3-aminophenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A preliminary toxicity test was performed to define the dose-levels of m-Aminophenol (A015) to be used for the mutagenicity study. The test material was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C).

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. Evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The test material did not induce any noteworthy increase in the number of revertants, in TA 1535, TA 1537, TA 100 and TA 102. A moderate genotoxicity was noted at dose-levels ≥ 2500 μg/plate in the TA 98 strain, without S9 mix.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

A preliminary toxicity test was performed to define the dose-levels of m-Aminophenol (A015) to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.
Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C).
Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other:

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

A S9 mix consisting of rat liver post-mitochondrial fraction (S9 enzyme fraction) and the necessary cofactors.

Test concentrations with justification for top dose:

PRELIMINARY TESTS:
0, 10, 100, 500, 1000, 2500 or 5000 μg/plate.

MUTAGENICITY EXPERIMENTS
Experiments without S9 mix:
The selected treatment-levels were:
• 156.3, 312.5, 625, 1250 or 2500 μg/plate, for the TA 98 and TA 1537 strains in the first experiment,
• 625, 1250, 2500, 3750 or 5000 μg/plate, for the TA 1535, TA 100 and TA 102 strains in the first experiment and for all the strains in the second experiment.

Experiments with S9 mix:
The selected treatment-levels were:
• 625, 1250, 2500, 3750 or 5000 μg/plate, for all the strains in both experiments.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO),
- Justification for choice of solvent/vehicle: substance is soluble in the current vehicle

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535 - TA 100

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

TA 102

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Anthramine (With S9 mix)

Remarks:

TA 1535 - TA 1537 - TA 98 - TA 100 - TA102

Details on test system and experimental conditions:

No data

Rationale for test conditions:

No data

Evaluation criteria:

No data

Statistics:

No data

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No data

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

The test item did not induce any noteworthy increase in the number of revertants, in any of the five tester strains (TA 1535, TA 1537, TA 100 and TA 102). A moderate toxicity was noted at dose-levels ≥ 2500 μg/plate in the TA 98 strain, without S9 mix.Under the experimental conditions, the test material m-Aminophenol (A015) (batch No. 220117) showed a slight mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Executive summary:

A preliminary toxicity test was performed to define the dose-levels of m-Aminophenol (A015) to be used for the mutagenicity study. The test material was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C).

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. Evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The test material did not induce any noteworthy increase in the number of revertants, in TA 1535, TA 1537, TA 100 and TA 102. A moderate genotoxicity was noted at dose-levels ≥ 2500 μg/plate in the TA 98 strain, without S9 mix.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo mammalian somatic cell study was performed to determine the mutagenic nature of the test chemical. The study was performed using male and female CFY strain rats. The test chemical was prepared as suspensions in 0.5% (w/v) gum tragacanth containing 0.05% (w/v) sodium sulphite to give a dose range of 0 or 5000 mg/Kg. The total dosages given were determined in preliminary studies to be close to the lethal doses, and were administered by gastric intubation as 2 equal doses separated by an interval of 24 h. 6 h after the 2nd dose the animals were killed by the i.p. injection of pentobarbitone sodium (Expiral) the femurs dissected out and bone-marrow smears prepared. The smears were fixed in methanol, defatted in xylene and stained with Giemsa stain. The stained smears were then examined microscopically to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. The group mean counts and ranges were then compared with the values obtained with the vehicle control group and with laboratory standard values.Agitation and/or convulsions, and/or lethargy were observed in the treated animals. Orange urine was also seen in treated animals.Based on the observations made,the test chemical did not induce micronuclei formation in the bone marrow of male and female rats.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

In vivo mammalian somatic cell study was performed to determine the mutagenic nature of the test chemical

GLP compliance:

not specified

Type of assay:

other: In vivo mammalian somatic cell study: Micronucleus study

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CFY - descendants

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Anglia Laboratory Animals, Alconbury, Cambs., U.K.
- Age at study initiation: No data
- Weight at study initiation: 130-160 g
- Assigned to test groups randomly: Random allocation
- Fasting period before study: No data
- Housing: No data
- Diet (e.g. ad libitum): No data
- Water (e.g. ad libitum): No data
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data

IN-LIFE DATES: From: To: No data

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: The test chemical was prepared as suspensions in 0.5% (w/v) gum tragacanth containing 0.05% (w/v) sodium sulphite to give a dose range of 0 or 5000 mg/Kg.
- Justification for choice of solvent/vehicle: The test chemical was soluble in the solvent
- Concentration of test material in vehicle: 0 or 5000 mg/Kg
- Amount of vehicle (if gavage or dermal): No data
- Type and concentration of dispersant aid (if powder): No data
- Lot/batch no. (if required): No data
- Purity: No data

Details on exposure:

For oral route
PREPARATION OF DOSING SOLUTIONS: The test chemical was prepared as suspensions in 0.5% (w/v) gum tragacanth containing 0.05% (w/v) sodium sulphite to give a dose range of 0 or 5000 mg/Kg.

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

Duration of treatment / exposure:

24 hrs

Frequency of treatment:

Twice separated by an interval of 24 h

Post exposure period:

No data

Remarks:

0 or 5000 mg/Kg

No. of animals per sex per dose:

Total: 10 males and 10 females
0 mg/Kg: 5 males and 5 females
5000 mg/Kg: 5 males and 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

No data

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The doses were selected on the basis of preliminary dose range finding study

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 6 h after the 2nd dose the animals were killed by the i.p.injection of pentobarbitone sodium (Expiral) the femurs dissected out and bone-marrow smears prepared.

DETAILS OF SLIDE PREPARATION: The smears were fixed in methanol, defatted in xylene and stained with Giemsa stain.

METHOD OF ANALYSIS: The stained smears were then examined microscopically to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. The group mean counts and ranges were then compared with the values obtained with the vehicle control group and with laboratory standard values.

OTHER: No data

Evaluation criteria:

The group means counts and ranges of of micronucleated cells were compared with the values obtained with the vehicle control group and with laboratory standard values.

Statistics:

No data

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not specified

Remarks on result:

other: No mutagenic potential

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: The total dosages given were determined in preliminary studies to be close to the lethal doses
- Solubility: No data
- Clinical signs of toxicity in test animals: No data
- Evidence of cytotoxicity in tissue analyzed: No data
- Rationale for exposure: No data
- Harvest times: No data
- High dose with and without activation: No data
- Other: No data

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): No data
- Induction of micronuclei (for Micronucleus assay): No data
- Ratio of PCE/NCE (for Micronucleus assay): No data
- Appropriateness of dose levels and route: No data
- Statistical evaluation: No data

Other: Agitation and/or convulsions, and/or lethargy were observed in the treated animals. Orange urine was also seen in treated animals.

Table:

Chemical	Dose over 24 hrs (mg/Kg)	Mortality	Incidence of micronucleated cells per 2000 polychromatic erythroeytes per rat
			Mean	Range
Vehicle control	-	0	1.8	0-5
Test chemical	5000	3	1.9	0-4

 

Table 2. Rat micronucleus test Laboratory standard values for negative control groups

Total number of animals examined	Mean micronucleated cell count	Range of group mean counts	Range of individual counts
175	1.19	0.7-2.3	0-6

 

Conclusions:

The test chemical did not induce micronuclei formation in the bone marrow of male and female rats.

Executive summary:

In vivo mammalian somatic cell study was performed to determine the mutagenic nature of the test chemical. The study was performed using male and female CFY strain rats. The test chemical was prepared as suspensions in 0.5% (w/v) gum tragacanth containing 0.05% (w/v) sodium sulphite to give a dose range of 0 or 5000 mg/Kg. The total dosages given were determined in preliminary studies to be close to the lethal doses, and were administered by gastric intubation as 2 equal doses separated by an interval of 24 h. 6 h after the 2nd dose the animals were killed by the i.p. injection of pentobarbitone sodium (Expiral) the femurs dissected out and bone-marrow smears prepared. The smears were fixed in methanol, defatted in xylene and stained with Giemsa stain. The stained smears were then examined microscopically to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. The group mean counts and ranges were then compared with the values obtained with the vehicle control group and with laboratory standard values.Agitation and/or convulsions, and/or lethargy were observed in the treated animals. Orange urine was also seen in treated animals.Based on the observations made,the test chemical did not induce micronuclei formation in the bone marrow of male and female rats.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in vitro:

Data available for the test chemical was reviewed to determine the mutagenic nature of 3 -aminophenol. The studies are as mentioned below:

A preliminary toxicity test was performed to define the dose-levels of m-Aminophenol (A015) to be used for the mutagenicity study. The test material was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. Evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test material did not induce any noteworthy increase in the number of revertants, in any of the five tester strains (TA 1535, TA 1537, TA 100 and TA 102). A moderate toxicity was noted at dose-levels ≥ 2500 μg/plate in the TA 98 strain, without S9 mix.

Another study consisted of a cytotoxicity range-finding experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S9). A 3-hour treatment incubation period was employed for all treatments in the absence and presence of S-9. The test article was dissolved in sterile anhydrous analytical grade dimethyl sulphoxide (DMSO). When tested up to 10 mM (in the absence of S-9) or up to toxic doses (in the presence of S-9), no statistically significant increases in mutant frequency were observed following treatment with m-Aminophenol (A015) at any dose level tested in Experiments 1, 2 or 3. It is concluded that, under the conditions employed in the current study, m-Aminophenol (A015) is not mutagenic in this test system.

The objective of the other study was to evaluate the potential of the test material m-Aminophenol (A015) to induce chromosome aberrations in cultured human lymphocytes. The test material was tested in two independent experiments, both with and without a liver metabolizing system (S9 mix), obtained from rats previously treated with Aroclor 1254. The test material m-Aminophenol (A015) was dissolved in dimethylsulfoxide (DMSO). Under our experimental conditions, the test material m-Aminophenol (A015) induced chromosome aberrations in cultured human lymphocytes, both in the presence and absence of metabolic activation (S9 mix).

In yet another toxicological study based on the OECD guideline 487 (A015) induction of micronuclei in cultured human peripheral blood lymphocytes was evident when the lymphocytes were exposed to m-aminophenol in different concentrations. m-Aminophenol (A015) induced micronuclei in cultured lymphocytes following 20 hour treatment in the absence of a rat liver metabolic activation system (S9) [20+28 hour -S9] where treatment commenced 48 hours following PHA stimulation. This increase in micro-nucleated cells were not apparent following treatment in the absence or presence of S9 [3+45 hour +S9] where treatment commenced 24 hour post PHA stimulation. Increased frequencies of micro-nucleated cells were observed in one out of three treatments in the presence of S9 (3+45 hour +S-9) following 48 hour PHA stimulation at concentrations up to and including 10 mM. However, due to the apparent absence of a dose-related effect in this experiment, two further experiments were performed at this time-point to clarify the nature of the response. In both of the subsequent experiments m-Aminophenol (A015) did not induce micronuclei at any test article concentration analyzed. The results of the first experiment (post 48 hour mitogen treatment) were therefore considered of highly questionable biological relevance.

Genetic toxicity of 3-aminophenol to Escherichia coli IC203 (deficient in OxyR) and Escherichia coli IC188 (OxyR+ parent) in a new bacterial reversion assay, the WP2 Mutoxitest, used in the evaluation of oxidative mutagenicity showed no effect at a dose concentration 750 μg/plate.

3-aminophenol did not induce chromosome aberrations in Chinese hamster lung (CHL/IU) cells, with S9 mix. Cells with structural chromosomal aberrations increased dose dependently with continuous treatment for 24 hr, without metabolic activation (frequency: 8.0-21.0%). Lowest concentration producing cytogenetic effects in vitro: Without metabolic activation (continuous treatment): 0.034 mg/mL (clastogenicity).

3-aminophenol did not induce gene mutations in S. typhimurium TA100, TA1535, TA98, TA1537 and E. coli WP2 uvrA strains, with or without S9 mix. Toxicity was not observed up to 5000 microgram/plate (five strains), with or without S9 mix.

3-aminophenol was studied for its ability to induce mutations in strains of Salmonella typhimurium. The test compound was dissolved in DMSO and was tested at concentration of 0, 100, 333, 1000, 3333 and 10000 µg/plate using Salmonella typhimurium TA100, TA1535, TA97 and TA98 in the presence and absence of 5%, 10 % and 30 % rat and hamster liver S9 metabolic activation system. Preincubation assay was performed with a preicubation for 20 mins. The plates were observed for histidine independence after 2 days incubation period. Concurrent solvent and positive controls were included in the study. 3-aminophenol is not mutagenic to the Salmonella typhimurium TA100, TA1535, TA97 in the presence and absence of rat and hamster liver S9 metabolic activation system. It however induced gene mutation in strain TA98 in the presence of 5% and 10% HLI.

Bacterial gene mutation assay was also performed to determine the mutagenic nature of 3-aminophenol. The study was performed usingSalmonella typhimurium strain TA98 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 10 or 30µg/plate. Spontaneous reversion rate was 22 rev/plate without S9 and 26 rev/plate with S9. Based on the observations made, test chemical did not induce gene mutation in Salmonella typhimurium strain TA98 in the presence and absence of S9 metabolic activation system.

In another study, Spot test was performed to determine the mutagenic nature of m-aminophenol. The study was performed usingSalmonella typhimuriumTA98, TA97, TA100 or TA1538. The test chemical was dissolved in suitable solvent and used at dose level of 0 or 1 mg/plate. Based on the observations made, test chemical did not induce gene mutation in Salmonella typhimurium strainTA98, TA97, TA100 or TA1538 in the spot test performed.

Salmonella typhimurium reverse mutation test was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strain TA100, (TA1535/pKM101) and TA98 (TA1538/pKM101) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 25, 50, 100, 250 or 500µg/plate. Concurrent solvent control plates were also included in the study.The test chemical did not induce gene mutation in Salmonella typhimurium strainTA98 and TA100 in the presence and absence of S9 metabolic activation system.

Gene mutation in vivo:

In vivo mammalian somatic cell study was performed to determine the mutagenic nature of the test chemical. The study was performed using male and female CFY strain rats. The test chemical was prepared as suspensions in 0.5% (w/v) gum tragacanth containing 0.05% (w/v) sodium sulphite to give a dose range of 0 or 5000 mg/Kg. The total dosages given were determined in preliminary studies to be close to the lethal doses, and were administered by gastric intubation as 2 equal doses separated by an interval of 24 h. 6 h after the 2nd dose the animals were killed by the i.p. injection of pentobarbitone sodium (Expiral) the femurs dissected out and bone-marrow smears prepared. The smears were fixed in methanol, defatted in xylene and stained with Giemsa stain. The stained smears were then examined microscopically to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. The group mean counts and ranges were then compared with the values obtained with the vehicle control group and with laboratory standard values.Agitation and/or convulsions, and/or lethargy were observed in the treated animals. Orange urine was also seen in treated animals.Based on the observations made,the test chemical did not induce micronuclei formation in the bone marrow of male and female rats.

Justification for classification or non-classification

In summary, 3-aminophenol has consistently tested negative for mutagenicity in Salmonella typhimurium TA100, TA 1002, TA1535, TA1537 and in E. coli WP2 uvrA. Furthermore, 3-aminophenol has tested negative for mutagenicity in a mammalian cell line study according to OECD Test Guideline 476. Therefore, despite inconclusive mutagenic data in Salmonella typhimurium TA98, the chemical is regarded to be non-mutagenic. 3-aminophenol has tested positive for clastogenic effects in vitro but negative for micronuclei-induction in polychromatic erythrocytes in vivo. By weight of evidence, 3-aminophenol is regarded to be Not Classified for Germ cell mutagenicity.