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Administrative data

Description of key information

One key study is available (Bradshaw J (2010) for the skin sensitisation endpoint. This study is considered to be a reliability 1 study (according to the Klimisch scale) as it has been conducted to the appropriate guideline (OECD 249, Local Lymph Node Assay) and under the conditions of GLP. Trisodium hydrogen diphosphate is estimated to be a non-sensitiser as found in the source study performed with sodium acid pyrophosphate.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 05 October 2009 and 21 October 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

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 or 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

(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-anion.
(3) Structurally the only difference between the two is the replacement of one hydrogen atom with an additional sodium atom to give trisodium hydrogen diphosphate. Removing one hydrogen atom from the anion will not enhance any sensitisation potential and the cation remains the same but is increased in quantity, the stimulation index results from the LLNA test with disodium dihydrogen pyrophosphate do not indicate that increasing the exposure levels has an impact on the results so increasing the levels of sodium is unlikely to increase the sensitisation potential. The difference between the two compounds will not have an impact on any sensitisation potential and therefore the negative LLNA results with disodium dihydrogen pyrophosphate can reliably be read across to trisodium hydrogen diphosphate.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of GLP inspection: 15 September 2009 Date of Signature on GLP certificate: 26 November2009
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 Limited, Bicester, 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 (e.g. ad libitum):
ad libitum (2014 Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK)

- Water (e.g. ad libitum):
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 deg 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:
acetone/olive oil (4:1 v/v)
Remarks:
Please see below for Vehicle Determination Record
Concentration:
Each group was exposed to concentrations of 10%, 5% or 2.5% w/w in propylene glycol
No. of animals per dose:
Groups of four mice were treated
Details on study design:
RANGE FINDING TESTS:
Using available information regarding the systemic toxicity/irritancy potential of the test material, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µl of the test material at a concentration of 10% 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. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.

- Lymph node proliferation response:
Clinical observations, bodyweight and mortality data are give in the results section (table 1).

No signs of systemic toxicity were noted.

Based on this information the dose levels selected for the main test were 10%, 5% or 2.5% w/w in propylene glycol.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT

- 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 at concentrations of 10%, 5% or 2.5% w/w in propylene glycol. This vehicle was chosen as it produced the most suitable formulation at the required concentration.

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.

The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The test material 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 material (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.
Positive control substance(s):
other: Phenylacetaldehyde (90%) as a solution in propylene glycol at a concentration of 2.5% v/v
Statistics:
None provided.
Positive control results:
A 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 control group of five animals was treated with propylene glycol alone.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:
Concentration (% v/v) in
propylene glycol Stimulation Index Result
2.5 8.00 Positive

Phenylacetaldehyde (90%) was considered to be a sensitiser under the conditions of the test.
Key result
Parameter:
SI
Value:
1.37
Test group / Remarks:
2.5% test material in vehicle, 4 animals tested
Key result
Parameter:
SI
Value:
0.92
Test group / Remarks:
5% test material in vehicle, 4 animals tested
Key result
Parameter:
SI
Value:
1.43
Test group / Remarks:
10% test material in vehicle, 4 animals tested

Table 1              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

10

S-1

19

17Ä

0

0

0

0

0

0

0

0

HDh


0=      No signs of systemic toxicity

Ä= Bodyweight loss considered to be due to animal’s inability to drink from blocked water spout and not as a result of the test material

H =    Hunched posture

Dh =   Dehydration

Table 2              Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration
(%w/w) in
propylene glycol

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

5730.44

716.31

na

na

2.5

7861.51

982.69

1.37

Negative

5

5247.37

655.92

0.92

Negative

10

8202.80

1025.35

1.43

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 3              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

2.5

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

5

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

10

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 4              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

21

20

-1

1-2

19

20

1

1-3

19

18

-1

1-4

20

21

1

2.5

2-1

19

19

0

2-2

20

21

1

2-3

19

21

2

2-4

20

20

0

5

3-1

19

19

0

3-2

18

19

1

3-3

18

19

1

3-4

19

19

0

10

4-1

20

20

0

4-2

19

20

1

4-3

20

19

-1

4-4

20

19

-1

 


Current Positive Control Study for the Local Lymph Node Assay

Introduction. A study was performed to assess the sensitivity of the strain of mouse used at these laboratories to a known sensitiser. The method was designed to meet the requirements of the following:

OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted24 April 2002)

Method B42 Skin Sensitisation (Local Lymph Node Assay) of CommissionRegulation (EC) No. 440/2008

Test Material: Phenylacetaldehyde (90%)

Project number:  0039/1079

Study dates:        20 March 2009to26 March 2009

Methods. A group of five animals was treated with 50 µl (25 µl per ear) of Phenylacetaldehyde (90%)as a solution inpropylene glycolata concentration of 2.5% v/v. A further control group of five animals was treated withpropylene glycolalone.

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

Concentration (% v/v) in
propylene glycol

Stimulation Index

Result

2.5

8.00

Positive

Conclusion. Phenylacetaldehyde (90%) was considered to be a sensitiser under the conditions of the test.

Interpretation of results:
GHS criteria not met
Conclusions:
The test material was considered to be a non-sensitiser under the conditions of the test. This study has been selected as the key study because the results are sufficient in order to derive a reliable conclusion on classification and labelling in accordance with Regulation EC (No.) 1272/2008 (EU CLP). Disodium dihydrogenpyrophosphate is not considered to be classified according to Regulation (EC) No. 1272/2008 (EU CLP).
Executive summary:

Introduction. A study was performed to assess the skin sensitisation potential of the test material in theCBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of the following:

OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 24 April 2002)

Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008

Methods. Following a preliminary screening test at a concentration of 10% 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 material as a suspension in propylene glycol at concentrations of 10%, 5% or 2.5% w/w. A further group of four animals was treated with propylene glycol alone.

Results. 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:

Concentration (%w/w) in
propylene glycol

Stimulation Index

Result

2.5

1.37

Negative

5

0.92

Negative

10

1.43

Negative

Conclusion. The test material was considered to be anon-sensitiser under the conditions of the test.

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

A negative LLNA test is available for disodium dihydrogenpyrophosphate, read-across is proposed from this to trisodium hydrogen diphosphate and is justified on the following basis:

Both are ionic inorganic compounds, structurally the only difference between the two is the replacement of one hydrogen atom with an additional sodium atom to give trisodium hydrogen diphosphate. Removing one hydrogen atom from the anion will not enhance any sensitisation potential and the cation remains the same but is increased in quantity, the stimulation index results from the LLNA test with disodium dihydrogen pyrophosphate do not indicate that increasing the exposure levels has an impact on the results so increasing the levels of sodium is unlikely to increase the sensitisation potential.

The difference between the two compounds will not have an impact on any sensitisation potential and therefore the negative LLNA results with disodium dihydrogen pyrophosphate can reliably be read across to trisodium hydrogen diphosphate.


Migrated from Short description of key information:
One key study (Bradshaw J, 2010) is available to assess the potential of the analogous substance disodium dihydrogenpyrophosphate to be a sensitizer when applied to the skin. This study is considered to be a reliability 2 study as it has been conducted to the current guideline (OECD Method 429) and under the conditions of GLP but has been conducted on an analogous substance.

Justification for selection of skin sensitisation endpoint:
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 and is therefore considered to be suitable for use as a key study.

Justification for classification or non-classification

Trisodium hydrogen diphosphate is not considered to be a sensitiser in accordance with Regulation (EC) No. 1272/2008 (EU CLP). No further testing is deemed necessary.