Tricalcium bis(orthophosphate)

Endpoint summary

Description of key information

Skin irritation (similar to OECD 404, RL1): not irritating 
Eye irritation (OECD 405, RL1): not irritating

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:

The study was performed between 19 January 2010 and 25 January 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EU Guideline Testing of Chemicals B46

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Test system:

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(TM) Model Kit
- Tissue batch number(s): no data
- Production date: no data
- Shipping date: no data
- Delivery date: 19 January 2010
- Date of initiation of testing: 20 January 2010

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: rinsing by filling and emptying each tissue insert for approximately 40 seconds at a constant stream of PBS
- Observable damage in the tissue due to washing: no data
- Modifications to validated SOP: no data

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 540 nm
- Filter: without reference filter
- Linear OD range of spectrophotometer: no data

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure period followed by the 42 hour post-exposure period is less than 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure period followed by the 42 hour post-exposure period is greater than or equal to 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 ± 2 mg (epidermis was moistened with 5 µL sterile distilled water before treatment)

NEGATIVE CONTROL
- Amount(s) applied: 10 µL PBS


POSITIVE CONTROL
- Amount(s) applied: 10 µL SDS
- Concentration: 5% w/v

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

test material

Value:

100

Vehicle controls validity:

not applicable

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: the test material did not directly reduce MTT

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

RESULTS

Direct MTT Reduction

The MTT solution containing the test material did not turn blue/purple which indicated that the test material did not directly reduce MTT.

Test Material, Positive Control Material and Negative Control Material

The individual and mean OD₅₄₀ values, standard deviations and tissue viabilities for the test material, negative control material and positive control material are given in Table 1. The mean viabilities and standard deviations of the test material and positive control, relative to the negative control are also given in Table 1.

The relative mean viability of the test material treated tissues was 100.0% after a 15 minute exposure.

The qualitative evaluation of tissue viability is given in Table 2.

Following the 15-minute exposure the test material treated tissues appeared blue/white which was considered indicative of viable tissue.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was ≤ 40% relative to the negative control treated tissues and the standard deviation value of the percentage viability was ≤ 20%. The positive control acceptance criterion was therefore satisfied.

The mean OD₅₄₀ for the negative control treated tissues was ≥0.6 and the SD value of the percentage viability was ≤ 20%. The negative control 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	0.787	0.825	0.054	95.4	100*	6.6
	0.887			107.5
	0.801			97.1
Positive Control Material	0.037	0.033	0.005	4.5	4.0	0.6
	0.034			4.1
	0.027			3.3
Test Material	0.790	0.825	0.040	95.8	100.0	4.8
	0.817			99.0
	0.869			105.3

SD=    Standard deviation

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

Table2 : Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material	Tissue 1	Tissue 2	Tissue 3
Negative Control Material	-	-	-
Positive Control Material	++	++	++
Test Material	-	-	-

MTT visual scoring scheme
-          =         blue tissue (viable)
+         =         blue/white tissue (semi-viable)
++       =         tissue is completely white (dead)

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). Tricalcium bis(orthophosphate) is not considered to be classified in accordance with Regulation (EC) No. 1272/2008 (EU CLP).

Executive summary:

Introduction: The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN^TMreconstituted human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 -[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 hour post-exposure incubation period is also determined for test materials which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.

Methods:

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for approximately 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96 -well plate. The optical density was measured at 540 nm.

Data are presented in the form of percentage viability (MTT reduction in the test material treated tissues relative to negative control tissues).

Results: 

The relative mean viability of the test material treated tissues was 100.0% after a 15-minute exposure.

Quality criteria: 

The quality criteria required for acceptance of results in the test were satisfied.

Conclusion:  The test material was considered to be Non-Irritant (NI).

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 06 May 2010 and 27 May 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Remarks:

Department of Health of the Government of the United Kingdom

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Limited, Bicester, Oxon, UK
- Age at study initiation: Twelve to twenty weeks old
- Weight at study initiation: 2.0 to 3.5 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 Teklad, Blackthorn, Bicester, 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

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL equal to approximately 99 mg


VEHICLE
none: used as supplied

Observation period (in vivo):

Approximately 1 hour and 24, 48 and 72 hours following treatment. An additional observation was made in one treated eye on Day 7 to assess the reversibility of the ocular effects.

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: eyes not washed

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:

animal #1

Remarks:

69200 Male

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

69242 Male

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

69200 Male

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: No effect observed

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

69242 Male

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: No effect observed

Irritation parameter:

conjunctivae score

Basis:

animal #1

Remarks:

69200 Male

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Remarks:

69242 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0.66

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

69200 Male

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

69242 Male

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

Individual and group mean scores for ocular irritation are given in Table 1 and Table 2.
No corneal or iridial effects were noted during the test.
Moderate conjunctival irritation was noted in both treated eyes one hour after treatment with minimal conjunctival irritation noted at the 24 and 48 hour observations. Minimal conjunctival irritation persisted 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:

Body weight
Both animals showed expected gain in bodyweight during the study.

Table1               IndividualScores and Individual Total Scoresfor Ocular Irritation

Rabbit Number and Sex	69200Male					69242Male
	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
E = Degree of Opacity	0	0	0	0	0	0	0	0	0
F = Area of Cornea Involved	0	0	0	0	0	0	0	0	0
Score (E x F) x 5	0	0	0	0	0	0	0	0	0
IRIS
D	0	0	0	0	0	0	0	0	0
Score (D x 5)	0	0	0	0	0	0	0	0	0
CONJUNCTIVAE
A = Redness	2	1	1	1	0	2	1	1	0
B = Chemosis	2	1	0	0	0	2	1	0	0
C = Discharge	2	1	0	0	0	2	1	0	0
Score (A + B + C) x 2	12	6	2	2	0	12	6	2	0
Total Score	12	6	2	2	0	12	6	2	0

IPR=  Initial pain reaction (scroes 0 - 5; 2= Rabbit blinks and tries to pen eye, but refelex closes it)

Interpretation of results:

GHS criteria not met

Conclusions:

The test material produced a maximum group mean score of 12.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.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 (EU ) 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).

Executive summary:

Introduction. 

The study was performed to assess the irritancy potential of the test material to the eye of the New Zealand White rabbit. The method was designed to meet the requirements of the following:

OECD Guidelines 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

Initial considerations.

Due to the high pH value of the test material a Rabbit Enucleated Eye Test (REET) was performed prior to thein vivo test. The results are given in Appendix 4 and indicated that the test material was unlikely to cause severe ocular irritancy.

Result. 

A single application of the test material to the non-irrigated eye of three rabbits produced 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 material produced slight and reversable conjunctivae and chemosis reactions.

The test material did not meet the criteria for classification as irritant according to 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:

In order to evaluate the skin irritating potential of tricalcium bisorthophosphate an in vitro skin irritation and an in vitro skin corrosion test is available.

The skin irritation potential of the test material was evaluated using the EPISKIN^TMreconstituted human epidermis model. 

Triplicate tissues were treated with 10 mg of test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for approximately 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96 -well plate. The optical density was measured at 540 nm.

The relative mean viability of the test material treated tissues was 100.0% after a 15-minute exposure.

The test material was considered to be non-irritant.

The skin corrosion potential of the test material was evaluated using the SkinEthic in vitro Reconstituted Human Epidermal (RHE) Model after treatment periods of 3 and 60 minutes (according to OECD 431 (adopted 2004)). Duplicate SkinEthic RHE tissues were topically treated with the test material for exposure periods of 3 minutes and 60 minutes.

Duplicate negative control and positive control treated tissues were concurrently exposed for 3 minutes and 60 minutes.

At the end of the exposure period each SkinEthic RHE tissue was rinsed using Dulbecco’s phosphate buffered saline (DPBS) and placed into a ‘holding plate’. They were then transferred to an MTT ‘loading plate’ and incubated (37°C, 5% CO₂) for 3 hours. At the end of this time, each SkinEthic RHE tissue was blotted dry and placed into an MTT ‘extraction plate’ to extract the reduced MTT from the tissues.

At the end of the extraction period, the reduced MTT solution was mixed for each SkinEthic RHE tissue and 3 x 200 µl samples for each tissue were transferred to the appropriate wells of a 96 well plate. The absorbency at 540 nm (OD₅₄₀) of each well was measured. Data are presented in the form of relative mean viability (MTT reduction in the test material treated tissues relative to negative control tissues) for each of the two exposure times. The relative mean viability of the test material treated tissues was 102.9% after a 3 minute exposure and 97.0% after a 60 minute exposure.

The test material was considered not to have the potential to be corrosive in vivo.

 

Eye Irritation:

One in vivo eye irritation test is available for tricalcium bis(orthophosphate). This eye irritation test was performed in 2010 according to OECD Guideline 404. The in vivo test was initially performed with one male New Zealand white rabbit and repeated with a second one after no severe effects were observed (2010). A volume of 0.1 mL of the test material, which was found to weigh approximately 99 mg was placed into the conjunctival sac of the right eye. After application the animals were observed 1, 24, 48, and 72 hours and 7 days after application. Evaluation of the two animals resulted in mean scores for the 24, 48 and 72 hour reading for cornea opacity, iritis, conjunctivae redness and chemosis 0.0, 0.0, 0.83 and 0.33, respectively. Conjunctivae redness and chemosis were reversible latest until day 7 after treatment. These effects leading to the conclusion that tricalcium bis(orthophosphate) is not irritating to the eyes.

Before the in vivo test was done in 2010, an in vitro Rabbit Enucleated Eye test was performed due to the low pH value of the test material. This test was performed before guidelines for in vitro tests were available. As this OECD guideline 492 is available since 2015 and the test was performed in 2010 a major deficiency is the too short incubation time of 10 minutes instead of 6 hours for solids. Furthermore, 30 mg instead of 50 mg were used for the test. The relative mean viability of the test material treated tissue was 99.6% after a 10-minute exposure which leads to the conclusion that the test material has no irritating potential. Due to the major deficiencies this study was disregarded.

 

Conclusion

Taken together, the available data on irritation/corrosion with tricalcium bis(orthophosphate) (CAS 7758 -87 -4) does not indicate any hazardous potential on skin and eyes. Thus, the test substance is not to be classified regarding skin and eye irritation.

Justification for classification or non-classification

The available data on skin and eye irritation with tricalcium bis(orthophosphate) do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.

There are no data (study or workplace) to support a classification for respiratory irritation.