4-amino-3-nitrophenol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2004-07-26 to 2004-12-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

Draft Guideline June 14, 2004 (1st version)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

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: batch No. 0508916
- 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: stored under nitrogen at 1-10°C in the dark
- Stability under test conditions: Stability data were provided by the Sponsor indicating that formulations of test item at 0.1 to 500 mg/mL in DMSO were stable for up to 4 hours following formulation when stored at room temperature, away from light and under nitrogen atmosphere (CIT study reference number 26965 AHS).
- Solubility and stability of the test substance in the solvent/vehicle: Preliminary solubility data indicated that test item was soluble in sterile anhydrous analytical grade DMSO at a concentration of approximately 172.2 mg/mL. A 100-fold dilution of this DMSO solution into culture medium at a final concentration of approximately 1722 mg/mL did not result in visible precipitation. A top concentration of 1540 mg/mL (approximately equivalent to 10 mM, molecular weight of
4-Amino-3-Nitrophenol (B051) = 154.12, information provided by the Sponsor) was selected as a suitable maximum for this study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test article stock solutions were prepared by dissolving the test item in DMSO, with the aid of vortex mixing, to give the top concentrations.Stock formulations were purged with Nitrogen gas. The stock solutions were membrane filter-sterilised in Experiment 1 (Pall Acrodisc CR, pore size, 0.2 μm) and subsequent dilutions made using sterile DMSO. The test article solutions were protected from light and used within 4 hours of initial formulation.
- Final dilution of a dissolved solid, stock liquid or gel: top concentration of 1540 mg/mL of test item in DMSO was selected as a suitable maximum for this study.

Method

Species / strain

Species / strain / cell type:

lymphocytes: blood culture from two healthy, non smoking female volunteers (under the age of 35 years) primary culture

Details on mammalian cell type (if applicable):

CELLS AND MEDIA USED
Blood from two healthy, non-smoking female volunteers (under the age of 35 years) was used in this study. No donor was suspected of any virus infection nor had been exposed to high levels of radiation or hazardous chemicals. For each experiment, an appropriate volume of whole blood was drawn from the peripheral circulation within two days prior to culture initiation. Blood was stored refrigerated and pooled prior to use. Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL heparinised blood into 9.0 mL Hepes-buffered RPMI medium containing 20% (v/v) foetal calf serum and 50 μg/mL gentamycin. Phytohaemagglutinin (PHA, reagent grade) was included at a concentration of approximately 2% of culture volume to stimulate the lymphocytes to divide. Blood cultures were incubated at 37°C and rocked continuously.
For treatments conducted as part of Experiment 1 blood cultures were incubated in the presence of PHA for approximately 24 hours prior to treatment.
For treatments conducted as part of Experiment 2 blood cultures were incubated in the presence of PHA for approximately 48 hours prior to treatment.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Cytochalasin B (at a final concentration of 6 μg/ml)

Metabolic activation:

with and without

Metabolic activation system:

The S-9 used was prepared from a rat liver post-mitochondrial fraction from Aroclor 1254 induced animals

Test concentrations with justification for top dose:

The top dose for analysis was to be one at which at least 60% (approximately) reduction in RI (Replication Index) occurred or the highest dose tested. The following doses were selected for analysis:
Experiment 1
Absence of -S-9 assay : 84.66, 132.3, 165.4, 206.7 μg/mL (69% reduction of RI at the highest dose tested)
Presence+S-9 assay : 788.5, 985.6, 1232 μg/mL (62% reduction of RI at the highest dose tested)
Experiment 2 :
Absence of -S-9 assay : 250.0, 300.0, 450.0 μg/mL (59% reduction of RI at the highest dose tested)
Presence+S-9 assay : 985.6, 1232, 1540 μg/mL (60% reduction of RI at the highest dose tested)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide DMSO
Preliminary solubility data indicated that the test item was soluble in sterile anhydrous analytical grade dimethyl sulphoxide (DMSO), with the aid of vortex mixing, at a concentration of approximately 172.2 mg/mL.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive control substance:

cyclophosphamide

other: Vinblastine, 4-Nitroquinoline 1-oxide

Details on test system and experimental conditions:

TREATMENT
S-9 mix or KCl (0.5 mL) was added. One set of quadruplicate cultures (A, B, C and D) for each of the treatment regimes was then treated with the solvent and one set of duplicate cultures with the test article (0.1 mL per culture). Additional duplicate cultures for treatments in the absence of S-9 and in its presence, were treated with 0.1 mL of the positive control chemicals. Final post-treatment volume was 10 mL per culture.
Experiment 1 comprised a 20 hour treatment - S-9 and a 3 hour treatment +S-9. The test chemical was added 24 hours following culture initiation and cells were harvested at 72 hours. The final 27 hours of incubation (approximately) was in the presence of Cytochalasin B (at a final concentration of 6 µg/mL).
In Experiment 2, cells were treated at 48 hours following culture initiation and harvested at 96 hours. Again, treatment in the absence of S-9 was for 20 hours and in the presence of S-9 for 3 hours and the final 27 hours of incubation (approximately) was in the presence of Cytochalasin B.

HARVESTING AND SLIDE PREPARATION
At the defined sampling time, cultures were centrifuged at approximately 300 x 'g' for 10 minutes; the supernatant carefully removed and cells resuspended in 4 mL pre-warmed hypotonic (0.075 M) KCl and incubated at 37°C for 5 minutes to allow cell swelling to occur. Cells were then fixed by dropping the KCl suspension into an equal volume of fresh, ice-cold methanol/glacial acetic acid (3:1, v/v). The fixative was changed by centrifugation (approximately 300 x 'g', 10 minutes) and resuspension. This procedure was repeated several times (centrifuging at
approximately 1250 x 'g', 2-3 minutes) until the cell pellets were clean. Lymphocytes were kept in fixative in the refrigerator before slides were prepared but slides were not made on the day of harvest to ensure cells were adequately fixed. Cells were pelleted and resuspended in a minimal amount of fresh fixative (if required) to give a milky suspension. Several drops of suspension were transferred to clean microscope slides. After the slides had dried the cells were stained for 5 minutes in 4% (v/v) filtered Giemsa stain in Gurr's pH 6.8 buffer. The slides were rinsed, dried and mounted with coverslips.

CITOTOXICITY AND REPLICATION INDEX
Slides were examined, uncoded, for proportions of mononucleate, binucleate and multinucleate cells and the replication index (RI) calculated based on the analysis of approximately 500 cells per replicate (approximately 1000 per dose).
Replication Index (RI) indicates the relative number of nuclei in treated cultures compared to control cultures. Individual replicate calculations are performed using the formulae below:
RI = (number binucleate cells + 2 x number multinucleate cells) / Total number of cells
Cytotoxicity (Cyt) is 100–RI where RI is calculated using the formulae below:
Cyt = [(number binucleate cells + 2 x number multinucleate cells) / total number of cells in treated cultures] / [(number binucleate cells + 2 x number multinucleate cells) / total number of cells in control cultures]

ANALYSIS OF RESULTS
After completion of scoring and decoding of slides, the numbers of binucleate cells with micronuclei in each culture were obtained. The proportions of micronucleated cells in each replicate were used to establish acceptable homogeneity between replicates by means of a binomial dispersion test. The proportion of cells with micronuclei for each treatment condition were compared with the proportion in solvent controls by using Fisher's exact test. Probability values of p <=0.05 were accepted as significant.
Additionally, the number of micronuclei per binucleate cell were obtained and recorded. This data was also used as a potential additional tool for the interpretation of the study data.

Rationale for test conditions:

The assay was to be considered valid if the following criteria were met:
1) the binomial dispersion test demonstrated acceptable heterogeneity between replicate cultures, particularly where no positive responses are seen, and
2) the frequency of cells with micronuclei in solvent controls fell within the laboratories historical negative control (normal) range, and
3) the positive control chemicals induced statistically significant increases in the proportion of cells with micronuclei.
4) a minimum of 50% binucleate cells was achieved in negative control cultures at the time of harvest.

Evaluation criteria:

A test chemical is considered as clearly positive in this assay if:
1) a statistically significant increase in the proportion of cells with micronuclei occurs at one or more concentrations, and
2) the incidence of micronucleated cells at such data points exceeds the normal range.

Statistics:

The proportions of micronucleated cells in each replicate were used to establish acceptable homogeneity between replicates by means of a binomial dispersion test. The proportion of cells with micronuclei for each treatment condition were compared with the proportion in solvent controls by using Fisher's exact test. Probability values of p <=0.05 were accepted as significant.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

lymphocytes: First experiment

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

lymphocytes: Second experiment

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Experiment 1 (24 hour PHA stimulation prior to treatment):
Treatment of cells with 4-Amino-3-Nitrophenol (B051) in the absence and presence of metabolic activation (S-9) resulted in frequencies of micronucleated binucleate (MNBN) cells, which were similar to, and not significantly different from, those observed in concurrent vehicle control cultures for the majority of concentrations analysed. The one exception to this was observed following treatment in the absence of S-9 at a concentration of 132.3 mg/mL where a small but statistically significant increase (p <= 0.01) was noted. The MNBN cell frequency of both replicate cultures at this concentration exceeded the historical negative control (normal) range. However, this increase was small, and was not dose-related such that higher and lower concentrations of 4-Amino-3-Nitrophenol (B051) analysed analysed (covering the range 84.66 to 206.7 mg/mL) exhibited normal (within historical range) frequencies of MNBN cells. This increase was therefore considered of questionable biological importance. The MNBN cell frequency of all other 4-Amino-3-Nitrophenol (B051) treated cultures (both treatment regimes) fell within normal values.

Experiment 2 (48 hour PHA stimulation prior to treatment):
Treatment of cells with 4-Amino-3-Nitrophenol (B051) in the absence and presence of S-9 in Experiment 2 (following 48 hour mitogen (PHA) stimulation), resulted in frequencies of MNBN cells, which were significantly elevated compared to those in concurrent vehicle controls for the majority of concentrations analysed. For treatment in the presence of S-9 significantly elevated frequencies of MNBN cells were observed for all three concentrations analysed. The MNBN cell frequency of both replicate cultures at the highest two concentrations analysed (1232 and 1540 mg/mL) and a single culture at the lowest (985.6 mg/mL) exceeded the normal range. These results were therefore considered of biological importance. For treatment in the absence of S-9 statistically significant increases in MNBN cells were observed for the two highest concentrations tested (300 and 450 mg/mL). MNBN cell frequencies that exceeded the historical negative control (normal) range were observed in single cultures at each of the three concentrations analysed. However, the increases observed were small such that group mean MNBN cells frequencies for the highest and lowest concentrations (250 and 450 mg/mL) fell within historical negative control values. No dose-response was apparent and for each concentration, the MNBN cell frequency fell within normal values in single replicate cultures. It was therefore considered that the increases observed were spurious and of no biological importance.

Any other information on results incl. tables

Experiment 1 (24 hour PHA) – Result summary

 

Treatment	Concentration (μg/mL)	Cytotoxicity (%)	Mean MNBN cell frequency (%)	Statistical significance
20+28 hour -S-9	Vehiclea	-	1.13	-
	84.66	14	1.00	NS
	132.3	39	2.10	P<=0.01
	165.4	51	1.10	NS
	206.7	69	1.00	NS
	*Vinblastine, 0.08	ND	1.80	P<=0.05
	*NQO, 5.00	ND	7.85	P<=0.001
3+45 hour +S-9	Vehiclea	-	0.95	-
	788.5	18	0.95	NS
	985.6	38	0.35	NS
	1232	62	0.65	NS
	*CPA, 6.25	ND	7.05	P<=0.001

aVehicle control was DMSO only

* Positive control

NS = notsignificant

ND = notdetermined

Experiment 2 (48 hour PHA) – Result summary

 

Treatment	Concentration (μg/mL)	Cytotoxicity (%)	Mean MNBN cell frequency (%)	Statistical significance
20+28 hour -S-9	Vehiclea	-	0.55	-
	250	26	0.90	NS
	300	33	1.30	P<=0.01
	450	59	1.00	P<=0.05
	*Vinblastine, 0.06	ND	4.20	P<=0.001
	*NQO, 5.00	ND	3.95	P<=0.001
3+45 hour +S-9	Vehiclea	-	0.30	-
	985.6	22	1.10	P<=0.01
	1232	20	2.55	P<=0.001
	1540	60	2.10	P<=0.001
	*CPA, 6.25	ND	14.2	P<=0.001

 

aVehicle control was DMSO only

* Positive control

NS = not significant

ND = not determined

Applicant's summary and conclusion

Conclusions:

It is concluded that 4-Amino-3-Nitrophenol (B051) induced micronuclei in cultured human peripheral blood lymphocytes following 3 hour treatment in the presence of a rat liver metabolic activation system (S-9) [3+45 hour +S-9] where treatment commenced 48 hours following PHA (mitogen) stimulation. No such increases in micronucleated cells were apparent following treatment in the absence of S-9 (20+28 hour –S-9) where treatment commenced 48 hours after PHA stimulation, or in the presence of S-9 where treatment commenced 24 hours after PHA stimulation. An isolated increase in micronucleated cells was observed following 20+28 hour treatment in the absence of S-9 where treatment commenced 24 hours post mitogen stimulation. This increase was not observed at higher or lower concentrations analysed and was therefore, considered of questionable biological importance.

Executive summary:

This GLP-compliant study was performed to assess the potential of the test item to induce micronuclei in the cytoplasm of the cultured human lymphocytes according to the draft version of OECD guideline 487 method for In vitro mammalian cell micronucleus test.

Lymphocytes from blood of two healthy humain donors were used for each two experiments. According to CIT/Study 26965 AHS data, the test item was used dissolved in DMSO at the top dose concentration at 1540μg/ml. Selection of doses for micronucleus analysis was perform with RI and Cytotoxicity calculation for each experiments as follow :

RI = number binucleate cells + 2 x number multinucleate cells / Total number of cells

Cyt = [(number binucleate cells + 2 x number multinucleate cells) / total number of cells in treated cultures] / [(number binucleate cells + 2 x number multinucleate cells) / total number of cells in control cultures]

Doses selectionned for experiments were :

Experiment 1

Absence of -S-9 assay : 84.66, 132.3, 165.4, 206.7 μg/mL (69% reduction of RI at the highest dose tested)

Presence+S-9 assay : 788.5, 985.6, 1232 μg/mL (62% reduction of RI at the highest dose tested)

Experiment 2 :

Absence of -S-9 assay : 250.0, 300.0, 450.0 μg/mL (59% reduction of RI at the highest dose tested)

Presence+S-9 assay : 985.6, 1232, 1540 μg/mL (60% reduction of RI at the highest dose tested)

Experiment 1 comprised a 20 hour treatment - S-9 and a 3 hour treatment +S-9. The test chemical was added 24 hours following culture initiation and cells were harvested at 72 hours. The final 27 hours (approximately) of incubation was in the presence of Cytochalasin B (at a final concentration of 6 µg/mL).

In Experiment 2, cells were treated at 48 hours following culture initiation and harvested at 96 hours. Again, treatment in the absence of S-9 was for 20 hours and in the presence of S-9 for 3 hours and the final 27 hours (approximately) of incubation was in the presence of Cytochalasin B.

Observations of micronucleus were made after deposition of cells on microscope slides.

Results of Experiment 1 (24 hour PHA stimulation prior to treatment):

Treatment of cells with 4-Amino-3-Nitrophenol (B051) in the absence and presence of metabolic activation (S-9) resulted in frequencies of micronucleated binucleate (MNBN) cells, which were similar to, and not significantly different from, those observed in concurrent vehicle control cultures for the majority of concentrations analysed. The one exception to this was observed following treatment in the absence of S-9 at a concentration of 132.3 mg/mL where a small but statistically significant increase (p <= 0.01) was noted. The MNBN cell frequency of both replicate cultures at this concentration exceeded the historical negative control (normal) range. However, this increase was small, and was not dose-related such that higher and lower concentrations of 4-Amino-3-Nitrophenol (B051) analysed (covering the range 84.66 to 206.7 mg/mL) exhibited normal (within historical range) frequencies of MNBN cells. This increase was therefore considered of questionable biological importance. The MNBN cell frequency of all other 4-Amino-3-Nitrophenol (B051) treated cultures (both treatment regimes) fell within normal values.

Rseults of Experiment 2 (48 hour PHA stimulation prior to treatment):

Treatment of cells with 4-Amino-3-Nitrophenol (B051) in the absence and presence of S-9 in Experiment 2 (following 48 hour mitogen (PHA) stimulation), resulted in frequencies of MNBN cells, which were significantly elevated compared to those in concurrent vehicle controls for the majority of concentrations analysed. For treatment in the presence of S-9 significantly elevated frequencies of MNBN cells were observed for all three concentrations analysed. The MNBN cell frequency of both replicate cultures at the highest two concentrations analysed (1232 and 1540 mg/mL) and a single culture at the lowest (985.6 mg/mL) exceeded the normal range. These results were therefore considered of biological importance.

For treatment in the absence of S-9 statistically significant increases in MNBN cells were observed for the two highest concentrations tested (300 and 450 mg/mL). MNBN cell frequencies that exceeded the historical negative control (normal) range were observed in single cultures at each of the three concentrations analysed. However, the increases observed were small such that group mean MNBN cells frequencies for the highest and lowest concentrations (250 and 450 mg/mL) fell within historical negative control values. No dose-response was apparent and for each concentration, the MNBN cell frequency fell within normal values in single replicate cultures. It was therefore considered that the increases observed were spurious and of no biological importance.

It is concluded that 4-Amino-3-Nitrophenol (B051) induced micronuclei in cultured human peripheral blood lymphocytes following 3 hour treatment in the presence of a rat liver metabolic activation system (S-9) [3+45 hour +S-9] where treatment commenced 48 hours following PHA (mitogen) stimulation. No such increases in micronucleated cells were apparent following treatment in the absence of S-9 (20+28 hour –S-9) where treatment commenced 48 hours after PHA stimulation, or in the presence of S-9 where treatment commenced 24 hours after PHA stimulation. An isolated increase in micronucleated cells was observed following 20+28 hour treatment in the absence of S-9 where treatment commenced 24 hours post mitogen stimulation. This increase was not observed at higher or lower concentrations analysed and was therefore, considered of questionable biological importance.