Reaction products of disodium 3,5-diamino-2-[(2-sulfonatophenyl)diazenyl]benzoate and  diazotised sodium 2-[(4-aminophenyl)sulfonyl]ethyl sulfate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment start date - 30 April 2003; Experiment end date - 14 May 2003; Study completion date - 12 June 2003.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Deviations:

no

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:

other: solid

Details on test material:

Identity: FAT 40’813/A (equivalent to FAT40810/A but granulated)
Description: Brown solid
Batch number: Br'10
Purity: approx. 75.0 %
Stability of test item: Stable under storage conditions
Expiry date: 1 1-APR-2010
Storage conditions: In the original container at room temperature (20 °C ±3 °C), away from direct sunlight.
Safety precautions: Routine hygienic procedures (gloves, goggles).

Specific details on test material used for the study:

Identity: FAT 40810/A
Batch: WP 6/02
Purity: approx. 75 %
Appearance: Solid, dark brownish powder
Expiration date: 12 December 2010
Storage: At room temperature at about 20 °C

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Test system: Mice, CBA/CaOlaHsd
Rationale: Recognized as the recommended test system.
Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst / The Netherlands
Number of animals for the pre-test (non-GLP): 2 females
Number of animals for the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) group: 1
Age: 8 - 12 weeks (beginning of acclimatization)
Body weight: 17.1 g - 20.1 g (beginning of acclimatization)
Identification: Each cage by unique cage card, in every cage each animal by individual code marked at tail with a permanent pen.
Randomization: Randomly selected by computer algorithm at time of delivery.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
The animals were distributed as follows:
Group 1: (Control Group), ethanol/water 7/3 v/v - 4 animals in cage 1
Group 2: 2.5 % w/v dose, 4 animals in cage 2
Group 3: 5 % w/v dose, 4 animals in cage 3
Group 4: 10 % w/v dose, 4 animals in cage 4
In a non-GLP conform pre-test in two mice, test item concentrations of 1 %, 2.5 %, 5 % and 10 % (w/v) were tested on one ear each. No irritation effects were observed at these concentrations after a single application. 10 % (w/v) was the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation in the chosen vehicle. The sensitivity and reliability of the experimental technique employed was assessed by use of a substance which is known to have skin sensitization properties in CBA/CaOlaHsd mice. The vaIidation / positive control study was performed with ALPHA -HEXYLCINNAMALDEHYDE in acetone/olive oil, 4:1 (vlv) using CBA/CaOlaHsd mice (RCC Study Number 846871) between 08-JAN-2003 and 22-JAN-2003.

HUSBANDRY
Room no. E21 / RCC ltingen
Conditions: Standard Laboratory Conditions. Air-conditioned with target 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.
Accommodation: In groups of four in Makrolon type-3 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
Diet: Pelleted standard Kliba 3433, batch no. 84/02 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst) available ad Iibitum. Results of analyses for contaminants are archived at RCC.
Water: Community tap water from ltingen, available ad Iibitum. Results of representative bacteriological, chemical and contaminant analyses are archived at RCC.

Study design: in vivo (LLNA)

Vehicle:

other: ethanol/water 7:3

Concentration:

0 (Vehicle control - Group 1),
2.5 (Group 2),
5 (Group 3) and
10 % w/v (Group 4)

No. of animals per dose:

4

Details on study design:

The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle (ethanol:water, 7:3 (v/v)) 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 freshly before each dosing occasion. To determine the highest non-irritant and technically applicable test item concentration, a non-GLP pre-test was performed in two mice with concentrations of 1 %, 2.5 %, 5 % and 10 % (w/v). 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 severe irritant effects were tolerated choosing the test concentrations.

TREATMENT PROCEDURES
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 2.5 %, 5 % and 10 % (w/v) in ethanol:water, 7:3 (vlv). The application volume, 25 µl, was spread over the entire dorsal surface (approx. 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). A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.

ADMINISTRATION OF 3H-METHYL THYMIDINE: 3H-methyl thymidine (3HTdR) was purchased from Amersham International (Amersham product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml). Five days after the first topical application, all mice were administered with 250 µl of 88.3 pCi/ml 3HTdR (equal to 22.1 pCi 3HTdR) by intravenous injection via a tail vein.

DETERMINATION OF INCORPORATED 3HTDR: Approximately five hours after treatment with 3HTdR all mice were euthanized by intraperitoneal injection of VETANARCOL (Veterinaria AG, Zurich). 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 pm mesh size). After washing two times 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 ‘Ultima Gold’ scintillation liquid and thoroughly mixed. The level of ³HTdR incorporation was then measured on a B-scintillation counter. Similarly, background ³HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The B-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

INTERPRETATION OF RAW DATA: The proliferative 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) (S.l.). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- First, that 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.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression. The decision to select a STIMULATION INDEX (S.l.) of 3 as an arbitrary indication of sensitizing activity was made on the basis of investigations performed with a wide range of chemicals.

Positive control substance(s):

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

Results and discussion

Positive control results:

In this study stimulation indices of 2.5, 3.7 and 9.7 were determined with the positive control substance alpha-hexylcinnamaldehyde at concentrations of 5 %, 10 % and 25 % (w/v) in acetone/olive oil, 4:1 (v/v). 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 stimulation index (S.l.).
The test item alpha-hexylcinnamaldehyde was found to be a sensitizer and an EC3 value of 7.08 % (w/v) was derived.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

2.4

Test group / Remarks:

Group 2

Results 2

Parameter:

SI

Value:

2.5

Test group / Remarks:

Group 3

Results 3

Parameter:

SI

Value:

2.6

Test group / Remarks:

Group 4

Any other information on results incl. tables

Mortality: No deaths occurred during the study period

Clinical signs: No test item-related clinical signs were observed in any animals of the control group  or Group 2 (2.5 %). On the third application day, a slight ear swelling was observed at both dosing sites in all mice of Group 3 (5 %) and Group 4 (10 %), persisting for two days.

Body weights: 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.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item FAT 40810/A was found to be a non-sensitizer when tested at concentrations of up to 10 % (w/v).

Executive summary:

In a GLP-compliant study conducted in accordance with OECD test guideline 429, the possible contact allergenic potential of FAT 40813/A was evaluated. Three groups each of four female mice were treated daily 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) for three consecutive days. 10 % was the highest technically achievable concentration in the vehicle. A control group of four mice was treated with the vehicle (ethanol: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 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 scintillation counter. No test item-related clinical signs were observed in any animals of the control group or Group 2 (2.5 %). On the third application day, a slight ear swelling was observed at both dosing sites in all mice of Group 3 (5 %) and Group 4 (10 %), persisting for two days. All treated animals survived the scheduled study period. 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 stimulation index (S.I.).

 

Group #	Test item concentration % (w/v)	S.I.
Group 2	2.5	2.4
Group 3	5	2.5
Group 4	10	2.6
A dose-response relation was observed. Calculation of the EC 3 value was not done because no test concentrations produced a stimulation index (S.I.) of 3 or higher.