Guanidinium nitrate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984-07-02 to 1984-07-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Reference 2

Reference Type:

publication

Title:

Ocular and Dermal Toxicity of Guanidine Nitrate

Author:

Korte D W et al

Year:

1993

Bibliographic source:

International Journal of Toxicology, Vol. 12, No. 6, p.592-593

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Health effects test guidelines, August 1982, EPA 560/6-82-001

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Guanidinium nitrate

EC Number:

208-060-1

EC Name:

Guanidinium nitrate

Cas Number:

506-93-4

Molecular formula:

CH6N4O3

IUPAC Name:

amino(imino)methanaminium nitrate

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

impurity 2

Reference substance name:

Ammonium nitrate

EC Number:

229-347-8

EC Name:

Ammonium nitrate

Cas Number:

6484-52-2

Molecular formula:

H3N.HNO3

IUPAC Name:

Ammonium nitrate

impurity 3

Reference substance name:

4,6-diamino-1,3,5-triazin-2(1H)-one

EC Number:

211-455-1

EC Name:

4,6-diamino-1,3,5-triazin-2(1H)-one

Cas Number:

645-92-1

Molecular formula:

C3H5N5O

IUPAC Name:

4,6-diamino-1,3,5-triazin-2(1H)-one

impurity 4

Reference substance name:

(4,6-Diamino-1,3,5-triazine-2-yl)guanidine

Cas Number:

4405-08-7

Molecular formula:

C4H8N8

IUPAC Name:

(4,6-Diamino-1,3,5-triazine-2-yl)guanidine

impurity 5

Reference substance name:

Melamine

EC Number:

203-615-4

EC Name:

Melamine

Cas Number:

108-78-1

Molecular formula:

C3H6N6

IUPAC Name:

1,3,5-triazine-2,4,6-triamine

impurity 6

Reference substance name:

Cyanoguanidine

EC Number:

207-312-8

EC Name:

Cyanoguanidine

Cas Number:

461-58-5

Molecular formula:

C2H4N4

IUPAC Name:

1-cyanoguanidine

Test material form:

solid: crystalline

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Elkhorn Rabbitry, 5265 Starr Way, Watsonville, CA 95076
- Age at study initiation: young adults
- Weight at study initiation: 3.9 - 4.5 kg
- Housing: individually in stainless steel, screen-bottomed (no bedding), battery-type cages with automatically flushing dump tanks
- Diet: approximately 150 g of Certified Purina Chow Diet 5322 (Ralston Purina Company, Checkerboard Square St. Louis, MD)
- Water (e.g. ad libitum): provided by contlnous drip from a central line
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8-22.2
- Humidity (%): 56-70 except for an 8-hour period when the steamline was being repaired during which the relative humidity rose to 76 %
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 1st group of 3 rabbits treated on 1984-07-03, 2nd group of 3 rabbits treated on 1984-07-10

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: the untreated eye served as a control.

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 ml (0.092 g) of 99.99 % pure substance

Duration of treatment / exposure:

single dose treatment, eyes were not rinsed after treatment

Observation period (in vivo):

21 days

Number of animals or in vitro replicates:

6 animals total

Details on study design:

NUMBER OF ANIMALS:
A total of 6 animals was treated, divided in two groups of 3, treatment of the second group took place 7 days after the first

REMOVAL OF TEST SUBSTANCE:
- Washing: no washing was done

SCORING SYSTEM: Scoring aud grading of ocular reactions were performed at 1 and 4 hours, 1, 2, 3, 7, 14, and 21 days. Fluorescein dye was used for scoring and grading at 24 hours, 7, 14 and 21 days.

GRADES FOR OCULAR LESIONS (* indicates minimum level for a positive response)
- CORNEA Opacity: degree of density (area of greatest density taken for reading)
0: No ulceration or opacity
1*: Scattered or diffuse area of opacity (other than slight dulling of normal luster) details of iris clearly visible
2: Easily discernible translucent areas, details of iris slightly obscured
3: Nacreous areas. no details of iris visible, size of pupil barely discernible
4: Opaque cornea. iris not discernible through opacity

- IRIS
0: Normal
1*: Markedly deepened rugae. congestion, swelling, moderate circumiridial hyperemia or injection, any of these or any combination thereof, iris still reacting to light (sluggish reaction is positive)
2: No reaction to light, hemorrhage, gross destruction (any or all of these)

- CONJUNCTIVA Redness (refers to palpebral and bulbar conjunctiva, excluding cornea and iris)
0: blood vessels normal
1: Some blood vessels definitely hyperemic (injected)
2*: Diffuse, crimson color, individual vessels not easily discernible
3: Diffuse beefy red

- CHEMOSIS: lids or nictitating membranes
0: no swelling
1: Any swelling above normal (including nictitating membranes)
2*: Obvious swelling with partial eversion of lids
3: Swelling with lids about half-closed
4: Swelling with lids more than half-closed

The EPA recommends that investigators follow the Consumer Product Safety Commission Guidelines on ocular irritation testing. These guidelines state that an animal is considered to have exhibited a positive reaction if the test substance produces one or more of the following signs: ulceration of the cornea (other than a fine stippling); opacity of the cornea (other than a slight dulling of the normal luster); inflammation of the iris (other than a slight deepening of the rugae or slight hyperemia of the circumcorneal blood vessels); an obvious swelling in the conjunctiva (excluding the cornea and iris) with partial eversion of the lids; or a diffuse crimson-red coloration in the conjunctiva with individual vessels not easily discernible. The test shall be considered positive if four or more of the six animals in the test group exhibit a positive reaction. The EPA also classifies irritation in terms of duration of response. A test compound which produces a response which is reversible in 21 days is classified as irritative, while a test compound that produces an irreversible response (present at 21 days) is classified as corrosive. This system requires that classification be dependent on the most severe responder.

TOOLS USED TO ASSESS SCORE: Fluorescein dye was used for scoring and grading at24 hours, 7, 14 and 21 days.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

animal: #1, #4

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Results 2

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 72 h

Results 3

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks:

7 d

Results 4

Irritation parameter:

cornea opacity score

Basis:

animal #5

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not reversible

Remarks:

21 d

Remarks on result:

other: score increases from 1 to 2 at day 21

Results 5

Irritation parameter:

cornea opacity score

Basis:

animal #6

Time point:

24/48/72 h

Score:

0

Max. score:

4

Results 6

Irritation parameter:

iris score

Basis:

animal: #1, #4

Time point:

24/48/72 h

Score:

0

Max. score:

2

Results 7

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

2

Reversibility:

fully reversible within: 7 d

Results 8

Irritation parameter:

iris score

Basis:

animal: #3, #6

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 14 d

Results 9

Irritation parameter:

iris score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0.67

Max. score:

2

Reversibility:

fully reversible within: 14 d

Results 10

Irritation parameter:

conjunctivae score

Basis:

animal: #1, #2, #3, #4, #5, #6

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 21 d

Results 11

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Results 12

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 7 d

Results 13

Irritation parameter:

chemosis score

Basis:

animal: #3, #4, #6

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 14 d

Results 14

Irritation parameter:

chemosis score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

not fully reversible within: 21 d

Irritant / corrosive response data:

Guanidin nitrate produced slight corneal opacity (score 1) in 5/6 animals which cleared in 3 animals within 72 hours and in 1 animal within 21 days. Whereas in one animal an increase of corneal opacity (score 2) as a result of the developing pannus was observed at day 21. A slight iritis (score 1) was observed in 4/6 animals and was fully reversible within 14 days. Conjunctival redness (score 1) and chemosis (score 1) was observed in all animals, an intermediate increase (score 2) for each effect was observed in one, but different animals. Conjuncitval effects were fully reversible within 21 days in all animals. Chemosis was fully reversible within 21days in 5/6 animals and persist in 1 animal at the end of observation.

Other effects:

TOXIC SYMPTOMS / MORTALITY: No toxic symptoms were observed in the animals during the test period and no mortality occurred.

CONTROLS: The control eye remained normal throughout the study.

Any other information on results incl. tables

Summary table: Irritant / corrosive response data for each animal at each observation time up to removal of each animal from the test

Score at time point / Reversibility	Cornea	Iris	Conjunctivae	Chemosis
	Max. score: 4	Max. score: 2	Max. score: 3	Max. score: 4
1 h	0/0/0/0/0/0	0/0/0/0/1/0	1/0/1/1/1/0	0/0/0/0/0/0
4 h	0/0/1/0/0/0	0/0/0/0/1/1	1/1/1/1/2/1	0/1/1/1/1/1
24 h	1/1/1/1/1/0	0/0/1/0/1/1	1/1/1/1/1/1	1/1/1/1/1/1
48 h	0/1/1/0/1/0	0/0/1/0/0/1	1/1/1/1/1/1	0/2/1/1/1/1
72 h	0/0/1/0/1/0	0/1/1/0/1/1	1/1/1/1/1/1	0/2/1/1/0/1
7 d	0/0/0/0/1/0	0/0/0/0/1/1	0/1/1/0/2/1	0/0/0/0/1/1
14 d	0/0/1/0/1/0	0/0/0/0/0/0	0/0/1/0/0/1	0/0/0/0/0/0
21 d	0/0/0/0/2/0	0/0/0/0/0/0	0/0/0/0/0/0	0/0/0/0/1/0
Average 24h, 48h, 72h	0.33/0.67/1/0.33/1/0	0/0.33/1/0/0.67/1	1/1/1/1/1/1	0.33/1,67/1/1/0.67/1
Area affected
Maximum average score (including area affected, max 110)
Reversibility*)	c/c/c/c/n/c	c/c/c/c/c/c	c/c/c/c/c/c	c/c/c/c/n/c
Average time for reversion	-	14 d	21 d	-

 

Reversibility: c=completely reversible, n=not reversible

Slight corneal opacity (score 1) was observed in five rabbits. This was first observed at 4 hours, reached a maximum incidence at 24 hours, and had cleared in 4 of the rabbits by 72 hours. The opacity in rabbit #3 persisted through day 14 but cleared by day 21. In rabbit #5 the opacity not only continued through day 21 when the study was terminated but also increased ln severity as a result of the developing pannus.

In four rabbits, slight (score 1) iritis (vascular injection) was observed. This was noted as early as the 1·hour observation and cleared by day 14.

In evaluating the conjunctiva for both redness and chemosis, all rabbits shoved a positive reaction at some point after dosing.

All rabbits exhibited a light conjunctival redness (score 1). This condition was present in 4 of 6 rabbits at the 1-hour observation. The redness in all animals cleared by the 14-day observation except for rabbits #3 and #6 in which redness had cleared by day 21. Rabbit #5 showed moderate redness (score 2) at the 4-hour and 7-day observation periods.

Conjunctival chemosis (score 1) was observed in all rabbits by the 24-hour observation. The swelling cleared by day 14 except one reading of slight chemosls st·day 2l (#5). In earlier observations, chemosis had been an edematous swelling, however, in rabbit #5 at day 21, both palpebra lids were about twice normal thickness and leathery. Rabbit #2 showed moderate (score 2) conjunctlval chemosis at the 48-hour and 72-hour observations.

In addition to the ocular reactions, all rabbits developed corneal erosions. In two of the animals the erosiuns cleared by the 7-day observation, but in one rabbit the erosions persisted through day 21. In the animal where the erosions remained, the areas of the cornea covered by the ulcerations were 5 mm. Also, a large area of the nictitatlng membrane became devitalized and was sloughed in three rabbits, #2, #3 and #5.

Animal #5 developed the most severe reaction to the test compound. 0ne hour after exposure, vascularization of the iris was observed. After 24 hours this rabbit showed a slight reaction (score 1) in all areas of the eye. These conditions persisted through day 7. By the 14-day observation, all conditions had cleared except the corneal opacity and erosion. However, pannus (vascularization of the cornea) was observed from day 14 through termination of the study on day 21. The pannus began as a slight vascularization in the left ventral portion of the cornea and spread until it covered the lower half of the eye. Other ocular reactions observed were slight-to-marked tearing and free-floating exudate.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Due to irreversible effects of the cornea and a persistent chemosis in one animal a classification category 1 (irreversible eye effects) is required. However, regarding mean scores following grading at 24, 48 and 72 h no classification for eye irritation would be required according to CLP, EU GHS (Regulation (EC) No 1272/2008).

Executive summary:

In a primary eye irritation study performed according to Health effects test guidelines, August 1982, EPA 560/6-82-001, 0.1 ml (0.092 g) of pure (99.99 %) Guanidine Nitrate was instilled into the conjunctival sac of six male White New Zealand rabbits. The eyes were not rinsed after treatment and animals were observed for 21 days. Irritation was scored according to Draize at 1, 4, 24, 48 and 72 hours and at 7, 14 and 21 days.

Guanidin nitrate produced slight corneal opacity (score 1) in 5/6 animals which cleared in 3 animals within 72 hours and in 1 animal within 21 days. Whereas in one animal an increase of corneal opacity (score 2) as a result of the developing pannus was observed at day 21. A slight iritis (score 1) was observed in 4/6 animals and was fully reversible within 14 days.

Conjunctival redness (score 1) and chemosis (score 1) was observed in all animals, an intermediate increase (score 2) for each effect was observed in one, but different animals. Conjuncitval effects were fully reversible within 21 days in all animals. Chemosis was fully reversible within 21days in 5/6 animals and persist in 1 animal at the end of observation.

Considering the mean scores following grading at 24, 48 and 72 h, no classification for eye irritation would be required according to CLP, EU GHS (Regulation (EC) No 1272/2008). However, if a substance produces at least in one animal effects on the cornea, iris or conjunctiva that are not expected to reverse or have not fully reversed within an observation period of normally 21 days the substance has to be classified as category 1for irreversible eye effects.

At day 21 in one animal an increase of corneal opacity (score 2) as a result of the developing pannus and a persistent chemosis (score 1) was observed. Therefore a classification category 1 according to CLP, EU GHS (Regulation (EC) No 1272/2008) is required.