9,10-Anthracenedione, 1,4-bis[(2-ethylhexyl)amino]-5,8-dihydroxy-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study was carried out in accordance with the OECD Guideline No. 429, "Skin Sensitisation:Local Lymph Node Assay", Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Directive 20041731EC, and with the Principles of Good Laboratory Practices (GLP).

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

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

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1,4-bis[(2-ethylhexyl)amino]-5,8-dihydroxy-9,10-dihydroanthracene-9,10-dione

EC Number:

807-717-5

Cas Number:

295800-70-3

Molecular formula:

C30H42N2O4

IUPAC Name:

1,4-bis[(2-ethylhexyl)amino]-5,8-dihydroxy-9,10-dihydroanthracene-9,10-dione

Test material form:

other: blue solid

Details on test material:

- Name of test material (as cited in study report): SpectraceTMM D-810 Marker (Solvent Free)
- Physical state: Blue solid
- Lot/batch No.: RWS-2-43
- Storage condition of test material: Stored at room temperature in the dark.

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBNCa (CBNCaBkl); CBNCa (CBNCa CruBR)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Twenty female CBNCa (CBNCaBkl) strain mice were supplied by B & K Universal Ltd, Hull, UK and one female CBNCa (CBNCa CruBR) strain mouse was supplied by Charles River UK Limited, Margate, Kent, UK.
- Age at study initiation: eight to twelve weeks old
- Weight at study initiation: weight range of 17 to 20 g
- Housing: individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet (e.g. ad libitum): Free access food (Certified Rat and Mouse Diet) was allowed throughout the study.
- Water (e.g. ad libitum):Free access to mains tap waterwas allowed throughout the study.
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 C
- Humidity (%): 30 to 70%
- Air changes (per hr): fifteen changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

2.5%, 5% or 10% wlw in acetone/olive oil 4: 1

No. of animals per dose:

5 animals per dose.

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: For the purpose of the study, the test material was fieshly prepared in acetonelolive oil 4:l. This vehicle was chosen as it produced the highest concentration that was suitable for dosing.
- Irritation: As no toxicological information was available regarding the systemic toxicitylirritancy potential of the test material, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 p1 of the test material at a concentration of 10% wlw 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. Any signs of toxicity or excessive local irritation noted during this period were recorded.
- Lymph node proliferation response: A stimulation index of greater than 3 was recorded for the two lower concentrations of the test material (2.5% and 5% wlw in acetonelolive oil 4: 1). A stimulation index of less than 3 was recorded for the highest concentration of the test material (10% WIW in acetonelolive oil 4:l).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Spectrace MD-810 Marker (Solvent Free)
- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph nodes from each individual animal and as the ratio of 'HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 'HT~R incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 'HT~R incorporation will be classified as a "non-sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of five mice were treated with the test material at concentrations of 2.5%, 5% or 10% wlw in acetonelolive oil 4: 1. The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 p1 of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test material 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 five mice received the vehicle alone in the same manner.

Positive control substance(s):

other: α-Hexylcinnamaldehyde, Tech, 85%

Statistics:

Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pailwise comparisons were performed between control and treated groups. For homogenous
datasets Dunnett's Multiple Comparison test was used and for non-homogenous datasets Dunnett's T3 Multiple Comparison Method was used.

Probability values (p) are presented as follows:
P<0.00l * * *
P<0.0l * *
P<0.05 *
P10.05 (not significant)

Results and discussion

Positive control results:

not applicable

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Vehicle: Simulation index: N/A Result: N/A Concentration of 2.5% of test material in acetone/olive oil 4:1: Simulation index: 3.39 Result: Positive Concentration of 5% of test material in acetone/olive oil 4:1: Simulation index: 4.03 Result: Positive Concentration of 10% of test material in acetone/olive oil 4:1: Simulation index: 2.59 Result: Negative

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Vehicle: Mean dpm/animal: 1710.62 (+ or - 737.41) Result: N/A Concentration of 2.5% of test material in acetone/olive oil 4:1: Mean dpm/animal: 5797.72 (+ or - 2800.11) Result: Positive Concentration of 5% of test material in acetone/olive oil 4:1: Mean dpm/animal: 6899.87 (+ or - 2213.83) Result: Positive Concentration of 10% of test material in acetone/olive oil 4:1: Mean dpm/animal: 4434.63 (+ or - 1704.88) Result: Negative

Any other information on results incl. tables

Individual disintegrations per Minute and Stimulation Indices

Concentration (% W/W) in Acetone/olive oil 4:1	Animal Number	dpd Animal	Mean dpm/ Animal (Standard Deviation)	Stimulation Index	Result
Vehicle	1-1	1253.07	1710.62 (±737.41)	N/A	N/A
	1-2	1307.02
	1-3	1918.99
	1-4	1157.60
	1-5	2916.31
2.5	2-1	3088.92	5797.72* (±2800.11)	3.39	Positive
	2-2	2714.84
	2-3	9159.93
	2-4	6648.35
	2-5	7376.56
5	3-1	10505.53	6899.87** (±2213.83)	4.03	Positive
	3-2	7389.10
	3-3	5628.76
	3-4	4870.93
	3-5	6105.03
10	4-1	7319.30	4434.63 (±1704.88)	2.59	Negative
	4-2	3613.09
	4-3	4194.71
	4-4	4197.79
	4-5	2848.27

 

dpm=Disintegrations per minute

a=Total number of lymph nodes per animal is 2

b=Stimulation Index of 3.0 or greater indicates a positive result

NIA=Not applicable

*=Significantly different fiom control group p<0.05

**=Significantly different fiom control group p<0.01

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the test, the test material, Spectrace TM MD-810 Marker (Solvent Free), was a sensitiser.

Executive summary:

Introduction: A study was performed to assess the skin sensitization potential of the test material, Spectrace® MD-810 Marker (Solvent Free) (Lot/Batch No. RWS-2-43; TD No. 04-049), in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of the following:

• OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitization: Local Lymph Node

Assay" (adopted 24 April 2002)

• Method B42 Skin Sensitization (Local Lymph Node Assay) of Commission Directive

2004/73/EC

Methods: Following a preliminary screening test, three groups, each of five animals, were treated with 50 μl (25 μl per ear) of the test material as a suspension in acetone/olive oil 4:1 at concentrations of 2.5%, 5% or 10% w/w. A further group of five animals was treated with acetone/olive oil 4:1 alone.

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 are as follows: Concentration (% w/w) in acetone/olive oil 4:1 Stimulation Index

 

Concentration (% w/w) in acetone/olive oil 4:1	Stimulation Index	Result
2.5	3.39	Positive
5	4.03	Positive
10	2.59	Negative

Conclusion: Under the conditions of the test, the test material, Spectrace® MD-810 Marker (Solvent

Free), was a sensitiser.