6,10-Dioxaspiro[4.5]decane-7-acetic acid, 9-[2-[2-(4-fluorophenyl)-5-(1-methylethyl)-3-phenyl-4-[(phenylamino)carbonyl]-1H-pyrrol-1-yl]ethyl]-, 1,1-dimethylethyl ester, (7R,9R)-

Endpoint summary

Description of key information

Skin irritation/corrosion
Two studies are available:
1) A primary skin irritation/corrosion study in rabbit (van den Bogaard, 2009) is available which is key study. This study showed that the test substance is not irritating.
2) An in vitro skin corrosion study using a human skin model (Buskens, 2009) is available which is supporting study. This study showed that the test substance is not corrosive.
Eye irritation/corrosion:
Two studies are available:
1) An acute eye irritation/corrosion study in rabbit (van den Bogaard, 2009) is available which is key study. This study showed that the test substance is not irritating.
2) An screening for the eye irritancy potential study with chicken eggs (Verspeek-Rip, 2009) is available which is supporting study. It is concluded that the test substance is not irritating.

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:

From 27 January to 06 February 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study run to a method comparable with current guidelines and to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan France SARL, Gannat, France
- Age at study initiation: 10-20 weeks old
- Weight at study initiation: 2-4 kg
- Housing: Animals were individually housed in labeld cages with perforated floors and shelters.
- Diet (e.g. ad libitum): Pelleted diet for rabbits approximately 100 grams per day. Hay was provided at least three times a week.
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0 ℃
- Humidity (%): 30-70% (actural range: 42-71%)
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artifical fluorescent light and 12 hours darkness per day

IN-LIFE DATES: From: To:

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

other: watery ethanol

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g

VEHICLE
- Amount(s) applied (volume or weight with unit): 1 mL

No additional data

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

3 males

Details on study design:

TEST SITE
- Area of exposure: 150 square centimeters (10×15 cm)
- Type of wrap if used: The patch was mounted on Micropore tape, which was wrapped around the abdomen and secured with Coban elastic bandage.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The skin cleaned of residual test substance using tap water.
- Time after start of exposure: four hours

No additional data

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: mean of 24, 48 and 72 hours

Score:

0

Max. score:

0

Remarks on result:

other: The exposure period is 4 hours.

Irritation parameter:

edema score

Basis:

mean

Time point:

other: mean of 24, 48 and 72 hours

Score:

0

Max. score:

0

Remarks on result:

other: The exposure period is 4 hours.

Irritant / corrosive response data:

Irritation
No skin irritation was caused by 4 hours exposure to the test substance.

Corrosion
There was no evidence of a corrosive effect on the skin.

Other effects:

Coloration / Remnants
No staining of the treated skin by the test substance was observed and no test substance remnants were seen.

Toxicity / Mortality
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Individual skin irritation scores

Animal	124^1			123			125
Time after exposure	Erythema	Oedema	comments	Erythema	Oedema	comments	Erythema	Oedema	comments
1 hour	0	0	-	0	0	-	0	0	-
24 hours	0	0	-	0	0	-	0	0	-
48 hours	0	0	-	0	0	-	0	0	-
72 hours	0	0	-	0	0	-	0	0	-

¹Sentinel.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Treatment with the test substance caused no skin irritation in three (3/3) rabbits.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 02 to 09 February 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study run to a method comparable with current guidelines and to GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute 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
- Source: Harlan France SARL, Gannat, France
- Age at study initiation: 10-20 weeks old
- Weight at study initiation: 2-4 kg
- Housing: Animals were individually housed in labeled cages with perforated floors and shelters.
- Diet (e.g. ad libitum): Pelleted diet for rabbits was approximately 100 grams per day. Hay was provided at least three times a week.
- Water (e.g. ad libitum): Free access to tap water
- Acclimation period: At least 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0 ℃ (actual range: 21.1-22.7 ℃)
- Humidity (%): 30-70% (actual range: 42-65%)
- Air changes (per hr): Approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day

No additional data

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): on average 37.0 mg (range 36.9-37.0 mg) of the test substance (a volume of approximately 0.1 mL)

No additional data

Duration of treatment / exposure:

72 h

Observation period (in vivo):

72 h

Number of animals or in vitro replicates:

3 males

Details on study design:

The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner 1 week later, after considering the degree of eye irritation observed in the first animal.

Animals were treated by instillation of the test substance in the conjunctival sac of one of the eyes 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 24-hour observation, a solution of 2% fluorescein in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage.

After the final observation, the animals were sacrificed by intra-venous injection of Euthasol® 20%.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: Mean of 24, 48 and 72 hours

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

mean

Time point:

other: mean of 24, 48 and 72 hours

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

other: Mean of 24, 48 and 72 hours

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Remarks on result:

other: Redness were observed at 1 hour and 24 hour in all of three animals.

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: Mean of 24, 48 and 72 hours

Score:

0

Max. score:

1

Reversibility:

fully reversible within: 24 hours

Remarks on result:

other: Chemosis were observed at 1 hour in all of three animals.

Irritant / corrosive response data:

Irritation
Instillation of approximately 37 mg of the test substance into one eye of each of three rabbits resulted in irritation of the conjunctivae, which consisted of redness, chemosis and discharge. The irritation completely resolved within 48 hours.
No iridial irritation or corneal opacity was observed, and treatment of the eyes with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage.

Corrosion
There was no evidence of ocular corrosion.

Other effects:

Coloration/Remnants
No staining of (peri) ocular tissues by the test substance was observed. Remnants of the test substance were present in the eye on day 1.

Toxicity/Mortality
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Individual eye irritation scores

Animal	Time after dosing	Cornea			Iris	Conjunctivae
		Opacity (0-4)	Area (0-4)	Fluor area (%)*	(0-2)	Redness (0-3)	Chemosis (0-4)	Discharge (0-3)
121#	1 h	0	0	0	0	1	1	1
	24 h	0	0		0	1	0	0
	48 h	0	0		0	0	0	0
	72 h	0	0		0	0	0	0
116	1 h	0	0	0	0	1	1	1
	24 h	0	0		0	1	0	0
	48 h	0	0		0	0	0	0
	72 h	0	0		0	0	0	0
128	1 h	0	0	0	0	1	1	1
	24 h	0	0		0	1	0	0
	48 h	0	0		0	0	0	0
	72 h	0	0		0	0	0	0

# Sentinel

* Green staining after fluorescein treatment (percentage of total corneal area)

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Treatment with the test substance caused reversible effects on the conjunctivae of three (3/3) rabbits.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation/corrosion

Two studies are available:

1) A primary skin irritation/corrosion study was conducted according to OECD 404 using rabbits (van den Bogaard, 2009). Key study.

This study showed that the test substance is not irritating.

2) An in vitro skin corrosion study was conducted according to OECD 431 using a human skin model (Buskens, 2009). Supporting study.

In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, a human skin model test was performed. This study showed that the test substance is not corrosive. Based on this, in vivo skin irritation study in rabbit was performed to establish the possible skin irritating properties of the test substance.

Eye irritation

Two studies are available:

1) An acute eye irritation/corrosion study was conducted according to OECD 405 using rabbits (van den Bogaard, 2009). Key study.

This study showed that the test substance is not irritating.

2) An screening for the eye irritancy potential study was conducted according to The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Method (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD) using chicken eggs (Verspeek-Rip, 2009). Supporting study.

In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, an hen's egg test-chorioallantoic membrane test (HET-CAM test) was performed. This study showed that the test substance is not irritating. Based on this, in vivo eye irritation study in rabbit was performed to establish the possible eye irritating properties of the test substance.

Justification for selection of skin irritation / corrosion endpoint:
This study was conducted according to OECD 404 under GLP.

Justification for selection of eye irritation endpoint:
This study was conducted according to OECD 405 under GLP.

Justification for classification or non-classification

Skin irritation/corrosion: Mean scores at 24, 48 & 72 hours for erythema were < 2.3 (actual value 0) for oedema were < 2.3 (actual value 0).

Serious eye damage/eye irritation: Mean scores at 24, 48 & 72 hours for corneal opacity were < 1 (actual value 0), for iritis were < 1 (actual value 0), for conjunctival redness were < 2 (actual value 0.3) and for conjunctival oedema were < 1 (actual value 0).

Therefore in accordance with Regulation (EC) No. 1272/2008 Tables 3.2.2 & 3.3.2 the substance is not classified for the skin corrosion/irritation and serious eye damage/eye irritation endpoint.