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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 22 - 25, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, a weight of evidence analysis was performed, prior to the start of this in vivo eye irritation study in the rabbit. As recommended in the test guidelines, all available information was evaluated (e.g. existing human and animal data, literature, substance data supplied by the sponsor, analysis of structure activity relationships (SAR), physicochemical properties and reactivity (pH, buffering capacity) and in vitro, ex vivo and in vivo tests) to determine the need for in vivo eye testing.
Based on the available information, it was concluded that there is need to perform this in vivo eye irritation study in rabbit in order to establish the possible eye irritating properties of the test substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Deviations:
no
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

Test material

Constituent 1
Chemical structure
Reference substance name:
calcium dinitrate tetrahydrate
EC Number:
603-865-8
Cas Number:
13477-34-4
Molecular formula:
Ca(NO3)2*4H20
IUPAC Name:
calcium dinitrate tetrahydrate
Test material form:
solid: flakes
Details on test material:
- Name of test material (as cited in study report): Calcium nitrate tetrahydrate
- Substance type: White flakes
- Physical state: Solid

Test animals / tissue source

Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L’Arbresle Cedex, France
- Age at study initiation: Animals used within the study were at least 6 weeks old *
- Weight at study initiation: body weights were at least 1.0 kg *.
- Housing: Individually housed in labeled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm).
- Diet (e.g. ad libitum): Pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) approximately 100 grams per day. Hay (TecniLab-BMI BV, Someren, The Netherlands) and wooden sticks (Swedish aspen wood, Uden, The Netherlands) were available during the study period.
- Water (e.g. ad libitum): Free access to tap water.
- Acclimatization period: at least 5 days before start of treatment under laboratory conditions.

* Animal specifications (sex, age and body weight) are specified in the attached table.

A health inspection was performed prior to commencement of treatment, to ensure that the animals were in a good state of health. Special attention was paid to the eyes, which were free from any abnormality.

Results of analysis for diet (nutrients and contaminants), hay and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9 - 19.4
- Humidity (%): 47 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN LIFE DATES: 22 February 2011

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: One eye of the animal remained untreated and served as the reference control.
Amount / concentration applied:
TEST MATERIAL- Amount(s) applied (volume or weight with unit): 98.2 mg (a volume of approximately 0.1 mL)

Duration of treatment / exposure:
Single instillation on Day 1.
Observation period (in vivo):
The eyes of each animal were examined approximately 1, 24, 48 and 72 hours after instillation of the test substance
Number of animals or in vitro replicates:
1 male
Details on study design:
STUDY DESIGN
The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). Based on the severity of the ocular lesions observed during the study, the animal was sacrificed for ethical reasons immediately after the 72 hours observation and the two further rabbits assigned to the study were not treated.

TREATMENT
On test Day 1, the test substance was instilled in the conjunctival sac of an eye of one animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test substance. The other eye remained untreated and served as the reference control.

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 the animal to quantitatively determine corneal epithelial damage. This procedure was repeated to assess recovery. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area.

After the final observation, the animal was sacrificed by intra-venous injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).

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

OBSERVATIONS
- Mortality/Viability: Twice daily.
- Toxicity: At least once daily.
- Body Weight: Day of treatment (prior to instillation) and at termination of the study.
- Necropsy: No necropsy was performed according to protocol.
- Irritation:
The irritation scores and a description of all other (local) effects were recorded.The irritation was assessed according to the following numerical scoring system. At each observation, the highest scores given were recorded:

CORNEAL IRRITATION
Opacity: degree of density (area most dense taken for reading)
0: No ulceration or opacity (may include slight dulling of normal luster)
1: Scattered or diffuse areas of opacity, 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

Area of cornea involved:
0: No ulceration or opacity
1: One quarter or less but not zero
2: Greater than one quarter, but less than half
3: Greater than half, but less than three quarters
4: Greater than three quarters, up to whole area

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

CONJUNCTIVAL IRRITATION
Redness (refers to palpebrae and sclera, excluding cornea and iris):
0: Blood vessels normal
1: Some blood vessels definitely hyperaemic (injected)
2: Diffuse, crimson color, individual vessels not easily discernible
3: Diffuse beefy red

Chemosis (refers to lids and/or nictitating membranes):
0: No swelling
1: Any swelling above normal (includes 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

Discharge:
0: No discharge (may include small amounts observed in inner canthus of normal animals)
1: Any amount different from normal and/or lacrimation
2: Discharge with moistening of the lids and hairs just adjacent to lids
3: Discharge with moistening of the lids and hairs (considerable area around the eye)

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

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other: outside of the eyelids were red and swollen
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other: outside of the eyelids were red and swollen
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
4
Max. score:
4
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other: outside of the eyelids were red and swollen
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
animal #1
Time point:
24/48/72 h
Score:
3
Max. score:
3
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other: outside of the eyelids were red and swollen
Irritant / corrosive response data:
Instillation of 98.2 mg of CALCIUM NITRATE TETRAHYDRATE (a volume of approximately 0.1 mL) into an eye of one rabbit resulted in effects on the cornea, iris and conjunctivae. The corneal injury consisted of opacity (maximum grade 2) and epithelial damage (maximum 100% of the corneal area). The corneal injury did not diminish within 72 hours. Iridial irritation grade 1 was observed and did not resolve within 72 hours. The irritation of the conjunctivae consisted of redness (grade 3), chemosis (grade 4) and discharge (grade 3) and did not diminish within 72 hours. In addition the outside of the eye lids were red and swollen during the observation period.
Due to the severity of effects and no signs of recovery, the animal was sacrificed after the 72-hour observation for ethical reasons. No further animals were treated.
Individual and mean eye irritation scores are specified in the attached table.
Other effects:
No staining of (peri) ocular tissues by the test substance was observed and no test substance remnants were seen.
No symptoms of systemic toxicity were observed in the animal during the test period and no mortality occurred.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye)
Remarks:
Classified as Category 1 according to Regulation (EC) 1272/2008
Conclusions:
Based on the degree and persistence of the corneal injury, it was concluded that severe ocular damage had occurred by instillation of CALCIUM NITRATE TETRAHYDRATE into the rabbit eye in one animal.

Based on these results:
- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007), CALCIUM NITRATE TETRAHYDRATE should be classified as: having irreversible effects on the eyes (Category 1).
- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures, CALCIUM NITRATE TETRAHYDRATE should be classified as Irreversible effects on the eye (Category 1) and labeled as H318: Causes serious eye damage.
Executive summary:

Acute eye irritation/corrosion study with Calcium nitrate tetrahydrate in the rabbit according to OECD 405 guideline and GLP principles.

Instillation of the test substance resulted in effects on the cornea, iris and conjunctivae. The corneal injury consisted of opacity (maximum grade 2) and epithelial damage (maximum 100% of the corneal area). The corneal injury did not diminish within 72 hours. Iridial irritation grade 1 was observed and did not resolve within 72 hours.

The irritation of the conjunctivae consisted of redness (grade 3), chemosis (grade 4) and discharge (grade 3) and did not diminish within 72 hours. In addition the outside of the eye lids were red and swollen during the observation period.

No staining of (peri) ocular tissues by the test substance was observed and no test substance remnants were seen.

Based on these results Calcium nitrate tetrahydrate should be classified as Irreversible effects on the eye (Category 1) and labeled as H318: Causes serious eye damage.