Sodium 1-amino-9,10-dihydro-9,10-dioxo-4-[[3-[(1-oxopropyl)amino]phenyl]amino]anthracene-2-sulphonate

Endpoint summary

Description of key information

The substance is concluded to be not 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:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Identification: FAT 20242/1 TE
Batch: AT-PD14-026Al
Purity:91%
Physical state - Appearance: dark blue solid
Expiry date: 20 January 2019
Storage Conditions: room temperature in the dark

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Horst, The Netherlands.
- Age at study initiation: 8 to 12 weeks old
- Weight at study initiation: 15 to 23 g
- Housing: The animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): Free access to food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK).
- Water (e.g. ad libitum): Free access to mains tap water.
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): The lighting was controlled by a time switch to give 12 hours continuous light (06.00 to 18.00) and 12 hours darkness.

Vehicle:

dimethylformamide

Concentration:

Preliminary Screening Test: 25 µL of the test item at a concentration of 50% w/w in dimethyl formamide.
Main Test: 50%, 25% or 10% w/w in dimethyl formamide.

No. of animals per dose:

Preliminary Screening Test: One mouse
Main Test: Four mice per group

Details on study design:

Preliminary Screening Test
Using available information regarding the systemic toxicity 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. 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 3008 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 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 (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):

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

Statistics:

No data

Positive control results:

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 gave a stimulation index of 2.1 (negative)
15 % v/v in dimethyl formamide gave a stimulation index of 6.26 (positive)*
25 % v/v in dimethyl formamide gave a stimulation index of 8.14 (positive)
*Based on four animals due to the death of one animal on Day 6

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.

Parameter:

SI

Value:

1.2

Test group / Remarks:

10 %

Remarks on result:

other: Negative

Parameter:

SI

Value:

1.23

Test group / Remarks:

25 %

Remarks on result:

other: Negative

Parameter:

SI

Value:

1.28

Test group / Remarks:

50 %

Remarks on result:

other: Negative

Preliminary Screening Test

Blue colored staining of the ears and fur was noted post dose on Days 1 to 3 and on Days 4 to 6.

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

Clinical Observations and Mortality Data

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 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 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

16.7

16.8

0

0Fs

0

0Fs

0

0Fs

0Fs

0Fs

0Fs

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 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	0.20	0.21	0.22	0.24	0.23	0.22
overall mean (mm)		0.205		0.230		0.225
overall mean ear thickness change (%)		na		12.195		9.756

na = Not applicable

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

Concentration (%w/w) in dimethyl formamide	dpm	dpm/Node [a]	Stimulation Index [b]	Result
Vehicle	8300.97	1037.62	na	na
10	9957.01	1244.63	1.20	Negative
25	10209.09	1276.14	1.23	Negative
50	10640.15	1330.02	1.28	Negative

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 = 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	20.3	20.7	0.4
	1-2	16.6	17.4	0.8
	1-3	19.8	21.1	1.3
	1-4	20.0	20.4	0.4
10	2-1	19.5	22.0	2.5
	2-2	20.8	20.2	-0.6
	2-3	19.5	20.6	1.1
	2-4	19.5	19.2	-0.3
25	3-1	18.7	20.0	1.3
	3-2	16.8	16.5	-0.3
	3-3	19.7	20.2	0.5
	3-4	19.5	19.9	0.4
50	4-1	19.5	19.9	0.4
	4-2	18.0	19.1	1.1
	4-3	17.8	20.1	2.3
	4-4	18.5	19.0	0.5

 

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:

FAT 20242/I was evaulated for skin sensitization potential according to OECD TG 429 following topical application to the dorsal surface of the mouse 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 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 dimethyl formamide at concentrations of 50 %, 25 % or 10 % w/w. A further group of four animals was treated with dimethyl formamide 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 a concentration of 10 % in dimethyl formamide the stimulation index (SI) was 1.20 (negative).

At a concentration of 25 % in dimethyl formamide the stimulation index (SI) was 1.23 (negative).

At a concentration of 50 % in dimethyl formamide the stimulation index (SI) was 1.28 (negative).

 

The test item was considered to be a non-sensitizer under the conditions of the test. The test item does not meet the criteria for classification according to the Globally Harmonized Classification System.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A key 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 concentration of 50 % 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 dimethyl formamide at concentrations of 50 %, 25 % or 10 % w/w. A further group of four animals was treated with dimethyl formamide 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 a concentration of 10 % in dimethyl formamide the stimulation index was 1.20 (negative).

At a concentration of 25 % in dimethyl formamide the stimulation index was 1.23 (negative).

At a concentration of 50 % in dimethyl formamide the stimulation index was 1.28 (negative).

 

The test item was considered to be a non-sensitizer under the conditions of the test. The test item does not meet the criteria for classification according to the Globally Harmonized Classification System.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

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

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