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Administrative data

Description of key information

Skin: Not classified as irritant, EpiSkin, OECD 439, Kiss 2014
Skin: Not classified as corrosive, EpiSkin, OECD 431, Kiss 2014
Eye: Not classified as irritant, ICE, OECD 438, Kiss 2014

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-12-18 until 2013-12-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: UN (2009), United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS), third revised edition, UN New York and Geneva.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Species:
other: Reconstructed human epidermis model EPISKIN (SM)
Strain:
other: Not applicable
Details on test animals or test system and environmental conditions:
HUMAN SKIN: EPISKIN (SM) (Manufacturer: SkinEthic, France, Batch No.:13-EKIN-045, Expiry Date: 23 December 2013) is a three-dimensional human epidermis model.

EXPERIMENTAL DATES: From: 18 December 2013 To: 20 December 2013
Type of coverage:
other: Not applicable
Preparation of test site:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: Not applicable
Duration of treatment / exposure:
15 mins
Observation period:
Not applicable
Number of animals:
Not applicable
Irritation / corrosion parameter:
other: other: optical density
Value:
0.731
Remarks on result:
other:
Remarks:
Basis: mean. (migrated information)
Irritation / corrosion parameter:
other: other: viability
Value:
99
Remarks on result:
other:
Remarks:
Basis: mean. Remarks: %. (migrated information)
Irritant / corrosive response data:
The optical density for the test item treated skin was 0.731 and showed a viability of 99%.
The optical density for the negative and positive controls were 0.740 and 0.088, respectively and showed viabilities of 100% and 12% respectively.

The test item is, therefore, considered not to be irritant to skin, as the mean relative viability after 15 minutes exposure and 42 hours post incubation, is greater than 50% of the negative control. The positive control was irritant.

Table 1: Optical density and calculated % viability of cells

Substance

Optical density

Viability (%)

Negative control

PBS

1

0.770

104

2

0.728

98

3

0.721

97

mean

0.740

100

Positive control

SDS 5%

1

0.100

14

2

0.095

13

3

0.070

9.5

mean

0.088

12

Test item

1

0.746

101

2

0.698

94

3

0.748

102

mean

0.731

99

 

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In an in vitro EPISKIN (SM) model test, the results indicate that the test item is Non Irritant (UN GHS: No Category).
Executive summary:

The reconstructed human epidermis model EPISKIN (SM) is designed to predict and classify the skin irritant potential of chemicals, by measuring its cytotoxic effect, as reflected in the MTT assay, on the EPISKIN reconstituted human epidermis. This method is approved by international regulatory agencies as a replacement for the identification of irritants / corrosives in the in vivo rabbit skin assay (OECD 404).

Disks of EPISKIN (three units / chemical) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with phosphate buffered saline (PBS). Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of the epidermis on each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified.

SDS 5% and PBS treated epidermis were used as positive and negative controls, respectively. An additional disk was used to provide an estimate of colour contribution from the test item. For each treated tissue viability was expressed as a % relative to negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test substance is considered to be irritant to skin.

Following exposure with the test item, the mean cell viability was 99% compared to the negative control and, therefore, non irritant. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

In the in vitro EPISKIN (SM) model test, the results indicate that the test item is Non Irritant [UN GHS: No category].

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-12-11 until 2013-12-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: INVITTOX (1994) Protocol 80: Chicken Enucleated Eye Test
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Species:
other: Isolated chicken eyes
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
ISOLATED CHICKEN'S EYES
- Source: Heads from chickens used for human consumption supplied by a commercial abattoir.
- Preparation: Eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 % (w/v) was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. The fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye still in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

The prepared eye was placed in a steel retainer with the cornea in the correct relative position. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The retainer holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline dripping from a stainless steel tube, at a rate of approximately 3-5 drops/minute.

The appropriate number of eyes (9-10) was selected, after being placed in the superfusion apparatus they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to clearly see the isotonic saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e. > 0.5) or corneal opacity score (i.e. > 0.5) were rejected. The cornea thickness was measured with an optical pachymeter on a slit-lamp microscope. Any eye with cornea thickness deviating by more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization was started and conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

EXPERIMENTAL DATES: 11 to 18 December 2013:
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg
Duration of treatment / exposure:
10 seconds
Observation period (in vivo):
Pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.
Number of animals or in vitro replicates:
3 test, 3 positive control, 1 negative control isolated eyes
Details on study design:
- BASE LINE ASSESSMENT: A zero reference measurement was recorded for cornea thickness and opacity to serve as a base line (t=0) for each individual eye.
- TREATMENT: The eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in a horizontal position, while the test item was applied onto the centre of the cornea. The test item was applied in an amount of 30 mg onto the entire surface of the cornea. The positive control eyes were treated in a similar way with 30 mg powdered imidazole and the negative control was treated with 30 µL sodium chloride solution.
- TEST ITEM REMOVAL: 10 seconds after application, the cornea surface was rinsed thoroughly with 20 mL isotonic saline at ambient temperature. The eye was returned to the chamber after rinsing. (The imidazole (in all eyes) was stuck on the corneas’ surface after the post-treatment rinse. Gentle rinsing with 20 mL saline was performed at each observation time point. The cornea surfaces treated were not cleared 240 minutes after the post-treatment rinse)..
- OBSERVATION / ASSESSMENT: Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at base line (t=0) and approximately 30 minutes after the post-treatment rinse using a slit-lamp microscope.

Endpoints evaluated were corneal opacity, swelling and fluorescein retention. Other observations which indicate damage, such as loss of epithelium were taken into account in making a classification.

Results from corneal swelling, opacity 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

Effects were divided into 4 categories (I = none, II = slight, III = moderate, IV = severe).
Irritation parameter:
other: Mean maximum corneal swelling (Run 1)
Basis:
mean
Time point:
other: 75 min
Score:
0
Remarks on result:
other: ICE Class I
Irritation parameter:
other: Mean maximum corneal swelling (Run 1)
Basis:
mean
Time point:
other: 240 min
Score:
0
Remarks on result:
other: ICE Class I
Irritation parameter:
other: Mean maximum corneal opacity change (Run 1)
Score:
0
Remarks on result:
other: ICE Class I
Irritation parameter:
other: Mean fluorescein retention change (Run 1)
Basis:
mean
Score:
0.17
Remarks on result:
other: ICE Class I
Irritation parameter:
other: Mean maximum corneal swelling (Run 2)
Basis:
mean
Time point:
other: 75 min
Score:
0
Remarks on result:
other: ICE Class I
Irritation parameter:
other: Mean maximum corneal swelling (Run 2)
Basis:
mean
Time point:
other: 240 min
Score:
0
Remarks on result:
other: ICE Class I
Irritation parameter:
other: Mean maximum corneal opacity change (Run 2)
Basis:
mean
Score:
0
Remarks on result:
other: ICE Class I
Irritation parameter:
other: Mean fluorescein retention change (Run 2)
Basis:
mean
Score:
0
Remarks on result:
other: ICE Class I
Irritant / corrosive response data:
No corneal swelling or corneal opacity was observed during the four hour observation period. Fluorescein retention (severity 0.5) was noted only in one eye in the first run. No other corneal effect was observed. The negative and positive control group results demonstrate that the study was valid.

Table 1: Eye irritation scores in the in vitro eye irritation test in isolated chicken eyes – Run 1

Observation

Test item

Positive control

Negative control

Value

ICE class

Value

ICE class

Value

ICE class

Mean maximum corneal swelling at up to 75 min

0%

I

4%

I

0%

I

Mean maximum corneal swelling at up to 240 min

0%

I

7%

II

0%

I

Mean maximum corneal opacity change

0

I

3.83

IV

0

I

Mean fluorescein retention change

0.17

I

2.83

IV

0

I

Other Observations

None

The imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared 240 minutes after the post-treatment rinse.

None

Overall ICE Class

3 x I

1 x II 2 x IV

3 x I

 

Table 2: Eye irritation scores in the in vitro eye irritation test in isolated chicken eyes – Run 2

Observation

Test item

Positive control

Negative control

Value

ICE class

Value

ICE class

Value

ICE class

Mean maximum corneal swelling at up to 75 min

0%

I

2%

I

0%

I

Mean maximum corneal swelling at up to 240 min

0%

I

5.3%

II

0%

I

Mean maximum corneal opacity change

0

I

3.83

IV

0

I

Mean fluorescein retention change

0

I

2.83

IV

0

I

Other Observations

None

The imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared 240 minutes after the post-treatment rinse.

None

Overall ICE Class

3 x I

1 x II 2 x IV

3 x I

 

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: other: UN GHS
Conclusions:
Based on this in vitro eye irritation study in isolated chicken eyes, the test item is non-irritating, GHS Classification: Non-classified.
Executive summary:

An in vitro eye irritation study was performed in isolated chicken’s eyes. After the zero reference measurements, the eye was held in a horizontal position and 30 mg of the test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with saline. The positive control eyes were treated in a similar way with 30 mg imidazole. The negative control eye was treated with 30 μL of 0.9% sodium chloride. Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (pitting or loosening of the epithelium) evaluated.

In line with the most recent OECD guideline, the negative effect was confirmed in a repeat run of 3 eyes, such that the guideline allows the test item to be classified as negative, without the need for an in vivo confirmatory study.

No corneal swelling or corneal opacity was observed during the four hour observation period. Fluorescein retention (severity 0.5) was noted only in one eye in the first run. No other corneal effect was observed. The negative and positive control group results demonstrate that the study was valid.

Based on this in vitro eye irritation study in isolated chicken eyes, the test item is non-irritating, GHS Classification: Non-classified.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation/Corrosion

In vivo data

Only one study (TSCAT 1970) to date conductedin vivoexperiments on guinea pigs, for which the original document was not available. No method details was provided and only a gross observation as "slightly irritant" was reported as result of the study. The information are insufficient to give a reliable conclusion.

In vitro data

Two studies are available, both done according to GLP, using guideline methods which are validated for regulatory use:

In order to assess the in vitro skin corrosion potential of the substance, EpiSkin disks (Kiss 2014) were treated with the test item, negative controls (0.9 % sodium chloride) and positive controls (glacial acetic acid) and incubated for 4 hours at room temperature (2 replicates each). The mean cell viability was 108 % compared to the negative control (100 %) and the positive control (0.9 %). All validity criteria were within acceptable limits and therefore the study can be considered as valid. The substance is not corrosive to the in vitro skin model EpiSkin.

In order to assess the in vitro skin irritation potential of the substance, EpiSkin disks (Kiss 2014) were treated with the test item,negative controls (PBS) and positive controls (5% SDS) for 15 min (3 replicates each). The mean cell viability was 99 % compared to the negative control (100 %) and the positive control (12 %). All validity criteria were within acceptable limits and therefore the study can be considered as valid. The substance is not irritant to the in vitro skin model EpiSkin.

Based on the available in vitro data which is considered to be adequate, reliable and conclusive for skin irritation/corrosion, the substance is considered to not be an irritant.

Eye Irritation

In vitro data

In order to assess the in vitro eye irritation potential of the substance, OECD Guideline 438 Isolated Chicken Eye Test Method was carried out. Simultaneously were performed 3 tests with the substance , 3 positive controls (imidazole) and 1 negative control (0.9 % sodium chloride) on isolated eyes. No corneal swelling or corneal opacity was observed during the four hour observation period. Fluorescein retention (severity 0.5) was noted only in one eye in the first run. No other corneal effect was observed. The negative and positive control group results demonstrate that the study was valid. Based on this in vitro eye irritation study in isolated chicken eyes, the test item is non-irritating.


Justification for selection of skin irritation / corrosion endpoint:
GLP compliant, guideline study, no restrictions, fully adequate for assessment.

Justification for selection of eye irritation endpoint:
GLP compliant, guideline study, no restrictions, fully adequate for assessment.

Justification for classification or non-classification

Skin Irritation / Corrosion

Results from validated in vitro studies for skin corrosion and irritation, conducted according to Regulation (EC) No. 440/2008, method B.4 demonstrated the substance to be non-corrosive and non-irritating to skin. Two EpiSkin studies were conducted following OECD guidelines and in compliance with GLP Directive 2004/10/EC. Both results are valid, reliable and adequate for the purpose of risk assessment, classification and labelling.

The substance does not fall into the criteria for classification as a corrosive or irritant substance according to Regulation (EC) 1272/2008, Part 3.2., and the substance is not considered to be classified under Directive 2001/59/EEC, 3.2.5 and 3.2.6. The result is conclusive.

Eye Irritation

Results from a validated in vitro study for eye irritation, conducted according to OECD Guideline 438 - Isolated Chicken Eye Test Method- demonstrated the substance to be non-irritating to the eye. The study was carried out in compliance with GLP Directive 2004/10/EC. The conclusion is considered valid, reliable and adequate for the purpose of risk assessment, classification and labelling.

The substance does not fall into the criteria for classification as irritant substance according to Regulation (EC) 1272/2008, Part 3.3.2., and the substance is not considered to be classified under Directive 2001/59/EEC, 3.2.6.2. The result is conclusive.

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