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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:
26 April 2010 to 17 May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

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:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
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 (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Nitric acid, ammonium calcium salt
EC Number:
239-289-5
EC Name:
Nitric acid, ammonium calcium salt
Cas Number:
15245-12-2
Molecular formula:
Ca.xH3N.xHNO3
IUPAC Name:
nitric acid, ammonium calcium salt
Test material form:
solid: granular
Details on test material:
Name of test material (as cited in study report): CN-Nitcal
Physical appearance: White granules
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: Harlan, Belton, Leics, England
- Age at study initiation: Animal used within the study was at least 6 weeks old
- Weight at study initiation: body weight was at least 1.0 kg.
- Housing: labeled cage with perforated floor (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelter (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) was provided at least three times a week.
- Water (e.g. ad libitum): Free access to tap water.
- Acclimatization period: at least 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.6 ¿ 19.4ºC
- Humidity (%): 43 - 68%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day

IN LIFE DATES: From: 26 April 2010 To:17 May 2010

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): 70.7 mg
Duration of treatment / exposure:
Single instillation on Day 1
Observation period (in vivo):
The eyes of the animal were examined approximately 1, 24, 48 and 72 hours and 7, 14 and 21 days 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 irreversibility of the ocular lesions observed during the study in the first animal (21 day of observation) and the two further rabbits assigned to the study were not treated.

TREATMENT
On test Day 1, 70.7 mg of the test substance (a volume of approximately 0.1 mL) 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

OBSERVATION
The irritation was assessed according to the following numerical scoring system. At each observation, the highest scores given were recorded:

SCORING SYSTEM:
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
Remarks:
opacity
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
not reversible
Irritation parameter:
cornea opacity score
Remarks:
area
Basis:
animal #1
Time point:
24/48/72 h
Score:
3
Max. score:
4
Reversibility:
not fully reversible within: 21 days, score 1
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 14 days
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: 21 days, score 2
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
not fully reversible within: 21 days, score 1
Irritation parameter:
conjunctivae score
Remarks:
discharge
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
not fully reversible within: 21 days, score 1
Irritant / corrosive response data:
Instillation of 70.7 mg of CN-Nitcal (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 4) and epithelial damage (maximum 75% of the corneal area). As a result of the corneal injury, pannus (neovascularization of the cornea) was apparent 7 days after instillation up to the end of the observation period. The corneal injury did not resolve within the observation period of 21 days.
Iridial irritation grade 1 was observed and resolved within 14 days.
The irritation of the conjunctivae consisted of redness, chemosis and discharge and did not resolve within the observation period of 21 days.

see also attached tables.
Other effects:
Corrosion:
There was no evidence of ocular corrosion.

Coloration / Remnants:
No staining of (peri) ocular tissues by the test substance was observed and no test substance remnants were seen. However, heavy discharge and swelling of the eyelids might have hampered the scoring of test substance remnants behind the lower eyelid.

Toxicity / Mortality:
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) based on GHS criteria
Remarks:
Labeled as H318: Causes serious eye damage
Conclusions:
Based on the persistence of the effects up to the end of the observation time of 21 days CN-Nitcal should be classified:
- according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007) as: having irreversible effects on the eyes (Category 1).

- according to the Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures, as Irreversible effects on the eye (Category 1) and labeled as H318: Causes serious eye damage.

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