Registration Dossier

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

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:

adverse effect observed (irritating)

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:

adverse effect observed (irritating)

Endpoint conclusion:

no study available

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

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).