2-Pyridinecarboxylic acid, 4-amino-3,6-dichloro-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 June 2001 to 30 August 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: JMAFF Primary Eye Irritation Study, 2000

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4-amino-3,6-dichloropyridine-2-carboxylic acid

EC Number:

604-721-7

Cas Number:

150114-71-9

Molecular formula:

C6H4Cl2N2O2

IUPAC Name:

4-amino-3,6-dichloropyridine-2-carboxylic acid

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: tan powder

Specific details on test material used for the study:

Purity: 94.5%

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Age at study initiation: 20 - 21 weeks
- Weight at study initiation: 2.7 - 2.8 kg
- Housing: animals were housed individually in stainless steel cages
- Diet: animals received 5 oz. per day
- Water: ad libitum
- Acclimation period: at least 2 weeks
- Other: The eyes of three adult New Zealand White rabbits were examined with 2 % aqueous fluorescein stain and established as being free of defects/irritation the day prior to the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 ± 3 °C
- Humidity: 40 - 60 % (relative)
- Air changes: 12 - 15 air changes per hour
- Photoperiod: 12 hours of darkness / 12 hours of light (06:00 to 18:00)

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: The left eye of each rabbit remained untreated and served as a control.

Amount / concentration applied:

A 0.1 g aliquot of the test material in powder form was instilled into each of the treated eyes.

Duration of treatment / exposure:

The eyelid of each rabbit was held closed for approximately one second after dosing.

Observation period (in vivo):

1, 24, 48 and 72 hours and 7, 14, 21, 28, and 35 days post-instillation

Number of animals or in vitro replicates:

Three

Details on study design:

Test material was instilled into the conjunctival sac of the right eye of three rabbits.
The behaviour of each rabbit was observed immediately post-treatment for indications of pain or discomfort. An ocular anaesthetic was used for both eyes of one rabbit on days 2, 3, and 4.

SCORING SYSTEM:
Irritation was scored according to the Draize scale (below):
> Cornea
Opacity: degree of density (area most dense is taken for reading).
0 = No ulceration or opacity
1 = Scattered or diffuse areas of opacity (except for slight dulling of normal lustre), details of iris clearly visible
2 = Easily discernible translucent area, details of iris slightly obscured
3 = Nacreous area, no details of iris visible, size of pupil barely discernible
4 = Opaque cornea, iris not discernible through the opacity

> Conjunctivae
Redness: (refers to the most severe effect of palpebral and bulbar conjunctivae in comparison to the control eye)
0 = Blood vessels normal
1 = Some blood vessels definitely hyperaemic (injected)
2 = Diffuse crimson colour, individual vessels not easily discernible
3 = Diffuse beefy red
Chemosis: lids and/or nictating membranes
0 = No swelling
1 = Any swelling above normal (includes nictating membranes)
2 = Obvious swelling with partial eversion of lids.
3 = Swelling with lids about half closed
4 = Swelling with lids more than half closed
Discharge
0 = No discharge
1 = Any amount different from normal (does not include small amounts observed in inner canthus of normal animals)
2 = Discharge with moistening of the lids and hairs just adjacent to lids
3 = Discharge with moistening of the lids and hairs a considerable area around the eye

> Iris
0 = Normal
1 = Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia or injection; any of these or combination of any thereof, iris still reacting to light (sluggish reaction is positive)
2 = No reaction to light, haemorrhage, gross destruction (any or of all these)

TOOL USED TO ASSESS SCORE:
Both eyes of the rabbits were examined with a binocular loupe and a white halogen light at approximately 1, 24, 48, and 72 hours and 7, 14, 21, 28, and 35 days post-instillation for conjunctival redness and chemosis, discharge, corneal opacity, and reddening of the iris. A cobalt blue light was used to assess the corneas of all rabbits following fluorescein stain application at various times during the study.

OTHER EXAMINATIONS
Rabbits were weighed on the day of treatment and study termination.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 35 days

Results 2

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 21 days

Results 3

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24 h

Score:

2

Max. score:

4

Results 4

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

other: 48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 21 days

Results 5

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24 h

Score:

0

Max. score:

2

Results 6

Irritation parameter:

iris score

Basis:

animal #1

Time point:

other: 48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 7 days

Results 7

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 14 days

Results 8

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 7 days

Results 9

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

not fully reversible within: 35 days

Results 10

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24 h

Score:

2

Max. score:

4

Results 11

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

other: 48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 21 days

Results 12

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24 h

Score:

3

Max. score:

4

Results 13

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

other: 48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 21 days

Irritant / corrosive response data:

One hour after dosing, all rabbits had slight conjunctival redness, moderate chemosis, and moderate discharge. Twenty-four hours after dosing, all rabbits had moderate to marked conjunctival redness, moderate chemosis, moderate to marked discharge, and slight corneal opacity. The two male rabbits also had slight irritation of the iris twenty-four hours after dosing. Forty-eight hours after dosing, all rabbits had moderate to marked conjunctival redness, slight to moderate chemosis, moderate to marked discharge, slight corneal opacity, and slight irritation of the iris. The ocular irritation slowly diminished over time. Corneal vascularisation was noted in all three animals starting on day 15 and continuing for one rabbit through day 36. Two rabbits had ocular irritation that continued through study termination.

Other effects:

All rabbits gained body weight over the duration of the study.

Any other information on results incl. tables

Table 1: Individual Animal Irritation Grades

Observation time after dosing	Animal no.	Redness	Conjunctival chemosis	Discharge	Corneal opacity	Reddening of iris
24 hours	01A3957	2	3	2	2	0
	01A3955	3	2	3	2	1
	01A3956	3	3	3	2	1
48 hours	01A3957	2	3	2	2	1
	01A3955	2	1	1	2	1
	01A3956	3	2	2	1	1
72 hours	01A3957	3	3	2	2	1
	01A3955	2	1	1	2	1
	01A3956	3	2	2	1	1

Irritation parameter	Time Point	Mean score	Max score	Reversibility
Corneal opacity score	24, 48, and 72 hours	1.78	4	Not fully reversible within 35 days
Iris score	24, 48, and 72 hours	0.89	2	Fully reversible within 14 days
Chemosis score	24, 48, and 72 hours	2.22	4	Not fully reversible within 35 days
Conjunctival redness	24, 48, and 72 hours	2.56	3	Not fully reversible within 35 days
Conjunctival discharge	24, 48, and 72 hours	2	3	Fully reversible within 28 days

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye)

Conclusions:

Under the conditions of the study, the animals displayed moderate to marked signs of ocular irritation which lessened over time but effects on the cornea, conjunctiva and chemosis persisted to study termination 35 days post-instillation.

Executive summary:

The eye irritation potential of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 405, EPA OPPTS 870.2400, EU Method B.5 and JMAFF (2000).

During the study aliquots of 0.1 g of neat test material in powder form were placed into the conjunctival sac of the right eye of one female and two male New Zealand White rabbits. An ocular anaesthetic was used for both eyes of one rabbit on days 2, 3, and 4.

One hour after dosing, all rabbits had slight conjunctival redness, moderate chemosis, and moderate discharge. Twenty-four hours after dosing, all rabbits had moderate to marked conjunctival redness, moderate chemosis, moderate to marked discharge, and slight corneal opacity. The two male rabbits also had slight irritation of the iris twenty-four hours after dosing. Forty-eight hours after dosing, all rabbits had moderate to marked conjunctival redness, slight to moderate chemosis, moderate to marked discharge, slight corneal opacity, and slight irritation of the iris. The ocular irritation slowly diminished over time. Corneal vascularisation was noted in all three animals starting on day 15 and continuing for one rabbit through day 36. Two rabbits had ocular irritation that continued through study termination. All rabbits gained body weight over the duration of the study.