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Administrative data

Description of key information

In differents skin sensitistaion studies on animals, the N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulphate induced skin sensitisation. Furthermore, the substance is a known skin sensitiser in humans.

A LLNA study could be used to quantify the elicitaion induction threshold.

N,N bis(2-hydroxyethyl)-p-phenylenediamine sulfate 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, the substance was categorised as a strong sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
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
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
according to German and OECD principles of GLP
Type of study:
mouse local lymph node assay (LLNA)
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
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
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.
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
Negative control
Key result
Parameter:
SI
Value:
0.7
Variability:
0.3 - 1.3
Test group / Remarks:
Test item at 0.5%
Key result
Parameter:
SI
Value:
9.1
Variability:
2.8- 13.9
Test group / Remarks:
Test item at 2.5%
Key result
Parameter:
SI
Value:
9.7
Variability:
6.2-14.9
Test group / Remarks:
Test item at 5.0%
Key result
Parameter:
SI
Value:
10.1
Variability:
8.2-11.2
Test group / Remarks:
Positive control
Key result
Parameter:
other: DPM (disintegration per minutes)
Value:
421.4
Variability:
SD 99.1
Test group / Remarks:
Negative Control
Key result
Parameter:
other: DPM (disintegration per minutes)
Value:
318.1
Variability:
SD 145.4
Test group / Remarks:
0.5% Test item
Key result
Parameter:
other: DPM (disintegration per minutes)
Value:
3 722
Variability:
SD 1649
Test group / Remarks:
2.5% Test item
Key result
Parameter:
other: DPM (disintegration per minutes)
Value:
3 996
Variability:
SD 1347
Test group / Remarks:
5% Test item
Key result
Parameter:
other: DPM (Disintegration per minute)
Value:
4 129
Variability:
SD 480.5
Test group / Remarks:
Positive control
Key result
Parameter:
EC3
Value:
1.04
Test group / Remarks:
EC3 value

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.

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 index3.0 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).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Sensitisation studies have been conducted with N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate in guinea pigs and mice. When tested in the open epicutaneous test in female Hartley albino guinea pigs, N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate was considered to be a sensitiser at an induction and challenge concentration of 3% N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate in a hydroalcoholic vehicle containing hydroxyethylcellulose.

In the guinea pig maximisation test in Hartley albino male and female guinea pigs, N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate was considered to be a sensitiser. N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate was tested at an induction concentration of 0.1% in aqueous solution (intradermal injection) and 25% in propylene glycol (topical patch concentration), and at a challenge concentration of 25% and 5% in propylene glycol.

A local lymph node assay in female CBA/CaJ mice with N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate produced evidence of a contact allergic response at concentrations up to 2% in dimethylsulfoxide (the highest concentration tested). Similarly, concentrations of 2.5% and 5% of N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate in dimethylsulfoxide produced allergic contact sensitisation in female CBA/Ca01HSD mice in a local lymph node assay. No evidence of stimulating allergic contact sensitisation was observed at concentrations of N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate up to 2.8% (the highest concentration tested) in acetone/aqua olive oil [4:1 (v/v)] in a local lymph node assay in female CBA/Ca01HSD mice.

The vehicle AAOO appears to be the reason for negative result, because positive results were obtained when DMSO was used as vehicle. Acetone/Aqua/Olive Oil (AAOO) is not a preferred vehicle. Its use was not explained. The range of test concentrations used was narrow. Also, higher concentrations should have been used.

Based on the results of these studies, N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate has the potential to induce allergic contact sensitisation in both guinea pigs and mice. A clinical repeat insult patch test with a hair dye base containing 3% N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate,  conducted under exagerated conditions with an occlusive patch and multiple exposures, showed evidence of sensitisation.

Justification for classification or non-classification

N,N bis(2-hydroxyethyl)-p-phenylenediamine sulfate 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, the substance was categorised as a strong sensitiser.

According to CLP ( 1272/2008/EC), the substance is considered as - Warning – sensitizer –category 1A – H317: May cause an allergic skin reaction.