Dinaphtho[1,2,3-cd:3',2',1'-lm]perylene-5,10-dione, (9,10-dihydro-9,10-dioxo-1-anthracenyl)amino derivs.

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 July, 2015 to 07 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

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:

None

Specific details on test material used for the study:

Identification: FAT 45082/I TE
Batch: JL1101V5C
Purity: not supplied
Physical state / Appearance: black solid
Expiry date: 27 January 2020
Storage Conditions: room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by 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 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.

IN LIFE PHASE: From: 15 July, 2015 to 04 August, 2015

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

Concentrations of 25 %, 10 % or 5 % w/w in propylene glycol.

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.

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 resuspended in 10 mL of PBS and repelleted. 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).

Interpretation of Results
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 ³HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in ³HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in ³HTdR incorporation will be classified as a "non-sensitizer". The results were also interpreted according to the Globally Harmonized Classification System.

Positive control substance(s):

other: Phenylacetaldehyde

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:
 
Concentration (% v/v) in propylene glycol Stimulation Index Result
5 14.82 Positive

Phenylacetaldehyde (>90 %) was considered to be a sensitizer under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.14

Test group / Remarks:

5 % concentration

Results 2

Parameter:

SI

Value:

0.86

Test group / Remarks:

10 % concentration

Results 3

Parameter:

SI

Value:

1.49

Test group / Remarks:

25 % concentration

Any other information on results incl. tables

Preliminary Screening Test

Black staining on the ears and fur was noted post dose on Days 1 to 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 25 %,10 % and 5 % w/w in propylene glycol.

Clinical Observations and Mortality Data

Black staining on the ears and fur was noted post dose on Days 1 to 3 in all test animals.

There were no deaths recorded. 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	17.9	17.7	0	0Fs	0	0Fs	0	0Fs	0	0	0

0 = No signs of systemic toxicity

Fs = Black staining

 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

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 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	0.20	0.22	0.20	0.23	0.20	0.22
overall mean (mm)		0.21		0.22		0.21
overall mean ear thickness change (%)		na		2.38		0.00

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	10336.15	1292.02	na	na
5	11771.84	1471.48	1.14	Negative
10	8865.74	1108.22	0.86	Negative
25	15377.40	1922.18	1.49	Negative

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	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 = Black staining

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	18.5	19.8	1.3
	1-2	14.7	16.3	1.6
	1-3	20.4	20.1	-0.3
	1-4	18.6	19.2	0.6
5	2-1	18.3	18.2	-0.1
	2-2	15.6	16.1	0.5
	2-3	17.6	19.3	1.7
	2-4	19.0	19.9	0.9
10	3-1	16.2	17.9	1.7
	3-2	18.7	19.3	0.6
	3-3	18.5	18.2	-0.3
	3-4	17.0	18.8	1.8
25	4-1	17.6	18.5	0.9
	4-2	19.4	20.2	0.8
	4-3	18.4	19.6	1.2
	4-4	16.5	17.6	1.1

Vehicle Determination Record

Test Item Concentration	Vehicle*
	acetone/ olive oil (4:1)	dimethyl formamide	butanone	dimethyl sulphoxide	acetone	1% pluronic L92 in distilled water	ethanol/ distilled water (7:3)	propylene glycol
50% 0.5 g (test item) + 0.5 g (vehicle)	X 1	X 1	X 1	X 1	X 1	X 1	X 1	X 1
25% 1 g of 50% dilution made up to 2 g	X 1	X 1	X 1	X 1	X 1	X 1	X 1	P suspension 1

* = Suitable for dosing if formulation is a solution or fine homogenous suspension which can be administered via a micropipette

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

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

Executive summary:

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.

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 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 showed negative results. The stimulation indices obtained were 1.14, 0.86 and 1.49 for concentrations 5, 10 and 25 % respectively. Hence, the test item was considered to be a non-sensitiser under the conditions of the test.

The test item does not meet the criteria for classification according to the Globally Harmonized Classification System.