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Description of key information

Study performed according to OECD 406 and under the conditions of GLP.

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:
supporting study
Study period:
01 February 2010 to 18 February 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

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

The proposed source chemical (diammonium hydrogenorthophosphate) is highly soluble in water (> 10000 mg/L). In aqueous media soluble inorganic orthophosphates will dissociate to their ionic constituents; in this case ammonium and orthophosphate ions. Diammonium dihydrogenpyrophosphate will dissociate to ammonium cations and pyrophosphate anions. The pyrophosphate anions are unstable in aqueous solutions with the degree of instability varying according to pH. In distilled water they will hydrolyse slowly via abiotic mechanisms to orthophosphate. In natural waters a number of different processes can occur; abiotic hydrolysis, biotic degradation (as a result of the action of phosphatases which cleave pyrophosphates into orthophosphate subunits) and assimilation by organisms in the water. Thus, the target substance (diammonium dihydrogenpyrophosphate) and the source substance (diammonium hydrogenorthophosphate) will be primarily absorbed as the same inorganic ions: ammonium and orthophosphate and are expected to behave in a similar manner under test conditions.
All (bio) transformation products of the source chemical are common to the target chemical and as such the data is considered to be adequate and reliable for use in the assessment of diammonium dihydrogenpyrophosphate for the toxicity hazard assessment.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report attached.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report attached.

4. DATA MATRIX
See read-across justification report attached.
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
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Preliminary irritation study: Charles River France, L'Arbresle Cedex, France; Main study: Harlan, Horst, The Netherlands
- Age at study initiation: Approximately 8-14 weeks
- Weight at study initiation: 17-22 g
- Housing: Individual housing in labelled Macrolon cages (MI type; height 12.5 cm) containing sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, WM. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied as cage-enrichment. The paper was removed on Day 1 prior to dosing and was supplied again after scoring of the ears on Day 3.
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiaten GmbH, Soest, Germany) provided ad libitum
- Water (e.g. ad libitum): Tap water provided ad libitum
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0 °C (actual range: 18.9 - 22.9 °C)
- Humidity (%): 40-70% (actual range 38-84%)
- Air changes (per hr): Approximately fifteen
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light
Vehicle:
propylene glycol
Concentration:
0, 10, 25, 50 % w/w
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:

Two test substance concentrations were tested; a 25% and 50% concentration.The test system, procedures and techniques were identical to those used during Days 1 to 3 of the main study unless otherwise specified. Two young adult animals were selected (8-14 weeks old). Each animal was treated with one concentration on three consecutive days. Approximately 3-4 hours after the last exposure, the irritation of the ears was assessed. Bodyweights were determined on Day 3. The animals were sacrificed after the final observation and no necropsy was performed.


MAIN STUDY

Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the preliminary irritation study.
One group of five animals was treated with vehicle and another group of five animals was treated with a positive control substance.
Group: 1 Animal numbers: 01-05 Induction: (test substance; % w/w) 0
Group: 2 Animal numbers: 06-10 Induction: (test substance; % w/w) 10
Group: 3 Animal numbers: 11-15 Induction: (test substance; % w/w) 25
Group: 4 Animal numbers: 16-20 Induction: (test substance; % w/w) 50
Positive Control - Group: 5 Animal numbers: 21-25 Induction: (test substance; % w/w) 25

TREATMENT PREPARATION AND ADMINISTRATION:

The test substance formulations (w/w) were prepared within 4 hours prior to each treatment at concentrations specified above. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.

The dorsal surface of both ears was epidermally treated (25 μL/ear) with the test substance concentration, at approximately the same time per day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing.

The vehicle and positive control animals were treated the same as the experimental animals, except that the vehicle and/or positive control substance was administered instead of the test substance.

Excision of the nodes - Day 6
All animals:
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
After approximately five hours, all animals were killed by intraperitoneal injection with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

Tissue processing for radioactivity - Day 6
A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) stored in the refrigerator until the next day.

Radioactivity measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Observations
Mortality/Viability Twice daily.
Toxicity At least once daily.
Body weights On Days 1 (pre-treatment) and 6.
Necropsy No necropsy was performed according to protocol.
Irritation On Day 3 (3-4 hours after treatment), the skin reactions were assessed. Skin reactions were graded according to Draize scoring system. Furthermore descriptions of all other (local) effects were recorded.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
No irritation was observed in any of the animals examined. Based on these results, the highest test substance concentration selected for the main study was a 50% concentration.
The positive control group added to the study showed that the vehicle is suitable for eliciting a SI of >3 in this batch of animals and procedures for this study used. The six monthly reliability check with Alpha-Hexylcinnamicaldehyde, indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 10, 25 and 50% were 0.7, 1.1 and 1.1 respectively.There was no indication that the test substance elicits an SI ≥ 3 when tested up to 50%.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The DPM values for the substance concentrations 10, 25 and 50% were 464, 740 and 718 respectively.

Skin reactions / Irritation (Table 2)

No irritation of the ears was observed in any of the animals examined. All positive control animals showed slight to well-defined erythema. No oedema was observed in any of the animals examined.

Macroscopy of the auricular lymph nodes and surrounding area (Table 2)

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size, except for one or both lymph nodes of one animal at 25% (no. 12) and one animal at 50% (no. 19). The auricular lymph nodes of the positive control group were all enlarged. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Body weights (Table 2)

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss, noted in some animals, was considered not toxicologically significant.

Radioactivity measurements (Tables 3 and 4)

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 464, 740 and 718 DPM respectively. The mean DPM/animal value for the vehicle control group was 672 DPM and a mean DPM/animal value of 5370 DPM was obtained from the positive control group.

Toxicity and mortality No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Table 1: Skin reactions after epidermal exposure and body weights

Animal number

Test substance (%w/w)

Day 1

Day 3

Body Weight (g)

skin reactions dorsal surface ear

Body weight (g)

left

right

erythema

oedema

erythema

oedema

1

252

19

0

0

0

0

18

2

502

20

0

0

0

0

19

2Applied using a pipette with the tip cut off

Table 2: Skin reactions, body weights and relative size auricular lymph nodes

Group

Animal number

Test substance (%w/w)

Day 1

Day 3

Day 6

Body Weight (g)

skin reactions dorsal surface ear

Body weight (g)

 

size nodes

left

right

left

right

erythema

oedema

erythema

oedema

 

 

1

1

 

 

0% (vehicle)

19

0

0

0

0

21

n

n

2

20

0

0

0

0

20

n

n

3

19

0

0

0

0

19

n

n

4

20

0

0

0

0

20

n

n

5

22

0

0

0

0

23

n

n

 

 

2

6

 

 

10%2

20

0

0

0

0

20

n

n

7

20

0

0

0

0

20

n

n

8

22

0

0

0

0

20

n

n

9

21

0

0

0

0

22

n

n

10

21

0

0

0

0

21

n

n

 

 

3

11

 

 

25%2

20

0

0

0

0

21

n

n

12

22

0

0

0

0

21

+

+

13

19

0

0

0

0

19

n

n

14

18

0

0

0

0

20

n

n

15

21

0

0

0

0

21

n

n

 

 

4

16

 

 

50%2

21

0

0

0

0

21

n

n

17

20

0

0

0

0

20

n

n

18

22

0

0

0

0

22

n

n

19

21

0

0

0

0

21

+

n

20

19

0

0

0

0

20

n

n

 

 

5

21

 

 

25%hexylcinnamicaldehyde

17

2

0

1

0

19

++

++

22

20

1

0

2

0

21

++

++

23

21

1

0

1

0

22

++

++

24

21

1

0

1

0

22

++

++

25

18

2

0

1

0

20

++

++

2Applied using a pipette with the tip cut off

Table 3: Radioactivity measurements (individual animals)

Group

Animal number

Test substance (%w/w)

DPM/animal

 

 

1

1

 

 

0% (vehicle)

386

2

579

3

516

4

1056

5

824

 

 

2

6

 

 

10%

536

7

377

8

482

9

167

10

759

 

 

3

11

 

 

25%

221

12

1011

13

1001

14

573

15

894

 

 

4

16

 

 

50%

491

17

1121

18

927

19

281

20

767

 

 

5

21

22

23

24

25

 

 

25%hexylcinnamicaldehyde

6018

6249

4119

5916

4547

 

Table 4: Disintegrations Per Minute (DPM) and Stimulation Index (SI)

group

test substance (% w/w)

mean

DPM ± SEM

SI ± SEM

2

10

464 ± 97

0.7 ± 0.2

3

25

740 ± 152

1.1 ± 0.3

4

50

718 ± 152

1.1 ± 0.3

5

25 % HCA

5370 ± 432

8.0 ± 1.6

1

0% (vehicle)

672 ± 119

1.0 ± 0.3

Interpretation of results:
GHS criteria not met
Conclusions:
The SI values calculated for the substance concentrations 10, 25 and 50% were 0.7, 1.1 and 1.1 respectively.
Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to 50%, Diammonium hydrogenorthophosphate was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test substance concentration that will give a SI =3) (if any) exceeds 50%.
The positive control group added to the study showed that the vehicle is suitable for eliciting an SI>3 in this batch of animals and procedures for this study used. The six monthly reliability check with Alpha-Hexylcinnamicaldehyde, indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.
Based on these results diammonium hydrogenorthophosphate would not be regarded as skin sensitizer according to the recommendations made in the test guidelines and does not have to be classified and has no obligatory labeling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures.
Executive summary:

In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (propylene glycol ). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 464, 740 and 718 DPM respectively. The mean DPM/animal value for the vehicle control group was 672 DPM and a mean DPM/animal value of 5370 DPM was obtained from the positive control group.

The SI values calculated for the substance concentrations 10, 25 and 50% were 0.7, 1.1 and 1.1 respectively.

Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to 50%, Diammonium hydrogenorthophosphate was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test substance concentration that will give a SI =3) (if any) exceeds 50%.

The positive control group added to the study showed that the vehicle is suitable for eliciting an SI>3 in this batch of animals and procedures for this study used. The six monthly reliability check with Hexylcinnamaldehyde, indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

Based on these results Diammonium hydrogenorthophosphate would not be regarded as skin sensitizer according to the recommendations made in the test guidelines and does not have to be classified and has no obligatory labeling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures.

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

A key study is provided on the substance Amgard LR-2. The identity of the test material is not reported within the study report itself, however the data is referred to in the Toxicological Risks of Selected Flame Retardant Chemicals (2000), Subcommittee on Flame-Retardant Chemicals, Committee on Toxicology, Board on Environmental Studies and Toxicology, National Research Council. ISBN: 0-309-59232-1. The substance LR-2 is an ‘ammonium polyphosphate’ and the author provides the following additional information with regards to the chemical identity of LR2: ‘Based on information provided by the manufacturer (Stewart Miller, Albright and Wilson, pers. commun., Nov. 1, 1999), a typical species distribution of polyphosphates in LR2 is 20% orthophosphate, 40% pyrophosphate, ’ The substance to be registered is ammonium pyrophosphate and is supplied into the EU in an aqueous solution containing <40% of the substance itself as such this data is considered to be appropriate for use in this dossier as it takes into consideration the ammonium ion in addition to pyrophosphate component of the substance to be registered.

Supporting data on similar substances is also provided to back up the conclusions made on the basis of this study.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In accordance with Regulation (EC) No. 1272/2008 (EU CLP) and on the basis of an assessment of the constituent ions, diammonium dihydrogenpyrophosphate is not considered to be a sensitiser.