Sodium nitrate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 May 2010 to 25 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 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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

Test guideline 4

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

Test material information

Constituent 1

Reference substance name:

Sodium nitrate

EC Number:

231-554-3

EC Name:

Sodium nitrate

Cas Number:

7631-99-4

Molecular formula:

HNO3.Na

IUPAC Name:

sodium nitrate

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): Natriumnitrat HQ unbehandelt (non-food grade)
- Substance type: White crystalline powder
- Physical state: Solid
- Stability under test conditions: Stable
- 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: 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) 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): 18.8 – 19.4ºC
- Humidity (%): 43 - 77%
- 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 03 May 2010 to 25 May 2010

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: One eye of each animal remained untreated and served as the reference control.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied weight with unit): 95.7 mg (range 88.7 - 99.2 mg)

Duration of treatment / exposure:

Single instillation on Day 1

Observation period (in vivo):

The eyes of each animal were examined approximately 1, 24, 48, 72 hours and 7 days after instillation of the test substance.

Number of animals or in vitro replicates:

3 Males

Details on study design:

STUDY DESIGN
The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner two weeks later, after considering the degree of eye irritation observed in the first animal.

TREATMENT
Animals were treated by instillation of, on average, 95.7 mg (range 88.7 - 99.2 mg) of the test substance (a volume of approximately 0.1 mL), in the conjunctival sac of one of the eyes 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 each animal to quantitatively determine corneal epithelial damage. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area.

After the final observation, the animals were sacrificed by intra-venous injection of Euthasol® 20%

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

OBSERVATION
The irritation was assessed according to a numerical scoring system. At each observation, the highest scores given were recorded. See attached table.

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

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Results 2

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Results 3

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Results 4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Results 5

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Results 6

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Results 7

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 7 days

Results 8

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 7 days

Results 9

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 7 days

Results 10

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 72 hrs

Results 11

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

48 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 hrs

Results 12

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Results 13

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 hrs

Results 14

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Results 15

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 hrs

Results 16

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritant / corrosive response data:

Irritation
Instillation of approximately 96 mg of Natriumnitrat HQ unbehandelt (non-food grade) (a volume of approximately 0.1 mL) into one eye of each of three rabbits resulted in effects on the cornea, iris and conjunctivae.
The corneal injury consisted of slightly dulling of the luster only. This was seen in two animals and was resolved within 24 hours after instillation in both animals.
Iridial irritation grade 1 was observed in two animals and resolved within 24 hours in both animals.
The irritation of the conjunctivae consisted of redness, chemosis and discharge and completely resolved within 7 days in all three animals. For redness the mean over 24, 48 and 72 hours was grade 2.0 in all three animals.

Treatment of the eyes with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage.

Corrosion
There was no evidence of ocular corrosion.

See also attached tables

Other effects:

Coloration / Remnants
Remnants of the test substance were present in the eye on Day 1. No staining of (peri) ocular tissues by the test substance was observed.

Toxicity / Mortality
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Applicant's summary and conclusion

Interpretation of results:

Category 2B (mildly irritating to eyes) based on GHS criteria

Remarks:

Classified as Category 2 according to Regulation (EC) 1272/2008

Conclusions:

Irritating.