phosphonic acid, [2-(4-aminophenyl)-1-hydroxyethylidene]bis-, monosodium salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 22 May 2019. Experimental completion date: 04 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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

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:

phosphonic acid, [2-(4-aminophenyl)-1-hydroxyethylidene]bis-, monosodium salt

EC Number:

828-215-2

Cas Number:

172796-84-8

Molecular formula:

C8H12NO7P2Na

IUPAC Name:

phosphonic acid, [2-(4-aminophenyl)-1-hydroxyethylidene]bis-, monosodium salt

impurity 1

Reference substance name:

4-aminophenylacetic acid

EC Number:

214-828-7

EC Name:

4-aminophenylacetic acid

Cas Number:

1197-55-3

Molecular formula:

C8H9NO2

IUPAC Name:

4-aminophenylacetic acid

impurity 2

Reference substance name:

EBP dimer

Molecular formula:

C16H22N2O12P4

IUPAC Name:

EBP dimer

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Identification: Phosphonic acid [2-(4-aminophenyl)-1-hydroxyethylidene]bis-, monosodium salt (“EBP“)
Batch: CHPC071917EBP
Purity: >98% pure
Physical state, appearance: Pale yellow powder
Expiry Date: 01 July 2019
Storage Conditions: Room temperature
Stability in Solvent: Not indicated by the Sponsor
Dose calculation was not adjusted to purity.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Test system: Mice, CBA/CaOlaHsd
Rationale: Recognised as the recommended test system.
Source: Envigo RMS B.V., Inc, Postbus 6174, 5960 AD Horst / The Netherlands
Number of animals for the pre-test: 2 females
Number of animals for the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 1
Age (beginning of treatment): 9 - 11 weeks
Identification: The animals were distributed into the test groups at random. All animals belonging to the same experimental group were kept in one cage. In the main experiment, the animals were identified by tail tags. In the pre-experiment, animals were identified by cage number.
Acclimation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: group
Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top
Bedding: granulated soft wood bedding
Feed: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
Water: tap water, ad libitum
Environment: temperature 22 ± 2°C
relative humidity approx. 45-65% (except for deviation)
artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

0 (control group), 5, 10, and 25%.
The highest concentration tested was the highest level that could technically be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

No. of animals per dose:

4

Details on study design:

Vehicle and Dose Selection
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was a 25% suspension in PG. Grinding of the test item in a mortar was used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. vortexing, sonicating, warming to 37°C).
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 25% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6.
At the tested concentrations the animals did not show any signs of systemic toxicity. The animals showed a very slight erythema of the ear skin (Score 1) and the animal treated with 25% showed scaly ears.
Thus, the test item in the main study was assayed at 5, 10, and 25%. The highest concentration tested was the highest level that could technically be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

Test Item Preparation
The test item was placed into an appropriate container on a tared balance and PG was added (weight per weight).
The different test item concentrations were prepared individually. Homogeneity of the test item in vehicle was maintained during treatment using a magnetic stirrer.
The preparations were made freshly before each dosing occasion.

Test Item Administration
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% in PG. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (Ø 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
Administration of 3H-methyl-thymidine
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 19.6 µCi of 3H-methyl thymidine (equivalent to 78.2 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
Terminal Procedure
Approximately five hours after treatment with 3HTdR all mice were euthanized by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.
Preparation of Single Cell Suspensions
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules.
Determination of cellular proliferation (incorporation of 3HTdR)
The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Positive control substance(s):

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

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.46

Test group / Remarks:

5 % concentration

Results 2

Parameter:

SI

Value:

1.01

Test group / Remarks:

10 % concentration

Results 3

Parameter:

SI

Value:

1.43

Test group / Remarks:

25 % concentration

Cellular proliferation data / Observations:

Calculation of the EC3 value
The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.
Viability / Mortality
No deaths occurred during the study period.
Clinical Signs
No signs of systemic toxicity were observed during the study period. On day 3 and day 4, the animals showed an erythema of the ear skin (Score 1).
Body Weights
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Any other information on results incl. tables

Calculation and Results of Individual Data

Test item concentration %	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b)	S.I.
---	BG I	14	---	---	---	---
---	BG II	10	---	---	---	---
0	1	5638	5626	8	703.2	1.00
5	2	8225	8213	8	1026.6	1.46
10	3	5717	5705	8	713.1	1.01
25	4	8037	8025	8	1003.1	1.43

1    =  Control Group

2-4=  Test Group

^a)   =  The mean value was taken from the figures BG I and BG II

^b)     =  Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

Results of Positive Control

Test item concentration %	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b)	S.I.
---	BG I	15	---	---	---	---
---	BG II	24	---	---	---	---
0	1	6866	6846.5	8	855.8	1.00
5	2	10186	10166.5	8	1270.8	1.48
10	3	15511	15491.5	8	1936.4	2.26
25	4	55457	55437.5	8	6929.7	8.10

1 = Control Group

2-4 = Test Group

^a)= The mean value was taken from the figures BG I and BG II

^b)= Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item Phosphonic acid [2-(4-aminophenyl)-1-hydroxyethylidene]bis-, monosodium salt (“EBP“) was not a skin sensitiser under the test conditions of this study.

Executive summary:

In the study the test item Phosphonic acid [2-(4-aminophenyl)-1-hydroxyethylidene]bis-, monosodium salt (“EBP“) formulated in PG (propylene glycol) was assessed for its possible skin sensitising potential.

For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 25% (w/w). The highest concentration tested was the highest concentration that could technically be achieved.

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. On day 3 and day 4, the animals (animals treated with 10% test item on day 3, animals treated with 25% test item on days 3 and 4) showed a very slight erythema of the ear skin (Score 1).

In this study Stimulation Indices (S.I.) of 1.46, 1.01, and 1.43 were determined with the test item at concentrations of 5, 10, and 25% in PG, respectively.

The test item Phosphonic acid [2-(4-aminophenyl)-1-hydroxyethylidene]bis-, monosodium salt (“EBP“) was not a skin sensitiser under the test conditions of this study.