Reaction mass of pentasodium 2-{[4-chloro-6-(ethyl{3-[(2-sulfonatoethyl)sulfonyl]phenyl}amino)-1,3,5-triazin-2-yl]amino}-5-hydroxy-6-({2-sulfonato-4-[(4-sulfonatophenyl)diazenyl]phenyl}diazenyl)naphthalene-1,7-disulfonate and tetrasodium 2-[(4-chloro-6-{ethyl[3-(vinylsulfonyl)phenyl]amino}-1,3,5-triazin-2-yl)amino]-5-hydroxy-6-({2-sulfonato-4-[(4-sulfonatophenyl)diazenyl]phenyl}diazenyl)naphthalene-1,7-disulfonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 December, 2005 to 22 March 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2004/73/EC, B.42: Skin Sensitization: Local Lymph Node Assay, 29 April 2004.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

-Batch No.: ROE 805 BOP 04/05
-Colour: Dark red
-Solubility in water: >100g/L at 20 °C
-Solubility in vehicle: Miscible
-Stability in solvent: 7 days in Water, Saline, Polyethylene Glycol, Carboxymethylcellulose, and 1 day in Vaseline and FCA at room temperature.
-Storage: At room temperature, in the desicator
-Expiration Date: October 01, 2010
-Purity: Approx. 82 % organic part (Na-salt),
all coloured components = 80.3 %;
Main component 1: 36.2 %,
Main component 2: 27.5 %, Oligomers: 10 %

Specific details on test material used for the study:

Identity: FAT 40824/A
Batch: Red ROE 805 BOP 04/05
Appearance: dark red powder
Purity: Organic part (Na-salt): approx. 82 %; Main component 1 : approx. 36.2 %; Main component 2: approx. 27.5 %; Oligomers: 10%
Expiration date: 01 October 2010
Storage: At room temperature at about 20°C in a desiccator (hygroscopic)

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
Test system: Mice, CBA/CaHsdRcc(SPF)
Source: RCC Ltd Laboratory Animal Service CH-4414 Füllinsdorf / Switzerland
Number of animals for the pre-test (non-GLP): 3 females
Number of animals for the main study: 16 females
Number of test groups: 3
Number of negative control group: 1
Age: 8-12 weeks (beginning of acclimatization)
Body weight: 16 g - 24 g (ordered)
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
Accommodation: Individual in Makrolon type-2 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
Diet: Pelleted standard Kliba 3433, batch no. 63/05 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst) available ad libitum.
Water: Community tap water from Itingen, available ad libitum.

ENVIRONMENTAL CONDITIONS
Standard Laboratory Conditions. Air-conditioned with ranges for room temperature 22 ±3 °C, relative humidity 30 - 70 % and 10 - 15 air changes per hour. Room temperature and humidity were monitored continuously and values outside of these ranges occasionally occurred, usually following room cleaning. These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at RCC. There was a 12 hour fluorescent light / 12 hour dark cycle with at least 8 hours music during the light period.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Remarks:

N,N-dimethylformamide (DMF)

Concentration:

2.5 %, 5 % and 10 % in N,N-dimethylformamide (DMF)

No. of animals per dose:

Number of animals per group: 4 females (nulliparous and non-pregnant)

Details on study design:

TEST ITEM FORMULATIONS PREPARATION
The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle N,N-dimethylformamide (DMF) was quantitatively added. The weight/volume (w/v) dilutions were prepared individually using a magnetic stirrer as homogenizer. Test item formulations were made freshly before each dosing occasion and no more than 4 hours prior to application to the ears.

TOPICAL APPLICATION
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with the test item at concentrations of 2.5 %, 5 % and 10 % in N,N-dimethylformamide (DMF). The application volume, 25µI, was spread over the entire dorsal surface ( 0 - 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

ADMINISTRATION OF 3H-METHYL THYMIDINE
Five days after the first topical application, all mice were administered 250 µI of 78.27 µCi/ml 3HTdR (equal to 19.6 µCi 3HTdR) by intravenous injection via a tail vein.

DETERMINATION OF INCORPORATED 3HTDR
Approximately five hours after treatment with 3HTdR all mice were euthanized by inhalation of CO2(dry ice).
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 /vm mesh size). After washing twice with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of 'Irga-Safe Plus' scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a ß-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 ml-aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (dpm).

OBSERVATIONS
In addition to the sensitizing reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: Once daily from acclimatization start to the termination of in-life phase.
Body weights: On the test day 1 (prior to the 1st application) and on the test day 6.
Clinical signs (local / systemic): Once daily on the day of animals delivery and from test day 1 (after the 1st application) to the termination of
in-life phase.

Positive control substance(s):

other: ALPHA-HEXYLCINNAMALDEHYDE

Statistics:

The mean values and standard deviations were calculated in the body weight tables. Body weights were recorded on-line in RCC-TOX LIMS. Clinical signs were compiled directly into the RCC computer system.

Results and discussion

Positive control results:

STIMULATION INDICES of 2.9, 3.9 and 8.7 were determined with the test item at concentrations of 5 %, 10 % and 25 % (w/v), respectively, in acetone:olive oil, 4:1 (v/v). The positive control item ALPHA-HEXYLCINNAMALDEHYDE showed an allergenic potential, and an EC3 value of 5.5 % (w/v) was derived.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

5

Test group / Remarks:

Group 2 (test item concentration (w/v):- 2.5)

Results 2

Parameter:

SI

Value:

3.1

Test group / Remarks:

Group 3 (test item concentration (w/v):- 5)

Results 3

Parameter:

SI

Value:

4.7

Test group / Remarks:

Group 4 (test item concentration (w/v):- 10)

Any other information on results incl. tables

No deaths occurred during the study period. Neither clinical / local signs nor other findings were observed in any animals of the control group. On the second application day, the residual test item was found at both dosing sites in all mice of Group 2 (2.5 %), Group 3 (5 %) and Group 4 (10 %), persisting for the remainder of the in-life phase of the study. The body weight of the animals, recorded prior to the first application and prior to necropsy, was within the range commonly recorded for animals of the strain and age.

The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured on a ß-scintillation counter.

 

	Test item concentration (w/v)	S.I.
Group 2	2.5	5.0
Group 3	5	3.1
Group 4	10	4.7
An EC3 value could not be determined because this calculation requires a S.I. value of less than 3.

 

CALCULATION AND RESULTS OF INDIVIDUAL DATA

The following results were obtained:

Vehicle: N,N-dimethylformamide (DMF)

 

Test item concentration (w/v)		Measurement dpm	Calculation			Result
			dpm- BGa)	number of lymph nodes	dpm per lymph nodeb)	S.I.
~	BGI	25	~	—	~	~
~	BGII	26	—	—	—	—
~	CG1	1683	1657	8	207	—
2.5	TG2	8257	8231	8	1029	5.0
5	TG3	5171	5145	8	643	3.1
10	TG4	7802	7776	8	972	4.7

 

BG = Background (1 ml 5 % trichloroacetic acid) in duplicate

CG = Control Group

TG = Test Item Group

S.I. = Stimulation Index

a) = The mean BG was calculated from the figures BG I and BG II values

b) = Since the lymph nodes of the animals of a dose group were pooled, dpm/node was

determined by dividing the measured value by the number of lymph nodes pooled

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In this study Stimulation indices of 5.0, 3.1 and 4.7 were determined with the test item at concentrations of 2.5 %, 5 % and 10 %, respectively, in N,N-dimethylformamide (DMF). FAT 40824/A was therefore found to be a skin sensitizer in the LLNA.

Executive summary:

A GLP compliant LLNA assay was carried with FAT 40824/A according to OECD guideline 429 and EU method B.42. In order to study a possible contact allergenic potential of FAT 40824/A, three groups each of four female mice were treated daily with the test item at concentrations of 2.5 %, 5 % and 10 % in N,N-dimethylformamide (DMF) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. 10% was the highest technically applicable concentration in the vehicle. A control group of four mice was treated with the vehicle N,Ndimethylformamide (DMF) 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 of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a ß scintillation counter.

All treated animals survived the scheduled study period. Neither clinical / local signs nor other findings were observed in any animals of the control group. On the second application day, the residual test item was found at both dosing sites in all mice of Group 2 (2.5%), Group 3 (5%) and Group 4 (10%), persisting for the remainder of the in-life phase of the study. The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value. A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the S.I.

In this study stimulation indices of 5.0, 3.1 and 4.7 were determined with the test item at concentrations of 2.5 %, 5 % and 10 %, respectively, in N,N-dimethylformamide (DMF). FAT 40824/A was therefore found to be a skin sensitizer in the LLNA.