Phenacetin

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro bacteria reverse mutation (Camus1982): Weight of evidence: Test method similar to OECD Guideline 471. Based on results, test item was found to be mutagenic for TA100 strain (+S9 in rats).

In vitro bacteria reverse mutation (Weinstein1981): Weight of evidence: Test method similar to OECD Guideline 471. Based on results, test item was found to be mutagenic for TA100 strain (+S9 in rats and hamsters). 

In vitro bacteria reverse mutation (Haworth1983): Weight of evidence: Test method similar to OECD Guideline 471. Based on results, mutagenic activity was not observed with this test substance in the presence or absence of S9 mix. 

In vitro bacteria reverse mutation (Nohmi1983): Weight of evidence: Test method similar to OECD Guideline 471. Based on results, mutagenic activity was observed with metabolic activation in hamsters.

In vitro mammalian cell micronucleus test (Dunn1987, Phenacetin): Weight of evidence: Test method similar to OECD Guideline 487. Based on results, test item was found not to induce chromosomal damage in NRK cells.

In vitro mammalian cell micronucleus test (Dunn1987, N-hydroxyphenacetin): Weight of evidence: Test method similar to OECD Guideline 487. Based on results, test item was found to induce chromosomal damage in NRK cells.

In vitro mammalian chromosome aberration test (Holme1986): Weight of evidence: Test method used was unscheduled DNA synthesis (UDS). Based on results, no marked increase in unscheduled DNA synthesis was observed. 

In vitro DNA damage (comet assay) (Robbiano2002): Weight of evidence: Test method similar to OECD Guideline 473. Based on results, test item producted cytotoxicity to rat and human urinary bladder cells.

 

 

 

 

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

mammalian cell line, other: NRK-49F

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

N-hydroxyphenacetin:5 mM
Since tets item sparingly soluble in aqueous solution, the maximum possible concentration was selected while minimizing the concentration of solvent used.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: (DMSO)

- Justification for percentage of solvent in the final culture medium: test item was first dissolved in DMSO, with the final DMSO concentration being less than 2% (v/v) in the incubation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

other: y-radiation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): duplicate.
- Number of independent experiments: 2.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 2-5 x 10^3 NRK-49F cells in 0.4 ml of growth medium were seeded into each chamber of Lab-Tek 8-chamber slides and incubated overnight (37ºC, 5%Co2).
- Test substance added in medium.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: the cytotoxicity of each of the compounds was assessed using Trypan Blue dye exclusion as a measure of cell viability. NRK-49F cells were trypsinized at approximately
80% confluence, and 1 x 105 cells were plated into 60-mm plastic dishes. 20 h later cells were exposed to the different compounds as described above. The dishes were then washed twice with Hanks balanced salt solution and 5 ml of growth medium added. At 24-h intervals, from 0 time to 72 h, cells were trypsinized, and the percentage of viable cells determined by Trypan Blue dye exclusion.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Method: An in vitro micronucleus assay using a normal rat-kidney fibroblast cell line, NRK-49F was used as an indicator of chromosomal damage induced by short-term exposure to test compounds.
Seeded cells were rinsed with Hanks balanced salt solution (prior adding 0.4 ml of RPMI 1640 medium containing 20 mM Hepes pH 7.5 and various concentrations of test compound). Test solution was dissolved in ethanol to prepare 2mM phenacetin solution in which ethanol was present at concentration 4% (v/v). Controls containg solvent only were included in each experiment. Cells were incubated with test solution (60 min, 37ºC, 5% CO2) and washed twice with Hanks balanced salt solution, Growth mediu was added and the cells were incubated (37ºC, 5%CO2). Cells were fixed with 3:1 (v(v) methanol-acetic acid, and slides stained with Giemsa and mounted. At least 2 experiments were performed, with duplicates for each data point.

Evaluation criteria:

Micronucleus assay: At least 4000 interphase cells with visible cytoplasm were scored per treatment. The criteria used to score micronuclei were as given by Lasne et al. (1984).

Key result

Species / strain:

mammalian cell line, other: Normal rat-kidney fibroblast cell line, NRK-49F

Metabolic activation:

not applicable

Genotoxicity:

positive

Remarks:

N-hydroxyphenacetin

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Table 1. Cytotoxic effect of analgesic compounds on NRK-49F cells: 

Compound	Concentration (mM)	% control viability*
		0	1	2	3 days
N-hydroxyphenacetin	5	100.1	83.4	85.1	96.2

*Values represent means of at least two separate experiments. Cell viability was determined by the Trypan blue dye exclusion
 method at 1-day intervals after treatment. Untreated cells were in log phase for the duration of these experiments and divided
 approximately 3 times. Zero time values represent viability determined immediately after 1 h exposure of cells to test compounds.

Conclusions:

In a micronucleus test, test item was found to induce chromosomal damage in NRK cells.

Executive summary:

The test item was evaluated for micronucleus test in normal rat-kidney fibroblast cell line (NRK-49F) and its cytotoxicity assessed using Trypan Blue dye exclusion as  ameasure of cell viability. Test was performed similar to OECD guideline 473. Based on the results of the solubility, the test item was formulated in DMSO and 5 mM as selected as highest concentration. The treatment was carried out in duplicates and in two different experiments. Based on the results of results, the test item is found to induce chromosomal damage under the presented test conditions.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

At least five strains of bacteria should be used in the test, but in the study, only one strain has been used.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Male Fischer rats (100-120 g) and male Golden syrian hamsters (80-100g)
- method of preparation of S9 mix: with a single intraperitoneal injection of PCB (Kanechlor KC-400, 500 mg/kg) dissolved in olive oil and killed 5 days later.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: olive oil

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

Assay method:
The incubation mixture (12 ml) contained 24 mg of hepatic microsomal protein prepared from PCB-treated or untreated rats or hamsters, 600 μmol of potassium phosphate buffer, 4 μmol of NADP, 60 μMOL of MgCl2, 124 μmol of G-6-P, 8 units of G-6-P dehydrogenase and 16.8 μmol of phenacetin. Sodium fluoride was included in the incubation mixture when phenacetin N-hydroxylation activity was determined. The incubation mixture contained 0.7 μmol pf p-phenetidine or 5.1 μmol of N-hydroxyphenacetine instead of phenacetin when p-phenetidine N-hydroxylation or N-hydroxyphenacetin deacylation activity was determined. The mixture was incubated for 20 min at 37ºC with shaking. The incubation time was shortened to 10 min when N-hydroxyphenacetin deacetylation activity was determined. the reaction was terminated by rapid cooling in an icebath. Extracts of metabolites and unmetabolized substrate were obatined by extracting the incubation mixture with three 24 ml aliquots of dischloromethane. the metabolites were analyzed by hplc using a reverse-phase column.

Evaluation criteria:

To investigate the metabolic activation route, metabolites of phenacetin by PCB-treated hamster liver microsomes were analyzed. The identification of each metabolite was performed by comparing its retention time of hplc and UV spectrum with those of the authentic sample. the amount of metabolites formed was determined from calibration curves constructed for each authentic sample by using a microcomputer Chromatopak C-R1A.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

hamsters

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

rats

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

hamster

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

rats

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

Interpretation of results: positive with metabolic activation in hamsters

Executive summary:

The mutagenic potency of the test item was investigated by bacterial reverse mutation assay with and without metabolic activation, similar to OECD 471. The species used were rats and hamsters and the starin of the bacteria was TA100 (S. typhimurium). The vehicle was olive oil. Under the experimental conditions reported, the test substance caused genotoxicity to strain TA100(+S9) in hamster. Therefore, the test substance is considered to be genotoxic in this salmonella mutagenicity test.

Reference 3

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Principles of method if other than guideline:

- Principle of test: comet assay
- Short description of test conditions: quantitating DNA damage expressed as single-strand breaks and alkali-labile sites
- Parameters analysed / observed: tail moment

GLP compliance:

not specified

Type of assay:

comet assay

Species / strain / cell type:

other: human cells from urinary bladder mucosa

Species / strain / cell type:

primary culture, other: Male Sprague-Dawley rats (200-250 g; about 2 months old)

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

Rat and human urinary cells (1 Mm, 2 Mm, 4Mm)

Concentrations to be tested were chosen on the basis of a preliminary cytotoxicity assay; in order to avoid toxicity-induced unspecific DNA damage, the maximum concentration tested of each compound was the highest producing a reduction of cell viability lower than 30%.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Negative solvent / vehicle controls:

yes

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of independent experiments: 3.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): cells were suspended in Williams’ Medium E (WME) supplemented with 10% fetal bovine serum and gentamicin (50μg/ml). Aliquotes of the suspension were plated in 60-mm uncoated dishes (1 x 10 ^6 cells/dish) for the DNA fragmentation Comet assay and in 35-mm dishes coated with rat tail collagen (0.5 x 10^6 cells/dish) for determination of cytotoxicity.
- Test substance added in medium. Cells were washed and incubated for 20 h with serial concentrations of the test compounds in serum-free Williams’ Medium E (WME).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: bladder cells of rats and humans were examined for cytotoxicity with the trypan blue assay.


METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Method: presence of DNA fragmentation with the Comet assay. Ten microliters of cellular suspension (>10 000 cells) were mixed with 75 ml of low melting point agarose at 37ºC
and then added to normal melting point agarosecoated microscope slides. The slides were immersed in cold lysing solution overnight, and then placed in an electrophoresis tray with an alkaline solution (300 mM NaOH, 1 mM Na2EDTA; pH 13) for 20 min to allow DNA to unwind. Electrophoresis was conducted at room temperature for 20 min at 25 V and 300 mA. The slides were washed, stained with ethidium bromide, and examined at 400 x magnification using a fluorescence microscope. Image analysis was performed with the software Cometa on 50 randomly chosen cells from two slides as routinely performed in this assay. The observer who selected the images for analysis was blinded as to treatment. Comets having well separated head and tail were not scored being presumably apoptotic cells. DNA damage was quantified by the increase of tail moment, that was defined as the product of the tail length and the amount of DNA in the tail.

Statistics:

For the cytotoxicity and DNA-damaging activity in primary cultures: Statistical analysis was performed by the use of analysis of variance followed by Dunnet’s test.

Key result

Species / strain:

mammalian cell line, other: human urinary bladder cells

Metabolic activation:

not applicable

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Key result

Species / strain:

primary culture, other: rat urinary bladder mucosa

Metabolic activation:

not applicable

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

- DNA-damaging concentrations ranged as from 2 to 4 mM
- The values (mean9/SD) of the relative survival (fraction of viable cells in treated cultures/ fraction of viable cells in control cultures) observed at the highest concentration tested in rat and human urinary bladder cells were respectively: 92.3±3.1 and 95.5±3.5

Table 1. Potency of test compounds in inducing DNA fragmentation in rat and human urinary bladder epithelial cells: 

Compound	DNA-damaging potency (xmM-1)
	Rat	Man
	Tail moment	Tail moment
Phenacetin	71±15	232±169

Conclusions:

In an in vitro comet assay performed in urinary bladder cells of rat and human, test item was considered to be genotoxic.

Executive summary:

The test item was tested for DNA damage using the comet assay with a preliminary cytotoxicity assay. DNA damage was quantified by the increase of tail lenght and tail moment. The experiments were carried out using primary cultures from rat and human urinary bladder cells. The concentrations to be tested were chosen on the basis on the preliminary cytotoxicity assay. Under the experimental conditions reported, the test substance produced genotoxicity. Therefore, the test substance is considered to be genotoxic in this comet assay.

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Principles of method if other than guideline:

- Principle of test: Unscheduled DNA synthesis measured by liquid scintillation of [3H] TdR incorporated intro nuclear DNA and measurement of lactate dehydrogenase for determining cytotoxicity.

GLP compliance:

not specified

Type of assay:

other: Unscheduled DNA synthesis (UDS)

Species / strain / cell type:

hepatocytes: Isolated from mouse, hamster, rat and guinea pig

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

0.1; 0.5; 1; 2.5; 5; 7.5 and 10 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 0.5% DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

0.1 mM and 1.0mM

Positive control substance:

2-acetylaminofluorene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of independent experiments: 5 (mean viability)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): hepatocytes were incubated as monolayer cultures (4x10^6 viable cells/dish).
- Test substance added in medium.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: by measurement of lactate dehydrogenase (LDH) released into the culture medium from injured cells 18-19 h after test chemicals were added. The plates were also examined microscopically for morphological alterations, and stained with trypan blue. The number of attached liver cells excluding trypan blue was counted in 15 different areas of each culture, and expressed as per cent of control cultures.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Genotoxicity was measured as DNA synthesis (UDS) by liquid scintillation counting of [3H]TdR incorporated into nuclear DNA.
The cells were rinsed with Tris-HC1- buffered saline, pH 7.4 containing 2.0 mM non-radioactive thymidine. Cells pooled from 2 dishes were treated with Nonidet P-40 (1%) and vortexed vigorously for 30 sec. After 15 min, the suspension was vortexed as above, and centrifuged at 800 × g for 5 min. The pellet was resuspended in 1 ml of 0.25 M sucrose buffer (50 mM Tris-HC1, 25 mM KC1, 15 mM MgC12, pH 8.0), and 1 ml of 0.88 M sucrose was layered underneath. The nuclei were then centrifuged at 1000 × g for 10 min. After the addition of 1 ml 10 mM Tris-HC1, pH 8.0 and 0.5 ml 1 M KOH, the nuclei were incubated for 45 min and neutralized with 0.5 ml 1 M HC1, DNA was precipitated in the presence of 10% TCA and 0.5% BSA. The precipitate was collected by centrifugation and DNA was hydrolysed by heating in 5% TCA at 90°C for 20 min. Remaining
undissolved material was recovered by centrifugation at 2000 × g for 15 min.

Key result

Species / strain:

hepatocytes: Isolated from the mouse, hamster, rat and guinea pig

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

In a Unscheduled DNA synthesis (UDS) test, test item was found not to increase DNA synthesis.

Executive summary:

The test item was evaluated in a primary monolayer culture of hepatocytes tes, using different animal species (mouse, hamster, rat and guinea pig). Test substance was dissolved in 0.5% DMSO. This test was conducted at concentrations of 0.1; 0.5; 1; 2.5; 5; 7.5 and 10 mM. It was first realised an isolation and culture of hepatocytes were mean viability was determined by trypan blue exclusion. Then was carried an unscheduled DNA synthesis and finally cytotoxicity was determined by measurement of lactate dehydrogenase. Based on results, no marked increase in unscheduled DNA synthesis (UDS) was observed.

Reference 5

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

mammalian cell line, other: NRK-49F

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

2 mM
Since test item is sparingly soluble in aqueous solution, the maximum possible concentration was selected while minimizing the concentration of solvent used.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: phenacetin (ethanol);

- Justification for choice of solvent/vehicle: phenacetin is sparingly soluble in aqueous solution

- Justification for percentage of solvent in the final culture medium: phenacetin was dissolved in ethanol in order to prepare a 2 mM phenacetin solution in which ethanol was present at a concentration of 4% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

other: y-radiation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): duplicate.
- Number of independent experiments: 2.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 2-5 x 10^3 NRK-49F cells in 0.4 ml of growth medium were seeded into each chamber of Lab-Tek 8-chamber slides and incubated overnight (37ºC, 5%Co2).
- Test substance added in medium.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: the cytotoxicity of each of the compounds was assessed using Trypan Blue dye exclusion as a measure of cell viability. NRK-49F cells were trypsinized at approximately
80% confluence, and 1 x 105 cells were plated into 60-mm plastic dishes. 20 h later cells were exposed to the different compounds as described above. The dishes were then washed twice with Hanks balanced salt solution and 5 ml of growth medium added. At 24-h intervals, from 0 time to 72 h, cells were trypsinized, and the percentage of viable cells determined by Trypan Blue dye exclusion.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Method: An in vitro micronucleus assay using a normal rat-kidney fibroblast cell line, NRK-49F was used as an indicator of chromosomal damage induced by short-term exposure to test compounds.
Seeded cells were rinsed with Hanks balanced salt solution (prior adding 0.4 ml of RPMI 1640 medium containing 20 mM Hepes pH 7.5 and various concentrations of test compound). Test solution was dissolved in ethanol to prepare 2mM phenacetin solution in which ethanol was present at concentration 4% (v/v). Controls containg solvent only were included in each experiment. Cells were incubated with test solution (60 min, 37ºC, 5% CO2) and washed twice with Hanks balanced salt solution, Growth mediu was added and the cells were incubated (37ºC, 5%CO2). Cells were fixed with 3:1 (v(v) methanol-acetic acid, and slides stained with Giemsa and mounted. At least 2 experiments were performed, with duplicates for each data point.

Evaluation criteria:

Micronucleus assay: At least 4000 interphase cells with visible cytoplasm were scored per treatment. The criteria used to score micronuclei were as given by Lasne et al. (1984).

Key result

Species / strain:

mammalian cell line, other: Normal rat-kidney fibroblast cell line, NRK-49F

Metabolic activation:

not applicable

Genotoxicity:

negative

Remarks:

Phenacetin

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Table 1. Cytotoxic effect of analgesic compounds on NRK-49F cells: 

Compound	Concentration (mM)	% control viability*
		0	1	2	3 days
Phenacetin	2	84.9	86.7	92.1	97.0

*Values represent means of at least two separate experiments. Cell viability was determined by the Trypan blue dye exclusion
 method at 1-day intervals after treatment. Untreated cells were in log phase for the duration of these experiments and divided
 approximately 3 times. Zero time values represent viability determined immediately after 1 h exposure of cells to test compounds.

Conclusions:

In a micronucleus test, test item was found not increase micronuclei frequency.

Executive summary:

The test item was evaluated for micronucleus test in normal rat-kidney fibroblast cell line (NRK-49F) and its cytotoxicity assessed using Trypan Blue dye exclusion as  ameasure of cell viability. Test was performed similar to OECD guideline 487. Based on the results of the solubility, the test item was formulated in ethanol and 2 mM as selected as highest concentration. The treatment was carried out in duplicates and in two different experiments. Based on the results of test, the test item is found not to increase micronuclei frequency.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only 4 Salmonella strains used, no E.coli

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Liver S-9 fractions were routinely prepared from male Sprague-Dawley rats and male Syrian hamsters
- method of preparation of S9 mix: animals were injected, ip, with Aroclor 1254 (200 mg/ml in corn oil) at 500 mg/kg. Five days after injection, the animals were sacrificed by
decapitation (EGG, SRI) or cervical dislocation (CWR) and the livers were removed aseptically. The animals were fasted for 12-24 hr immediately preceding sacrifice. Liver homogenates were prepared aseptically at 04°C. Excised livers were rinsed with 0.15 M KCI, then minced and homogenized (3 ml of 0.15 M KCl/g wet tissue) in a Potter-Elvehjem apparatus with a teflon pestle (EGG, SRI) or in a Waring blender (CWR). The homogenate was centrifuged for 10 min at 9,OOOg at 4°C. The supernatant (S-9) was decanted and distributed into freezing ampules and stored at -70ºC.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 ml of S-9 or 0. I M PO4 buffer was dispensed into an appropriate number of 13 X 100 mm
culture tubes maintained at 37°C in a dry-bath. Then, 0.05 ml of cells and 0.05 ml of solvent or chemical dilution were added to each tube
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): not specified

Test concentrations with justification for top dose:

TEST CONCENTRATIONS:
0.0, 33.0, 100.0, 333.0, 1000.0, 2730.0 μg.
To select the dose range for the mutagenesis assay, the test chemicals were checked for toxicity to TA100 up to a concentration of 10 mg/plate or the limit of solubility, both in the presence and absence of S-9 mix. One or more parameters were used as an indication of toxicity: viability on complete medium (EGG) and reduced numbers of revertant colonies per plate and/or thinning or absence of the bacterial lawn (CWR, EGG, SRI). If toxicity was not apparent in the preliminary toxicity
determination, the highest dose tested was 10 mg/plate; otherwise the upper limit of solubility was used. If toxicity was observed, the doses of test chemical were chosen so that the high dose exhibited some degree of toxicity. Occasionally, in the earlier tests, the high dose was greater than 10 mg/plate.

Vehicle / solvent:

DMSO (used if the chemical was insoluble or not sufficiently soluble in water).

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Remarks:

(choline chloride, glycerol, glycine, mannitol, and sodium phosphate)

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine: 12.0 μg/plate (TA98, -S9) // 2-aminoanthracene: (TA98, +S9); (TA100, +S9); (TA1537, +S-9)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate.
- Number of independent experiments: 2.

METHOD OF TREATMENT/ EXPOSURE:
1. Preincubation methodology: 0.5 ml of S-9 mix or 0. 1 M PO4 buffer was dispensed into an appropriate number of 13 X 100 mm culture tubes maintained at 37°C in a dry-bath. Then, 0.05 ml of cells and 0.05 ml of solvent or chemical dilution were added to each tube. The mixture was vortexed and allowed to incubate for 20 min at 37ºC. Following the preincubation period, 2.5 ml) of molten top agar (45°C) supplemented with 0.5 mM L-histidine and 0.5 mM d-biotin was pipetted into the tubes, which were immediately vortexed, and their contents poured onto 25 ml of minimal glucose
bottom agar. After the overlay solidified, the plates were inverted and incubated at 37°C for 48 h.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 min.
- Exposure duration/duration of treatment: 48h.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
TA100 up to a concentration of 10 mg/plate or the limit of solubility, both in the presence and absence of S-9 mix. One or more parameters were used as an indication of toxicity: viability on complete medium and reduced numbers of revertant colonies per plate and/or thinning or absence of the bacterial lawn. If toxicity was not apparent in the preliminary toxicity determination, the highest dose tested was 10 mg/plate; otherwise the upper limit of solubility was used. If toxicity was observed, the doses of test chemical were chosen so that the high dose exhibited some degree of toxicity. Occasionally, in the earlier tests, the high dose was greater than 10 mg/plate.

Statistics:

The data were subsequently evaluated using an analysis based on the models presented by Margolin et a1 [1981]; this analysis will be described elsewhere [Risko et al, manuscript in preparation]

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

valid

Positive controls validity:

valid

Table 1. Summary results

TA100

TA1535

TA1537

TA98

Dose

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

0.0

97±6.8

121±6.8

122±11.6

6±.9

6±2.1

6±3.0

2±1.2

6±0.0

6±1.2

15±1.9

22±1.2

19±.6

33.0

96±3.8

121±4.2

107±6.9

3±.3

7±1.2

6±1.3

3±.7

5±.3

5±1.2

9±1.9

21±3.3

20±2.1

100.0

94±6.8

103±5.6

118±9.3

3±1.2

6±1.2

7±.9

2±.9

5±1.0

4±1.5

14±1.3

18±2.4

21±2.3

333.0

90±2.6

110±7.4

109±4.8

4±.3

4±.9

7±.7

3±1.2

5±1.7

7±1.5

14±1.5

15±4.3

17±4.6

1000.0

87±7.4

114±2.7

124±3.7

3±1.3

4±.9

5±.9

3±.6

4±.3

7±1.2

13±4.3

18±1.5

19±4.7

2730.0

90±5.9

105±4.3

123±11.6

3±.6

3±.9

7±.6

2±.6

8±1.2

5±.6

15±1.0

23±2.4

25±2.9

Doses are in pg/plate; 0.0 dose is the solvent control.

DMSO, Dimethyl sulfoxide (solvent); NA, not activated; RLI, rat liver S-9, Aroclor 1254 induced; HLI, hamster liver S-9, Aroclor 1254 induced

Conclusions:

Mutagenic activity was not observed with this test substance in the presence or absence of S9 mix.

Executive summary:

The genetic toxicity in vitro of the test item has been tested following a method similar to guideline OECD 471. It has been tested in S. typhimurium strains: TA 98, TA 100, TA 1535 and TA 1537. Mutagenic activity was not observed with this test substance with and without metabolic activity.

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

At least five strains of bacteria should be used in the test

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Young adult, Sprague-Dawley male rats and male Syrian golden hamsters (from Charles River Breeding Laboratories)
- method of preparation of S9 mix: The S-9s from both species were prepared according to the method of Ames et a1 [ 19751 . Rats (250 gm) or hamsters (100 gm) were given a single intraperitoneal injection of Aroclor 1254 (500 mg/kg) 5 days before sacrifice to induce hepatic monooxygenases. The livers were removed under sterile conditions and homogenized in a Polytron with three volumes of 0.1 5 M KCl. The homogenate was centrifuged for 10 minutes at 9,000g, and the supernatant
fluid (S-9) was quickly frozen and stored in liquid nitrogen.
- concentration or volume of S9 mix and S9 in the final culture medium: This solution contains per ml: S-9 (6.25, 12.5, 25.0, or 50 mg wet weight of liver or livers), MgCl2 (8 μmoles), KCI (33 μmoles), glucose-6-phosphate (5 μmoles), NADP (4 μmoles), and sodium phosphate (100 μmoles).
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Not specified.

Test concentrations with justification for top dose:

0, 25, 250, 800, 1400, 2000, 2500 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Negative solvent / vehicle controls:

yes

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Three replicate plates were used for each dosage group and six plates were used for the control samples
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation): The bacterial suspension of an overnight culture (0.1 ml), the appropriate concentration of the test compound in 0.1 ml of dimethylsulfoxide or dimethylsulfoxide alone and 0.5 ml of the S-9 solution (equivalent to 0, 3.125, 6.25, 12.5, or 25 mg wet weight of liver of a single species or of each species), were added to 1.9 ml of molten top agar (0.6% agar, 0.5% NaC1,0.5 mM I-histidine, and 0.5 mM biotin) and mixed. This mixture was immediately poured onto the minimal glucose agar plates (approximately 12 ml of 2.0% agar in Vogel-Bonner medium with 0.5% glucose). Three replicate plates were used for each dosage group and six plates were used for the control samples, which contained no test compound and no S-9. The plates were incubated for 48 hours at 37ºC in a humidified incubator and the number of revertant bacterial colonies formed were counted. The colonies that were observed in the negative control plates were recorded as spontaneous revertants.

Statistics:

A variance stabilization was made by taking the sum of the square root of the colony counts plus the square root of the colony counts plus one. A series of “Many One t Tests” [Miller, 19661 was performed for each bacterial strain. Significance level for a given t-test was chosen at a “critical value” of P = 0.05/n, where ti equals the number of groups compared to the reference group (no compound but the comparable amount of S-9).

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Standard Ames assay (Rat S-9)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Standard Ames assay (Rat S-9)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Standard Ames assay (S-9)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Hamster uninduced and induced-Aroclor

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Rat/hamster S-9

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Table 1. Lack of mutagenic effect of phenacetin in the standard ames assay 

Compound (µg/plate)	S-9a (mg wet weight per plate)	Salmonella typhimurium (Reverant colonies per plate)
		TA1535	TA100		TA1537	TA98
			A	B		A	B
0	0	17±3 b	102±26 b	88±13 b	8±4 b	17±5 b	22±8 b
	3.125	15 ±1	93±10	97±6	8±3	26±10	35±6
	6.25	13 ±4	121±23	108±13	14±5	32±7	39±6
	12.5	18 ±7	134±11	110±2	7±1	44±10	42±5
Phenacetin 25	0	15±1	93±11		8±1	13±5
	3.125	16±5	98±8		9±1	31±3
	6.25	14±4	94±5		11±5	28±2
	12.5	18±7	114±8		9±2	35±4
250	0	14±7	78±8		8±2	11±3
	3.125	14±3	94±16		10±3	33±2
	6.25	18±1	115±28		9±1	30±7
	12.5	23±1	99±28		12±6	31±5
800	0			88±21			19±4
	3.125			87±11			37±8
	6.25			93±7			34±5
	12.5			111±19			41±11
1400	0			63±9			22±4
	3.125			78±7			23±1
	6.25			94±8			45±9
	12.5			90±19			34±4
2000	0			72±13			18±1
	3.125			68±6			27±1
	6.25			95±5			41±7
	12.5			111±13			37±6
2500	0	12±4	66±3		6±3	8±5
	3.125	11±1	86±15		11±3	13±5
	6.25	15±8	79±10		5±1	30±6
	12.5	11±10	87±26		10±2	28±2

a 9,OOOxg supernatant fluid of liver homogenate from Aroclor 1254 induced Sprague-Dawley rats.
b Mean 5 SD (6 plates, all others: 3 plates).
*P < Critical P (0.0042). 

Table 2. Mutagenic Effect of phenacetin on Salmonella Typhimurium  Ta 100 with uninduced Aroclor-Induced Hamster S-9, and Aroclor-Induced Rat/Hamster S-9 Mixture 

Compound (µg/plate)	S-9 (mg wet weight per plate) a	Reverant colonies per plate
		Hamster S-9		Rat/hamster S-9
0	0	112±12 b	78±16 b	94±11 b	127±11 b
	3.125		71±14		135±13
	6.25	118±2	82±12	122±15	142±17
	12.5	144±10	100±6	149±27	139±4
	25.0	116±4		118±12
Phenacetin 25.0	0	110±9	91±15	101±32	121±6
	3.125		102±19		161±11
	6.25	138±6	94±15	134±12	184±26*
	12.5	170±37	149±29*	138±22	151±8
	25.0	126±11		116±8
250	0	108±4	73±15	89±10	115±9
	3.125		162±11*		146±11
	6.25	122±38	100±7	161±34	161±11
	12.5	152±16	192±10*	256±35*	240±18*
	25.0	183±4*		312±118*
2500	0	110±4	61±12	88±12	115±6
	3.125		89±10		104±2
	6.25	127±32	44±13	151±25	182±16*
	12.5	140±23		235±31*	262±42*
	25.0	226±27*		332±43*

a "In mixture = wet weight of cach species' liver (therefore total wet weight of livers = 2 X indicated
weight).
b Mean k SD (6 plates, all others: 3 plates).
*P < Critical P (0.0042).

Conclusions:

In a bacterial reverse mutation assay, the test substance caused gene mutations at strain TA 100 in hamster uninduced S-9 and rat/hamster induced S-9

Executive summary:

The test item was tested for genotoxicity using gene mutation study in bacteria. Three sidderent experiments were carried out: standard ames assay (rat S-9), hamster S-9 and rat/hamster S-9. The experiments were carried out using S. typhimurium TA 98, 100, 1535 and 1537. The metabolic activation system was with and without and the source of S9 were male Sprague-Dawley rats and male Syrian golden hamsters livers. The vehicle/solvent was DMSO. The test concentrations were: 0, 25, 250, 800, 1400, 2000, 2500 µg/plate. Under the experimental conditions reported, the test substance caused genotoxicity TA100 strains in hamster S-9 and rat/hamster S-9. Therefore, the test substance is considered to be genotoxic in this salmonella mutagenicity tests.

Reference 8

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

At least five strains of bacteria should be used in the test

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA100 NR

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 98

Metabolic activation:

with

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: The pooled liver of 2 to 3 animals that had received an i.p. injection of Aroclor (500 mg/kg body weight) 5 days before their sacrifice were homogenized in 3 ml of 0.15 M potassium chloride:5 mM sodium phosphate buffer, pH 7.4, per g of animal liver at 0-4C°.
- method of preparation of S9 mix: by centrifugation at 9000 x g for 10 min. Microsomal suspensions and 100,000 x g supernatant were prepared and stored at -70C°. Protein concentrations were determined by the method of Lowry ef al., using bovine serum albumin as standard.
- concentration or volume of S9 mix and S9 in the final culture medium: The test compound dissolved in 100µL DMSO, 0.5 ml of a mixture containing 50 to 150 µL of liver S9 from Aroclortreated animals,
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Not specified.

Test concentrations with justification for top dose:

4x100 mg/animal at 12-hourly intervals; 2x100 mg/animal at 24-hourly intervals and 0 mg/animal.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

MUTAGENICITY ASSAYS:

- Principle of test: Plate incorporation assay (method of Ames ef al.)
- Short description of test conditions: The test compound dissolved in 100 μL of DMSO, 0.5 ml of a mixture containing 50 to 150 μL of liver S9 from Aroclortreated animals, cofactors (2 μmol of NADP+ and 2.5 μmol of glucose 6-phosphate), 4 μmol of MgCI2, 50 μmnol of Sorensen phosphate buffer (pH 7.4), and 0.1 ml of an overnight culture medium (1.2 x 108 bacteria) were combined with 2 ml of histidine-poor soft agar. For assays with urine concentrates, 50 to 500 units of each deconjugating enzyme, /8-glucuronidase and arylsulfatase, were added. The agar mixture was agitated and immediately poured onto plates of minimal glucose agar.

- Principle of test: Liquid incubation assay was adapted from the method of Mailing
- Short description of test conditions: The incubation medium (final volume, 460μL) contained 40 to 120 μL of the various subcellular liver fractions (microsomes, S9, or 100,000 x g supernatant), 3.2 ,μmol of MgCI2, 0.32 fimo\ of NADP+, 2 μmol of glucose 6-phosphate, 8 μmol of Sorensen phosphate buffer (pH 7.4). 1 unit of glucose-6-phosphate dehydrogenase when necessary, 80 μL of a concentrated overnight culture medium (3 to 5 x 108 bacteria), and the test compound added as a solution in 20 μL of DMSO. After incubation for 15, 30, or 60 min at 37° under air, the medium was diluted with ice-cold 0.9% NaCI solution and 5 mM Sorensen phosphate buffer, pH 7.4, and the bacteria were separated by centrifugation at 1900 x g for 10 min. The bacterial pellet was suspended in 1 ml buffered 0.9% NaCI solution, and 500μL of this bacterial suspension were incorporated in 2 ml of histidine-poor soft agar and plated onto minimal glucose agar. The survivors were determined by plating on selective medium after appropriate dilution, as described previously. For some assays, incubation was carried out under nitrogen.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

Liquid incubation assay

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

Rat (plate incorporation assay)

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Hamster (plate incorporation assay)

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Conclusions:

In a bacterial reverse mutation assay, the test substance caused gene mutations at strain TA 100 +S9 in hamsters

Executive summary:

The test item was tested for genotoxicity using the Bacterial Reverse Mutation Assay similar to OECD Guideline 471 and identification of N-hydroxyphenacetin as a promutagen in the urine. Two types of mutagenicity assays were performed: plate incorporation and liquid incubation. The experiments were carried out using Salmonella typhimurium strains TA100, TA98 and TA100 NR in the presence of metabolic activation S9 mix prepared from the livers of rats and hamsters). Under the experimental conditions reported, the test substance caused gene mutations at strain TA 100 +S9 in hamsters. Therefore, the test substance is considered to be mutagenic in this bacterial reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo mammalian somatic cell study: mammalian erythrocite micronucleus test. (Hachiya1989).Weight of evidence. Test method similar to guideline OECD 474. Based on results, test item was positive in micronucleus induction by intraperitoneal route administration in CD-1 mouse strain. 

In vivo mammalian somatic cell study: mammalian erythrocite micronucleus test. (Asanami1995).Weight of evidence. Test method similar to guideline OECD 474. Based on results, test item was genotoxic in the rat peripheral blood and bone marrow micronucleus test. 

In vivo mammalian dell study (commet assay). (Robbiano2002). Weight of evidence. Test method similar to OECD 489. Based on results, test item was genotoxic in the rat urinary bladder mucosa, liver and kidney by comet assay.

In vivo mammalian dell study (commet assay). (Sasaki1997). Weight of evidence. Test method similar to OECD 489. Based on results, test item caused genotoxicity to mouse kidney by comet assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

other: MS/Ae

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: MS/A ( Charles River Japan, Inc.)
- Age at study initiation: 9 week old
- Diet (e.g. ad libitum): fed commercial pellets (not specified)
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: 1 week or more

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: olive oil (henacetin was suspended homogeneously in the vehicle but could not be dissolved.)
- Concentration of test material in vehicle: was suspended in olive oil at concentrations of 1-500 mg/ml to make an injection volume of 10 ml/kg body weight for all doses

Duration of treatment / exposure:

24 hours

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

600 mg/kg bw/day (actual dose received)

Dose / conc.:

1 200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

4 mice per group were used

Control animals:

yes, concurrent vehicle

Positive control(s):

none

Tissues and cell types examined:

Micronucleated polychromatic erythrocytes (MNPCEs) in mouse bone marrow

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Table 2. Micronucleus induction of phenacetin in MS/Ae and CD-1 mice 24h after administration

Strain	Dose (mg/kg)	i.p (intraperitoneal injection)				p.o (gastric incubation)
		survival	Number of PCEs observed	Frequency of PCEs (%±SD)	Frequency of MNPCEs (%±SD)	Survival	Number of PCEs observed	Frequency of PCEs (%±SD)	Frequency of MNPCEs (%±SD)
MS/Ae	0	4/4	4000	35.4±3.7	0.53±0.10	4/4	4000	42.1±4.5	0.40±0.24
	150	4/4	4000	30.4±12.4	0.35±0.31	4/4	4000	41.1±9.0	0.48±0.10
	300	4/4	4000	34.4±4.2	0.55±0.13	4/4	4000	37.5±1.4	0.33±0.25
	600	4/4	4000	36.2±6.3	1.18±0.56	4/4	4000	37.1±8.2	1.30±0.52**
	1200	0/4	0			3/4	3000	18.4±5.9	0.43±0.35
CD-1	0	4/4	4000	47.3±5.0	0.08±0.10	4/4	4000	45.6±5.2	0.15±0.13
	150	4/4	4000	51.8±3.8	0.15±0.17	4/4	4000	44.6±3.1	0.08±0.10
	300	4/4	4000	57.2±1.2	0.25±0.13	4/4	4000	55.2±4.4	0.15±0.13
	600	4/4	4000	43.5±4.1	0.45±0.52	4/4	4000	50.8±2.4	0.43±0.10
	1200	3/4	3000	44.5±6.5	1.53±0.92	4/4	4000	44.2±5.2	1.75±0.33

Conclusions:

Test item was positive in micronucleus induction by intraperitoneal route of administration in MS/Ae mouse strain.

Executive summary:

In a micronucleus test conducted, test item was administered intraperitoneal to MS/Ae mouse strain (male). Administration doses were 0 (vehicle), 150, 300, 600 amd 1200 (mg/kg). The frequency of MNPCEs increased at doses up to 1200 mg/kg.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

other: MS/Ae

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CD-1 ( Charles River Japan, Inc.)
- Age at study initiation: 9 week old
- Diet (e.g. ad libitum): fed commercial pellets (not specified)
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: 1 week or more

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: olive oil (henacetin was suspended homogeneously in the vehicle but could not be dissolved.)
- Concentration of test material in vehicle: was suspended in olive oil at concentrations of 1-500 mg/ml to make an injection volume of 10 ml/kg body weight for all doses

Duration of treatment / exposure:

24 hours

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

600 mg/kg bw/day (actual dose received)

Dose / conc.:

1 200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

4 mice per group were used

Control animals:

yes, concurrent vehicle

Positive control(s):

none

Tissues and cell types examined:

Micronucleated polychromatic erythrocytes (MNPCEs) in mouse bone marrow

Key result

Sex:

male

Genotoxicity:

ambiguous

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Table 2. Micronucleus induction of phenacetin in MS/Ae and CD-1 mice 24h after administration

Strain	Dose (mg/kg)	i.p (intraperitoneal injection)				p.o (gastric incubation)
		survival	Number of PCEs observed	Frequency of PCEs (%±SD)	Frequency of MNPCEs (%±SD)	Survival	Number of PCEs observed	Frequency of PCEs (%±SD)	Frequency of MNPCEs (%±SD)
MS/Ae	0	4/4	4000	35.4±3.7	0.53±0.10	4/4	4000	42.1±4.5	0.40±0.24
	150	4/4	4000	30.4±12.4	0.35±0.31	4/4	4000	41.1±9.0	0.48±0.10
	300	4/4	4000	34.4±4.2	0.55±0.13	4/4	4000	37.5±1.4	0.33±0.25
	600	4/4	4000	36.2±6.3	1.18±0.56	4/4	4000	37.1±8.2	1.30±0.52**
	1200	0/4	0			3/4	3000	18.4±5.9	0.43±0.35
CD-1	0	4/4	4000	47.3±5.0	0.08±0.10	4/4	4000	45.6±5.2	0.15±0.13
	150	4/4	4000	51.8±3.8	0.15±0.17	4/4	4000	44.6±3.1	0.08±0.10
	300	4/4	4000	57.2±1.2	0.25±0.13	4/4	4000	55.2±4.4	0.15±0.13
	600	4/4	4000	43.5±4.1	0.45±0.52	4/4	4000	50.8±2.4	0.43±0.10
	1200	3/4	3000	44.5±6.5	1.53±0.92	4/4	4000	44.2±5.2	1.75±0.33

Conclusions:

Test item was inconclusive in micronucleus induction by gastric intubation route of administration in MS/Ae mouse strain.

Executive summary:

In a micronucleus test conducted, test item was administered intraperitoneal to MS/Ae mouse strain (male). Administration doses were150, 300, 600 amd 1200 (mg/kg). The non-linear dose-response reltionship of MNPCE induction phenacetin made test be inconclusive.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CD-1 ( Charles River Japan, Inc.)
- Age at study initiation: 9 week old
- Diet (e.g. ad libitum): fed commercial pellets (not specified)
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: 1 week or more

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: olive oil (henacetin was suspended homogeneously in the vehicle but could not be dissolved.)
- Concentration of test material in vehicle: was suspended in olive oil at concentrations of 1-500 mg/ml to make an injection volume of 10 ml/kg body weight for all doses

Duration of treatment / exposure:

24 hours

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

600 mg/kg bw/day (actual dose received)

Dose / conc.:

1 200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

4 mice per group were used

Control animals:

yes, concurrent vehicle

Positive control(s):

none

Tissues and cell types examined:

Micronucleated polychromatic erythrocytes (MNPCEs) in mouse bone marrow

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Table 2. Micronucleus induction of phenacetin in MS/Ae and CD-1 mice 24h after administration

Strain	Dose (mg/kg)	i.p (intraperitoneal injection)				p.o (gastric incubation)
		survival	Number of PCEs observed	Frequency of PCEs (%±SD)	Frequency of MNPCEs (%±SD)	Survival	Number of PCEs observed	Frequency of PCEs (%±SD)	Frequency of MNPCEs (%±SD)
MS/Ae	0	4/4	4000	35.4±3.7	0.53±0.10	4/4	4000	42.1±4.5	0.40±0.24
	150	4/4	4000	30.4±12.4	0.35±0.31	4/4	4000	41.1±9.0	0.48±0.10
	300	4/4	4000	34.4±4.2	0.55±0.13	4/4	4000	37.5±1.4	0.33±0.25
	600	4/4	4000	36.2±6.3	1.18±0.56	4/4	4000	37.1±8.2	1.30±0.52**
	1200	0/4	0			3/4	3000	18.4±5.9	0.43±0.35
CD-1	0	4/4	4000	47.3±5.0	0.08±0.10	4/4	4000	45.6±5.2	0.15±0.13
	150	4/4	4000	51.8±3.8	0.15±0.17	4/4	4000	44.6±3.1	0.08±0.10
	300	4/4	4000	57.2±1.2	0.25±0.13	4/4	4000	55.2±4.4	0.15±0.13
	600	4/4	4000	43.5±4.1	0.45±0.52	4/4	4000	50.8±2.4	0.43±0.10
	1200	3/4	3000	44.5±6.5	1.53±0.92	4/4	4000	44.2±5.2	1.75±0.33

Conclusions:

Test item was positive in micronucleus induction by gastric intubation route of administration in CD-1 mouse strain.

Executive summary:

In a micronucleus test conducted, test item was administered intraperitoneal to CD-1 mouse strain (male). Administration doses were 0 (vehicle), 150, 300, 600 amd 1200 (mg/kg). The frequency of MNPCEs increased at doses up to 1200 mg/kg.

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CD-1 ( Charles River Japan, Inc.)
- Age at study initiation: 9 week old
- Diet (e.g. ad libitum): fed commercial pellets (not specified)
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: 1 week or more

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: olive oil (henacetin was suspended homogeneously in the vehicle but could not be dissolved.)
- Concentration of test material in vehicle: was suspended in olive oil at concentrations of 1-500 mg/ml to make an injection volume of 10 ml/kg body weight for all doses

Duration of treatment / exposure:

24 hours

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

600 mg/kg bw/day (actual dose received)

Dose / conc.:

1 200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

4 mice per group were used

Control animals:

yes, concurrent vehicle

Positive control(s):

none

Tissues and cell types examined:

Micronucleated polychromatic erythrocytes (MNPCEs) in mouse bone marrow

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Table 2. Micronucleus induction of phenacetin in MS/Ae and CD-1 mice 24h after administration

Strain	Dose (mg/kg)	i.p (intraperitoneal injection)				p.o (gastric incubation)
		survival	Number of PCEs observed	Frequency of PCEs (%±SD)	Frequency of MNPCEs (%±SD)	Survival	Number of PCEs observed	Frequency of PCEs (%±SD)	Frequency of MNPCEs (%±SD)
MS/Ae	0	4/4	4000	35.4±3.7	0.53±0.10	4/4	4000	42.1±4.5	0.40±0.24
	150	4/4	4000	30.4±12.4	0.35±0.31	4/4	4000	41.1±9.0	0.48±0.10
	300	4/4	4000	34.4±4.2	0.55±0.13	4/4	4000	37.5±1.4	0.33±0.25
	600	4/4	4000	36.2±6.3	1.18±0.56	4/4	4000	37.1±8.2	1.30±0.52**
	1200	0/4	0			3/4	3000	18.4±5.9	0.43±0.35
CD-1	0	4/4	4000	47.3±5.0	0.08±0.10	4/4	4000	45.6±5.2	0.15±0.13
	150	4/4	4000	51.8±3.8	0.15±0.17	4/4	4000	44.6±3.1	0.08±0.10
	300	4/4	4000	57.2±1.2	0.25±0.13	4/4	4000	55.2±4.4	0.15±0.13
	600	4/4	4000	43.5±4.1	0.45±0.52	4/4	4000	50.8±2.4	0.43±0.10
	1200	3/4	3000	44.5±6.5	1.53±0.92	4/4	4000	44.2±5.2	1.75±0.33

Conclusions:

Test item was positive in micronucleus induction by intraperitoneal route of administration in CD-1 mouse strain.

Executive summary:

In a micronucleus test conducted, test item was administered intraperitoneal to CD-1 mouse strain (male). Administration doses were 0 (vehicle), 150, 300, 600 amd 1200 (mg/kg). The frequency of MNPCEs increased at doses up to 1200 mg/kg.

Reference 5

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: charles River Japan Inc.
- Age at study initiation: 9 weeks
- Acclimation period: 10 days

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 5% solution gumic arabic
- Justification for choice of solvent/vehicle: to prepare stable suspensions of test item.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item was administered daily by oral gavage in a volume of 10 ml/kg.

Duration of treatment / exposure:

2 days

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Just solvent 5% Gum arabic

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 animals for solvent solution and 6 animals per each dose concentration

Control animals:

yes, concurrent vehicle

Positive control(s):

none

Tissues and cell types examined:

Peripheral blood samples and bone marrow.

Evaluation criteria:

One thousand polychromatic erythrocytes (PCEs) were scored for each animal. The ratio of PCEs to total erythrocytes, based on 200 PCEs per animal, was also recorded, The table of Kastenbaum and Bowman (1970) was used to test for significance. The day 1 value served as the control for the peripheral blood assay, and a vehicle control was used for the bone marrow assay.

Statistics:

Spleen weights were analyzed statistically by the Dunnett (1955) test.

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Table 2. Micronucleus frequencies in bone marrow cells in rats treated orally for 2 days with phenacetin:

Compound	Dose (mg/kg)	Number of mice	% PCE (mean ±SD)	MNPCEs based on 1000 PVEs per animal
				individual animal data	Mean ± SD
5% Gum arabic		6	54.1±10.2	1,3,2,2,1,3	2.0±0.9
Phenacetin	500	6	41.5±16.9	1,3,3,4,2,1	2.3±1.2
	1000	6	29.3±8.6	0,7,5,2,8,5	4.5±3.5*
	2000	6	30.5±18.2	10,6,2,14,5,11	8.0±4.4**

a Three of seven rats died.
* P < 0.05, * * P < 0.01 by the table of Kastenbaum and Bowman (1970).

Conclusions:

Test item was mutagenic in the rat peripheral blood and bone marrow micronucleus test.

Executive summary:

In a peripheral blood and bone marrow micronucleus test, test item was administered by oral gavage in a volume of 10 ml/kg at dose levels 500, 1000, 20000 mg/kg. MNPCE frequencies increased significantly at 1000 (P <0.05) and 2000 (P <0.011 mg/kg following phenacetin treatment. It was not shown spleen enlargement after the test treatment. 

Reference 6

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: charles River Japan Inc.
- Age at study initiation: 6 weeks
- Acclimation period: 10 days

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 5% solution gumic arabic
- Justification for choice of solvent/vehicle: to prepare stable suspensions of test item.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item was administered daily by oral gavage in a volume of 10 ml/kg.

Duration of treatment / exposure:

14 days

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Just solvent (5% gum arabic)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

750 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 animals for solvent solution and 6 animals per each dose concentration

Control animals:

yes, concurrent vehicle

Positive control(s):

none

Tissues and cell types examined:

Peripheral blood samples and bone marrow.

Evaluation criteria:

One thousand polychromatic erythrocytes (PCEs) were scored for each animal. The ratio of PCEs to total erythrocytes, based on 200 PCEs per animal, was also recorded, The table of Kastenbaum and Bowman (1970) was used to test for significance. The day 1 value served as the control for the peripheral blood assay, and a vehicle control was used for the bone marrow assay.

Statistics:

Spleen weights were analyzed statistically by the Dunnett (1955) test.

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Table 2. Micronucleus frequencies in peripheral blood and bone marrow cells in rats treated orally for 14 days with phenacetin:

Compound	Dose (mg/kg)	Number of mice	MNRETs/1000 RETs (peripheral blood)						MNPCEs/1000 PCEs (bone marrow cells)
			day 1	day 3	day 6	day 9	day 12	day 15	day 15
5% Gum arabic		5	0.6±0.5 (3.9)a	0.4±0.5 (3.2)	1.4±1.3(3.6)	0.6±0.5(3.0)	0.8±1.3(3.1)	0.2±0.4(4.2)	1.0±0.7(48.8)b
Phenacetin	250	6	0.7±0.8(23)	0.5±0.5(3.5)	1.2±1.2(3.8)	0.7±0.8(3.2)	0.5±0.8(2.9)	1.7±1.8(4.3)	2.1±2.1(57.5)
	500	6	0.5±0.5(2.7)	1.0±1.7(3.0)	1.0±1.1(3.4)	2.0±2.3*(4.7)	2.2±1.3*(4.9)	4.3±2.0*(6.2)	3.2±1.3*(63.2)
	750	6	1.0±1.1(2.4)	1.3±1.0(2.8)	3.0±1.7*(3.8)	3.2±2.1*(3.2)	3.2±2.1*(4.8)	5.5±2.0**(9.4)	7.0±2.0**(64.6)
	1000	6	0.8±0.8(2.7)	1.5±1.5(2.3)	2.3±1.4*(2.5)	2.8±1.5*(6.0)	6.8±3.2**(6.7)	6.3±4.3**(9.2)	8.5±5.2**(65.0)

a Mean reticulocyte ratio (o/o).
b Mean polychromatic erythrocyte ratio (%).
c Ah animals died.
N.S., not scored because of low reticulocyte frequency.
* P < 0.05, * * P < 0.01 by the table of Kastenbaum and Bowman (1970).

Conclusions:

Test item was mutagenic in the rat peripheral blood and bone marrow micronucleus test.

Executive summary:

In a peripheral blood and bone marrow micronucleus test, test item was administered by oral gavage in a volume of 10 ml/kg at dose levels 250, 500, 750, 1000 mg/kg. Animals receiving only the vehicle did not show any induction of MNRETs at
any sample time in peripheral blood and of MNPCEs in bone marrow on day 15. Test item significantly induced MNRETs at 750 and 1000 mg/kg starting on day 6 (P < 0.05), and at 500 mg/kg starting on day 9 (P < 0.05). Statistically significant (P
< 0.01) spleen enlargement was observed after 14 daily treatments at 500, 750, and 1000 mg/kg. Histopathological examination revealed congestion and dilation of the sinuses (> 250 mg/kg), atrophy of the white pulp (> 750 mg/kg), increased extramedullary hematopoiesis (> 250 mg/kg), and hemosiderin deposits ( > 250 mg/kg) in the spleens of animals treated for 14 days.

Reference 7

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

not specified

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Italy (Carezzana, Italy)
- Age at study initiation: about two months old
- Weight at study initiation: 200-250g
- Housing: air conditioned room
- Diet (e.g. ad libitum): ad libitum (rat chow, TRM, Harlan, Italy).
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2ºC
- Photoperiod (hrs dark / hrs light): 12hlight/dark

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: DMSO
- Concentration of test material in vehicle: compount was dissolved and administered in a volume of 0.01 ml/g body weight;

Frequency of treatment:

Once

Post exposure period:

Rats were sacrificed for the evaluation of DNA fragmentation 20 h after treatment

Remarks:

Kidney

Remarks:

Liver

Remarks:

Urinary bladder

Remarks:

Urinary bladder

No. of animals per sex per dose:

- Each dose level was tested in 3 rats.
- 18 rats given an equal volume of the vehicle were used as controls for the urinary bladder
- 9 animals were used as controls for the liver and the kidney

Control animals:

yes, concurrent vehicle

Positive control(s):

none

Tissues and cell types examined:

Urinary bladder cells, liver and kidney

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

urinary bladder, liver and kidney

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Conclusions:

Test item was mutagenic in the raturinary bladder mucosa and kidney by comet assay (DNA fragmentation).

Executive summary:

The test item was tested for DNA damage using the comet assay, test item was administered oral by gastric intubation to male rat. The experiments were carried out using rat urinary bladder cells, liver and kidney. The vehicle was DMSO. The administration doses were 1/2 LD50 (kidney, liver and urinary bladder) and 1/4 (urinary bladder). Under the experimental conditions reported, the test substance caused genotoxicity. Therefore, the test substance is considered to be genotoxic in this comet assay.

 

Reference 8

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

not specified

Type of assay:

mammalian comet assay

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Co. (Tokyo.
- Age at study initiation: 7 weeks
- Diet (e.g. ad libitum): commercial pellets MF Oriental Yeast Industries Co., Tokyo, Japan.
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 55-65%
- Photoperiod (hrs dark / hrs light): 12-h light-dark cycle.

Route of administration:

intraperitoneal

Vehicle:

Saline, 2% Tween 80

No. of animals per sex per dose:

2 males assigned to each tretament group and 8 male were assigned to each control group

Control animals:

yes, concurrent no treatment

Positive control(s):

none

Tissues and cell types examined:

liver,, lung, spleen, kidney, and bone marrow

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION: After weighing the organs, they were minced, suspended at a concentration of 1 g/ml in chilled homogenizing buffer (pH 7.5) containing 0.075 M NaCl and 0.024 M Na2EDTA, and then homogenized gently using a Potter-type homogenizer at 500–800 rpm, in ice. To obtain nuclei, the homogenate was centrifuged at 700xg for 10 min at 8ºC, and the precipitate was re-suspended in chilled homogenizing buffer at 1 g organ weight per ml. Seventy-five ml of agarose 1% GP-42 was quickly layered on a fully frosted slide (Matsunami Glass Ind., Ltd., Osaka, Japan) and covered with another slide. The slide sandwiches were placed on ice to allow the agarose to gel. The nucleus suspension was mixed 1:1 (v/v) with 2%, 45ºC agarose–LGT, and 75 μl of the nucleus mixture was quickly layered in the same manner after removal of the covering slide. Finally, 75 μl of agarose GP-42 was quickly layered on again. The slides were placed immediately into a chilled lysing solution (pH 10) of 2.5 M NaCl, 100 mM Na EDTA, 10 mM Trizma, 1% sarkosyl, 10% 2 DMSO, and 1% Triton X-100 and kept at 08C in the dark for 60 min

METHOD OF ANALYSIS: The slides were placed on a horizontal gel electrophoresis platform and covered with chilled alkaline solution made up of 300 mM NaOH and 1 mM Na EDTA (pH 13). The slides were left in the 2 solution for 10 min in the dark at 0ºC to allow DNA unwinding and expression of alkali-labile sites. Electrophoresis was conducted simultaneously at 0ºC in the dark for 15 min at 25 V and approximately 250 mA and the slides were rinsed gently 2 times with 400 mM Trizma (pH 7.5) to neutralize the excess alkali. Each slide was stained with 50 ml ethidium bromide (Wako Pure Chemical Industries, Ltd)

Statistics:

Statistical analysis was carried out using StatView (Abacus Concepts, Inc., USA). The significance of
differences was assayed by Mann–Whitney test. For the comparison of the data between zero time control (untreated) and each of the treatment groups (3 and 24 h), a p-value less than 0.025 was considered statistically significant based on the multiplication rule for independent events. Tolerance limits were calculated according to Goldstein.

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

not examined

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: at 800 mg/kg increased DNA damage significantly at 3 h, but not at 24 h, in the kidney

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: No significant increase in migration was observed for liver, lung, spleen, and bone marrow DNA.

Table 1. Migration of nuclear DNA from 5 organs of mice treated with mutagens 

Mutagens	Dose (mg/kg)	Sampling time (h)	animal no.	Migration (μm, mean leangth ± SD)
				Liver	Kidney	Lung	Spleen	Bone marrow
Phenacetin	800	0	1	1.70±8.37	1.19±8.31	2.06±10.1	0.88±6.14	0.00±0.00
			2	3.56±14.1	2.84±12.2	3.66±14.7	1.08±5.46	1.70±8.44
			Pooled	2.63±11.7	2.01±14.5	2.86±12.6	0.98±5.81	0.85±6.03
		3	1	2.63±10.2	10.50±19.4	6.19±14.0	0.77±5.42	5.99±5.99
			2	2.22±2.22	8.84±15.4	5.73±13.6	0.00±0.00	4.23±11.5
			Pooled	2.43±8.82	9.67±18.3 a	5.96±13.8	0.39±3.85	5.11±13.7
		24	1	1.50±6.11	0.93±4.58	3.15±9.49	1.86±6.45	0.00±0.00
			2	1.91±7.67	2.89±10.5	2.37±9.53	1.65±6.62	0.00±0.00
			Pooled	1.70±6.94	1.91±8.15	2.76±9.52	1.75±6.54	0.00±0.00

Conclusions:

Interpretation of results: positive in the kidney

Executive summary:

The test item was tested for genotoxicity using single-cell gel electrophoresis (comet assay) in liver, lung, spleen, kidney and bone marrow. The experiments were carried out using male mouse from CD-1 strain. The route of administration was intraperitoneal. The test concentration was 800 mg/kg. The vehicles were: saline and 2% Tween 80. Under the experimental conditions reported, the test substance caused genotoxicity to kidney. Therefore, the test substance is considered to be genotoxic in this comet assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Justification for classification or non-classification

Genetic toxicity. Based on the available data, the substance is considered to be mutagenic and clasified as Category 2 according to GLP regulation 1272/2008.