Sodium 2-[[2-amino-8-hydroxy-6-[(methylanilino)sulphonyl]-1-naphthyl]azo]-5-(chloroacetamido)benzenesulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 July 2014 to 24 October 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 2-[[2-amino-8-hydroxy-6-[(methylanilino)sulphonyl]-1-naphthyl]azo]-5-(chloroacetamido)benzenesulphonate

EC Number:

274-395-5

EC Name:

Sodium 2-[[2-amino-8-hydroxy-6-[(methylanilino)sulphonyl]-1-naphthyl]azo]-5-(chloroacetamido)benzenesulphonate

Cas Number:

70209-97-1

Molecular formula:

C25H22ClN5O7S2.Na

IUPAC Name:

sodium 2-({2-amino-8-hydroxy-6-[(2-methylphenyl)sulfamoyl]-1-naphthyl}diazenyl)-5-[(chloroacetyl)amino]benzenesulfonate

Test material form:

other: solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: AT-0027506800
- Expiration date of the lot/batch: 20 January 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Solubility in water: 10 to 50 g/L at 30 °C

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories GmbH, 5800 AN Venray,
The Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 17-22 g
- Housing: Full barrier in an air-conditioned room
- Diet: Free access to Altromin 1324 maintenance diet for rats and mice (prescreen test and main study: lot no. 1526)
- Water: Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): at least 10 x / hour
- Photoperiod (hrs dark / hrs light): sequence being 12 hours light, 12 hours dark
- IN-LIFE DATES: From: 11 July 2014 to 24 October 2014

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Remarks:

Applichem, lot no. 2P003474, expiry date: 09/2014

Concentration:

The maximum technically applicable concentration of the test item in the vehicle was found to be 12.5 % in DMSO.
Based on the results observed in the preliminary test the following test item concentrations were selected for the main study:
3 %, 6.25 % and 12.5 % (w/v)
DMSO was used as vehicle and served as negative control.

No. of animals per dose:

5 mice / group; 3 mice / prescreen test

Details on study design:

Preparation of the Test Item
Based on the results observed in the prescreen test the following test item concentrations were selected for the main study:
3 %, 6.25 % and 12.5 % (w/v)
The preparations were made immediately prior to each dosing.

Preparation of the Animals
The animals were randomly selected. Identification was ensured by cage number and individual marking (tail).
 
Clinical Observation
Prior to the application and once a day thereafter all animals were observed in order to detect signs of toxicity, including dermal irritation at site of application.

Weight Assessment
The animals were weighed prior to the application and at the end of the test period (prior to the treatment with ³HTdR).

Dose Groups
3 test groups (3 different concentrations) and 1 negative control group (vehicle) were tested.

Test Regime

Topical Application
Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear. Topical applications were performed once daily over three consecutive days.
Administration of ³H-Methyl thymidine
Five days after the first topical application all mice were dosed with 20 µCi ³H-methyl thymidine by intravenous injection (tail vein) of 250 µL of 3H-methyl thymidine, diluted to a working concentration of 80 µCi/mL.
Preparation of Cell Suspension
Approximately 5 hours after the injection of ³H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approx. 1 mL 5 % TCA at approx. 4 °C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5 % TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.
Determination of Incorporated ³H-Methyl Thymidine:
The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background ³H-methyl thymidine levels were also measured (5 % TCA). Determination of radioactivity was performed individually for each animal.

Evaluation of Results
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of ³H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined,
background values were subtracted. EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation, EC3=c+[(3-d)/(b-d)]x(a c), between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation
index of three, no EC3 value can be stated.
A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in ³H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0). On the basis of the test results, the test substance may be classified into one of the following categories in conformity with the criteria given in Commission Regulation (EU) No 286/2011 as well as in GHS - Globally Harmonised System of Classification and Labelling of Chemicals, fifth revised edition, 2013: Skin sensitiser
Category 1:
A substance is classified as a skin sensitiser
a) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons, or
b) if there are positive results from an appropriate animal test.
WARNING, exclamation mark. May cause an allergic skin reaction.
Sub-category 1A:
Substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Severity of reaction may also be considered.
EC3 value ≤2 %
WARNING, exclamation mark. May cause an allergic skin reaction.
Sub-category 1B:
Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.
EC3 value >2 %
WARNING, exclamation mark. May cause an allergic skin reaction.

Positive control substance(s):

other: Phenylenediamine

Statistics:

Outlier tests according to Dixon, Grubbs and Nalimov were performed for the values measured for the number of disintegrations per minute (DPM).
If outliers were identified, these values were not included in the calculation of the stimulation indices. As at least four values per group are required for the evaluation of the results, the outlier test was not repeated to detect further outliers.

Results and discussion

Positive control results:

The recent reliability check was performed in May 2014. The raw data of this study are kept in the BSL archives (BSL Project ID 142473 A).
Positive-control substance: P-Phenylenediamine (CAS 106-50-3, Sigma, purity >98 %; Lot No.: SLBC7171V) 1 %
Vehicle: DMSO
Species/strain: healthy CBA/CaOlaHsd mice
Source: Harlan Laboratories GmbH, 5800 AN Venray, The Netherlands
Concentrations: 1 % on three consecutive days

The Stimulation Index was 10.0

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

2.4

Test group / Remarks:

Low dose

Remarks on result:

other: 3 % concentration

Results 2

Parameter:

SI

Value:

2.2

Test group / Remarks:

Middle dose

Remarks on result:

other: 6.25% concentration

Results 3

Parameter:

SI

Value:

3.3

Test group / Remarks:

High dose

Remarks on result:

other: 12.5 % concentration

Any other information on results incl. tables

All animals survived throughout the test period without showing any adverse clinical signs. All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study. 

Radioactive Determination of the Test Substance Groups

 

POS	CPM	Test Item	Conc. [%]	Animal number	DPM	DPM- mean back- ground	DPM/ Node	Stimulation Index
16	1382.0	Negative Control	100	16	3672.0	3645.8	1822.9
17	760.0			17	1993.0	1966.8	983.4
18	1144.0			18	2979.0	2952.8	1476.4
19	1690.0			19	4479.0	4452.8	2226.4
20	1050.0			20	2783.0	2756.8	1378.4
MV	1205.2			MV	3181.2	3155.0	1577.5	1.0
SD	313.8			SD	841.0	841.0	420.5
56	4179.0	FAT 20075/C in DMSO	3	1	10942.0	10915.8	5457.9	3.5
57	2520.0			2	6714.0	6687.8	3343.9	2.1
58	1637.0			3	4321.0	4294.8	2147.4	1.4
59	3691.0			4	9827.0	9800.8	4900.4	3.1
60	2519.0			5	6647.0	6620.8	3310.4	2.1
MV	2909.2			MV	7690.2	7664.0	3832.0	2.4
SD	910.6			SD	2388.8	2388.8	1194.4	0.8
61	5570.0*	FAT 20075/C in DMSO	6.25	6	14914.0*	n.d.	n.d.	n.d.
62	2179.0			7	5698.0	5671.8	2835.9	1.8
63	2488.0			8	6566.0	6539.8	3269.9	2.1
64	2474.0			9	6460.0	6433.8	3216.9	2.0
65	3493.0			10	9245.0	9218.8	4609.4	2.9
MV	2658.5			MV	6992.3	6966.1	3483.0	2.2
SD	497.3			SD	1343.0	1343.0	671.5	0.4
66	4299.0	FAT 20075/C in DMSO	12.5	11	11242.0	11215.8	5607.9	3.6
67	2878.0			12	7560.0	7533.8	3766.9	2.4
68	3558.0			13	9385.0	9358.8	4679.4	3.0
69	5584.0			14	14616.0	14589.8	7294.9	4.6
70	3594.0			15	9522.0	9495.8	4747.9	3.0
MV	3982.6			MV	10465.0	10438.8	5219.4	3.3
SD	918.3			SD	2380.3	2380.3	1190.2	0.8
73	11.0	Background Szinti and TCA			28.0
74	9.0				24.0
75	10.0				25.0
76	11.0				27.0
77	11.0				27.0
MV	10.4			MV	26.2	0.0	0.0	0.0
SD	0.8			SD	1.5
* = outlier, failed Nalimov;  n.d. = not determined

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

FAT 20075/C is classified into Category 1 skin sensitiser.

Executive summary:

Local Lymph Node Assay with FAT 20075/C was carried out according to OECD guideline 429 and EU method B.42. Assay was carried out in CBA female mice. Prior to the application and once a day thereafter all animals were observed in order to detect signs of toxicity, including dermal irritation at site of application. The animals were weighed prior to the application and at the end of the test period (prior to the treatment with ³HTdR). Three test groups (3 different concentrations) and 1 negative control group (vehicle) were tested. Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear. Topical applications were performed once daily over three consecutive days. Five days after the first topical application all mice were dosed with 20 µCi ³H-methyl thymidine by intravenous injection (tail vein) of 250 µL of ³H-methyl thymidine, diluted to a working concentration of 80 µCi/mL.

Approximately 5 hours after the injection of ³H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated. After the final wash each pellet was resuspended in approx. 1 mL 5 % TCA at approx. 4° C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5 % TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight. The ³H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background ³H-methyl thymidine levels were also measured (5 % TCA). Determination of radioactivity was performed individually for each animal. All animals survived throughout the test period without showing any adverse clinical signs. All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study. The stimulation index at a concentration of 3, 6.25 and 12.5 % was 2.4, 2.2 and 3.3, respectively. One of the tested concentrations exceeded the stimulation index of 3. The EC₃ value could not be calculated due to the lack of a dose-response relationship of the data obtained. So, FAT 20075/C is considered to be a skin sensitizer.