature.
After the extraction period was completed the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken. Afterwards the insert was discarded. 24 well plates were placed on a shaker for 15 minutes until solution was homogeneous in colour.
Per each tissue 3 x 200 µL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate from the 15 minutes exposure. OD was read in a microplate reader (Versamax(R) Molecular Devices, D-85737 Ismaning) at 570 nm without reference filter. Mean values were calculated from the 3 wells per tissue.


VIABILITY CALCULATION:
- A decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of orange formazan formed, as monitored by the absorbance. The results for the dose-response cytotoxicity curves can be presented as the arithmetic mean ± standard deviation. To calculate the concentration of toxican needed to reduce absorbance of the ET50% (ET50) the following formula is used:
ET50 = time > 50 - [(time > 50 - time < 50) x (% > 50 - 50) / (% > 50 - % < 50)]
a) time > 50 = max. measured time in min with the % of negative control > 50%
b) time < 50 = min. measured time in min with the % of negative control < 50%
c) % > 50 = relative absorbance at a) in %
d) % < 50 = relative absorbance at b) in %

- Data for each individual test item and control were reported in Table 7.3.2/1.

PREDICTION MODEL / DECISION CRITERIA
The lower the ET50 value, the higher is the eye irritant/cytotoxic potential of the test item.
Those calculated ET50-values was used for classification according to the following prediction model:
Non/minimal irritant: ET50 > 60
Mild irritant: 31 < ET50 < 60
Moderate irritant: 3 < ET50 < 30
Severe irritant: ET50 < 3

ACCEPTABILITY OF THE ASSAY
- The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the two tissues is ≥ 0.8.
The ET50 value of the positive control should be under 30 minutes.
Irritation parameter:
mean percent tissue viability 
Run / experiment:
30 minutes tretament interval
Value:
101.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
After treatment with the negative control the absorbance values were well above the required acceptability criterion of mean OD ≥ 0.8 for the 60 minutes treatment interval thus showing the quality of the tissues.
Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 46.5% thus ensuring the validity of the test system. The calculated ET50 value was 14.0 min.
No relevant irritating effects were observed following incubation with the test item up to the longest treatment period. Due to the lack of cytotoxicity, a ET50 value could not be calculated.

The relative absorbance (% of negative control) of the RhCE replicates treated with the test item at the 30 minutes treatment interval was 101.8% versus a range of 19.8-46.5% % in the positive control at the 15 and 45 minutes treatment intervals.

Table 7.3.2/1: Results after treatment with test item



 
















































Dose group 



Treatment interval



Mean absorbance of 2 tissues*



Rel. Absorbance [% of negative control]**



Negative control



60 minutes



1.8443



100.0



Positive control



15 minutes



0.8569



46.5



Positive control



45 minutes



0.3655



19.8



Test item



3 minutes



1.8550



100.6



Test item



30 minutes



1.8766



101.8



Test item



60 minutes



1.8700



101.4







* Mean of three replicate wells after blank correction


** Relative absorbance (rounded values): (100 x ABSORBANCEtest item)/(ABSORBANCEnegative control)




 


Acceptability criteria:


- ET50 value of the test item: could not be determined as viability was not reduced


- ET50 value of the positive control: 14.0 min.


- Optical evaluation of the MTT-reducing capacity of the test item after 1 hour 


Interpretation of results:
GHS criteria not met
Conclusions:
The relative absorbance (% of negative control) of the RhCE replicates treated with the test item at the 30 minutes treatment interval was 101.8% versus a range of 19.8-46.5% % in the positive control at the 15 and 45 minutes treatment intervals.
In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item does not possess any eye irritating potential.
Executive summary:

This in vitro study was performed to assess the eye irritation potential of the test item by means of the Human Cornea Model Test.


Tissues of the human cornea model EpiOcular (TM) were treated with the test item for 3, 30 and 60 minutes in duplicate. The cells for the negative control were treated for 60 minutes and the positive control for 15 and 45 minutes each in duplicate.


 


100 µL of the liquid test item were applied to each tissue, spread to match the tissue size.


100 µL of either the negative control (deionised water) or the positive control (0.3 % Triton X-100) were applied to each tissue 


 


After treatment with the negative control the absorbance values were well above the required acceptability criterion of mean OD ≥ 0.8 for the 60 minutes treatment interval thus showing the quality of the tissues.


Treatment with the positive control induced a sufficient decrease in the relative absorbance after both treatment intervals as compared to the negative control thus ensuring the validity of the test system.


No irritating effects were observed  following incubation with the test item up to the longest treatment period (60 min). Due to the lack of cytotoxicity, a ET50 value could not be calculated.


 


In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item does not possess any eye irritating potential.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:



  • An in vivo key study was identified (RCC, 2008).


In a dermal irritation study performed according to the OECD Guideline No. 404, and in compliance with GLP, 0.5 mL of test material was applied on the clipped skin of the dorsal flank area of 1 male and 2 females New Zealand rabbits. Test sites were covered with a semi-occlusive dressing for 4 hours, followed by several observations at approximately 1, 24, 48 and 72 hours and 7 days after removal of dressing, gauze patch and test substance.


The observed skin irritation consisted of a very slight erythema observed in all animals 1 hour after test item exposure and persisted up to the 24- and 72-hour reading, respectively. All skin reactions were clear within 7 days after treatment.


No abnormal findings were observed on the treated skin of any animal 48 hours and 7 days after treatment, respectively. No staining produced by the test item of the treated skin was observed. Neither alterations of the treated skin were observed nor were corrosive effects evident on the skin. The body weights of all rabbits were considered to be within the normal range of variability.


The mean erythema/eschar score of the three animals was 1.00, 0.33 and 0.33, respectively and the mean oedema score was 0.00 for each of the three animals.


Under the test conditions, test substance is not classified as irritating to skin according to the criteria of the Annex I of the Regulation EC No. 1272/2008 (CLP) and to the GHS. This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.


 



  • An in vitro supporting study is also provided (RCC, 2008).


An in vitro skin irritation test using the Reconstructed human Epidermis (Episkin Standard model) was performed in accordance with the ECVAM international validation study on in vitro tests which followed the current OECD Guideline 439 originally adopted in 2010. The study was compliant with GLP to predict the acute skin irritation potential of the test substance.


Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues was 98.6%. Since the mean relative tissue viability for the test item was above 50% after 15 minutes treatment, the test item is considered to be non-irritant.


The positive control had a mean cell viability after 15 minutes exposure of 28.1%. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean viability of positive control tissues was within the laboratory historical control data range.


Finally, it is concluded that this test is valid and that the test item is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.


 


Eye irritation:



  • An in vivo key study was identified (RCC, 2008).


In an eye irritation study performed according to the OECD Guideline No. 405, and in compliance with GLP, 0.1 mL of undiluted test material was instilled into one eye of one male and two females New Zealand White rabbits. The other eye remained untreated and served as control. The upper and lower eyelids were held together for about one second immediately after application, to prevent loss of the test material, and then released. The eyes were examined and the changes were observed at 1, 24, 48 and 72 h after treatment and graded according to the Draize method.


The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately. The individual mean scores for corneal opacity and iris were 0.00 for all three animals. The individual mean scores for the conjunctivae were 0.00, 0.67 and 0.67 for reddening and 0.00 for chemosis for all three animals.


No abnormal findings were observed in the cornea of iris of any animal at any of the measurement intervals. A slight to moderate reddening of the conjunctivae was noted at the 1-hour reading and persisted as slight in two animals up to 48 hours after treatment. A slight swelling (chemosis) of the conjunctivae was observed in one animal 1 hour after treatement.


No abnormal findings were observed in the treated eye of any animal 72 hours after treatment, the end of the observation period for all animals.


No staining produced by the test item of the treated eye was observed.
No corrosion of the cornea was observed at any of the reading times.
The body weight of all rabbits were considered to be within the normal range of variability.


Mean individual scores at 24, 48 and 72 h after exposure for the 3 animals were 0 / 0 / 0 for cornea score; 0 / 0 / 0 for iris score; 0 / 0.67 / 0.67 for conjunctivae score and 0 / 0 / 0 for chemosis score.


Under the test conditions, test substance is not classified as irritating to eyes according to the criteria of the Annex I of the Regulation EC No. 1272/2008 (CLP) and to the GHS. This study is considered as acceptable and satisfies the requirement for eye irritation endpoint.


 



  • An in vitro supporting study is also provided (RCC, 2008).


This in vitro study was performed to assess the eye irritation potential of the test item by means of the Human Cornea Model Test.


Tissues of the human cornea model EpiOcular (TM) were treated with the test item for 3, 30 and 60 minutes in duplicate. The cells for the negative control were treated for 60 minutes and the positive control for 15 and 45 minutes each in duplicate.


100 µL of the liquid test item were applied to each tissue, spread to match the tissue size.


100 µL of either the negative control (deionised water) or the positive control (0.3 % Triton X-100) were applied to each tissue 


After treatment with the negative control the absorbance values were well above the required acceptability criterion of mean OD ≥ 0.8 for the 60 minutes treatment interval thus showing the quality of the tissues.


Treatment with the positive control induced a sufficient decrease in the relative absorbance after both treatment intervals as compared to the negative control thus ensuring the validity of the test system.


No irritating effects were observed  following incubation with the test item up to the longest treatment period (60 min). Due to the lack of cytotoxicity, a ET50 value could not be calculated.


In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item does not possess any eye irritating potential.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008 (CLP).

 

Self classification:

Based on the available data:

No additional self-classification is proposed regarding skin and eye irritation and the test substance is not classified according to the criteria of the Regulation EC No. 1272/2008 (CLP) and to the GHS.

- No data was available regarding respiratory irritation.