Xanthylium, 3,6-bis(diethylamino)-9-[2-(ethoxycarbonyl)phenyl]-, molybdatetungstatephosphate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 October 2017 to 08 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In silico and in vitro alternative testing method not appplicable for UVCB substances such as PV2.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

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:

Xanthylium, 3,6-bis(diethylamino)-9-[2-(ethoxycarbonyl)phenyl]-, molybdatetungstatephosphate

EC Number:

215-414-9

EC Name:

Xanthylium, 3,6-bis(diethylamino)-9-[2-(ethoxycarbonyl)phenyl]-, molybdatetungstatephosphate

Cas Number:

1326-04-1

IUPAC Name:

Reaction product of 6-(diethylamino)-9-(2-ethoxycarbonylphenyl)xanthen-3-ylidene]-diethylazanium chloride with phosphotungstomolybdic acid

Test material form:

solid: nanoform

Details on test material:

Shape
Shape Category: spheroidal
Shape: spherical
Pure Shape:no
Typical Composition: ≤100%
range: >0; ≤100%

Particle size distribution & range
Shape Category: spheroidal
Percentile D10, typical value: 40nm
Percentile D10, range: ≥10; ≤60nm
Percentile D50, typical value: 60nm
Percentile D50, range: ≥40; ≤100nm
Percentile D90, typical value: 90nm
Percentile D90, range: ≥60; ≤150nm
Fraction in size range 1-100nm:≥50; ≤100%

Crystallinity
structure: Amorphous
Pure structure: Yes

Specific Surface Area
Typical specific surface area: ca. 40m2/g
Range: ≥10; ≤200m2/g
Skeletal Density: 2.1 g/cm3

Surface Functionalisation/treatment
surface treatment applied no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Source Sponsor, batch no 280PE310516
- Expiration date of the lot/batch: 01 July 2022
- Purity : UVCB treat as 100%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: Prepared as a solution in DMSO, Stable for the duration of the test Test item formulated within 2 hours of being applied to the test system

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by reputable commercial supplier. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, a nd were 8 to 12 weeks old.
Animal Care and Husbandry
The animals were housed in suspended solid floor polypropylene cages furnished with softwood wood flakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 #C and 30 to 70%, respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not tocontain any contaminant of a level that might have affected the purpose or integrity of the study.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

5, 10 or 25% (w/w)

No. of animals per dose:

4 mice per dose

Details on study design:

PRE-SCREEN TESTS:
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the test item at a concentration of 25% w/w in dimethyl formamide, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the scale included in the report. Any clinical signs of toxicity, if present, were also recorded. The body weight of the was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation), pre-dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.

MAIN STUDY

Groups of four mice were treated with the test item at concentrations of 25%, 10% or 5% w/w in dimethyl formamide. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of four mice received the vehicle alone in the same manner.

Observations
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

3.78

Test group / Remarks:

5%

Remarks on result:

other: positive

Results 2

Key result

Parameter:

SI

Value:

5.03

Test group / Remarks:

10%

Remarks on result:

other: positive

Results 3

Key result

Parameter:

SI

Value:

9.11

Test group / Remarks:

25%

Remarks on result:

other: positive

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

The test item was considered to be a sensitizer under the conditions of the test.