m-toluic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reliable data on the mutagenicity of m-toluic acid are available from bacterial and mammalian cells gene mutation assays and an in vivo micronucleus tests in rats. m-Toluic acid showed in none of these tests a genotoxic potential.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

yes

Remarks:

evaluation criteria not in accordance with OECD guidelines; tests were conducted partially above limit dose of 0.01 M (equivalent to 1361 µg/mL for m-toluic acid), namely 1500 and 2000 µg/mL; less than 3 analysable test concentrations due to cytotoxicity

GLP compliance:

yes

Type of assay:

other: in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored in a cool and dark place
- Stability of test material confirmed by the supplier

Target gene:

not applicable

Species / strain / cell type:

mammalian cell line, other: CHL/IU

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: derived from female Chinese hamster lung obtained from Pharmaceutical Co., Ltd., Japan (assumed)
- Number of passages if applicable: 5
- Methods for maintenance in cell culture if applicable: incubation at 37 °C, in a humidified 5 % CO2 atmosphere

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: culture medium consisted of Eagle's minimum medium (MEM) supplemented with calf serum to a final concentration of 10 %

Cytokinesis block (if used):

Colcemid

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (containing 30 % S9; 5 mM D-glucose-6-phosphate, approx. 4,5 mM NADP+, 4 mM HEPES (pH 7.2), 5 mM MgCl x 6H2O, 33 mM KCl, sterile water),

Test concentrations with justification for top dose:

A cell proliferation inhibition test was conducted for purpose of dose range finding.
Experiment 1 (short time exposure): 250, 500, 1000 and 2000 µg/mL with and without S9
Experiment 2 (continuous exposure): 250, 500, 1000 and 2000 µg/mL for 24 hour treatment; 62.5, 125, 250, 500 and 1000 µg/mL for 48 hour treatment
Confirmation test: 1000, 1500 and 2000 µg/mL without S9; 500, 750 and 1000 µg/mL with S9

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: m-toluic acid was not well soluble in physiological saline and did not reach a solubility of 50 mg/mL. Dissolution of 50 mg m-toluic acid in 1 % carboxymethylcellulose aqueous solution resulted in an inhomogenous suspension. m-Toluic acid was soluble in DMSO up to the limit concentration of 50 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

TOXICITY TEST:
- 4 x 10 ^4 cells/mL were seeded and cultured for 3 days
- cells were treated with test substance as described in experiment 1 (short term exposure, 6 hours, with and without metabolic activation) and 2 (continuous exposure, 24 or 48 hours, without metabolic activation)
- each concentration was tested in duplicates
- pH was recorded during the experiments
- cells were harvested (please see below) and counted with a hemocytometer

MUTAGENICITY TEST (Experiment 1 and Experiment 2):
- 4 x 10 ^4 cells/mL were seeded and cultured for 3 days
- cells were treated with test substance as described in experiment 1 (short term exposure for 6 hours, 18 hours recovery, with and without metabolic activation) and 2 (continuous exposure, 24 or 48 hours, without metabolic activation)
- each concentration was tested in duplicates
- pH was recorded during the experiments

NUMBER OF REPLICATIONS: duplicate

CELL HARVESTING:
- 2 hours before the end of the incubation period, the cell division was arrested by the addition of 0.1 μg/ml Colcemid solution and incubated for further 2 hours.
- 24 hours or 48 hours after beginning of the treatment the cells were harvested: cells were washed with PBS, trypsinised and centrifuged
- the cell pellet was resuspended in 0.075 M KCl solution (hypotonic) and incubated 15 minutes at 37 °C
- cells were fixed by adding 4 mL of a cooled methanol/acetic acid solution (3:1). After centrifugation, supernatant was discarded and fixation was repeated 2-3 times.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- cells were centrifuged and resuspended in fresh fixative.
- two drops of this suspension were transferred to a microscopic slide and left to air-dry.
- at least two slides/culture were prepared.
- slides were stained in 3 % Giemsa solution for 20 minutes, washed with water, left to air-dry and were sealed

CONFIRMATION TEST:
- the test was conducted as described for mutagenicity tests above.
- cells were exposed to the test substance for 6 hours in the absence of metabolic activation and had a recovery time of 18 hours


NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE:
- slides were observed under a microscope
- for a preliminary confirmation of suitability of the test, 50 cells from highest dose were analysed
- for evaluating the mitotic index/ mitotic activity, 1000 cells/slide. Mitotic index/ mitotic activity was calculated from a total of 2000 cells (duplicate)
- for evaluation of chromosomal aberration, 100 cells/slide. Mitotic index/ mitotic activity was calculated from a total of 200 cells (duplicate)

Rationale for test conditions:

According to the preliminary toxicity test the dose levels were chosen for the main study.

Evaluation criteria:

A cell having one or more structural abnormality was regarded as a chromosomal abnormal cell.
The test substance was regarded negative for induction of chromosomal aberrations, when the frequency of appearance of structural and numerically aberrant cells is < 5 %.
The test substance was regarded positive for induction of chromosomal aberrations, when the frequency of appearance of structural and numerically aberrant cells is >= 10 %.
The test substance was regarded equivocal for induction of chromosomal aberrations, when the frequency of appearance of structural and numerically aberrant cells is >= 5 % and < 10 %.

Statistics:

no statistical method was used

Species / strain:

mammalian cell line, other: CHL/IU

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

please refer to the field "Additional information on results"

Vehicle controls validity:

not specified

Untreated negative controls validity:

not examined

Positive controls validity:

not specified

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: the pH of cell culture medium wasaffected by test substance, the lowest pH observed while inducing chromosomal aberrations was 6.2
- Precipitation: test substance precipitated at test concentration of 1000 µg/mL and above
- the culture medium changed to yellow colour at test concentrations of 1000 µg/mL and above

RANGE-FINDING/SCREENING STUDIES/ CYTOTOXICITY TESTS:
- the calculated IC50 from preliminary testing were the following:
- for continuous treatment (without metabolic activation): 812 µg/mL after 24 hours and 455 µg/mL after 48 hours
- for short term exposure (6 hours): 863 µg/mL (with metabolic activation) and 1182 µg/mL without metabolic activation)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: cells were counted with a hemocytometer and IC50 was calculated by the probit method


CHROMOSOMAL ABERRATION TEST:
- the mimimum dose at which aberrations were found in 20 % of metaphases was calculated:
D20 (continuous exposure, 48 hours, without metabolic activation) = 2 mg/mL
D20 (short term exposure, 6 hours, without metabolic activation) = 1.2 mg/mL

- experiment 1 (short term exposure, with and without metabolic activation):
2000 µg/mL (without metabolic activation): equivocal result for chromosomal aberration was found at a frequency of 7 % (only one replicate analysed due to cytotoxicity)
1000 µg/mL (with metabolic activation): equivocal result for chromosomal aberration was found at a frequency of 5 %

-experiment 2 (continuous exposure, without metabolic activation):
1000 µg/mL (after 48 hours): positive result for chromosomal aberration was found at a frequency of 11 % (only one replicate analysed due to cytotoxicity)
500 µg/mL (after 48 hours): equivocal result for chromosomal aberration was found at a frequency of 5 %

- confirmation test (short term exposure, without metabolic activation)
2000 µg/mL: positive result for chromosomal aberration was found at a frequency of 86.6 %
1500 µg/mL: equivocal result for chromosomal aberration was found at a frequency of 8.5 %

For additional information please refer to "Any other information on results incl. tables"

Table1: continuous treatment
Substance	S9 mix	Incubation time (hours)	Concentration (µg/mL)	Number of observed cells (mean of two experiments)	Cells with aberrations (%, mean of two experiments)		Mitotic index (2000 cells observed)	Mitotic activity (%)
					- gap	+ gap
DMSO	-	24	0	200	0.5	0.5	5.9	100
m-toluic acid	-	24	250	200	1.5	1.5	2.6	43
	-	24	500	200	1.5	1.5	2.2	36
	-	24	1000°*	200	3.5	3.5	3.7	63
Mitomycin C	-	24	0.03	200	19.5	19.5	2.4	40
° precipitation of test substance * culture medium turned yellow with test substance - Cytotoxicity marked with grey colour

Table2: Short time treatment
Substance	S9 mix	Incubation time (hours)	Concentration (µg/mL)	Number of observed cells (mean of two experiments)	Cells with aberrations (%, mean of two experiments)		Mitotic index (2000 cells observed)	Mitotic activity (%)
					- gap	+ gap
DMSO	-	6	0	200	1.5	1.5	4.7	100
	+	6	0	200	0.0	0.0	6.5	100
m-toluic acid	-	6	250	200	0.5	0.5	3.7	78
	-	6	500	200	1.0	2.0	4.6	98
	-	6	1000°*	200	1.5	1.5	4.9	104
	+	6	250	200	0.0	0.5	6.5	101
	+	6	500	200	1.0	1.5	9.4	145
	+	6	1000°*	200	5.0	5.0	2.9	44
Benzo-[a]-pyren	-	6	20	200	1.5	1.5	7.3	155
	+	6	20	200	82.0	82.5	1.4	21
° precipitation of test substance * culture medium turned yellow with test substance - Cytotoxicity marked with grey colour Table 3:Confirmation test (short time)     Substance S9 mix Incubation time (hours) Concentration (µg/mL) Number of observed cells (mean of two experiments) Cells with aberrations (%, mean of two experiments) Mitotic index (2000 cells observed) Mitotic activity (%)     - gap + gap     DMSO - 6 0 200 0.5 1.0 5.2 100     + 6 0 200 0.5 0.5 8.6 100     m-toluic acid - 6 1000° 200 0.5 0.5 6.7 129     - 6 1500°* 200 8.5 8.5 4.8 92     + 6 500 200 0.5 0.5 6.6 77     + 6 750° 200 1.0 1.0 5.8 68     + 6 1000°* 200 4.5 4.5 3.3 38     Benzo-[a]-pyren - 6 20 200 1.0 1.0 5.2 101     + 6 20 200 83.5 83.5 0.9 11     ° precipitation of test substance * culture medium turned yellow with test substance - Cytotoxicity marked with grey colour

Conclusions:

The authors concluded that the test substance increased the frequency of of cells with chromosomal aberrations. However, these effects might be related to cytotoxicity or osmotic/ionic effects. Data from dosages above the limit concentration of 0.01 M (according to 1361 mg/L for m-toluic acid) stated by OECD 473 cannot be clearly related to the test substance.
The substance tested equivocal under the conditions of the study. Thus, classification according to Regulation (EC) No 1272/2008 and subsequent adaptations is not possible.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-02-23 to 1998-03-27

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

2-aminoanthracene was used as the sole indicator of the efficacy of the S9 mix. Only 4 analysable results due to cytotoxicity in TA100, TA1535 and TA1537.

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerated in the dark

Target gene:

TA98: hisD
TA100: hisG
TA1535: hisG
TA1537: hisC
WP2uvrA: trpE

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: uvrB, rfa

Remarks:

TA98 and TA100: pKM101

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (containing 10 % S9, 8 µM MgCl2 x 6 H2O, 33 µM KCl, 5 µM D-Glucose-6-phosphat, 4 µM NADPH, 4µM NADH, 100 µM sodium buffer (pH 7.4), sterile water))

Test concentrations with justification for top dose:

A preliminary test (preincubation, with and without metabolic activation) was conducted for purpose of dose range finding with the following concentrations: 1.22, 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate.
The following concentrations were selected for the main tests (preincubation method, without metabolic activation): 156, 313, 625, 1250, 2500 and 5000 µg/plate and (preincubation method, with metabolic activation): 313, 625, 1250, 2500 and 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance was insoluble in water at a concentration of 50 mg/mL. Thus, the test substance was dissolved in DMSO and diluted with DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

furylfuramide

other: 2-aminoanthracene

Remarks:

positive control substances were dissolved in DMSO, except sodium azide (dissolved in water for injection)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

MAIN EXPERIMENT (two independent experiments with and without metabolic activation)
The test item was preincubated with the test strain (containing approximately 10^9 viable cells/mL) and sterile buffer (0.5 mL of 0.1 M sodium phosphate buffer, pH 7.4) or the metabolic activation system (0.5 mL of S9 mix) for 20 minutes at 37°C prior to mixing with the overlay agar and pouring onto the surface of a minimal agar plate.
0.1 mL of test item (or 0.1 mL solvent or 0.1 mL positive control), 0.1 mL of bacteria, and 0.5 mL of S9 mix or sterile buffer, were mixed with 2 mL of overlay agar. Tubes were aerated during preincubation by using a shaker.
The plates were incubated at 37°C for 48 hours. The revertant colonies on the test plates and on the control plates were counted with a colony counter or visually.

NUMBER OF REPLICATIONS: 3 replicates for each experimental point in the main experiment, one sample in the preliminary test

DETERMINATION OF CYTOTOXICITY
Prior to the main test a preliminary test (preincubation method, with and without metabolic activation) was performed in the test strains TA98, TA100, TA1535, TA1537 and WP2uvrA. The following concentrations were tested: 1.22, 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate.

Rationale for test conditions:

The test concentrations were chosen based on a preliminary test. Cytotoxicity was observed at 5000 µg/plate (with and without metabolic activation). Precipitation was observed for a concentration of 5000 µg/plate (with metabolic activation). No mutagenicity was observed.

Evaluation criteria:

A test item is considered to show a positive response if
- the number of revertants is increased compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and WP2uvrA
- positive results have to be reproducible
Biological relevance of the results should be considered first.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

Statistics:

no statistical method was used

Key result

Species / strain:

other: TA98, TA100, TA1535, TA1537, and WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

please refer to the field "Additional information on results"

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

please refer to the field "Additional information on results"

Vehicle controls validity:

not specified

Untreated negative controls validity:

not examined

Positive controls validity:

not specified

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
- test item precipitation was noted at a concentration 5000 μg/plate in the presence of S9 mix in all test strains.
- cytotoxicity was observed at a concentration 5000 μg/plate in the absence of S9 mix in all test strains.

MAIN STUDY (2 independent experiments)
1) Test-specific confounding factors:
- Precipitation: test item precipitation was noted in both experiments with metabolic activation, at a concentration of 5000 μg/plate in all test strains.

2) Cytotoxicity:
- cytotoxicity (reduction of the number of revertants by more than 50%) was noted in both experiments with metabolic activation in all test strains at 5000 μg/plate and in test strains TA100, TA1535 and TA1537 at 2500 µg/plate.

3) Genotoxicity:
- no increase in revertant colony numbers as compared with control counts was observed for the test item in any of the two independent experiments without and with metabolic activation.
- positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

Conclusions:

The substance tested non-mutagenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance is not considered to have a mutagenic potential.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: Gene mutation (hprt locus)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-09-27 to 2018-03-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Type of assay:

other: mammalian cell gene mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 151226
- Expiration date of the lot/batch: 2018-05-20

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15-25°C, protected from light
- Stability under test conditions: Confirmed by the Sponsor

Target gene:

hprt

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co.
- Cell doubling time: 10-12 hours (average)


MEDIA USED
- Type and identity of media: RPMI 1640 media containing 2.5 µg/mL Amphotericin B, 100 µg/mL Streptomycin, 100 units/mL Penicillin, 0.5 µg/mL Pluronic (except for RPMI 20) and 0%, 10% or 20% (v/v) heat inactivated horse serum for RPMI A, RPMI 10 and RPMI 20,
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Range-Finder Experiment:
with and without S9-mix: 42.56, 85.13, 170.3, 340.5, 681, and 1362 µg/mL

Mutation Experiment:
with and without S9-mix: 50, 100, 200, 400, 600, 800, 1000, and 1362 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: anhydrous analytical dimethyl sulphoxide (DMSO)

- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that m-toluic acid was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at concentrations up to at least 193.12 mg/mL. The solubility limit in culture medium was in excess of 1931.2 μg/mL, as indicated by the absence of precipitation at this concentration following 3 hours of incubation at 37±1°C.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO diluted 100-fold in the treatment medium.

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO diluted 100-fold in the treatment medium.

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours at 37°C
- Expression time (cells in growth medium): Cultures were maintained in flasks for a period of 7 days during which the hprt mutation would be expressed.
- Selection time (if incubation with a selection agent): Using a multichannel pipette, 0.2 mL of each suspension was placed into each well of 4 x 96-well microtitre plates (384 wells at 2 x 104 cells/well). Plates were incubated at 37±1°C in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (12 days) and wells containing clones were identified as above and counted.

SELECTION AGENT (mutation assays): 6-thioguanine (6TG)

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative survival
- Any supplementary information relevant to cytotoxicity: Treatment of cell cultures for the cytotoxicity Range-Finder Experiment was as described below for the Mutation Experiment. However, single cultures only were used and positive controls were not included. The final treatment volume was 20 mL. Following 3 hour treatment, cells were centrifuged (200 g), washed with tissue culture medium and resuspended in 20 mL RPMI 10. Cell concentrations were adjusted to 8 cells/mL and, for each concentration, 0.2 mL was plated into each well of a 96-well microtitre plate for determination of relative survival. The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air for 10 days. Wells containing viable clones were identified by eye using background illumination and counted.

EVLAUATION: Wells containing viable clones were identified by eye using background illumination and counted.

- OTHER:
- Probable number of clones/well (P) = -ln (empty wells (without clones) /total of TW),
- Plating efficiency (PE) = P/No of cells plated per well,
- Percentage relative survival (% RS) = % RS = [PE (test)/PE (control)] x 100,
- Adjusted %RS = %RS x post-treatment cell concentration for test article treatment/post-treatment cell concentration for vehicle control,
- Mutant frequency (MF) = [PE (mutant)/PE (viable)] x 10^6.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF at one or more concentrations was significantly greater than that of the
negative control (p≤0.05)

2. There was a significant concentration-relationship as indicated by the linear trend
analysis (p≤0.05)

3. If both of the above criteria were fulfilled, the results should exceed the upper
limit of the last 20 studies in the historical negative control database (mean MF
+/- 2 standard deviations.

Results that only partially satisfied the assessment criteria described above were
considered on a case-by-case basis.

Statistics:

Statistical significance of mutant frequencies was assessed according to the UKEMSguidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

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

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: Osmolality (measured using a Fiske Osmometer) and pH (measured using a Denver pH 215A) measurements on post-treatment media were taken in the cytotoxicity Range-Finder Experiment.No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentration tested (1362 μg/mL) as compared to the concurrent vehicle controls (measured data not reported).

- Precipitation:
Eight concentrations were tested in the absence and presence of S-9 ranging from 50 to 1362 μg/mL. Upon addition of the test article to the cultures, precipitate was observed at the highest four concentrations tested in the absence and presence of S-9 (600 to 1362 μg/mL). Following the 3 hour treatment incubation period, no precipitate was observed at any concentration tested in the absence or presence of S-9. (Mutation experiment)

Six concentrations were tested in the absence and presence of S-9 ranging from 42.56 to 1362 μg/mL (equivalent to 10 mM at the highest concentration tested). Upon addition of the test article to the cultures, precipitate was observed at the highest two concentrations tested in the absence and presence of S-9 (681 and 1362 μg/mL). Following the 3 hour treatment incubation period, no precipitate was observed at any concentration tested in the absence or presence of S-9. (Range-Finder Experiment)


RANGE-FINDING/SCREENING STUDIES:
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 42.56 to 1362 μg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration tested in the absence and presence of S-9 (1362 μg/mL) gave 60% and 61% RS.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the Mutation Experiment, eight concentrations were tested in the absence and presence of S-9, ranging from 50 to 1362 μg/mL. Seven days after treatment all
concentrations in the absence and presence of S-9 were selected to determine viability and 6TG resistance. The highest concentration tested in the absence and presence of S-9 (1362 μg/mL) gave 62% and 57% RS, respectively.

Conclusions:

It is concluded that m-toluic acid did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to a concentration equivalent to 10 mM, an acceptable maximum concentration for this type of study according to current regulatory test guidelines, in the absence and presence of a rat liver metabolic
activation system (S-9).

Executive summary:

The test article, m-toluic acid, was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical dimethyl sulphoxide (DMSO). A 3 hour treatment incubation period was used for each experiment. In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 42.56 to 1362 μg/mL (equivalent to 10 mM at the highest concentration tested and an acceptable maximum concentration for this type of study according to current regulatory test guidelines). The highest concentration tested (1362 μg/mL) gave 60% and 61% Relative Survival (RS), in the absence and presence of S-9, respectively. In the Mutation Experiment, eight concentrations were tested in the absence and presence of S-9, ranging from 50 to 1362 μg/mL. Seven days after treatment all concentrations in the absence and presence of S-9 were selected to determine viability and 6TG resistance. The highest concentration tested in the absence and presence of

S-9 (1362 μg/mL) gave 62% and 57% RS, respectively. Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures

fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid. Following 3 hour treatment in the absence and presence of S-9, no statistically significant increases in MF, compared to the vehicle control MF values, were observed at any concentration analysed and there were no statistically significant positive linear trends. All values were within the historical ranges generated by the last twenty experiments performed in this laboratory (1.22 to 7.60 mutants per 106 viable cells in the absence of S-9 and 2.37 to 7.03 mutants per 106 viable cells in the presence of S-9).

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990 - 1992

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)

Version / remarks:

1997

Deviations:

yes

Remarks:

not tested up to the limit concentration of 5 mg/plate. No strain for detection of certain oxidising mutagens, cross-linking agents and hydrazines.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Target gene encoding histidine

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

S. typhimurium TA 97

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

with and without

Metabolic activation system:

induced male Sprague-Dawley rat liver S9 (10 % and 30 %), induced Syrian hamster liver S9 (10 % and 30 %)

Test concentrations with justification for top dose:

0, 33, 100, 333, 1000, 3333 µg/plate, justification for top dose was not specified

Vehicle / solvent:

- Vehicle used: DMSO

Untreated negative controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine

Remarks:

positive control substances were dissolved in DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

MAIN EXPERIMENT (two independent experiments with (10 % RLI, 30 % RLI, 10 % HLIand 30 % HLI respectively) and without metabolic activation)
Test solution (0.05 mL) was incubated with 0.05 or 0.1 mL of overnight culture plus 0.5 mL of S9 mix for 20 min at 37 °C, then top agar was added and the cells were plated on petri dishes containing Vogel-Bonner medium.
The plates were incubated at 37°C for 48 hours. Colonies were machine counted unless precipitate or colour interfered.

NUMBER OF REPLICATIONS: 3 replicates for each experimental point

DETERMINATION OF CYTOTOXICITY
Yes

OTHER
Precipitation and contamination was determined

Rationale for test conditions:

The conduction of this study is part of NTPs evaluation for health-related effects using study protocols designed to characterise the toxic potential of a substance.

Evaluation criteria:

A positive response for the assay is defined as a reproducible, dose-related increase in histidine-independent or tryptophan-independent (revertant) colonies in any one strain/activation combination. An equivocal response is defined as an increase in revertants that is not dose-related, is not reproducible, or is not of sufficient magnitude to support a determination of mutagenicity. A negative response is obtained when no increase in revertant colonies is observed following chemical treatment in any strain/activation combination. There is no minimum percentage or fold-increase required for a chemical to be judged positive or weakly positive.

References

Mortelmans K, Zeiger E. The Ames Salmonella/microsome mutagenicity assay. Mutat Res. 2000 Nov 20;455(1-2):29-60.

Mortelmans K, Riccio ES. The bacterial tryptophan reverse mutation assay with Escherichia coli WP2. Mutat Res. 2000 Nov 20;455(1-2):61-9. Review. PMID: 11113467

Statistics:

not specified

Key result

Species / strain:

other: TA97, TA98, TA100, T1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Please refer to the field " Any other information results incl. tables".

Table 1: Individual strain data is presented as mean ± standard error. Trial summary calls are shown in parentheses.

Strain: TA100
Dose	No Activation		No Activation		30% RLI		30% HLI		10% RLI		10% HLI
	(Negative)		(Negative)		(Negative)		(Negative)		(Negative)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
ug/Plate	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM
0	110	4.5	155	9.5	122	3.9	121	4.3	155	5	139	4.9
33	116	3.4	153	5.6	122	4.8	113	4.3	164	.3	165	4.6
100	114	4.6	155	6.8	112	1.8	119	6	156	2.7	149	10.7
333	113	.9	154	4.3	103	12	113	4.6	148	7	166	9.5
1000	116	2.9	140	3	116	1.5	114	3	148	11.6	152	8.7
3333	111	2.2	120	6.2	114	.7	115	5	140	9	125	9
Positive Control	646	12.6	906	20.2	311	18	431	5.8	339	8.7	474	19.9
Strain: TA1535
Dose	No Activation		No Activation		30% RLI		30% HLI		10% RLI		10% HLI
	(Negative)		(Negative)		(Negative)		(Negative)		(Negative)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
ug/Plate	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM
0	13	2.1	14	2.7	14	.3	12	1.5	18	0	11	.9
33	14	.6	17	1.5	13	.9	12	1.2	16	3.3	16	2.9
100	13	.7	16	1	15	3	17	2.2	13	1.3	16	2.3
333	14	1	14	1.3	12	1.5	14	3.4	13	1.5	15	2.1
1000	12	.6	14	2.5	13	2.4	18 c	0	11	1	13	.3
3333	13	.3	15	1.9	15	2.1	13	1.5	13	2.7	13	4.1
Positive Control	948	25.6	808	9.8	117	6.4	504	51.7	112	6.6	124	10.5
Strain: TA1537
Dose	No Activation
	(Negative)
Protocol	Preincubation
ug/Plate	Mean	±SEM
0	6	.6
33	5	1.2
66	6	.9
100	4	1.9
116	5	.7
333	3	.7
1000	5	.6
3333	4	1.9
Positive Control	227	20.1
Strain: TA97
Dose	No Activation		No Activation		30% RLI		30% HLI		10% RLI		10% HLI
	(Equivocal)		(Negative)		(Negative)		(Negative)		(Negative)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
ug/Plate	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM
0	138	1.2	121	5.5	151	12.1	143	19.1	178	4.9	176	6.8
33	132	4.5	129	3.3	166	11.8	168	2.9	185	9.3	175	6.3
66			152	17.2
100	211	7.2	154	14.6	170	6.7	151	5	176	12	171	4.6
116			157	10.2
333	201	24.1	136	10.7	144	4.4	162 c	28	173	7.8	192	1.8
1000	174 c	.5	141	6	159	3.2	166	5.8	172	5.5	174	.6
3333	89	21.4	107	10.3	160	11.9	128	11.4	92	6.7	58	3.1
Positive Control	393	7.2	456	12.6	311	17.3	393	17.5	369	10.4	475	15.5
Strain: TA98
Dose	No Activation		No Activation		30% RLI		30% HLI		10% RLI		10% HLI
	(Negative)		(Negative)		(Negative)		(Negative)		(Negative)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
ug/Plate	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM
0	12	.3	18	.6	16	1.5	17	1.8	24	2.1	21	1.3
33	12	1.2	18	2.2	19	4.1	15	.6	23	2	22	.9
100	13	1.3	21	1.5	17	.6	18	5.6	23	1.2	23	3.9
333	11	1.5	18	2.3	15	2.3	17	1.3	26	1.3	18 c	1
1000	10	.3	17	1.5	12	.6	15	.7	25	2	23	.6
3333	9	2	18	1	11	2	8	.6	17	2.3	19	.9
Positive Control	346	17.6	415	13.9	188	12.7	289	19.1	258	19.5	465	24.2
Abbreviations:
RLI = induced male Sprague Dawley rat liver S9
HLI = induced male Syrian hamster liver S9
s = Slight Toxicity; p = Precipitate; x = Slight Toxicity and Precipitate; t = Toxic; c = Contamination

Conclusions:

The substance tested non-mutagenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance is not considered to have a mutagenic potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

m-Toluic acid has been tested in an vivo cytogenicity test (bone marrow micronucleus test) in rats following oral exposure up to the limit dose. The test revealed a negative response, thus m-toluic acid is considered non-clastogenic and non-aneugenic.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-02-20 to 2002-03-27

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

yes

Remarks:

source of test material not stated

GLP compliance:

yes

Type of assay:

other: Micronucleus Assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lot No. 1015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in a light-resistant container at room temperature
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- pulverisation to a fine powder in an agate mortar

Species:

rat

Strain:

Crj: CD(SD)

Remarks:

IGS

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS - Crj: CD(SD), IGS
- Source: Charles River Japan, Inc. (IGS refers to animals bred using the Charles River International Genetic Standardisation system)
- Age at study initiation (at administration): 7 weeks
- Weight at study initiation (at administration): 259 - 277 g (confirmation test: 266 - 289 g)
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing:
- Diet (ad libitum): pellet diet for experimental animals (MF, Oriental Yeast Co., Ltd.)
- Water (ad libitum): tap water, filtered through 5-µm filter and irradiated with UV light
- Acclimation period: 5 days
- Levels of contaminants were below acceptable upper limits specified at the test facility

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 15 %
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 1 w/v % MC (methylcellulose)
- Concentration of test material in vehicle: serial dilution: 200, 100, 50, 25 and 12.5 mg/mL (for usage in the main test and the confirmation test)
- Lot/batch no.: Metolose ® SM-100, Shin-Etsu Chemical, Inc., Lot no. 002658

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- suspended by addition of 1 w/v % methylcellulose
- adjusting a concentration of 200 mg/mL
- in an stepwise dilution procedure by adding 1 w/v % methylcellulose, suspensions of 100 mg/mL and 50 mg/mL were prepared
- in the confirmation test the test material was prepared as described previously, but diluted to a concentration of 50 mg/mL, 25 mg/mL and 12.5 mg/mL
- the dose solutions were prepared under yellow light

Duration of treatment / exposure:

2 days

Frequency of treatment:

twice at 24 hour intervals

Post exposure period:

24 hours (after the final administration)

Dose / conc.:

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

Remarks:

confirmation tes

Dose / conc.:

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

Remarks:

confirmation test

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 (male) per dose

Control animals:

yes, concurrent vehicle

yes, historical

Positive control(s):

cyclophosphamide (obtained from Sigma Chemical Company, Lot no. 86F-0101)
- Route of administration: intraperitoneally administered (once)
- Doses / concentrations: 10 mg/kg

Tissues and cell types examined:

1000 erythrocytes were scored from each slide

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- doses were set according to a single dose toxicity study by the sponsor with LD50 >= 2000 mg/kg
- dose selection in the confirmation test: doses were set according to the main test which resulted in an unusual value in 1 of 5 animals of the low dose group (500 mg/kg)

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- the test substance and the negative control was administered by oral gavage twice at 24 hour intervals.
- the positive control was administered once intraperitoneally
- 24 hours after the last administration rats were sacrificed by exsanguination from the abdominal aorta under anesthesia (thiopental sodium) and femurs were dissected


DETAILS OF SLIDE PREPARATION:
- bone marrow cells were collected with phosphate buffered saline (PBS) and suspensions were centrifuged at 200 rpm for 5 min
- supernatant was transferred to 10 % neutral buffered formalin solution and centrifuged at 1000 rpm for 5 min
- cells were twice washed with 10 % neutral buffered formalin solution
- cells were resuspended in 10 % neutral buffered formalin solution
- suspension was stained with acridine orange and transferred to slides for observation

METHOD OF ANALYSIS:
- slides were observed and scored under blind condition with fluorescent microscope with B-2 excitation filter
- 1000 erythrocytes were scored from each slide in order to determine the ratio of polychromatic erythrocytes (PCEs) to the total erythrocytes (PCEs and normochromatic erythrocytes (NCEs))
- PCEs were further scored up to 2000 cells, the number of micronucleated PCEs (MNPCEs) in a slide were examined
- PCEs and NCEs were identified according to the method of Hayashi et al.*

*Reference:
Hayashi M, Sofuni T, Ishidate M Jr. (1983): An application of acridine orange fluorescent staining to the micronucleus test. Mutat. Res. 120, 241 - 247

Evaluation criteria:

When test substance induced a significant increase in the total number of micronucleated polychromatic erythrocytes with dose-dependency, the test is considered positive

Statistics:

- for the analysis of the percentage of polychromatic erythrocytes (PCEs): Student's t-test
- for the incidence of micronucleated PCEs (MNPCEs): tables of Kastenbaum and Bowman*
- statistics were conducted at the significance levels of 5 % and 1 %

*Reference:
Kastenbaum MA, Bowman KO (1970): Tables for determining the statistical significance of mutation frequencies. Mutat Res. 9, 527 - 549

Key result

Sex:

male

Genotoxicity:

negative

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
- significant induction of micronuclei in the 500 mg/kg dose group: mean incidence of MNPCE = 0.68 ± 1.19

- Ratio of PCE/NCE (for Micronucleus assay):
- 500 mg/kg dose group: mean ratio PCE/NCE: 54.0 ± 1.0
- 1000 mg/kg dose group: mean ratio PCE/NCE: 53.5 ± 2.3
- 2000 mg/kg dose group: mean ratio PCE/NCE: 52.2 ± 2.6

RESULTS OF CONFIRMATION TEST
- Induction of micronuclei (for Micronucleus assay): no significant induction observed

- Ratio of PCE/NCE (for Micronucleus assay):
- 125 mg/kg dose group: mean ratio PCE/NCE: 50.4 ± 3.3
- 250 mg/kg dose group: mean ratio PCE/NCE: 49.1 ± 5.1
- 500 mg/kg dose group: mean ratio PCE/NCE: 50.5 ± 2.8

Please refer to the field 'Any other information on results incl. tables'

Further observation:

body weight: no effects observed

clinical signs: no effects observed

Table 1: Results of micronucleus test

Treatment group	Dosage (mg/kg) x times	Animal no.	Number of PCEs scored	MNPCE		PCE/(PCE+NCE)^5
				Number (total)	Incidence Mean ± SD	(%) Mean ± SD
Negative control 0 x 2^1		1	2000	2	0.10	53.9
		2	2000	3	0.15	55.2
		3	2000	1	0.05	54.9
		4	2000	2	0.10	56.0
		5	2000	1	0.05	54.0
				(9)	0.09 ± 0.04	54.8 ± 0.9
Test substance 500 x 2^1		1	2000	3	0.15	53.8
		2	2000	3	0.15	55.5
		3	2000	3	0.15	54.0
		4	2000	3	0.15	52.7
		5	2000	56	2.80	53.9
				(68)^3**	0.68 ± 1.19	54.0 ± 1.0
Test substance 1000 x 2^1		1	2000	2	0.10	50.9
		2	2000	4	0.20	55.7
		3	2000	1	0.05	55.6
		4	2000	4	0.20	54.1
		5	2000	5	0.25	51.2
				(16)	0.16 ± 0.08	53.5 ± 2.3
Test substance 2000 x 2^1		1	2000	3	0.15	48.4
		2	2000	1	0.05	55.0
		3	2000	4	0.20	54.2
		4	2000	4	0.20	52.0
		5	2000	4	0.20	51.3
				(16)	0.16 ± 0.07	52.2 ± 2.6
Positive control 10 x 1^2		1	2000	40	2.00	50.4
		2	2000	35	1.75	53.6
		3	2000	44	2.20	50.4
		4	2000	62	3.10	48.3
		5	2000	48	2.40	50.9
				(229)^3**	2.29 ± 0.51	50.7 ± 1.9^4##

PCE: polychromatic erythrocytes, MNPCE: micronucleated PCE, NCE: normochromatic erythrocytes

¹twice administered by oral gavage at 24 hour interval

²once administered by intraperitoneal injection

³significantly different from the negative control (**p<0.01) by Kastenbaum & Bowman’s method

⁴significantly different from the negative control (##p<0.01) by Student’s t-test

⁵1000 erythrocytes were scored

 

 

Table 2: Results of confirmation test

Treatment group	Dosage (mg/kg) x times	Animal no.	Number of PCEs scored	MNPCE		PCE/(PCE+NCE)^5
				Number (total)	Incidence Mean ± SD	(%) Mean ± SD
Negative control 0 x 2^1		1	2000	1	0.05	57.7
		2	2000	3	0.15	50.8
		3	2000	2	0.10	53.9
		4	2000	1	0.05	58.2
		5	2000	1	0.05	52.9
				(8)	0.08 ± 0.04	54.7 ± 3.2
Test substance 125 x 2^1		1	2000	2	0.10	46.5
		2	2000	1	0.05	19.4
		3	2000	1	0.05	52.3
		4	2000	1	0.05	48.8
		5	2000	3	0.15	55.0
				(8)	0.08 ± 0.04	50.4 ± 3.3
Test substance 250 x 2^1		1	2000	1	0.05	51.9
		2	2000	1	0.05	52.3
		3	2000	2	0.10	48.9
		4	2000	2	0.10	40.3
		5	2000	4	0.20	51.9
				(10)	0.10 ± 0.06	49.1 ± 5.1
Test substance 500 x 2^1		1	2000	3	0.15	47.8
		2	2000	2	0.10	50.5
		3	2000	2	0.10	54.9
		4	2000	2	0.10	48.2
		5	2000	1	0.05	51.1
				(10)	0.10 ± 0.04	50.5 ± 2.8
Positive control 10 x 1^2		1	2000	50	2.50	45.6
		2	2000	28	1.40	43.1
		3	2000	31	1.55	42.6
		4	2000	44	2.20	37.3
		5	2000	33	1.65	39.2
				(186)^3**	1.86 ± 0.47	41.6 ± 3.3

PCE: polychromatic erythrocytes, MNPCE: micronucleated PCE, NCE: normochromatic erythrocytes

¹twice administered by oral gavage at 24 hour interval

²once administered by intraperitoneal injection

³significantly different from the negative control (**p<0.01) by Kastenbaum & Bowman’s method

⁴significantly different from the negative control (##p<0.01) by Student’s t-test

⁵1000 erythrocytes were scored

 

Conclusions:

Negative
The test item did not induce biologically relevant increases in micronucleated polychromatic erythrocytes in the bone marrow of rats when administered up to the limit dose of 2000mg/kg bw via oral route.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity – in vitro results

 

Gene mutation in bacterial test systems

In the NTP study (NTP, 1992) m-toluic acid was assessed in regard to its mutagenic potential using the Ames test. Five Salmonella typhimurium strains (TA97, TA98, TA100, TA1535 and TA1537) were tested at 5 different concentrations (33-3333 µg/plate) using the preincubation method. The cells were either tested without metabolic activation or in presence of either induced male Sprague-Dawley rat liver S9 (10 % and 30 %) or induced Syrian hamster liver S9 (10 % and 30 %). The test substance was not tested at the recommended top dose (OECD 471; EU: B.13/B.14), although no signs of precipitation and cytotoxicity were observed. In two independent experiments, the treated tester strains showed no increased revertant colony number under the conditions tested.

This negative result was confirmed and further extended in a GLP study (Mitsubishi Chemical Safety Institute (Japan); 1999) using two different bacterial genera. A preincubation test was performed using four Salmonella typhimurium tester strains (TA98, TA100, TA1535 and TA1537) and one Escherichia coli strain (WP2uvrA). The cells were treated either without metabolic activation at six concentrations (156-5000 µg/plate) or with metabolic activation at six concentrations, ranging from 313-5000 µg/plate. m-Toluic acid was tested up to the recommended top dose. Precipitation and cytotoxicity (decrease of revertant number of over 50%) were observed in combination with metabolic activation at the highest dose applied. Additionally, cytotoxicity occurred in Salmonella tester strains TA100, TA1535, and TA1537 at a dose of 2500 µg/plate in presence of a metabolic activation system. The study is compliant with the current guidelines (OECD 471; EU: B.13/B.14) and is well-suited for risk assessment purposes. None of the triplicates showed an increase in the revertant colony number, when compared to controls both in presence and absence of a metabolic activation system.

 

Gene mutation in mammalian cells

In a GLP study (Hargreaves, 2018), the mutagenic potential of m-toluic acid was evaluated at the hprt locus in mouse L5178Y lymphoma cells. The cells were tested at eight concentrations (50-1362 µg/mL), up to highest concentration recommended (equivalent to 10 mM; OECD TG476 and EU: B.17), either in absence or in presence of a metabolic activation system (S-9 rat liver). The cells were incubated for 3 hours and mutant frequency was scored after 12 days in duplicate experiments. Neither significant increases nor positive linear trends in mutant frequency were observed, independent of metabolic activation status. Thus, the test substance did not induce mutations at the hprt locus in mouse lymphoma cells being exposed up to top concentrations recommended by current regulatory testing guidelines.

 

Chromosomal aberrations (CA) in mammalian cells

Only one in vitro cytogenicity study is present, which do not fulfil the relevance, reliability and adequacy criteria as foreseen by the ECHA ‘Guidance on Information Requirements and Chemical Safety Assessment – Chapter R.7a’:

Mitsubishi Chemical Safety Institute (Japan), 1999: CHL/IU (Chinese hamster lung) cells were treated in a ‘short time exposure’ experiment at concentration of 250, 500, 1000 and 2000 µg/mL with and without S9, and additionally, in a second ‘continuous exposure’ experiment either for 24 hours at 250, 500, 1000 and 2000 µg/mL or for 48 hours at 62.5, 125, 250, 500 and 1000 µg/mL. Eventually, a confirmation test was performed at either 1000, 1500 and 2000 µg/mL without S9 or 500, 750 and 1000 µg/mL with S9. Positive results were found, in absence of S9, in the ‘continuous exposure’ experiment (exposure for 48 hours) and in the confirmation test at concentrations of 1000 and 2000 µg/ml, respectively. The authors concluded that the test substance induces elevated CA frequencies. However, following methodological deficiencies hamper the interpretation of these findings:

-Tests have been conducted partially above limit dose of 10 mM (equivalent to 1361 µg/mL for m-toluic acid), namely 1500 and 2000 µg/mL;

- Eventually, less than 3 test concentrations were analysable due to cytotoxicity;

- Furthermore, other confounding factors are noticed: The pH of cell culture medium was affected by test substance, the lowest pH observed while inducing chromosomal aberrations was at pH 6.2; the test substance precipitated at test concentrations of 1000 µg/mL and above; the culture medium changed to yellow colour at test concentrations of 1000 µg/mL and above.

 

Genetic toxicity – in vivo somatic cell results

Micronucleus (MN) test

A GLP study (Watabe, 2002 (Mitsubishi Chemical Safety Institute (Japan)) has been conducted to measure MN induction in bone marrow erythrocytes upon m-toluic acid treatment. Five male SD (Cry: CD (SD), IGS) rats per group received, within 24 hours, two oral gavages to reach total doses of 500, 1000, and 2000 mg/kg bw m-toluic acid. The top dose was chosen based on the findings of a prior single dose toxicity study showing a LD₅₀of greater than 2000 mg/kg bw. 24 hours after treatment, 2000 polychromatic erythrocytes were scored for MN occurrence. No significant increase in MN induction was observed, except for one rat showing a highly significant increased MN number at a dose of 500 mg/kg bw. However, this finding was not reproduced in a second confirmation test, in which five animals were exposed to 125, 250, and 500 mg/kg bw m-toluic acid. The confirmation test showed no elevated MN levels at all doses tested, and consequently, the prior positive finding was proven as chance finding. Concurrent positive and negative controls constituted the study being valid. Therefore, m-toluic acid is concluded to induce no micronucleation under the conditions tested.

 

Discussion

The gene mutagenic potential of m-toluic acid was evaluated in three independent studies, including two studies which are GLP compliant. The test substance was tested in two in vitro studies (NTP, 1992; and MHLW, 1999) exploiting bacterial test systems and in one GLP study (Hargreaves 2018) using mammalian cells. All of the studies showed that m-toluic acid does not have potential to induce gene mutations under the conditions tested. Thus, it is assessed to be non-mutagenic in in vitro gene mutation systems.

Two cytogenicity studies were performed to evaluate the potential of m-toluic acid to induce clastogenicity, both of them were GLP compliant. However, the in vitro test performed (Mitsubishi Chemical Safety Institute (Japan), 1999) is due to significant methodological deficiencies not suitable for risk assessment purposes. In a subsequent in vivo cytogenicity study performed as bone marrow micronucleus test in rats (Watanabe, 2002) m-toluic acid induced no clastogenicity.

According to Regulation (EC) No 1907/2006 Annex VIII 8.4.2. column 1, an in vitro cytogenicity study is a standard information requirement. However, according to the specific rules for adaption from column 1, in column 2 is clearly stated that the in vitro cytogenicity test could be replaced by an in vivo cytogenicity test provided that the data is adequate. The in vivo MN study (Watabe, 2002) present does satisfactorily meet the requirements according to Regulation (EC) No 440/2008 EU: B.12 and OECD TG474. Thus, according to Regulation (EC) No 1907/2006 Annex VIII 8.4.2., this in vivo cytogenicity study could be used as substitute for the in vitro cytogenicity test required. Moreover, according to the ECHA ‘Guidance on Information Requirements and Chemical Safety Assessment – Chapter R.7a’, in vivo studies indicate a higher degree of reliability, and thus, they are more appropriate as surrogate for human health risk assessment. Under these conditions m-toluic acid is considered to induce no cytogenic damage.

Justification for classification or non-classification

Genetic toxicity, in vivo

The GLP and guideline compliant in vivo cytogenicity study (micronucleus test) performed for m-toluic acid (up to limit dose) did not show test item induced genetic toxicity.

 

Genetic toxicity, in vitro

None of the in vitro genotoxicity studies rated as reliable showed bacterial gene mutation or mammalian cell gene mutation.

According to the criteria of REGULATION (EC) No 1272/2008 and its subsequent adaptations, m-toluic acid does not have to be classified and has no obligatory labelling requirement for germ cell mutagenicity.