Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin irritation

The results obtained from the in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. This result is supported by a skin irritation study in vivo.
Eye irritation

Application of the test item into the rabbits' eye mucosa caused slight to severe conjunctival and slight to moderate corneal irritant effects, fully reversible within 2 weeks. This result is supported by a second eye irritation study in vivo. The test item showed no corrosive potential in an Isolated chicken eye test in vitro.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-10-03 to 2012-11-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
22 July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
06 July 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
other: reconstructed human epidermis
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EpiSkinTM-SM model has been validated for irritation testing in an international trial and is considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTMSM, EPISKIN SNC Lyon, France
- Tissue batch number: 12-EKIN-038
- Expiry date: 22 October 2012
- Date of initiation of testing: 17 October 2012

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the incubation time, the EPISKIN-SM units were removed and rinsed thoroughly with PBS 1 x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging the epidermis).
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL MTT per well
- Incubation time: 3 h
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irrtating to skin if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: The test item is a viscous liquid therefore exact weighting of treatment volume was not performed. While avoiding infinite dose a sufficient amount to cover evenly all the epidermal surface (approx. 20 μL) was spread gently with a flat curved spatula onto the skin.

NEGATIVE CONTROL
- Amount applied: 20 µL
- Concentration: 1 x

POSITIVE CONTROL
- Amount applied: 20 µL
- Concentration: 5 %
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Value:
70
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control, therefore the test item was considered to be non-irritant to skin.

- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.
- Colour interference with MTT: The test item showed no ability to become coloured in contact with water therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method TOXI-COOP ZRT. demonstrated the technical proficiency, using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The mean OD value of the three negative control tissues was 0.648.
- Acceptance criteria met for positive control: yes. The mean OD value obtained for the positive control was 0.119 and this result corresponds to 18 % viability when compared to the results obtained from the negative controls.
- Acceptance criteria met for variability between replicate measurements: yes. Each calculated standard deviation value (SD) for the % viability was below 18.
All validity criteria were within acceptable limits and therefore the study can be considered as valid.

VALIDITY OF THE TEST:
The mean OD value of the three negative control tissues was 0.648. The mean OD value obtained for the positive control was 0.119 and this result corresponds to 18 % viability when compared to the results obtained from the negative controls. Each calculated standard deviation value (SD) for the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Indicator for potential false viability

Possible direct MTT reduction with test substance:
No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

Colouring potential of test substances:
The test item showed no ability to become coloured in contact with water therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.
Interpretation of results:
GHS criteria not met
Conclusions:
The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. According to the current OECD Guideline No. 439, Incozol 4 is considered as non-irritant to skin and is therefore not classified.
Executive summary:

The purpose of this study was to determine the skin irritation potential of the test item Incozol 4 on reconstituted human epidermis in the EPISKIN model in vitro. Disks of epidermal units (three units / chemical) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with PBS 1x solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. The resulting formazan crystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically. SDS 5 % aq. and 1 x PBS treated epidermis units were used as positive and negative controls, respectively. For each treated tissue viability was expressed as a percentage relative to negative control. The test item is considered to be a skin irritant, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less than or equal to (≤) 50% when compared to the viability values obtained from the negative control. In this in vitro skin irritation test using the EPISKIN model, the test item Incozol 4 did not show significantly reduced cell viability in comparison to the negative control. All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control, therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. According to the current OECD Guideline No. 439, Incozol 4 is considered as non-irritant to skin and is therefore not classified.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1978-10-24 to 1978-10-30
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Only basic information available. Documentation not sufficient for evaluation.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The test substance was applied on the ear of rabbits. After 24 hours, the test substance was washed off and skin reaction was evaluated within an observation period of 7 days.
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 3 - 4 kg
- Housing: Single housing
- Sex: Male and female
Type of coverage:
not specified
Preparation of test site:
clipped
Vehicle:
not specified
Controls:
not specified
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 500 µL/animal
Duration of treatment / exposure:
24 hours
Observation period:
7 days
Number of animals:
2
Details on study design:
TEST SITE
- Area of exposure: Ear
- Type of wrap: Adhesive bandage

REMOVAL OF TEST SUBSTANCE
- Washing: Water and soap and veg oil
- Time after start of exposure: 7 days
Irritation parameter:
other: No irritation parameters given.
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Not irritating (no information about scores available).
Interpretation of results:
study cannot be used for classification
Conclusions:
The test item was considered not to be skin irritating.
Executive summary:

The skin irritation test of the test item was conducted with White New Zealand rabbits. One female and one male rabbit were tested. The test item (500 µL/animal) has been fixed with an adhesive bandage at the inside area of the ear and the animals were exposed for 24 hours. After the exposure period the application sites were washed with water, soap/veg oil and the test animals were observed for 7 days. There were no observations of irritations to the skin. According to the results of this test, the test item Incozol 4 was considered to be not irritating to the skin.

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:
26 September 2012 to 06 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
24 April 2002
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: S & K LAP Kft. 2173 Kartal, Császár út 135, Hungary
- Age at study initiation: young adult male rabbits
- Weight at study initiation: 2625 - 3356 g
- Housing: Animals were housed individually in metal cages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3 °C
- Humidity: 30 - 70 %
- Air changes (per hr): 9 - 12
- Photoperiod (hrs dark / hrs light): 12 hours artificial light, from 6 a.m. to 6 p.m.

IN-LIFE DATES: From: 01 October 2012 To: 15 October 2012
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.1 mL
Duration of treatment / exposure:
The eyes of the test animals were not washed out after the application of test item.
Observation period (in vivo):
The eyes were examined at 1, 24, 48 and 72 hour(s) and then at 1 and 2 weeks after the application.
Number of animals or in vitro replicates:
3
Details on study design:
SCORING SYSTEM: The eye irritation scores were evaluated according to the scoring system by Draize (1979) and OECD 405 (24 April 2002)

TOOL USED TO ASSESS SCORE: hand-slit lamp / biomicroscope
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 2 weeks
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Max. score:
4
Reversibility:
fully reversible within: 2 weeks
Remarks on result:
other: No calculation of score due to severely conjunctivae irritation after 24 and 48 h
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 2 weeks
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Max. score:
2
Remarks on result:
other: No calculation of score due to severely conjunctivae irritation after 24 and 48 h.
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.66
Max. score:
4
Reversibility:
fully reversible within: 2 weeks
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
3.33
Max. score:
4
Reversibility:
fully reversible within: 2 weeks
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 2 weeks
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1.33
Max. score:
3
Reversibility:
fully reversible within: 2 weeks
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Max. score:
3
Reversibility:
fully reversible within: 2 weeks
Remarks on result:
other: No calculation of score due to severely conjunctivae irritation after 24 and 48 hr.
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 2 weeks
Irritant / corrosive response data:
One hour after the treatment, moderate conjunctival redness, moderate to severe chemosis and discharge, as well as slight cornea opacity were observed. The area of cornea involved was one quarter in one animal and was greater than half, but less than three quarters in other animal. Redness of conjunctivae, corneal and iris alterations could not be observed in third animal treated due to the swelling the lids were completely closed.

72 hours after the treatment, slight to moderate redness and discharge and moderate chemosis, as well as slight to moderate cornea opacity were detected. The area of cornea involved was greater than one quarter, but less than half or was greater than half, but less than three quarters.

1 week after the treatment, slight to moderate redness, slight chemosis and discharge, as well as slight cornea opacity were found. The area of cornea involved was one quarter. One animal became free of symptoms.

2 weeks after treatment the study was terminated, as all animals were free of symptoms of irritation.
Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
In conclusion, test item applied to the rabbits' eye mucosa, caused slight to severe conjunctival and slight to moderate corneal irritant effects, fully reversible within 2 weeks.
Executive summary:

The acute eye irritation study of the test item was performed in three New Zealand White rabbits. The irritation effect of the test item was evaluated according to the Draize method (OECD No.: 405, 2002).

The test item was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. A weight of 0.1 ml of the test item was used in pure state, as a single dose. The eyes of the test animals were not washed out after the application of test item.

The eyes were examined at 1, 24, 48 and 72 hour(s) and then at 1 and 2 weeks after the application.

One hour after the treatment, moderate conjunctival redness, moderate to severe chemosis and discharge, as well as slight cornea opacity were observed. The area of cornea involved was one quarter in one animal and was greater than half, but less than three quarters in other animal. Redness of conjunctivae, corneal and iris alterations could not be observed in third animal treated due to the swelling the lids were completely closed.

72 hours after the treatment, slight to moderate redness and discharge and moderate chemosis, as well as slight to moderate cornea opacity were detected. The area of cornea involved was greater than one quarter, but less than half or was greater than half, but less than three quarters.

1 week after the treatment, slight to moderate redness, slight chemosis and discharge, as well as slight cornea opacity were found. The area of cornea involved was one quarter. One animal became free of symptoms.

2 weeks after treatment the study was terminated, as all animals were free of symptoms of irritation.

The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after the treatment) were as follows:

cornea opacity: 1.00, n.d., 2.00

iris: 0.00, n.d., 0.00

redness: 1.33, n.d., 2.00

chemosis: 0.66, 3.33, 2.00

 

(n.d.: The respective mean values of redness of conjunctivae, cornea opacity and iris could not be calculated, due to the severe conjunctivae irritation as the swelling the lids were completely closed.)

 

In conclusion, test item applied to the rabbits' eye mucosa, caused slight to severe conjunctival and slight to moderate corneal irritant effects, fully reversible within 2 weeks.

According to Regulation (EC) No 1272/2008, the test item should be classified into “Category 2 (reversible effects on the eye/irritating to eyes)” on basis of corneal and conjunctivae irritation sign as cornea opacity and chemosis.

However, it is to be noted that the test item caused severe conjunctivae irritation sign as chemosis in one animal in 24 and 48 hours after the treatment, so the redness, cornea opacity and iris irritation could not be scored.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1978-10-24 to 1978-10-30
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Only basic information available. Documentation not sufficient for evaluation.
Qualifier:
no guideline followed
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 3 - 4 kg
- Housing: Single housing
- Sex: Male and female
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 100 µL/animal
Duration of treatment / exposure:
24 hours/Single dosing
Observation period (in vivo):
7 days
Number of animals or in vitro replicates:
2
Details on study design:
TEST SITE
- Area of exposure: Conjunctival sac

REMOVAL OF TEST SUBSTANCE
- Time after start of exposure: 7 days
Irritation parameter:
other: No irritation parameters given.
Remarks on result:
positive indication of irritation
Irritant / corrosive response data:
Slightly irritating (no scores given).
Interpretation of results:
study cannot be used for classification
Conclusions:
The test item was considered to be slightly irritating to the eyes.
Executive summary:

The eye irritation test of the test item Incozol 4 was conducted with White New Zealand rabbit. One female and one male rabbit were tested. The test item (100 µL/animal) has been applied in the conjunctival sac and has been exposed for 24 hours. After exposure the test animals were observed for 7 days. The test substance showed irritation effects to the eyes. According to the results the test item Incozol 4 can be considered to be slightly irritating to the eyes.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-08-21 to 2012-08-21
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 Ocular Corrosives and Severe Irritants)
Version / remarks:
adopted 07 Sep 2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Version / remarks:
adopted 08 Dec 2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
TEST SYSTEM
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129., Hungary

Head collection was performed by a slaughter house technician. The heads were transported to TOXI-COOP ZRT. at the earliest convenience for use approximately within 2 hours from collection. All eyes used in the assay were from the same groups of eyes collected on one specific day.
After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4 - 5 heads/box).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.1 mL
Duration of treatment / exposure:
10 seconds
Observation period (in vivo):
30, 75, 120, 180 and 240 min after washing
Number of animals or in vitro replicates:
Three isolated chicken eyes
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: 20 mL saline solution at ambient temperature
- Time after start of exposure: 10 seconds

SCORING SYSTEM: The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium). Results from corneal opacity, swelling, and fluorescein retention were evaluated separately to generate an Isolated Chicken Eye (ICE) class for each endpoint. The ICE classes for each endpoint were then combined to generate an Irritancy Classification for each test substance. The effects were divided into four categories:
I = none
II = slight
III = moderate
IV = severe

TOOL USED FOR EXAMINATION OF ISOLATED EYES: fluorescein
Irritation parameter:
percent corneal swelling
Run / experiment:
at up to 75 min
Value:
2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
at up to 240 min
Value:
3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Value:
0.33
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The negative control NaCl (9 g/L saline) had no significant effects on the chicken eye in this study.
- Acceptance criteria met for positive control: yes. The positive control Trichloroacetic acid 30 % (w/v) was classed as corrosive/severely irritating, GHS Classification: Category 1.

Positive Control: Trichloroacetic acid 30 % (w/v)

 

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

-23 %

(severe2) IV

Mean maximum corneal swelling at up to 240 min

-24 %

(severe2) IV

Mean maximum corneal opacity

4.00

IV

Mean fluorescein retention

2.83

IV

Other Observations

Immediate cornea opacity score 4. Severe thinning was observed in all eyes (3/3) suggesting epithelial loss

Overall ICE Class

3 x IV

 

The positive control Trichloroacetic acid 30 % (w/v) was classed as corrosive/severely irritating, GHS Classification: Category 1.

 

Negative Control: NaCl (9 g/L saline)

 

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

2 %

I

Mean maximum corneal swelling at up to 240 min

4 %

I

Mean maximum corneal opacity

0.00

I

Mean fluorescein retention

0.50

I

Other Observations

None

Overall ICE Class

3 x I

 

The negative control NaCl (9 g/L saline) had no significant effects on the chicken eye in this study.

Interpretation of results:
GHS criteria not met
Conclusions:
In this in vitro eye corrosives and severe irritants study, using the Isolated Chicken Eyes model with Incozol 4, no ocular corrosion or severe irritation potential was observed. Thus, according to the guideline OECD 438, Incozol 4 is not classified as an ocular corrosive or severe eye irritant. However, to obtain a definitive classification in relation to the irritation potential, a further in vivo rabbit study or other in vitro studies are required.
Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity or severe irritancy of the test item Incozol 4 by its ability to induce toxicity in enucleated chicken eyes. The test compound was applied in a single dose onto the cornea of isolated chicken eyes in order to potentially classify the test compound as ocular corrosive and/or severe irritant. The ICET does not fully replace thein vivorabbit eye test (OECD 405); however, the ICET is used as part of a tiered testing strategy for regulatory purposes. Test substances that are positive in this assay can be classified as ocular corrosives or severe irritants without further testing in rabbits. A substance that tests negative would need to be tested according to OECD 405. In this ICET, Incozol 4 did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. Moreover, the results suggest that the test item has no irritation potential according to the ICET classification. Positive and negative controls showed the expected results. The experiment was considered to be valid. In this in vitro eye corrosives and severe irritants study, using the Isolated Chicken Eye model with Incozol 4, no ocular corrosion or severe irritation potential was observed. Thus, according to the guideline OECD 438, Incozol 4 is not classified as an ocular corrosive or severe eye irritant. However, to obtain a definitive classification in relation to the irritation potential, a furtherin vivo rabbit study or other in vitro studies are required.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation

The purpose of this study was to determine the skin irritation potential of the test item on reconstituted human epidermis in the EPISKIN model in vitro.

Disks of epidermal units (three units / chemical) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with PBS 1x solution. Epidermis units were then incubated at 37 °C for 42 hours. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically.

SDS 5 % aq. and 1 x PBS treated epidermis units were used as positive and negative controls, respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control. All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control, therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions and is therefore not classified.

 

In a supporting study the skin irritation potential of the test item was conducted with White New Zealand rabbits. One female and one male rabbit were tested. The test item (500 µL/animal) has been fixed with an adhesive bandage at the inside area of the ear and the animals were exposed for 24 hours. After the exposure period the application sites were washed with water, soap/veg oil and the test animals were observed for 7 days.

There were no observations of irritations to the skin. According to the results of this test, the test item was considered to be not irritating to the skin.

 

Eye irritation

The acute eye irritation study of the test item was performed in three New Zealand White rabbits. The irritation effect of the test item was evaluated according to the Draize method (OECD No.: 405, 2002).

The test item was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. A weight of 0.1 mL of the test item was used in pure state, as a single dose. The eyes of the test animals were not washed out after the application of test item.

The eyes were examined at 1, 24, 48 and 72 hour(s) and then at 1 and 2 weeks after the application.

One hour after the treatment, moderate conjunctival redness, moderate to severe chemosis and discharge, as well as slight cornea opacity were observed. The area of cornea involved was one quarter in one animal and was greater than half, but less than three quarters in other animal. Redness of conjunctivae, corneal and iris alterations could not be observed in third animal treated due to the swelling the lids were completely closed.

72 hours after the treatment, slight to moderate redness and discharge and moderate chemosis, as well as slight to moderate cornea opacity were detected. The area of cornea involved was greater than one quarter, but less than half or was greater than half, but less than three quarters.

The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after the treatment) were as follows:

 

cornea opacity: 1.00, n.d., 2.00

iris: 0.00, n.d., 0.00

redness: 1.33, n.d., 2.00

chemosis: 0.66, 3.33, 2.00

 

1 week after the treatment, slight to moderate redness, slight chemosis and discharge, as well as slight cornea opacity were found. The area of cornea involved was one quarter. One animal became free of symptoms. 2 weeks after treatment the study was terminated, as all animals were free of symptoms of irritation.

In conclusion, test item applied to the rabbits' eye mucosa, caused slight to severe conjunctival and slight to moderate corneal irritant effects, fully reversible within 2 weeks.

 

In a supporting study the eye irritation potential of the test item was conducted with White New Zealand rabbits. One female and one male rabbit were tested. The test item (100 µL/animal) was applied in the conjunctival sac and the animals were exposed for 24 hours. After exposure the test animals were observed for 7 days. The test substance showed irritation effects to the eyes. According to the results the test item was considered to be slightly irritating to the eyes.

 

In an Isolated Chicken Eye Test (ICET) according to OECD TG 438 the test item did not cause ocular corrosion or severe irritation.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin and eye irritation or corrosion, the test item is not classified as skin irritant or corrosive according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776. However, based on in vivo data available on eye irritation, the substance is classified as Category 2 (H319) "Causes serious eye irritation" eye irritant according to Regulation (EC) No1272/2008 (CLP), as amended for tenth time in Regulation (EU) No 2017/776.