Reaction mass of potassium sodium (2R,3R)-2-hydroxy-3-(phosphonatooxy)butanedioate and potassium sodium (2S,3S)-2-hydroxy-3-(phosphonatooxy)butanedioate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Feb 2016 - 01 Mar 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Deviations:

yes

Remarks:

(no retention sample)

GLP compliance:

yes (incl. QA statement)

Remarks:

Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- name of test material (as cited in study report): Reaction mass of tetra (potassium and sodium) (2R,3R)-2-hydroxy-3-(phosphonatooxy)succinate and tetra (potassium and sodium) (2S,3S)-2-hydroxy-3-(phosphonatooxy)succinate
- physical state: White to slightly yellowish powder of crystalline appearance

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/JN

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier Labs., Cedex, France
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: 17-25 g
- Housing: individual in Type III Makrolon cages, granulated textured wood bedding (Granulat A2, J. Brandenburg, Goldenstedt, Germany), change of cages once a week
- Diet (ad libitum): Commercial diet ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, Soest, Germany), analysed for contaminants twice a year
- Water (ad libitum): tap water, analysed at least once a year
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 01 Feb 2016 To: 01 Mar 2016

Study design: in vivo (LLNA)

Vehicle:

other: highly purified water

Concentration:

10, 25 and 50% (w/w)

No. of animals per dose:

5

Details on study design:

Pre-screening test:
In a preliminary solubility assessment, the following vehicles were assessed: acetone / olive oil (4 : 1 v/v), N,N-dimethylformamide, methyl ethyl ketone, dimethyl sulfoxide, propylene glycol, ethanol and highly purified water. The test item could not be dissolved or suspended in these above mentioned vehicles except highly purified water. Only highly purified water gave homogenous solutions suitable for application of the test item and was used as the vehicle for the LLNA. A 50% solution was the highest possible concentration resulting in a formulation applicable onto the mouse ear.

RANGE FINDING TESTS:
- Compound solubility: 50% (w/w) in water, highest technically feasable concentration
- Concentrations tested: 0.5, 1, 2.5, 5, 10, 25 and 50% test item (2 animals each)
- Irritation: No irritation observed up to 50%; no differences in ear punch weight and ear thickness, Erythema score: 0
- Clinical signs (observed daily): None oberved
- Body weights (recorded before administration of test item and prior to termination on Day 6): No effect oberved.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Concentrations tested in the main study: 10, 25 and 50% of the test item
- Name of test method: LLNA: BrdU-ELISA: determination of cellular proliferation via incorporation of the thymidine analogue BrdU, incorporation measured by ELISA (commercial kit from Roche Applied Science, Mannheim, Germany)
- The BrdU labelling index is defined as: BrdU labelling index = (ABS/em – ABS blank/em) – (ABS/ref – ABS blank/ref) (em = emission wavelength; and ref = reference wavelength)
- Results of each treatment group are expressed as the mean SI. SI is derived by dividing the mean BrdU labelling index/mouse within each test item group and the positive control group by the mean BrdU labelling index for the vehicle control group. The average SI for the vehicle control is then one.
- Criteria used to consider a positive response: SI ≥ 1.6 (borderline result: SI value between 1.6 and 1.9)

TREATMENT PREPARATION AND ADMINISTRATION: On Day 1 25 µL of test item or positve control item or the respective vehicle control was applied to the entire dorsal surface of each ear of each mouse. The application was repeated on Day 2 and 3; local irritation reactions were assessed. On Day 5 0.5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution was injected interperitoneally. On Day 6 (approx. 24 hours after BrdU injection) the draining auricular lymph node of each ear was excised into PBS. A single cell suspension of lymph node cells was prepared from each mouse. The optimised volume was based on achieving a mean absorbance of the negative control group within 0.1 - 0.2 (measured with a spectrophotometer at λ =570 nm).

Positive control substance(s):

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

Statistics:

Student's t-test

Results and discussion

Positive control results:

SI = 2.469
A statistically significant increase was observed for the positive control (p ≤ 0.01) compared to the respective vehicle control (Acetone/ olive oil (4 : 1 v/v)).

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

RESULTS OF THE MAIN STUDY BrdU Integration: Stimulation Indices (SI values) (Mean of 5 animals per group) - Group 1 (vehicle control/water): 1.000 - Group 2 (10% test item): 1.069 - Group 3 (25% test item): 1.073 - Group 4 (50% test item): 1.033 No increase in SI values in any of the test item treatment groups compared to the vehicle control.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

RESULTS OF THE MAIN STUDY BrdU Integration: Absolute values (Mean of 5 animals per group) - Group 1 (vehicle control/water): 0.274 - Group 2 (10% test item): 0.293 - Group 3 (25% test item): 0.294 - Group 4 (50% test item): 0.283 No increase in BrdU values in any of the test item treatment groups compared to the vehicle control.

Any other information on results incl. tables

RESULTS OF THE MAIN STUDY

 

Parameter (n=5)	Negative control (highly purified water)	Test item 10%	Test item 25%	Test item 50%	Positive control	Vehicle of the positive control
BrdU labelling index	0.274	0.293	0.294	0.283	0.684^**	0.277
Stimulation index	1.000	1.069	1.073	1.033	2.469^**	1.000
Ear punch weight [mg]	14.7	13.5	13.3	14.1	17.9^**	14.9
Ear thickness[µm] (TD1)	202.0	206.0	214.0	210.0	210.0	206.0
Ear thickness [µm] (TD3)	202.0	206.0	214.0	214.0	230.0^**	210.0
Ear thickness[µm] (TD6)	204.0	208.0	214.0	216.0	254.0^**	210.0

^**significantly increased compared to control at p ≤ 0.01

 

No increase in ear punch weights in any of the test item treatment groups compared to the vehicle control.

A statistically significant increase was observed for the positive control (p ≤ 0.01) compared to the respective vehicle control.

Body weight (Mean and SD per group): Day 1 (TD1) / Day 6 (TD6) / change [%]:

- Group 1 (vehicle control/water): 21.4 g ± 1.1 / 21.4 g ± 1.1 / +0.1

- Group 2 (10% test item): 20.4 g ± 2.3 / 20.4 g ± 2.1 / +0.1

- Group 3 (25% test item): 21.2 g ± 1.1 / 21.2 g ± 1.3 / +0.0

- Group 4 (50% test item): 22.8 g ± 1.3 / 22.0 g ± 1.0 / -3.4

- Group 5 (PC): 21.2 g ± 0.8 / 21.8 g ± 0.8 / +2.9

- Group 6 (Vehicle Control of PC): 21.6 g ± 1.3 / 21.4 ± 2.1 / -1.1

Erythema:

- Group 1 (vehicle control/water): no erythema

- Group 2 (10% test item): no erythema

- Group 3 (25% test item): no erythema

- Group 4 (50% test item): no erythema

- Group 5 (PC): no erythema

- Group 6 (Vehicle Control of PC): no erythema

Clinical signs:

- Group 1 (vehicle control/water): no signs of systemic intolerance / no signs of dermal irritation

- Group 2 (10% test item): no signs of systemic intolerance / no signs of dermal irritation

- Group 3 (25% test item): no signs of systemic intolerance / no signs of dermal irritation

- Group 4 (50% test item): no signs of systemic intolerance / no signs of dermal irritation

- Group 5 (PC): no signs of systemic intolerance / no signs of dermal irritation

- Group 6 (Vehicle Control of PC): no signs of systemic intolerance / no signs of dermal irritation

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified