Alkyl 4-(6-(6-bromo-8-cycloalkyl-5-alkyl-7-oxo-7,8-dihydropyirdo[2,3-d]pyrimidin-2-ylamino)pyridin-3-yl)piperazine-1-carboxylate

Endpoint summary

Description of key information

Skin irritation/corrosion
Two studies are available:
1) An acute dermal irritation study (Pooles A, 2013) is available which is key study. This study showed that the test substance is not irritating.
2) An in vitro skin corrosion study (Warren N, 2013) is available which is supporting study. This study showed that the test substance is not corrosive.
Eye irritation
Two studies are available:
1) An acute eye irritation study (Pooles A, 2013) is available which is key study. This study showed that the test substance is slightly irritating.
2) An in vitro BCOP study (Warren N, 2013) is available which is supporting study. The test item was considered not to be an ocular 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 23 April to 01 June 2013

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 (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Leicestershire, UK
- Age at study initiation: twelve to twenty weeks old
- Weight at study initiation: 2.38 to 2.58 kg
- Housing: individually housed in suspended cages
- Diet (e.g. ad libitum): Free access to food (2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): at least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light (06:00 to 18:00) and twelve hours darkness

IN-LIFE DATES:

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): 0.5g
- Concentration (if solution):

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

Duration of treatment / exposure:

First animal: 3 minutes, 1 hour and 4 hours
Additional two animals: 4 hours

Observation period:

72 hours

Number of animals:

3 males

Details on study design:

TEST SITE
- Area of exposure: 2.5 cm*2.5 cm
- % coverage: Each patch was secured in position with a strip of surgical adhesive tape.
- Type of wrap if used: To prevent the animal interfering with the patches, the trunk of the rabbit was wrapped in an elasticated corset for the duration of the exposure period.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Any residual test item was removed by gentle swabbing with cotton wool soaked in distilled water.
- Time after start of exposure: 4 hours

SCORING SYSTEM: Draize score 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:

3-Minute and 1-Hour semi-occluded applications of the test item to the intact skin of one rabbit produced no evidence of skin irritation.
A single 4-Hour, semi-occluded application of the test item to the intact skin of three rabbits produced no evidence of skin irritation.

Other effects:

All animals showed expected gain in body weight during the study.

Individual Skin Reactions Following 4-Hour Exposure

Observation period (hours)	Rabbit No.
	73144 Male		73152 Male		73216 Male
	Erythema	Oedema	Erythema	Oedema	Erythema	Oedema
1	0	0	0	0	0	0
24	0	0	0	0	0	0
48	0	0	0	0	0	0
72	0	0	0	0	0	0

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item produced a primary irritation index of 0.0 and was classified as non-irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.
The test item was not classified as an irritant according to GHS and EC CLP 1272/2008.

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 28 May to 13 June 2013

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 (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Leicestershire, UK
- Age at study initiation: twelve to twenty weeks old
- Weight at study initiation: 2.68 to 3.20 kg
- Housing: individually housed in suspended cages
- Diet (e.g. ad libitum): Free access to mains food (2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study.
- Water (e.g. ad libitum): Free access to mains drinking water
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): at least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

IN-LIFE DATES: From To:

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): A volume of 0.1 mL of the test item, which was found to weigh approximately 59 mg
- Concentration (if solution):

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

Duration of treatment / exposure:

8 hours

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3 males

Details on study design:

Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.
Initially, a single rabbit was treated. A subcutaneous injection of buprenorphine 0.01 mg/kg was administered 60 minutes prior to test item application to provide a therapeutic level of systemicanalgesia. Five minutes prior to test item application, a pre-dose anaesthesia of ocular anesthetic (two drops of 0.5% tetracaine hydrochloride) was applied to each eye.
A volume of 0.1 mL of the test item, which was found to weigh approximately 59 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball.The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to the six point scale .
Eight hours after test item application, a subcutaneous injection of post-dose analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg, was administered to provide a continued therapeutic level of systemic analgesia. The treated animal was checked for signs of pain and suffering approximately 12 hours later. No further analgesia was required.
After consideration of the ocular responses produced in the first treated animal, two additional animals were similarly treated.
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation (Draize, J.H, 1977).
Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.
Any clinical signs of toxicity, if present, were also recorded.
Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

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:

1

Reversibility:

fully reversible within: 24 hours

Remarks on result:

other: fully reversible within 24 hours in all three animals

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

other: mean of 24, 48 and 72 hours

Score:

ca. 0.67

Max. score:

2

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: fully reversible within 72 hours in all three animals

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: mean of 24, 48 and 72 hours

Score:

ca. 0.33

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Remarks on result:

other: fully reversible within 48 hours in all three animals

Irritant / corrosive response data:

Yellow colored staining of the fur around the treated eye was noted in all animals.
No corneal effects were noted during the study.
Iridial inflammation was noted in all treated eyes one hour after treatment.
Moderate conjunctival irritation was noted in two treated eyes and minimal conjunctival irritation was noted in one treated eye one hour after treatment. Minimal conjunctival irritation was noted in all treated eyes at the 24 and 48-Hour observations.
All treated eyes appeared normal at the 72-Hour observation.

Other effects:

One animal showed body weight loss and two animals showed expected gain in body weight during the study.

Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex	Time after application	Cornea opacity	Iris	Conjunctivae
				Redness	Chemosis
73202 Male	1 hours 24hours 48hours 72hours	0 0 0 0	1 0 0 0	1 1 1 0	1 1 0 0
73239 Male	1 hour 24 hours 48 hours 72 hours	0 0 0 0	1 0 0 0	2 1 1 0	1 1 0 0
73240 Male	1 hour 24 hours 48 hours 72 hours	0 0 0 0	1 0 0 0	2 1 1 0	1 1 0 0

Interpretation of results:

slightly irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item produced a maximum group mean score of 11.7 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.
The test item was not classified as an irritant according to GHS and EC CLP 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin irritation/corrosion

Two studies are available:

1) An acute dermal irritation study was conducted according to OECD 404 using rabbits under GLP (Pooles A, 2013). 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 (Warren N, 2013). Supporting study. This study showed that the test substance is not corrosive.

Eye irritation

Two studies are available:

1) An acute eye irritation study was conducted according to OECD 405 using rabbits under GLP (Pooles A, 2013). Key study.

This study showed that the test substance is slightly irritating.

2) An in vitro BCOP study was conducted according to OECD 437 using bovine cornea (Warren N, 2013). Supporting study.

The test item was considered not to be an ocular corrosive or severe irritant.

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

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

Effects on eye irritation: slightly irritating

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 0.67) and for conjunctival oedema were < 1 (actual value 0.33).

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.