Tetrasodium 4,4'-bis[[4-morpholino-6-(p-sulphonatoanilino)-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 April 22 to 14 May 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 24 April 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:

Tetrasodium 4,4'-bis[[4-morpholino-6-(p-sulphonatoanilino)-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

EC Number:

249-323-0

EC Name:

Tetrasodium 4,4'-bis[[4-morpholino-6-(p-sulphonatoanilino)-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

Cas Number:

28950-61-0

Molecular formula:

C40H40-xN12NaxO14S4

IUPAC Name:

tetrasodium 2,2'-ethene-1,2-diylbis[5-({4-morpholin-4-yl-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)benzenesulfonate]

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 0582900
- Expiration date of the lot/batch: 25-FEB-2013
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands.
- Age at study initiation: 8 - 12 weeks (beginning of treatment).
- Housing: single
- Cage Type: Makrolon Type I, with wire mesh top.
- Bedding: granulated soft wood bedding.
- Diet: pelleted standard diet, ad libitum.
- Water: tap water, ad libitum.
- Acclimation period: at least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: relative humidity 30-70 %
- Photoperiod: artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Remarks:

purity: 99 %

Concentration:

0 (vehicle group), 5, 10, 25 %

No. of animals per dose:

4 animals per dose group.

Details on study design:

RANGE FINDING TESTS
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used was a 25 % suspension in dimethylformamide. Warming and sonicating could not achieve a higher concentration. With other vehicles used, e.g actone:olive oil (4+1), methyl ethyl ketone, DMSO, ethanol:deionised water (7+3) or propylene glycol, higher concentrations could also not be achieved. Upon sponsor's consent dimethylformamide was used as a vehicle. To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 2.5, 5, 10, and 25 % on one ear each on three consecutive days. Clinical signs were recorded 24 ± 4 hours after each application. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity. The test item in the main study was assayed at 5, 10, and 25 %. The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.

MAIN STUDY
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 different test item concentrations of 5, 10, and 25 % (w/v) in dimethylformamide. The application volume, 25 µl, 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 with 250 µl of 78.9 µCi/ml ³HTdR (corresponds to 19.7 µCi ³HTdR per mouse) by intravenous injection via a tail vein.

DETERMINATION of INCORPORATED ³HTdR
Approximately five hours after treatment with ³HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium (Release®, WOT, 0-30827 Garbsen). The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 1 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 plastic scintillation vials with 10 ml of 'Ultima Gold' scintillation liquid (Perkin Elmer (LAS) GmbH, 0-63110 Rodgau) and thoroughly mixed. The level of ³HTdR incorporation was then measured on a β-scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, 0-63110 Rodgau). Similarly, background ³HTdR levels were also measured in two 1 ml-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (OPM).

OBSERVATIONS
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality/Viability: once daily (week day) from experimental start to necropsy.
Body weights: prior to the first application and prior to treatment with ³HTdR.
Clinical signs (local I systemic): once daily (week day). Especially the treatment sites were observed carefully.

Positive control substance(s):

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

Results and discussion

Positive control results:

EC3 = 15.7 % (w/v)

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.64

Test group / Remarks:

5 % test item concentration

Results 2

Parameter:

SI

Value:

1.25

Test group / Remarks:

10 % test item concentration

Results 3

Parameter:

SI

Value:

0.86

Test group / Remarks:

25 % test item concentration

Results 4

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: DPM measured Background I: 29 Background II: 30 Control: 5370 Group 2: 3446 Group 3: 6695 Group 4: 4612

Any other information on results incl. tables

Vehicle: dimethylformamide

Test item conc. % (w/v)	Group	Measurement DPM	Calculation			Result
			DPM-BG*	number of lymph nodes	DPM per lymph nodes**	S.I.
-	BG I	29	-	-	-	-
-	BG II	30	-	-	-	-
-	1	5370	5341	8	667.6
5	2	3446	3417	8	427.10	0.64
10	3	6695	6666	8	833.20	1.25
25	4	4612	4583	8	572.80	0.86

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

1 = Control Group

2-4 = Test Group

S.I. = Stimulation Index

* = The mean value was taken from the figures BG I and BG "

** = 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.

The EC3 value could not be calculated, since all S.I.'s are below 3.

Other results

Viability/Mortality: no deaths occurred during the study period.

Clinical Signs: the animals did not show any clinical signs during the course of the study and no cases of mortality were observed.

Body Weights: the body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 66042/A was not found to be a skin sensitizer in LLNA in mice study.

Executive summary:

FAT 66042/A was assessed for its possible contact allergenic potential, according to the OECD Guideline 429 Skin Sensitisation: Local Lymph Node Assay (adopted 24 April 2002). For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 25 %. Each group had 4 animals per test concentration. Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 5, 10, and 25 % (w/v) in dimethylformamide. Five days after the first topical application, all mice were administered with 250 µl of 78.9 µCi/ml ³HTdR (corresponds to 19.7 µCi ³HTdR per mouse) by intravenous injection via a tail vein. In addition to the sensitising reactions the mortality, body weights and clinical signs were recorded during the test and observation period. The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. In this study Stimulation Indices (S.I.) of 0.64, 1.25, and 0.86 were determined with the test item at concentrations of 5, 10, and 25 % in dimethylformamide, respectively. The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3. Based on the study results, FAT 66042/A was found to be not a skin sensitizer under the described conditions.