N-[2-[(2-cyano-4,6-dinitrophenyl)azo]-5-(diethylamino)phenyl]acetamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 September 2015 to 29 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

N-[2-[(2-cyano-4,6-dinitrophenyl)azo]-5-(diethylamino)phenyl]acetamide

EC Number:

246-058-2

EC Name:

N-[2-[(2-cyano-4,6-dinitrophenyl)azo]-5-(diethylamino)phenyl]acetamide

Cas Number:

24170-60-3

Molecular formula:

C19H19N7O5

IUPAC Name:

N-{2-[(2-cyano-4,6-dinitrophenyl)diazenyl]-5-(diethylamino)phenyl}acetamide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

None

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 20140804
- Expiration date of the lot/batch: 21 August 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source:
Envigo RMS B.V., Inc., Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
8 to 12 weeks
- Weight at study initiation:
15 to 23 g
- Housing:
suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet: 2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK)ad libitum
- Water: tap water ad libitum
- Acclimation period:
5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness.

- IN-LIFE DATES: From: 08 September 2015 to 29 September 2015

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Preliminary Screening Test: 25 % w/w
Main Test: 25 %, 10 % or 5 % w/w

No. of animals per dose:

4

Details on study design:

Test Item Formulation and Experimental Preparation:
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 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 acetone/olive oil 4:1, 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 25 %, 10 % or 5 % w/w in acetone/olive oil 4:1. 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 labelled 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):

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

Results and discussion

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 methodology for the LLNA is detailed in the OECD Guideline for the Testing of Chemicals No. 429 and Method B.42 of Commission Regulation (EC) No. 440/2008. The study described in this document is based on these test methods but has been refined in order to reduce the number of animals required. The reduced LLNA (rLLNA) has been endorsed by the non Commission members of the European Centre for the Validation of Alternative Methods (ECVAM) Scientific Advisory Committee (ESAC) at its 26th meeting held on 26 – 27 April 2007 at ECVAM, Ispra, Italy.

Test item: α Hexylcinnamaldehyde, tech., 85%
Study number: 41500394
Study dates: 21 February 2015 to 27 February 2015

Methods
A group of five animals was treated with 50 µL (25 µL per ear) of α Hexylcinnamaldehyde, tech., 85% as a solution in acetone/olive oil 4:1 at a concentration of 25% v/v. A further control group of five animals was treated with acetone/olive oil 4:1 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:

25 (% v/v) in acetone/olive oil 4:1 gave a Stimulation Index of 13.93 (positive).

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

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

3.76

Test group / Remarks:

5% concentration

Results 2

Parameter:

SI

Value:

3.21

Test group / Remarks:

10% concentration

Results 3

Parameter:

SI

Value:

3.01

Test group / Remarks:

25% concentration

Any other information on results incl. tables

Preliminary Screening Test

Blue colored staining of the ears and fur was noted on Days 1 to 4 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. A greater than 5% body weight loss was noted but as there was no visual evidence of systemic toxicity, local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness the 25 % concentration was considered acceptable for use in the main test. Based on this information the dose levels selected for the main test were 25 %,10 % and 5 % w/w in acetone/olive oil 4:1.

 

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:

 

Concentration (%w/w) in acetone/olive oil 4:1	Stimulation Index	Result
5	3.76	Positive
10	3.21	Positive
25	3.01	Positive

 

Clinical Observations and Mortality Data

Blue colored staining of the ears and fur was noted in all test animals, post dose on Days 1 to 3.  There were no deaths. No signs of systemic toxicity were noted in the test or control animals during 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 acetone/olive oil 4:1	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	21.7	20.1	0	0Fs	0	0Fs	0	0Fs	0Fs	0	0

0 = No signs of systemic toxicity

Fs = Blue colored staining of the ears and fur 

 

Local Skin Irritation – Preliminary Screening Test

Concentration (%w/w) in acetone/olive oil 4:1

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=   Blue colored staining prevented evaluation of erythema

 

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

Concentration (%w/w) in acetone/olive oil 4:1	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	0.22	0.21	0.23	0.23	0.21	0.23
overall mean (mm)		0.215		0.230		0.220
overall mean ear thickness change (%)		na		6.977		2.326

na = Not applicable

 

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

Concentration (%w/w) in acetone/olive oil 4:1	dpm	dpm/Nodea	Stimulation Indexb	Result
Vehicle	16848.59	2106.07	na	na
5	63354.21	7919.28	3.76	Positive
10	54058.98	6757.37	3.21	Positive
25	50747.64	6343.46	3.01	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 acetone/olive oil 4:1	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	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
25	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 =Blue colored staining of the ears and fur

 

Individual Body Weights and Body Weight Change

Concentration (% w/w) in acetone/olive oil 4:1	Animal Number	Body Weight (g)		Body Weight Change (g)
		Day 1	Day 6
Vehicle	1-1	17.2	18.3	1.1
	1-2	18.0	17.4	-0.6
	1-3	19.5	18.9	-0.6
	1-4	18.2	19.8	1.6
5	2-1	17.6	19.0	1.4
	2-2	19.3	20.4	1.1
	2-3	20.2	20.9	0.7
	2-4	20.3	21.7	1.4
10	3-1	18.9	19.7	0.8
	3-2	19.0	18.7	-0.3
	3-3	18.6	19.6	1.0
	3-4	19.4	20.8	1.4
25	4-1	19.6	19.7	0.1
	4-2	19.8	19.9	0.1
	4-3	19.8	20.1	0.3
	4-4	18.7	18.6	-0.1

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

FAT 36156/D is considered as a skin sensitizer from the findings of this LLNA study.

Executive summary:

A study was performed to assess the skin sensitization potential of FAT 36156/D in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The study was carried out according to OECD Guideline 429 and EU Method B42.

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) of the test item as a suspension in acetone/olive oil 4:1at concentrations of 25 %, 10 % or 5 % w/w. A further group of four animals was treated with acetone /olive oil 4:1 alone. All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6 for clinical observations. Any signs of toxicity or signs of ill health during the test were recorded. The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination). Five hours following the administration of test item 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. The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of test item incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). Blue colored staining of the ears and fur was noted in all test animals, post dose on Days 1 to 3. There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. 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. The Stimulation Index for each 5, 10 and 25 % test concentration was 3.76, 3.21 and 3.01 respectively and based on these results FAT36156/D was considered to be a skin sensitizer under given conditions of the test.