Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

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 Apr. 27, 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:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
animals were housed in groups instead of individual housing
GLP compliance:
yes (incl. certificate)
Remarks:
according to OECD principles of GLP
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Name of test material : 2-Methyl-1,4-Benzenediamine Sulphate (Haarbraun) (Code: A00165)

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 - 20 g- Housing: The animals were kept in groups in Macrolon- cages on Altromin saw fiber bedding. Animals were barrier maintained (semi-barrier) in air conditioned rooms- Diet : Altromin 1324 maintenance diet for rats and mice, totally- pathogen- free- TPF, ad libitum- Water : Free access to Tap water (drinking water, municipal residue control, microbiol. controlled periodically), ad libitum- Acclimation period: Not reportedENVIRONMENTAL CONDITIONS- Temperature : 22 ± 3°C- Humidity : 55 ±10%- Air changes : At least 10 per hour - Photoperiod : 12 h dark/12 h lightEXPERIMENTAL START DATE: Apr 27, 2000; EXPERIMENTAL COMPLETION DATE: May 3, 2000

Study design: in vivo (LLNA)

Vehicle:
other: Acetone/Aqua/Olive Oil (AAOO)
Concentration:
Dose concentration: 0, 2.8%, 1.5% and 0.5%Positive control (P-Phenylenediamine): 1% in vehicle (AAOO)
No. of animals per dose:
5 mice per test group
Details on study design:
RANGE FINDING TESTS: No range-finding study was performedMAIN STUDYANIMAL ASSIGNMENT AND TREATMENT- Name of test method: Local Lymph Node Assay (LLNA) - Criteria used to consider a positive response: A positive response was defined as a 3-fold or greater increase in3-H-methyl thymidine-incorporation into lymph node cells of the lymph nodes of the test group animals relative to that recorded for the lymph nodes of vehicle group animals. (SI) = ≥ 3 TREATMENT/VEHICLE PREPARATION AND ADMINISTRATION:- Treatment preparation: Dosing solutions were prepared daily, immediately prior to dosing. The preparation were prepared according to sponsor's protocol in order to gain 2.8%, 1.5% and 0.5%concentrations:1: 2-Methyl-1,4-Benzenediamine Sulphate (Haarbraun)2: 2.5 g of (1) adjusted to 15 mL NaOH ad 50 mL H2O bidest3: 15 mL of (2) + 5 mL Acetone4: 3 mL of (3) + 1 mL Olive oil = 2.8% test concentration5: 5.35 mL of (3) + 4.65 mL H2O/Acetone (4:1)6: 3 mL of (5) + 1 mL Olive Oil = 1.5% test concentration7: 1.8 mL of (3) + 8.65 mL H2O/Acetone (4:1)8: 3 mL of (7) + 1 mL Olive oil = -0.5% test concentration - Vehicle preparation: The vehicle AOO (4:1 (v/v) Acetone/Olive oil) was modified to gain AAOO:Acetone/Aqua (1:1 v/v) = AAAA/Olive Oil (4:1 v/v) = AAOOAdministration of the test preparations: Animals received 25 µL vehicle, positive control or test substance once daily over three consecutive days on the dorsal surface of each ear 0 OBSERVATIONS: Weight assessment: The animals were weighed prior to first application and at the end of the test period.Clinical observation: Prior to the first application all animals were observed for special clinical signs or reactions to treatment.- Evaluation of cell proliferation: On Day 5, 250 µL of [3H]-methyl thymidine containing 20.6 µCi of [3H] methyl thymidine was injected intravenously to each experimental mouse Approx. 5 h later, all mice were sacrificed. The draining auricular lymph nodes were excised, pooled for each animal separately and collected in PBS. A single cell suspension of pooled lymph nodes was prepared by gentle mechanical disaggregation through polyamide gauze of 200 mesh size. The gauze was then washed with Phosphate buffered saline (PBS) and cells were pelleted in a centrifuge. The supernatant was discarded and pallets were resuspended in PBS. This washing procedure was repeated twice. After the final wash pallet was suspended in approx. 3 mL of 5% TCA (trichloroacetic acid) and transferred in to scintillation vials. The [3H]-methyl thymidine incorporation was measured in a β-counter and expressed as the number of DPM (disintegration per minute)
Positive control substance(s):
other: 1% P-Phenylenediamine in AAOO (CAS # 106-50-3; purity:>98%; Lot # 69H3638)
Statistics:
No statistical analyses were performed

Results and discussion

Positive control results:
Mean DPM: 984±664.7Mean DPM/node: 485.6Mean stimulation index: 5.3. (The mean SI is greater than 3 thus validated the experimental conditions of OECD 429)For details, refer to ’Table 1’ under ‘Any other information on results’ section

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The mean stimulation indices for at the concentrations of 0, 0.5 %, 1.5 % and 2.8 % in were 1, 4.4, 10.4 and 19.4, respectively. Calculation of EC3 value was not useful, as all stimulation index were found to be over 3. For details, refer to’ table 1’ under ‘Any other information on results’ section.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Treatment of mice with 2-Methyl-1,4-Benzenediamine Sulphate (Haarbraun) in Acetone/Aqua/Olive oil (AAOO) vehicle produced statistically significant increases thymidine incorporation in the auricular lymph nodes in comparison to vehicle control. Mean DPM: 197.7, 826.4, 1938 and 3595 at 0, 0.5, 1.5, and 2.8% test substance concentration respectively. For details, refer to’ Table 1’ under ‘Any other information on results’ section

Any other information on results incl. tables

Table 1: Measure of Disintegrations per minute (DPM) and Stimulation Index (SI) of individual animals (study # 70772)

Group

Animal no.*

DPM

DPM/ Node

SI

Vehicle control

1

154.7

70.9

 

92

140.9

64.1

 

93

162.7

75.0

 

94

338.5

162.8

 

95

191.9

89.6

 

Mean value

197.7

92.5

1.0

2-Methyl-1,4-Benzenediamine Sulphate (Haarbraun)0.5%

1

444.5

215.9

2.3

2

811.8

399.5

4.3

3

884.2

435.7

4.7

4

1608.7

797.9

8.6

5

383.0

185.1

2.0

Mean value

826.4

406.8

4.4

2-Methyl-1,4-Benzenediamine Sulphate (Haarbraun)1.5%

6

1829.2

908.2

9.8

7

2362.7

1174.9

12.7

8

11147.7

567.5

6.1

9

2897.1

1442.1

15.6

10

1454.8

721

7.8

Mean value

1938

962.8

10.4

2-Methyl-1,4-Benzenediamine Sulphate (Haarbraun)2.8%

11

4390

2188.6

23.7

12

3230.4

1608.8

17.4

13

4794.2

2390.7

25.9

14

3110.4

1548.8

16.7

15

2451.0

1219.1

13.2

Mean value

3595

1791

19.4

positive control

96

300.1

143.8

1.6

97

834.2

410.7

4.4

98

1193.1

590.1

6.4

99

435.7

211.4

2.3

100

2156.7

1072.0

11.6

Mean value

984.0

485.6

5.3

*as mentioned in the report

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
2-Methyl-1,4-Benzenediamine Sulphate (Haarbraun) in acetone/aqua/olive oil ) was considered as sensitizer in Local Lymph Node Assay (LLNA).
Executive summary:

The skin sensitization potential of 2-Methyl-1,4-Benzenediamine Sulphate (Haarbraun) was determined following OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay).

A total of 5 mice per test group, age 8-12 wk, weighing 16-20 g from source: Harlan Winkelmann GmbH, D-33178 Borchen were kept in groups in Macrolon- cages on Altromin saw fiber bedding and were barrier maintained (semi-barrier) under standard laboratory conditions (temperature: 22 ±3°C; relative humidity: 55 ±10%; Air Changes: 10 per hour; light/dark cycle: 12h light / 12 h dark cycle). The feed used was Altromin 1324 maintenance diet for rats and mice, totally- pathogen- free- TPF, ad libitum.

The test substance (Haarbraun) was prepared at three different test concentrations (i.e. 0.5, 1.5 and 2.8 mg/L) in the vehicle Acetone/Aqua/Olive oil (AAOO). The vehicle group received AAOO. Positive control animals received 1% P-Phenylenediamine in AAOO. The animals received 25 µL of appropriate formulations vehicle on dorsal surface of each ear for three consecutive days. 5 d after first topical application 250 µL of 20.6 µCi [3H] methyl thymidine (diluted to a working concentration of 80µCi/mL) was injected intravenously to each experimental mouse. Approx. 5 h later, the animals were sacrificed and the draining auricular lymph nodes were excised, pooled for each animal and collected in Phosphate buffered saline (PBS). A single cell suspension of lymph node cells for each animal was prepared by gentle mechanical disaggregation. The cell suspension was then centrifuged to pellets, supernatant was discarded and cells were resuspended in PBS. This washing procedure was repeated twice.

After final wash the pellets were resuspended in 3 mL of 5% Trichloroacetic acid (TCA) at approx 4°C, left over night for precipitation of macromolecules, centrifuged, resuspended in 1 mL of 5% TCA and transferred into the scintillation vials. [3H]-methyl thymidine incorporation was measured in a β-counter and expressed as number of disintegration per minute (DPM).The positive control (1% P-Phenylenediamine) induced a 5.3-fold increase in isotope incorporation in the draining auricular lymph nodes relative to the vehicle. The mean stimulation index was above 3 i.e. 5.3.

Treatment of mice with all the three concentrations of test substance produced significant increase in thymidine incorporation in the auricular lymph nodes in comparison to vehicle control.. The stimulation indices of the 2.8%, 1.5% and 0.5% 2-Methyl-1,4-Benzenediamine Sulphate (Haarbraun) treatments were 19.4, 10.4 and 4.4 fold respectively.

Thus the test substance, 2-Methyl-1,4-Benzenediamine Sulphate (Haarbraun) in the vehicle Acetone/Aqua/Olive oil (AAOO) was considered as sensitizer in Local Lymph Node Assay (LLNA).

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