Trimanganese bis(orthophosphate)

Endpoint summary

Description of key information

Skin irritation/corrosion: not corrosive and not irritating (OECD 431 and 439, GLP)
Eye irritation: not irritating (OECD 405, GLP)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-10-23 to 2012-10-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

2004-04-13

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method 40 bis: In vitro skin corrosion: human skin model test

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2012-11-30

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: room temperature in the dark

Test system:

human skin model

Source species:

human

Cell type:

other: human-derived epidermal keratinocytes

Cell source:

other: not specified

Source strain:

not specified

Details on animal used as source of test system:

not applicable

Justification for test system used:

Validation studies have shown that tests employing human skin models are able to discriminate between known skin corrosives and non-corrosives.

Vehicle:

other: 0.9% w/v sodium chloride solution

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin TM tissues (purchased from SkinEthic Laboratories, Lyon, France)
- Batch No.: 13-EKIN-039
- Delivery date: 2012-10-23
- Date of initiation of testing: 2012-10-24 (following overnight pre-incubation)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature of pre-incubation: approx. 37 °C (incubated overnight)
- Temperature used during treatment / exposure: approx. 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
At the end of the exposure period the tissues were removed from the the plate and rinsed with DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL MTT solution (2.0 mL/well)
- Incubation time: 3 hours
At the end of the 3-hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was taken uand the epidermis was separated from the collagen matrix. Both parts (epidermis and collagen matrix) were placed into micro tubes containing acidified isopropanol. Each tube was plugged, mixed thoroughly and stored overnight at room temperature, protected from light, to extract formazan crystals out of the MTT-loaded tissues.
At the end of the formazan extraction period each tube was mixed to produce a homogenous coloured solution. For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using a microplate reader.

TEST FOR DIRECT MTT REDUCTION
The test item should be evaluated for their potential to interfere with MTT assay. 20 mg of the test item was added to 2.2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at room temperature for 3 hours. Untreated MTT solution was used as a control. If the MTT solution containing the test item turns blue relative to the control, the test item was presumed to have reduced the MTT.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: tissues pass analysis for tissue functionality
- Contamination: absence of bacteria, fungus and mycoplasma as well as absence of HIV1 antibodies, HIV2 antibodies, Hepatitis B antigen, Hepatitis C antibodies
Please also refer to the field "Attached background material" below.

QUANTITATIVE MTT ASSESSMENT
The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3, 60 and 240-Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with 0.9% w/v sodium chloride solution.
The remative mean viabilities were calculated in the following way: relative mean viability (%) = (mean OD540 of test item / mean OD540 of negative control) x 100

PREDICTION MODEL / DECISION CRITERIA
Classification of corrosivity potential was based on relative viabilities for each exposure time according to the prediction model as shown in the field "Any other information on materials and methods incl. tables" below (Table 1).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 20 mg of the solid test item

VEHICLE
- Amount(s) applied: 100 μl of 0.9% w/v sodium chloride solution

NEGATIVE CONTROL
- Amount(s) applied: 50 µL of 0.9% w/v sodium chloride solution

POSITIVE CONTROL
- Amount(s) applied: 50 µL of glacial acetic acid

Duration of treatment / exposure:

3, 60 and 240 minutes

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

Test item: duplicate
Negative control: duplicate
Positive control: duplicate

Irritation / corrosion parameter:

% tissue viability

Remarks:

(mean)

Run / experiment:

3 minute exposure

Value:

115.8

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

not examined

Irritation / corrosion parameter:

% tissue viability

Remarks:

(mean)

Run / experiment:

60 minute exposure

Value:

102.1

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

not examined

Irritation / corrosion parameter:

% tissue viability

Remarks:

(mean)

Run / experiment:

240 minute exposure

Value:

102.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS
- Direct MTT reduction: the MTT solution containing the test item did not turn blue. This was taken to indicate the test item did not reduce MTT.

ACCEPTANCE OF RESULTS
- Acceptance criteria met for positive control: the relative mean tissue viability for the positive control treated tissues was 2.5% relative to the negative control treated tissues following the 240-Minute exposure period (acceptibility criteria: relative mean tissue viability for the positive control should be 0 to 20 % relative to the negative control following 240 minute exposure).
- Acceptance criteria met for negative control: the mean OD540 for the negative control treated tissues was 0.812 (acceptability criteria: ≥ 0.600 and ≤ 1.500).

Table 3: Results after treatment with trimanganese bis(orthophosphate) and the controls

Dose Group	Exposure Interval	Mean OD540 of individual tissue 1	Mean OD540 of individual tissue 2	Mean Absorbance (OD540) of duplicate tissues	Rel. mean viability (%)
Test Item	3 minutes	0.955	0.925	0.940	115.8
Test Item	60 minutes	0.800	0.858	0.829	102.1
Negative Control	240 minutes	0.767	0.856	0.812	100*
Positive Control		0.015	0.025	0.020	2.5
Test Item		0.846	0.811	0.829	102.1

* The mean viability of the negative control tissues is set at 100%.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not corrosive to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is not corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-11-06 to 2012-11-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2010-07-22

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2012-11-30

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: room temperature in the dark

Test system:

human skin model

Source species:

human

Cell type:

other: normal, human-derived epidermal keratinocytes

Cell source:

other: not specified

Source strain:

not specified

Details on animal used as source of test system:

not applicable

Justification for test system used:

In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiSkin™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.

Vehicle:

other: distilled water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin™ model (source: SkinEthic Laboratories, Lyon, France)
- Batch No.: 12-EKIN-041

TEMPERATURE USED FOR TEST SYSTEM
- Temperature of pre-incubation: approx. 37 °C (overnight)
- Temperature used during treatment / exposure: approx. 37 °C
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
Tissues were rinsed with DPBS with Ca and Mg. Rinsing was achieved by filling and emptying each tissue insert for approx. 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.
The rinsed tissues were transferred to wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT/ EXPOSURE
- MTT concentration: 0.3 mg/ml MTT solution
- Incubation time: 3 hours
- Extraction of Formazan: after the incubation period, each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10°C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution. For each tissue, duplicate 200 μlL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μl of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured using an Anthos 2001 microplate reader.
- Spectrophotometer:
- Wavelength: 540 nm (without a reference filter)

TEST FOR DIRECT MTT REDUCTION
Eeach test item is checked for the ability to directly reduce MTT according to the following procedure: 10 mg of the test item was added to 2 mL of a 0.3 mg/ml MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37ºC, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control. If the MTT solution containing the test item turns blue, the test item is presumed to have reduced the MTT.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: tissues pass analysis for tissue functionality
- Contamination: absence of bacteria, fungus and mycoplasma as well as absence of HIV1 antibodies, HIV2 antibodies, Hepatitis B antigen, Hepatitis C antibodies
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
For the test item the relative mean tissue viabilities obtained after the 15-minute exposure period followed by the 42-hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way: relative mean viability(%) = (mean OD540 test item/mean OD540 of negative control) x 100.
Classification of irritation potential is based upon relative mean tissue viability following the 15-minute exposure period followed by the 42-hour post-exposure incubation period according to the following prediction model: if the mean relative tissue viability of three individual tissues is less or equal to 50% of the negative control, the test item needs to be classified and labeled for its skin irritauion potential: Category 2 – irritant, H315 according to Regulation (EC) No 1272/2008.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 10 mg of the test item, wetted with vehicle

VEHICLE
- Amount(s) applied (volume or weight with unit): 5 µL of sterile distilled water

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL of a 5% w/v Sodium Dodecyl Sulphate (SDS) solution

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Test item: triplicates
Negative control: triplicates
Positive control: triplicates

Irritation / corrosion parameter:

% tissue viability

Remarks:

(mean)

Value:

105.4

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:

OTHER EFFECTS
- Direct-MTT reduction: MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the mean OD540 for the negative control treated tissues was 0.697 and the standard deviation value of the percentage viability was 8.2%. The negative control acceptance criterion was therefore satisfied (acceptability criteria: mean OD540 ≥0.6; standard deviation value of the percentage viability: ≤18%).
- Acceptance criteria met for positive control: the relative mean tissue viability for the positive control treated tissues was 5.4% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 2.2%. The positive control acceptance criterion was therefore satisfied (acceptability criteria: relative mean tissue viability ≤40%; standard deviation value of the percentage viability: ≤18%).
- Acceptance criteria met for variability between replicate measurements: standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 2.1%. The test item acceptance criterion was therefore satisfied (acceptability criteria: individual percentage tissue viabilities of the three identically treated tissues: ≤18%).

Table 1: Results after treatment with Trimanganese bis(orthophosphate) and controls

Dose Group	Treatment Interval	OD540 of Tissue 1	OD540 of Tissue 2	OD540 of Tissue 3	Mean Absorbance of 3 Tissues	Mean Rel. Absorbance [% of Negative Control]
Negative Control	15 min	0.728	0.631	0.732	0.697	100*
Positive Control	15 min	0.032	0.055	0.026	0.038	5.4
Test Item	15 min	0.751	0.723	0.731	0.735	105.4

*The mean viability of the negative control tissues is set at 100%.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-irritant.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance does not require classification as skin irritant.

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:

14 - 31 Jan 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2002-04-24

Deviations:

yes

Remarks:

only 1 animal (instead of 2) was tested in the confirmatory test

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

2008

Deviations:

yes

Remarks:

only 1 animal (instead of 2) was tested in the confirmatory test

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Leicestershire, UK
- Age at study initiation: 12-20 weeks
- Weight at study initiation: 2.14-2.62 kg
- Housing: animals were individually housed in suspended cages.
- Diet: 2930C Teklad Global Rabbit diet (Harlan Laboratories UK Ltd., Oxon, UK), ad libitum
- Water: mains drinking water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23
- Humidity (%): 30-70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL (ca. 93 mg)

Duration of treatment / exposure:

single instillation without washing

Observation period (in vivo):

up to 7 days
Reading time points: 1, 24, 48 and 72 h and 7 days

Number of animals or in vitro replicates:

2

Details on study design:

SCORING SYSTEM: Draize scoring system

TOOL USED TO ASSESS SCORE: ophthalmoscope

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal '2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0.7

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

Individual scores for ocular irritation are given in Table 1. The individual and mean scores as required for EC and GHS classification and labelling are presented in Table 2.
No corneal effects were noted during the study.
Iridial inflammation was noted in both treated eyes one hour after treatment.
Moderate conjunctival irritation was noted in both treated eyes one hour after treatment. Minimal conjunctival irritation was noted in both treated eyes at the 24 and 48-Hour observations and in one treated eye at the 72-Hour observation.
One treated eye appeared normal at the 72-Hour observation and the other treated eye appeared normal at the 7-Day observation.

Other effects:

Individual bodyweights and bodyweight change are given in Table 3.
Both animals showed expected gain in bodyweight during the study.
No further local or systemic effects were observed during the study period.

Table 1 Individual Scores for Ocular Irritation

Rabbit Number and Sex	72876 Male					72927 Male
	IPR = 2					IPR = 2
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	7 Days	1 Hour	24 Hours	48 Hours	72 Hours
CORNEA
Degree of Opacity	0	0	0	0	0	0	0	0	0
Area of Cornea Involved	0	0	0	0	0	0	0	0	0
IRIS	1	0	0	0	0	1	0	0	0
CONJUNCTIVAE
Redness	2	1	1	1	0	2	1	1	0
Chemosis	2	1	1	1	0	2	1	0	0
Discharge	1	0	0	0	0	1	1	0	0

IPR = Initial Pain Reaction

Table 2 Individual and Mean Scores for Cornea, Iris and Conjunctivae

Rabbit Number and Sex	Time After Treatment	Corneal Opacity	Iridial Inflammation	Conjuctival Redness	Conjunctival Chemosis
72876 Male	24 Hours	0	0	1	1
	48 Hours	0	0	1	1
	72 Hours	0	0	1	1
Mean		0.0	0.0	1.0	1.0
72827 Male	24 Hours	0	0	1	1
	48 Hours	0	0	1	0
	72 Hours	0	0	0	0
Mean		0.0	0.0	0.7	0.3

Table 3 Individual Bodyweights and Bodyweight Change

Rabbit Number and Sex	Individual Bodyweight (kg)		Bodyweight Change (kg)
72876 Male	Day 0	Day 7	0.14
	2.62	2.76
72972 Male	Day 0	Day 3	0.04
	2.14	2.18

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is not irritating to the eyes.
According to the EC Regulation No. 1272/2008 and subsequent adaptations, the substance is not classified as an eye irritant.

Executive summary:

Introduction

The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit. The method was designed to be compatible with the following:

 OECD Guideline for the Testing of Chemicals No. 405 “Acute Eye Irritation/Corrosion” (adopted 24 April 2002)

 Method B5 Acute Toxicity (Eye Irritation) of Commission Regulation (EC) No. 440/2008

Result

A single application of the test item to the non-irrigated eye of two rabbits produced iridial inflammation and moderate conjunctival irritation. One treated eye appeared normal at the 72-Hour observation and the other treated eye appeared normal at the 7-Day observation.

Conclusion

The test item produced individual scores of 0/0 for corneal opacity, 0/0 for iritis, 1.0/0.7 for conjunctival redness and 1.0/0.3 for chemosis, calculated as the mean scores following grading at 24, 48 and 72 hour after instillation in both tested animals. Observed effects were fully reversible within the observation period.

The test item does not meet the criteria for classification according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

The test item does not meet the criteria for classification according to the Globally Harmonized System of Classification and Labelling of Chemicals.

The test item is thus considered to be non-irritating to rabbit eyes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation/corrosion

Skin corrosion (in vitro)

The corrosivity potential of trimanganese bis(orthophosphate) was tested using the EPISKIN™ in vitro Reconstituted Human Epidermis (RHE) Model following OECD Guideline 431 and complying with GLP (Warren, 2014). Duplicate tissues were treated with 20 mg of the test material for 3, 60 and 240 min. Duplicate tissues treated for 240 min with 50 µL 0.9% w/v sodium chloride or glacial acetic acid served as negative and positive controls, respectively. At the end of the exposure period, tissues were rinsed prior to MTT loading. Formazan crystals were extracted from the MTT loaded tissues by acidic isopropanol extraction. The optical density of the extracts was measured at 540 nm. The relative mean viability (MTT reduction to formazan in treated vs. negative control tissues) was calculated as percent mean optical density of the isopropanol extracts from treated tissues relative to the negative control.

The relative mean viability of tissues treated with the test material was 115.8, 102.1 and 102.1% after 3, 60 and 240 min, respectively. The relative mean viability of the positive control treated tissues was 2.5% after 240 min.

Therefore, based on the study results, the test material does not meet the criteria for classification for Skin corrosion Category 1 according to Regulation (EC) No 1272/2008 (CLP) or the Globally Harmonized System (GHS), and is thus considered to be not skin corrosive in vitro.

Skin irritation (in vitro)

In another GLP-compliant in vitro study, the skin irritation potential of trimanganese bis(orthophosphate) was evaluated using the EPISKIN™ RHE Model according to OECD Guideline 439 and EU Method B.46 (Warren, 2014). Triplicate tissues were treated with 10 mg of the test substance for 15 min, followed by rinsing and a 42 h post-exposure incubation period. Triplicate tissues concurrently treated with 10 µL of Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++ or Sodium Dodecyl Sulphate (SDS) 5% w/v served as negative and positive controls, respectively. Following the post-exposure period, MTT tissue loading and determination of relative mean viability was performed as described above under ‘Skin corrosion (in vitro)’.

The relative mean viability of the test substance-treated tissues was 105.4% of the negative control value, while the relative mean tissue viability of the positive control was 5.4%.

Therefore, based on the study results, the test item does not meet the criteria for classification according to Regulation (EC) No 1272/2008 (CLP) or the Globally Harmonized System (GHS), and is thus considered to be not skin irritating in vitro.

In support of this notion, no skin irritation was observed in guinea pigs topically treated with trimanganese bis(orthophosphate) in a skin sensitisation study (Grümmer, 2014). This suggests that the substance is likely to be not skin irritating in vivo.

Based on the negative results of the above results from valid in vitro skin corrosion and irritation studies, along with supporting evidence from an in vivo skin sensitisation study, the test material does not fulfil the criteria for classification according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), and is thus considered to be not skin irritating.

Eye irritation/corrosion

A Bovine Corneal Opacity and Permeability (BCOP) Assay was conducted with trimanganese bis(orthophosphate) following OECD Guideline 437 and in accordance with GLP (Warren, 2014). Three corneas were treated with the test substance at 20% w/v in 0.9% w/v sodium chloride solution for 240 min at 32 °C. Two groups of three corneas each treated with 0.9% w/v sodium chloride and 20% w/v imidazole in 0.9% w/v sodium chloride served as negative and positive controls, respectively. Following opacity and permeability measurements, the in vitro irritancy score was calculated.

Two corneas treated with the test item were cloudy and one was slightly cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment. The in vitro irritancy scores of the test substance-treated, negative and positive control corneas were 19.1, 4.2 and 105.5.

Therefore, the test material does not meet the criteria for classification for Severe Eye Damage Category 1 according to Regulation (EC) No 1272/2008 (CLP) or the Globally Harmonized System (GHS), and is thus considered not to be an ocular corrosive or severe irritant in vitro.

Trimanganese bis(orthophosphate) was tested for its irritancy potential to the rabbit eye in a GLP-compliant study conducted according to OECD Guideline 405 (Bradshaw, 2014). Two New Zealand White rabbits were sequentially tested. In each case, 0.1 mL (ca. 93 mg) of the test material was placed into the conjunctival sac of one eye, the untreated eye serving as control. The treated eyes were not rinsed after exposure, and ocular effects were assessed at 1, 24, 48 and 72 h and 7 days post-instillation. No corneal effects were noted during the study. Iridial inflammation was noted in both treated eyes one hour after treatment. Moderate conjunctival irritation was noted in both treated eyes one hour after treatment. Minimal conjunctival irritation was noted in both treated eyes at the 24 and 48-Hour observations and in one treated eye at the 72-Hour observation. One treated eye appeared normal at the 72-Hour observation and the other treated eye appeared normal at the 7-Day observation. The test item produced individual scores of 0/0 for corneal opacity, 0/0 for iritis, 1.0/0.7 for conjunctival redness and 1.0/0.3 for chemosis, calculated as the mean scores following grading at 24, 48 and 72 hour after instillation in both tested animals. Observed effects were fully reversible within the observation period.

Therefore, the test material does not fulfil the criteria for classification according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), and is thus considered to be not eye irritating.

Justification for classification or non-classification

Skin corrosion

The substance does not possess a skin corrosive potential based on an in vitro OECD 431 (2004) test and does not require classification as skin corrosive according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Skin irritation

The substance does not possess an skin irritating potential based on an in vitro OECD 439 (2010) test and does not require classification as skin irritating according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Eye irritation

The substance does not possess an eye irritating potential based on an in vivo OECD 405 (2002) test and does not require classification as eye irritating according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Respiratory irritation

No signs of respiratory tract irritation were observed in an acute inhalation toxicity study in rats, up to the limit concentration. No classification required as respiratory irritatant according to regulation (EC) 1272/2008.