Superphosphates

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 April 2010 to 06 May 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Diammonium phosphate (DAP)

IUPAC Name:

Diammonium phosphate (DAP)

Constituent 2

Reference substance name:

Diammonium hydrogenorthophosphate

EC Number:

231-987-8

EC Name:

Diammonium hydrogenorthophosphate

Cas Number:

7783-28-0

Molecular formula:

H3N.1/2H3O4P

IUPAC Name:

diammonium hydrogen phosphate

Test material form:

solid: crystalline

Details on test material:

Name of test material (as cited in study report): Diammonium hydrogenorthophosphate
- Substance type: White crystals
- Physical state: Solid

In vivo test system

Test animals

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: Young adult animals (approx. 10 weeks old) were selected.
- Weight at study initiation: 17-22 gram.
- Housing: Individual housing in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & 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): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap water.
- Acclimatization period: at least 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9 – 22.9ºC
- Humidity (%): 38 - 84%. Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day

IN LIFE DATES: From: 24 April 2010 to 06 May 2010

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

0-10-25-50%
Positive control: 25% (Alpha-Hexylcinnamicaldehyde in propylene glycol)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- The vehicle was selected based on trial formulations performed at NOTOX and on test substance data
supplied by the sponsor.
- A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (maximally grade 2; see section 6.6) at the highest concentration. Two test substance concentrations were tested; a 25% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids, 50% for solids).
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.
- Lymph node proliferation response: Not determined in the preliminary irritation study.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A
Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group.
If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer, based on the test guideline and
recommendations done by ICCVAM. The results were evaluated 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 of the European
Parliament and of the Council of 16 December 2008 on classification, labeling and packaging of substances and mixtures.
Consideration was given to the EC3 value (the estimated test substance concentration that will give a SI =3).

TREATMENT PREPARATION AND ADMINISTRATION:
The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for
specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.

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..

Induction - Days 1, 2 and 3
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 the
numerical scoring system described in 'Any other information on materials and methods incl. tables'. Furthermore descriptions of
all other (local) effects were recorded.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Not performed

Results and discussion

Positive control results:

A mean DPM/animal value of 5370 DPM was obtained from the positive control group.

The six-month 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. See attached document 'Reliability check'.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.7

Test group / Remarks:

10%

Results 2

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

25%

Results 3

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

50%

Results 4

Parameter:

other: DPM

Value:

464

Test group / Remarks:

10%

Remarks on result:

other: mean DPM for vehicle group: 672

Results 5

Parameter:

other: DPM

Value:

740

Test group / Remarks:

25%

Remarks on result:

other: mean DPM for vehicle group: 672

Results 6

Parameter:

other: DPM

Value:

718

Test group / Remarks:

50%

Remarks on result:

other: mean DPM for vehicle group: 672

Any other information on results incl. tables

Preliminary irritation study:

The results of the epidermal exposures for the selection of highest test substance concentration to be tested in the main study are described in Table 1 of the attached document 'Figures and tables'. Based on these results, the highest test substance concentration selected for the main study was a 50% concentration.

Main study(see Table 2 of attached document 'Figures and tables'):

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.

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 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.

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Not classified according to Regulation (EC) No. 1272/2008

Conclusions:

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%.
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.