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Description of key information

The test substance was found to be irritating to both skin and eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation / corrosion
Remarks:
in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-documented experiment according to GLP and EC and OECD guidelines.
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Species: New Zealand White, SPF-quality
- Source: Broekman Institute, Someren, The Netherlands
- Age at study initiation: approx. 15 weeks
- Weight at study initiation: 2489-2733 g
- Housing: individually, cages with perforated floors and equiped with automated drinking system
- Diet (e.g. ad libitum): Standard laboratory diet (LKK-20, pellet diameter 4mm, Hope Farms, Woerden, The Netherlands); approx. 100 g/day
- Water (e.g. ad libitum): Tap water diluted with decalcified water, ad libitum
- Acclimation period: at least 5 days before start of treatment under test conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 55%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (artificial fluorescent light)
Fluctuations from these optimal conditions were noted, but were considered to not have affected the study integrity.

Type of coverage:
semiocclusive
Preparation of test site:
shaved
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.5 mL
Duration of treatment / exposure:
4 hours
Observation period:
14 days
Number of animals:
3 (all females)
Details on study design:
Approx. 24 hours before treatment, the dorsal fur was shaved with electric clippers, exposing an area of approx. 100 square centimeters (10 cm x 10 cm).

Prior to test substance administration, the animals were physically examined and the shaved area of skin to be treated was normal.

On test day 1, 0.5 mL of the test substance was applied to the intact skin of the shaved area on one flank, using a surgical gauze patch 2 x 3 cm mounted on Micropore tape (3M, St. Paul, USA). The contralateral flank was similarly prepared, but without test substance, to act as a procedural control. The dressing was wrapped around the abdomen and secured with an elastic bandage (Corban, 3M, St. Paul, USA).

Four hours after the application, the dressing was removed and the remaining test substance removed using a tissue moistened with tap water and subsequently a dry tissue. However, the treated skin area remained sticky after removal of the test substance.

In order to facilitate the scoring, the skin area concerned of all animals was re-shaved before the observation on days 2, 3, 4, 7 and 14.
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Time point:
other: 24-48-72h
Score:
4.2
Max. score:
8
Reversibility:
fully reversible within: 14d
Irritant / corrosive response data:
There was no evidence of a corrosive effect on the skin.
No staining of the treated skin by the test substance was observed.
Other effects:
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.
Interpretation of results:
moderately irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The observed irritation resulted in a PPDI of 4.2 when applied to the intact rabbit skin. When compared to the EEC criteria for classification and labelling for dangerous susbtances (DPD), the test substance should be classified as a skin irritant (moderately irritating).
Executive summary:

The study assesses the possible irritation or corrosion potential of the test substance in the rabbit according to OECD and EEC guidelines. The test substance was applied onto shaved skin of 3 female albino rabbits using semi-occlusive dressing for 4 hours, followed by 6 observations at approx. 1, 24, 48 and 72 hours, and 7 and 14 days after removal of the dressings and remaining test substance.

 

Under the conditions of the test, the test substance resulted in well-defined erythema and slight or moderate oedema. The skin irritation was reversed within 14 days after exposure in all 3 animals. No corrosive effect occurred on the skin in all 3 rabbits. In the area of application, no staining of the treated skin by the test substance was observed. No signs of systemic intoxication were observed during the study period.

 

The test substance resulted in a primary irritation index of 4.2 (moderately irritating) when applied to intact rabbit skin.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-documented experiment according to GLP and EC and OECD guidelines.
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Species: New Zealand White, SPF-quality
- Source: Broekman Institute, Someren, The Netherlands
- Age at study initiation: approx. 15 weeks
- Weight at study initiation: 2489-2733 g
- Housing: individually, cages with perforated floors and equiped with automated drinking system
- Diet (e.g. ad libitum): Standard laboratory diet (LKK-20, pellet diameter 4mm, Hope Farms, Woerden, The Netherlands); approx. 100 g/day
- Water (e.g. ad libitum): Tap water diluted with decalcified water, ad libitum
- Acclimation period: at least 5 days before start of treatment under test conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 55%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (artificial fluorescent light)
Fluctuations from these optimal conditions were noted, but were considered to not have affected the study integrity.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
0.1 mL
Duration of treatment / exposure:
one single treatment
Observation period (in vivo):
22 days
Number of animals or in vitro replicates:
3
Details on study design:
STUDY DESIGN:
On day 1, prior to test substance administration, the animals were physically examined and the eyes were found to be normal. The test substance (0.1 mL per animal) was instilled in the conjunctival sac of one eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test substance. The other eye remained untreated and served as the reference control.
Immediately after the 24 hour observation, a solution of 2% fluorescein in water (adjusted to pH 7) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. This procedure was repeated in all three animals on days 4, 8 and 15, and in two of three animals also on day 22. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area.

OBSERVATIONS:
- Viability / mortality / toxicity: daily
- Body weight: day 1 of test
- Irritation: 1h, 24h, 48h and 72 h and 7d, 14d and 21d after instillation of the test substance
Irritation parameter:
cornea opacity score
Remarks:
opacity
Basis:
mean
Time point:
other: mean over 24-48-72h
Score:
1.44
Max. score:
4
Reversibility:
not fully reversible within: 21d
Irritation parameter:
iris score
Basis:
mean
Time point:
other: mean over 24-48-72h
Score:
0.3
Max. score:
2
Reversibility:
fully reversible within: 48h
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
other: mean over 24-48-72h
Score:
3
Max. score:
3
Reversibility:
not reversible
Irritation parameter:
chemosis score
Basis:
mean
Time point:
other: mean over 24-48-72h
Score:
1.7
Max. score:
4
Reversibility:
fully reversible within: 7d

Draize score calculation

#

Tissue

1h

24h

48h

72h

7d

14d

21d

673

Cornea

0

15

20

20

10

5

5

Iris

5

5

0

0

0

0

0

Conjunct.

10

14

14

10

2

0

0

Subtotal

15

34

34

30

12

5

5

674

Cornea

0

20

10

10

20

0

0

Iris

5

5

0

0

0

0

0

Conjunct.

10

16

16

12

4

2

0

Subtotal

15

41

26

22

24

2

0

675

Cornea

0

20

10

10

15

20

10

Iris

5

5

0

0

0

0

0

Conjunct.

12

16

14

12

2

2

2

Subtotal

17

41

24

22

17

22

12

Calculations for EEC Classification

#

Time

Corneal Opacity

Iris Lesion

Conjunctival

Redness

Conjunctival

Chemosis

673

24h

1

1

3

2

48h

1

0

3

2

72h

1

0

3

1

Mean

1.0

0.3

3.0

1.7

674

24h

1

1

3

2

48h

2

0

3

2

72h

2

0

3

1

Mean

1.7

0.3

3.0

1.7

675

24h

1

1

3

2

48h

2

0

3

2

72h

2

0

3

1

Mean

1.7

0.3

3.0

1.7

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the test, the test substance was found to be an eye irritant.
Executive summary:

The study assesses the possible irritation or corrosion potential of the test substance to the rabbit eye according to OECD and EEC guidelines. A single dose of the test substance (0.1 mL) was instilled into one eye of each of 3 female albino rabbits, followed by 7 observations at approx. 1, 24, 48 and 72 hours, and 7, 14 and 21 days after test substance administration.

Under the conditions of the test, the test substance resulted in adverse effects on the cornea, iris and the conjunctivae in all 3 animals. The opacity of the cornea was reversed within 14 days in one animal and was not reversed within the study period in the other two animals. The injection of the iris was reversed within 48 hours in all three animals. The irritation of the conjunctivae was reversed within 14 days in one animal and within 21 days in a second animal, but was not reversed within the study period in the third animal. Ocular corrosion was not observed in any of the three animals. No signs of systemic intoxication were observed during the study period.

 

In conclusion, according to the EEC criteria for classification and labelling requirements for dangerous substances (67/548/EEC), the test substance is to be classified as an eye irritant.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

A total of 3 studies are available that assess the possible irritation or corrosion potential of the test substance. All 3 studies were performed according to GLP and internationally accepted guidelines.

 

In the key study (Pels Rijcke, 1991) the test substance was applied onto the shaved skin of 3 female albino rabbits using semi-occlusive dressing for 4 hours, followed by 6 observations at approx. 1, 24, 48 and 72 hours, and 7 and 14 days after removal of the dressings and remaining test substance. Under the conditions of the test, the test substance resulted in well-defined erythema and slight or moderate oedema. The skin irritation was reversed within 14 days after exposure in all 3 animals. No corrosive effect occurred on the skin in all 3 rabbits.

The test substance resulted in a primary irritation index of 4.2 (moderately irritating) when applied to intact rabbit skin. The mean erythema and oedema scores were:

 

 

Rabbit n° 658

Rabbit n° 660

Rabbit n° 653

Time

Erythema

Oedema

Erythema

Oedema

Erythema

Oedema

24h

2

2

1

2

2

2

48h

2

2

2

3

2

3

72h

2

2

2

3

2

3

Mean

2

2

1.7

2.7

2

2.7

 

As the mean oedema score is 2.3 in 2 of the 3 tested animals, the substance is to be classified as a Skin irritant Cat. 2 according to the criteria described in EU Regulation No. 1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures (CLP).

Furthermore, in a supporting study (Skydsgaard, 1991) 5 different concentrations ranging from 100% to 1% of test substance in ethanol:DEP 1:4 were tested. The results of this experiment demonstrate that, under the experimental conditions of the test, the test substance is to be classified as a skin irritant in its pure form, whereas solutions of the test substance in concentrations of 25% or less shall not be classified.

 

The results obtained in a third study report (Dyring Jacobsen, 1992) are disregarded for the purpose of the hazard assessment and classification, as in this test report insufficient information is available on the composition and concentration of the tested material.

Eye irritation

Three studies are available that assess the possible irritation or corrosion potential of the test substance to the eye. Two of those studies were performed according to GLP and internationally accepted guidelines.

 

In the key study (Pels Rijcke, 1991) the test substance was instilled into one eye of each of 3 rabbits followed by 7 observations at approx. 1, 24, 48 and 72 hours, and 7, 14 and 21 days. Under the conditions of the test, the test substance resulted in adverse effects on the cornea, iris and conjunctivae in all 3 animals.The opacity of the cornea was reversed within 14 days in one animal and was not reversed within the study period in the other two animals. The injection of the iris was reversed within 48 hours in all three animals. The irritation of the conjunctivae was reversed within 14 days in one animal and within 21 days in a second animal, but was not reversed within the study period in the third animal. Ocular corrosion was not observed in any of the three animals. No signs of systemic intoxication were observed during the study period.

As the observed effects regarding the opacity of the cornea and the irritation of the conjunctivae were not reversed during the 21 day observation period, the substance is to be classified as having Irreversible effects on the eye (Cat. 1) according to the criteria described in EU Regulation No. 1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures (CLP).

In a first supporting study (Skydsgaard, 1991), a single dose of 0.1 mL of the test substance resulted in the following mean values: corneal opacity (1.0), iris lesions (0.0), chemosis (0.0) and conjunctival redness (1.5). Based on these results classification. Comparing these results with the criteria described in the CLP regulation would classify the test substance as Irritating to eyes (Cat. 1) based on the corneal opacity value of 1.0.

The second supporting study (Lüpke, 1994) is an in vitro study (no OECD guideline available yet) in which the test substance is administered to the chorio-allantoic membrane of a fertile chicken egg (HET-CAM test). The test substance exerted clear effects of vascular injection, hemorrhage, lysis and coagulation when administered to the CAM in concentrations of 50% and 25%. At a concentration of 10%, only weak reactions were observed.

As the three available study reports result in different conclusions regarding the classification and labelling, the classification for Irreversible effects on the eye (Cat. 1) is retained based on the precautionary principle.


Justification for selection of skin irritation / corrosion endpoint:
Well documented study according to GLP and internationally accepted guidelines.

Justification for selection of eye irritation endpoint:
Well documented study according to GLP and internationally accepted guidelines.

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: irritating

Justification for classification or non-classification

Skin irritation

As the mean oedema score is ≥ 2.3 in 2 of the 3 tested animals, the substance is to be classified as a Skin irritant Cat. 2 according to the criteria described in EU Regulation No. 1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures (CLP).

Likewise, the observed effects require classification as a skin irritant (R38) according to the criteria described in section 3.2.6.1 of Annex VI to Directive 67/548/EEC (Dangerous Substances Directive).

 

Eye irritation

Based on the irreversibility of the effects observed in the key study, the substance is to be classified as having Irreversible effects on the eye (Cat. 1) according to the criteria described in EU Regulation No. 1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures (CLP).

Likewise, the observed effects require classification as a substance having risk of serious damage to the eyes (R41) according to the criteria described in section 3.2.6.2 of Annex VI to Directive 67/548/EEC (Dangerous Substances Directive).

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