Anthraquinone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9.6.-15.7.2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was carried out in accordance with internationally valid GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

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

Reference substance name:

Anthraquinone

EC Number:

201-549-0

EC Name:

Anthraquinone

Cas Number:

84-65-1

Molecular formula:

C14H8O2

IUPAC Name:

9,10-dihydroanthracene-9,10-dione

Test material form:

solid: particulate/powder

Details on test material:

Batch No.: V 1161
Purity: main component: 9,10-Anthraquinone 98.9 wt.%
Significant impurities: Anthracene 0.01 wt.%, 2,3-Naphthalic anhydride 0.25%, Naphtho[2,3-b]thiphenquinone 0.33%, 1,4- Anthraquinone 0.32%
Appearance: Yellow crystalline powder
Expiration date: 06/2024
Storage: The test substance was stored in the dark place at laboratory temperature during the study.

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760118
- Age at study initiation: 8 to 10 weeks (at start of dosing)
- Weight at study initiation (range): 21.1 to 24.0 g (at start of dosing)
- Animal caging: Animals in groups of maximum six in macrolon cages with sterilized softwood shavings.
- Diet: Pelleted standard diet for experimental animals ad libitum.
- Water: Drinking tap water ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Room temperature 22 +/- 3°C, permanently monitored
- Humidity (%): Relative humidity 30 – 70 %, permanently monitored
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle: 6am-6pm/6pm-6am

TIME SCHEDULE
Animal arrival/ start of acclimatization: 6.5.2009
Pilot experiment: 9.-12.6 2009
Main study:
First day of administration: 17.6.2009
End of treatment period: 19.6.2009
Application of radionuclide and necropsy: 22.6.2009

Study design: in vivo (LLNA)

Vehicle:

other: DAE 433 - mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol

Concentration:

30% (w/v) 300 mg/mL
3% (w/v) 30 mg/mL
0.3% (w/v) 3 mg/mL

No. of animals per dose:

5 animals

Details on study design:

RANGE FINDING TESTS.
The highest concentration 30% (maximum technically practicable concentration) was administered to three animals to assess a possible systemic toxicity. The route of administration was same as in the main study. During pilot experiment no clinical symptoms of systemic toxicity and no macroscopic changes (after necropsy) were found out in all three animals.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Animals were subjected to a clinical examination (health check) shortly after arrival. Study animals were randomly allocated to the dose groups manually and assigned animal numbers

EVALUATION OF RESULTS
Cell proliferation: When the SI for any single treatment dose group is ≥ 3 (exceeded threshold) and clear concentration dependence is recorded, the test substance is regarded as a skin sensitizer.

Ear weight – irritation effect: When a statistical significant increase of ear weight after treatment by the test substance together with clear concentration dependence of the effect is recorded, the end point is considered positive – test substance may cause the irritation of skin.

TREATMENT PREPARATION AND ADMINISTRATION
Dosage volume: 25μL / ear / animal
Preparation for administration: All suspensions were prepared by suspending an appropriate amount of Anthraquinone in the vehicle to obtain a concentration of 30%, 3% or 0.3% (w/v). The suspensions were prepared before the start of application by mixing on magnetic stirrer and then were still mixed duringapplication.
Application
The volume of the application form was constant at all groups of animals - 25μl of the appropriate dilution to the dorsum of each ear once a day morning for 3 consecutive days. The application was performed very slowly by micropipette. Losses caused by draining from the ear must be minimized.
This route of administration is listed in the guideline and it is similar to expected exposure conditions at the workplace. The application form of test substance was prepared immediately before administration.

Positive control substance(s):

other: Dinitrochlorobenzene (DNCB)

Statistics:

For statistical calculations the software Statgraphic ® Centurion (version XV, USA) was used. At first the global comparison of all three values of the concentration groups with vehicle control was performed by applying the non-parametric Kruskal-Wallis test, and then the non-parametric two-group Mann-Whitney rank test (probability level 0.05) was applied to all two-group comparisons.

Results and discussion

Positive control results:

All animals in the positive control group showed these symptoms: hyperaemia of skin and clonospasm.
In the positive control group, the SI was ≥ 3 (10.46) – test LLNA was efficient.
In the positive control group, the weight of ear target was statistically significantly increased against negative control group – test LLNA was efficient.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

>= 6.53

Test group / Remarks:

30%

Remarks on result:

positive indication of skin sensitisation based on QSAR/QSPR prediction

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: See table No. 6

Results 3

Key result

Parameter:

SI

Value:

ca. 1.6

Test group / Remarks:

0,3%

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Results 4

Key result

Parameter:

SI

Value:

ca. 1.8

Test group / Remarks:

3%

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Cellular proliferation data / Observations:

The SI for the test groups treated by the test substance was increased with dependence on the dose level. In the highest dose level the SI was 6.53 (exceeded threshold). SI for the middle and lowest dose groups was below threshold.

Any other information on results incl. tables

Table No.6.  Summary of results

Group	Radioisotope incorporation		Ear weight
	Mean DPM	SI	Mean (mg)
NC	1026.2	1.00	22.84
PC	10738.8	10.46+	27.50*
30%	6705.4	6.53+	28.22*
3%	1845.6	1.80	24.84*
0.3%	1637.4	1.60	23.80*

Figures with asterisk = values statistically significant on probability level 0.05 (Mann-Whitney test)

Figures with cross = values ≥ 3

NC – Negative control group

PC – Positive control group

 

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

The test substance Anthraquinone gave a positive result in LLNA test. Positive results in cell proliferation and no clinical symptoms of systemic toxicity revealed that the test substance Anthraquinone could be a contact allergen in mice but potential irritation effect does not rule out the possibility that it could be false positive result.

Executive summary:

The test substance, Anthraquinone, was tested for the assessment of skin sensitisation potential with the murine local lymph node assay. This study is a part of the test substance health hazard evaluation.

The Local Lymph Node Assay (LLNA) with radionuclides was used. The testing was conducted according to the EU Method B.42, Skin sensitization: Local Lymph Node Assay, Council Regulation (EC) No. 440/2008, published in O.J. L142, 2008.

In this study the contact allergenic potential of Anthraquinone was evaluated after topical application to female BALB/c mice. Five mice per group were exposed by test and control substances on the dorsum of both ears once a day during 3 consecutive days. Primary proliferation of lymphocytes in the lymph node draining the site of application was evaluated on the base on using of radioactive labelling. The ratio of the proliferation in treated groups to that in vehicular controls, termed the Stimulation Index, was determined. Statistical evaluation of ear weight was performed for elimination of false positive findings with certain skin irritants.

Concentrations: positive control DNCB (dinitrochlorobenzene): 0.5% (w/v) and Antraquinone: 30%, 3%, 0.3% (w/v) in DAE 433.

The animals exposed to the test substance at the lowest and highest dose levels showed no pathological skin reactions and no other negative clinical symptoms of intoxication throughout the experiment. Slight hyperaemia of skin and swelling of ears was recorded at the highest dose level. There was no significant difference in body weight increment of all groups in comparison to the vehicle control.

The positive control substance DNCB elicited a reaction pattern with statistically significant increase in ear weight and Stimulation Index of cell proliferation 10.5, which was in congruence with his expected mode of action as a contact allergen.                 

The test substance Anthraquinone showed a tendency to increase ear weight with concentration-dependence. Ear weight of animals in all dose level was statistically significantly increased. The result of skin irritation effect was considered as positive – it means the test substance caused irritation of skin.

Comparison of Stimulation Indexes between treated groups and control group revealed that the test substance Anthraquinone caused significant increase in radioisotope incorporation into the DNA of dividing lymphocytes. This effect was dose dependent.

The test substance Anthraquinone had a positive result in LLNA test. Positive results in cell proliferation and no clinical symptoms of systemic toxicity revealed that the test substance Anthraquinone could be a contact allergen in mice but potential irritation effect does not rule out the possibility that it could be false positive result.

Based on the positive results of LLNA study, for the test substance Anthaquinone, it could be recommended to be assigned by symbol “Xi“ and the risk phrase R43 “may cause sensitisation by skin contact”.