Superphosphates, concd.

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 June 2010 - 14 July 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

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:

Superphosphates, concd.

EC Number:

266-030-3

EC Name:

Superphosphates, concd.

Cas Number:

65996-95-4

Molecular formula:

not applicable

IUPAC Name:

Superphosphate, concd.

Constituent 2

Reference substance name:

Superphosphate, concd. (TSP powder)

IUPAC Name:

Superphosphate, concd. (TSP powder)

Details on test material:

- Name of test material (as cited in study report): Superphosphate, concd. (TSP powder)
- Substance type: Brownish powder
- Physical state: Solid
- Analytical purity: >70%
- Lot/batch No.: FB200110s
- Expiration date of the lot/batch: 20 January 2012
- Stability under storage conditions: Stable
- Storage condition of test material: At room temperature in the dark
- Stability under storage conditions: Stable
- pH: 3.0 at concentration of 1%

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: 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.

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.

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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1-23.4ºC
- Humidity (%): 38-83.2%
Temporary deviations from the maximum level of relative humidity occurred. Laboratory historical data do not indicate an effect of the deviations.
- 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: 10 June 2010 To: 14 July 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 (volume or weight with unit) : 68.6 mg (range 68.4 – 68.8 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 and 7, 14 and 21 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 approximately 2 weeks later, after considering the degree of eye irritation observed in the first animal.

TREATMENT
Animals were treated by instillation of, on average, 68.6 mg (range 68.4 – 68.8 mg) of the test substance (a volume of approximately 0.1 mL) as the maximum required amount 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. 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 animals were 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 after the final observation.
- Necropsy: No necropsy was performed according to protocol.
- Irritation:
The eyes of each animal were examined approximately 1, 24, 48 and 72 hours and 7, 14 and 21 days after instillation of the test substance.
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
0: Opacity: degree of density (area most dense taken for reading)
1: No ulceration or opacity (may include slight dulling of normal luster)
2: Scattered or diffuse areas of opacity, details of iris clearly visible
3: Easily discernible translucent area, details of iris slightly obscured
4: Nacreous area, no details of iris visible, size of pupil barely discernible
5: 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
Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia,
or injection, any of these or combination thereof, iris still reacting to light
1: (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

Remarks:

(opacity)

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility not applicable

Results 2

Irritation parameter:

cornea opacity score

Remarks:

(opacity)

Basis:

animal #2

Time point:

24/48/72 h

Score:

2.3

Max. score:

4

Reversibility:

not fully reversible within: 21 days

Results 3

Irritation parameter:

cornea opacity score

Remarks:

(opacity)

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 24 h

Remarks on result:

other: Slight dulling of normal luster at 1 h after instillation.

Results 4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: reversibility not applicable

Results 5

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.7

Max. score:

2

Reversibility:

fully reversible within: 72 h

Results 6

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

fully reversible within: 24 h

Results 7

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.3

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:

3

Max. score:

3

Reversibility:

not reversible

Remarks:

within 21 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: 14 days

Results 10

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 h

Results 11

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2.7

Max. score:

4

Reversibility:

fully reversible within: 21 days

Results 12

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritant / corrosive response data:

Instillation of approximately 69 mg of Superphosphate, concd. (TSP powder) (a volume of approximately 0.1 mL) into one eye of each of three rabbits resulted in effects on the cornea, iris and/or conjunctivae.
Corneal injury was observed in one animal behind the lower eyelid and consisted of opacity (maximum grade 3) and epithelial damage (maximum 25% of the corneal area). As a result of the corneal injury, pannus (neovascularization of the cornea) was apparent 14 and 21 days after instillation. In this same animal irritation of the conjunctivae (consisting of redness, chemosis and discharge) remained present up to the end of the 21-day observation period, along with gray/white discolouration of the nictating membrane and lower eyelid (sign of necrosis). Reduced elasticity of both eyelids was also observed for this animal. Irritation of the conjunctivae among the other two animals also consisted of redness, chemosis and discharge, and had completely resolved within 7 or 14 days.
Iridial irritation grade 1 was observed in two animals and had resolved within 24 or 72 hours.

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 animals during the test period and no mortality occurred.

Applicant's summary and conclusion

Interpretation of results:

corrosive

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on these results:
- according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007), Superphosphate, concd. (TSP powder) should be classified 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, Superphosphate, concd. (TSP powder) should be classified as Irreversible effects on the eye (Category 1) and labeled as H318: Causes serious eye damage.