Calcium dibenzoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The in vitro genetic toxicity of Calcium dibenzoate has been evaluated in a bacterial reverse gene mutation assay (acc. to OECD TG 471) and in a mammalian cell gene mutation assay (acc. to OECD TG 476). These studies were performed according to the current guidelines and in accordance with GLP and were evaluated to be reliable without restrictions. The cytogenetic potential in mammalian cells is addressed with data on the individual moieties, calcium and benzoic acid. The cytogenetic potential in mammalian cells for the moiety benzoic acid is addressed with a micronucleus test and a chromosome aberration assay, both of which were performed in the pre-guideline and pre-GLP era but are considered to be reliable with restrictions. For the moiety calcium, the data is considered to be not scientifically necessary.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 April 2021 - 14 June 2021

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:

corrected June 26, 2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate signed on 23 Oktober 2019

Type of assay:

bacterial reverse mutation assay

Target gene:

hisD (TA 98), hisG (TA 100, TA 1535), hisC (TA 1537), and trpE (WP2 uvr A pKM101)

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: The S9 was prepared in-house. Phenobarbital/β-naphthoflavone induced male Wistar rat liver S9 was used as the metabolic activation system.
- method of preparation of S9 mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 10% (v/v) in the S9 mix. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, and 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
- concentration or volume of S9 mix and S9 in the final culture medium: 500 μL S9 mix (containing 10% (v/v) S9)
- quality controls of S9: sterility and metabolic capability

Test concentrations with justification for top dose:

- Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
- Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate
The top concentration was the recommended maximum test concentration for soluble non-cytotoxic substances.

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria (Maron et al.; 1981)*.

*References:
- Maron, D.M., J. Katzenellenbogen, and B.N. Ames (1981) Compatibility of organic solvents with the Salmonella/Microsome Test. Mutation Res. 88, 343-350.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

CONCENTRATION SELECTION
In the pre-experiment the concentration range of the test item was 3 - 5000 μg/plate. The pre-experiment is reported as experiment I. Since no toxic effects were observed 5000 μg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades.
The following concentrations were tested in experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate

BACTERIAL REVERSE MUTATION ASSAY
For each strain and dose level, including the controls, three plates were used.

Experiment I (Plate Incorporation):
The following materials were mixed in a test tube and poured onto the selective agar plates: 100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)), 500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation), 100 μL bacteria suspension, 2000 μL overlay agar

Experiment II (Pre-Incubation):
The following materials were mixed in a test tube and incubated at 37°C±1.5°C for 60 minutes: 100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)), 500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation), 100 μL Bacteria suspension. After pre-incubation 2 mL overlay agar (45°C) was added to each tube.

The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37°C±1.5°C in the dark.
In parallel to each test a sterile control of the test item was performed and documented in the raw data. Therefore, 100 μL of the stock solution, 500 μL S9 mix / S9 mix substitution buffer were mixed with 2 mL overlay agar and poured on minimal agar plates.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Toxicity of the test item results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The colonies were counted using a validated computer system (Petri Viewer Sorcerer Colony Counter 3.0 (Instem, Suffolk IP33 3TA, UK) with the software program Ames Study Manager (v1.24) and Ames Archive Manager (v1.01)).

Evaluation criteria:

- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA (pKM101)) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
- A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
- An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
- A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

Statistical analysis of the data is not mandatory

Key result

Species / strain:

S. typhimurium TA 98

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

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate. No precipitation of the test item occurred in the overlay agar on the incubated agar plates.

TOXICITY
- The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.
- No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

GENOTOXICITY RESULTS
- No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Calcium dibenzoate at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix) (please refer to "Attached background material: Results"). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

ASSAY VALIDITY
- Appropriate reference mutagens were used as positive controls. They showed a distinct increase in the number of revertant colonies, which fell in the expected range (please refer to "Attached background material: Historical control data").
- Vehicle and untreated control treatments were included for all strains in both experiments. The mean number of revertant colonies fell within acceptable ranges of the historical control database.
Thus, the controls demonstrated sensitivity of the test systems and the validity of the assay.

Conclusions:

No toxicity (thinning of the background lawn or a reduction in the number of revertants) was found in both experiments. No precipitation was observed on the test plates. Calcium dibenzoate, tested up to the recommended maximum concentration, did not induce biologically relevant increases in the number of revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. All validity criteria were met. The study was fully compliant with OECD 471 (2020).

Therefore, Calcium dibenzoate (CAS: 2090-05-3; EC: 218-235-4) is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 May 2021 - 07 October 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate signed on 23 Oktober 2019

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment (immediately before treatment), the test item was formulated in culture medium under sonification.

Target gene:

Hprt

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Chinese hamster lung fibroblasts (supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany)
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate and a good cloning efficiency of untreated cells both necessary for the appropriate performance of the study, recommend the use of this cell line.

For cell lines:
- Absence of Mycoplasma contamination: Each master cell stock is screened for mycoplasma contamination.
- Number of passages if applicable: Large stocks of the V79 cell line are stored in liquid nitrogen in the cell bank of ICCR-Roßdorf GmbH allowing the repeated use of the same cell culture batch in experiments.
- Cell doubling time: 12-16 hours in stock cultures
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes


MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Thawed stock cultures were propagated at 37°C in 75 cm² plastic flasks. About 2-3x10^6 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10% FBS, neomycin (5 μg/mL) and amphotericin B (1%). The cells were sub-cultured once or twice weekly. All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: The S9 was prepared in-house. Phenobarbital/β-naphthoflavone induced male Wistar rat liver S9 was used as the metabolic activation system.
- method of preparation of S9 mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 10% (v/v) in the S9 mix. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, and 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
- concentration or volume of S9 mix and S9 in the final culture medium: 500 μL S9 mix (containing 10% (v/v) S9)
- quality controls of S9: sterility and metabolic capability

Test concentrations with justification for top dose:

- Main experiment I (without metabolic activation): 62.5, 125, 250, 500, 1000, and 2000 µg/mL
- Main experiment I (with metabolic activation): 62.5, 125, 250, 500, 1000, and 2000 µg/mL
- Main experiment IA (without metabolic activation): 62.5, 125, 187, 250, 500, 1000, and 2000 µg/mL
The maximum test item concentration of the pre-experiment and the main experiments (2000 μg/mL) was chosen with respect to the OECD guideline 476 (2016).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium

- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures (Easterbrook et al., 2001)*.

*References:
- Easterbrook, J., Lu, C., Sakai, Y. and Li, A.P. (2001) Effects of organic solvents on the activities of cytochrome P450 isoforms, UDP-dependent glucuronyl transferase, and phenol sulfotransferase in human hepatocytes Drug Metabolism and Disposition, 29, 141-144.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

PRE-EXPERIMENT ON TOXICITY
A pre-test was performed in order to determine the toxicity of the test item. The osmolarity and the pH value were determined in culture medium of the solvent control and of the highest concentration.
The general culturing and experimental conditions in this pre-test were the same as described below for the mutagenicity experiment.
In this pre-test approximately 1.5 million cells were seeded in 25 cm² flasks 24 hours prior to treatment. After approximately 24 hours the test item was added and the treatment proceeds for 4 hours (with and without metabolic activation) (duplicate cultures per concentration level).
Immediately after treatment the test item was removed by rinsing with PBS. Subsequently, the cells were trypsinised and suspended in complete culture medium. After an appropriate dilution the cell density was determined with a cell counter. Toxicity of the test item is evident as a reduction of the cell density compared to a corresponding solvent control. A cell density of approximately 1.5 million cells in 25 cm² flasks is about the same as approximately 10 million cells seeded in 175 cm² bottles 24 hours prior to treatment with the main experiment.

CONCENTRATION SELECTION
Based on the results of the pre-experiment the following concentrations were applied in the main experiment: 62.5; 125; 250; 500; 1000; and 2000 μg/mL.

HPRT MUTAGENICITY EXPERIMENT
- Seeding: Two to four days after sub-cultivation stock cultures were trypsinised at 37°C for approximately 5 to 10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10% FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2% in saline. Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7-1.2x10^7 cells were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.
- Treatment: After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μL/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was replaced with complete medium following two washing steps with PBS.
Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2x10^6 cells per experimental point (concentration series plus controls) were sub-cultivated in 175 cm² flasks containing 30 mL medium.
Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (RS) as measure of test item induced cytotoxicity. The cultures were incubated at 37±1.5°C in a humidified atmosphere with 1.5%±0.5 CO2.
The colonies used to determine the relative survival (RS) were fixed and stained approximately 8±2 days after treatment as described below.
Three or four days after the first sub-cultivation, at least 2x10^6 cells per experimental point were again, sub-cultivated in 175 cm² flasks containing 30 mL medium.
Following the expression time of 7 days, five 75 cm² cell culture flasks were seeded with 4-5x10^5 cells each in medium containing 6-TG (11 μg/mL). Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated at 37°C±1.5°C in a humidified atmosphere with 1.5%±0.5 CO2.
After 8 days (evaluation for viability) and 9±2 days (mutation analysis) the colonies were stained with 10% methylene blue in 0.01% KOH solution.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative survival (RS)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

METHODS USED TO DETERMINE pH, OSMOLALITY AND PRECIPITATION
- The osmolarity and pH of the test item formulated in culture medium was determined by using an osmometer (Gonotec, Model Osmomat 030) or a pH meter (TW, Model Vario pH), respectively, in the pre-experiment without metabolic activation.
- Precipitation or phase separation was be evaluated at the beginning and at the end of treatment by the unaided eye.

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is concentration-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits) (please refer to “Attached background material: Historical control data”).

A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits) (please refer to “Attached background material: Historical control data”).

There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.
In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results, and therefore the test chemical response will be concluded to be equivocal.

Statistics:

The statistical analysis was performed on the mean values of culture I and II for the main experiments.

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value is below 0.05.

A t-test was performed to evaluate a significant increase of the mutation frequency at test points exceeding the 95% control limit. Again, a t-test is judged as significant if the p-value is below 0.05.

However, both, biological and statistical significance will be considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Calcium dibenzoate was tested up to the recommended maximum concentration in absence of metabolic activation or precipitating concentrations in presence of metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRE-EXPERIMENT ON TOXICITY
- Test item concentrations between 15.6 μg/mL and 2000 μg/mL were used in the pre-experiment with and without metabolic activation following 4 hours treatment. The maximum concentration in the pre-experiment was chosen corresponding with respect to the OECD guideline 476 (2016).
- The test medium was checked for phase separation and precipitation at the end of the treatment period (4 hours) before the test item was removed. Precipitation occurred at 1000 μg/mL and above with and without metabolic activation.
- No relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed.

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: There was no relevant shift of osmolarity and pH of the medium even at the maximum concentration of the test item measured in the pre-experiment.
- Precipitation and time of the determination: Precipitation of the test item was observed by the unaided eye at ≥1000 μg/mL (with metabolic activation) and at 2000 µg/mL (without metabolic activation) after the 4 hours exposure period.

HPRT MUTAGENICITY ASSAY
Main Experiment I (without metabolic activation):
- Based on the results of the pre-experiment the following concentrations were applied in the main experiment: 62.5; 125; 250; 500; 1000; and 2000 μg/mL
- Precipitation of the test item was observed by the unaided eye at 2000 μg/mL after the 4 hours exposure period.
- No relevant cytotoxic effects indicated by a relative adjusted cloning efficiency I (survival rate) below 50% (mean value of both parallel cultures) were noted up to the maximum concentration.
- Consequently, the concentrations of 125 to 2000 μg/mL were evaluated for mutagenicity in the absence of metabolic activation.
- The MF (25.5 mutant colonies per 10^6 cells) at the lowest evaluated concentration (125 μg/mL) exceeded the 95% control limit of the solvent historical control data (2.9-22.4 mutant colonies per 10^6 cells), but the value was still within the range of historical control data (5.0-30.5 mutant colonies per 10^6 cells; please refer to “Attached background material: Historical control data”). The t-test performed at this concentration showed a significant difference, when compared to the concurrent solvent control. However, no concentration dependency was noted in the absence of metabolic activation as linear regression analysis showed no statistically significant trend. Since the increased mutation frequency was observed at the lowest concentration and the value was still within the historical control data this observation is judged as biologically irrelevant. To verify this result, a repeat experiment (IA) was performed under the same conditions (4-hour treatment in the absence of metabolic activation) including an additional test concentration close to the concentration showing the statistically significant value.

Main Experiment IA (Repeat-experiment; without metabolic activation):
- Based on the results of Experiment I the following concentrations were applied in Experiment IA without metabolic activation: 62.5; 125; 187.5; 250; 500; 1000; and 2000 μg/mL
- Precipitation of the test item was observed by the unaided eye at 2000 μg/mL after the 4-hour exposure period.
- No relevant cytotoxic effects, indicated by a relative adjusted cloning efficiency I (survival rate) below 50% (mean value of both parallel cultures), were noted up to the maximum concentration.
- Consequently, the concentrations of 125 to 2000 μg/mL were evaluated for mutagenicity in the absence of metabolic activation.
- No increases in the numbers of mutant colonies were observed after treatment with the test item. The observed mean mutant frequency of all evaluated concentrations was within the 95% control limits of the solvent historical control data (please refer to “Attached background material: Historical control data”).
- Linear regression analysis showed no statistically significant trend. Thus, the repeat experiment confirmed the biological irrelevance of the isolated statistically significant increase in mutant frequency obtained in the first experiment (see above: Main Experiment I).

Main Experiment I (with metabolic activation):
- Based on the results of the pre-experiment the following concentrations were applied in the main experiment: 62.5; 125; 250; 500; 1000; and 2000 μg/mL
- Precipitation of the test item was observed by the unaided eye at 1000 μg/mL and above after the 4-hour exposure period.
- No relevant cytotoxic effects indicated by a relative adjusted cloning efficiency I (survival rate) below 50% (mean value of both parallel cultures) were noted.
- Consequently, the concentrations of 62.5 to 1000 μg/mL were evaluated for mutagenicity in the absence of metabolic activation.
- The mutant frequency (24.7 mutant colonies per 10^6 cells) at the concentration of 500 μg/mL marginally exceeded the 95% control limit of the solvent historical control data (2.9-23.7 mutant colonies per 10^6 cells) but the value was still well within the historical control data range (5.3-27.5 mutant colonies per 10^6 cells; please refer to “Attached background material: Historical control data”). Compared to the solvent control the value is not statistically significantly increased as confirmed by the t-test. Moreover, no concentration dependency was observed as confirmed by linear regression analysis of the MF. Since the increased MF was still within the historical control data range and attained no statistical significance, this observation is judged as biologically irrelevant.

The outcome of the experiments was clearly negative in the presence and absence of metabolic activation.

ASSAY VALIDITY
- The observed mean mutant frequencies of the solvent controls were within the 95% control limits of the solvent historical control data.
- EMS and DMBA were used as positive controls and showed a distinct increase in the mutant frequency, which fell well within the historical control data range. The concurrent solvent control cultures showed mutant frequency, which were well within the acceptable ranges of the historical control data base.
Thus, the controls demonstrated sensitivity of the test systems and the validity of the assay.

Conclusions:

No relevant toxicity (10 to 20% relative survival) was found in the experiments. Precipitation of the test item was observed at ≥1000 μg/mL (with metabolic activation) and at 2000 µg/mL (without metabolic activation). Calcium dibenzoate, tested up to the recommended maximum concentration (without metabolic activation) or precipitating concentrations (with metabolic activation), did not induce biologically relevant increases in the mutant frequency. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the Hprt locus in V79 cells. All validity criteria were met. The study was fully compliant with OECD 476 (2016).

Therefore, Calcium dibenzoate (CAS: 2090-05-3; EC: 218-235-4) is considered to be non-mutagenic in this HPRT assay.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Limited report, non-GLP, the omission of information on status of cell division does not lower the reliability

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

yes

Remarks:

no method included to check cell division status

Principles of method if other than guideline:

Mouse lymphoma L5178Y cells were used in this study. Three types of treatment were carried out parallel: two treatments without metabolic activation with or without a recovery period after 24 h continuous treatment and one treatment with metabolic activation by S9 mix. The concentrations of the test substance were 1000, 500, 250 μg/mL in the three type of treatment. Mitomycin C was used as a positive control in the absence of S9 mix and cyclophosphamide was used as a positive control in the presence of S9 mix.

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

None stated

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

1000, 500, 250 μg/mL (cytotoxicity at 200 ug/mL)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 10-12 h
- Exposure duration: 24 h without S9 mix, 4 h with S9 mix
- Expression time (cells in growth medium): 0 or 20 hours without S9 mix, 24 hours with S9 mix
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): 10 min

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays): ethanol/acetic acid
STAIN (for cytogenetic assays): with 2% Giemsa water solution

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 1000

DETERMINATION OF CYTOTOXICITY
- Method: colometric method

Evaluation criteria:

The criteria for determining a positive result were a concentration-related increased in the number of micronucleated cells and a statistically significant increase over the spontaneous level in at least one treatment schedule. Cells with more than 5 micronuclei were excluded from the evaluation to prevent nuclear fragmentation prvent.

Statistics:

The statistical significance of differences between groups was determined using the X2 test.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

None stated

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

The test substance gave negative responses both with or without metabolic activation.

Executive summary:

Mouse lymphoma L5178Y cells were used in this study. Three types of treatment were carried out parallel: two treatments without metabolic activation with or without a recovery period after 24 h continuous treatment and one treatment with metabolic activation by S9 mix. The concentrations of the test substance were 1000, 500, 250 μg/mL in the three type of treatment. Mitomycin C was used as a positive control in the absence of S9 mix and cyclophosphamide was used as a positive control in the presence of S9 mix.

The test substance gave unequivocal negative responses both with or without metabolic activation or cytotoxicity.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Review article, results are given for one concentration only, non-GLP

Qualifier:

no guideline followed

Principles of method if other than guideline:

Chromosomal aberration test was carried out on the test substance, using a Chinese hamster fibroblast cell line, CHL.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

None stated

Species / strain / cell type:

other: Chinese hamster fibroblast cell line, CHL

Metabolic activation:

without

Test concentrations with justification for top dose:

1.5 mg/mL

Vehicle / solvent:

Dimethylsulphoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 4 days
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 13 mins
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): 12-15 mins

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa solution

NUMBER OF REPLICATIONS: not indicated

NUMBER OF CELLS EVALUATED: A hundred well-spread metaphases

DETERMINATION OF CYTOTOXICITY; no data

No further details provided

Evaluation criteria:

The results were considered to be negative if the incidence of the incidence was less than 4.9%, equivocal if it was between 5.0% and 9.9%, and positive if it was more than 10.0%.

Statistics:

None stated

Species / strain:

other: Chinese hamster fibroblast cell line, CHL

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Additional information on results:

No information provided

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

The test substance was ambiguous in the chromosomal aberration test.

Executive summary:

Chromosomal aberration test was carried out on the test substance, using a Chinese hamster fibroblast cell line, CHL.

The test substance was ambiguous in the chromosomal aberration test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo genetic toxicity study with Calcium dibenzoate is available, thus the genetic toxicity will be addressed with existing data on the individual moieties calcium and benzoate.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro gene mutation in bacteria:

 

Calcium dibenzoate:

Chang (2021) investigated the mutagenic potential of Calcium dibenzoate in a bacterial reverse mutation assay according to OECD TG 471 (2020) under GLP. The test was performed in S. typhimurium TA 98, TA 100, TA 1535, and TA 1537 as well as E. coli WP2 uvrA (pKM101) in two independent experiments using triplicate cultures. The cell cultures were exposed to the test material, both in absence and presence of metabolic activation, up to the recommended maximum concentration of 5 mg/plate. The two independent experiments were performed according to the plate incorporation and pre-incubation procedure. Treatment with Calcium dibenzoate did not result in a mutagenic response at all concentrations tested both in absence and presence of S9 metabolic activation. Appropriate positive controls demonstrated the activity of the metabolic activation system and the sensitivity of the test system.

 

In vitro gene mutation in mammalian cells:

 

Calcium dibenzoate:

Sokolowski (2021) examined the gene mutation potential of Calcium dibenzoate in an in vitro mammalian cell gene mutation test at the Hprt locus according to OECD TG 476 (2016) and under GLP. Chinese hamster lung fibroblasts (V79) were exposed for 4 hours to Calcium dibenzoate up to a precipitating concentration of 2 mg/mL (recommended maximum concentration) without metabolic activation and 1 mg/mL with metabolic activation (S9 fraction from phenobarbital/β-naphthoflavone induced rat livers). The outcome of this study was judged as clearly negative both in the absence and presence of S9 metabolic activation. Appropriate positive controls demonstrated the activity of the metabolic activation system and the sensitivity of the test system.

 

In vitro cytogenicity:

 

Calcium:

Considering the overall weight of evidence, such as in vitro data and the crucial role of calcium in cellular processes such as DNA-polymerase and cell cycle functioning as well as its role in approved in vitro genotoxicity experiments show that calcium does not exert any genotoxic or mutagenic effects to mammalian cells. Based on the above existing information on the genotoxicity of calcium substances and the weight of evidence information, calcium is considered as not genotoxic.

 

Benzoic acid:

In vitro studies

For cytogenetic effects, a chromosome aberration test was ambiguous without metabolic activation (Ishidate, 1983). Benzoic acid was clearly negative in an in vitro micronucleus test (with and without metabolic activation) (Nesslany, 1999).

Additional studies were identified, but were not available for review. The available results confirm that benzoic acid is not likely to induce effects on the chromosomal level.

 

In vivo studies

A battery of in vivo tests (Fabrizio, 1974) was conducted with the structurally related Sodium benzoate and these all clearly indicated no cytogenetic effects. These studies are considered to fully address and over-ride the ambiguous results found in the in vitro data set. The toxicokinetic evaluation on Sodium benzoate concluded that the substance will not be taken up as the salt, but rather as its moieties benzoate and sodium (NOTOX 2010). Therefore it can be concluded that the results for benzoic acid will not differ from those found in in vivo tests with Sodium benzoate.

 

Based on the clear negative in vivo results, it is concluded that benzoic acid does not induce genetic toxicity.

 

 

Overall conclusion:

Calcium dibenzoate is not expected to be genotoxic, since Calcium dibenzoate has neither shown any gene mutation potential in bacteria or mammalian cells. A cytogenetic potential is not anticipated, since the two moieties, calcium and benzoate are either considered not cytogenic due to their crucial role in cell function, as in the case of calcium, or, as in the case of benzoate, are clearly negative in existing in vivo studies. Further testing is not required. Thus, Calcium dibenzoate is not to be classified according to regulation (EC) 1272/2008 and its subsequent amendments as genetic toxicant. For further information on the toxicity of the individual moieties, please refer to the relevant sections in the IUCLID and CSR.

Justification for classification or non-classification

Calcium dibenzoate showed no genotoxic effect neither in a bacterial reverse mutation test nor in a mammalian cell gene mutation test (Hprt assay). Moreover, data from both moieties, calcium and benzoate, indicated no cytogenetic potential.

 

The classification criteria acc. to regulation (EC) 1272/2008 and its subsequent amendments as germ cell mutagen are not met, thus no classification is required.