N-(4-amino-9,10-dihydro-3-methoxy-9,10-dioxo-1-anthryl)benzenesulphonamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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:

yes

Remarks:

Due to a Study Director oversight, the Study Plan was not sent to QA for review prior to performance of the study, in error. This deviation was considered not to affect the purpose or integrity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

Due to a Study Director oversight, the Study Plan was not sent to QA for review prior to performance of the study, in error. This deviation was considered not to affect the purpose or integrity of the study.

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-(4-amino-9,10-dihydro-3-methoxy-9,10-dioxo-1-anthryl)benzenesulphonamide

EC Number:

274-040-4

EC Name:

N-(4-amino-9,10-dihydro-3-methoxy-9,10-dioxo-1-anthryl)benzenesulphonamide

Cas Number:

69563-51-5

Molecular formula:

C21H16N2O5S

IUPAC Name:

N-(4-amino-3-methoxy-9,10-dioxo-9,10-dihydroanthracen-1-yl)benzenesulfonamide

Test material form:

other: Solid

Details on test material:

None

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 12-45
- Expiration date of the lot/batch: 05 March 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: Harlan Laboratories B.V., 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: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

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

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

25 %, 10 % and 5 % W/W

No. of animals per dose:

Four female mice

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. 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 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. 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 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

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:
At 5 % (v/v) concentration in propylene glycol the stimulation index was 14.82 (positive).
Conclusion
Phenyl acetaldehyde (>90%) was considered to be a sensitizer under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

2.6

Test group / Remarks:

5%

Remarks on result:

other: Variability not specified

Results 2

Parameter:

SI

Value:

1.96

Test group / Remarks:

10%

Remarks on result:

other: Variability not specified

Results 3

Parameter:

SI

Value:

4.37

Test group / Remarks:

25%

Remarks on result:

other: Variability not specified

Any other information on results incl. tables

Preliminary Screening Test

Red colored staining of the ears and fur was noted post dose on Day 1 and at all subsequent observations. Red colored staining of the ears prevented accurate evaluation of erythema on Days 2 and 3. No visual local skin irritation was noted on Day 1 and 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

Clinical Observations and Mortality Data

Red colored staining of the ears and fur was noted in all test animals. 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 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

16.6

16.7

0

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0 = No signs of systemic toxicity

Fs = Red colored staining of the ears and 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

0

0

Rs

Rs

Rs

Rs

0

0

0

0

0

0

Rs = Red colored staining of the ears prevented accurate 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	0.19	0.21	0.22	0.23	0.21	0.22
overall mean (mm)		0.20		0.23		0.22
overall mean ear thickness change (%)		na		12.50		7.50

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	4111.34	513.92	na	na
5	10680.40	1335.05	2.60	Negative
10	8068.34	1008.54	1.96	Negative
25	17973.29	2246.66	4.37	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 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	0Fs	0Fs	0Fs	0Fs	0	0	0
	3-2	0	0Fs	0Fs	0Fs	0Fs	0Fs	0	0	0
	3-3	0	0Fs	0Fs	0Fs	0Fs	0Fs	0	0	0
	3-4	0	0Fs	0Fs	0Fs	0Fs	0Fs	0	0	0
25	4-1	0	0Fs	0Fs	0Fs	0Fs	0Fs	0Fs	0	0
	4-2	0	0Fs	0Fs	0Fs	0Fs	0Fs	0Fs	0	0
	4-3	0	0Fs	0Fs	0Fs	0Fs	0Fs	0Fs	0	0
	4-4	0	0Fs	0Fs	0Fs	0Fs	0Fs	0Fs	0	0

0=    No signs of systemic toxicity

Fs = Red colored staining of the ears and 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	21.1	20.2	-0.9
	1-2	17.1	17.5	0.4
	1-3	20.1	21.5	1.4
	1-4	20.6	21.6	1.0
5	2-1	18.0	19.1	1.1
	2-2	20.0	20.2	0.2
	2-3	19.7	20.3	0.6
	2-4	21.2	21.4	0.2
10	3-1	18.3	17.9	-0.4
	3-2	20.3	21.1	0.8
	3-3	21.2	21.6	0.4
	3-4	17.8	18.0	0.2
25	4-1	18.6	19.8	1.2
	4-2	18.1	17.7	-0.4
	4-3	16.5	17.3	0.8
	4-4	17.7	17.7	0.0

Applicant's summary and conclusion

Interpretation of results:

sensitising

Conclusions:

FAT 36034/H was considered to be a sensitizer at concentration of 25% under the conditions of the test. However, the test item did not showed stimulation index of > 3 at concentration of 10%. The substance is not a sensitiser at concentration of 10% in LLNA assay.

Executive summary:

A study was performed as per OECD 429 guideline to assess the skin sensitization potential of FAT 36034/H 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 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 propylene glycol at concentrations of 25 %, 10 % or 5 % w/w. A further group of four animals was treated with propylene glycol alone.

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:

At 5 % (w/w) concentration in propylene glycol the stimulation index was 2.60 (negative)

At 10 % (w/w) concentration in propylene glycol the stimulation index was 1.96 (negative)

At 25 % (w/w) concentration in propylene glycol the stimulation index was 4.37 (positive)

The test item was considered to be a sensitizer at concentration of 25 % under the conditions of the test. However, the test item did not showed stimulation index of > 3 at concentration of 10 %. The substance is not a sensitiser at concentration of 10 % in LLNA assay.