(p-ammoniophenyl)bis(2-hydroxyethyl)ammonium sulphate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From May 03, 2000 to May 22, 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed guideline, GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to German and OECD principles of GLP

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

58262-44-5

Cas Number:

58262-44-5

IUPAC Name:

58262-44-5

Constituent 2

Reference substance name:

N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate

IUPAC Name:

N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate

Details on test material:

- Name of test material: N,N-bis(2-hydroxyethyl)-P-Phenylenediamine sulfate; Blauentwickler; Sample number: R98006523
- Molecular formula: C10H16N2O2.H2O4S
- Molecular weight: 294.3 g/mol
- Substance type: Pure active substance
- Physical state: Crème colored solid
- Stability under test conditions: Years
- Storage condition of test material: At room temperature, protected from light

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca01 aHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D-33178 Borchen
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 16-19 g
- Housing: Kept in groups in Macrolon- cages on Altromin saw fiber bedding. The animals were barrier maintained (semi-barrier) in air conditioned rooms.
- Diet: Altomin 1324 maintainance diet for rats and mice, totally-pathogen-free TPF, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: Not reported

ENVIRONMENTAL CONDITIONS
- Temperature : 22 ± 3°C
- Humidity: 55 ± 10 %
- Air changes : At least 10 x/ hour
- Photoperiod : 12 h dark/ 12 h light, 6:30-18:30

IN-LIFE DATES: From: May 03, 2000 To: May 09, 2000

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Remarks:

Fluka; Lot # 392116/1

Concentration:

5.0, 2.5 and 0.5%

No. of animals per dose:

5 mice per group

Details on study design:

RANGE FINDING TESTS: No range-finding study was performed

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node assay (LLNA)
- Criteria used to consider a positive response: A substance will be regarded as a 'sensitizer' in the LLNA if at least one concentration of the test substance results in a 3-fold or greater increase in 3H-methyl thymidine incorporation into lymph node cells relative to the control group (stimulation indices equal to or greater than 3.0).

TREATMENT PREPARATION AND ADMINISTRATION:
- Dose preparation: The dosing solutions were prepared using DMSO according to sponsor's protocol in order to gain the required concentrations. The preparations were made immediately prior to each dosing.
The details of preparation are provided in the study report.
- Application of the test preparations: Each mouse was treated by the topical application of 25 µL of the selected solution onto the entire dorsal surface of each ear.
Topical applications were performed once daily over 3 consecutive days.

OBSERVATIONS:
- Clinical signs: Mice were observed prior to the application and once a day thereafter following application of test substances to detect special clinical signs or reactions to treatment.
- Weight assessment: The animals were weighed prior to first application and at the end of the test period.

EVALUATION OF CELL PROLIFERATION: 5 d after the first topical application treatment all mice were dosed with 20 µCi of [3H]-methyl thymidine by intravenous injection (tail vein) of 250 µL of 3H-methyl thymidine (working concentration of 80 µCi/mL). Approx. 5 h later, the animals were sacrificed, and the draining auricular lymph nodes were removed (2 lymph nodes for each animal). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamid 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 twice. After the final wash each pellet was resuspended in approx. 3 mL 5% TCA at approx. 4°C overnight for precipitation of macromolecules. Each precipitate was recovered by centrifugation, resuspended in 1 mL 5% TCA and transferred into scintillation vials.

DETERMINATION OF INCORPORATED 3H-METHYL THYMIDINE:
- The 3H-methyl thymidine - incorporation was measured in a β-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA).
- Radioactivity was determined individually for each animal.

Positive control substance(s):

other: P-Phenylenediamine (at 1% concentration in DMSO)

Statistics:

Not reported

Results and discussion

Positive control results:

The stimulation index for the positive control (1% P-Phenylenediamine) was 10.1.
The mean DPM of the positive control was 4129.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

Negative control

Results 2

Key result

Parameter:

SI

Value:

0.7

Variability:

0.3 - 1.3

Test group / Remarks:

Test item at 0.5%

Results 3

Key result

Parameter:

SI

Value:

9.1

Variability:

2.8- 13.9

Test group / Remarks:

Test item at 2.5%

Results 4

Key result

Parameter:

SI

Value:

9.7

Variability:

6.2-14.9

Test group / Remarks:

Test item at 5.0%

Results 5

Key result

Parameter:

SI

Value:

10.1

Variability:

8.2-11.2

Test group / Remarks:

Positive control

Results 6

Key result

Parameter:

other: DPM (disintegration per minutes)

Value:

421.4

Variability:

SD 99.1

Test group / Remarks:

Negative Control

Results 7

Key result

Parameter:

other: DPM (disintegration per minutes)

Value:

318.1

Variability:

SD 145.4

Test group / Remarks:

0.5% Test item

Results 8

Key result

Parameter:

other: DPM (disintegration per minutes)

Value:

3 722

Variability:

SD 1649

Test group / Remarks:

2.5% Test item

Results 9

Key result

Parameter:

other: DPM (disintegration per minutes)

Value:

3 996

Variability:

SD 1347

Test group / Remarks:

5% Test item

Results 10

Key result

Parameter:

other: DPM (Disintegration per minute)

Value:

4 129

Variability:

SD 480.5

Test group / Remarks:

Positive control

Results 11

Key result

Parameter:

EC3

Value:

1.04

Test group / Remarks:

EC3 value

Any other information on results incl. tables

Body weight:No effect on body weight or body weight gain was observed. The details on body weight gain are provided in the study report (Table 1: weight gain data).

Table 1: Local lymph node assay with N,N-Bis(2Hydroxyethyl)-P-Phenylenediamine Sulphate (Study # 70775)

Test substance Dose Mean DPM Stimulation index DMSO Vehicle control 421.4 1.0 N,N-Bis(2Hydroxyethyl)-P-Phenylenediamine Sulphate(Blauentwickler) 0.5% 318.1 0.7 2.5% 3722 9.1 5.0% 3996 9.7 1% P- Phenylenediamine in DMSO Positive control 4129 10.1

- Calculated EC3 value (derived by linear interpolation) was at a concentration of 1.04% of test substance.

- Based on the above, it was stated that the test substance caused reactions identified as sensitization up from a concentration of 1.04% of test substance, where the stimulation index was equal to 3.0.

Applicant's summary and conclusion

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

N,N bis(2-hydroxyethyl)-p-phenylenediamine sulfate (Blauentwickler) caused reactions identified as sensitization i.e. demonstrating a stimulation index ≥3.0 in a Local lymph Node Assay. Calculated EC3 value (derived by linear interpolation) was at a concentration of 1.04% of test substance.
Under the conditions of this test, Blauentwickler was categorised as a strong sensitiser, Category 1A according to CLP criterias.

Executive summary:

Thein-vivoskin sensitisation test of N,N-Bis(2Hydroxyethyl)-P-Phenylenediamine Sulphate (Blauentwickler) was performed following the methods comparable to OECD 429 guideline (Skin Sensitisation: Local Lymph Node Assay).

The test material was assayed at concentrations of 0, 0.5, 2.5 and 5.0%. The vehicle group received dimethylsulfoxide (DMSO). P- Phenylenediamine at 1% concentration in DMSO served as the positive control.

25 µL of test material or vehicle was applied to the dorsal surface of each ear of each mouse consecutively for 3 d. Five days after the first topical application treatment all mice were injected intravenously with [3H]-methyl thymidine. Approx. 5 h after [3H]-methyl thymidine injection all mice were sacrificed and the draining auricular lymph nodes were excised, in order to prepare single cell suspension of the lymph node cells.

[3H]-methyl thymidine incorporation was measured in a β- scintillation counter and expressed as the number of disintegrations per minute (DPM). The proliferative response of lymph node cells was calculated as the ratio of 3H·methyl thymidine· incorporation into lymph node cells of test group animals relative to that recorded for control group animals. A stimulation index (SI), ratio of test substance / vehicle control, was calculated for each concentration.

The stimulation index for the positive control (1% P·Phenylenediamine) was 10.1 (>3).

The mean stimulation index for the test substance was 1.0, 0.7, 9.1 (>3) and 9.7 (>3) at 0 (vehicle control), 0.5, 2.5 and 5.0% concentration of test substance.

A substance is regarded as a 'sensitizer' in the LLNA if at least one concentration of the test substance results in a 3-fold or greater increase in 3H-methyl thymidine incorporation into lymph node cells relative to the control group(stimulation indices ≥3.0).

N,N bis(2-hydroxyethyl)-p-phenylenediamine sulfate (Blauentwickler) caused reactions identified as sensitization i.e. demonstrating a stimulation index≥3.0 in a Local lymph Node Assay. Calculated EC3 value (derived by linear interpolation) was at a concentration of 1.04% of test substance.

Therefore, under the conditions of this test Blauentwickler was categorised as a strong sensitiser, Category 1A according to CLP criterias.

This LLNA study is classified as acceptable, and satisfies the guideline requirements of OECD 429 (Skin Sensitisation: Local Lymph Node Assay).