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Description of key information

In the key study (Sachsse, 1973), the average oedema score at 24 hours for intact skin is 2.67. The average oedema score at 72 hours is 2.33. As the mean score at both of these time points is greater than 2, the test material can be said to be irritating to skin. The primary Irritation Index of the test material was 1.7.

In the key study, Sanders (2010), a study was performed to assess the ocular irritancy potential of the test material in the rabbit following application onto the cornea of the enucleated eye. The results of the study are believed to be of value in predicting the ocular irritation potential of the test material in man.  Following assessment of the data for all endpoints, the test material was considered to have the potential to cause severe ocular irritancy in vivo.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No GLP or purity info. Similar to OECD 404, well reported, but with deviations. No scores recorded at 48 hours.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
yes
Remarks:
See principles of method if other than guideline
Principles of method if other than guideline:
Deviations:
- Shaved 2 days before testing, normally 24 hours.
- Test material applied for 24 hours. Normally only applied for 4 hours.
- Observation period only 7 days, should be 14 days.

The procedure adopted was the patch-test technique described in the "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" (1959) of the US Association of Food and Drug Officials (AFDO).
GLP compliance:
not specified
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Six rabbits (3 males/3 females) of the New Zealand White strain, weighing 2 to 3 kg, were shaved on the whole back and flanks two days before treatment with an electric clipper. In order to prevent injuries by bites the rabbits were kept separately in V2A wire cages, where they had access to food (NAFAG, Gossau SG, rabbit food) and water ad libitum.
Type of coverage:
occlusive
Preparation of test site:
shaved
Vehicle:
not specified
Controls:
not specified
Amount / concentration applied:
The test material was applied to each side in quantities of 0.5 mL.
Duration of treatment / exposure:
24 hours
Observation period:
72 hours upon removal of patch.
Number of animals:
6 (3 males and 3 females)
Details on study design:
The shaven skin on the left side was slightly scarified immediately before treatment.
A gauze patch 2.5 x 2.5 cm was soaked with the test material and immediately applied to the prepared skin. The patch was covered with a plastic film 5 x 5 cm, which was fixed to the body with adhesive tape. The gauze patches were removed 24 hours after the application. The reaction of the skin was appraised upon removal and 72 hours after it, on the basis of the evaluation scheme.
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
not fully reversible within: 72 hours
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.5
Max. score:
2
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
not fully reversible within: 72 hours
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.5
Max. score:
1
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
edema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
3.5
Max. score:
4
Reversibility:
not fully reversible within: 72 hours
Irritation parameter:
erythema score
Basis:
animal #4
Time point:
24/48/72 h
Score:
0.5
Max. score:
1
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
edema score
Basis:
animal #4
Time point:
24/48/72 h
Score:
3.5
Max. score:
4
Reversibility:
not fully reversible within: 72 hours
Irritation parameter:
erythema score
Basis:
animal #5
Time point:
24/48/72 h
Score:
0.5
Max. score:
1
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
edema score
Basis:
animal #5
Time point:
24/48/72 h
Score:
3.5
Max. score:
4
Reversibility:
not fully reversible within: 72 hours
Irritation parameter:
erythema score
Basis:
animal #6
Time point:
24/48/72 h
Score:
0.5
Max. score:
1
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
edema score
Basis:
animal #6
Time point:
24/48/72 h
Score:
3.5
Max. score:
4
Reversibility:
not fully reversible within: 72 hours
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Score:
1.7
Irritant / corrosive response data:
The average oedema score at 24 hours for intact skin is 2.67. The average edema score at 72 hours is 2.33. As the mean score at both of these time points is greater than 2, the test material can be said to be irritating to skin.
Other effects:
The skin of the rabbits treated with the test material, showed a white to yellow colour and a slight to medium oedema.
These symptoms did not diminish or disappear during the 7-day observation period.

The results of the skin Irritation test are summarised in the attached table.

The primary Irritation Index as the measure of the acute skin irritation provoked by the test material is defined as the average of all the ratings found after 24 and 72 hours on the intact and slightly scarified skin. The primary Irritation Index of the test material was 1.7.

Interpretation of results:
other: EU Criteria: H315: Cause skin irritation
Conclusions:
Looking at the table, the average oedema score at 24 hours for intact skin is 2.67. The average oedema score at 72 hours is 2.33. As the mean score at both of these time points is greater than 2, the test material can be said to be irritating to skin. The primary Irritation Index of the test material was 1.7.
Executive summary:

The primary Irritation Index as the measure of the acute skin irritation provoked by the test material is defined as the average of all the ratings found after 24 and 72 hours on the intact and slightly scarified skin.

The primary Irritation Index of the test material was 1.7.

This study is considered the most relevant and reliable study for this endpoint, although technically the results are unable to be used for classification purposes as readings were only taken at 24 and 72 hours and also the test material was left on longer than specified in the guideline. However the mean oedema scores at 24 and 72 hours are above the EU C&L cut-off criteria according to Regulation (EC) no 1272/2008 and so it is highly likely that the 48 hours reading would also be above this cut-off value, and so this study would be considered to be the worst case scenario used to classify as irritating to the skin, therefore this study will be used for classification purposes for this endpoint.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
30 June 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Validated in vitro study conducted to GLP.
Qualifier:
no guideline available
Principles of method if other than guideline:
A study was performed to assess the ocular irritancy potential of the test material in the rabbit following application onto the cornea of the enucleated eye. The results of the study are believed to be of value in predicting the ocular irritation potential of the test material in man.
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
not specified
Details on test animals or tissues and environmental conditions:
not applicable
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
0.1 mL
Duration of treatment / exposure:
The test material was applied as evenly as possible to the surface of the cornea. After ten seconds the test material was washed off the cornea using a minimum of 20 mL of saline solution (approximately 32 °C).
Observation period (in vivo):
Assessment of corneal cloudiness was made pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment
Number of animals or in vitro replicates:
None, in vitro test. Three eyes were treated with test material, two additional eyes remained untreated for control purposes.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): After ten seconds the test material was washed off the cornea using a minimum of 20 mL of saline solution (approximately 32 °C). Immediately following washing of the corneal surface, the treated eye was returned to the superfusion chamber and the saline drip repositioned to irrigate the eye. The untreated eyes were similarly washed and used for control purposes.
- Time after start of exposure: 10 seconds

SCORING SYSTEM:
Method for Evaluation of Ocular Irritation by Slit-Lamp Biomicroscopic Examination - McDonald - Shadduck Score System:

CORNEA
The scoring scheme measures the severity of corneal cloudiness and the area of the cornea involved. Severity of corneal cloudiness is graded as follows:
0 = Normal cornea. Appears with the slit-lamp as having a bright grey line on the epithelial surface and a bright grey appearance of the stroma.
1 = Some loss of transparency. Only the anterior half of the stroma is involved as observed with an optical section of the slit-lamp. The underlying
structures are clearly visible with diffuse illumination, although some cloudiness can be readily apparent with diffuse illumination.
2 = Moderate loss of transparency. In addition to involving the anterior stroma, the cloudiness extends all the way to the endothelium. The stroma has lost its marble-like appearance and is homogeneously white. With diffuse illumination, underlying structures are clearly visible.
3 = Involvement of the entire thickness of the stroma. With optical section, the endothelial surface is still visible. However, with diffuse illumination the underlying structures are just visible.
4 = Involvement of the entire thickness of the stroma. With the optical section cannot clearly visualise the endothelium. With diffuse illumination, the underlying structures cannot be seen.

The surface of the cornea relative to the area of cloudiness is divided into five grades from 0 to 4.
0 = Normal cornea with no area of cloudiness
1 = 1 to 25 % area of stromal cloudiness
2 = 26 to 50 % area of stromal cloudiness
3 = 51 to 75 % area of stromal cloudiness
4 = 76 to 100 % area of stromal cloudiness

FLUORESCEIN
The use of fluorescein is a valuable aid in defining epithelial damage for fluorescein staining. The area can be judged as a 0 to 4 scale using the same terminology as for corneal cloudiness. The intensity of fluorescein staining can be divided into a 0 to 4 scale.
0 = Absence of fluorescein staining
1 = Slight fluorescein staining confined to a small focus. With diffuse illumination the underlying structures are clearly visible, although there is some loss of detail.
2 = Moderate fluorescein staining confined to a small focus. With diffuse illumination the underlying structures are clearly visible, although there is some loss of detail.
3 = Marked fluorescein staining. Staining may involve a larger portion of the cornea. With diffuse illumination underlying structures are barely visible but are not completely obliterated.
4 = Extreme fluorescein staining. With diffuse illumination the underlying structures cannot be seen.

REFERENCE:
Hackett R B and McDonald T O, Eye Irritation. In: Advances in Modern Toxicology: Dermatoxicology. 4th ed. (F Marzulli and H Maibach, eds) Hemisphere Publishing Corporation, Washington DC, 1991, pp 749 815.


TOOL USED TO ASSESS SCORE:
Examination of the eye was facilitated by use of a slit-lamp biomicroscope. The thickness of the cornea was measured using an ultrasonic pachymeter. For each enucleated eye a measurement was made at the optical centre, and at a further four locations at the apex of the cornea. A mean value for corneal thickness was then calculated. Measurements for corneal thickness were carried out pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment.
The condition of the corneal epithelium was assessed approximately 60, 120, 180 and 240 minutes following treatment. Assessment was facilitated by the use of the slit-lamp biomicroscope.
The uptake of fluorescein by the corneal epithelium was assessed pre-enucleation, post equilibration and approximately 240 minutes following treatment, according to the numerical evaluation. This was carried out using the cobalt blue filter of the slit lamp biomicroscope, following application of Fluorescein Sodium drops.
Irritation parameter:
cornea opacity score
Run / experiment:
Mean 240 minutes
Value:
>= 8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Irritation parameter:
fluorescein retention score
Run / experiment:
Mean 240 minutes
Value:
>= 4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Irritation parameter:
percent corneal swelling
Run / experiment:
Mean 60 minutes
Value:
>= 28.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Irritation parameter:
percent corneal swelling
Run / experiment:
Mean 120 minutes
Value:
>= 59.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Irritation parameter:
percent corneal swelling
Run / experiment:
Mean 240 minutes
Value:
>= 166.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Other effects / acceptance of results:
Corneal Opacity:
Individual scores for corneal opacity are given in Table 1.
Some loss of transparency was noted in all test eyes 60 and 120 minutes following treatment with moderate loss of transparency 180 and 240 minutes following treatment.
No corneal effects were noted in the control eyes during the study period.

Corneal Thickness:
Individual and mean corneal thickness measurements and corneal swelling calculations are given in Table 2 and Table 3.
Corneal swelling of the test eyes during the study period was considerably greater than that observed in the control eyes over the same period.

Corneal Condition:
The condition of the corneal epithelium following treatment is given in Table 4.
Pitting of the corneal epithelium was noted in all test eyes 60 and 120 minutes following treatment with sloughing of the corneal epithelium 180 and 240 minutes following treatment.
The condition of the corneal epithelium of the control eyes appeared normal during the study period.

Fluorescein Uptake:
Individual scores for fluorescein uptake are given in Table 5.
Slight fluorescein uptake was noted in the test eyes 240 minutes following test material application. No fluorescein uptake was noted in the control eyes 240 minutes following treatment.

Interpretation of Results:

The data for all endpoints was assessed and an estimate of the test material ocular irritancy potential was made based on the following cut-off values:

REET Parameter*

REET Cut‑Off Value

Maximum Corneal Opacity (Corneal Cloudiness x Area)

> or = 4

Maximum Fluorescein Uptake (Intensity x Area)

> or = 4

Mean Corneal Swelling (mins): 60, 120, 240

> or = 25 %

Corneal Epithelium Observations

Any with pitting, mottling or sloughing

Endpoints included corneal opacity, condition of the corneal epithelium, fluorescein uptake (240 minutes following treatment) and the percentage change in corneal thickness (corneal swelling). For each test and control eye, the percentage change in corneal thickness following treatment (60, 120, 180 and 240 minutes) was calculated based upon the pre‑treatment value as follows:

((mean corneal thickness post-treatment) – (mean corneal thickness post equilibration) /(mean corneal thickness post equilibration)) x 100

- A mean value for corneal swelling was then calculated for the test and control eyes for the 60, 120 and 240 Minute post treatment observation periods.

- A negative ocular irritancy potential may require further investigation using an in vivo ocular irritation study.


*= Any parameter that meets or exceeds the cut-off values indicates a severe eye irritant

Maximal ocular irritation observations recorded for the test eyes were as follows:

Corneal Opacity

Fluorescein Uptake

Corneal Swelling (%)

Condition of Corneal Epithelium

Test Eyesa

Control Eyesb

Cldy x Area

Int x Area

60mins

120 mins

240 mins

60 mins

120 mins

240 mins

8+

4+

28.5+

59.3+

166.2+

3.1

3.6

3.9

pitting/sloughing+

Interpretation of results:
other: EU Criteria:
Conclusions:
Following assessment of the data for all endpoints, the test material was considered to have the potential to cause severe ocular irritancy in vivo.
Executive summary:

A study was performed to assess the ocular irritancy potential of the test material in the rabbit following application onto the cornea of the enucleated eye. The results of the study are believed to be of value in predicting the ocular irritation potential of the test material in man. 

0.1 mL of the test material was applied onto the cornea of each of three enucleated eyes which had been maintained at a temperature of 32 ± 1.5 °C within the superfusion chamber. A further two enucleated eyes were treated, for control purposes, with saline solution (0.9 % Sodium Chloride).

Following assessment of the data for all endpoints, the test material was considered to have the potential to cause severe ocular irritancy in vivo.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin:

In the key study (Sachsse, 1973), the purpose of the test was to determine the primary-irritation index, which serves as a measure of the acute irritation, provoked by the compound on the skin of the rabbits.

The average oedema score at 24 hours for intact skin is 2.67. The average oedema score at 72 hours is 2.33. As the mean score at both of these time points is greater than 2, the substance can be said to be irritating to skin. The primary Irritation Index of the test material was 1.7. The reliability rating for this study is 2, according to the criteria of Klimisch, 1997 as the study was conducted according to methods comparable to OECD 404 wtih the following deviations:

- Shaved 2 days before testing, normally 24 hours.

- Test material applied for 24 hours. Normally only applied for 4 hours.

- Observation period only 7 days, should be 14 days.

This study is considered the most relevant and reliable study for this endpoint, although technically the results are unable to be used for classification purposes as readings were only taken at 24 and 72 hours and also the test material was left on longer than specified in the guideline. However the mean oedema scores at 24 and 72 hours are above the EU C&L cut-off criteria according to Regulation (EC) no 1272/2008 and so it is highly likely that the 48 hours reading would also be above this cut-off value, and so this study would be considered to be the worst case scenario used to classify as irritating to the skin, therefore this study will be used for classification purposes for this endpoint.

- Supporting information is available from a test material safety data sheet, Atofina Chemicals Inc. (2001). A reliability rating of 4 was assigned to this study, according to the criteria of Klimisch, 1997 as this information is from a secondary source. The test material was severely irritating to rabbits.

- In the Lawrence et al (1996) paper, the PDII for the test material on rabbit skin is 2.99, this is considered to be moderately irritating. A reliability rating of 4 was assigned to this paper, according to the criteria of Klimisch, 1997 as it was secondary source information, the mean value was calculated including observations at 1 hr to 72 hrs (instead of 24, 48 & 72 hrs), no individual values are given and it is considered unsuitable for classification purposes.

 

Eye:

- In the key study, Sanders (2010), in vitro study, a study was performed to assess the ocular irritancy potential of the test material in the rabbit following application onto the cornea of the enucleated eye. The results of the study are believed to be of value in predicting the ocular irritation potential of the test material in man. The reliability rating for this study is 1, according to the criteria of Klimisch, 1997 as the test was performed to a validated model, for eye irritation and the study was performed in compliance with GLP, with a good level of detail in the reporting of the methodology and the results.

The following supporting studies corroberate with the key study:

- In the Warren, 2010 in vitro study, the eye irritation potential of the test material was assessed in the in vitro test system using the SkinEthic HCE Model (reconstituted human corneal epithelium). The test material was found to be irritating under the conditions of the test. The reliability rating for this study is 2, according to the criteria of Klimisch, 1997 as the test was performed to a pre-validated model, for eye irritation and the study was performed in compliance with GLP, with a good level of detail in the reporting of the methodology and the results.

- In the Sachsse, 1973 study, the test material was tested on the eyes of 6 rabbits (3 males/3 females) of the New Zealand White strain. The primary-irritation Index, which serves as a measure of the acute irritation provoked by the test material, was found to be

48 for the cornea, 8 for the iris and 19.8 for the conjunctivae. The test material is to be considered as a severe irritant to the eye of rabbits. The reliability rating for this study is 2, according to the criteria of Klimisch, 1997 as the study was conducted according to methods comparable to OECD 405 wtih the following deviations:

- In the test the eye was left open and not closed as it should be.

- Eyes were rinsed straight away and not left 24 hours.

- Observations were only done up to 7 days, not 21 as advised.

The results are unable to be used for EU classification purposes as the conjunctivae scores (redness and chemosis) were combined.

- Supporting information is available from a test material material safety data sheet, Atofina Chemicals Inc. (2001). A reliability rating of 4 was assigned to this study, according to the criteria of Klimisch, 1997 as this information is from a secondary source.

The test material was severely irritating to rabbits.

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: irritating

Justification for classification or non-classification

Skin:

Based on the available data for skin irritation this sustance is considered to be irritating to skin and would be classified as 'H315: Cause skin irritation, according to Regulation (EC) No 1272/2008.

Eyes:

Following assessment of the in vitro data for all endpoints, the test material was considered to have the potential to cause severe ocular irritancy in vivo.

The data indicates that the substance may be considered severely irritating to the eyes and so it will therefore be classified as a Category 1 irritant and so will have the hazard statement H318: causes serious eye damage and the 'Danger' symbol.