Disodium 3-[[4-amino-9,10-dihydro-9,10-dioxo-3-[sulphonato-4-(1,1,3,3-tetramethylbutyl)phenoxy]-1-anthryl]amino]-2,4,6-trimethylbenzenesulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 20 to December 04, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

adopted 24 June 2002

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:

Acid Violet 048

IUPAC Name:

Acid Violet 048

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands B.\/. Postbus 6174 NL - 5960 AD Horst/ The Nelherlands.
- Females: nulliparous and non-pregnant.
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 16.7 - 20.8 g
- Housing: in groups of four in Makrolon type-3 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet: pelleted standard Klibe 3433, batch no. 67/02 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst) available ad Ilbitum.
- Water: community tap water from Itigen, ad libitum.
- Acclimation period: under test conditions after health examination.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: 10 - 15 air change per hour.
- Photoperiod: 12 hrs dark / 12 hrs light.
- Other: 8 hours music during the light period.

Study design: in vivo (LLNA)

Vehicle:

other: ethanol:water, 7:3 (v/v)

Concentration:

2.5, 5 and 10 % (w/v).

No. of animals per dose:

4 females

Details on study design:

PRE-SCREEN TESTS
To determine the highest non-irritant and technically applicable test item concentration, a non-GLP pretest. The test item in the main study was assayed at three consecutive concentrations. The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation.
- No animals: 2 mice.
- Concentrations: 1, 2.5, 5 and 10 %(w/v).

MAIN STUDY
FORMULATION
The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle was quantitatively added. The weight/volume dilutions were prepared individually using a magnetic stirrer as homogenizer. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.
The preparations were made shortly before each dosing occasion.

TOPlCALAPPLlCATlON
- Application volume: 25 µl
- Application: topical (epidermal) application to the dorsal surface of each ear lobe (lett and right); test item was spread over the entire dorsal surface (Ø - 8 mm).
- Frequence of application: once daily, for three consecutive days.
- Control: a further group of mice was treated with an equivalent volume of the relevant vehicle alone.

ADMINISTHATEON OF 3H-METHYL THYMIDINE
3H-methyl thymidine (3HTdR) was purchased from Amersham International (specific activity, 2 Ci/mmol; concentration, 1 mCi/ml).
Five days after the first topical application, all mice were administered with 250 µl of 77.6 µCi/ml 3HTdR (equal to 19.4 µCi 3HTdR) by intravenous injection via a tail vein.

DETERMINATION OF lNCORPORATED 3HTDR
- Sacrifice: approximately five hours after treatment with 3HTDR all mice were euthanized by intraperitoneal injection of \/ETANARCOL.
- Lymph node: 8 nodes per group; the draining lymph nodes were rapidly excised and pooled for each experimental group.
- Cell suspension: single cell suspensions (phosphate buttered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size).
- Incubation: after washing two times with phosphate buttered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 “C for approximately 18 hours for precipitation of macromolecules.
- Resuspension: the precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of "Ultima Gold" scintillation liquid and thoroughly mixed.
- 3HTdR measurements: levels were measured on a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 ml-aliquots of 5 % trichloroacetlc acid. The β-scintillation counter expresses 3HTdFt incorporation as the number of radioactive disintegrations per minute (DPM).

INTERPRETATION OF RAW DATA
The proliterative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (Stimulation Index). Before DPM/NODE values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

OBSERVATIONS
- Mortality/Viability: twice daily from acclimatization start to the termination of in-life phase.
- Body weights: at acclimatization start and prior to necropsy.
- Clinical signs (local/systemic): daily from acclimatization start to the termination of in-lite phase. Especially the treatment sites were recorded carefully.

POSITIVE CONTROL
Three groups of four female mice each were treated with the alpha-hexylcinnamaldehyde at concentrations oi 5, 10 and 25 % (w/v) in acetone:olive oil, 4:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) on three consecutive days. A control group of four mice was treated with the vehicle (acetone:olive oil, 4:1 (v/v)) only. Five days alter the first topical appplication, the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were washed subsequently and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation oi 3H-rnethyl thymidine measured in a β-scintillation counter.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated for the body weight.

Results and discussion

Positive control results:

No test item-related clinical signs were observed. All treated animals survived the scheduled study period.
Stimulation index resulted to be 2.9, 2.6 and 7.1 at the test item concentration of 5, 10 and 25 % (w/v), respectively. the EC3 was calculated to be 11.3 % (w/v).

In vivo (LLNA)

Results

Results 1

Parameter:

EC3

Remarks on result:

not determinable

Remarks:

because stimulation index was lower than 3

Results 2

Parameter:

SI

Value:

1

Test group / Remarks:

test item concentrations 2.5 %

Results 3

Parameter:

SI

Value:

0.8

Test group / Remarks:

test item concentrations 5 %

Results 4

Parameter:

SI

Value:

1.1

Test group / Remarks:

test item concentrations 10 %

Cellular proliferation data / Observations:

Stimulation index resulted to be 1.0, 0.8 and 1.1 at the test item concentration of 2.5, 5 and 10 % (w/v), respectively.

OBSERVATIONS
VlABlLlTY / MORTALITY: no deaths occurred during the study period.
CLINICAL SIGNS: no symploms of local toxicity at ihe ears of the animals and no sysiemlo findings were observed during the study period.
BODY WEEGHTS: the body weight of the animals, recorded at the start of acclimatization period and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

Any other information on results incl. tables

Individual data

Test item concentration % (W/v)	Measurement dpm	No lymph nodes	dpm per lymph nodes	Stimulation index
Background I	--	--	--	--
Background II	--	--	--	--
Control group	2525	8	316	--
Test group, 2.5 %	2645	8	331	1
Test group, 5 %	1925	8	241	0.8
Test group, 10 %	2769	8	346	1.1

Applicant's summary and conclusion

Interpretation of results:

other: not classified, according to the CLP Regulation (EC) No 1272/2008

Conclusions:

Not skin sensitising

Executive summary:

In order to study a possible allergenic potential of test item three groups of four fernale mice were each treated with the test item at concentrations of 2.5, 5 % and 10 % (w/v) in ethanol:water, 7:3 (v/v) by topical application to the dorsum of each ear lobe (left and right) on three consecutive days. A control group of four mice was treated with the vehicle (ethano:water, 7:3 (v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine).

Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions ot lymph node cells were prepared from pooled lymph nodes which were washed subsequently and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of SH-methyl thymidine measured in a β-scintillation counter.

No test item-related clinical signs were observed. All treated animals survived the scheduled study period. A test item is regarded as a sensitizer in the LLNA if the exposure to at least one concentration of the test item resulted in an incorporation ot 3HTdR at least 3 -fold or greater than that recorded in control mice, as indicated by the stimulation index.

Stimulation index resulted to be 1.0, 0.8 and 1.1 at the test item concentration of 2.5, 5 and 10 % (w/v), respectively, therefore, the EC3 cannot be estimated.

A test item is regarded as a sensitizer in the LLNA if the exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.

Based on these criteria, the test item was found to be a non-sensitizer when tested up to 10 % (w/v) in ethanol:water, 7:3 (v/v).

Conclusion

An EC3 value could not be calculated because the calculation requires data lying immediately above and below Stimulation Index value of 3; therefore, the substance does not meet the criteria to be classified as a skin sensitiser, in accordance with the CLP Regulation (EC) 1272/2008.