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Administrative data

Description of key information

Skin corrosivity (in vitro, Epiderm): Not corrosive.
Skin irritation (in vitro, Episkin): Not irritating.
Eye irritation/corrosivity (ex vivo, BCOP): non-corrosive/non-severe eye irritant
Eye irritation (in vivo, rabbit): Not irritating.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 June 2010 - 18 June 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted according to official EC and OECD test guidelines, and in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Remarks:
EpiDerm™ three-dimensional human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37±1°C
- Temperature of post-treatment incubation (if applicable): 37±1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the required dosing period, each tissue was rinsed with Dulbecco’s Phosphate Buffered Saline (DPBS), for a minimum of 20 rinses, and then blotted on absorbent paper. The inserts were then transferred to the holding plate. After rinsing with DPBS and gently swabbing with a sterile swab, a small quantity of the test material remained on the surface of each of the tissues treated with test material Ferrophosphorus (Fe3P).
- Post-incubation: transferred to the MTT assay plate which was then incubated for three hours at 37±1°C in a humidified atmosphere of 5% CO2 in air. After three hours, the tissues were rinsed with DPBS and transferred to 1 ml per well extraction solution (isopropanol) in 24-well plates. A further 1 ml was then added to each insert. The tissues were extracted overnight at room temperature, without shaking. After the extraction period, triplicate 200 μl aliquots of extractant from each tissue were pipetted into the wells of flat-bottomed 96-well plates and the absorbance read at 540 nm.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mL of 1 mg/mL MTT solution in duplicate
- Incubation time: 3 minutes and 1 hour
- Spectrophotometer: yes
- Wavelength: 540 nm

NUMBER OF REPLICATE TISSUES: duplicate tissues; triplicate measurements

PREDICTION MODEL / DECISION CRITERIA
If the tissue viability from the three minute exposure is less than 50% of the negative control value, then the sample is classified as corrosive. If the tissue viability from the three minute exposure is greater than or equal to 50% of the negative control value but the one hour value is less than 15% of the negative control value, the sample is classified as corrosive. If the tissue viability from the three minute exposure is greater than or equal to 50% and the one hour value is greater than or equal to 15% of the negative control value, then the sample is classified as non-corrosive.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
Nominal weights of 25 mg of the test material was dispensed over each tissue. The test material was then wetted with 25 μl purified water.
The positive and negative controls were in liquid form and were applied by dispensing a volume of 50 μl over each tissue using a pipette.
Duration of treatment / exposure:
3 minutes; 1 hour
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
duplicate tissues; triplicate measurements
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour
Value:
97.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes
Value:
109.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Other effects / acceptance of results:
Negative control:
The mean optical density of each duplicate negative control value for the three minute and one hour contact were 1.707 and 1.805, respectively. Both values were above the minimum assay acceptance value of 0.8.

Positive control:
The mean relative tissue viability of the positive control, 8.0 N potassium hydroxide, for the one hour application was 7.0%. This value was below the maximum acceptable value of 15%.

Inter-tissue viability difference:
The coefficient of variation (CV) was not applicable for the positive control one hour application, as the mean percentage viability, 7.0%, was below the 20% - 100 % viability range. All other values did not exceed the CV value of 0.3.

Results

Reduction of MTT by test material

After the one hour incubation, the colour of the test material, Ferrophophorus (Fe3P) / MTT mixture (orange with black powder) and MTT solution control (orange) remained unchanged, from the start of incubation. Therefore, the test material had not reduced the MTT.

Interpretation of results:
GHS criteria not met
Conclusions:
The test material, Ferrophosphorus (Fe3P), elicited a mean tissue viability of 109.1% for three minute contact and 97.5% for one hour contact and was non-corrosive in the Epiderm™ Skin Corrosivity test.
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 June 2011 - 27 June 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted according to official EC and OECD test guidelines, and in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (L'Oreal, 2009).
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Remarks:
EPISKIN three-dimensional human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN (SNC, Lyon, France)
- Tissue batch number(s): 11-EKIN-025
- Production date: not specified (expiration date: 27 June 2011)
- Date of initiation of testing: 21 June 2011

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (not further specified)
- Temperature of post-treatment incubation (if applicable): 37 ± 2°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After 15 ± 0.5 minutes, each tissue was rinsed with 25 ml sterile Dulbeccos Phosphate Buffered Saline (DPBS) to remove residual test substance.
- Post-incubation: The tissues treated with the test substance, FE3P, were gently swabbed to remove the remaining test material. Inserts were then blotted on absorbent paper to remove remaining DPBS. Each insert was then transferred to a well containing 2 ml maintenance medium and incubated for 42 ± 1 hour at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air. After 42 ± 1 hour each insert was transferred to a well containing 2 ml of 0.3 mg/ml MTT and incubated for 3 hours ± 5 minutes at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air. At the end of 3 hours ± 5 minutes the triplicate inserts were blotted on absorbent paper. The epidermis was removed from the insert using a biopsy punch, the epidermis separated from the collagen matrix using forceps and both parts placed in a micro tube. When all tissues had been punched, the tissues were vortexed with 500 μL of acidic isopropanol (0.04 N HCl final concentration). The tissues were extracted by storing at 2 - 8 °C, protected from light, for 70 hours. After formazan extraction, duplicate 200 μL aliquots of the extractant from each micro tube were pipetted into the wells of flat-bottomed 96-well plates.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL MTT
- Incubation time: 15 ± 0.5 minutes
- Spectrophotometer: yes
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Morphology: Well differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.

NUMBER OF REPLICATE TISSUES: triplicate tissues; duplicate measurements

PREDICTION
If the mean tissue viability was equal to or less than 50% of the negative control value, the sample was classed as Irritant. If the mean tissue viability was greater than 50% of the negative control value, the sample was considered as Non-irritant.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): A weight of 10 ± 2 mg was dispensed over each tissue using glass weighing boats. The tissues were wetted with 5 μL of distilled water prior to application of the test substance. The test substance was spread over the surface of the tissue using a curved spatula.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): 5% SDS
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 ± 1 hours + 3 hours ± 5 minutes
Number of replicates:
triplicate tissues
Irritation / corrosion parameter:
% tissue viability
Value:
99.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
EPISKIN results
THe mean tissue viability was 99.1 ± 2.9% as compared to the negative control.

Possible reduction of MTT by test substance
There was no change in the test substance, FE3P/MTT solution or the water control/MTT solution after three hours incubation in the dark at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air. The test substance had not interacted with the MTT.

Check for colouring potential of test substance
The test substance, FE3P/water solution and water control were colourless after the 15 minute shaking period. The test substance, FE3P, had not shown any potential for colouring water.

Negative control
The mean absorbance of the triplicate negative control values was 0.758 which was between the minimum and maximum values of 0.6 and 1.5. The standard deviation (SD) of the % viability was 3.7 which was below the maximum value of 18.

Positive control
The percentage mean viability of the positive control was 14.0 ± 4.2 of the negative control. These were below the maximum acceptance values of 40% viability and SD of 18.

Deviations from Protocol

Check for colouring potential of the test substance, FE3P.

The incorrect quantity of test substance was used in the test for checking the colouring potential of the test sample. Usually 10 μg of test substance is shaken with 90 μl of distilled water (section 2.6 of protocol) however in the actual test a nominal 10 mg was used in error.

As the test substance did not show any potential for colouring of the water and the quantity of test substance used was more than required for the test, the error in the amount of test substance used was not considered to affect the scientific integrity of the test.

Measurement of pH

It was not possible to measure pH as the test substance was a solid. This was not considered to affect the scientific integrity of the study.

Interpretation of results:
GHS criteria not met
Conclusions:
It was concluded that the test substance, FE3P, with a mean tissue viability of 99.1 ± 2.9%, was predicted as non-irritant to the skin.
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:
experimental study
Adequacy of study:
key study
Study period:
01 December 2011 - 22 December 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in compliance with EC, OECD, EPA, and Japanese test guidelines, and in compliance with GLP.
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
2008-05-30
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
2002-04-24
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, Test Data for Registration of Agricultural Chemicals, Eye Irritation (2-1-5), 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Age at study initiation: 44 - 47 weeks
- Weight at study initiation: 3.98 - 4.52 kg
- Housing: Housed individually in plastic cages with perforated floors
- Diet (e.g. ad libitum): Ad libitum access to standard laboratory diet.
- Water (e.g. ad libitum): Ad libitum access to drinking water.
- Acclimation period: At least 25 weeks.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 - 20°C
- Humidity (%): 40 - 70%
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark
Vehicle:
unchanged (no vehicle)
Controls:
other: No dedicated control animals were used; in each animal the right eye was treated; the left eye remained untreated.
Amount / concentration applied:
a single ocular dose of approximately 100 mg
Duration of treatment / exposure:
Not applicable. A single instillation of the test material was made; eyelids were held together for one second before releasing.
Observation period (in vivo):
72 hours; observations were made 1, 24, 48, and 72 hours after treatment.
Duration of post- treatment incubation (in vitro):
not applicable
Number of animals or in vitro replicates:
Three females
Details on study design:
TREATMENT PROCEDURE
The eyes of each animal were examined prior to instillation of the test substance to ensure that there was no pre-existing corneal damage, iridial inflammation or conjunctival irritation. Each animal was gently restrained. The dose was instilled into the right eye by pulling the lower eyelid away from the eyeball to form a cup into which the test substance was dropped. The eyelids were then gently held together for one second before releasing; the left eye remained untreated.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): None

SERIAL OBSERVATIONS
- Clinical signs:
The behaviour of each rabbit was observed immediately following instillation of the test substance to allow assessment of the initial pain response by the pain scoring system. The animals were returned to their cages and checked at least twice during the first hour after dosing and at regular intervals throughout the day to ensure no severe injury passed unnoticed.

- Ocular responses:
Ocular reactions to treatment were assessed 1, 24, 48 and 72 hours after treatment, according to the Draize scale. Consistent with the system described in the EU test method (Commision regulation 440/2008, Part B, method B5). The untreated eye was used as a comparison with the treated eye during assessment of ocular lesions.

TOOL USED TO ASSESS SCORE: An opthalmoscope or pencil beam torch was available.

INTERPRETATION OF RESPONSES
In addition to the classification according to UN GHS (EU CLP) the classification system of Kay and Calandra (1962) was employed for classification of ocular irritants.

TERMINATION
Following completion of the observation period the animals were humanely killed by an intravenous injection of sodium pentobarbital.



Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no effects
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: no effects
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
3
Reversibility:
other: no effects
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no effects
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no effects
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: no effects
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
3
Reversibility:
other: no effects
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no effects
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no effects
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: no effects
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
3
Reversibility:
other: no effects
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no effects
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
- Injection of the conjunctival blood vessels was apparent in one animal one hour after instillation; no reaction to treatment was evident in the other animals. The treated eyes of all animals were overtly normal 24 hours after instillation
- Instillation of the test substance gave rise to practically no initial pain response.
- Accordingl to the criteria of Kay and Calandra (1962) Ferrophosphorus (Fe3P) was classified as “practically non-irritating” to the eye.
Other effects:
There was no sign of toxicity or ill health in any rabbit during the observation period.
Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, in this study and under the experimental conditions reported, Ferrophosphorus (Fe3P) does not require classification and labelling for eye irritation or serious eye damage according to UN GHS and EU CLP.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Two in-vitro tests were conducted to assess the effect of exposure of Fe3P to the skin. An Epiderm study (Huntingdon Life Sciences, 2010, study number FGE0004) assessed the potential corrosivity of the test material, whereas an Episkin study (Huntingdon Life Sciences, 2011, study number FGE0018) was performed to assess potential skin irritancy. Both studies were conducted according to official OECD test guidelines and in compliance with GLP. The overall conclusion of the two studies was that Fe3P shows no evidence of skin irritation or skin corrosion. On the basis of these two studies, it was concluded that an in-vitro test was not necessary.


 


An in-vitro study (Bovine Corneal Opacity and Permeability, BCOP, Huntingdon Life Sciences, 2011, study number FGE0017) was conducted to assess the potential of Fe3P to damage the eye. The BCOP test was negative (did not indicate any potential to cause eye irritation). This result alone was not considered sufficient to reach a definitive conclusion on the eye irritant properties of Fe3P, and so an in-vivo test was conducted (Huntingdon Life Sciences, 2012, study number FGE0019). Both studies were conducted according to official test guidelines (OECD and / or EC) and in compliance with GLP. Mild conjunctival redness was seen in one rabbit (out of three) one hour after instillation, but this was fully resolved by the 24-hour observation timepoint, and all treated animals' eyes were considered normal from the 24-hour timepoint onwards. It was concluded that Fe3P is not an eye irritant.



Justification for selection of skin irritation / corrosion endpoint:
Note that no single study has been selected, as the conclusion as "not corrosive and not irritating" is based on the results of two in-vitro tests (Episkin and Epiderm).

Justification for selection of eye irritation endpoint:
The in-vivo eye irritation test was selected as the Key study as in-vivo data is considered more reliable than in-vitro data.

Justification for classification or non-classification

Fe3P did not demonstrate any irritant properties in any of the skin or eye irritation studies conducted. On this basis it is concluded that Fe3P does not meet the criteria for classification as a skin or eye irritant under either the Classification, Labelling and Packaging (CLP) Regulation.