Reaction mass of Tetrasodium 3-{[5-({4-[alkyl(3-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)amino]-6-fluoro- heteromonocycle-2-yl}amino)-2-sulfonatophenyl]diazenyl}-4-hydroxy-5-(alkanoylamino)naphthalene-2,7-disulfonate and Trisodium 3-[{5-[(4-{[3-(ethenylsulfonyl)phenyl](alkyl)amino}-6-fluoro-heteromonocycle-2-yl)amino]-2-sulfonatophenyl}diazenyl]-4-hydroxy-5-(alkanoylamino)naphthalene-2,7-disulfonate

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 20 August 2015 and 08 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatization period of at least 5 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 8 to 12 weeks old.

The animals were 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 Envigo RMS (UK) Limited, 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 12 hours continuous light and 12 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.


Justification
Mice are the preferred species of choice since quantitative methods have been developed for the measurement of skin sensitization responses in the mouse and are specified in the appropriate test guidelines.

Vehicle:

propylene glycol

Concentration:

Concentrations of 25%, 10% or 5% w/w

No. of animals per dose:

Four

Details on study design:

Test Item Formulation and Experimental Preparation
For the purpose of the study, the test item was freshly prepared as a suspension in propylene glycol. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. The concentrations used are given in the procedure section.

The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.


Procedure
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 maximum attainable concentration of 25% w/w in propylene glycol, 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 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 25%, 10% or 5% w/w in propylene glycol. 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 followed by cervical separation. 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 10 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 18 hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR 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 to stand in darkness for approximately 20 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 20 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):

other: Phenylacetaldehyde (>90%)

Statistics:

No data

Positive control results:

See below

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Preliminary Screening Test Residual test item on the ears was noted post dose on Days 1 and 2. Red colored staining of the fur was noted post dose on Day 1 and at all subsequent observations and prevented evaluation of erythema on both ears, on Days 1 to 3. No visual local skin irritation was noted on Days 4 to 6. No signs of systemic toxicity 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 25%, 10% and 5% w/w in propylene glycol. Main Test Estimation of the Proliferative Response of Lymph Node Cells 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: 5 % concentration (% w/w) in propylene glycol gave a stimulation index of 3.10 (positive) 10 % concentration (% w/w) in propylene glycol gave a stimulation index of 5.43 (positive) 25 % concentration (% w/w) in propylene glycol gave a stimulation index of 6.18 (positive)

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: No data

Clinical Observations and Mortality Data

Red colored staining of the fur was noted in all test animals during the observation period. Residual test item on the ears was noted on Days 1 to 3 in animals treated with the test item at a concentration of25% w/winpropylene glycol. 

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

 

Body Weight

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

EC₃= 2^ {log₂(c) + (3-d)/(b-d) x [log₂(a)-log₂(c)]}

a = 10

b = 5.43

c = 5

d = 3.10

 

EC₃= 2^ {log₂(25) + (3-3.10)/(5.43-3.10) x [log₂(10)-log₂(5)]} =4.9

The concentration of test item expected to cause a 3 fold increase in³HTdR incorporation (extrapolated EC₃value) was calculated to be 4.9%.

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

Concentration (%w/w) in propylene glycol

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

25

S-1

19.7

20.6

0

0RtFs

0Fs

0RtFs

0Fs

0Fs

0Fs

0Fs

0Fs

0 = No signs of systemic toxicity

Rt = Residual test item on the ears

Fs = Red colored staining of the fur

Local Skin Irritation – Preliminary Screening Test

Concentration (%w/w) in propylene glycol

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

25

S-1

?s

?s

?s

?s

?s

?s

0

0

0

0

0

0

?s = Dark redcolored staining prevented evaluation of erythema

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

Concentration (%w/w) in propylene glycol	Animal Number	Ear Thickness Measurement (mm)
		Day 1		Day 3		Day 6
		pre‑dose		post dose
		left	right	left	right	left	right
25	S-1	0.21	0.23	0.19	0.20	0.21	0.20
overall mean (mm)		0.220		0.195		0.205
overall mean ear thickness change (%)		na		-11.36		-6.82

na = Not applicable

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

Concentration (%w/w) in propylene glycol	dpm	dpm/Nodea	Stimulation Indexb	Result
Vehicle	10133.40	1266.68	na	na
5	31397.56	3924.70	3.10	Positive
10	55034.30	6879.29	5.43	Positive
25	62624.63	7828.08	6.18	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

Individual Clinical Observations and Mortality Data

Concentration (% w/w) in propylene glycol	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	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
10	3-1	0	0Fs	0	0Fs	0Fs	0Fs	0Fs	0	0
	3-2	0	0Fs	0	0Fs	0Fs	0Fs	0Fs	0	0
	3-3	0	0Fs	0	0Fs	0Fs	0Fs	0Fs	0	0
	3-4	0	0Fs	0	0Fs	0Fs	0Fs	0Fs	0	0
25	4-1	0	0FsRt	0FsRt	0FsRt	0FsRt	0FsRt	0Fs	0Fs	0
	4-2	0	0FsRt	0FsRt	0FsRt	0FsRt	0FsRt	0Fs	0Fs	0
	4-3	0	0FsRt	0FsRt	0FsRt	0FsRt	0FsRt	0Fs	0Fs	0
	4-4	0	0FsRt	0FsRt	0FsRt	0FsRt	0FsRt	0Fs	0Fs	0

0 = No signs of systemic toxicity

Rt = Residual test item on the ears

Fs = Red colored staining of the fur

Individual Body Weights and Body Weight Change

Concentration (% w/w) in propylene glycol	Animal Number	Body Weight (g)		Body Weight Change (g)
		Day 1	Day 6
Vehicle	1-1	16.3	20.8	4.5
	1-2	19.8	19.2	-0.6
	1-3	21.2	21.0	-0.2
	1-4	20.2	22.6	2.4
5	2-1	18.8	19.9	1.1
	2-2	19.7	20.5	0.8
	2-3	21.5	23.4	1.9
	2-4	19.6	19.7	0.1
10	3-1	19.0	19.9	0.9
	3-2	18.1	20.3	2.2
	3-3	18.3	21.9	3.6
	3-4	21.0	19.9	-1.1
25	4-1	19.7	21.2	1.5
	4-2	19.9	20.5	0.6
	4-3	20.3	18.7	-1.6
	4-4	17.9	21.9	4.0

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 classified as a contact sensitizer (Category 1, sub-category 1B) according to the Globally Harmonized System of Classification and Labelling of Chemicals.

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 a maximum attainable concentration of 25% 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) ofthe test item as a suspension in propylene glycol at concentrations of 25%,10% or 5% w/w. A further group of four animals was treated with propylene glycol 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 propylene glycol	Stimulation Index	Result
5	3.10	Positive
10	5.43	Positive
25	6.18	Positive

The concentration of test item expected to cause a 3 fold increase in³HTdR incorporation (extrapolated EC₃value) was calculated to be 4.9%.

 

Conclusion

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

The test item was classified as a contact sensitizer (Category 1, sub-category 1B) according to the Globally Harmonized System of Classification and Labelling of Chemicals.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Following a preliminary screening test, three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test item as a suspension in propylene glycol at concentrations of 25%,10% or 5% w/w. A further group of four animals was treated with propylene glycol 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 propylene glycol	Stimulation Index	Result
5	3.10	Positive
10	5.43	Positive
25	6.18	Positive

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

The test item was classified as a contact sensitizer (Category 1, sub-category 1B) according to the GloballyHarmonized Systemof Classification and Labelling of Chemicals.

Migrated from Short description of key information:
The 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.

Justification for selection of skin sensitisation endpoint:
Only this study is available and the study is GLP Guideline study (Study Klimisch 1).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the above stated assessment of the skin sensitisation potential, the substance does needs to be classified as Skin sensitiser according to CLP (Regulation (EC) No 1272/2008 Of The European Parliament And Of The Council)as implementation of UN-GHS in the EU.