Aluminium tribenzoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An AMES test, performed according to OECD guideline 471, a chromosome aberration study, performed according to OECD guideline 473, and an in vitro mammalian cell gene mutation test, performed according to OECD guideline 490, are available. All studies were performed with aluminium tribenzoate and under GLP principles (Klimisch 1 studies). Aluminium tribenzoate was shown not to be genotoxic in any of the test systems, without and with metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 February 2017- 20 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd 03 November 2015

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 17, 52, 164, 512, 1600 and 5000 µg/plate
Experiment 2 (all strains):
Without and with S9-mix: 17, 52, 164, 512, 1600 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: DMSO is accepted and approved by authorities and international guidelines. The test item was tested for solubility in DMSO and at concentrations of 0.17 mg/mL and higher the test item was suspended in DMSO.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: ICR-191 2.5 µg/plate in DMSO for TA1537

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 ± 4 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Experiment 1: Precipitation of Aluminium tribenzoate on the plates was observed at the start of the incubation period at the concentration of 5000 μg/plate in all tester strains. At the end of the incubation period the test item precipitated slightly at 5000 μg/plate in the tester strains TA100 and WP2uvrA only.
Experiment 2: Precipitation of Aluminium tribenzoate on the plates was observed at the start of the incubation period at the concentration of 5000 μg/plate. No precipitation was observed at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The following acceptability criteria were met, therefore the study was considered valid:

a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) did exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.

b) The selected dose range extended to 5 mg/plate.

c) No plates were lost throughout the study.

The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for WP2uvrA in the absence of S9-mix in the second experiment. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study.

Conclusions:

In an AMES test, performed according to OECD guideline 471 and GLP principles, Aluminium tribenzoate was found not to be mutagenic with or without metabolic activation.

Executive summary:

An AMES test was performed according to OECD guideline 471 and GLP principles. All bacterial strains showed negative responses up to 5000 µg/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. No cytotoxicity and/or precipitation of the test substance was observed. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that Aluminium tribenzoate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation. Since all acceptability criteria were met, the study was considered valid.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 January 2017 - 20 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd. 03 November 2015

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Correction factor for purity: 1.164

Species / strain / cell type:

lymphocytes: Peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: healthy adult, non-smoking volunteers
- Cell cycle length, doubling time or proliferation index: AGT = 14 hours (Dose range finding/First cytogenic assay; 25 years old donor) and 13.8 hours (Second cytogenic assay; 25 years old donor).
- Sex, age and number of blood donors if applicable: 18-35 years old
- Whether whole blood or separated lymphocytes were used: whole blood
- Number of passages if applicable:
- Methods for maintenance in cell culture: per culture 0.1 mL phytohaemagglutinin was added.

MEDIA USED
- Type and identity of media: RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) and 30 U/ml heparin.
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 prepared from male rats treated with phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).

Test concentrations with justification for top dose:

Dose range finding test (without S9): 3.9, 7.9, 15.7, 31.3, 62.5 and 125 μg/mL
First cytogenic assay (with and without S9): 7.9, 15.7 and 31.3 μg/mL
Second cytogenic assay (without S9, 24 h exposure time): 7.8, 15.7 and 31.3 μg/mL
Second cytogenic assay (without S9, 48 h exposure time): 2.0, 3.9 and 7.8 μg/mL
The top dose (31.3 μg/mL) was selected based on the solubility of the test substance in culture medium.

Vehicle / solvent:

- Solvent used: dimethyl sulfoxide
- Justification for choice of solvent: a homogenous suspension could be obtained after treatment with ultrasonic waves. Also, the solvent is according to OECD guidelines.
- Concentration of solvent in the culture medium: 1.0% (v/v)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 +/- 2 hours culture period
- Exposure duration: 3h, 24 h and 48 h
- Fixation time: 3 h and 24 h exposure period: 24 h fixation time; 48 h exposure period: 48 h fixation time.

SPINDLE INHIBITOR: colchicine (0.5 μg/mL medium)

STAIN: 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. Slides were marked and allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper.

NUMBER OF CELLS EVALUATED: 1000 cells

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 150

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Any supplementary information relevant to cytotoxicity: no cytotoxicity observed in the slides of the 3 h exposure time

ACCEPTABILITY CRITERIA
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the number of cells with chromosome aberrations. The positive control data will be analysed by the Fisher’s exact test (one-sided, p < 0.05).

Evaluation criteria:

A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, onesided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.

Key result

Species / strain:

lymphocytes: Peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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: no
- Effects of osmolality: no
- Evaporation from medium: no
- Precipitation: at and above 31.3 μg/mL

RANGE-FINDING/SCREENING STUDIES: yes, no statistically significant increase in the number of cells with chromosome aberrations was observed.

NUMBER OF METAPHASES OBSERVED
- Dose range finding test (without S9) 24 h exposure: 68-82% of control; 48 h exposure: 65-85% of control
- First cytogenetic assay (without S9): 3 h exposure: 75-80% of control
- First cytogenetic assay (with S9): 3 h exposure: 87-101% of control
- Second cytogenetic assay (without S9): 24 h exposure: 72-81% of control; 48 h exposure: 78-93% of control

HISTORICAL CONTROL DATA (see Table 1 and Table 2)
- The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.
- 1 endoreduplicated chromosome was observed at the 48 h exposure time, at the highest concentration tested, which is outside the 95% control limits of the distribution of the historical negative control database, this was considered an isolated event and therefore considered not biologically relevant.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No effects of the test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

Table 1 Historical negative control data for lymphocyte chromosome aberration studies

	3 h exposure time				24 h exposure time		48 h exposure time
	Gaps incl		Gaps excl		Gaps incl	Gaps exl	Gaps incl	Gaps excl
	+S9 mix	-S9 mix	+S9 mix	-S9 mix	+S9 mix	-S9 mix	+S9 mix	-S9 mix
Mean number of aberrant cells per 100 cells	0.77	0.77	0.71	0.76	0.76	0.64	0.96	0.74
SD	1.00	1.09	1.02	1.12	1.05	1.02	1.23	1.12
n	284	282	284	282	278	278	270	270
Upper limit (95% control limit)	3.25	3.14	3.23	3.14	3.13	2.79	3.97	3.13
Lower limit (95% control limit)	-1.71	-1.60	-1.80	-1.61	-1.61	-1.51	-2.06	-1.66

SD = Standard deviation

n = Number of observations

Distribution historical negative control data from experiments performed between January 2012 and November 2016.

Table 2 Historical positive control data for lymphocyte chromosome aberration studies

	3 h exposure time				24 h exposure time		48 h exposure time
	Gaps incl		Gaps excl		Gaps incl	Gaps exl	Gaps incl	Gaps excl
	+S9 mix	-S9 mix	+S9 mix	-S9 mix	+S9 mix	-S9 mix	+S9 mix	-S9 mix
Mean number of aberrant cells per 100 cells	32.63	29.33	31.98	28.86	30.90	30.00	35.95	34.96
SD	13.05	13.32	13.09	13.39	13.79	14.09	15.06	15.19
n	280	280	280	280	276	276	268	268
Upper limit (95% control limit)	57.86	53.16	57.12	52.57	58.23	57.87	66.11	65.15
Lower limit (95% control limit)	7.41	5.50	6.84	5.15	3.58	2.13	5.79	4.78

SD = Standard deviation

n = Number of observations

Distribution historical positive control data from experiments performed between January 2012 and November 2016.

Conclusions:

The results of a chromosome aberration study, performed according to OECD guideline 473 and GLP principles, showed that Aluminium tribenzoate is not clastogenic in human lymphocytes.

Executive summary:

A chromosome aberration study was performed, according to OECD guideline 473 and GLP principles, to assess the possible clastogenicity of Aluminium tribenzoate. Cultured peripheral human lymphocytes were used as a test system. The test substance was tested in the presence and absence of a metabolic activation system (S9 -mix) in two independent experiments up to a precipitating concentration (at and above 31.3 μg/mL). Aluminium tribenzoate did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments. No effects of Aluminium tribenzoate on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. The acceptability criteria were met and therefore it was concluded that the test conditions were adequate and the metabolic activation system functioned properly. It is concluded that Aluminium tribenzoate is not clastogenic in human lymphocytes.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 May 2017 - 07 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

26 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro mammalian cell gene mutation test

Specific details on test material used for the study:

- Correction factor for purity: 1.164

Target gene:

- Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: L5178Y/TK+/- -3.7.2C mouse lymphoma cells from American Type Culture Collection, (ATCC, Manassas, USA), (2001)
- Suitability of cells: recommended test system in international guidelines

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
* Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin
* Growth medium: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
* Exposure medium: 3 hr exposure: basic medium, supplemented with 5% (v/v) heat-inactivated horse serum (=R5 medium); 24 hr exposure: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
* Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 μg/mL trifluorothymidine
* Non-selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium)
- Properly maintained: yes, stock cultures of the cells were stored in liquid nitrogen (-196°C).
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not indicated
- Periodically 'cleansed' against high spontaneous background: yes, prior to dose-range finding and mutagenicity testing

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital and ß-naphthoflavone.

Test concentrations with justification for top dose:

Dose-range finding test: 3.13, 6.25, 12.5, 25 and 50 μg/mL
Since the test substance was poorly soluble in the exposure medium, the highest tested concentration was 50 μg/mL exposure medium.

Experiment 1 (with and without S9): 0.1, 0.2, 0.39, 0.78, 1.56, 3.13, 6.25 and 12.5 μg/mL
Experiment 2: 0.2, 0.39, 0.78, 1.56, 3.13, 6.25 and 12.5 μg/mL
Since the test item was not toxic and difficult to dissolve in aqueous solutions the highest concentration was determined by the solubility in the culture medium. The highest test item concentrations showed slight precipitate in the exposure medium.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: as recommended in OECD test guideline 490. A solubility test showed that the test substance suspended in DMSO at concentrations of 0.313 mg/mL and higher and the test substance dissolved in DMSO at concentrations of 0.156 mg/mL and lower.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): below 1 x 10^6 cells/mL
- Cell density used for the test: 8 x 10^6 cells (10^6 cells/ml for 3 hour treatment) or 6 x 10^6 cells (1.25 x 10^5 cells/ml for 24 hour treatment) per culture

DURATION
- Cleansing period: prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10^-4 M hypoxanthine, 2 x 10^-7 M aminopterine and 1.6 x 10^-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.
- Exposure duration: 3 hours (experiment 1; with and without S9-mix); 24 hours (experiment 2; without S9-mix).
- Expression time: 2 days in which at least 4 x 10^6 cells were subcultured every day
- Selection time (if incubation with a selection agent): 11 or 12 days

SELECTION AGENT: TFT

STAIN: 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)

NUMBER OF REPLICATIONS:
- test concentrations: 1
- positive control: 1
- solvent control: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: For determination of the cloning efficiency the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium. For determination of the mutation frequency (MF) a total number of 9.6 x 10^5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). In the dose level of 0.39 μg/ml in the second experiment a total number of 384 wells was used for determination of the mutation frequency (since only one plate out of five fall out, at a mid-dose and the mutation frequency was well within the range and clear negative results were obtained, this deviation had no effect on the results of the study).
The microtiter plates for cloning efficiency and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFTselection were stained for 2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the cloning efficiency and MF were scored with the naked eye or with the microscope.

NUMBER OF CELLS EVALUATED: 9.6 x 10^5 cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (see 'Any other information on materials and methods incl. tables' for details on calculations)

Evaluation criteria:

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
- A test item is considered positive (mutagenic) in the mutation assay if it induces a mutation frequency of more than the mutation frequency in the controls + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
- A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
- A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of the mutation frequency in the controls + 126.

ACCEPTABILITY CRITERIA:
a) The absolute cloning efficiency of the solvent controls is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10^-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10^-6).

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility in medium: the test item precipitated in the exposure medium at concentrations of 12.5 μg/mL and above. The test item was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test item concentration for the dose-range finding test was 50 μg/mL.

RANGE-FINDING/SCREENING STUDIES:
- After 3 hours of treatment, both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test item concentration of 50 μg/mL compared to the suspension growth of the solvent control.
- After 24 hours of treatment, in the absence of S9-mix, no significant toxicity in the relative suspension growth was observed up to and including the highest test item concentration of 50 μg/mL compared to the suspension growth of the solvent control.

MUTAGENICITY TESTS:
- No significant toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix in both experiments.
- No significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test substance treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

HISTORICAL CONTROL DATA: see table 1 and table 2
- The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.
- Positive control chemicals both produced significant increases in the mutation frequency and the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.

ACCEPTABILITY:
- The absolute cloning efficiency of the solvent controls was between 65 and 120% (i.e., 97% and 105%, without S9 (exp.1); 97% and 111%, without S9 (exp. 2) and 79% and 91%, with S9)
- The spontaneous frequency in the solvent control was ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
- The suspension growth over the two-day expression period for solvent control groups was between 8 and 32 for the 3 hour treatment (23-27) and between 32 and 180 for the 24 hour treatment (40-41).
- Increase in total mutation frequency: Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.

For detailed results, see attached illustration.

Table 1Historical Control Data of the Spontaneous Mutation Frequencies of the Solvent Controls for the Mouse Lymphoma Assay

	Mutation frequency per 10^6survivors
	- S9-mix		+ S9-mix
	3 hour treatment	24 hour treatment	3 hour treatment
Mean	86	81	87
SD	23	26	28
n	220	202	273
Upper control limit (95% control limits)	135	135	145
Lower control limit (95% control limits)	37	28	28

SD = Standard deviation

n = Number of observations

Table 2Historical Control Data of the Mutation Frequencies of the Positive Controls for the Mouse Lymphoma Assay

	Mutation frequency per 10^6survivors
	- S9-mix		+ S9-mix
	3 hour treatment	24 hour treatment	3 hour treatment
Mean	857	688	1710
SD	246	187	815
n	110	102	139
Upper control limit (95% control limits)	1425	1124	4214
Lower control limit (95% control limits)	289	253	-793

SD = Standard deviation

n = Number of observations

Distribution historical positive control data from experiments performed between January 2013 and November 2016. 

Conclusions:

Based on the results of a in vitro mammalian cell gene mutation test, performed according to OECD guideline 490 and GLP principles, aluminium tribenzoate is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

In an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells, performed according to OECD test guideline 490 and GLP principles, aluminium tribenzoate was tested for its mutagenic potential. The test consisted out of two independent experiments. In the first experiment, the test substance was tested up to concentrations of 12.5 μg/mL for an exposure time of 3 hours, with and without S9 -mix (metabolic activation system). In the second experiment, the test substance was tested up to 12.5 μg/mL for an exposure time of 24 hours, without S9 -mix.

The test substance precipitated at the highest dose level tested. No cytotoxicity was observed up to this dose level (12.5 μg/ml) but based on the results of the range finding study this concentration was considered to be the limit of cytotoxicity. In the absence of S9 -mix, the test substance did not induce a significant increase in the mutation frequency. This results was confirmed in the second experiment with a longer exposure time (24 hours). In the presence of S9 -mix, the test substance did not induce a significant increase in the mutation frequency either.
Results of the solvent control and the positive controls were within the 95% control limits of the historical data range generated at the test facility.

Based on the results of this study, aluminium tribenzoate is not mutagenic in the mouse lymphoma L5178Y test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

An AMES test was performed according to OECD guideline 471 and GLP principles. All bacterial strains showed negative responses up to 5000 µg/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. No cytotoxicity and/or precipitation of the test substance was observed. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that Aluminium tribenzoate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation. Since all acceptability criteria were met, the study was considered valid.

A chromosome aberration study was performed, according to OECD guideline 473 and GLP principles, to assess the possible clastogenicity of Aluminium tribenzoate. Cultured peripheral human lymphocytes were used as a test system. The test substance was tested in the presence and absence of a metabolic activation system (S9 -mix) in two independent experiments, up to a dose level of 31.3 μg/mL. Aluminium tribenzoate did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments. No effects of Aluminium tribenzoate on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. The acceptability criteria were met and therefore it was concluded that the test conditions were adequate and the metabolic activation system functioned properly. It is concluded that Aluminium tribenzoate is not clastogenic in human lymphocytes.

In an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells, performed according to OECD test guideline 490 and GLP principles, aluminium tribenzoate was tested for its mutagenic potential. The test consisted out of two independent experiments. In the first experiment, the test substance was tested up to concentrations of 12.5 μg/mL for an exposure time of 3 hours, with and without S9 -mix (metabolic activation system). In the second experiment, the test substance was tested up to 12.5 μg/mL for an exposure time of 24 hours, without S9 -mix. The test substance precipitated at the highest dose level tested. No cytotoxicity was observed up to this dose level (12.5 μg/ml) but based on the results of the range finding study this concentration was considered to be the limit of cytotoxicity. In the absence of S9 -mix, the test substance did not induce a significant increase in the mutation frequency. This results was confirmed in the second experiment with a longer exposure time (24 hours). In the presence of S9 -mix, the test substance did not induce a significant increase in the mutation frequency either. Results of the solvent control and the positive controls were within the 95% control limits of the historical data range generated at the test facility. Based on the results of this study, aluminium tribenzoate is not mutagenic in the mouse lymphoma L5178Y test system.

Justification for classification or non-classification

Based on the available data, aluminium tribenzoate is not classified for genotoxicity according to CLP Regulation (EC) No. 1272/2008.