Potassium sodium amino-hydroxy-[(3-{[2-(substituted)ethyl]sulfonyl}phenyl)diazenyl]-[(2-sulfonato-4-{[2-(substituted)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-disulfonate

Endpoint summary

Description of key information

The test item was considered a skin sensitiser.

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 12 February 2015 and 25 February 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Principles of method if other than guideline:

None

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

None

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied Harlan Laboratories B.V., 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 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 12 hours continuous light (06.00 to 18.00) 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:

dimethylformamide

Concentration:

50%, 25% or 10% w/w

No. of animals per dose:

Groups of four mice were treated with the test item.

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 dimethyl formamide. 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 concentration of 50% 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. 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 dimethyl formamide. 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 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 (Optiphase 'Trisafe'). 3HTdR incorporation was measured by  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 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):

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

Statistics:

No data

Positive control results:

Introduction
A study was performed to assess the sensitivity of the strain of mouse used at these laboratories to a known sensitizer. 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 22 July 2010)
 Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) 440/2008

Test item: α Hexylcinnamaldehyde, tech., 85%
Study number: 41500209
Study dates: 06 February 2015 to 12 February 2015

Methods
Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of α Hexylcinnamaldehyde, tech., 85% as a solution in dimethyl formamide at concentrations of 5%, 15% or 25% v/v. A further group of five animals was treated with dimethyl formamide alone and served as the vehicle control group.

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 is as follows:

5 % v/v in dimethyl formamide the Stimulation Index was 2.81 (positive)
15 % v/v in dimethyl formamide the Stimulation Index was 6.26 (positive)
25 % v/v in dimethyl formamide the Stimulation Index was 8.14 (positive)

The concentration of α Hexylcinnamaldehyde, tech., 85% expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 6%.

Conclusion
α Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.

Key result

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Preliminary Screening Test Dark blue coloured staining of the ears and fur was noted post dose on Days 2 and 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 were 50%, 25% and 10% w/w in dimethyl formamide. 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: 10 % (w/w) in dimethyl formamide the Stimulation index was 5.58 (positive) 25 % (w/w) in dimethyl formamide the Stimulation index was 5.40 (positive) 50 % (w/w) in dimethyl formamide the Stimulation index was 5.86 (positive)

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: No data

Estimation of the Proliferative Response of Lymph Node Cells

Concentration (%w/w) in dimethyl formamide	Stimulation Index	Result
10	5.58	Positive
25	5.40	Positive
50	5.86	Positive

Clinical Observations and Mortality Data

Dark blue colored staining of the ears and fur was noted, post dose on Days 1 to 3, in animals treated with the test item at concentrations of 50% or 25% w/w in dimethyl formamide. 

 

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.

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

Concentration (%w/w) in dimethyl formamide	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	18.1	19.6	0	0	0	0Fs	0	0Fs	0	0	0

0=    No signs of systemic toxicity

Fs = Dark blue colored staining of the ears and fur

Local Skin Irritation – Preliminary Screening Test

Concentration (%w/w) in dimethyl formamide

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

 

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

Concentration (%w/w) in dimethyl formamide	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	0.21	0.22	0.22	0.22	0.23
overall mean (mm)		0.21		0.22		0.23
overall mean ear thickness change (%)		na		4.76		7.14

na = Not applicable

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

Concentration (%w/w) in dimethyl formamide	dpm	dpm/Nodea	Stimulation Indexb	Result
Vehicle	10590.42	1323.80	na	na
10	59126.27	7390.78	5.58	Positive
25	57138.24	7142.28	5.40	Positive
50	62088.44	7761.06	5.86	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 dimethyl formamide	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 of the ears and fur

Individual Body Weights and Body Weight Change

Concentration (% w/w) in dimethyl formamide	Animal Number	Body Weight (g)		Body Weight Change (g)
		Day 1	Day 6
Vehicle	1-1	18.2	18.3	0.1
	1-2	20.6	19.9	-0.7
	1-3	17.9	18.5	0.6
	1-4	20.8	20.7	-0.1
10	2-1	18.6	19.8	1.2
	2-2	20.2	21.0	0.8
	2-3	18.2	18.9	0.7
	2-4	19.9	21.6	1.7
25	3-1	19.4	20.5	1.1
	3-2	17.5	19.0	1.5
	3-3	18.1	19.9	1.8
	3-4	18.1	19.0	0.9
50	4-1	16.8	17.1	0.3
	4-2	21.9	21.8	-0.1
	4-3	18.3	19.4	1.1
	4-4	18.3	20.1	1.8

Summary of Positive Control Data for the Local Lymph Node Assay

Study Number	Start Date	Finish Date	Test Item	Concentration	Vehicle	Stimulation Index^a	Classification^b
41403324	03/10/14	09/10/14	α‑Hexylcinnamaldehyde, tech., 85%	50% v/v	cottonseed oil	8.37	Positive
41403326	09/10/14	15/10/14	α‑Hexylcinnamaldehyde, tech., 85%	15% v/v	butanone	6.50	Positive
41403327	24/10/14	30/10/14	α‑Hexylcinnamaldehyde, tech., 85%	25% v/v	dimethyl sulphoxide	5.19	Positive
41403329	24/10/14	30/10/14	α‑Hexylcinnamaldehyde, tech., 85%	15% v/v	ethanol/distilled water 7:3	8.21	Positive
41403330	14/11/14	20/11/14	α‑Hexylcinnamaldehyde, tech., 85%	15% v/v	acetone	12.40	Positive
41404270*	14/01/15	20/01/15	α‑Hexylcinnamaldehyde, tech., 85%	5%, 15%, 25% v/v	acetone/olive oil 4:1	1.63, 2.28, 5.45	Positive
41500120*	21/01/15	27/01/15	α‑Hexylcinnamaldehyde, tech., 85%	5%, 10%, 25% v/v	1% pluronic L92 in distilled water	1.67, 2.45, 11.62	Positive
41500210•	04/02/15	10/02/15	α‑Hexylcinnamaldehyde, tech., 85%	15% v/v	butanone	5.27	Positive
41500209•	06/02/15	12/02/15	α‑Hexylcinnamaldehyde, tech., 85%	5%, 15%, 25% v/v	dimethyl formamide	2.81, 6.26, 8.14	Positive
41500211•	06/02/15	12/02/15	Phenylacetaldehyde (>90%)	5% v/v	propylene glycol	14.82	Positive

a= Ratio of test to control lymphocyte proliferation

b= Stimulation index greater than 3.0 indicates a positive result

*= Animals supplied by Charles River

• = Animals supplied by Harlan Laboratories B.V., The Netherlands

Interpretation of results:

sensitising

Conclusions:

The test item was considered to be a sensitiser.

Executive summary:

A Local Lymph Node Assay according to OECD Guideline 429 and EU Method B.42, 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. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50% w/w, this concentration was selected as the highest dose investigated in the main test. Three groups, consisting four animals each, were treated with 50 µL (25 µL per ear) of the test item as asuspensionindimethyl formamideat concentrations of 50%, 25% or 10% w/w. The stimulation indices, expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group, were 5.58, 5.40 and 5.86 for 10, 25 and 50% concentrations respectively, indicating a positive response at all the tested concentrations. Hence the test item was considered as a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A Local Lymph Node Assay according to OECD Guideline 429 and EU Method B.42, 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. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50% w/w, this concentration was selected as the highest dose investigated in the main test. Three groups, consisting four animals each, were treated with 50 µL (25 µL per ear) of the test item as a suspension in dimethyl formamide at concentrations of 50%, 25% or 10% w/w. The stimulation indices, expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group, were 5.58, 5.40 and 5.86 for 10, 25 and 50% concentrations respectively, indicating a positive response at all the tested concentrations. Hence the test item was considered as a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In a mouse lymph node assay, all three concentrations of the test substance gave a stimulation index of greater than 3. Hence the substance is therefore considered to be a sensitiser. Therefore, the test substance can be classified as a skin sensitiser cat 1 according to the CLP (Regulation 1272/2008).