4-amino-3-nitrophenol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2003-12-12 to 2005-02-07

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:

4-amino-3-nitrophenol

EC Number:

210-236-8

EC Name:

4-amino-3-nitrophenol

Cas Number:

610-81-1

Molecular formula:

C6H6N2O3

IUPAC Name:

4-amino-3-nitrophenol

Test material form:

solid: particulate/powder

Remarks:

Dark red powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:0508916
- Expiration date of the lot/batch: September 2005
- Purity test date: 2004-08-31

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 stability (data not shown)
- Solubility and stability of the test substance in the solvent/vehicle: known stability (data not shown)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:The test item was ground to a fine powder, using a mortar and pestle
- Final dilution of a dissolved solid, stock liquid or gel: Dissolved at 25%, 10%, 5%, 2.5%, 1%, 0.5%, 0.25%, 0.1% and 0.05% in a mixture acetone/olive oil (4/1, v/v) (acetone: Merck, Chelles, France; olive oil: Sigma, St-Quenti-Fallavier, France).
The test item dosage forms were prepared extemporaneously undernitrogen atmosphere and were stored protected from light and under nitrogen gas before use. They were used within 4 hours following the preparation according to the known stability results.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, France
- Females (if applicable) nulliparous and non-pregnant: Yes
- Microbiological status of animals, when known: not specified
- Age at study initiation: 9 weeks old
- Weight at study initiation: 20.8±1.3g
- Housing:The animals were housed individually in disposable crystal polystyrene cages (22.00 cm x 8.50 cm x 8.00 cm). Each cage contained autoclaved sawdust (SICSA, Alfortville, France). Sawdust is analysed by the supplier for composition and contaminant levels.
- Diet (e.g. ad libitum): All animals had free access to A04 C pelleted diet (SAFE, France) and tap water (filtered using a 0.22 micron filter) contained in bottles. Each batch of food is analyzed by the supplier for composition and contaminant levels.
- Water (e.g. ad libitum): Bacteriological and chemical analyses of water, including the detection of possible contaminants (pesticides, heavy metals and nitrosamines) are performed regularly by external laboratories. The results of these analyses are archived at CIT. No contaminants are known to be present in the diet, drinking water or sawdust at levels which may be expected to interfere with or prejudice the outcome of the study.
- Acclimation period: at least 5 days before the beginning of the study.
- Indication of any skin lesions: Not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 30 to 70%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12 h/12 h

- IN-LIFE DATES: From: 6 March 2004 To: 15 March 2004 (First experiment)
From: 9 May 2004 To: 18 May 2004

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Remarks:

The vehicle used in the study was chosen based on the results of solubility assay

Concentration:

The concentrations chosen from the preliminary test were 25, 10, 5 and 2.5%
In the second experiment, concentrations ranged from 0.05 to 2.5%

No. of animals per dose:

4 animals per dose group

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: known stability (data not shown) according to the solubility assay CIT/Study No. 26965
- Irritation: not irritant
- Systemic toxicity: not specified
- Ear thickness measurements: done
- Erythema scores: not specified

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: animals were assigned to the treatment groups by hand procedure.
- Criteria used to consider a positive response: The study is considered valid if all the following criteria are met:
• the incorporation of 3H-TdR expressed as disintegration's/mn (dpm) in the vehicle control group should be at least 2 fold higher than that of the control blank,
• the SI for the positive controls should be higher than the threshold positive value of 3.
Stimulation Indices (SI) were calculated according to the following formula: SI = dpm of treated group / dpm of control group
The same calculation was made for the positive control group.
The test item was considered as a skin sensitizer when the SI for a dose group is ≥ 3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results.
Ear thickness measurements and recording of local reactions were performed in order to assess any possible irritant effect of the test item, as possible irritancy may be involved in false positive lymphoproliferative responses.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was prepared as a solution in the vehicle at the chosen concentrations.The test item dosage forms were prepared extemporaneously under nitrogen atmosphere and were stored protected from light and under nitrogen gas before use. They were used within the 4 hours following the preparation according to the known stability results.
Preliminary test:
To assess the irritant potential of the test item, a preliminary test was performed on a small number of animals as follows:
- the test item was prepared at the concentrations of 2.5, 5, 10 and 25%,
- for 3 consecutive days, the animals received applications of 25 µL 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.
Main test:
The test item was tested in 2 independent experiments.
In the 1st experiment, the concentrations of the test item were selected according to the criteria of the OECD GL and on the basis of the results of the solubility and preliminary assays: 1-2.2-5-10-25%
At the request of the sponsor, as positive results were obtained in the 1st exp., the 2nd exp. was performed as follows: 0.05-0.1-0.5-1-2.5%
On days 1, 2 and 3 of each experiment, a dose-volume of 25 μL 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.

CLINICAL EXAMINATIONS
Clinical signs, morbidity and mortality: the animals were observed at least once a day during the study for clinical signs, signs of morbidity or mortality.
Body weight: in each experiment, the animals were weighed individually on the first day of the study and on the day of the sacrifice (day 6).
Ear thickness measurements and recording of local reactions: ear thickness measurements and recording of local reactions were performed in order to assess any possible irritant effect of the test item, as possible irritancy may be involved in false positive lymphoproliferative responses.
On days 1, 2 and 3 (before application) as well as on day 6 (after sacrifice) of each experiment, the thickness of the left ear of each animal groups 1 to 6 was measured using a micrometer. No measurement of ear thickness was performed for the animals of the positive control group. Any irritation reaction (erythema and oedema) was recorded in parallel. Any observation (coloration, presence of residual test item, ...) was noted.
The irritation level of the test item was determined according to the following:
% increase in ear thickness between day 1 and day 3 or 6: <10%: level I, non irritant; 10-30%: level II, slightly irritant; >30%: level III, irritant

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 Dearman (1991). On day 6 of each experiment, all animals of all groups received a single intravenous injection of 250 µL of 0.9% NaCl containing 20 µCi of 3H-TdR (specific activity of 25 Ci/mmol) via the tail vein. Approx. 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.

Preparation of auricular lymph node cell suspensions and determination of proliferation:
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. Cell suspensions were washed with 15 mL of 0.9% NaCl and pellets obtained were re-suspended in 0.9% NaCl for numeration of lymphocytes (cellularity) and determination of their viability by exclusion of Trypan blue. Each cell suspension was then centrifuged and pellets were precipitated with 3 mL of 5% (w/v) trichloroacetic acid in purified water at +4°C overnight. After a last centrifugation, the pellets were precipitated with 1 mL of 5% TCA. 3 mL of Ultima Gold scintillation fluid were added in order to measure incorporation of 3H-TdR using beta-scintillation counting.

Positive control substance(s):

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

Statistics:

Statistical approach not specified.

Results and discussion

Positive control results:

1st exp: at 25% HCA, no signs of local irritation, Stimulation Index=9.72.
2nd exp: at 25% HCA, no signs of local irritation, Stimulation Index=8.44.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Remarks:

second experiment

Value:

0.2

Results 2

Key result

Parameter:

SI

Remarks:

second experiment

Value:

>= 1.65 - <= 27.67

Test group / Remarks:

From 0.05% to 2.5%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
viability = viable cells/viable cells + dead cells
cellularity index = amount of cells (x10E6 cells) in the treated group / amount of cells (x10E6 cells) in the vehicle group
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.

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation index = dpm of treated group / dpm of control group.
In the 1st exp., positive lymphoproliferative responses were noted at all tested concentrations. The SI values increased in a dose-dependent manner in the treated groups given the test item concentrations comprised between 1 and 10% and decreased thereafter at the highest concentration (without evidence of a cellular toxicity). In the absence of local irritation, these positive lymphoproliferative responses were attributed to delayed contact hypersensitivity.
In the second exp. (0.05-2.5%), a dose-related increase in the SI was noted and the threshold positive value of 3 exceeded at the conc. >=0.5%.

EC3 CALCULATION
EC3 value = theorical concentration resulting in a SI value of 3.
The EC3 value for the test item calculated on the basis of the results obtained in the second exp. is 0.2%

CLINICAL OBSERVATIONS:
In the first exp., 1/4 animals of the treated group 5 (10%) was found on day 6; no clinical signs were observed prior to death. In addition hypoactivity or sedation and piloerection were noted on day 6 in 1/4 animals of the treated groups 2 (1%) and 3 (2.5%). No mortality and no clinical signs were observed in the second exp.

BODY WEIGHTS
The body weight changes of the treated animals was similar to that of controls.

LOCAL IRRITATION
In both exp., no cutaneous reactions and no noteworthy increase in ear thickness were observed. An orange coloration of the skin of the ears was noted in all animals given the test item at the conc. >=1%.

Any other information on results incl. tables

B051- Skin sensitization

First experiment :

Groups	Treatment andconc.	Cell count		Viability %	Amount of cells (10^6cells)	Cellularity index	Nb of nodes per group	Dpm per group	Dpm per node	SI	Increase ear thickness	EC3	Irritation class
		viable	dead
1	AOO, 0	51	6	89.47	5.10	/	8	392.31	49.04	/	-2.91	/	/
2	B051 1%	144	25	85.21	14.40	2.82	8	3021.08	377.64	7.7	-3.03	NA	I
3	B051 2.5%	156	40	79.59	15.60	3.06	8	8190.27	1023.78	20.88	-6.80
4	B051 5%	314	50	86.26	31.40	6.16	8	13052.30	1631.54	33.27	0.00
5	B051 10%	262	37	87.63	26.20	6.85	8	11297.01	1882.84	38.93	0.00
6	B051 25%	168	49	77.42	16.80	3.29	8	13721.20	1715.15	34.98	0.00
7	HCA 25%	244	40	85.92	24.40	4.78	8	3814.84	476.86	9.72	/	/	/

 

Second experiment :

Groups	Treatment and conc.	Cell count		Viability %	Amount of cells (10^6cells)	Cellularity index	Nb of nodes per group	Dpm per group	Dpm per node	SI	Increase ear thickness	EC3	Irritation class
		viable	dead
1	AOO, 0	49	7	87.5	5.10	/	4	197.47	49.37	/	6.12	/	/
2	B051 0.05%	64	16	80.0	14.40	0.87	6	488.88	81.48	1.65	4.23	0.2	I
3	B051 0.1%	102	28	78.46	15.60	1.04	8	719.79	89.97	1.82	7.29
4	B051 0.5%	239	8	96.76	31.40	2.44	8	2727.62	340.95	6.91	1.02
5	B051 1%	298	11	96.44	26.20	3.04	8	3509.19	438.65	8.88	8.25
6	B051 2.5%	279	72	79.49	16.80	2.85	8	10927.94	1365.99	27.65	0.00
7	HCA 25%	201	50	80.16	24.40	4.12	8	3331.59	416.45	8.44	/	/	/

 

NA : not applicable

AOO : acetone/oliveoil

Dpm : disintegrations per minute

Viability : viable cells/ (viable cells+dead cells)

Cellularity index : amount of cells (x10⁶cells) in the treated group / amount of cells (x10⁶cells) in the vehicle group

Stimulation index: dpm of treated group /dpm of control group

EC3 value: theoretical concentration resulting in a SI value of 3

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Under the experimental conditions, the test item induces delayed contact hypersensitivity in the murine Local Lymph Node Assay. According to the EC3 value obtained, the test item should be considered as a extreme sensitizer.

Executive summary:

This GLP-compliant study was performed to evaluate the potential sensitization of the test item according to OECD Guideline 429 method (adopted the 24th April 2004) for Local Lymph Node Assay. This assessment was made through two independent experiments using 28 animals each: 5 treated groups received the test item at the chosen concentration, one negative control group received the vehicle (a mixture of acetone/olive oil (4/1, v/v)), one positive control received the reference item (HCA) at the concentration of 25% (v/v) in AOO. In the first exp. The test item was tested at 1, 2.5, 5, 10 and 25%. As positive results were obtained, the test item was tested in a second exp. at the concentrations of 0.05, 0.1, 0.5, 1 and 2.5%. In each experiment, the substances were applied for 3 consecutive days. After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine. The obtained values were used to calculate the 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. n the 1st exp., positive lymphoproliferative responses were noted at all tested concentrations. The SI values increased in a dose-dependent manner in the treated groups given the test item concentrations comprised between 1 and 10% and decreased thereafter at the highest concentration (without evidence of a cellular toxicity). In the absence of local irritation, these positive lymphoproliferative responses were attributed to delayed contact hypersensitivity. In the second exp. (0.05-2.5%), a dose-related increase in the SI was noted and the threshold positive value of 3 exceeded at the conc. >=0.5%. The EC3 value for the test item calculated on the basis of the results obtained in the second exp. is 0.2%. Under the experimental conditions, the test item induces delayed contact hypersensitivity in the murine Local Lymph Node Assay. According to the EC3 value obtained, the test item should be considered as a extreme sensitizer.