4-amino-m-cresol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Nov. 7, 2003 to April 13, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed OECD 429 guideline, GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Remarks:

according to OECD and US FDA principles of GLP

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

4-amino-m-cresol

EC Number:

220-621-2

EC Name:

4-amino-m-cresol

Cas Number:

2835-99-6

Molecular formula:

C7H9NO

IUPAC Name:

4-amino-m-cresol

Constituent 2

Reference substance name:

Oxyrot

IUPAC Name:

Oxyrot

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material: 4-amino-m-cresol ( Oxyrot)
- TSIN #23533
- Substance type: Pure active substance
- Physical state: Beige-brown crystalline powder
- Stability under test conditions: The substance on storage in dryness and darkness is considered to be stable more than 5 years.
- Storage condition of test material: At room temperature, kept away from light and humidity
- Solubility: Solubility of test substance in different solvents is as follows:
2 weight% in acetonitrile
0.6 weight% in water
4 weight% in acetone/water 1:1
> 10 weight% in DMSO

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: CBA/J mouse were obtained Centre d'Elevage Janvier, route des chenes secs, 53940 Le Genest Saint Isle, France.
- Age at study initiation: 10 weeks
- Weight at study initiation: 18 to 24 g (on Day - 2). The body weight at initiation of treatment was performed on Day -2 because of technical constraints.
- Housing: Animals were housed in groups of 5 of the same dose group in plastic cages (265 x 160 x 140 mm). Animals were group-housed for animal welfare reasons. Dust-free sawdust (Parisienne des Sciures) made from spruce tree wood, analysed at least twice a year for chemical and bacterial contaminants.
- Diet: Mouse pelleted complete diet, ad-libitum (Diet ref: A04-C10). Diet was sterilised by irradiation and analysed for chemical and bacterial contaminants.
- Water: Softened and filtered (0.2 µm) drinking water, ad-libitum (via bottles). Water was analysed at least once a year for chemical contaminants and at least twice a year for bacterial contaminants.
- Acclimation period: 8 days. All animals received a clinical inspection for ill-health on arrival.

- No known contaminants were present in bedding, diet or water at levels which might have interfered with achieving the objective of the study.


ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25 °C
- Humidity: >40 %
- Air changes: 8 air changes/hour
- Photoperiod: 12 hours artificial light / 12 hours dark

IN-LIFE DATES: From: Nov. 4, 2003 To: Nov. 17, 2003

Study design: in vivo (non-LLNA)

No. of animals per dose:

5 females

Study design: in vivo (LLNA)

Vehicle:

other: Vehicle 1:- Dimethyl sulfoxide (DMSO) , Vehicle 2: 3 Parts of aceotne/water (1:1) mixed with 1 part of olive oil (=3:1)

Concentration:

-0, 0.5, 1.5, 5.0 and 10.0% in vehicle 1 i.e. DMSO.
- 0, 0.5, 1.5, 3.0 and 5.0% in vehicle 2 i.e. 3 parts of acetone/water (1:1) mixed with 1 part of Olive oil.
- The positive control was prepared at the concentration of 1% (w/v) in DMSO.

No. of animals per dose:

5 female animals/dose 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 based on previous results with structurally related substances in vehicle 1, and due to maximum solubility in vehicle 2.

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 in 3-H-methyl thymidine-incorporation relative to the vehicle. Thus, a stimulation index ≥ 3.0 was regarded as a positive response. The EC3 value (concentration inducing a stimulation index of 3) was derived by linear interpolation between two points on the SI (Stimulation index) axis, one immediately above and one immediately below the SI value of 3.

TREATMENT PREPARATION AND ADMINISTRATION

A) Treatment Preparation: Test substance and positive control preparation: The test item was prepared as a solution in DMSO at the following concentrations: 0.5, 1.5, 5 & 10% and in acetone/water (1:1); olive oil was added to obtain the following conc. 0.5, 1.5, 3 and 5%. (NaOH 2M was added to 5% conc. to obtain a homogenous suspension. HCl 1 N was then added to lower the pH. The final pH was approximately 11).The positive control was prepared as a 1% (w/v) solution in DMSO. The formulations were used within 5 hours of preparation.

B) Administration of the test preparations: 25 µL of test substance, positive control or vehicle was applied to the dorsal surface of each ear of each mouse on Days 0, 1 and 2. A hair dryer was used for about 1 minute to dry ears.
Animals were assigned to the following groups:
Group 1 (Vehicle Control 1): Animals received dimethyl sulfoxide (DMSO).
Group 2 (Vehicle Control 2): Animals received acetone/water (1: 1) mixed with olive oil (3: 1).
Group 3 (Positive control): Animals received p-phenylenediamine (PPD) at a concentration of 1% in DMSO.
Group 4 to Group 7 (Test substance in vehicle 1): Animals received test substance as a solution at a concentration of 0.5, 1.5, 5 and 10% respectively in DMSO.
Group 8 to Group 11 (Test substance in vehicle 2): 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 (3: 1)

OBSERVATIONS
- Morbidity/mortality: All animals were observed at least once daily for morbidity or mortality.
- Clinical signs: Animals were observed daily. On treatment days, animals were examined before and at least once after dosing to detect any clinical signs or reaction to treatment (especially at the treatment sites).
- Body weight: All animals were weighed on Days -2 and 5.

- Evaluation of cell proliferation: On Day 5, 250 µL of phosphate buffered saline (PBS) containing 23.6 µCi of [3H] methyl thymidine was injected intravenously (using Harvard type P44 infusion pumps). Approximately 5 hours later the mice were sacrificed by carbon dioxide inhalation and the draining auricular lymph node taken and weighed. A single cell suspension was prepared for each animal Cells were washed twice with PBS and precipitated with ice cold 5% trichloro-acetic acid (TCA). Approximately 18 hours later the pellets were resuspended in 1 mL of TCA and transferred into the scintillation cocktail 3H-methyl thymidine incorporation was measured by liquid scintillation counting in a TRI-CARB 2700TR counter (Packard).

Positive control substance(s):

other: p-phenylenediamine (PPD) Batch:100K3462, Purity: 98%, CAS 106-50-3

Results and discussion

Positive control results:

-Mean DPM: 3911 ± 1207
- Stimulation index: 4.3 (The SI is greater than 3 thus validated the experimental conditions of OECD 429)
- EC3 value for the positive control was not estimated (one single value).
- Mean lymph node weight was 8.4 ± 1.2 and the ratio of mean lymph nodes weight (positive control)/mean lymph nodes weight (vehicle 1) was 1.3 at the concentrations of 1%, indicating an immune response.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

- Mean DPM (test substance in vehicle 1): 849 ± 362, 2767 ± 1498, 5884 ± 2070, 6027 ± 2459 at 0.5, 1.5, 5 and 10% test substance concentration respectively. - Mean DPM (test substance in vehicle 2): 556 ± 296, 609 ± 108, 1695 ± 535, 2509 ± 1185 at 0.5, 1.5, 3 and 5% test substance concentration respectively. - Vehicle control mean DPM: 901 ± 486 (vehicle 1) and 363 ± 80 (vehicle 2). - Individual animal results are provided in the study report.

Results 2

Key result

Parameter:

EC3

Value:

1.45

Test group / Remarks:

Test item in DMSO

Results 3

Key result

Parameter:

EC3

Value:

2.45

Test group / Remarks:

Test item in AOO

Results 4

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

0.5% in DMSO

Results 5

Key result

Parameter:

SI

Value:

3.1

Test group / Remarks:

1.5% in DMSO

Results 6

Key result

Parameter:

SI

Value:

6.5

Test group / Remarks:

5% in DMSO

Results 7

Key result

Parameter:

SI

Value:

6.7

Test group / Remarks:

10% in DMSO

Results 8

Key result

Parameter:

SI

Value:

1.5

Test group / Remarks:

0.5% in AOO

Results 9

Key result

Parameter:

SI

Value:

1.7

Test group / Remarks:

1.5% in AOO

Results 10

Key result

Parameter:

SI

Value:

4.7

Test group / Remarks:

3% in AOO

Results 11

Key result

Parameter:

SI

Value:

6.9

Test group / Remarks:

5% in AOO

Results 12

Parameter:

SI

Value:

4.3

Test group / Remarks:

PPD - Positive Control

Any other information on results incl. tables

a) Evaluation of Lymph Nodes Weight

- In animals given the test substance in DMSO, the ratio of mean lymph nodes weight (test substance)/mean lymph nodes weight (vehicle 1) was 0.9, 1.1, 1.6 and 1.2 at the concentrations of 0.5, 1.5, 5.0 and 10.0% respectively. Lymph node weights of the test substance group at the concentration of 5% were therefore considered to indicate an immune response comparable to the positive control.

- In animals given the test substance in in 3 parts of acetone/water (1:1) mixed with 1 part of olive, the ratio of mean lymph nodes weight (test substance)/meanlymph nodes weight (vehicle 2) was 2.0, 1.5, 1.7 and 1.6 at the concentrations of 0.5, 1.5, 3.0 and 5.0% respectively. Lymph node weights of the test substance groups at the four concentrations were therefore considered to indicate an immune response comparable to the positive control.

-Mean lymph node weight for vehicle 1 was 6.3 ±1.6 and for vehicle 2 was 3.5 ± 0.4.

b) Mortality:No mortality was observed during study period.

c) Clinical Signs:There were no clinical signs in the treated animals during the study.

d) 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:

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

Conclusions:

4-amino-m-cresol induced a biologically relevant immune response in local lymph nodes after dermal application to the mouse ear with either vehicle (DMSO or aqua/acetone/olive oil ) used. EC3 values of 1.45 % and 2.15 % were calculated for DMSO and aqua/acetone/olive oil, respectively.

Based on these findings 4-amino-m-cresol is evaluated to be a skin-sensitiser under the described test conditions. 4-amino-m-cresol would be categorised as a strong sensitizer Category 1A according to CLP criterias.

Executive summary:

The study was performed to assess the skin sensitization potential of 4-amino-m-cresol by following the OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay).

A total of 55 Female CBA/J mice of 10 week age, weighing 18 to 24 g (source: Centre d'Elevage Janvier, route des chenes secs, 53940 Le Genest Saint Isle, France) were used in the study. Animals were housed in groups of 5 of the same dose group in plastic cages (265 x 160 x 140 mm) and dust-free sawdust bedding made from spruce tree wood was used. Animals were maintained under standard laboratory conditions (temperature: 19 to 25 °C, relative humidity: >40 %; 8 air change/ hour and 12 hours light/12 hours dark cycle/day). Prior to the treatment, animals were acclimatized under laboratory conditions for a minimum period of 8 days. The animals received mouse pelleted complete diet, ad libitum (Diet reference A04-C10) and filtered (0.2 µm) drinking water ad libitum.

25 μL of 0 (vehicle only), 0.5, 1.5, 5 and 10 % of 4-amino-m-cresol in DMSO and 0.5, 1.5, 3 and 5 % in a mixture of aqua/acetone (1:1) with olive oil (3:1) (equal to the maximum solubility) were applied to the surface of the ear to each of five female CBA/J mice per group for three consecutive days. After application, the ears were dried by means of a hair dryer for 5 minutes. p-Phenylenediamine (PPD) at 1 % in DMSO was used as the positive control in parallel under identical test conditions. Although this is not the conventional material used as a positive control, it was regarded as the most adequate control for oxidative hair dye precursors.

Animals were checked for morbidity/mortality at least once daily. Observation for clinical signs was done daily before and at least once after dosing. Body weight was determined on Day -2 and on Day 5.

On Day 5, the mice received an intravenous injection of 250 μL phosphate buffered saline containing 23.6 μCi of [H3] methyl thymidine. Approximately 5 hours later, the mice were sacrificed by CO2-inhalation and the draining auricular lymph nodes were removed and weighed. 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).

There were no abnormal clinical signs or mortality throughout the study period. Body weight development was not affected by the treatment. Lymph node weights were increased after treatment with 4-amino-m-cresol as compared to the vehicle controls indicating an immune response in both vehicles. The mean stimulation indices were affected in a dose-dependent manner by the treatment with 4-amino-m-cresol. With the test substance in DMSO, mean stimulation indices of 0.9, 3.1, 6.5 and 6.7 were obtained for the 4 test concentrations of 0.5, 1.5, 5 and 10 %, respectively. An EC3 value (equal to the concentration inducing a stimulation index of 3) of 1.45 % was calculated. In the second vehicle (aqua/acetone/olive oil), the indices were 1.5, 1.7, 4.7 and 6.9 for the 4 test concentrations of 0.5, 1.5, 3 and 5 %, respectively. An EC3 value of 2.15 % was calculated from these findings.

The responses noted in both groups for 4-amino-m-cresol are considered positive with EC3 values of 1.45 % and 2.15% indicative of a moderate sensitiser.

The positive control (PPD, 1 % in DMSO) caused a stimulation index of 4.3 and an increase in lymph node weight by a factor of 1.3 which demonstrated the sensitivity of the test system used.

4-amino-m-cresol induced a biologically relevant immune response in local lymph nodes after dermal application to the mouse ear with either vehicle (DMSO or aqua/acetone/olive oil ) used. EC3 values of 1.45 % and 2.15 % were calculated for DMSO and aqua/acetone/olive oil, respectively. The concurrent positive control demonstrated the sensitivity of the assay.

Based on these findings 4-amino-m-cresol is evaluated to be a skin-sensitiser under the described test conditions. 4-amino-m-cresol would be categorised as a strong sensitizer Category 1A according to CLP criterias.