2-methyl-p-phenylenediamine sulfate

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Apr. 27, 2000 To May 22, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed guideline, GLP

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to 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 - 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 reported

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

EXPERIMENTAL START DATE: Apr 27, 2000; EXPERIMENTAL COMPLETION DATE: May 3, 2000

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 performed

MAIN 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 bidest
3: 15 mL of (2) + 5 mL Acetone
4: 3 mL of (3) + 1 mL Olive oil = 2.8% test concentration
5: 5.35 mL of (3) + 4.65 mL H2O/Acetone (4:1)
6: 3 mL of (5) + 1 mL Olive Oil = 1.5% test concentration
7: 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) = AA
AA/Olive Oil (4:1 v/v) = AAOO

Administration 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

Positive control results:

Mean DPM: 984±664.7
Mean DPM/node: 485.6
Mean 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

Parameter:

other: disintegrations per minute (DPM)

Value:

826.4

Test group / Remarks:

0.5 % test substance in vehicle; 5 animals

Parameter:

other: disintegrations per minute (DPM)

Value:

1 938

Test group / Remarks:

1.5 % test substance in vehicle; 5 animals

Parameter:

other: disintegrations per minute (DPM)

Value:

3 595

Test group / Remarks:

2.8 % test substance in vehicle; 5 animals

Parameter:

SI

Value:

4.4

Test group / Remarks:

0.5 % test substance in vehicle; 5 animals

Parameter:

SI

Value:

10.4

Test group / Remarks:

1.5% test substance in vehicle; 5 animals

Parameter:

SI

Value:

19.4

Test group / Remarks:

0.5 % test substance in vehicle; 5 animals

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

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

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 %) 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 0.5, 1.5 and 2.8% 2-Methyl-1,4-Benzenediamine Sulphate (Haarbraun) treatments were 4.4, 10.4 and 19.4 fold respectively. The lowest concentration used in this test was too high.An extrapolated EC3 value was calculated from the study report by linear regression and found to be 0.31% ( Aeby, P.; Wyss, C.; Beck, H.; Griem, P.; Scheffler, H.; Goebel, C.; Characterization of the sensitizing potential of chemicals by in vitro analysis of dendritic cell activation and skin penetration; J. INVEST. DERMATOL.; 122, 1154-1164; 2004).

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)

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

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 %) 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 0.5, 1.5 and 2.8% 2-Methyl-1,4-Benzenediamine Sulphate (Haarbraun) treatments were 4.4, 10.4 and 19.4 fold respectively.The lowest concentration used in this test was too high.An extrapolated EC3 value was calculated from the study report by linear regression and found to be 0.31% ( Aeby, P.; Wyss, C.; Beck, H.; Griem, P.; Scheffler, H.; Goebel, C.; Characterization of the sensitizing potential of chemicals by in vitro analysis of dendritic cell activation and skin penetration; J. INVEST. DERMATOL.; 122, 1154-1164; 2004).

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).

Justification for selection of skin sensitisation endpoint:
reliability 1

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

This LLNA study is classified as acceptable, and satisfies the guideline requirements of OECD 429 (Skin Sensitisation: Local Lymph Node Assay), the substance is classified as skin sensitiser according to CLP criteria.