Piperidine, 4-(4-iodo-1H-pyrazol-1-yl)-

Endpoint summary

Description of key information

Skin irritation/corrosion
Two studies are available:
1)  A skin irritation study in rabbit (Beerens - Heijnen, 2010) is available which is key study. This study showed that the test substance is not irritating.
2)  A in vitro skin corrosion study using a human skin model (Buskens, 2010) is available which is supporting study. This study showed that the test substance is not corrosive.
Eye irritation:
A eye irritation with an isolated bovine (Verspeek-Rip, 2010)  is available which is key study. It is concluded that the test substance is corrosive or severe irritant.

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 24 Aug to 02 Sep 2010

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: Charles River France, L'Arbresle Cedex, France
- Age at study initiation: between 10-20 weeks old
- Weight at study initiation: 2-4 kg
- Housing: housed in labeled cages with perforated floors and shelters
- Diet (e.g. ad libitum): Pelleted diet 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 ± 3.0ºC (actual range: 19.0-19.9ºC)
- Humidity (%): 40-70% (actual range: 49 - 87%)
- 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

IN-LIFE DATES: From: 2010-08-24 To: 2010-09-02

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

water

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 grams
- Concentration (if solution):

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.5 mL
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

3 males

Details on study design:

TEST SITE
- Area of exposure: approximately 150 square centimeters (10x15 cm)
- % coverage:
- Type of wrap if used: Micropore tape

REMOVAL OF TEST SUBSTANCE
- Washing (if done): tap water
- Time after start of exposure: 4 hours

SCORING SYSTEM: numerical scoring system

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:

No skin irritation was caused by 4 hours exposure to the test substance.
There was no evidence of a corrosive effect on the skin.

Other effects:

No staining of the treated skin by the test substance was observed and no test substance remnants were seen.
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Individual skin irritation scores

Animal	472		487		488
Time after exposure	Erythema (0-4)	Oedema (0-4)	Erythema (0-4)	Oedema (0-4)	Erythema (0-4)	Oedema (0-4)
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

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

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

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:

From 12 to 13 Jul 2010

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:

other: OECD Guideline 437: "Bovine comeal opacity and permeablility (BCOP) test method for identifying ocular corrosives and severe irritants" (adopted September 07, 2009)

Deviations:

no

GLP compliance:

yes

Species:

other: cattle

Strain:

other: an isolated bovine cornea

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse, where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Age at study initiation:
- Weight at study initiation:
- Housing:
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period: minimium of 1 hour

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 301.8 to 302.4 mg

Duration of treatment / exposure:

240±10 minutes

Observation period (in vivo):

Immediate opacity measurement and permeability evaluation of the cornea

Number of animals or in vitro replicates:

Three corneas for each treatment group (total 9 corneas)

Details on study design:

Negative control: Physiological saline
Positive control: 20% (w/v) Imidazole in physiological saline

Treatment of corneas and opacity measurements:
The medium from the anterior compartment was removed and 750 l of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. Three corneas were covered with 301.8 to 302.4 mg the test substance. The holder was slightly rotated, with the cornea maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240±10 minutes at 32±1℃. After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red. Possible pH effects of the test substance on the corneas were recorded. The anterior and the posterior compartment were refilled with fresh cMEM and an opacity determination was performed without any further incubation. After the completion of the incubation period each cornea were inspected visually for dissimilar opacity patterns and the opacity determination was performed.

Opacity measurement:
The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post treatment reading. The corrected opacity for each positive control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test substance treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

Application of sodium fluorescein:
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck) was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 5 mg Na-fluorescein/ml cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90±5 minutes at 32±1℃.

Permeability determinations:
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 l of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader. Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

Irritation parameter:

other: In vitro irritancy score

Basis:

mean

Time point:

other: 240±10 minutes

Score:

144

Irritation parameter:

other: Opacity score

Basis:

mean

Time point:

other: 240±10 minutes

Score:

41

Irritation parameter:

other: Permeability score

Basis:

mean

Time point:

other: 240±10 minutes

Score:

6.866

Summary of opacity, permeability and in vitro scores

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation^1,2
Negative control	0	0.000	0
Positive control	75	2.236	109
The test substance	41	6.866	144

1 Calculated using the negative control mean opacity and mean permeability values.

2 In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490value).

Interpretation of results:

Category 1 (irreversible effects on the eye)

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

It is concluded that the test substance is corrosive or severe irritant in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Two studies are available:

1)A skin irritation study was conducted according to OECD 404 using rabbits (Beerens - Heijnen, 2010). Key study.

This study showed that the test substance is not irritating.

2) A in vitro skin corrosion study was studied according to OECD 431 using a human skin model (Buskens, 2010). 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 resulted 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:

One eye irritation with an isolated bovine cornea (Verspeek-Rip, 2010) was carried out according to OECD 437. This study is considered as reliable and key study. It is concluded that the test substance is corrosive or severe irritant.

Justification for selection of skin irritation / corrosion endpoint:
Study run to a method comparable with current guidelines and to GLP

Justification for selection of eye irritation endpoint:
Study run to a method comparable with current guidelines and to GLP

Effects on eye irritation: corrosive

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). In addition all reactions were fully reversible within the observation period (actual value within 72hours).

Serious eye damage/eye irritation: An eye irritation study with an isolated bovine cornea showed that the test substance is corrosive or severe irritant. The test substance induced an IVIS ≥ 55.1.

Therefore in accordance with Regulation (EC) No. 1272/2008 Tables 3.2.2 & 3.3.1 the substance is not classified for the skin corrosion/irritation and classified for Category 1 (irreversible effects on the eye) for the serious eye damage/eye irritation endpoint.