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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Nov. 23, 2000 to Nov. 29, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline, GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2001

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

Constituent 1
Chemical structure
Reference substance name:
5-amino-o-cresol
EC Number:
220-618-6
EC Name:
5-amino-o-cresol
Cas Number:
2835-95-2
Molecular formula:
C7H9NO
IUPAC Name:
5-amino-2-methylphenol
Constituent 2
Reference substance name:
HAARPURPUR
IUPAC Name:
HAARPURPUR
Constituent 3
Reference substance name:
5-Amino-2-methyl-phenol
IUPAC Name:
5-Amino-2-methyl-phenol
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: 5-Amino-2-methyl-phenol (HAARPURPUR)
- TSIN # 23032
- Substance type: Pure active substance
- Physical state: Beige crystalline powder
- Stability under test conditions: The substance stored in dryness and darkness is considered to be stable for more than 5 years.
- Stability in solution: The stability of the test substance (5%, w/v) in acetone/water (1: 1, v/v) and DMSO was monitored over a time period of 2 hours using HPLC chromatography at the 274 nm. During the test procedure all stock solutions were stored at ambient temperature in the absence of light. The results in acetone/water 1:1 (99.5%) and DMSO (100.3%) point to an excellent stability under the conditions applied.
- Storage condition of test material: Room temperature, protected from light
- Solubility: Solubility of test substance in different solvents is as follows:
7.3 weight% in acetonitrile
0.6 weight% in water ~ 4.11 g/L (pH-value 7.3)
6 weight% in acetone/water 1:1
10 weight% in DMSO
6.4 weight% in ethanol

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CBA/Ca01aHsd mice were obtained from Harlan Winkelmann GmbH, D-33178 Borchen.
- Age at study initiation: 8-12 weeks
- Weight at study initiation: Between 16-21 g
- Housing: The animals were housed in Macrolon cages on Altromin saw fiber bedding under barrier maintained (semi-barrier) in air conditioned rooms.
- Diet: Altromin 1324 maintenance 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 air change/hour
- Photoperiod: 12 hours artificial light/ 12 hours dark

IN-LIFE DATES: From: Nov. 23, 2000 To: Nov. 29, 2000

Study design: in vivo (LLNA)

Vehicle:
other: Acetone/aqua (1: 1) i.e. AA mixed with olive oil (4: 1)
Concentration:
0, 0.5, 1.5, 3 and 5% test substance in aqua/acetone (1:1) mixed with olive oil at a ratio of 4:1
- The positive control was prepared at the concentration of 1% (w/v) in aqua/acetone (1:1) mixed with olive oil at a ratio of 4:1.
No. of animals per dose:
5 female animals/group
Details on study design:
RANGE FINDING TESTS: No range-finding study was performed. The test concentrations of the study were chosen by the Study Sponsor.

MAIN STUDYANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: A substance was considered a skin sensitizer if at least one concentration of the test substance resulted in a 3 fold or greater increase in 3H-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 control group animals. Thus, a stimulation index (SI) ≥ 3.0 was regarded as a positive response.

TREATMENT PREPARATION AND ADMINISTRATION
A) Treatment Preparation:
Test substance and positive control preparation: The test substance was prepared immediately prior to each dosing at the concentrations of 0, 0.5, 1.5, 3 and 5% in acetone/aqua (1: 1) mixed with olive oil (4: 1). The positive control was prepared as a 1% solution in acetone/aqua (1: 1) mixed with olive oil (4: 1).
B) Administration of the test preparations: Each mouse was treated by topical application of 25 µL of test substance, positive control or vehicle was applied to the entire dorsal surface of each ear. Topical applications were performed once daily over three consecutive days.
Animals were assigned to the following groups:
Group 1 (Vehicle Control): Animals received acetone/water (1: 1) mixed with olive oil (4: 1).
Group 2 (Positive control): Animals received p-phenylenediamine (PPD) at a concentration of 1% in acetone/water (1: 1) mixed with olive oil (4: 1).
Group 3 to Group 6 (Test substance in vehicle): Animals received test substance as a solution at a concentration of 0.5, 1.5, 3 and 5% respectively in acetone/water (1: 1) mixed with olive oil (4: 1).

OBSERVATIONS
- Clinical observation: Animals were observed prior to the first application and once a day thereafter in order to detect special clinical signs or reactions to treatment.
- Body weight: The animals were weighed prior to first application and at the end of the test period.

EVALUATION OF CELL PROLIFERATION: Five days after the first topical application treatment all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250 µL of 3H-methyl thymidine, diluted to a working concentration of 80 µCi/mL.
Approximately 5 hours after 3H-methyl thymidine-injection all mice were sacrificed. The draining auricular lymph nodes were excised, pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). 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 approximately 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.
The 3H-methyl thymidine - incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.
Positive control substance(s):
other: p-Phenylenediamine

Results and discussion

Positive control results:
-Mean DPM: 2072.9 ± 910.6
- Stimulation index: 31.2 (The SI is greater than 3 thus validated the experimental conditions of OECD 429)
- EC3 value for the positive control was not estimated (all stimulation indices were above 3).

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
- Mean stimulation indices for test substance at the concentrations of 0.5, 1.5, 3 and 5% were 3.2, 5.9, 5.3 and 9.4 respectively. - As all stimulation indices were found to be over 3, no EC3 value could be calculated. As requested by the sponsor a surrogate EC3 (sEC3) was calculated by extrapolation to an SI of 3 using the two lowest concentrations. The EC3 estimated by extrapolation was 0.44%.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: - Mean DPM: 217.42 ± 31.6, 400.54 ± 202.54, 359.87 ± 107.38, 631.06 ± 275.36 at 0.5, 1.5, 3 and 5% test substance concentration respectively. - Vehicle control mean DPM: 73.8 ± 24.8 Individual animal results are provided in the study report.
Key result
Parameter:
SI
Value:
3.2
Test group / Remarks:
Test item at 0.5%
Key result
Parameter:
SI
Value:
5.9
Test group / Remarks:
Test item at 1.5%
Key result
Parameter:
SI
Value:
5.3
Test group / Remarks:
Test item at 3%
Key result
Parameter:
SI
Value:
9.4
Test group / Remarks:
Test item at 5.0%

Any other information on results incl. tables

Body Weights: No treatment-related effects were observed in body weight or body weight gains in all groups during the study.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information strong Criteria used for interpretation of results: expert judgment
Conclusions:
In LLNA, 5-Amino-2-methyl-phenol produced a biologically relevant immune response in local lymph nodes when tested at the concentration of 0.5, 1.5, 3 and 5% in aqua/acetone (1:1) with olive oil (4:1). As an EC3 of 0.44% was calculated by linear extrapolation, 5-Amino-2-methyl-phenol is evaluated to be a strong skin sensitizer.
Executive summary:

The study was performed to assess the skin sensitization potential of 5-Amino-2-methyl-phenol by following the OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay).

A total of 30 Female CBA/Ca01aHsd mice of 8-12 weeks, weighing between 16 to 21 g (source: Harlan Winkelmann GmbH, D-33178 Borchen) were used in the study. The animals were housed in Macrolon cages on Altromin saw fiber bedding under barrier maintained (semi-barrier) in air conditioned rooms. Animals were maintained under standard laboratory conditions (temperature: 22 ± 3°C, relative humidity: 55 ± 10%; 10 air change/ hour and 12 hours light/12 hours dark cycle/day). The animals received Altromin 1324 maintenance diet for rats and mice totally pathogen free (TPF) and tap water ad libitum.

25 μL of 0 (vehicle only), 0.5, 1.5, 3 and 5 % of 5-Amino-2-methyl-phenol in a mixture of aqua/acetone (1:1) with olive oil (4:1) was applied to the surface of the ear to each of five female mice per group for three consecutive days. As a positive control, p-phenylenediamine (PPD) at 1% in acetone/aqua/olive oil was investigated in parallel under identical test conditions.

On Day 5, the mice received an intravenous injection of 250 μL solution containing 20 μCi of [H3]  methyl thymidine. Approximately five hours later, the mice were killed, and the draining auricular lymph nodes were removed and collected in PBS. After preparing a single cell suspension for each mouse, cells were precipitated by TCA, and the radioactivity was determined (incorporation of [H3] methyl thymidine in the pellets) by means of liquid scintillation counting as disintegration per minute (dpm).

Animals were observed prior to the first application and once a day thereafter in order to detect special clinical signs or reactions to treatment. The animals were weighed prior to first application and at the end of the test period. No treatment-related effects were observed in body weight or body weight gains in any group during the study.

Mean stimulation indices for test substance at the concentrations of 0.5, 1.5, 3 and 5% were 3.2, 5.9, 5.3 and 9.4 respectively. An EC3 was estimated by extrapolation to be 0.44%.

The sensitivity of the test system was demonstrated by the reaction of the positive control p-phenylenediamine (1%) which exhibited a stimulation index of 31.2.

Based on these findings, 5-Amino-2-methyl-phenolproduced a biologically relevant immune response in local lymph nodes after dermal application even at the lowest test concentration of 0.5%. As an EC3 of 0.44% was calculated by linear extrapolation,5-Amino-2-methyl-phenolisevaluated to be astrong skin sensitizerunder the described test conditions.

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