reaction mass of calcium bis(dihydrogenorthophosphate) and calcium hydrogenorthophosphate

Endpoint summary

Description of key information

Skin irritation: In accordance with the testing strategy detailed in Annex VIII, column 1 of Regulation (EC) No. 1907/2006 the assessment of the endpoint ‘skin irritation or skin corrosion’  has been performed following the consecutive steps detailed in the Regulation. As such an in vitro skin corrosion study has been performed. This study is not considered as the key study because it is not sufficient for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP) and is therefore submitted as supporting data.  The key study (Warren N, 2010) 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).

Eye irritation/corrosion:

- In accordance with the testing strategy detailed in Annex VIII, column 1 of Regulation (EC) No. 1907/2006 an in vitro study has been performed prior to conducting an in vivo study. This study is not considered as the key study because it is not sufficient for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP).

- A key study according to OECD guideline 405 (in vivo) has been performed and is considered to be adequate and reliable for use as a key study.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 26 May 2010 and 28 May 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 15-09-2009 Date of Signature: 26-11-2009

Test system:

other: reconstituted human epidermal model

Source species:

other: reconstituted human epidermal model

Cell type:

non-transformed keratinocytes

Cell source:

other: reconstituted human epidermal model

Source strain:

other: reconstituted human epidermal model

Details on animal used as source of test system:

Not applicable

Justification for test system used:

Test method is validated for this system

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
EPISKIN(TM)
- Tissue batch number(s):
Not given
- Delivery date:
26 May 2010 (date received)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
37°C
- Temperature of post-treatment incubation (if applicable):
37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test material
- Observable damage in the tissue due to washing:
None recorded

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
0.3 mg/mL
- Incubation time:
3 hours
- Spectrophotometer: Anthos 2001 miscoplate reader
- Wavelength - 540 nm



NUMBER OF REPLICATE TISSUES:
2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
N/A, no direct MTT interference


NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
One pre-incubation test and one test material

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to to skin if the mean tissue viability is:
- <35% after the 3-minute exposure period
- >=35% after the 3-minute exposure period and <35 % after the 60-minute exposure period
- >=35% after the 60-minute exposure period and <35 % after the 240-minute exposure period

- The test substance is considered to be non-corrosive to skin if the mean tissue viability is >=35% after the 240-minute exposure period

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg
- Concentration (if solution): N/A

VEHICLE
- N/A

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 0.9% w/v

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): ca. 100%

Duration of treatment / exposure:

3, 60 or 240 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

2

Species:

other: reconstituted human epidermis model

Strain:

other: reconstituted human epidermis model

Details on test animals or test system and environmental conditions:

Not applicable

Type of coverage:

other: topical

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL

- The test Material was applied neat.

- Amount(s) applied (volume or weight with unit):
20 mg of the test material was applied to the epidermis surface.

- Concentration (if solution):
The test material was used as supplied.

VEHICLE
No vehicle used

Duration of treatment / exposure:

3, 60, 240 minute treatments

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

TEST SITE
- Area of exposure:
20 mg of the test materialwas applied to the epidermis surface.

- % coverage:
The test material was applied topically to the corresponding tissues ensuring uniform covering.

- Type of wrap if used:
None used

REMOVAL OF TEST SUBSTANCE
- Washing (if done):
At the end of each exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Phosphate Buffered Saline Dulbeccos (PBS) with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test material. Each rinsed tissue was placed into the third column of the 12-well plate until all tissues were rinsed.


SCORING SYSTEM:
Quantitative MTT Assessment (percentage tissue viability)
The corrosivity potential of the test material was predicted from the relative mean tissue viabilities obtained after the 3, 60 and 240 minute treatments, compared to the mean of the negative control tissues (n=2) treated with 0.9% w/v sodium chloride solution. The relative mean viabilities were calculated in the following way:

mean OD540 of test material / mean OD540 of negative control x 100 = Relative mean tissue viability (percentage of negative control)

Classification of corrosivity potential was based upon relative viabilities for both exposure times according to the following:

3 minute exposure : <35 Corrosive (EU R35)

3 minute exposure : ≥35
and 60 minute exposure : <35 Corrosive (EU R34)

60 minute exposure : ≥35
and 240 minute exposure : <35 Corrosive (EU R34)

240 minute exposure : <35 Non-corrosive

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean - after 240 minutes

Value:

109.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean - after 60 minutes exposure

Value:

121.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean - after 3 minutes exposure

Value:

0.278

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Direct MTT Reduction

The MTT solution containing the test material did not turn blue/purple. This was taken to indicate the test material did not reduce MTT.

6.2Test Material, Positive Control Material and Negative Control Material

Mean OD540values and viabilities for the negative control, positive control and test material are given in Table 1.

 

The relative mean viability of the test material treated tissues was as follows:

240 minutes exposure:          109.6%

60 minutes exposure:121.9%

3 minutes exposure:              156.2%

 

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

Following the 3, 60 and 240-Minute exposure periods the test material treated tissues appeared blue which was considered to be indicative of viable tissue.

 

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 6.20/0 relative to the negative control treated tissues following the 240-minute exposure period. The positive control acceptance criterion was therefore satisfied.

Table 1. Mean OD₅₄₀Values and Viabilities for the Negative Control Material,

Positive Control Material and Test Material

Material	Exposure period	Mean OD540if duplicate tissues	Relative mean viability (%)
Negative control material	240 mins	0.178	100*
Positive control material	240 mins	0.011	6.2
Test material	240 mins	0.195	109.6
	60 mins	0.217	121.9
	3 mins	0.278	156.2

 

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

 

Table 2. Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

 

Material	Exposure period	Tissue 1	Tissue 2
Negative control material	240 mins	-	-
Positive control material	240 mins	++	++
Test material	240 mins	-	-
	60 mins	-	-
	3 mins	-	-

 

- = Blue tissue (viable)

+ = Blue/white tissue (semi-viable)

++ = Tissue completely white (dead)

Interpretation of results:

GHS criteria not met

Conclusions:

The test material was considered to be Non-Corrosive to the skin.

In accordance with the testing strategy detailed in Annex VIII, column 1 of Regulation (EC) No. 1907/2006 the assessment of the endpoint ‘skin irritation or skin corrosion’ has been performed following the consecutive steps detailed in the Regulation. As such an in vitro skin corrosion study has been performed. This study is not considered as the key study because it is not sufficient for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP). However, the study does support the conclusion that the reaction mass of calcium bis(dihydrogenorthophosphate) and calcium hydrogenorthophosphate has a low overall potential for skin irritation in vivo and the data can therefore be used to support the conclusions made in the key study.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 08 June 2010 and 14 June 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD TG Draft guideline (Version 7.6)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 15 September 2009, Date of signature: 26 November 2009

Test system:

other: reconstituted human epidermal model

Source species:

other: reconstituted human epidermal model

Cell type:

non-transformed keratinocytes

Cell source:

other: not applicable

Source strain:

other:

Justification for test system used:

This method has been validated for the assessment of skin irritation

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN(TM)
- Tissue batch number(s): Not given
- Delivery date: 08 June 2010 (date received)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test material
- Observable damage in the tissue due to washing: None recorded

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

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
N/A, no direct MTT interference


NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One pre-incubation test and one test material

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the mean tissue viability is <=50% after the 15-minute exposure period followed by the 42-hour post-exposure incubation period
- The test substance is considered to be non-irritant to skin if the mean tissue viability is >50% after the 15-minute exposure period followed by the 42-hour post-exposure incubation period

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.
- Concentration (if solution): N/A

VEHICLE
- N/A but the epidermis surface had previously been moistened with 5 ul of sterile distilled water to improve contact between the solid test material and the epidermis

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): Used as supplied

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5% w/v aqueous dilution

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

other: viability of cells

Run / experiment:

mean

Value:

100.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

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 OD₅₄₀values, 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 94.1% 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 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 OD₅₄₀for 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.

 

Table 1. Mean OD₅₄₀Values and Percentage Viabilities for the Negative Control Material, Positive Control Material and Test Material

Material	OD540of tissues	Mean OD540of triplicate tissues	±SD of OD540	Relative individual tissue viability (%)	Relative mean viability (%)
Negative Control Material	0.811	0.824	0.024	98.4	100*
	0.809			98.2
	0.852			103.4
Positive Control Material	0.053	0.033	0.018	6.4	3.9
	0.025			3.0
	0.020			2.4
Test Material	0.819	0.825	0.045	99.4	100.1
	0.784			95.1
	0.873			105.9

 

Table 2.  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)

 

SD=    Standard deviation

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

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). The reaction mass of calcium bis(dihydrogenorthophosphate) and calcium hydrogenorthophosphate 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

Remarks:

Carried out before the entry into force of the amendments to Annexes VII and VIII

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In vivo study was carried out before the entry into force of the amendments to Annexes VII and VIII

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: 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 U.K. Ltd., Loughborough, UK
- Age at study initiation: twelve to twenty weeks old
- Weight at study initiation: 2.63 kg
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): 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:

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): 0.1 mL, weighing approximately 93 mg.

Duration of treatment / exposure:

7 days

Observation period (in vivo):

7 days

Number of animals or in vitro replicates:

1

Details on study design:

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

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). Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope. Any clinical signs of toxicity, if present, were also recorded. Additional observations were made on Days 7 and 11 to assess the reversibility of the ocular effects. The animal’s bodyweight was recorded on Day 0 (the day of dosing) and at the end of the observation period.

TOOL USED TO ASSESS SCORE: ophthalmoscope

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: a score of 3 for corneal opacity was recorded on day 11

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

not fully reversible within: 11 days

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

not fully reversible within: 11 days

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 11 days

Other effects:

Individual scores for ocular irritation are given in Table 1. Opalescent areas of the cornea were noted in the treated eye at the 11-Day observation.
Iridial inflammation and moderate conjunctival irritation were noted in the treated eye one hour after treatment and at the 24, 48, 72-Hour, 7 and 11-Day observations.
A pale area on the nictitating membrane, approximately 5 mm x 5 mm in size, was noted in the treated eye at 7-Day observation. Additional ocular effects noted in the treated eye at the 11-Day observation were vascularisation, with a generalised ingrowth of vessels for approximately 3 mm, over lower half of cornea, pale areas on the remainder of the nictitating membrane and lower conjunctival membrane, blood stained discharge, a white area on the nictitating membrane, approximately 10 mm x 10 mm in size, and also scattered areas on the lower conjunctival membrane, and a small area of sloughing on lower edge of the nictitating membrane, approximately 1 mm x 4 mm in size, which had almost completely detached. The reactions noted were considered to be indicative of irreversible ocular damage. Due to worsening reactions the animal was killed for humane reasons immediately after the 11-Day observation in accordance with current UK Home Office guidelines.

Table 1: Individual scores for ocular irritation

Rabbit number and sex	69653 male
	IPR = 2
Time after treatment	1 hour	2 hours	48 hours	72 hours	7 days	11 days K
CORNEA
Degree of opacity	0	0	0	0	0	3
Area of cornea involved	0	0	0	0	0	2
IRIS	1	1	1	1	1	1
CONJUNCTIVA
Redness	2	2	2	2	2	2
Chemosis	2	2	2	2	2	2
Discharge	2	3	3	3	1	2
Other reactions	-	-	-	-	P	VBsWP*Sl

 

IPR = Initial pain reaction V = Vascularisation, with a generalised ingrowth of vessels for approximately 3 mm, over lower half of cornea

P = Pale area on nictitating membrane, approximately 5 mm x 5 mm in size

Bs = Blood stained discharge

P* = Pale area on remainder of nictitating membrane and lower conjunctival membrane

W = White area on nictitating membrane, approximately 10 mm x 10 mm in size, and scattered areas on lower conjunctival membrane

Sl = Sloughing, small area on lower edge of nictitating membrane approximately 1 mm x 4 mm in size, almost completely detached

K = Due to the persistence of severe reactions, animal killed for humane reasons in accordance with current UK Home Office guidelines

- = Not applicable

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

In accordance with Regulation (EC) No. 1272/2008 the reaction mass of calcium bis(dihydrogenorthophosphate) and calcium hydrogenorthophosphate is classified as Category 1: irreversible effects on the eye/serious damage to eyes due to the severe reactions noted at day 7 which led to the animal being euthanized for humane reasons.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

Skin irritation/corrosion: The substance 'a reaction mass of calcium bis(dihydrogenorthophosphate) and calcium hydrogenorthophosphate is not considered to be classified for skin irritation/corrosion in accordance with Regulation (EC) No. 1272/2008 (EU CLP). The key study is considered to be adequate and reliable for the purposes of classification and therefore further testing is not considered to be scientifically justified.

Eye irritation/corrosion: An in vivo study showed serious effects at day 11 which were not expected to be reversed by day 21 and as such the animal was euthanised. In accordance with Regulation (EC) No. 1272/2008 the substance is classified as corrosive to the eyes (category 1).

There are no data (workplace or study data) to suggest that the reaction mass of calcium bis(dihydrogenorthophosphate) and calcium hydrogenorthophosphate is irritating to the respiratory tract.