Phosphonium, tetraphenyl-, salt with 4,4'-sulfonylbis[phenol], compd. with 4,4'-sulfonylbis[phenol] (2:2:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 21 October 2003 and 21 January 2004.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

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:

-

EC Number:

484-440-2

EC Name:

-

Cas Number:

502157-74-6

Molecular formula:

C84H68O12S3P2

IUPAC Name:

4-(4-hydroxybenzenesulfonyl)phenol bis(tetraphenylphosphanium) bis(4-(4-hydroxybenzenesulfonyl)benzen-1-olate)

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Sponsor's identification SBCAT-03
Description white powder
Batch number UK.030622B
Date received 09 October 2003
Storage conditions room temperature in the dark

Data relating to the identity, purity and stability of the test material are the responsibility of the
Sponsor.

For the purpose of the study the test material was ground to a powder and freshly prepared in
dimethyl formamide. This vehicle was chosen as it produced the highest concentration that was
suitable for dosing. Groups of four mice were treated with the test material at concentrations of 2.5%, 5% or 10% w/w in dimethyl formamide.
Determination, by analysis, of the concentration, homogeneity and stability of the test material
preparations was not appropriate because it was not specified in the Study Plan and is not a
requirement of the Test Guideline.
Five days following the first topical application of the test material (Day 6) all mice were injected
via the tail vein with 250 μl of phosphate buffered saline containing 3H-methyl thymidine
(3HTdR: 80 μCi/ml, specific activity 2.0 Ci/mmol, Amersham Biosciences UK Ltd) giving a total
of20 μCi to each mouse.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaBkl) strain mice were supplied by B & K Universal Ltd, Hull, UK. On
receipt the animals were randomly allocated to cages. The animals were nulliparous and
non-pregnant. After an acclimatisation period of at least five 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, and were eight to twelve weeks old.
The animals were individually housed in suspended solid-floor polypropylene cages furnished
with softwood woodflakes. Free access to mains tap water and food (Certified Rat and Mouse
Diet (Certified Rabbit Diet (Code 5322) supplied by International Product Supplies Limited,
Wellingborough, Northants, UK) was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 19 to
25°C and 30 to 70% respectively. Any occasional deviations from these targets were considered
not to have affected the purpose or integrity of the study. The rate of air exchange was
approximately fifteen changes per hour and the lighting was controlled by a time switch to give
twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to
contain any contaminant of a level that might have affected the purpose or integrity of the study.
The strain of mouse used in these laboratories has been shown to produce satisfactory responses using known sensitisers and non-sensitisers during the in-house validation. The results of routine positive control studies are shown in "Any other information on materials". The results of the study are believed to be of value in predicting the sensitisation potential of the test material to man.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Groups of four mice were treated with the test material at concentrations of 2.5%, 5% or 10% w/w
in dimethyl formamide.

No. of animals per dose:

4

Details on study design:

Preliminary Screening Test
As no toxicological information was available regarding the systemic toxicity/irritancy potential
of the test material a preliminary screening test was performed using one mouse. The mouse was
treated by daily application of 25 μI of the test material at a concentration of 10% 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. Any signs of
toxicity or excessive local irritation noted during this period were recorded. The bodyweight of
the mouse was recorded on Day 1 (prior to dosing) and on Day 6.

Main Test
Test Material Administration
Groups of four mice were treated with the test material at concentrations of 2.5%, 5% or 10% w/w
in dimethyl formamide. The preliminary screening test suggested that the test material would not
produce systemic toxicity or excessive local irritation at the highest suitable concentration. The
mice were treated by daily application of 25 μl of the appropriate concentration of the test
material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test
material 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 Administration
Five days following the first topical application of the test material (Day 6) all mice were injected
via the tail vein with 250 μl of phosphate buffered saline containing 3H-methyl thymidine
(3HTdR: 80 μCi/ml, specific activity 2.0 Ci/mmol, Amersham Biosciences UK Ltd) giving a total
of20 μCi to each mouse.

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 study were
recorded.

Bodyweights: The bodyweight 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. The draining auricular lymph nodes from the four mice were excised and
pooled for each experimental group. For each group 1 ml of phosphate buffered saline (PBS) was
added to the pooled lymph nodes.

Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells
was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 ml of PBS into a petri dish labelled with the project number and dose concentration. The lymph node cell suspension was transferred to a 10 ml centrifuge tube. The petri dish was washed with an additional 5 ml of PBS to remove all
remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node
cells were pelleted at 1400 rpm ( approximately 190 g) for ten minutes. The pellet was
resuspended in 10 ml of PBS and re-pelleted. To precipitate out the radioactive material, the
pellet was resuspended in 3 ml of 5% Trichloroacetic acid (TCA).

Determination of 3 HTdR Incorporation: After overnight incubation at 4°C, the precipitates
were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended
in 1 ml of TCA and transferred to 10 ml of scintillation fluid (Optiphase 'Trisafe'). 3 HTdR
incorporation was measured by β-scintillation counting. The "Poly Q™" vials containing the
samples and scintillation fluid were placed in the sample changer of the scintillator and left for
approximately twenty minutes. The purpose of this period of time in darkness was to reduce the
risk of luminescence, which has been shown to affect the reliability of the results. After
approximately twenty minutes, the vials were shaken vigorously. The number of radioactive
disintegrations per minute was then measured using the Beckman LS6500 scintillation system
(Beckman Instruments Inc, Fullerton, CA).

Interpretation of Results
The proliferation response of lymph node cells was expressed as the number of radioactive
disintegrations per minute per lymph node (dpm/node) and as the ratio of 3 HTdR incorporation
into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation
Index).
The test substance will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation, will be classified as a "non-sensitiser".

Positive control substance(s):

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

Statistics:

not specified

Results and discussion

Positive control results:

The Stimulation Index (SI) expressed as the mean radioactive incorporation for each
treatment group divided by the mean radioactive incorporation of the vehicle control group are as
shown in "Any other information on results"

a-HEXYLCINNAMALDEHYDE was considered to be a sensitiser under the
conditions of the test.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2.13

Variability:

not specified

Test group / Remarks:

2.5 % w/w in dimethyl formamide

Results 2

Key result

Parameter:

SI

Value:

2.6

Variability:

not specified

Test group / Remarks:

5 % w/w in dimethyl formamide

Results 3

Key result

Parameter:

SI

Value:

4.11

Variability:

not specified

Test group / Remarks:

10 % w/w in dimethyl formamide

Cellular proliferation data / Observations:

Preliminary Screening Test
Clinical observations, bodyweight and mortality data are given in "Any other information on results".
No signs of systemic toxicity were noted.

Based on this information the dose levels selected for the main test were 2.5%, 5% and 10% w/w
in dimethyl formamide.

Main Test
Estimation of the Proliferative Response of Lymph Node Cells
The radioactive disintegrations per minute (dpm) per lymph node and the stimulation index (SI)
are given in "Any other information on results".
A stimulation index of greater than 3 was recorded for the highest concentration of the test
material (10% w/w).
A stimulation index of less than 3 was recorded for the two lower concentrations of the test
material (2.5% and 5% w/w).

Clinical Observations and Mortality Data
Individual clinical observations and mortality data for test and control animals are given in
"Any other information on results".
There were no deaths. No signs of systemic toxicity were noted in the test or control animals
during the study.

Bodyweight
Individual bodyweights and bodyweight changes for test and control animals are given in "Any other information on results".
Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those
observed in the corresponding control group animals over the same period.

Any other information on results incl. tables

Latest Positive Control Study for the Local Lymph Node Assay

Test Material: a-HEXYLCINNAMALDEHYDE

Concentration (% w/w) in Acetone/Olive Oil 4: 1	Stimulation Index (SI)	Result
5	1.76	Negative
10	2.78	Negative
25	5.06	Positive

Clinical Observations, Bodyweight and Mortality Data -

Preliminary Screening Test

 

Concentration (% w/w) in dimethyl formamide	Animal Number	Bodyweight (g)		Day
				1		2		3		4	5	6
		Day 1	Day 6	Pre-Dose	1 Hr Post Dose	Pre-Dose	1 Hr Post Dose	Pre-Dose	1 Hr Post Dose
10	S-1	21	22	0	0	0	0	0	0	0	0	0

 

Hr = Hour

0 = No signs of system toxicity

 

Dpm, Dpm/Node and Stimulation Index (SI)

 

Concentration (% w/w) in dimethyl formamide	Dpm	Dpm/Node^a	Stimulation Index (SI)^b	Result
Vehicle	3482.84	435.36	N/A	N/A
2.5	7404.89	925.61	2.13	Negative
5	9066.44	1133.31	2.60	Negative
10	14308.57	1788.57	4.11	Positive

^a = Dpm/node obtained by dividing the Dpm value by 8 (total number of lymph nodes)

^b = Stimulation Index of3.0 or greater indicates a positive result

N/A = Not applicable

 

Individual Clinical Observations and Mortality Data

 

Concentration (% w/w) in dimethyl formamide	Animal Number	Day 1		Day 2		Day 3		Day 4	Day 5	Day 6
		Pre-Dose	1 Hr Post Dose	Pre-Dose	1 Hr Post Dose	Pre-Dose	1 Hr 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
2.5	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
5	3-1	0	0	0	0	0	0	0	0	0
	3-2	0	0	0	0	0	0	0	0	0
	3-3	0	0	0	0	0	0	0	0	0
	3-4	0	0	0	0	0	0	0	0	0
10	4-1	0	0	0	0	0	0	0	0	0
	4-2	0	0	0	0	0	0	0	0	0
	4-3	0	0	0	0	0	0	0	0	0
	4-4	0	0	0	0	0	0	0	0	0

Hr = Hour

0 = No signs of systemic toxicity

 

Individual Bodyweights and Bodyweight Changes

 

Concentration (% w/w) in dimethyl formamide	Animal Number	Bodyweight (g)		Bodyweight change (g)
		Day 1	Day 6
Vehicle	1-1	18	20	2
	1-2	21	23	2
	1-3	21	22	1
	1-4	20	22	2
2.5	2-1	22	23	1
	2-2	21	22	1
	2-3	22	22	0
	2-4	23	24	1
5	3-1	21	23	2
	3-2	18	19	1
	3-3	21	22	1
	3-4	19	19	0
10	4-1	21	22	1
	4-2	20	21	1
	4-3	19	20	1
	4-4	21	21	0

 

Summary of Positive Control Data for the Local Lymph Node Assay

 

Project Number	Start Date	Finish Date	Test Material	Concentration	Vehicle	Stimulation Index^a	Classification^b
039/503*	03/07/01	09/07/01	2-Mercapto-benzothiazole	1, 5, 25% w/v	Dimethyl Formamide	2.0, 2.8, 5.4	Positive
039/586*	13/08/02	19/08/02	α-Hexylcinnamaldehyde	5, 10, 50% w/v	4:1 acetone/olive oil	5.7, 5.5, 33.5	Positive
039/629*	13/03/03	19/03/03	α-Hexylcinnamaldehyde	5, 10, 25% v/v	4:1 acetone/olive oil	2.8, 2.3, 5.5	Positive
039/630	13/03/03	19/03/03	α-Hexylcinnamaldehyde	5, 10, 25% v/v	4:1 acetone/olive oil	2.0, 1.9, 6.8	Positive
039/656*	10/10/03	16/10/03	α-Hexylcinnamaldehyde	5, 10, 25% v/v	4:1 acetone/olive oil	1.76, 2.78, 5.06	Positive
039/658	16/10/03	22/10/03	α-Hexylcinnamaldehyde	5, 10, 25% v/v	4:1 acetone/olive oil	1.49, 1.73, 5.26	Positive

 

a= Ratio of test to control lymphocyte proliferation

b = Stimulation index greater than 3.0 indicates a positive result

*=Standard Test Method 595 ('Pooled' nodes)

- Standard Test Method 599 ('Individual' nodes)

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test material was considered to be a sensitiser under the conditions of the test.

Executive summary:

Introduction. A study was performed to assess the skin sensitisation potential of the test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of the following:
• OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 24 April 2002)

Methods. Following a preliminary screening test, three groups, each of four animals, were treated with 50 μl (25 μl per ear) of the test material as a solution in dimethyl formamide at concentrations of 2.5%, 5% and 10% w/w. A further group of four animals was treated with dimethyl formamide alone.

Results. The Stimulation Index (SI) expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (% w/w) in dimethyl formamide	Stimulation Index (SI)	Result
2.5	2.13	Negative
5	2.60	Negative
10	4.11	Positive

 

Conclusion. The test material was considered to be a sensitiser under the conditions of the test.