3-aminophenol

Endpoint summary

Description of key information

The skin sensitization potential of target chemical 3-aminophenol (CAS No: 591-27-5) was assessed in various experimental studies which were conducted on guinea pigs and humans. Based on the available key data and supporting studies, it can be concluded that chemical 3-aminophenol (CAS No: 591-27-5) is able to cause skin sensitization and considered as sensitizing. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Skin Sensitizer”.

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 1 April 2004 to 18 May 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

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).

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.

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)

Positive control results:

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

Key result

Parameter:

EC3

Value:

0.24

Key result

Parameter:

SI

Value:

1.41

Test group / Remarks:

Concentration of the test item at 0.1%

Key result

Parameter:

SI

Value:

5.88

Test group / Remarks:

Concentration of the test item at 0.5%

Key result

Parameter:

SI

Value:

9

Test group / Remarks:

Concentration of the test item at 1%

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

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

 

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Various studies have been investigated for the test chemical3-aminophenol (CAS No: 591-27-5)to observe the potential for skin sensitization to a greater or lesser extent. The studies are based LLNA studies in mice and in non-LLNA experiments in rabbits, guinea pigs and humans target chemical material m-Aminophenol /3-aminophenol (CAS No: 591-27-5) which have been summarized as below;

 

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.

 

The skin sensitizing potential of the test chemical 3-aminophenol (CAS No: 591-27-5) was assessed by using the Mouse Local Lymph node Assay. Groups of mice (n = 4) received 25 µl of one of three concentrations of the test chemical on the dorsum of both ears daily for three consecutive days. Control mice received an equal volume of vehicle alone. Three to 6 days after the first topical application, all mice were injected intravenously via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 20 µCi of [3H]methylthymidine (3HTdR: specific activity of 2.0 Ci/mmol; Amersham International, Amersham, U.K.). Five hours later, the mice were killed and the draining auricular lymph nodes excised and pooled for each experimental group. Single cell suspensions of lymph node cells (LNCs) were prepared by gentle mechanical disaggregation through stainless steel gauze (200 mesh size). The pooled LNCs were pelleted by centrifugation at approximately 190g for 10 min, washed twice with 10 ml of PBS, and resuspended in 3 ml of 5% trichloroacetic acid (TCA). After overnight incubation at 4 °C, the precipitates were recovered by Centrifugation, resuspended in 1 ml of TCA, and transferred to 10 ml of scintillation fluid (Optiphase MP; LKB, Surrey, U.K.). 3HTdR incorporation was measured by β-scintillation counting. The proliferative response of LNCs was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node). The ratio of 3HTdR incorporation into LNCs of test lymph nodes relative to that recorded for control lymph nodes (T/C ratio) was calculated for each test group. A test chemical was regarded as positive in the local lymph node assay if the following criteria were fulfilled: First, exposure to at least one concentration of the test chemical resulted in an incorporation of 3HTdR at least threefold greater than that recorded in the control mice. Second, the data were not incompatible with a conventional biological dose response, although on occasion allowance was made for local toxicity at the highest dose(s). If the test chemical that failed to fulfill these criteria were regarded as unlikely to be strong sensitizers. The resulted test: control (T:C) ratios were greater than 3 at each concentration. Hence the chemical 3-aminophenol (CAS No: 591-27-5) was considered to be sensitizing in Local Lymph Node Assay (LLNA).

 

The Guinea pig maximization study of test chemical 3-aminophenol (CAS No: 591-27-5) was conducted on guinea pigs to determine its skin sensitizing potential. The induction involved intradermal injection of 1% and topical application of 10% of test chemical in 4 :1 acetone:olive oil (AOO) for 24 and 48 hours. After a rest period, animals were challenged with 5% of test chemical in 4 :1 acetone:olive oil (AOO) and observed for skin reaction. The potential of a test chemical to cause skin sensitization was determined by visual assessment of the erythema present at the test site(s) 24 and 48 h after removal of topical challenge patches. The potential of a test chemical to cause skin sensitization was determined by visual assessment of the erythema present at the test site(s) 24 and 48 h after removal of topical challenge patches. Since the test chemical caused skin sensitization in all treated animals, the test chemical 3-aminophenol (CAS No: 591-27-5) was considered to be sensitizing on guinea pig skin.

 

Another Mouse Local Lymphnode Assay was conducted for 3-aminophenol (CAS No: 591-27-5) on groups of CBA mice (7-12 weeks of age) by mean of topical application of chemical on the dorsum of both ears at a dose of 25µl or to an equal volume of relevant vehicle (Acetic acid in olive oil (4:1))only. Treatment was performed daily for 3 consecutive days. Five days after initiation of exposure all mice were injected via the tail vein with 250µl of PBS containing 20µCi of tritiatied thymidine. The mice were sacrificed 5 hours later, and draining the auricular lymph nodes were excised and pooled for each experimental group or each individual animal. The incorporation of tritiated thymidine measured by β-scintillation counting and was reported in disintegrations /minute. An SI was calculated for each chemical group as the ratio of disintegrations/minute of the treated group to the disintegrations/minute of the concurrent vehicle control group. A substance was classified skin sensitizer, if at one or more than one concentrations, it induced a three-fold or greater increase in local lymph node proliferative activity when treated with the concurrent vehicle treated controls (SI ≥3) .The approach to estimation of the relative skin sensitization potential is based on the mathematical estimation of the concentration of chemical necessary to obtain a threshold positive response (SI = 3); this is termed as the EC3 value. The relative potency index of 3-aminophenol (CAS No: 591-27-5) was estimated to be 3.2. Based on the relative potency index 3-aminophenol (CAS No: 591-27-5) was considered as skin sensitizer.

   

The above results were supported by the skin sensitization test conducted to study the frequency of allergic reactions of test chemical in eczema populations. Two thousand nine hundred and thirty-nine consecutive eczema patients attending 12 dermatology clinics during a 6-month period (in 2007/2008) were patch tested with test chemical. The chemical was applied on the upper back of the patients with 8-mm Finn Chambers® (Epitest Ltd Oy, Tuusla, Finland) on Scanpor® tape (Alpharma AS, Norway), using 20mg of test material, in all centers. Readings were performed on D2, D3/4, and D7. Reactions were scored according to the scale of the International Contact Dermatitis Research Group. The 1% of tested patients had developed positive reaction. Hence the test material 3-aminophenol (CAS No: 591-27-5) was considered to be sensitizing to skin of human patients.

 

The above results were further supported by another sensitization study which was conducted by using an open epicutaneous method. Daily induction applications of the test materials for 3 weeks at 3% concentrations in a water-based vehicle to the shaved flanks of 20 guinea pigs were followed by a non-treatment period and a single challenge application to a separate site. The chemical did not induce any signs of contact sensitization, thus the chemical was considered to be not sensitizing to skin of treated rabbits.

 

Although negative skin reactions were observed in last study but on the basis of results obtained in key and majority of supporting studies, the chemical 3-aminophenol (CAS No: 591-27-5) can considered as potent skin sensitizer. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Skin Sensitizer”.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The skin sensitization potential of test substance 3-aminophenol (CAS No: 591-27-5) were observed in various studies. On the results obtained in key and supporting studies the test chemical3-aminophenol (CAS No: 591-27-5) was considered to be sensitizing on skin. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Skin Sensitizer”.