Guanidinium nitrate

Endpoint summary

Description of key information

Data on Guanidine Nitrate are available from two studies for skin irritation / corrosion and from one for eye irritation.

The mild skin irritation effects observed after 4 hours of treatment are below the threshold level for classification and

are fully reversible. The effects observed in the eye indicate mild irritation effects but since they are not reversible in

one animals, corrosive properties have to be declared.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988-06-22 to 1988-06-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

adopted May 12, 1981

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: The Broekman Institute, Someren, The Netherlands.
- Age at study initiation: 12 weeks
- Weight at study initiation: 2065 - 2390 g
- Housing: individually housed in a plastic cage with a perforated floor
- Diet: 100 g per day (LKK-20, diameter 4 mm) of standard laboratory animal diet obtained from Hope Farms, Woerden, The Netherlands
- Water: ad libitum, tap-water (via automatic nozzles):
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-21
- Humidity (%): 55-75
- Photoperiod (hrs dark / hrs light): 12/12

One day before treatment, the fur was removed from the dorsal flanks of the animals by clipping, exposing an area of the skin of
approximately 10 cm x 10 cm.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

water

Remarks:

Milli-RO water (Millipore Corp., Bedford, Mass., USA)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.5 g, moistened with 0.3 mL Milli-RO water

Duration of treatment / exposure:

4 h

Observation period:

72 h
The exposed skin areas were examined for signs of erythema and oedema and the responses were scored approximately 60 minutes and 24, 48 and 72 hours after removal of the patches.

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: approx. 6 cm²
- Coverage: Each portion of the prepared test substance was spread on a 6 cm2 patch of Metalline (Lohmann, FRG) and mounted on permeable tape (Micropore, 3M, St.Paul, USA). This was applied to the left flank of each animal, the right flank being covered with the same dressing without test substance.
- Type of wrap used: flexible bandage (Coban, 3M, St. Paul, USA).

REMOVAL OF TEST SUBSTANCE
- Washing: The remaining test substance was removed using a dry tissue and subsequently a tissue moistened with tap-water.
- Time after start of exposure: 4 h

SCORING SYSTEM:

Erythema and eschar formation
0: No erythema
1: Very slight erythema (barely perceptible)
2: Well—defined erythema
3: Moderate to severe erythema
4: Severe erythema (beef redness) to slight eschar formation (injuries in depth)

Oedema formation
0: No oedema
1: Very slight oedema (barely perceptible)
2: Slight oedema (edges of area well defined by definite raising)
3: Moderate oedema (raised approximately 1 millimetre)
4: Severe oedema (raised more than 1 millimetre and extending beyond area of exposure)

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 24 h

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 24 h

Irritant / corrosive response data:

The treated skin of all three animals was scored for erythema and oedema. The test substance caused no adverse effects to the skin, except very
slight erythema and very slight oedema in one animal 60 minutes after removal of the bandage.

Other effects:

No signs of systemic intoxication were observed in any of the rabbits.

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	Erythema	Oedema
	Max. score: 4	Max. score: 4
60 min	0/0/1	0/0/1
24 h	0/0/0	0/0/0
48 h	0/0/0	0/0/0
72 h	0/0/0	0/0/0
Average 24h, 48h, 72h	0/0/0	0/0/0
Reversibility	c/c/c	c/c/c
Average time (unit) for reversion	24 h	24 h

Reversibility: c=completely reversible

Interpretation of results:

GHS criteria not met

Conclusions:

Classification is based on erythema/eschar and oedema calculated as mean scores following grading at 24, 48 and 72 hours after removal of the test material, in consideration of reversibility. In this study, the test substance Guanidine Nitrate is considered not to be irritant to the skin according to the criteria of:
- CLP - (EU GHS), Regulation (EC) No. 1272/2008
- OECD GHS (2nd rev. edition, UN, 2007)
- Directive 67/548/EEG .

Executive summary:

In a primary dermal irritation study according to OECD Guideline 404 1981, three White New Zealand rabbits were semi-occlusive dermally exposed to 0.5 g of Guanidine Nitrate for 4 hours to approximately 6 cm² of body surface area. Animals then were observed for 72 hours. Irritation was scored by the method of Draize as stipulated by OECD Guideline 404.

Very slight erythema and oedema was observed in 1/3 animals 60 minutes after patch removal and was fully reversible after 24 hours.

 

No classification for skin irritation is required for Guanidine Nitrate according to CLP, EU GHS (Regulation (EC) No 1272/2008).

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

3 May 1984 - 5 Jun 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Supporting information for read-across, structural analogue approach.

Qualifier:

according to guideline

Guideline:

other: EPA TS-792 Dermal irritation. Health effects test guidelines, EPA, August 1982; EPA 560/6-82-001

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Elkhorn Rabbitry, 5265 Starr Way, Watsonville, CA 95076
- Age at study initiation: young adults
- Weight at study initiation: 3.1 to 3.6 kg
- Housing: individually
- Diet (e.g. ad libitum): 150 g of Certified Purina Chow Diet 5322
- Water (e.g. ad libitum): d libitum
- Acclimation period: 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16.7 to 18.9°C
- Humidity (%): 40 to 70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Preparation of test site:

other: one test site shaved, one test site shaved+abraded

Vehicle:

physiological saline

Controls:

other: vehicle-treated sites on the same animals

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g

VEHICLE
The test compound was moistened with a few drops physiological saline (0.9%) to make a thick paste.

Duration of treatment / exposure:

24 h

Observation period:

14 d

Number of animals:

6 (3 males, 3 females)

Details on study design:

TEST SITE
- Area of exposure: 2.5x2.5 cm
- Type of wrap if used: Blenderm (semi-impervious hypoallergenic surgical tape)

REMOVAL OF TEST SUBSTANCE
- Washing (if done):skin was wiped, if the material adhered
- Time after start of exposure: 24 h

SCORING SYSTEM: according to Draize scale

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: intact skin

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

3.67

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: this animal has severe razor burns, thus should be regarded as abraded

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: intact skin

Irritation parameter:

erythema score

Basis:

animal #4

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: intact skin

Irritation parameter:

erythema score

Basis:

animal #5

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: intact skin

Irritation parameter:

erythema score

Basis:

animal #6

Time point:

24/48/72 h

Score:

2.67

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: intact skin

Irritation parameter:

edema score

Basis:

animal: #1, #3, #5

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: intact skin

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 2 d

Remarks on result:

other: this animal has severe razor burns, thus should be regarded as abraded

Irritation parameter:

edema score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 3 d

Remarks on result:

other: intact skin

Irritation parameter:

edema score

Basis:

animal #6

Time point:

24/48/72 h

Score:

0.66

Max. score:

4

Reversibility:

fully reversible within: 14 d

Remarks on result:

other: intact skin

Irritant / corrosive response data:

One rabbit was severely razor-burned during preclipping and still exhibited very slight erythema (score 1) at dosing. Consequently, the "non-abraded" sites were actually abraded and these sites exhibited necrosis and sloughling earlier than the non-abraded sites of the other animals. However, four of the remaining five rabbits possessed sites which exhibited necrosis and sloughing; therefore, the razor burns on this rabbit did not affect the overall final irritation rating of the compound.

Intact skin:
- erythema scores:
Erythema scores for the intact sites were first obtained after wrap removal (after 24 h of exposure). The erythema scores for this period ranged from slight (score 1) in one animal, to welldefined (score 2) in three animals, and to moderate-to-severe (score 3) in two animals. The scores remained the same or improved slightly through Day 7 for all except two animals, of which animal number 2 had severe razor burns. In this animal the application site had progressed
to necrosis by the second day, followed by eschar formation and sloughing within the first week.
During the second week, the test compound application site for three of the remaining four animals had also progressed to necrosis with eschar formation and sloughing.
- edema scores:
Edema at the intact sites was negligible. Three animals exhibited very slight edema formation (score 1). This had cleared by the third day after application in two animals and by Day 7 in the third rabbit.

Abraded skin:
The erythema at the abraded sites of all six animals were evaluated as moderate tn severe (score 3) when wraps were removed 24 h after application of the test compound. In four of the animals this had progressed to necrosis by the third day with eschar formation and sloughing developing within the first week. The application site on a fifth animal progressed to eschar formation during the second week.
Edema at the abraded sites was more prevalent and slightly more severe, but still reversible within 3 days.

Other effects:

none reported

Animal	Intact skin		Abraded skin
	Erythema scores Day 1/2/3/7/14	Edema scores Day 1/2/3/7/14	Erythema scores Day 1/2/3/7/14	Edema scores Day 1/2/3/7/14
1	2/2/1/1/4	0/0/0/0/0	3/3/4/4/4	0/0/0/0/0
2*	3/4/4/4/4	1/0/0/0/0	3/4/4/4/4	2/0/0/0/0
3	2/1/1/2/4	0/0/0/0/0	3/4/4/4/4	1/0/0/2/0
4	3/3/3/3/4	0/1/0/0/0	3/3/3/3/4	1/1/0/0/0
5	1/1/1/1/1	0/0/0/0/0	3/4/4/4/4	2/1/0/0/0
6	2/3/3/3/3	1/1/0/1/0	3/3/3/3/3	2/0/0/0/0

* this animal has severe razor burns, thus should be regarded as abraded

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Guanidine hydrochloride is legally classified as irritating to the skin (CLP, EU GHS (Regulation (EC) No 1272/2008)).

Executive summary:

In a primary dermal irritation study according to EPA guideline 560/6-82-001 (Primary Dermal Irritation), August 1982, 6 young adult New Zealand White rabbits were dermally exposed to 0.5 mg of Guanidine hydrochloride (>98%) for 24 hours to 2.5 cm x 2.5 cm body surface area to intact and abraded skin using semi-occlusive dressing. Animals then were observed for 14 days. The scoring system was according to Draize.

On intact skin, erythema scores after 24 h ranged from slight (score 1) in one animal, to well-defined (score 2) in three animals, and to moderate-to-severe (score 3) in two animals. The scores remained the same or improved slightly through day 7 for all except two animals, of which one animal had severe razor burns. In this animal the application site had progressed to necrosis by the second day, followed by eschar formation and sloughing within the first week.

During the second week, the test compound application site for three of the remaining four animals had also progressed to necrosis with eschar formation and sloughing.

Edema at the intact sites was negligible. Three animals exhibited very slight edema formation (score 1). This had cleared by the third day after application in two animals and by Day 7 in the third rabbit.

The erythema at the abraded sites of all six animals were moderate to severe (grade 3) after 24 h. In four of the animals this had progressed to necrosis by the third day with eschar formation and sloughing developing within the first week. The application site on a fifth animal progressed to eschar formation during the second week. Edema at the abraded sites was more prevalent and slightly more severe, but still reversible within 3 days.

The irritation response on intact skin sites was not reversible within 14 days.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1984-06-21 to 1984-07-24

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Elkhorn Rabbitry, 5265 Starr Way, Watsonville, CA 95076
- Age at study initiation: young adults
- Weight at study initiation: 2.65 - 3.33 kg
- Housing: individually in stainless steel, battery-type cages with screened bottoms and 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: 19 days
- Earmite prevention: during acclimation the animals were given one application of Canex mineral oil (Pitman-Moore, Inc., Washington Crossing, NJ)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 14.1-21.1
- Humidity (%): 40-70 except for a steam outage when it increased to 80 % for seferal hours
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 1 group of 4 male and 4 female rabbits was treated on 1984-07-10

Type of coverage:

occlusive

Preparation of test site:

clipped

Vehicle:

physiological saline

Remarks:

pH 5.2

Controls:

other: each animal had two sites treated with the test compound, one site was treated with the vehicle, one was a sham patch

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.5 g test substance was moistened with a few drops of physiological saline (0.9 %) to make a thick paste
- pH of a saturated solution of Guanidine Nitrate in vehicle: 4.74

VEHICLE
- Amount applied: 0.5 ml physiol. saline (0.9 %) for control sites;

Duration of treatment / exposure:

24 h

Observation period:

14 d

Number of animals:

8 (4 males + 4 females)

Details on study design:

The backs of eight rabbits were close-clipped and divided into 4 quadrants designated I - IV. Areas I and IV were intact on all animals, and areas II and III were abraded by making two perpendicular scratches in the stratum corneum of the skin about 1 inch long with an escarifier. The Guanidine Nitrate paste was placed on a 1-inch square gauze patch which was taped to the appropriate site. Blenderm® (Medical Products Division of 3M, St. Paul, MN), a semi-impervious hypoallergenic surgical tape, was used to hold the patches in place. Vet Wrap® (Animal Care Products, Division f 3M, St. Paul, MN) was then wrapped securely around the animal, followed by Conform® elastic tape (The Kendall Company, Boston, MA). The test compound was left in contact with the skin for 24 hr. At the end of the exposure period, the wrapping and patches were removed, the skin was wiped if the material adhered, and the areas were scored.

Scoring and grading of dermal reactions were performed at 1, 2, 3, 7, and 14 days after application. Animals were sacrificed on day 15.

Evaluation of skin reactions
Erythema and eschar formation
0: No erythema
1: Very slight erythema (barely perceptible)
2: Well-defined erythema
3: Moderate-to-severe erythema
4: Severe erythema (beef redness to slight eschar formation injuries in depth)

Oedema formation
0: No oedema
1: Very slight oedema (barely perceptible)
2: Slight oedema (edges of area well defined by definite raising)
3: Moderate oedema (raised approximately 1 mm)
4: Severe oedema (raised more than 1 mm and extending beyond area of exposure)

Irritation parameter:

erythema score

Remarks:

Intact Sites

Basis:

mean

Remarks:

Female

Time point:

24/48/72 h

Score:

0 - 4

Max. score:

4

Reversibility:

not specified

Irritation parameter:

erythema score

Remarks:

Intact Sites

Basis:

mean

Remarks:

Male

Time point:

24/48/72 h

Score:

ca. 0 - 2

Max. score:

2

Reversibility:

not specified

Irritation parameter:

edema score

Remarks:

Intact Sites

Basis:

mean

Remarks:

Female

Time point:

24/48/72 h

Score:

0 - 2

Max. score:

2

Reversibility:

fully reversible within: 14 d

Irritation parameter:

edema score

Remarks:

Intact Sites

Basis:

mean

Remarks:

Male

Time point:

24/48/72 h

Score:

0 - 2

Max. score:

2

Reversibility:

fully reversible within: 14 d

Irritation parameter:

erythema score

Remarks:

Abraded Sites

Basis:

mean

Remarks:

Female

Time point:

24/48/72 h

Score:

2 - 4

Max. score:

4

Reversibility:

not specified

Irritation parameter:

erythema score

Remarks:

Abraded Sites

Basis:

mean

Remarks:

Male

Time point:

24/48/72 h

Score:

1 - 4

Max. score:

4

Reversibility:

not specified

Irritation parameter:

edema score

Remarks:

Abraded Sites

Basis:

mean

Remarks:

Female

Time point:

24/48/72 h

Score:

0 - 2

Max. score:

2

Reversibility:

fully reversible within: 14 d

Irritation parameter:

edema score

Remarks:

Abraded Sites

Basis:

mean

Remarks:

Male

Time point:

24/48/72 h

Score:

1 - 4

Max. score:

4

Reversibility:

fully reversible within: 14 d

Summary Tables: 

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

Intact Sites	Females		Males
Score at time point / Reversibility	Erythema	Oedema	Erythema	Oedema
	Max. score: 4	Max. score: 4	Max. score: 4	Max. score: 4
24 h	4/2/1/1	1/1/1/1	2/1/1/1	1/2/1/1
48 h	4/4/0/1	2/0/0/0	2/0/2/2	0/2/1/2
72 h	3/4/0/1	1/1/0/0	1/1/1/1	1/0/0/0
7 d	4/4/1/1	0/2/0/0	4/2/1/1	1/0/0/0
14 d	4/4/0/1	0/0/0/0	4/1/1/1	0/0/0/0
Average 24h, 48h, 72h	3.67/3.33/0,.33/1	1.33/0.67/0.33/0.33	1.67/0.67/1.33/1.33	0.67/1.33/0.67/1
Reversibility*)	n.d.	c/c/c/c	n.d.	c/c/c/c
Average time (unit) for reversion		14 d		14 d

 

Abraded Sites	Females		Males
Score at time point / Reversibility	Erythema	Oedema	Erythema	Oedema
	Max. score: 4	Max. score: 4	Max. score: 4	Max. score: 4
24 h	4/2/2/2	1/1/1/2	3/2/3/4	2/2/4/1
48 h	4/4/4/2	2/0/0/2	4/2/4/4	1/1/4/4
72 h	4/4/1/2	1/2/0/0	4/1/4/4	1/2/4/2
7 d	4/4/4/4	0/1/0/0	4/4/4/4	0/0/0/0
14 d	4/4/4/4	0/0/0/0	4/2/4/4	0/0/0/0
Average 24h, 48h, 72h	4/3.33/2.33/2	1.33/1/0.33/1.33	3.67/1.67/3.67/4	1.33/1.67/4/2.33
Reversibility*)	n.d.	c/c/c/c	n.d.	c/c/c/c
Average time (unit) for reversion		14 d		7 d

 

Reversibility: c=completely reversible; n.d.=not determined

Interpretation of results:

GHS criteria not met

Conclusions:

After 24 hours exposure, severe irritation with corrosive properties was observed. Due to the extended exposure of 24 hours instead of 4 hours, as required by current guidelines, the results are not suitable for classification purpose according to CLP - (EU GHS), Regulation (EC) No 1272/2008.

Executive summary:

The primary dermal irritation potential of Guanidine Nitrate was examined in male and female New Zealand White rabbits using a modified Draizee method. Following 24 hours of exposure with 500 mg of substance on intact and abraded skin, severe primary irritancy with corrosive properties was observed. Erythema, edema, and eschar formation (injuries in depth) were detected at 24, 48, and 72 hours after dosing. After 14 days no further edema was observed. Irreversible skin damage was apparent in 7/8 animals at the time of sacrifice, 14 days after dosing..

The test conditions applied produce a significantly higher and more invasive exposure than foreseen by current guidelines. Consequently, the results cannot be used for classification according to CLP - (EU GHS), Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

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

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.

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

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

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

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

Irritation parameter:

cornea opacity score

Basis:

animal #6

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal: #1, #4

Time point:

24/48/72 h

Score:

0

Max. score:

2

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

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

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

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

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

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

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

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.

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.

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.

 

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1984-05-03 to 1984-06-09

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

Supporting information for read-across, structural analogue approach.

Qualifier:

according to guideline

Guideline:

other: EPA 560/6-82-001 (Primary Eye Irritation), August 1982

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

Observation was not continued until fully reversibility, or 21 days.

GLP compliance:

yes

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: 2.3 - 2.9 kg
- Housing: individually
- Diet (e.g. ad libitum): 150 g of Certified Purina Chow Diet 5322
- Water (e.g. ad libitum): d libitum
- Acclimation period: 14 d minimum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16.1 to 20.0°C, except for a spike to 21.1°C during a steam outage
- Humidity (%): 40 to 70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated left eye served as control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL was recalculated to mg: 81.4 mg ± 1.0 mg

Duration of treatment / exposure:

no removal of the test substance

Observation period (in vivo):

14 d

Number of animals or in vitro replicates:

6

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no

SCORING SYSTEM: Draize

TOOL USED TO ASSESS SCORE: fluorescein for scoring at 24 h, 7 and 14 d

Irritation parameter:

cornea opacity score

Basis:

animal: #1, #5, #6

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 d

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 7 d

Irritation parameter:

cornea opacity score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritation parameter:

iris score

Basis:

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

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 7 d

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

2

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.33

Max. score:

3

Reversibility:

fully reversible within: 14 d

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 14 d

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 14 d

Irritation parameter:

conjunctivae score

Basis:

animal #4

Time point:

24/48/72 h

Score:

1.67

Max. score:

3

Reversibility:

fully reversible within: 14 d

Irritation parameter:

conjunctivae score

Basis:

animal #5

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 7 d

Irritation parameter:

conjunctivae score

Basis:

animal #6

Time point:

24/48/72 h

Score:

1.67

Max. score:

3

Reversibility:

not fully reversible within: 14 d

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 14 d

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 d

Irritation parameter:

chemosis score

Basis:

animal #4

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 14 d

Irritation parameter:

chemosis score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritation parameter:

chemosis score

Basis:

animal #6

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 14 d

Irritant / corrosive response data:

- slight corneal opacity (grade 1) was observed in 3/6 rabbits for up to 4 days
- 2/6 animals showed slight iritis (vascular injection; grade 1) after 24 h, this was reversible after 48 h and 4 d, respectively
- slight to moderate conjunctival redness (grade 1 - 2) was observed in all animals, reversibility was achieved in 5/6 animals within 14 d
- in one animal slight redness (grade 1) was still present on day 14, but this can be judged as resolution of the initial lesion and can therefore expected to be reversible within 21 d
- slight to moderate chemosis (grade 1 - 2) was observed in all animals being fully reversible within 14 d

Animal number	Cornea score1 h/ 1/2/3/4/7/14 d	Iris score 1 h/ 1/2/3/4/7/14 d	Conjunctival redness score 1 h/ 1/2/3/4/7/14 d	Conjuntival chemosis score 1 h/ 1/2/3/4/7/14 d
1	0/0/0/0/0/0/0	0/0/0/0/0/0/0	1/1/2/1/1/1/0	1/0/0/0/0/0/0
2	1/1/1/1/1/0/0	0/1/1/1/1/0/0	1/2/2/2/2/1/0	2/1/1/1/2/1/0
3	0/0/1/1/1/0/0	0/1/0/0/0/0/0	0/1/1/1/0/1/0	1/2/1/0/1/0/0
4	0/0/1/0/0/0/0	0/0/0/0/0/0/0	1/1/2/2/2/1/0	2/2/2/1/1/1/0
5	0/0/0/0/0/0/0	0/0/0/0/0/0/0	1/1/1/1/1/0/0	1/1/1/0/0/0/0
6	0/0/0/0/0/0/0	0/0/0/0/0/0/0	1/1/2/2/2/1/1	2/1/2/1/1/1/0

Interpretation of results:

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

Conclusions:

Guanidine hydrochloride is legally classified as irritating to the eye (CLP, EU GHS (Regulation (EC) No 1272/2008)).

Executive summary:

In a primary eye irritation study similar to OECD guideline 405, 81.4 ± 1.0 mg (corresponding to 0.1 mL) of Guanidine hydrochloride (98% purity) was instilled into the conjunctival sac of 6 male, young adult New Zealand White rabbits. The eyes were not rinsed. Animals then were observed for 14 days. Irritation was scored similar to OECD guideline 405, at 1, 4, 24, 48 and 72 hours and at 7, 14 and 21 days. At 24, 48, 72 hours as well as 7 and 14 days post application the eyes were further examined with fluorescein to look for cornea damage.

Slight corneal opacity (grade 1) was observed in 3/6 rabbits for up to 4 days. 2/6 animals showed slight iritis (vascular injection; grade 1) after 24 h, which was reversible within 4 d. Slight to moderate conjunctival redness (grade 1 - 2) was observed in all animals; reversibility was achieved in 5/6 animals within 14 d. In one animal slight redness (grade 1) was still present on day 14, but this was judged as resolution of the initial lesion by the study authors and can therefore be expected to be fully reversible within 21 d, although observation was not continued until full reversibility was achieved. Slight to moderate chemosis (grade 1 - 2) was observed in all animals being fully reversible within 14 d.

Guanidine hydrochloride is legally classified as irritating to the eye.

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro eye irritation study does not need to be conducted because adequate data from an in vivo eye irritation study are available

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin irritation / corrosion:

In the key primary dermal irritation study performed according to OECD Guideline 404 1981, three White New Zealand rabbits were semi-occlusive dermally exposed to 0.5 g of Guanidine Nitrate for 4 hours to approximately 6 cm² of body surface area. Animals then were observed for 72 hours. Irritation was scored by the method of Draize as stipulated by OECD Guideline 404.

Very slight erythema and oedema was observed in 1/3 animals 60 minutes after patch removal and was fully reversible after 24 hours.

 No classification for skin irritation is required for Guanidine Nitrate according to CLP, EU GHS (Regulation (EC) No 1272/2008).

In the supporting study for the primary dermal irritation potential of Guanidine Nitrate, male and female New Zealand White rabbits were treated using a modified Draizee method. Following 24 hours of exposure with 500 mg of substance on intact and abraded skin, severe primary irritancy with corrosive properties was observed. Erythema, edema, and eschar formation (injuries in depth) were detected at 24, 48, and 72 hours after dosing. After 14 days no further edema was observed. Irreversible skin damage was apparent in 7/8 animals at the time of sacrifice, 14 days after dosing..

The test conditions applied produce a significantly higher and more invasive exposure than foreseen by current guidelines. Consequently, the results cannot be used for classification according to CLP - (EU GHS), Regulation (EC) No 1272/2008.

Eye irritation:

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.

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

Supporting data

Additional supporting data from the read-across substance guanidine chloride were included to demonstrate the similar toxicological profile of both substances. Data were used to substantiate the justification for read-across, outlined in IUCLID chapter 13 of technical dossier and chapter 1.1.2 Justification for Read-across (Analogue approach) of CSR.

Effects on eye irritation: corrosive

Justification for classification or non-classification

According to CLP, EU GHS (Regulation (EC) No 1272/2008),

- Guanidine Nitrate is not classified for skin irritation / corrosion since the effects observed are only slight and reversible after 24 hours.

- In spite of the mean scores after 24, 48 and 72 hours which would not require classification, Guanidine Nitrate is classified with Category 1 for eye irritation due to irreversible effects on the cornea and persistent chemosis in one animal.