hexasodium 5-[(4-{3-[(8-benzamido-1-hydroxy-3,6-disulfonato-2-naphthyl)diazenyl]-4-sulfonatoanilino}-6-[(2-sulfonatoethyl)amino]-1,3,5-triazin-2-yl)amino]isophthalate

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 conducted between 16 July 2007 and 31 July 2007.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

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:

Magenta T-43

IUPAC Name:

Magenta T-43

Test material form:

solid: particulate/powder

Details on test material:

Sponsor's identification: MAGENTA T-43
Description: dark red solid
Storage conditions: room temperature in the dark
Batch number: H186-5

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Animals and Animal Husbandry:

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) 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.

Study design: in vivo (LLNA)

Vehicle:

other: 1% pluronic L92 in distilled water

Concentration:

Groups of four mice were treated with the test material at concentrations of 5%, 10% or 25% w/w in 1% pluronic L92 in distilled water. The mice were treated by daily application of 25 µl per ear of the appropriate concentration of the test material for three consecutive days.

A further group of four mice received the vehicle alone in the same manner.

No. of animals per dose:

Three groups each of four mice were treated with the test material at concentrations of 5%, 10% or 25% w/w .

Details on study design:

Preparation of Test Material:
For the purpose of the study, the test material was freshly prepared in 1% pluronic L92 in distilled water. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. The concentrations used are given in the procedure section. 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.

Procedure
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 µl of the test material at a concentration of 25% w/w in 1% pluronic L92 in distilled water, 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 5%, 10% or 25% w/w in 1% pluronic L92 in distilled water. 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 (PBS) containing 3H-methyl thymidine 3HTdR: 80 µCi/ml, specific activity 2.0 Ci/mmol) giving a total of 20 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 test 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 mlof 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 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 3HTdR Incorporation:
After approximately eighteen hours incubation at approximately 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’). 3HTdR incorporation was measured by β-scintillation counting. The "Poly QTM" 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.

Positive control substance(s):

other: 2,4-Dinitrobenzenesulfonic acid, sodium salt

Results and discussion

Positive control results:

A positive control study was conducted to assess the sensitivity of the strain of mouse used to test material 2,4-dinitrobenzenesulfonic acid, sodium salt; a known sensitizer, following OECD Test Guideline 429 and Method B42 of Commission Directive 2004/73/EC.

Three groups, each of five animals were treated with 50 µl (25 µl per ear) of 2,4-dinitrobenzenesulfonic acid, sodium salt, as a solution in 1% pluronic L92 in distilled water at concentrations of 1, 10 and 20% w/w. A further control group of five animals were treated with 1% pluronic L92 solution in distilled water alone.

The results of the stimulation index were expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group.

At 1% w/w concentration, the stimulation index was 1.39 producing a negative result. However, at 10 and 20 %w/w, the stimulation index results were 11.33 and 19.34 respectively, producing positive results for both concentration levels.

From these results it was concluded that 2,4-dinitrobenzenesulfonic acid, sodium salt was considered to be a sensitizer under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.36

Test group / Remarks:

5 % test material in 1 % pluronic L92 in distilled water

Remarks on result:

other: Negative

Results 2

Parameter:

SI

Value:

1.29

Test group / Remarks:

10 % test material in 1 % pluronic L92 in distilled water

Remarks on result:

other: Negative

Results 3

Parameter:

SI

Value:

3.21

Test group / Remarks:

25 % test material in 1 % pluronic L92 in distilled water

Remarks on result:

other: Positive

Any other information on results incl. tables

Results

Preliminary Screening Test:

Clinical observations, bodyweight and mortality data are given below:

 

No signs of systemic toxicity were noted. Red-coloured staining on the ears was noted post-dose on Day 1 and for the remainder of the test.

 

Based on this information the dose levels selected for the main test were 5%, 10% and 25% w/w in 1% pluronic L92 in distilled water.

Clinical Observations, Bodyweight and Mortality Data Preliminary Screening Test

 

Concentration (% w/w) in 1% Pluronic L92 In Distilled Water

Animal Number

Bodyweight (g)

Day

1

2

3

4

5

6

Day 1

Day 6

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Pre-Dose

Post Dose

25

S-1

17

17

0

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

 

0 = No signs of systemic toxicity

Fs = Red-coloured staining on the ears

 

Main Test:

Clinical Observations and Mortality Data:

Individual clinical observations and mortality data for test and control animals are given.

 

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Red-coloured staining on the ears was noted at all test sites post dose on Days 1 and 2 and for the remainder of the test.

 

Bodyweight:

Individual bodyweights and bodyweight changes for test and control animals are given. 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.

Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

 

Concentration (%w/w) in 1% pluronic L92 in distilled water	dpm	Dpm/Node^a	Stimulation Index^b	Result
Vehicle	3975.92	496.99	na	na
5	5415.41	676.80	1.36	Negative
10	5134.74	641.84	1.29	Negative
25	12781.72	1597.72	3.21	Positive

 

Dpm = Disintegrations per minute

a=Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

b= Stimulation Index of 3.0 or greater indicates a positive result

na= Not applicable

Individual Clinical Observations and Mortality Data

 

Concentration (% w/w) in 1% pluronic L92 in distilled water	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
5	2-1	0	0Fs	0	0Fs	0Fs	0Fs	0Fs	0Fs	0Fs
	2-2	0	0Fs	0	0Fs	0Fs	0Fs	0Fs	0Fs	0Fs
	2-3	0	0Fs	0	0Fs	0Fs	0Fs	0Fs	0Fs	0Fs
	2-4	0	0Fs	0	0Fs	0Fs	0Fs	0Fs	0Fs	0Fs
10	3-1	0	0Fs	0	0Fs	0Fs	0Fs	0Fs	0Fs	0Fs
	3-2	0	0Fs	0	0Fs	0Fs	0Fs	0Fs	0Fs	0Fs
	3-3	0	0Fs	0	0Fs	0Fs	0Fs	0Fs	0Fs	0Fs
	3-4	0	0Fs	0	0Fs	0Fs	0Fs	0Fs	0Fs	0Fs
25	4-1	0	0Fs	0	0Fs	0Fs	0Fs	0Fs	0Fs	0Fs
	4-2	0	0Fs	0	0Fs	0Fs	0Fs	0Fs	0Fs	0Fs
	4-3	0	0Fs	0	0Fs	0Fs	0Fs	0Fs	0Fs	0Fs
	4-4	0	0Fs	0	0Fs	0Fs	0Fs	0Fs	0Fs	0Fs

 

0 = No signs of systemic toxicity

Fs = red-coloured staining on the ears

Individual Bodyweights and Bodyweight Changes

 

Concentration (% w/w) in 1% pluronic L92 in distilled water	Animal Number	Bodyweight		Bodyweight Change (g)
		Day 1	Day 6
Vehicle	1-1	19	18	-1
	1-2	16	15	-1
	1-3	17	17	0
	1-4	20	19	-1
5	2-1	17	17	0
	2-2	17	17	0
	2-3	18	18	0
	2-4	18	17	-1
10	3-1	17	18	1
	3-2	20	21	1
	3-3	20	20	0
	3-4	19	18	-1
25	4-1	17	17	0
	4-2	20	18	-2
	4-3	18	18	0
	4-4	19	17	-2

 

Applicant's summary and conclusion

Interpretation of results:

other: Not classified according to EU 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)

• Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Directive 2004173/EC

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 1% pluronic L92 in distilled water at concentrations of 5%, 10% or 25%w/w. A further group of four animals was treated with 1% pluronic L92 in distilled water alone.

 

Results

The Stimulation Index 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 1% pluronic L92 in distilled water	Stimulation Index	Result
5	1.36	Negative
10	1.29	Negative
25	3.21	Positive

 

Conclusion

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