1,1'-{(phenylmethanediyl)bis[(3-substituted benzene-4,1-diyl)diazene-2,1-diyl{1-[3-(dimethylamino)propyl]-2-hydroxy-4-methyl-6-oxo-1,6-dihydropyridine-5,3-diyl}]}dipyridinium chloride lactate salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 19 to 29, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study, conducted according to internationally accepted technical guidelines and in compliance with GLP in recognized contract research organization.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

of 2002

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

of 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000, including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

- Age at study initiation (treatment day): 9 - 11 weeks.
- Number and Sex: 3 males
- Animal supplier: Harlan France SARL, Gannat, France
- Weight at study initiation( before application): Minimum 1950 g, maximum 2201 g.
- Housing: Individual housing in cages with perforated floor and with shelters.
- Diet (approx. 100 g/day): Commercially available pelleted rabbit diet (K-H from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Hay (at least 3 times/week): Supplier: TecniLab-BMO BV, Someren, The Netherlands)
- Water (ad libitum): Tap water
- Acclimation period: At least 5 days before treatment under laboratory conditions.

During health inspection of the animals prior to treatment, special attention was paid to the eyes, which were free from any abnormality.

ENVIRONMENTAL CONDITIONS

Animal housing and environmental conditions were appropriate for eye irritation / corrosion testing in the rabbit: Controlled environment with approximately 15 air changes per hour, 12 hours artificial fluorescent light and 12 hours darkness per day and 21 – 23ºC. The relative humidity during the study period was 41 – 71%.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

Single dose of 100 mg test substance per animal instilled into the conjunctival sac of one eye. The other eye remained untreated and served as a control.

Explanatory Note Regarding Administered Amount of Test Substance:

Each animal was treated with approximately 116 mg (range: 115.7 - 116.4) of the test substance as received by the testing laboratory corresponding to 100 mg of the test substance expressed as water- and minor impurity-free test substance. Dosing was based on water- and minor impurity-free test substance. Therefore, a multiplication factor of 1.16 was applied to calculate the amount of test substance required for administration. This correction resulted in the administration of a larger amount than the 100 mg prescribed by the guidelines. However, since this correction almost exclusively accounts for the water content of the test substance, it was considered that animal welfare was not adversely affected by this.

Duration of treatment / exposure:

72 hours, as the eyes were not rinsed with tap-water or physiological saline after treatment.

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3

Details on study design:

Eyes were evaluated at: 1, 24, 48 and 72 hours after application. Grades attained at 24, 48 and 72 hours after application were included in the mean gradings of ocular lesions used for eye irritation/corrosion classification.

Immediately after the 24-hour observation, a solution of 2% fluorescein (Merck, Darmstadt, Germany) in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage.

Where standard lighting was considered inadequate for observing minor effects, eye examinations were performed using an ophthalmic examination lamp. In cases of equivocal results when comparing the treated and untreated eyes, the illustrated guide from the Consumer Product Safety Commission, Washington, D.C. 20207 was used for additional control purposes.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

over 3 animals and 3 time points

Time point:

other: 24, 48, 72 h

Score:

0

Max. score:

4

Reversibility:

not specified

Remarks:

corneal opacity was always grade 0

Results 2

Irritation parameter:

iris score

Basis:

mean

Remarks:

over 3 animals and 3 time points

Time point:

other: 24, 48, 72 h

Score:

0

Max. score:

2

Reversibility:

not specified

Remarks:

Iris score was always grade 0

Results 3

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

mean

Remarks:

over 3 animals and 3 time points

Time point:

other: 24, 48, 72 h

Score:

0.55

Max. score:

3

Reversibility:

fully reversible within: 72 h

Results 4

Irritation parameter:

chemosis score

Remarks:

swelling of lids and/or nictitating membranes

Basis:

mean

Remarks:

over 3 animals and 3 time points

Time point:

other: 24, 48, 72 h

Score:

0.22

Max. score:

4

Reversibility:

fully reversible within: 48 h

Results 5

Irritation parameter:

other: Area of corneal opacity

Basis:

mean

Remarks:

over 3 animals and 3 time points

Time point:

other: 24, 48, 72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 24 h. Reversibility referring to slight dulling of the normal lustre of the cornea (grade 0) in two of three animals

Results 6

Irritation parameter:

other: discharge

Basis:

mean

Remarks:

over 3 animals and 3 time points

Time point:

other: 24, 48, 72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritant / corrosive response data:

Ocular lesions of each individual animal are listed in Table 2 under "Remarks on results including tables and figures".

Other effects:

No other effects were reported. Mortality, symptoms of systemic toxicity or adverse effects on bodyweight were not evident.

Any other information on results incl. tables

Table 2: OCULAR LESIONS OF EACH ANIMAL

					Cornea				Iris		Conjunctivae				Comments
Animal	Time after dosing				Opacity (0-4)	Area (0-4)	Fluor area (%)^2		(0-2)		Redness (0-3)	Chemosis (0-4)	Discharge (0-3)
1^1	1 hour				0	0			0		1	1	2		a b e
	24 hours				0	0	0		0		1	0	1		e
	48 hours				0	0			0		0	0	0		e
	72 hours				0	0			0		0	0	0		e
2	1 hour				0	2			0		1	2	2		a b e g
	24 hours				0	0	0		0		1	1	1		e
	48 hours				0	0			0		1	0	0		e
	72 hours				0	0			0		0	0	0		e
3	1 hour				0	1			0		1	2	2		a b e g
	24 hours				0	0	0		0		1	1	1		e
	48 hours				0	0			0		1	0	0		e
	72 hours				0	0			0		0	0	0		e

 

¹     Animal 1 was treated one week before Animals 2 and 3.
(Animal identification numbers stated in the original report were altered to nos. 1, 2 and 3 for convenience)

²     Green staining after fluorescein treatment (percentage of total corneal area)

 

Comments:

a      Yellow/orange staining by the test substance hampered scoring for redness of the upper and/or lower eyelids and third eyelid.

b      Remnants of the test substance in the eye.

e      Yellow/orange staining of the head and paws by the test substance.

g      Slight dulling of the normal lustre of the cornea (grade 0).

Adverse effects on iris and cornea were not evident during the 72 hour observation period, and staining with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage. Conjunctival redness, chemosis and discharge were seen in all animals. These signs of irritation had completely resolved within 48 hours after test substance administration in animal No. 1 and within 72 hours in the other two animals. Yellow/orange staining of the fur on the head and paws, caused by the test substance, was noted during the observation period in all animals, but this finding was considered to be toxicologically irrelevant.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In the present study, the overall mean eye irritation scores derived from 3 rabbits 24, 48 and 72 hours post test substance instillation were 0.55 for conjunctival redness, 0.22 for chemosis and 0.0 for both corneal opacity and iridal irritation. In view of the absence of relevant effects on iris and cornea and the presence of only minor conjunctival irritation, which was completely reversible within 72 hours post test substance instillation, and according to EU classification rules [DIRECTIVE 67/548/EEC and REGULATION (EC) 1272/2008], the test substance was considered "not irritating to eyes" and not having any labelling requirement regarding eye irritation.

Executive summary:

the test substance was tested for eye irritation according to OECD Guideline 405 and the corresponding EC, EPA-OPPTS and JMAFF Technical Guidelines in compliance with GLP. Reliability grade 1 was assigned to the study.

 

Each of three New Zealand White rabbits was treated for 4 hours by instillation of a single dose of 100 mg test substance* into the conjunctival sac of one eye. The other eye remained untreated and served as a control. Eyes were evaluated for ocular lesions at 1, 24, 48 and 72 hours after instillation.

 

Conjunctival redness grade 1, chemosis grades 1 or 2 and discharge grade 2 were seen in all animals at 1 hour after test substance instillation. By 24 hours afterwards grades 0 or 1 were assigned to these signs of irritation and by 48 hours they had completely resolved in one animal and by 72 hours in the other two animals. Adverse effects on iris and cornea were not evident throughout the observation period, and staining with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage. Yellow/orange staining of eyelids and remnants of test substance in the treated eyes were only recorded at 1 hour after test substance instillation and were no longer evident thereafter. Mortality, signs of systemic toxicity or adverse effects on bodyweight were not evident.

 

In view of the absence of relevant effects on iris and cornea and the presence of only minor conjunctival irritation completely reversible within 72 hours post test substance instillation in the present study, and according to EU classification rules [DIRECTIVE 67/548/EEC and REGULATION (EC) 1272/2008], the test substance was considered "not irritating to eyes" and not having any labelling requirement regarding eye irritation.

________________________________________________________________

* Expressed as water- and minor impurity-free test substance