Reaction mass of Phosphorous(1+), (N-ethylethanaminato) diphenyl(phenylmethyl)-(T-4)-salt with 4, 4' - [2,2,2-trifluoro-1-(trifluoromethyl) ethylidene bis[phenol] (1:1) and (4,4'-[2,2,2-Trifluoro-1-(trifluoromethyl)ethylidene]diphenol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2014 - September 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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:

Reaction mass of Phosphorous(1+), (N-ethylethanaminato) diphenyl(phenylmethyl)-(T-4)-salt with 4, 4' - [2,2,2-trifluoro-1-(trifluoromethyl) ethylidene bis[phenol] (1:1) and (4,4'-[2,2,2-Trifluoro-1-(trifluoromethyl)ethylidene]diphenol

EC Number:

943-265-6

Molecular formula:

not applicable for a multi constituent substance

IUPAC Name:

Reaction mass of Phosphorous(1+), (N-ethylethanaminato) diphenyl(phenylmethyl)-(T-4)-salt with 4, 4' - [2,2,2-trifluoro-1-(trifluoromethyl) ethylidene bis[phenol] (1:1) and (4,4'-[2,2,2-Trifluoro-1-(trifluoromethyl)ethylidene]diphenol

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 37060B
- Expiration date of the lot/batch:
- Purity test date: >99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Females nulliparous and non-pregnant: yes
- Age at study initiation: eight to twelve weeks old
- Weight at study initiation: weight range of 15 to 23 g
- Housing: suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet / Water: Free access to mains tap water and food was allowed throughout the study.
- Acclimation period: at least five days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%):30 to 70%
- Air changes (per hr): approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Preliminary Screening Test: 50% w/w in dimethyl formamide

Main Test: 50%, 25% or 10% w/w in dimethyl formamide.

No. of animals per dose:

Preliminary Screening Test: 1 animal

Main test:
4 per group / 4 groups (3 test concentrations plus vehicule)

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: 50%
- Systemic toxicity: No signs of systemic toxicity
- Irritation / ear thickness measurements: no visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness.

Based on this information the dose levels selected for the main test were 50%, 25% and 10% w/w in dimethyl formamide.

MAIN TEST:
- Criteria used to consider a positive response: The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitizer."

TREATMENT PREPARATION AND ADMINISTRATION:
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.

3H-Methyl Thymidine (HTdR) Administration:
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H-methyl thymidine giving a total of 20 µCi to each mouse.

Termination: Five hours following the administration of HTdR all mice were killed.

Positive control substance(s):

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

Results and discussion

Positive control results:

Alfa-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.
Alfa-Hexylcinnamaldehyde at 15% in dimethyl formamide gave a SI of 6.13.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.15

Test group / Remarks:

10% w/w in dimethyl formamide

Results 2

Key result

Parameter:

SI

Value:

1.07

Test group / Remarks:

25% w/w in dimethyl formamide

Results 3

Key result

Parameter:

SI

Value:

1.17

Test group / Remarks:

50% w/w in dimethyl formamide

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION:
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

CLINICAL OBSERVATIONS: (See table 1 in " Any other informations on results includ. Tables")
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.
Slight off white residual test item on the ears was noted in animals treated with the test item at concentrations of 50% or 25% w/w in dimethyl formamide.

BODY WEIGHTS: (See table 2 in " Any other informations on results includ. Tables")
Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period

Any other information on results incl. tables

Table 1: Individual Clinical Observations and Mortality Data (Main test)

Concentration (% w/w) in dimethyl formamide	Animal Number	Day 1		Day 2		Day 3		Day 4	Day 5	Day 6
		Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
Vehicle	1-1	0	0	0	0	0	0	0	0	0
	1-2	0	0	0	0	0	0	0	0	0
	1-3	0	0	0	0	0	0	0	0	0
	1-4	0	0	0	0	0	0	0	0	0
10	2-1	0	0	0	0	0	0	0	0	0
	2-2	0	0	0	0	0	0	0	0	0
	2-3	0	0	0	0	0	0	0	0	0
	2-4	0	0	0	0	0	0	0	0	0
25	3-1	0	0	0	0Rt	0	0Rt	0	0	0
	3-2	0	0	0	0Rt	0	0Rt	0	0	0
	3-3	0	0	0	0Rt	0	0Rt	0	0	0
	3-4	0	0	0	0Rt	0	0Rt	0	0	0
50	4-1	0	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt	0
	4-2	0	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt	0
	4-3	0	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt	0
	4-4	0	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt	0

0 = No signs of systemic toxicity

Rt = Slight off white residual test item on ears

Table 2:    Individual Body Weights and Body Weight Change (Main test)

Concentration (% w/w) in dimethyl formamide	Animal Number	Body Weight (g)		Body Weight Change (g)
		Dayl	Day 6
Vehicle	1-1	18.8	21.0	2.2
	1-2	18.4	19.8	1.4
	1-3	22.2	21.5	-0.7
	1-4	20.2	19.8	-0.4
10	2-1	16.7	17.8	1.1
	2-2	16.7	17.6	0.9
	2-3	19.1	19.4	0.3
	2-4	17.6	18.4	0.8
25	3-1	19.5	18.1	-1.4
	3-2	19.1	20.5	1.4
	3-3	18.8	19.9	1.1
	3-4	19.2	19.1	-0.1
50	4-1	20.5	18.6	-1.9
	4-2	18.1	18.0	-0.1
	4-3	16.2	17.4	1.2
	4-4	17.7	18.0	0.3

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The stimulation Index were 1.15, 1.07 and 1.17 for the 10%, 25% and 50% of test item in dimethyl formamide, respectively.
Under the experimental conditions of this study, the test item did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay and therefore was considered to be a non-sensitizer.

Executive summary:

A study was performed to assess the skin sensitization potential of the test item XA 31 in the CB A/Ca strain mouse following topical application to the dorsal surface of the ear.The potential of the test item to induce delayed contact hypersensitivity was assessed according to the OECD guideline 429 and to the EU Method B.42. The study was conducted in compliance with the principles of Good Laboratory Practice.

Based on a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in dimethyl formamide at concentrations of 50%, 25% or 10% w/w. A further group of four animals was treated with dimethyl formamide alone.

No noteworthy lymphoproliferation was noted with the test substance at any tested concentration. The Stimulation Index was below 3 (1.15, 1.07 and 1.17 for the concentration of 10%, 25% and 50% respectively) and no dose response reaction was observed.

No systemic clinical signs and mortality, neither changes in body weight, nor local irritation was observed for all tested concentrations. As a result, XA 31 was considered as not sensitising.