Aluminium tris(dihydrogen phosphate)

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). 
 - Three other studies were not considered to be acceptable and reliable for use as key data.
Eye irritation/corrosion: Five studies are available for this endpoint;
- The key study (Warren 2010) is conducted under the conditions of GLP and according to OECD 437 (ex vivo). Under the conditions of this test, and in accordance with Regulation (EC) 1272/2008, aluminium tris(dihydrogen phosphate) is classified for irreversible effects on the eye/ serious damage to eyes (Category I). 
- A supporting study conducted in vitro, using Skinethic(TM) reconstructed human corneal models and under the conditions of GLP indicates that there is potential for eye irritation. As in vitro models are not currently accepted for the assessment of eye irritation potential in accordance with Regulation (EC) 1272/2008, this study is to be used to support the conclusions found in the key study.
 - Three other studies were not considered to be acceptable and reliable for use as key data.

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 30 March 2010 and 05 April 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:

according to guideline

Guideline:

other: OECD Draft Test Guideline (version 4)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

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:

other: No vehicle used

Controls:

no

Amount / concentration applied:

TEST MATERIAL

- The test Material was applied neat.
- Amount(s) applied (volume or weight with unit): Approximately 10 mg of the test material was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 µl of sterile distilled water to improve contact between the solid test material and the epidermis.

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

VEHICLE

- No vehicle used

Duration of treatment / exposure:

15 Minutes & 42 hour post exposure incubation

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

TEST SITE

- Area of exposure: Approximately 10 mg of the test material was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 µl of sterile distilled water to improve contact between the solid test material and the epidermis.
- % 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 the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing 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.

- Time after start of exposure: 15 Minutes post exposure

SCORING SYSTEM:

Quantitative MTT Assessment (percentage tissue viability)
For the test material the relative mean tissue viabilities obtained after the 15 minute treatment 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:

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

Classification of irritation potential is based upon relative tissue viability following the 15 minute exposure period followed by the 42 hour post-exposure incubation period according to the following:

Mean tissue viability is ≤50% : Irritant (I) R38

Mean tissue viability is >50% : Non-Irritant (NI)

Irritation parameter:

other: Viability of cells (%)

Basis:

mean

Remarks:

Viability of cells

Time point:

other: Post exposure (exposed for 15 mins)

Score:

84.2

Max. score:

100

Reversibility:

other: Not applicable

Remarks on result:

other: See relative mean viability below.

Irritant / corrosive response data:

The relative mean viability of the test material treated tissues was 84.2% after a 15 minute exposure.

Other effects:

No

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 84.2% 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	0.839	0.874	0.066	96.0	100*	7.6
	0.832			95.2
	0.950			108.7
Positive Control Material	0.084	0.071	0.013	9.6	8.1	1.5
	0.058			6.6
	0.071			8.1
Test Material	0.596	0.736	0.123	68.2	84.2	14.1
	0.785			89.8
	0.827			94.6

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:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

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

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 and acceptable for classification and labelling purposes, 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 84.2% 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 vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed on 29 May 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results. This study is conducted according to an appropriate guideline and under the conditions of GLP. In accordance with Regulation (EC) No. 1272/2008 and as detailed in the Guidance on the application of the CLP criteria, a positive BCOP assay is sufficient for the purposes of classification and labelling and as such 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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

Study performed in 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Species:

other: Excised Bovine Cornea

Strain:

other: Not Applicable

Details on test animals or tissues and environmental conditions:

Not applicable

Vehicle:

physiological saline

Controls:

no

Amount / concentration applied:

TEST MATERIAL

-Amounts(s) applied (volume or weight with unit):
Triplicate corneas were treated with 0.75 ml of the test material.

-Concentration (if solution):
The test material was used as a 20% dilution.

VEHICLE
0.9% w/v sodium chloride solution.

Duration of treatment / exposure:

240 Minutes.

Observation period (in vivo):

Not Applicable.

Number of animals or in vitro replicates:

Not Applicable.

Details on study design:

TEST SITE:
-Area of exposure:
Triplicate corneas were treated with 0.75 ml of the test material.

-% coverage:
The test material preparation was topically applied to the cornea. The corneal holders were gently tilted back and forth to ensure a uniform application of the material over the entire cornea.

-Type of wrap used:
None used.

REMOVAL OF TEST SUBSTANCE:
-Washing (if done):
At the end of the exposure period the test material preparation was removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM.

-Time after start of exposure:
240 Minutes post exposure.

SCORING SYSTEM:
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.
Opacity Measurement: The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting from each the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
Permeability Measurement: The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

The following formula was used to determine the in vitro score:
In Vitro Irritancy Score = mean opacity value + (15 x mean OD492 value)

Data Interpretation:
A test material that induces an in vitro irritancy score >55.1 is defined as an ocular corrosive or severe irritant.

Additionally, the opacity and permeability values were evaluated independently to determine whether the test material induced a response through only one of the two endpoints.

Visual Observation: The condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

mean viability of opacity & Permeability (%)

Time point:

other: 240 Minutes post exposure

Score:

86.7

Max. score:

100

Reversibility:

other: not investigated

Remarks on result:

other: See below

Irritant / corrosive response data:

This BCOP is a short term assay which does not assess reversibility of reactions.

Other effects:

Not applicable.

RESULTS

Corneal Opacity and Permeability Measurement

Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Table 1. 

Corneal Epithelium Condition

The condition of the cornea immediately after rinsing is given in Table 2.

The corneas treated with the test material or positive control material were cloudy post treatment. The corneas treated with the negative control material were clear post treatment.

In Vitro Irritancy Score

The results are summarised as follows:

Treatment	In VitroIrritancy Score
Test Material	86.7
Negative Control	5.2
Positive Control	115.4

Criteria for an Acceptable Test

The positive contro lIn Vitro irritancy Score was within the range of 55.8 to 126.1. The positive control acceptance criterion was therefore satisfied.

Table 1          Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity				Permeability (OD)		In vitroIrritancy Score
		Pre-Treatment	Post-Treatment	Post-Treatment-Pre‑Treatment	Corrected Value		Corrected Value
Negative Control	1	4	8	4		0.083
	2	3	7	4		0.065
	3	3	7	4		0.090
				4.0*		0.079^		5.2
Positive Control	4	4	96	92	88.0	2.715	2.636
	5	3	69	66	62.0	3.175	3.096
	6	3	69	66	62.0	3.295	3.216
					70.7·		2.982·	115.4
Test Material	7	3	102	99	95.0	0.396	0.317
	8	4	88	84	80.0	0.346	0.267
	9	1	77	76	72.0	0.364	0.285
					82.3·		0.289·	86.7

 

OD= Optical density                 * = Mean of the post treatment-pre‑treatment values      ^= Mean permeability             ·= Mean corrected value

Table 2          Corneal Epithelium Condition

Treatment	Cornea Number	Observation
		Immediately after Rinsing
Negative Control	1	clear
	2	clear
	3	clear
Positive Control	4	cloudy
	5	cloudy
	6	cloudy
Test Material	7	cloudy
	8	cloudy
	9	cloudy

Interpretation of results:

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

Conclusions:

The test material was considered to be an ocular corrosive or severe irritant.

Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.
This study is conducted according to an appropriate guideline and under the conditions of GLP. In accordance with Regulation (EC) No. 1272/2008 and as detailed in the Guidance on the application of the CLP criteria, a positive BCOP assay is sufficient for the purposes of classification and labelling and as such 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.

Executive summary:

Introduction. A study was performed to assess the ocular irritancy potential of the test material to the isolated bovine cornea.

Method. The test material was applied at a concentration of 20% w/v for 240 minutes. Negative and positive control materials were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). 

Results. The in vitro Irritancy scores are summarised as follows:

Treatment	In Vitro Irritancy Score
Test Material	86.7
Negative Control	5.2
Positive Control	115.4

Conclusion.  The test material was considered to be an ocular corrosive or severe irritant.

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: Aluminium tris(dihydrogen phosphate) 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: Aluminium tris(dihydrogen phosphate) is classified for Irreversible effects on the eye/serious damage to eyes (Category I) 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.

Respiratory irritation: there are no data (workplace or study) to suggest that aluminium tris(dihydrogen phosphate) is irritating to the respiratory tract.