(4-amino-1-hydroxy-1-phosphonobutyl)phosphonic acid;hydrate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-19 to 2017-07-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

not applicable

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

not applicable

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

not applicable

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:

(4-amino-1-hydroxy-1-phosphonobutyl)phosphonic acid hydrate

EC Number:

680-378-7

Cas Number:

138624-11-0

Molecular formula:

C4H15NO8P2

IUPAC Name:

(4-amino-1-hydroxy-1-phosphonobutyl)phosphonic acid hydrate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor and 610-GTH
- Expiration date of the lot/batch: 2019-03-31
- Purity test date: 2016-04-16

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Solubility and stability of the test substance in the solvent/vehicle: Soluble except for the 25% and 50% w/w concentrations, which saw scaling on the test animals ears. This was determined to not have affected the validity of the results.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dissolve in propylene glycol to concentrations of 0, 10, 25, and 50% test item / solvent (w/w)

FORM AS APPLIED IN THE TEST (if different from that of starting material)
0, 10, 25, and 50% test item / solvent (w/w)

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known:
- Age at study initiation: ~8-10 weeks
- Weight at study initiation: 19.3 to 23.6 g
- Housing: Polycarbonate cages containing sterilized sawdust as bedding material.
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: >=5 days
- Indication of any skin lesions: none

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): >10/hr
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

Preliminary Test #1: 5 and 10 % test item / solvent (w/w)
Preliminary Test #2: 25 and 50% test item / solvent (w/w)
Main Test: 0, 10, 25, and 50% test item / solvent (w/w)

No. of animals per dose:

Preliminary Test #1: 2
Preliminary Test #2: 2
Main Test: 5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: White test item remnants were present on the dorsal surface of the ears of animals at 25% (between Days 2-4) and 50% (between Days 1 and 5), which did not hamper scoring of the skin reactions.
- Irritation: At a 10% test item concentration very slight erythema was noted on the dorsal surface of the ears between Days 2 and 5, no further erythema was noted.
- Systemic toxicity: None
- Ear thickness measurements: Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values.
- Erythema scores: (Left ear/right ear)
TS (%) Animal # Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
5 1 0/0 0/0 0/0 0/0 0/0 0/0
2 0/0 0/0 0/0 0/0 0/0 0/0
10 3 0/0 1/1 1/1 1/1 1/1 1/1
4 0/0 1/1 1/1 1/1 1/1 1/1
25 5 0/0 0/0 0/0 0/0 0/0 0/0
6 0/0 0/0 0/0 0/0 0/0 0/0
50 7 0/0 0/0 0/0 0/0 0/0 0/0
8 0/0 0/0 0/0 0/0 0/0 0/0

MAIN STUDY
General Test Procedure:
Excision of the Nodes - Day 6
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 five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of 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 PBS.
Tissue Processing for Radioactivity - Day 6
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 µm, diameter: ± 1.5 cm). 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) and then 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. Radioactivity 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).

Mortality was observed twice daily at the beginning and end fo the day. Post dose observations were performed once daily on Days 1-6. All animals were examined for reaction to dosing. Bodyweights were weight on Day 1 and 6. Irritation was scored based on erythema and eschar formation on Days 1-6 according to the Draize scoring system.



ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: mouse LLNA study
- 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 using the individual SI values. The individual SI is the ratio of the DPM/anima compared to the DPM/vehicle control group mean. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer. The EC3 value (the estimated test item concentration that will give a SI =3) was determined using linear interpolation.

TREATMENT PREPARATION AND ADMINISTRATION:
Preperation:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item.
The dosing formulations were kept at room temperature until dosing. The dosing formulations were stirred until and during dosing.
No adjustment was made for specific gravity of the vehicle and no correction was made for the purity/composition of the test item, since the test method requires a logical concentration range rather than specific dose levels.
Administration:
The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.

Positive control substance(s):

other: Historical

Results and discussion

Positive control results:

Positive control was a historical comparison, and not run concurrently.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

ca. 30.2

Test group / Remarks:

Overall calculation

Results 2

Key result

Parameter:

SI

Value:

ca. 1.5

Test group / Remarks:

10% test substance (w/w)

Results 3

Key result

Parameter:

SI

Value:

ca. 1.2

Test group / Remarks:

25% test ingredient (w/w)

Results 4

Key result

Parameter:

SI

Value:

ca. 9.9

Test group / Remarks:

50% test substance (w/w)

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.

EC3 CALCULATION
The EC3 value (the estimated test item concentration that will give a SI =3) was determined using linear interpolation.

CLINICAL OBSERVATIONS:
No erythema was noted for any of the animals. Scaliness was noted on the ears of three animals treated at 50% on Day 6.
White test item remnants were present on the dorsal surface of the ears of animals at 25% (Days 1-5) and 50% (Days 1-4), which did not hamper scoring of the skin reactions.
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.

BODY WEIGHTS
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Any other information on results incl. tables

Main Study:  Radioactivity Counts (DPM) and Stimulation Index (SI)

Group	Test substance % w/w	Animal	DPM/animal	Mean DPM±SEM	Mean SI±SEM
1	0	1	206	450 ± 146	1.0 ± 0.5
		2	236
		3	1004
		4	478
		5	325
2	10	1	459	658 ± 98	1.5 ± 0.5
		2	1023
		3	663
		4	536
		5	607
3	25	1	423	561 ± 147	1.2 ± 0.5
		2	269
		3	532
		4	461
		5	1122
4	50	1	4538	4461 ± 1203	9.9 ± 4.2
		2	6197
		3	6906*
		4	0*
		5	4662

 *

Inadvertently, the lymph nodes of animal nos. 18 and 19 were pooled during processing, the nodes of these

animals are processed together and reported under animal no. 18. The DPM value of animal no. 19 is

therefore set to 0.  

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

This test substance met the criteria to be a Category 1B skin sensitizer based on GHS guidelines.