Registration Dossier

Administrative data

Description of key information

Read-across data of the components of the submission substance indicate that it is not irritating to skin, but causes irreversible effects to the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
10 June 2010 - 14 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study has been performed according to OECD and/or EC guidelines and according to GLP principles. The rationale to use data from individual constituents and components of the complex is explained in chapter 1 of the CSR and in the adjacent "read-across document".
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Deviations:
no
Qualifier:
according to
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. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Kisslegg, Germany (animal no. 1) and Harlan, Belton, Leics, England (animal nos. 2 and 3)
Use of an animal from a different source than protocolled (Harlan France or UK) is considered to have no adverse effect on the study outcome or interpretation of results since the same strain of rabbit was used and the animal was of an appropriate age and weight.
- 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): 19.1-20.0ºC
- Humidity (%): 37-84%
Temporary deviations from the maximum and minimum 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
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 (volulme or weight with unit): average: 89.3 mg (range 89.1-89.5 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, 89.3 mg (range 89.1-89.5 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. 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.
Irritation parameter:
cornea opacity score
Remarks:
(opacity)
Basis:
animal #1
Remarks:
(mean)
Time point:
other: 24, 48 and 72 hrs and 1, 14 and 21 days
Score:
0
Max. score:
4
Remarks on result:
other: Slight dulling of the normal luster of the cornea at 1 hour after instillation.
Irritation parameter:
cornea opacity score
Remarks:
(opacity)
Basis:
animal #2
Remarks:
(mean)
Time point:
other: 24, 48 and 72 hrs and 1, 14 and 21 days
Score:
0
Max. score:
4
Remarks on result:
other: Slight dulling of the normal luster of the cornea at 1 hour after instillation.
Irritation parameter:
cornea opacity score
Remarks:
(opacity)
Basis:
animal #3
Remarks:
(mean)
Time point:
other: 24, 48 and 72 hrs and 1, 14 and 21 days
Score:
0
Max. score:
4
Remarks on result:
other: Slight dulling of the normal luster of the cornea at 1 hour after instillation.
Irritation parameter:
iris score
Basis:
animal #1
Remarks:
(mean)
Time point:
other: 24, 48 and 72 hrs and 1, 14 and 21 days
Score:
0
Max. score:
2
Reversibility:
fully reversible within: 24 hr
Irritation parameter:
iris score
Basis:
animal #2
Remarks:
(mean)
Time point:
other: 24, 48 and 72 hrs and 1, 14 and 21 days
Score:
0.3
Max. score:
2
Reversibility:
fully reversible within: 48 hrs
Irritation parameter:
iris score
Basis:
animal #3
Remarks:
(mean)
Time point:
other: 24, 48 and 72 hrs and 1, 14 and 21 days
Score:
0.3
Max. score:
2
Reversibility:
fully reversible within: 48 hrs
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
animal #1
Remarks:
(mean)
Time point:
other: 24, 48 and 72 hrs and 1, 14 and 21 days
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 72 hrs
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
animal #2
Remarks:
(mean)
Time point:
other: 24, 48 and 72 hrs and 1, 14 and 21 days
Score:
3
Max. score:
3
Reversibility:
not reversible
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
animal #3
Remarks:
(mean)
Time point:
other: 24, 48 and 72 hrs and 1, 14 and 21 days
Score:
3
Max. score:
3
Reversibility:
not reversible
Irritation parameter:
chemosis score
Basis:
animal #1
Remarks:
(mean)
Time point:
other: 24, 48 and 72 hrs and 1, 14 and 21 days
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 72 hrs
Irritation parameter:
chemosis score
Basis:
animal #2
Remarks:
(mean)
Time point:
other: 24, 48 and 72 hrs and 1, 14 and 21 days
Score:
1.3
Max. score:
4
Reversibility:
fully reversible within: 14 days
Irritation parameter:
chemosis score
Basis:
animal #3
Remarks:
(mean)
Time point:
other: 24, 48 and 72 hrs and 1, 14 and 21 days
Score:
1.3
Max. score:
4
Reversibility:
fully reversible within: 14 days
Irritant / corrosive response data:
Instillation of approximately 89 mg of Single super phosphate (SSP) (a volume of approximately 0.1 mL) into one eye of each of three rabbits resulted in effects on the cornea, iris and conjunctivae.
Corneal injury consisted of slight dulling of the normal luster and had resolved within 24 hours in all animals. Iridial irritation grade 1 was observed in all animals and had resolved within 24 hours in one animal and within 48 hours in the other animals.
The irritation of the conjunctivae consisted of redness, chemosis and discharge. These findings had completely resolved within 72 hours in one animal, but remained present up to the end of the 21-day observation period in the other two animals, along with gray/white discolouration of the nictating membrane and/or lower eyelid (sign of necrosis).

Individual eye irritation scores are specified in the attached table.

Remnants of the test substance were present in the eye of all animals on Day 1.

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

Interpretation of results:
Category 1 (irreversible effects on the eye)
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), Single super phosphate (SSP) 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, Single super phosphate (SSP) should be classified as Irreversible effects on the eye (Category 1) and labeled as H318: Causes serious eye damage.
Executive summary:

Eye irritation of the test substance was tested in rabbits according to OECD TG 405 / EU B.5. One of three animals had conjunctival score of 1 (reversible) and 2/3 animals had conjunctival score of 3, irreversible within 21 days. The cornea score was zero for all animals, the iris score was zero in 1/3 animal and 0.3 in 2/3 animals (reversible) and the chemosis score was 1 in 1/3 animals and 1.3 in 2/3 animals (reversible). No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Therefore, the test substance is rated to have irreversible effects to the eyes.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Read-across data of the components of the submission substance or similar substances is available for skin irritation:

Monoammoniumphosphate:

The skin irritation potential of the test substance was tested in 6 rabbits exposed to 0.5 g test item in water (80%) for 24 h. 3/6 animals had an erythema score of 0 at the 24-h-reading and the other 3/6 animals had an erythema score of 1 at the 24-h-reading. These effects were fully reversible as 6/6 animals showed an erythema score of 0 at the 72-h-reading and at the 9-d-reading. The edema score was zero in all animals at any observation time point (24 h, 72 h, 9 d). Therefore, the test item is not irritating to skin.

Calcium superphosphate:

In a study with calcium superphosphate, the test substance proved to be not irritating to skin when tested in rabbits exposed to doses of 20 or 300 mg/kg for 24 h.

Calcium sulfate

Calcium sulfate is not irritating to skin under the conditions of an OECD TG 404 study. The substance was applied to the skin of 3 New Zealand white rabbits for 4 hours under semi-occlusvie conditions. Two animals showed slight erythema after 24 hours, which were completely reversible within 48 hours. Based on the results of this study calcium sulfate is not irritating to rabbit skin.

Humic acid potassium salt:

No irritating effects on skin were detected in a guideline study (EU B.4) with humic acid, potassium salt: The test substance was tested for acute dermal irritation in rabbits (New Zealand Albino breed).Test was performed according to Method B.4 - Acute Toxicity: Dermal Irritation/Corrosion, Directive 2004/73/EC, published in O.J. L 152, 2004. Three rabbits were exposed to of test substance, applied onto clipped skin for 4 hours using a semi-occlusive dressing. Skin reactions were evaluated after patch removal and observations were made 1, 24, 48 and 72 hours after exposure. No skin irritation was caused by 4-hour exposure. No other signs of intoxication were observed.

Read-across data of the components or similar substances is available also for eye irritation:

Single superphosphate:

Eye irritation of the test substance was tested in rabbits according to OECD TG 405 / EU B.5. One of three animals had conjunctival score of 1 (reversible) and 2/3 animals had conjunctival score of 3, irreversible within 21 days. The cornea score was zero for all animals, the iris score was zero in 1/3 animal and 0.3 in 2/3 animals (reversible) and the chemosis score was 1 in 1/3 animals and 1.3 in 2/3 animals (reversible). No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred. Therefore, the test substance is rated to have irreversible effects to the eyes.

Spporting studies on phosphates:

Single superphosphate:

Single superphosphate was tested in the BCOP (bovine cornea opacity and permeability assay). The positive and negative controls were within the historical control data. The mean in vitro irritancy score was 47 after 240 minutes of treatment with Single super phosphate (SSP). Although the corneas were turbid after the 240 minutes of treatment with Single super phosphate (SSP), the mean in vitro irritancy score (IVIS) for Single super phosphate (SSP) was below 55.1 after 240 minutes treatment. A substance producing an IVIS equal to or higher than 55.1 is defined as corrosive or severely irritating. Reaction mass of calcium bis(dihydrogenorthophosphate) and calcium hydrogenorthophosphate: Severe eye reactions were noted in a OECD 405 study at day 7 which led to the animal being euthanized for humane reasons. In accordance with Regulation (EC) No. 1272/2008 the reaction mass of calcium bis(dihydrogenorthophosphate) and calcium hydrogenorthophosphate therefore is considered to be severely irritating to eyes. Calcium sulfate:

The test substance was tested for eye irritation in an OECD 405 study with 3 New Zealand white rabbits. Slight effects on cornea, conjunctivae and regarding chemosis were observed (highest individual score 0.33 for 24 to 72 h), which were reversible within 72 hours. Slight hyperemia of conjunctiva was still visible in one animal at 72 hours, but from the (non-)severeness of the effect it can be concluded that this effect was reversible as well. Based on the results from this study the substance is considered to be not irritating to the rabbit eye.

.

Humic acid potassium salt:

The test substance was tested for eye irritation/corrosion using albino rabbits (New Zealand Albino breed). The test was performed according to the EU Method B.5. The test was performed initially using one animal. Because neither a corrosive nor a severe irritant effect was observed in initial test, the negative response was confirmed using two additional animals. The following changes were observed: At 1 hour after application in all animals: conjunctivae - some blood vessels hyperaemic (injected) or diffuse, crimson colour, individual vessels not easily discernible, in one animal chemosis: some swelling above normal. At 24 hours after application changes were observedin two animals: conjunctivae - some blood vessels hyperaemic (injected). At 48, 72 hours after application no other signs of eye irritation were observed. No clinical signs of systemic intoxication were detected. Examination of eye irritation after single application demonstrated that the test substance is not irritating to rabbit eyes.

The data summarised above indicate that the submission substance is not irritating to skin, but causes irreversible effects to the eyes.

No information on respiratory irritation is available.


Effects on eye irritation: highly irritating

Justification for classification or non-classification

Due to the findings explained above, the submission substance is not classified for skin irritation according to Regulation (EC) No. 1272/2008, but for eye irritation as Category 1: irreversible effects on the eye/serious damage to eyes.