Tetrasodium {5-[(4-chloro-6-{[4-(alkenylsulfonyl)phenyl]amino}-heteromonocycl-2-yl)amino]-4-(hydroxy-κO)-3-{[2-(hydroxy-κO)phenyl]diazenyl-κN}naphthalene-disulfonato(4-)}[3-(hydroxy-κO)-4-{[2-(hydroxy-κO)naphthalen-1-yl]diazenyl-κN}naphthalene-sulfonato(3-)]chromate(4-)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 4th March 2014 Experimental completion date: 26th March 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP 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

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification: FAT 20341/A TE
Physical State/Appearance: black powder, solid at 20 °C
Batch: BOP 02-12 (Navy PLK 241, BS)
Purity: 80.1%
Expiry date: 21 November 2017
Storage Conditions: room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatization 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 (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., 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 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:

Preliminary screening test: 50% w/w in 1% Pluronic L92 in distilled water

Main test: 50%, 25% and 10% w/w in 1% Pluronic L92 in distilled water

No. of animals per dose:

Four mice per dose group

Details on study design:

Preliminary Screening Test
Using available information 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 50% 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. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The body weight 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 Test
Test Item Administration
Groups of four mice were treated with the test item at concentrations of 50%, 25% or 10% w/w in 1% pluronic L92 in distilled water. 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.

3H-Methyl Thymidine 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 (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 µCi to each mouse

Positive control substance(s):

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

Statistics:

Not applicable

Results and discussion

Positive control results:

The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:

Concentration (% v/v) in
1% pluronic L92 in distilled water Stimulation Index Result
25 4.66 Positive


Conclusion
alpha Hexylcinnamaldehyde was considered to be a sensitizer under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Concentration (% w/w) in Stimulation Index Result 1% pluronic L92 in Distilled water Vehicle na na 10 1.73 Negative 25 1.83 Negative 50 7.24 Positive

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Concentration (% w/w) in dpm dpm/Node Result 1% pluronic L92 in Distilled water Vehicle 12468.88 1558.61 na 10 21523.70 2690.46 Negative 25 22801.10 2850.14 Negative 50 90320.17 11290.02 Positive

Any other information on results incl. tables

Clinical observations, body weight and mortality data are given inTable 1 and local skin irritation is given inTable 2. The ear thickness measurements and mean ear thickness changes are given inTable 3.

 

Dark blue colored staining on the ears was noted post dose on Days 1 to 3. 

 

No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.

 

Based on this information the dose levels selected for the main test were50%,25% and10% w/win1% pluronic L92 in distilled water.

Table1               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
50	S-1	18	18	0	0Fs	0	0Fs	0	0Fs	0	0	0

0= No signs of systemic toxicity

Table2     Local Skin Irritation – Preliminary Screening Test

Concentration (%w/w) in 1% pluronic L92 indistilled water

Animal Number

Local Skin Irritation

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

left

right

left

right

left

right

left

right

left

right

left

right

50

S-1

0

0

0

0

0

0

0

0

0

0

0

0

 

Table3     Measurement of Ear Thickness and Mean Ear Thickness Changes – Preliminary Screening Test

Concentration (%w/w) in 1% pluronic L92 indistilled water	Animal Number	Ear Thickness Measurement (mm)
		Day 1		Day 3		Day 6
		pre‑dose		post dose
		left	right	left	right	left	right
50	S-1	0.21	0.22	0.22	0.22	0.23	0.23
overall mean (mm)		0.22		0.22		0.23
overall mean ear thickness change (%)		na		2.33		6.98

 

na=        Not applicable

Main Test

Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute per lymph node and the stimulation index are given inTable 4.

 

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
10	1.73	Negative
25	1.83	Negative
50	7.24	Positive

 

 

Table4     Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (%w/w) in 1% pluronic L92 indistilled water	dpm	dpm/Nodea	Stimulation Indexb	Result
Vehicle	12468.88	1558.61	na	na
10	21523.70	2690.46	1.73	Negative
25	22801.10	2850.14	1.83	Negative
50	90320.17	11290.02	7.24	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

 Clinical Observations and Mortality Data

Individual clinical observations and mortality data for test and control animals are given inTable 5.

 

Dark blue colored staining on the ears was noted post dose on Days 1 to 3 in animals treated with the test item at concentrations of50% or25% w/win1% pluronic L92 in distilled water. 

 

There were no deaths. No signs of systemic toxicity were noted in the test or control animalsduring the test.

Table5     Individual Clinical Observations and Mortality Data

Concentration (%w/w) in 1% pluronic L92 indistilled 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
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		0Fs		0		0Fs		0		0Fs		0		0		0
		3-2		0		0Fs		0		0Fs		0		0Fs		0		0		0
		3-3		0		0Fs		0		0Fs		0		0Fs		0		0		0
		3-4		0		0Fs		0		0Fs		0		0Fs		0		0		0
50		4-1		0		0Fs		0		0Fs		0		0Fs		0		0		0
		4-2		0		0Fs		0		0Fs		0		0Fs		0		0		0
		4-3		0		0Fs		0		0Fs		0		0Fs		0		0		0
		4-4		0		0Fs		0		0Fs		0		0Fs		0		0		0

0=          No signs of systemic toxicity

Fs =        Dark blue colored staining on the ears

  Body Weight

Individual body weights and body weight change for test and control animals are given inTable 6.

 

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.

Table6     Individual Body Weights and Body Weight Change

Concentration (%w/w) in 1% pluronic L92 indistilled water	Animal Number	Body Weight (g)		Body Weight Change (g)
		Day 1	Day 6
Vehicle	1-1	20	19	-1
	1-2	19	20	1
	1-3	19	20	1
	1-4	20	21	1
10	2-1	17	18	1
	2-2	16	18	2
	2-3	18	18	0
	2-4	17	18	1
25	3-1	18	20	2
	3-2	16	16	0
	3-3	19	20	1
	3-4	19	22	3
50	4-1	20	20	0
	4-2	19	20	1
	4-3	18	20	2
	4-4	18	18	0

 

Calculation of EC₃Value

aEC₃= c + [[(3-d)/(b-d)] x (a-c)]

 

a	=	50
b	=	7.24
c	=	25
d	=	1.83
EC3=25+ [[(3-1.83)/(7.24-1.83)] x (50-25)] =30

 

The concentration of test item expected to cause a 3 fold increase in³HTdR incorporation (EC₃value) was calculated to be30%.

a=   lowest concentration giving stimulation index >3

b =  actual stimulation index caused by ‘a’

c =  highest concentration failing to produce a stimulation index of 3

d =  actual stimulation index caused by ‘c’

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

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

Executive summary:

Introduction

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

 

 

Methods

This study was designed to be compatible with the procedures indicated by the following internationally accepted guidelines and recommendations:

 

·     OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitization: Local Lymph Node Assay" (adopted 22 July 2010)

·     Method B42 Skin Sensitization (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008

Following a preliminary screening test in which no clinical signs of toxicity were noted at aconcentration 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 asolution in 1% pluronic L92 in distilled water at concentrations of 50%,25% or 10% w/w. A further group of four animals was treated with1% 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
10	1.73	Negative
25	1.83	Negative
50	7.24	Positive

 

The concentration of test item expected to cause a 3 fold increase in³HTdR incorporation (EC₃value) was calculated to be30%.

 

 

Conclusion

The test item was considered to be asensitizerunder the conditions of the test.

 

The test item was also classified as a contact sensitizer (Category 1) according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. It is reasonable to assume that the Signal Word “Warning” and the Hazard Statement “H317: May cause an allergic skin reaction” are therefore required.