Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
The study was performed between 03 April 2012 and 17 April 2012.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
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. The reliability has been amended in accordance with 'practical guide 6: How to report read-across and categories' which states that the maximum reliability for a read-across study is 2. The study is considered to be adequate and reliable for the purpose of registration under REACH (Regulation (EC) No. 1907/2006) and for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP). Read-across is justified on the basis that the sensitisation potential of aluminium dihydrogen triphosphate will be determined by the Al cation. Triphosphate itself is not considered to be a sensitiser, in addition, the ultimate breakdown product of triphosphate (orthophosphate) is a natural component of blood and cellular fluids. As aluminium dihydrogen triphosphate has a lower water solubility than aluminium orthophosphate it is considered to be less bioavailable and therefore aluminium orthophosphate is considered to be a worst case for sensitisation potential of the Al cation. The study reports that aluminium orthophosphate is a non-sensitiser under the conditions of the study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection: 19-21 July 2011. Date of signature: 31 August 2011
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS

- Source: Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Ltd., Oxon, UK.

- Age at study initiation: At the start of the study the animals were eight to twelve weeks old.

- Weight at study initiation: At the start of the study the animals were in the weight range of 15 to 23g.

- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.

- Diet: ad libitum (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK)

- Water: ad libitum.

- Acclimation period: At least five days.


ENVIRONMENTAL CONDITIONS

- Temperature (°C): The temperature was controlled to remain within the target ranges of 19 to 25°C.

- Humidity (%): The humidity was controlled to remain within the target ranges of 30 to 70%.

- Air changes (per hr): The rate of air exchange was approximately fifteen changes per hour.

- Photoperiod (hrs dark / hrs light): The lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

IN-LIFE DATES: From: Day 1 To: Day 6
Vehicle:
propylene glycol
Remarks:
Please see below for Vehicle Determination Record
Concentration:
Each group was exposed to concentrations of 25 &, 10 or 5% w/w (in propylene glycol)
No. of animals per dose:
Groups of four mice were treated
Details on study design:
Preliminary Screening Test
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μl of the test item at a concentration of 25% w/w in propylene glycol, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the scale included as Appendix 4. Any clinical signs of toxicity, if present, were also recorded. The
bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre-dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
-animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card

- Name of test method:
Local Lymph Node Assay in the Mouse. The assay has undergone extensive inter-laboratory validation and has been shown to reliably detect test materials that are moderate to strong sensitisers.

- Criteria used to consider a positive response:
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node(dpm/node) and as the ratio of 3HTdR incorporation in lymph node cells of test nodes relative to that recorded for the control nodes (stimulation Index).

The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitier".

TREATMENT PREPARATION AND ADMINISTRATION:
For the purpose of the study, the test material was used undiluted and also freshly prepared in propylene glycol. This vehicle was chosen as it produced the most suitable formulation at the required concentration. The concentrations used are given above.

Determination, by analysis, of the concentration, homogeneity and stability of the test material preparations was not appropriate because it was not specified in the Study Plan and is not a requirement of the Test Guidelines.

Test Material Administration
Groups of four mice were treated with the test item at concentrations of 25%, 10% or 5% w/w in propylene glycol. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 μl of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the
pipette.
A further group of four mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration:
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 μl of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/ml, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20 μCi to each mouse.

OBSERVATIONS
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the Test were recorded.
Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

TERMINAL PROCEDURES
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 ml of PBS was added to the pooled lymph nodes.

Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 ml of PBS into a petri dish labelled with the project number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 ml of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 ml of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 ml of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation: After approximately eighteen hours incubation at approximately 4°C, the precipitates were recover ed by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by b-scintillation counting. The "Poly QTM" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

Positive control substance(s):
other: Phenylacetaldehyde (90%)
Positive control results:
One group of five animals was treated with 50 µl (25 µl per ear) of Phenylacetaldehyde (90%) as a solution in propylene glycol at a concentration of 2.5% v/v. A further group of five animals was treated with propylene glycol alone.

The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:
Concentration (% v/v) in Stimulation Index Result
propylene glycol

2.5 3.49 Positive

Phenylacetaldehyde (90%) was considered to be a sensitiser under the conditions of the test.
Parameter:
SI
Remarks on result:
other: A stimulation index of less than 3 was recorded for the test material at concentrations of 10%, 5% and 2.5% w/w in propylene glycol
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The radioactive disintegrations per minute (dpm) per lymph node and the stimulation index (SI) are given in Table 2.

Preliminary Screening Test

Clinical observations, bodyweight and mortality data are given in Table 2 and local skin irritation is given in Table 3. The ear thickness measurements and mean ear thickness changes are given in Table 4.

No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.

Based on this information the dose levels selected for the main test were 25%, 10% and 5% w/w in propylene glycol.

Main Test

Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 1.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Table 1.

Concentration (%w/w) in
propylene glycol

Stimulation Index

Result

5

1.06

Negative

10

1.01

Negative

25

0.89

Negative

Clinical Observations and Mortality Data

Individual clinical observations and mortality data for test and control animals are given in Table 5.

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Bodyweight

Individual bodyweights and bodyweight changes for test and control animals are given in Table 6.

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Table 2             Clinical Observations, Bodyweight and Mortality Data – Preliminary Screening Test

Concentration (%w/w) in
propylene glycol

Animal Number

Bodyweight (g)

Day

1

2

3

4

5

6

Day 1

Day 6

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Pre-Dose

Post Dose

25

S-1

20

19

0

0

0

0

0

0

0

0

0


0=      No signs of systemic toxicity

Table 3. Local skin irritation - preliminary screening test

Concentration

(%w/w) in

propylene glycol

Animal

number

Local skin irritation

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

25

S-1

0

0

0

0

0

0

0

0

0

0

0

0


Table 4. Measurement of Ear Thickness and Mean Ear Thickness Changes – Preliminary Screening Test

Concentration

(% w/w) in

propylene

glycol

Animal number

Ear thickness measurement (mm)

Day 1

Day 3

Day 6

Pre-dose

Post-dose

Left

Right

Left

Right

Left

Right

25

S-1

0.230

0.230

0.240

0.235

0.230

0.235

Overall mean (mm)

0.230

0.238

0.233

Overall mean ear thickness change (%)

n/a

3.261

1.087

Table 5. Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration
(%w/w) in
propylene glycol

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

6112.62

764.08

na

na

5

6450.73

806.34

1.06

Negative

10

6192.92

774.12

1.01

Negative

25

5463.95

682.99

0.89

Negative

 

dpm = Disintegrations per minute

a = Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8

(total number of lymph nodes)

b = Stimulation Index of 3.0 or greater indicates a positive result

na = Not applicable

Table 6. Individual Clinical Observations and Mortality Data


Concentration
(% w/w) in
propylene glycol

Animal Number

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Vehicle

1-1

0

0

0

0

0

0

0

0

0

1-2

0

0

0

0

0

0

0

0

0

1-3

0

0

0

0

0

0

0

0

0

1-4

0

0

0

0

0

0

0

0

0

5

2-1

0

0

0

0

0

0

0

0

0

2-2

0

0

0

0

0

0

0

0

0

2-3

0

0

0

0

0

0

0

0

0

2-4

0

0

0

0

0

0

0

0

0

10

3-1

0

0

0

0

0

0

0

0

0

3-2

0

0

0

0

0

0

0

0

0

3-3

0

0

0

0

0

0

0

0

0

3-4

0

0

0

0

0

0

0

0

0

25

4-1

0

0

0

0

0

0

0

0

0

4-2

0

0

0

0

0

0

0

0

0

4-3

0

0

0

0

0

0

0

0

0

4-4

0

0

0

0

0

0

0

0

0


0=      No signs of systemic toxicity

Table 7. Individual Bodyweights and Bodyweight Changes

Concentration
(% w/w) in
propylene glycol

Animal Number

Bodyweight (g)

Bodyweight Change (g)

Day 1

Day 6

Vehicle

1-1

20

19

-1

1-2

18

17

-1

1-3

23

22

-1

1-4

18

18

0

5

2-1

21

20

-1

2-2

21

22

1

2-3

20

20

0

2-4

20

21

1

10

3-1

20

21

1

3-2

19

20

1

3-3

19

19

0

3-4

21

21

0

25

4-1

20

20

0

4-2

20

21

1

4-3

18

19

1

4-4

20

20

0

Please see attachment "Appendix 1 Current Positive Control Study for the Local Lymph Node Assay”

Please see attachment "Appendix 2 Summary of Positive Control Data for the Local Lymph Node Assay”

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test material was considered to be a non-sensitiser under the conditions of the test. The study is considered to be reliable and acceptable for use as a key study.

Read-across is justified on the basis that the sensitisation potential of aluminium dihydrogen triphosphate will be determined by the Al cation. Triphosphate itself is not considered to be a sensitiser, in addition, the ultimate breakdown product of triphosphate (orthophosphate) is a natural component of blood and cellular fluids. As aluminium dihydrogen triphosphate has a lower water solubility than aluminium orthophosphate it is considered to be less bioavailable and therefore aluminium orthophosphate is considered to be a worst case for sensitisation potential of the Al cation. The study reports that aluminium orthophosphate is a non-sensitiser under the conditions of the study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In accordance with Article 13(1) of Regulation (EC) No 1907/2006, information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met. This includes the use of alternative methods, for example, in vitro methods or qualitative or quantitative structure-activity relationship models or from information from structurally related substances (grouping or read-across).

In accordance with Annex XI, Section 1.5, substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. Application of the group concept requires that physicochemical properties, human health effects and environmental effects or environmental fate may be predicted from data for reference substance(s) within the group by interpolation to other substances in the group (read-across approach).

The similarities may be based on:

(1) a common functional group;

(2) the common precursors and/or the likelihood of common breakdown products via physical and biological processes, which result in structurally similar chemicals; or

(3) a constant pattern in the changing of the potency of the properties across the category.

There are no or no adequate and reliable studies available in which the skin sensitisation of Aluminium dihydrogen triphosphate has been investigated. Therefore, in order to fulfil the standard information requirements, assessment of this endpoint/property is conducted by means of read-across from the following reference substance:

-          Aluminium orthophosphate (EC 232-056-9)

The similarity between the source and target substance is based on:

(1) common functional groups: aluminium orthophosphate and aluminium dihydrogen triphosphate are both inorganic salts of aluminium (Al3+) cations and orthophosphate (PO43-) or dihydrogen triphosphate (H2P3O103-) anions, respectively; the latter anion being composed of orthophosphate moieties.

(2) common precursors and/or the likelihood of common breakdown products via physical and biological processes, which result in structurally similar chemicals: both the source and target substance are ionic compounds resulting from the neutralization reaction of an aluminium base and orthophosphoric acid. Condensation of orthophosphate to diphosphate, triphosphate, etc. is achieved by heat-induced removal of molecular water. The reverse reactions, i.e. chemical or biological hydrolysis of condensated phosphates into orthophosphate and dissolution of the ionic bonds, lead to the common breakdown products aluminium (Al3+) cations and orthophosphate (PO43-) anions.

(3) constant pattern in the changing of the potency of the properties across the category: in general, independently of the cation under consideration, the water solubility of phosphates decreases with increasing degree of phosphate condensation (orthophosphate > diphosphate > triphosphate > polyphosphate).

In accordance with the provisions set out in Annex XI, Section 1.5, the results of the studies used for assessment and read-across are adequate for the purpose of classification and labelling and/or risk assessment; have adequate and reliable coverage of the key parameters addressed in the corresponding test method; cover an exposure duration comparable to or longer than the corresponding test method; and adequate and reliable documentation of the applied method is provided in the technical dossier.

A detailed read-across justification is provided in Section 13 of the technical dossier.

Skin sensitisation

The skin sensitisation potential of aluminium orthophosphate was assessed in CBA/Ca mice in a Local Lymph Node Assay conducted in accordance with OECD Guideline 429 and under GLP conditions (Bradshaw, 2013). Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 μl (25 μl per ear) of the test item as a suspension in propylene glycol at concentrations of 25%, 10% or 5% w/w. A further group of four animals was treated with propylene glycol alone.

No deaths occurred and no clinical signs of systemic toxicity or adverse effects in body weight (development) were observed during the study. The Stimulation Index (SI) expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group was 1.06, 1.01 and 0.89 for the 5, 10 and 25% w/w groups, respectively.

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 skin sensitising.


Migrated from Short description of key information:
Skin sensitisation: not sensitising (OECD 429, GLP; read-across from Aluminium orthophosphate EC 232-056-9)

Justification for selection of skin sensitisation endpoint:
Hazard assessment is conducted by means of read-across from the results of a GLP and OECD Guideline compliant study conducted with an analogue substance. The read-across approach is justified in accordance with Annex XI, Section 1.5 of Regulation (EC) No 1907/2006 based on common functional groups and common precursors/breakdown products, as both the source and target substances are inorganic salts of aluminium and orthophosphoric or triphosphoric acid, respectively. Furthermore, the source substance has a higher water solubility, thus representing a worst case approach for assessment of the breakdown products.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

This information is not available.


Migrated from Short description of key information:
Respiratory sensitisation: no study available

Justification for classification or non-classification

The available data indicate that the substance does not meet the classification criteria in accordance with Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

CLP

Skin sensitisation: not classified

Respiratory sensitisation: data lacking

GHS

Skin sensitisation: not classified

Respiratory sensitisation: data lacking