Registration Dossier

Administrative data

Description of key information

SKIN IRRITATION
Skin corrosion: Not corrosive (in vitro), OECD 431, EU Method B40.Bis, Kiss 2012a.
Skin irritation: Not irritating (in vitro), OECD 439, EU Method B.46, Kiss 2012b.
EYE IRRITATION
Eye irritation: Should not be clasified (in vitro), OECD 438, EU Method B.48, Kiss 2012c.

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:
09 May 2012 to 11 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg.
Duration of treatment / exposure:
15 minutes
Other effects:
CELL VIABILITY
- The viability results of the test material treated disks was 88 % of the negative control, since this is > 50 % of the negative control the test material is considered to be a non-irritant.

VALIDITY OF THE TEST
The mean OD value of the three negative control tissues was 0.756. The positive control result showed a mean of 6.9 % viability. Each standard deviation value (SD) of 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 the test material: No colour change was observed after three hours of incubation of the test item in MTT solution. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability due the MTT interaction can be precluded.
> Colouring potential of the test material: As the test material is coloured, one additional chemical-treated tissue was used for the non specific OD evaluation. Optical Density (measured at 540 nm) of this tissue was determined as 0.034, Non Specific Colour % was calculated as 4.5 %, below the threshold of 50 %. Therefore additional data calculation was not necessary.

Table 1: Optical Density (OD) and the Calculated % Viability

 Substance

Optical Density (OD)

Viability (%)

Negative Control

1

0.820

108

2

0.659

87

3

0.788

104

Mean

0.756

100

(SD)

 

11.15

Positive Control

1

0.040

5.3

2

0.079

10

3

0.041

5.4

Mean

0.053

6.9

(SD)

 

2.69

Test Material

1

0.721

95

2

0.536

71

3

0.746

99

Mean

0.668

88

(SD)

 

15.14

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the test, the results indicate that the test material is not a skin irritant.
Executive summary:

The potential for the test material to cause skin irritation was predicted in vitro using the EPISKIN Model in a study conducted under GLP conditions and in line with OECD 439 and EU Method B.46. The test material was applied topically to the surface of the skin for 15 minutes, which was terminated by rinsing with PBS 1 x solution (0.9 %). 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 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified. Positive and negative controls were run concurrently and tissue viability was expressed as a % relative to the negative control.

Under the conditions of the study, exposure to the test material resulted in a mean relative cell viability of 88 %. Since the cell viability was determined to be > 50 % of the negative control the test material was considered to be non-irritating.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg
Duration of treatment / exposure:
Single application, removed after 10 seconds.
Irritant / corrosive response data:
The results suggest that the test material is moderately irritating but not a severe irritant.
Other effects:
Controls:
The positive control imidazole was classed as severely irritating.
The negative control Sodium chloride (Salsol solution 0.9%) had no significant effects on the chicken eye in this study.

Table 4: Test Material Results 

Observations

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

1 %

I

Mean maximum corneal swelling at up to 240 min

1 %

I

Mean maximum corneal opacity

2.00

III

Mean fluorescein retention

2.00

III

Other Observations

The test material was stuck on the cornea surface after the post-treatment rinse. All corneal surfaces were cleared 75 minutes after the post-treatment rinse.

Overall ICE Class

1 x I, 2 x III

Table 5: Positive Control Results

Observations

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

3 %

I

Mean maximum corneal swelling at up to 240 min

8 %

II

Mean maximum corneal opacity

4.00

IV

Mean fluorescein retention

2.83

IV

Other Observations

The Imidazole was stuck on the cornea surface after the post treatment rinse. The cornea surface was not cleared 240 minutes after the post-treatment rinse.

Overall ICE Class

1 x II 2 x IV

 

Table 6: Negative Control Results

Observations

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

0 %

I

Mean maximum corneal swelling at up to 240 min

0 %

I

Mean maximum corneal opacity

0.00

I

Mean fluorescein retention

0.00

I

Other Observations

None

Overall ICE Class

3 x I

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the test, the results suggest that the test material is moderately irritating.
Executive summary:

An in vitro eye irritation study in isolated chicken eyes was conducted under GLP conditions and according to the OECD guideline 438 and EU Method B.48 to determine if the test material caused corrosion or severe eye irritation. Three chicken eyes were exposed to the test material (30 mg) for 10 seconds, rinsed with isotonic saline, and then assessed for irritation at intervals for up to 240 minutes. Toxic effects to the cornea were measured by a qualitative assessment of opacity, damage to epithelium based on application of fluorescein to the eye (fluorescein retention), and measurement of cornea thickness (swelling). Damage was assessed individually and then combined to derive an Eye Irritancy Classification. Positive and negative controls were run concurrently to assess the viability of the test system.

Under the conditions of the study, exposure to the test material resulted in an overall ICE class of 1 x I and 2 x III, which corresponds to a classification of moderately irritating in accordance with the OECD guideline. The results suggest that the test material is moderately irritating, but should not be classified as a severe eye irritant.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation

The potential for the test material to cause skin corrosion (Kiss, 2012a) was predicted in vitro using the EPISKIN Model, in a study conducted under GLP conditions and in line with OECD 431 and EU Method B.40Bis. The test material was applied topically to the surface of the skin for 4 hours. Exposure was terminated by rinsing with PBS 1 x solution (0.9 %). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. Positive and negative controls were run concurrently and tissue viability was expressed as a % relative to the negative control.

The mean cell viability, after adjustment for colour, was 98 % compared to the negative control. Since this was > 35 % the test material is considered to be non-corrosive. Therefore under the conditions of the test, the results indicate that the test material is not a skin corrosive.

 

The potential for the test material to cause skin irritation (Kiss, 2012b) was predicted in vitro using the EPISKIN Model in a study conducted under GLP conditions and in line with OECD 439 and EU Method B.46. The test material was applied topically to the surface of the skin for 15 minutes, which was terminated by rinsing with PBS 1 x solution (0.9 %). 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 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified. Positive and negative controls were run concurrently and tissue viability was expressed as a % relative to the negative control.

Under the conditions of the study, exposure to the test material resulted in a mean relative cell viability of 88 %. Since the cell viability was determined to be > 50 % of the negative control the test material was considered to be non-irritating.

 

Based on the results from the in vitro models the test material is considered to be non-irritating and non-corrosive to the skin. Both studies have been assigned a reliability score of 1 in line with the principles for assessing data quality as defined by Klimish (1997). The data is considered complete and sufficient for classification and labelling purposes.

Eye Irritation

In the key study (Kiss, 2012c), the potential of the test material to cause corrosion or severe eye irritation was determined in vitro in isolated chicken eyes. The study was conducted under GLP conditions and according to the OECD guideline 438 and EU Method B.48.

Three chicken eyes were exposed to the test material (30 mg) for 10 seconds, rinsed with isotonic saline, and then assessed for irritation at intervals for up to 240 minutes. Toxic effects to the cornea were measured by a qualitative assessment of opacity, damage to epithelium based on application of fluorescein to the eye (fluorescein retention), and measurement of cornea thickness (swelling). Damage was assessed individually and then combined to derive an Eye Irritancy Classification. Positive and negative controls were run concurrently to assess the viability of the test system.

Under the conditions of the study, exposure to the test material resulted in an overall ICE class of 1 x I and 2 x III, which corresponds to a classification of moderately irritating in accordance with the OECD guideline. The results suggest that the test material is moderately irritating, but should not be classified as a severe eye irritant.


Justification for selection of skin irritation / corrosion endpoint:
The key study was performed in vitro, in line with GLP and standardised guidelines. It was assigned a reliability score of 1 in accordance with the criteria outlined in Klimisch (1997). It was therefore considered suitable to be the key study for this endpoint.

Justification for selection of eye irritation endpoint:
The key study was performed in vitro, in line with GLP and standardised guidelines. It was assigned a reliability score of 1 in accordance with the criteria outlined in Klimisch (1997). It was therefore considered suitable to be the key study for this endpoint.

Justification for classification or non-classification

Skin Irritation

According to the criteria outlined in Regulation (EC) No. 1272/2008, the test material does not meet the criteria for classification as a skin irritant.

Eye Irritation

In accordance with OECD guideline 438, the available information is sufficient to rule out a severe eye damage/irritation classification in accordance with Regulation (EC) No. 1272/2008. However, the study suggests that the test material is moderately irritating. In vitro data is not sufficient to determine a Category 2 classification, and further testing is beyond the requirements of an Annex VII Registration in accordance with Regulation (EC) No. 1907/2006.