3-aminophenol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 1 April 2004 to 18 May 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted : 24th April 2002

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

3-aminophenol

EC Number:

209-711-2

EC Name:

3-aminophenol

Cas Number:

591-27-5

Molecular formula:

C6H7NO

IUPAC Name:

3-aminophenol

Constituent 2

Reference substance name:

m-Aminophenol

IUPAC Name:

m-Aminophenol

Specific details on test material used for the study:

- Name of test material (as cited in study report): m-aminophenol
- Molecular formula (if other than submission substance): C6H7NO
- Molecular weight (if other than submission substance): 109.13
- Substance type: Organic
- Physical state: Solid
- storage conditions: at +4°C, protected from light and under nitrogen gas
- batch number: 220117
- titre: 99%
- expiry date: September 2005.
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: batch number: 220117
- Expiration date of the lot/batch: September 2005.
- Purity test date: 31 August 2004

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at +4°C, protected from light and under nitrogen gas
- Stability under test conditions: known but not reported on this study (According to results of CIT/Study No. 26941)
- Solubility and stability of the test substance in the solvent/vehicle: known but not reported on this study (According to results of CIT/Study No. 26941)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was prepared as a solution in the vehicle at the chosen concentration. The test item dosage forms were prepared extemporaneously under nitrogen atmosphere and were stored protected from light and under nitrogen gas before use. The were used within the 4 hours following the preparation according to the known stability (results of CIT/Study No. 26941).

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: on the first day of treatment, the animals were approximately 9 weeks old
- Weight at study initiation: had a mean body weight ± standard deviation of 21.4 ± 1.2 g.
- Housing: The animals were housed individually in disposable crystal polystyrene cages
- Diet (e.g. ad libitum): All animals had access to A04 C pelleted diet (SAFE, Villemoisson, Epinay-sur-Orge, France)
- Water (e.g. ad libitum): tap water (filtered using a 0.22 micron filter) contained in bottles.
- Acclimation period: at least 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 30 to 70% relative humidity
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12 h/ 12 h. The temperature and relative humidity were under continuous control and recording. The records were checked daily and filed. In addition to these daily checks, the housing conditions and corresponding instrumentation and equipment were verified and calibrated at regular intervals.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Preliminary test : 0, 2.5, 5, 10, 25 or 50%
Main test : 0, 0.05, 0.1, 0.5, 1, 2.5 or 25 %

No. of animals per dose:

4 females for the preliminary test, 56 females for the main test : 4 females (in total 28 animals are used in the first LLNA experiment and 28 in the second LLNA experiment)

Details on study design:

Preliminary irritation test was first performed in order to define the concentrations of test material to be used in the Main test.

The test material was tested in two independent experiments, both on twenty-eight female CBA/J mice allocated to seven groups of four animals each:
five treated groups receiving the test material m-Aminophenol (A015) at the chosen concentrations, one negative control group receiving the vehicle (dimethylformamide = DMF), one positive control group receiving the reference material, a-hexylcinnamaldehyde (HCA), at the concentration of 25% (v/v).

For the first experiment, the highest tested concentration (25% in DMF) was selected, according to the criteria described in the OECD guideline and on the basis of the solubility (C/T/Study No. 26941 AHS) and preliminary assays. For the second experiment, the tested concentrations were selected in agreement with the Sponsor on the basis of the results of the first experiment.

In the first experiment, the test material was tested at the concentrations of 1, 2.5, 5, 10, 25%. As positive results were obtained in the first experiment and in order to determine more precisely the EC3 value, the test material was then tested in the second experiment at the concentrations of 0.05, 0.1, 0.5, 1 2.5%.

In each experiment, the test material, vehicle or reference material was applied over the ears (25 mL per ear) for three consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of the lymph node cells in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate stimulation indices (SI).

The irritant potential of the test item was assessed in parallel by measurement of ear thickness
on days 1, 2, 3 and 6.


TREATMENT PREPARATION AND ADMINISTRATION:

Preliminary test
To assess the irritant potential of the test material (through ear thickness measurement), a preliminary test was performed on a small number of animals, as follows:
the test item was prepared at the concentrations of 50, 25, 10 and 5%, for 3 consecutive days, the animals received applications of 25 mL of the dosage form preparations to the external surface of both ears (one concentration per ear), measurement of the ear thickness (using a micrometer) was performed each day before treatment and 24 hours after the last application.

Administration of the dosage forms
On day 1, 2 and 3 of each experiment, a dose-volume of 25 mL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip. In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration. No massage was performed but the tip was used to spread the preparation over the application site. No rinsing was performed between each application.

PROLIFERATION ASSAY

Intravenous injection of 3H-TdR and sampling of auricular lymph nodes
Lymph node cell proliferative responses were measured as described by Kimber and boatman (1991). On day 6 of each experiment, all animals of all groups received a single intravenous injection of 250 mL of 0.9% NaCl containing 20 mCi of 3H-TdR (specific activity of 25 Ci/mmol), via the tail vein. Approximately 5 hours later, the animals were killed by cervical dislocation and the auricular lymph nodes were excised. The lymph nodes were pooled for each experimental group. A single cell suspension of auricular lymph node cells (ALNC) was prepared by mechanical dissagregation in Petri dishes with the plunger of a syringe.

Clinical examinations were performed for clinical signs, mortality and morbidity. Body weight and Ear thickness and recording of local reaction measurements were perform too. The irritation level of the test item was determined according the following table ;

% increase in ear thickness between day 1 and day 3 or 6 Irritation level Interpretation
<10% I Non irritant
10-30% II Slighty irritant
>30% III Irritant

Auricular lymph node cell suspensions were sampled after Tritiated thymidine injection (after 5 hours later, mice were sacrified). Viability was measured by tryptan blue approach. After suspension of these cells, they were centrifugated and pellet were precipitated with 1 mL of 5% TCA. Three mL of scintillation fluid were added in order to measure incorporation of tritiated thymidine using beta-scintillation counting.
The results were expressed as disintigration per min (dpm) per group per node.
Stimulation Index were calculated according the following formula : SI = dpm of treated group / dpm of control group

Viability was calculated as follows : viability = viable cells / viable cells + dead cells
cellular index = amount of cells (x10E6 cells) in treated group/ amount of cells (x10E6 cells) in control group

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:

Postive control 25% HCA induced increases of stimulation index (4.11 in first experoment and 5.77 in the second experiment)

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

0.24

Results 2

Key result

Parameter:

SI

Value:

1.41

Test group / Remarks:

Concentration of the test item at 0.1%

Results 3

Key result

Parameter:

SI

Value:

5.88

Test group / Remarks:

Concentration of the test item at 0.5%

Results 4

Key result

Parameter:

SI

Value:

9

Test group / Remarks:

Concentration of the test item at 1%

Results 5

Key result

Parameter:

SI

Value:

11.01

Test group / Remarks:

Test item concentration at 2.5%

Cellular proliferation data / Observations:

In both experiments, the incorporation of 3H-TdR in the vehicle control group was as specified in acceptance criteria. The quantity of cells obtained in each group was satisfactory, the cellularity was correlated with incorporation of 3H-TdR, the cell viability in each group was higher than 70% and the threshold positive value of 3 for the SI was exceeded in the positive control group. The study was therefore considered valid. In the first experiment, positive lymphoproliferative responses were noted at all tested concentrations but without clear evidence of a dose-response relationship. The SI value was maximum at the concentration of 2.5% (SI = 12.57) and decreased in a dose-dependent manner. In the absence of local irritation, these positive lymphoproliferative responses were attributed to delayed contact hypersensitivity. In the second experiment, a dose-related increase in the SI was noted and the threshold positive value of 3 was exceeded at the concentrations 0.5%. SI for first Experiment: m-aminophenol at concentration 1, 2.5, 5, 10 and 25 % are 7.62, 12.57, 10.38, 7.19 and 6.00, respectively HCA : at 25 % concetration SI is 4.11 These SI shows no local irritation In the second experiment, a dose-related increase in the SI was noted and the threshold positive value of 3 was exceeded at the concentrations For the test item; the SI at concentration 0.05, 0.1, 0.5, 1 and 2.5% is found to be 1.04, 1.41, 5.88, 9.00 and 11.01 respectively. For HCA, SI value was determined at 5.77 at concentration 25% (v/v). The EC3 value for the test item m-Aminophenol (A015) calculated on the basis of the results obtained in the second experiment is equal to 0.24%.

EC3 was calculated as follows : EC3 = c + [(3-d)/(b-d)]x(a-c) where a = the lowest concentration giving stimulation>3 ; b=tbe actual stimulation indew caused by a; c= the highest concentration failing to produce a stimulation index of 3; and d = the actual stimulation index caused by c concentration.
Stimulation Index were calculated according the following formula : SI = dpm of treated group / dpm of control group

Viability was calculated as follows : viability = viable cells / viable cells + dead cells
cellular index = amount of cells (x10E6 cells) in treated group/ amount of cells (x10E6 cells) in control group

Any other information on results incl. tables

Table 1 :Study Results

 

Treatment	Concentration (%)	Signs of local irritation	Stimulation Index (SI)
m-Aminophenol (A015)	1	no	7.62
m-Aminophenol (A015)	2.5	no	12.57
m-Aminophenol (A015)	5	no	10.38
m-Aminophenol (A015)	10	no	7.19
m-Aminophenol (A015)	25	no	6.00
HCA	25	no	4.11
Treatment	Concentration (%)	Signs of local irritation	Stimulation Index (SI)
m-Aminophenol (A015)	0.05	no	1.04
m-Aminophenol (A015)	0.1	no	1.41
m-Aminophenol (A015)	0.5	no	5.88
m-Aminophenol (A015)	1	no	9.00
m-Aminophenol (A015)	2.5	no	11.01
HCA	25	no	5.77

 

Applicant's summary and conclusion

Interpretation of results:

other: Skin sensitizing

Conclusions:

Under the experimental conditions, the test material m-aminophenol (A0l5) (batch No. 220117) induces delayed contact hypersensitivity in the murine Local Lymph Node Assay , indicating on being a strong sentiziser when exposed to mice. According to GHS and CLP classification critera, the test item m-aminphenol was defined as strong sensitizer.

Executive summary:

The skin sensitization study was performed to evaluate the potential of the test material m-Aminophenol (A015) (batch No. 220117) in mice to induce delayed contact hypersensitivity using the marine Local Lymph Node Assay (LLNA) according to OECD guideline method 429 (Adopted : 24th April 2002).This study was conducted in female CBA/J mice to investigate the sensitisation potential of m-aminophenol. Concentrations up to 25% (the maximum soluble concentration) in dimethylformamide were tested in two successive experiments. A positive control group received 25% alpha-hexylcinnamaldehyde in dimethylformamide.  No mortality, clinical signs or body weight changes could be attributed to compound administration. An increase in stimulation index was observed with increasing dose and the threshold positive value of 3 was exceeded at concentrations of 0.5% and higher. The EC3 value (0.24%) indicated a strong sensitisation potential for m-aminophenol. Under the experimental conditions, the test material m-aminophenol (A0l5) (batch No. 220117) induced delayed contact hypersensitivity in the murine Local Lymph Node Assay. According to the EC3 value obtained in this experiment, the test item m-Aminophenol (A0l5) can be considered as a strong sensitizer.