Ammonium 3-nitrobenzoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-04-25 - 2017-04-27 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

under GLP

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Remarks:

OECD 490

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-13 - 2017-08-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

Reason / purpose for cross-reference:

reference to other study

Remarks:

OECD 471

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

OECD Guideline for the Testing of Chemicals, Part 490, adopted 29. Jul. 2016 ”In vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene“

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

EU-Method B.17 of the Commission Regulation (EC) No. 440/2008, adopted 30 May 2008: “Mutagenicity – In vitro Mammalian Cell Gene Mutation Test”

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell forward mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored in the test facility at room temperature (20 ±◦5 °C) protected from light and humidity.

Target gene:

tk+/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The cells were purchased by ATCC (Wesel, Germany) and were sold under the name L5178Y TK+/- clone (3.7.2C) [TK+/- (clone 3.7.2C)] (ATCC® CRL-9518™).
- Suitability of cells: Reasons for the Choice of the Cell Line L5178Y: The L5178Y is a murine T-cell lymphoma cell line, which grows as single or aggregated round cells in suspension. This cell line is characterized by a high sensitivity to chemical mutagens, by a high proliferation rate (doubling time 10-12 h in stock cultures), a high cloning efficiency (CE) and a stable spontaneous mutant frequency. The L5178Y consists of a stable karyotype and shows a diploid chromosome number (40 ± 2).
- Cell cycle length, doubling time or proliferation index: doubling time 10-12 h in stock cultures
- Modal number of chromosomes: 40 ± 2
- Normal (negative control) cell cycle time: doubling time 10-12 h in stock cultures

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cleansed and for mycoplasma contamination screened stocks of cells were stored in liquid nitrogen in the cell bank of the laboratory to allow a continuous stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells.
Cells were thawed 6 d prior treatment and cultivated in RPMI 1640 complete culture medium with 5 % HS in cell culture flasks at 37.0 ± 1.0 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

According to the OECD guideline 490 the highest concentration should be 0.01 M or 2 mg/mL or 2 µL/mL (whichever is lowest), unless limited by the solubility or toxicity of the test item. In reference to the results of the pre-test, 6 concentrations were chosen for experiment: 10, 5, 2.5, 1.25, 0.63, 0.31, 0.16 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 1640 medium without supplements
- Justification for choice of solvent/vehicle: Solubility was tested for the determination of a suitable solvent for the test item in a non-GLP pre-test with RPMI 1640. The test item was soluble in this medium at the required concentration (100 mM).

Untreated negative controls:

yes

Remarks:

solvent control, i.e. medium

Negative solvent / vehicle controls:

yes

Remarks:

medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h resp. 24h
- Expression time (cells in growth medium): 48h
- Selection time (if incubation with a selection agent): 10-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13-15 days

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2 independent experiments, two 96 well microtiter plates per concnetration

DETERMINATION OF CYTOTOXICITY
- Method: relative cloning efficiency

Rationale for test conditions:

as stipulated by the guideline; concentrations were based on the content of solids in the test item which was tested as aqueous solution

Evaluation criteria:

Due to limitations of this free-text field, please see "Any other information on materials and methods".

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Remarks:

medium

Untreated negative controls validity:

valid

Remarks:

medium

Positive controls validity:

valid

Conclusions:

The study was conducted under GLP according to OECD guideline 490 (mouse lymphoma assay) on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation. Positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of Ammonium-3-nitrobenzoate to induce gene mutations in mammalian cells. In all evaluated concentrations of the test item no substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item. The mutation frequency did not reach or exceed the threshold of 126 above the corresponding solvent control. In conclusion, it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the thymidine kinase locus (Tk1) in heterozygous mouse lymphoma L5178Y Tk+/- cells. Therefore, the test item Ammonium-3-nitrobenzoate is considered to be “not mutagenic under the conditions of the mouse lymphoma assay”.
In the available Ames test, Ammonium-3-nitrobenzoate showed an increase in the number of revertants in three bacteria strains in the experiment. Based on the results of this study it is concluded that Ammonium-3-nitrobenzoate is mutagenic in the Salmonella typhimurium strains TA98, TA100 and TA102 in the absence and presence of metabolic activation under the experimental conditions. As the present forward mutation assay in mammalian cells can be considered as higher tier test, as the test system is more related to humans than bacteria, the negative results in the present OECD 490 can be considered more relevant for the assessment of Ammonium-3-nitrobenzoate than the positive one in the bacteria assay. Hence, it can be concluded that Ammonium-3-nitrobenzoate does not bear a relevant genotoxic potential for humans.

Executive summary:

This study was performed to investigate the potential ofAmmonium-3-nitrobenzoateto induce mutations at the thymidine kinase locus (Tk1) on chromosome 11 and/or structural chromosomal aberrations in mouse lymphoma L5178YTk^+/-cells according to OECD 490 under GLP.

The assay was performed in a pre-test and two independent experiments (experiment I and II). The pre-test was performed to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the two experiments were determined.

Experiment I was performed with and without metabolic activation (liver enzyme S9 fraction / “liver S9 mix from male rats, treated with Aroclor 1254”) and a treatment period of 4 h. Experiment II was performed with a treatment period of 24 h in the absence of metabolic activation.

The highest nominal concentration (10 mM) applied was chosen with regard to the solubility of the test item in organic solvents and aqueous media and the cytotoxicity.

Precipitation or turbidity of the test item was not visible in all experimental parts at the maximum concentration of the test item.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.

Conclusion:In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Therefore, the test itemAmmonium-3-nitrobenzoateis considered to be “non-mutagenic under the conditions of the mouse lymphoma assay”.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for the Testing of Chemicals Part 471, adopted 21. Jul. 1997 “Bacterial Reverse Mutation Test“

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

Commission Regulation (EC) No. 440/2008, EU-Method B.13/14 adopted 30. May 2008 “Mutagenicity –Reverse mutation test using bacteria”

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

by the Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium 3-nitrobenzoate

EC Number:

242-965-2

EC Name:

Ammonium 3-nitrobenzoate

Cas Number:

19328-56-4

Molecular formula:

C7H5NO4.H3N

IUPAC Name:

ammonium 3-nitrobenzoate

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

liquid

Details on test material:

- Storage: Room Temperature (20 ± 5 °C)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored in the test facility in a closed vessel at room temperature (18.0 – 22.5 °C).

FORM AS APPLIED IN THE TEST (if different from that of starting material)
aqueous solution

Method

Target gene:

his-

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA97a

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: obtained from TRINOVA BioChem GmbH, batch 4997D

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: obtained from TRINOVA BioChem GmbH, batch 5011D

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: obtained from TRINOVA BioChem GmbH, batch 4996D

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: obtained from TRINOVA BioChem GmbH, batch 4982D

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: obtained from TRINOVA BioChem GmbH, batch 5012D

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

5 / 1.5 / 0.5 / 0.15 / 0.05 µg/plate (based on the content of Ammonium-3-nitrobenzoate in the test item)
Top dose as required from the guideline.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: demin. water
- Justification for choice of solvent/vehicle: In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 mL/L in demineralized water, dimethyl sulfoxide (DMSO) and ethanol. Demin. water was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Controls

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

demin. water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene Diamine, CAS-No.: 99-56-9 / 2-Amino-anthracene, CAS-No.: 613-13-8

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 100 µL bacteria suspension per plate

DURATION
- Preincubation period: none
- Exposure duration: 48h

SELECTION AGENT (mutation assays): Histidine minimal medium

NUMBER OF REPLICATIONS:
Per strain and dose, three plates with and three plates without S9 mix were used.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Rationale for test conditions:

as described in OECD TG 471

Evaluation criteria:

Evaluation
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Statistics:

The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

at 1.5 and 5 mg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Remarks:

vehicle controls

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

at 0.5, 1.5, and 5 mg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Remarks:

vehicle controls

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

at 0.15, 0.5, 1.5, and 5 mg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Remarks:

vehicle controls

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

at 5 mg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Remarks:

vehicle controls

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium, other: 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Remarks:

vehicle controls

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Remarks:

vehicle controls

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none stated
- Water solubility: not exceeded
- Precipitation: The test item showed no precipitates on the plates in any tested concentration.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
- Negative (solvent/vehicle) historical control data: All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No signs of toxicity towards the bacteria strains could be observed. The bacterial back-ground lawn was visible and not affected. The number of revertant colonies was not reduced.

Applicant's summary and conclusion

Conclusions:

The study was conducted under GLP according to OECD guideline 471 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. the substance was tested in the form in which it is distributed, i.e. the marketed aqueous solution. However, the applied amounts were corrected for the actual content of the registered substance, Ammonium-3-nitrobenzoate, in the aqueous solution, so that the limit dose of 5 mg/plate of Ammonium-3-nitrobenzoate was reached and the study can be considered valid. Hence, the results can be considered as reliable to assess the potential of Ammonium-3-nitrobenzoate to induce reverse mutations in bacteria.
The test item Ammonium-3-nitrobenzoate showed an increase in the number of revertants in three bacteria strains in the experiment. Based on the results of this study it is concluded that Ammonium-3-nitrobenzoate is mutagenic in the Salmonella typhimurium strains TA98, TA100 and TA102 in the absence and presence of metabolic activation under the experimental conditions.
In consequence, a gene mutation test in mammalian cells is required as a follow up to evaluate the toxicological significance of this positive result. In the additionally conducted forward mutation assay in mammalian cells, no indication for a genotoxic activity of the test item was found. The OECD 490 study can be considered as higher tier test, as the test system is more related to humans than bacteria, the negative results in the present OECD 490 can be considered more relevant for the assessment of Ammonium-3-nitrobenzoate than the positive one in the bacteria assay. Hence, it can be concluded that Ammonium-3-nitrobenzoate does not bear a relevant genotoxic potential for humans.

Executive summary:

The mutagenic potential of Ammonium-3-nitrobenzoate was determined under GLP with the Bacterial Reverse Mutation Test following OECD 471 and EU B.13/14, most recent versions.

One valid experiment was performed.

The test item Ammonium-3-nitrobenzoate was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535).

The test was performed in one experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

In the experiment, the test item (dissolved in demin. water) was tested up to concentrations of 5 mg/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains.

The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiment showed an increase in the number of revertants in the following tested strains and concentrations:

TA98 with metabolic activation: 5 mg/plate and 1.5 mg/plate

TA98 without metabolic activation: 5 mg/plate, 1.5 mg/plate and 0.5  mg/plate

TA100 with and without metabolic activation: 5 mg/plate, 1.5 mg/plate, 0.5 mg/plate and 0.15  mg/plate

TA102 with and without metabolic activation: 5 mg/plate

Based on the results of this study it is concluded that Ammonium-3-nitrobenzoate is mutagenic in the Salmonella typhimurium strains TA98, TA100 and TA102 in the absence and presence of metabolic activation under the experimental conditions.