Trimagnesium bis(orthophosphate)

Endpoint summary

Description of key information

Skin Irritation: not irritating in vitro (OECD 439, GLP, RL1)
Eye irritation: not irritating in in vivo (OECD 405, GLP, RL1)

Respiratory irritation: no study available

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 July to 30 July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. This study is conducted according to an appropriate validated in vitro guideline and under the conditions of GLP and therefore the study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for this endpoint. In addition, the data is considered to be adequate and reliable for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP).

Qualifier:

according to guideline

Guideline:

other: EU Guideline Testing of Chemicals B46

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2010)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult donors

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ model kit
- Tissue batch number(s): not specified
- Production date: not specified
- Shipping date: not specified
- Delivery date: 12 June 2012
- Date of initiation of testing: exposure: 11 July 2012; assessment of repsonse: 26 July

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Dulbecco's Phosphate Buffered Saline (DPBS) with Ca2+ and Mg2+. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL MTT solution
- Incubation time: 3 hours at 37°C
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 540 nm
- Filter: without reference filter

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA
For the test item, the relative mean tissue viabilities obtained after the 15 min exposure period followed by the 42 h post-exposure incubation period were compared to the mean of the negative control tissues (n = 3). The relative mean viabilities were calculated as follows:

Relative mean viability (%) = (mean OD540 of test item / mean OD540 of negative control) x 100

Classification of irritation potential was based upon relative mean tissue viability following the 15 min exposure period followed by the 42 h post-exposure incubation period according to the following criteria:

Relative mean tissue viability ≤ 50%: Irritant (Category 2 according to Regulation (EC) No. 1272/2008 (CLP) and UN-GHS)
Relative mean tissue viability ≥ 50%: Non-irritant (Not classified according to Regulation (EC) No. 1272/2008 (CLP) and UN-GHS)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 mg

VEHICLE
-no vehicle used

NEGATIVE CONTROL
- Amount(s) applied: 10 µL Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca2+ and Mg2+


POSITIVE CONTROL
- Amount(s) applied: 10 µl sodium dodecylsulfate
- Concentration: 5% (w/v)

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

triplicates

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 min

Value:

118.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

8.6% tissue viability

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: not specified
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

The individual and mean OD540 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Table 1. The mean viability values and standard deviations of the test item and positive control, relative to the negative control are also given in Table 1.The relative mean viability of the test item treated tissues was 118.8% after a 15-minute exposure period.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 8.6% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.0%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was 0.659 and the standard deviation value of the percentage viability was 6.2%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 7.6%. The test item acceptance criterion was therefore satisfied.

Table1 : Mean OD₅₄₀ Values and Percentage Viabilities for the Negative Control Material, Positive Control Material and Test Material

Material	OD540of tissues	Mean OD540of triplicate tissues	±SDof OD540	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Material¤	0.666	0.659	0.041	101.1	100*	6.2
	0.696			105.6
	0.615			93.3
Positive Control Material¤	0.059	0.056	0.006	9.0	8.6	1.0
	0.061			9.3
	0.049			7.4
Test Material	0.823	0.783	0.050	124.9	118.8	7.6
	0.727			110.3
	0.799			121.2

SD=    Standard deviation

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

¤ = Control group shared with Harlan Laboratories Ltd, Project numbers 41200853, 41200860, 41200866, 41200871, 41200880, 41200884 and 41201543

Interpretation of results:

GHS criteria not met

Conclusions:

The test material was considered to be Non-Irritant (NI).
This study is conducted according to an appropriate validated in vitro guideline and under the conditions of GLP and therefore the study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for this endpoint. In addition, the data is considered to be adequate and reliable for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP). Trimagnesium bis(orthophosphate) is not considered to be classified in accordance with Regulation (EC) No. 1272/2008 (EU CLP).

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:

The study was performed between 30 July 2012 and 09 August 2012.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in compliance with agreed protocols, with no deviations from standard test guidelines and no minor methodological deficiencies. This study is conducted according to an appropriate guideline and under the conditions of GLP and therefore the study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement (Regulation (EC) No. 1907/2006; REACH) as a key study for this endpoint. In addition, this study is considered to be acceptable for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP).

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

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
- Source:
Harlan Laboratories UK Ltd., Leicestershire, UK.

- Age at study initiation:
Twelve to twenty weeks old

- Weight at study initiation:
2.33 or 2.65 kg

- Housing:
The animals were individually housed in suspended cages. The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

- Diet:
ad libitum (2030 Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK)

- Water:
ad libitum.

- Acclimation period:
At least five days


ENVIRONMENTAL CONDITIONS

- Temperature (°C):
17 to 23°C

- Humidity (%):
30 to 70%

- Air changes (per hr):
At least fifteen changes per hour

- Photoperiod (hrs dark / hrs light):
Twelve hours continuous light (06:00 to 18:00) and twelve hours darkness


IN-LIFE DATES:
From: day 1 To:day 3

Vehicle:

unchanged (no vehicle)

Controls:

other: The left eye remained untreated and was used for control purposes.

Amount / concentration applied:

TEST MATERIAL

- Amount(s) applied (volume or weight with unit): A volume of 0.1 mL of the test item, which was found to weigh approximately 57 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball.

- Concentration (if solution):
Undiluted and used as supplied

VEHICLE

- Amount(s) applied (volume or weight with unit):
Not applicable

- Concentration (if solution):
Not applicable

- Lot/batch no. (if required):
Not applicable

- Purity:
Not reported

Duration of treatment / exposure:

single treatment, eyes were not washed out

Observation period (in vivo):

Approximately 1 hour and 24, 48 and 72 hours following treatment.

Number of animals or in vitro replicates:

2 animals were tested in total. (After consideration of the ocular responses produced in the first treated animal, one additional animals was treated. )

Details on study design:

REMOVAL OF TEST SUBSTANCE

- Washing (if done):
Not applicable

- Time after start of exposure:
Not applicable


SCORING SYSTEM:
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation given in Appendix 2, (from Draize J H (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington DC p.48 to 49).


TOOL USED TO ASSESS SCORE:

Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: Initial pain reaction = 2

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0.66

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritant / corrosive response data:

Individual and group mean scores for ocular irritation are given in Table 1 and Table 2.
No corneal effects were noted during the test.
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. Moderate conjunnctival irritation was one in one treated eye and minimal conjunctival irritation was noted in the other treated eye at the 24-hour observation with minimal conjunctival irritation noted in both treated eyes at the 48-hour observation.
Both treated eyes appeared normal by the 72-hour observation.

Other effects:

Bodyweight
Both animals showed expected gain in bodyweight during the study.

Interpretation of Results

The numerical values corresponding to each animal, tissue and observation time were recorded. The data relating to the conjunctivae were designated by the letters A (redness), B (chemosis) and C (discharge), those relating to the iris designated by the letter D and those relating to the cornea by the letters E (degree of opacity) and F (area of cornea involved). For each tissue the score was calculated as follows:

Score for conjunctivae =         (A + B + C) x 2
Score for iris                            =         D x 5
Score for cornea                      =         (E x F) x 5

Using the numerical data obtained a modified version of the system ( Modified Kay and Calandra Interpretation of Eye Irritation Test was used to classify the ocular irritancy potential of the test material. This was achieved by adding together the scores for the cornea, iris and conjunctivae for each time point for each rabbit. The group means of the total scores for each observation were calculated. The highest of these group means (the maximum group mean score) together with the persistence of the reactions enabled classification of the eye irritancy potential of the test material.

If evidence of irreversible ocular damage is noted, the test material will be classified as corrosive to the eye.

 

Table1               IndividualScores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex	72304Male				72338Male
	IPR= 2				IPR = 2
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	1 Hour	24 Hours	48 Hours	72 Hours
CORNEA
E = Degree of Opacity	0	0	0	0	0	0	0	0
F = Area of Cornea Involved	0	0	0	0	0	0	0	0
Score (E x F) x 5	0	0	0	0	0	0	0	0
IRIS
D	1	0	0	0	1	0	0	0
Score (D x 5)	5	0	0	0	5	0	0	0
CONJUNCTIVAE
A = Redness	2	2	1	0	2	2	1	0
B = Chemosis	2	1	1	0	2	2	0	0
C = Discharge	2	0	0	0	2	1	0	0
Score (A + B + C) x 2	12	6	4	0	12	10	2	0
Total Score	17	6	4	0	17	10	2	0

 

IPR=  Initial pain reaction

 

Table 2               Individual Total Scores and Group Mean Scores for Ocular Irritation

 

Rabbit Number and Sex	Individual Total Scores At:
	1 Hour	24 Hours	48 Hours	72 Hours
72304 Male	17	6	4	0
72338 Male	17	10	2	0
Group Total	34	16	6	0
Group Mean Score	17.0	8.0	3.0	0.0

 

 

Interpretation of results:

GHS criteria not met

Conclusions:

The test material did not meet the criteria for classification as irritant according to Regulation (EC) No. 1272/2008 (EU CLP). This study is conducted according to the appropriate guidelines (OECD 405 ) and under the conditions of GLP and therefore the study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for this endpoint. Study is sufficient for classification and labelling purposes, in accordance with Regulation (EC) No. 1272/2008 (EU CLP).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

Skin irritation was evaluated in vitro and in vivo.

In a GLP-compliant in vitro study (Harlan, 2012), the skin irritation potential of trimagnesium bis(orthophosphate) was evaluated using the SKINETHIC™ RHE Model according to OECD Guideline 439. 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. 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 the test substance-treated tissues was 118.8% of the negative control value, while the relative mean tissue viability of the positive control was 8.6%.

This result is supported by in vivo tests done before GLP and guidelines were available (Stauffer, 1972 and Younnger, 1975).

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.

Eye irritation/corrosion

A Bovine Corneal Opacity and Permeability (BCOP) Assay was conducted with trimagnesium bis(orthophosphate) following OECD Guideline 437 and in accordance with GLP (Harlan, 2012). 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 sligthly cloudy and one was clear 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 9.0, 3.9 and 87.7.

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 but no conclusion could be made regarding eye irritating potential.

Trimagnesium bis(orthophosphate) was tested for its irritancy potential to the rabbit eye in a GLP-compliant study conducted according to OECD Guideline 405 (Harlan, 2012). Two New Zealand White rabbits were sequentially tested. In each case, 100 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 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 and 24 hours after treatment. Minimal conjunctival irritation noted in both treated eyes at the 48-hour observation. Both treated eyes appeared normal at the 72-hour observation. The test item produced individual scores of 0/0 for corneal opacity, 0/0 for iritis, 1.0/1.0 for conjunctival redness and 0.67/0.67 for chemosis, calculated as the mean scores following gradings at 24, 48 and 72 hour after instillation. Observed effects were fully reversible within the 72 h observation period.

Therefore, the test material does not fulfill 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

No classification is proposed for skin or eye irritancy of trimagnesium bis(orthophosphate) in accordance with Regulation (EC) No. 1272/2008 (EU CLP). This conclusion is based on reliable (Klimisch 1) and adequate data and as such no further testing is anticipated.