Reaction mass of Potassium sodium 3-{[4-({3-(substitutedamino)-4-[(2-sulfonato-4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]phenyl}amino)-6-chloro-heteromonocyl-2-yl]amino}-2-substituted-5-{[2-(sulfonatooxy)ethyl]sulfonyl}benzenesulfonate (1:3:1) and Potassium sodium 3-[(4-{[3-(subsitutedamino)-4-{[4-(ethenylsulfonyl)-2-sulfonatophenyl]diazenyl}phenyl]amino}-6-chloro-heteromonocy-2-yl)amino]-2-substituted-5-{[2-(sulfonatooxy)ethyl]sulfonyl}benzenesulfonate (3:9:3)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 February 2014 to 25 March 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

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:

other: solid

Details on test material:

Identification: FAT 40863/A TE
Sponsor description: yellowish to brown solid
Harlan description: red solid
Batch: BOP 02-13 (BS-ROE1670)
Purity: 82.1%
Expiry date: 05 June 2018
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:

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

No. of animals per dose:

Groups of four mice were treated

Details on study design:

The assay has undergone extensive inter-laboratory validation and has been shown to reliably detect test items that are moderate to strong sensitizers.

The strain of mouse used in these laboratories has been shown to produce satisfactory responses using known sensitizers and non sensitizers during the in house validation. The results of routine positive control studies are shown in Appendix 1 and Appendix 2. The results of the study are believed to be of value in predicting the sensitization potential of the test item to man.

Preliminary Screening Test
Using available information regarding the 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 according to the scale included as Appendix 4. 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.

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. 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.

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 labeled with the study 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
re-suspended in 10 mL of PBS and re pelleted. To precipitate out the radioactive material, the pellet was re suspended 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, re suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by beta 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, USA).

Positive control substance(s):

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

Statistics:

None provided.

Results and discussion

Positive control results:

Methods
A group of five animals was treated with 50 µL (25 µL per ear) of alpha Hexylcinnamaldehyde as an emulsion in 1% pluronic L92 in distilled water at a concentration of 25% v/v. A further control group of five animals was treated with 1% pluronic L92 in distilled water alone.


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:

See Table 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, 1.58, Negative 25, 2.20, Negative 50, 4.79, Positive

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

See Table 4 Disintegrations per Minute, Disintegrations per Minute/Node are as follows Concentration (% w/w) in 1% pluronic L92 in distilled water, dpm, dpm/Node Vehicle, 14392.92, 1799.12 10, 22669.58, 2833.70 25, 31632.62, 3954.08 50, 68870.23, 8608.78

Any other information on results incl. tables

Preliminary Screening Test

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

 

Red/pink colored staining of the ears and fur was noted on Days 2 and 3. The staining did not affect evaluation of erythema on the ears.

 

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 were 50%, 25% and 10% w/w in 1% pluronic L92 in distilled water.

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 in Table 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	1.58	Negative
25	2.20	Negative
50	4.79	Positive

Clinical Observations and Mortality Data

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

 

Red/pink colored staining of the ears and fur was noted in all test animals, post dose on Days 1 to 3. Fur loss was also noted in animals treated with the test item at a concentration of 50% w/w in 1% pluronic L92 in distilled water.

 

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

 

Body Weight

Individual body weights and body weight change for test and control animals are given in Table 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.

Calculation of EC₃Value

aEC₃= c + [[(3-d)/(b-d)] x (a-c)]

 

a	=	50
b	=	4.79
c	=	25
d	=	2.20
EC3=25+ [[(3-2.20)/(4.79-2.20)] x (50-25)] =33

 

The concentration of test item expected to cause a 3 fold increase in ³HTdR incorporation (EC₃value) was calculated to be 33%.

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’

Table 1     Clinical Observations, Body Weight and Mortality Data – Preliminary Screening Test

Concentration (%w/w) in 1% pluronic L92 indistilled water	Animal Number	Body Weight (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	19	0	0	0	0Fs	0	0Fs	0	0	0

0 =          No signs of systemic toxicity

Fs =        Red/pink colored staining of the ears and fur

Table 2     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 STA

0 STA

0 STA

0 STA

0

0

0

0

0

0

STA = Red/pink colored staining of the ears

Table 3     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	0.22	0.21	0.23	0.23	0.21	0.21
overall mean (mm)		0.22		0.23		0.21
overall mean ear thickness change (%)		na		6.98		-2.33

na=        Not applicable

Table 4     Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (% w/w) in 1% pluronic L92 indistilled water	dpm	dpm/Node ^a	Stimulation Index ^b	Result
Vehicle	14392.92	1799.12	na	na
10	22669.58	2833.70	1.58	Negative
25	31632.62	3954.08	2.20	Negative
50	68870.23	8608.78	4.79	Positive

dpm = Disintegrations per minut

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

Table 5     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	0Fs	0	0Fs	0	0Fs	0	0	0
	2-2	0	0Fs	0	0Fs	0	0Fs	0	0	0
	2-3	0	0Fs	0	0Fs	0	0Fs	0	0	0
	2-4	0	0Fs	0	0Fs	0	0Fs	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	0FsFl	0Fl	0Fl	0Fl
	4-2	0	0Fs	0	0Fs	0Fl	0FsFl	0Fl	0Fl	0Fl
	4-3	0	0Fs	0	0Fs	0	0FsFl	0Fl	0Fl	0Fl
	4-4	0	0Fs	0	0Fs	0	0FsFl	0Fl	0Fl	0Fl

0 =          No signs of systemic toxicity

Fs =        Red/pink colored staining of the ears and fur

Fl =        Fur loss

Table 6     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	19	20	1
	1-2	21	21	0
	1-3	16	16	0
	1-4	18	19	1
10	2-1	17	17	0
	2-2	18	19	1
	2-3	17	18	1
	2-4	19	19	0
25	3-1	19	17	-2
	3-2	18	19	1
	3-3	18	18	0
	3-4	18	20	2
50	4-1	17	17	0
	4-2	20	20	0
	4-3	18	18	0
	4-4	22	21	-1

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.

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.

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

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 a solution in1% 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	1.58	Negative
25	2.20	Negative
50	4.79	Positive

 

The concentration of test item expected to cause a 3 fold increase in ³HTdR incorporation (EC₃value) was calculated to be 33%.

 

 

Conclusion

The test item was considered to be a sensitizer under 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.