Diguanidinium carbonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 1998-07-21 to 1999-01-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study performed under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Diguanidinium carbonate

EC Number:

209-813-7

EC Name:

Diguanidinium carbonate

Cas Number:

593-85-1

Molecular formula:

CH5N3.1/2CH2O3 (one guanidine species, as denoted in the ESIS database) CH5N3.CH5N3.CH2O3 (as in the crystalline from, basis for the molecular weight 180.1658 g/mol as given below)

IUPAC Name:

bis(amino(imino)methanaminium) carbonate

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): Guanidine Carbonate Pure
- Substance type: Salt with organic kation and inorganic anion
- Physical state: White crystalline powder
- Analytical purity: 99.7 %
- Impurities: Sulphate ash <0.05 %, heavy metals calculated as lead, <10 ppm (mg/L), iron 1 ppm, lead <1 ppm, arsenic <3 ppm
- Purity test date: 1998-06-09
- Lot/batch No.: 1867 of Agrolinz Melamin GmbH, St.-Peter-Strasse 25, A-4021 Linz
- Expiration date of the lot/batch: Not specified but considered stable under conditions of storage for at least 5 years
- Storage condition of test material: At room temperature in the dark

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: J. Schitkovits, A-2620 Natschbach
- Age at study initiation: not reported
- Weight at study initiation: 2.9 - 3.2 kg
- Housing: Individual caging in metal wire cages, Ehret GmbH, D-79312 Emmendingen, type KK 016R, 79 cm x 59 cm x 35 cm.
- Diet (e.g. ad libitum): Altromin 2023 diet for rabbits, ad libitum.
- Water (e.g. ad libitum): Tap water from an automatic watering system, ad libitum.
- Acclimation period: 4 days (animal No. 51), 11 days (animals No. 52 and No. 53).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 (average)
- Humidity (%): 60 (average)
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: The left eyes remained untreated and served as negative controls.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The equivalent of 0.1 mL of the test substance was administered. The weights of the administered doses were: 85 mg, 98 mg and 95 mg.

Duration of treatment / exposure:

single dose (no washing)

Observation period (in vivo):

Both eyes of the animals were examined within 24 hours before instillation and approximately 1, 24,48 and 72 hours p.a.
Additional examinations were performed 6, 8, 10, 13, 15, 17 and 21 days after the instillation, ending individually for each animal.

Number of animals or in vitro replicates:

3 females

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): not done

SCORING SYSTEM:
CORNEA
Opacity : degree of density (area most dense area 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.

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 of all these or all together).

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.


TOOL USED TO ASSESS SCORE: otoscope lamp

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

animal #1

Remarks:

(animal No. 51)

Time point:

other: mean of scores at 24, 48 and 72 h

Score:

1.7

Max. score:

4

Reversibility:

not fully reversible within: 15 d

Remarks on result:

other: cornea was not examinable on day 17 and 21 due to severe chemosis

Results 2

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

(animal No. 52)

Time point:

other: mean of scores at 24, 48 and 72 h

Score:

2.7

Max. score:

4

Reversibility:

not reversible

Remarks:

until death on day 14

Remarks on result:

other: euthanized on day 14 for humane reasons

Results 3

Irritation parameter:

cornea opacity score

Basis:

animal #3

Remarks:

(animal No. 53)

Time point:

other: mean of scores at 24, 48 and 72 h

Score:

1

Max. score:

4

Reversibility:

not reversible

Remarks:

until death on day 14

Remarks on result:

other: found dead on day 14

Results 4

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

(animal No. 51)

Time point:

other: mean of scores at 24, 48 and 72 hours

Score:

0.3

Max. score:

2

Reversibility:

not fully reversible within: 72 h

Remarks on result:

other: iris was not discernible from day 6 on due to the opacity of the cornea.

Results 5

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

(animal No. 52)

Time point:

other: mean of 24 and 48 h

Score:

1

Max. score:

2

Reversibility:

not fully reversible within: 48 h

Remarks on result:

other: iris was not discernible from 72 h on due to the opacity of the cornea.

Results 6

Irritation parameter:

iris score

Basis:

animal #3

Remarks:

(animal No. 53)

Time point:

other: mean of scores at 24, 48 and 72 h

Score:

0

Max. score:

2

Reversibility:

fully reversible within: 10 d

Remarks on result:

other: iris was not discernible on day 13 due to the opacity of the cornea.

Results 7

Irritation parameter:

conjunctivae score

Basis:

animal #1

Remarks:

(animal No. 51)

Time point:

other: mean scores from 24, 48 and 72 h

Score:

3

Max. score:

3

Reversibility:

not reversible

Remarks:

until end of test (day 21)

Remarks on result:

other: mucopurulent ocular discharge from 24 h p.a. until terminal examination.

Results 8

Irritation parameter:

conjunctivae score

Basis:

animal #2

Remarks:

(animal No. 52)

Time point:

other: mean scores from 24, 48 and 72 h

Score:

3

Max. score:

3

Reversibility:

not reversible

Remarks:

until death on day 14

Remarks on result:

other: mucopurulent ocular discharge from 24 h p.a. until terminal examination and swelling dorsal of the instilled eye from 48 h until 14 d p.a.

Results 9

Irritation parameter:

conjunctivae score

Basis:

animal #3

Remarks:

(animal No. 53)

Time point:

other: mean scores of 24, 48 and 72 hours

Score:

2

Max. score:

2

Reversibility:

not reversible

Remarks:

until death on day 14

Remarks on result:

other: mucopurulent ocular discharge from 24 h p.a. until terminal examination.

Results 10

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

(animal No. 51)

Time point:

other: mean of scores at 24, 48 and 72 h

Score:

4

Max. score:

4

Reversibility:

not reversible

Remarks:

until end of test (day 21)

Results 11

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

(animal No. 52)

Time point:

other: mean of scores at 24, 48 and 72 h

Score:

4

Max. score:

4

Reversibility:

not reversible

Remarks:

until death on day 14

Results 12

Irritation parameter:

chemosis score

Basis:

animal #3

Remarks:

(animal No. 53)

Time point:

other: mean of scores at 24, 48 and 72 h

Score:

3

Max. score:

4

Reversibility:

not reversible

Remarks:

until death on day 14

Irritant / corrosive response data:

Cornea: In all animals corneal abnormalities were noted. Diffuse areas of opacity (score " 1 ") raised to easily discernible translucent area, details of iris slightly obscured (score "2") and to nacreous area (score "3") and opaque cornea (score "4") were the maximum grade of severity reached. The whole corneae were affected most of the time.

Iris: In animal No. 51 score "1" was noted 24 h. p.a. followed by 2 normal findings. Animal No. 52 reached score "2" 48 h p.a. and animal No. 53 score "1" was seen from 6 to 10 d p.a. Later on, no examinations were possible due to the opaque cornea in all animals.

Conjunctivae, redness: In animals No. 51 and No. 52 a diffuse beefy red (score "3") was noted from 1 h p.a. onwards. In animal No. 53 a diffuse crimson colour (score "2") from 1 h to 10 d raised also to a diffuse beefy red 13 d p.a.

Conjunctivae, chemosis: In animals No. 51 and No. 52 swelling with lids more than half closed (score "4") was present at each reading time. In animal No. 53 changes from an obvious swelling to closed eyes (scores "2", "3" and "4") were noted from 1 h onwards. A mucopurulent discharge from the instilled eye of all animals was noted from 24 p.a. until the end of the examinations.

Other effects:

Deaths: Animal No. 53 was found dead 14 days p.a. A moderate fibinous to fibrous pericarditis and foci of enteritis in the small intestine were noted post mortem. Severe ocular lesions may have worsened a pre-existing poor health condition and therefore have contributed to the death. No direct influence of the test substance is considered to have been the cause of death.
In animal No. 52 a swelling dorsal of the instilled eye was noted from 48 h until 13 d p.a. The nature of the swelling was not identified. In the same animal a total destruction of the cornea was noted 14 d p.a. after rinsing of the eye. This animal was subsequently euthanized for humane reasons.

Any other information on results incl. tables

Scores of the test eyes. Examination of cornea and iris.

Time after instillation (p.a.)	Cornea			Iris
	Animal No.			Animal No.
	51	52	53	51	52	53
l h	0	1^a	0	0	0	0
24 h	1^a	1^a	1^a	1	0	0
48 h	2^a	3^a	1^b	0	2	0
72 h	2^a	4^a	1^b	0	^c	0
Mean (24 – 72 h)	1.7	2.7	1.0	0.3	^c	0.0
6 d	4^b	4^a	2^a	^c	^c	1
8 d	4^b	4^a	3^a	^c	^c	1
10 d	4^b	4^a	3^a	^c	^c	1
13 d	4^b	4^a	4^b	^c	^c	^c
14 d	-	4^a	-	-	^c	-
15 d	4^b	-	-	^c	-	-
17 d	^h	-	-	^c	-	-
21 d	^h	-	-	^c	-	-

a) 1/2 of the cornea affected.

b) whole cornea affected.

c) iris was not discernible due to the opacity of the cornea.

h) cornea was not examinable due to severe chemosis

-) not examined.

Scores of the test eyes. Examination of conjunctivae.

Time after instillation (p.a.)	Conjunctivae (Redness)			Conjunctivae (Chemosis)
	Animal No.			Animal No.
	51	52	53	51	52	53
l h	3	3^de	2	4	4	2
24 h	3^e	3^ef	2^e	4	4	3
48 h	3^e	3^ef	2^e	4	4	3
72 h	3^e	3^ef	2^e	4	4	3
Mean(24 – 72 h)	3.0	3.0	2.0	4.0	4.0	3.0
6 d	3^e	3^ef	2^e	4	4	3
8 d	3^e	3^ef	2^e	4	4	4
10 d	3^e	3^ef	2^e	4	4	4
13 d	3^e	3^ef	3^e	4	4	4
14 d	-	3^ef	-	-	4	-
15 d	3^e	-	-	4	-	-
17 d	3^e	-	-	4	-	-
21 d	3^e	-	-	4	-	-

-) not examined.

d) turbid ocular discharge 24 h p.a.

e) mucopurulent ocular discharge from 24 h p.a. until terminal examination. swelling dorsal of the instilled eye from 48 h until 14 d p.a.

f) swelling dorsal of the instilled eye from 48 h until 14 d p.a.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye)

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance Guanidine carbonate is proposed to be classified with R41 Risk of serious damage to eyes) according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and Category 1 (Danger; H318: Causes serious eye damage) according to CLP (Regulation (EC) No 1272/2008).

Executive summary:

The aim of this study was to investigate possible irritating or corrosive effects of the undiluted test substance following a single administration into a conjunctival sac of the eye of New Zealand White rabbits. Methods and investigations were performed in conformance with the OECD TG 405.

The approximate equivalent of 0.1 mL Guanidine carbonate was instilled into the conjunctival sac of one eye of each of 3 rabbits. The body weight was measured at the start and at the termination of the test. The animals were examined for general signs of toxicity once daily. Eye examinations were performed 1, 24, 48,7 2 h and 6, 8, 10, 13, 15, 17, 21 d after the administration (p.a.) of the test substance (ending individually).

One animal died 14 days p.a. A moderate fibinous to fibrous pericarditis and foci of enteritis in the small intestine were noted post mortem. A direct influence of the test substance on the death is not likely. In 2/3 animals no general toxic effects were observed.

In all animals irreversible corneal alterations were observed with the maximum score of 4 with the whole cornea affected. The irises were affected in varying severities at the beginning. Later on, an examination was made impossible by opacity of the cornea in all animals. Pronounced redness was observed in all animals with the maximum score of 3. An obvious to a severe swelling was seen in all animals, reaching the maximum score of 4. A mucopurulent ocular discharge was present in all animals from 24 h p.a. until the end of the examinations. In 1/3 animals additionally a swelling dorsal of the test eye was noted from 48 h until 13 d p.a. In the same animal a total destruction of the cornea was noted 14 d p.a. after rinsing of the eye. This animal was subsequently euthanized for humane reasons.

The following mean scores were calculated from the individual examinations performed 24 h, 48 h and 72 h p.a.:

	Animal No.
	51	52	53
Cornea:	1.7	2.7	1.0
Iris:	0.3	*	0.0
Conjunctivae, redness:	3.0	3.0	2.2
Conjunctivae, chemosis:	4.0	4.0	3.3

*Iris was not discernible at all examination times

Ocular lesions were not reversible in all animals.

The classification of R41 (Risk of serious damage to eyes) according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and Category 1 (Danger; H318: Causes serious eye damage) according to CLP (Regulation (EC) No 1272/2008) is proposed.