Sodium benzoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Limited data available on methods and results, no guidelines and no GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene in S. typhimurium
Tryptophan gene in E. coli

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain / cell type:

E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0.033, 0.1, 0.33, 1.0, 3.3 and 10 mg/plate.

Vehicle / solvent:

0.067M potassium or sodium phosphate buffer (pH 7.0)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-anthramine

Remarks:

In the presence of S9 mix for all strains.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

In the absence of S9 mix for strains TA 98 and TA 1538.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

In the absence of S9 mix for strains TA100 and TA1535.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

In the absence of S9 mix for strain TA1537.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

furylfuramide

Remarks:

In the absence of S9 mix for strain E. coli WP2.

Migrated to IUCLID6: or N-methyl-N'-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

The standard S. typhimurium plate-incorporation assay was performed in the presence and in the absence of S9 mix, contained 10% Aroclor 1254 induced liver S9 from male Sprague-Dawley rats. The E. coli test was performed by the same procedure as the S. typhimurium assay except that each liter of base agar was supplemented with 10 ml (1% v/v) of Oxoid nutrient broth to provide a trace of tryptophan.

Evaluation criteria:

Test results were considered valid only if the positive control compounds induced increases in mutant counts to at least twice background.

Statistics:

None stated.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with and without S9

Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Reliability of this study can be demonstrated as 2 (reliable with restrictions) due to limited data available on methods and results, no guidelines and no GLP.

Executive summary:

Reverse mutation assays using S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537, TA 1538 and E. coli WP2 were carried out with and without metabolic activation system of S9 mix. The test was performed using doses of 0.033, 0.1, 0.33, 1.0, 3.3 and 10 mg/plate. All platings were performed in duplicate and concurrent positive controls were run with the test.

The negative result indicated that the test substance was not mutagenic in any tested S. typhimurium and E. coli strains with or without metabolic activation.

Reliability of this study can be demonstrated as 2 (reliable with restrictions) due to limited data available on methods and results, no guidelines and no GLP.

Genetic toxicity in vivo

Description of key information

Genetic toxicity in vitro:

The reverse mutation assays were negative. Most of chromosome aberration assays (except one equivocal and one negative result) and sister chromosome exchange assays (except one equivocal result) were positive.

Genetic toxicity in vivo:

All the in vivo genotoxicity tests were negative.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study predates approved guidelines and GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

number of metaphases counted

Principles of method if other than guideline:

Study predates approved guidelines.
This in vivo chromosome aberration study was conducted using rats. In acute study, animals were killed 6 hours, 24 hours and 48 hours after administration. In subacute study, five doses 24 hours apart, animals were killed 6 hours after last dose. Four hours af ter the last compound administration, and two hours prior to killing, each animal was given 4 mg/kg of colcemid. The chromosomes of bonemarrow cells were counted and only diploid cells were analyzed. Fifty metaphase spreads were scored per animal. Mitotic indices were obtained by counting at least 500 cells.

GLP compliance:

no

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: A closed colony (random-bred)
- Age at study initiation: 10 to 12 weeks old
- Weight at study initiation: 280 to 350 g
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: Housed one to five to a cage, sanitary cages and bedding were used.
- Diet (e.g. ad libitum): Commercial 4% fat diet and provided ad libitum.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 4-11 days

Route of administration:

oral: gavage

Vehicle:

0.85% saline

Duration of treatment / exposure:

Acute study: Immediately
Subacute study: 96h

Frequency of treatment:

Acute study: Single dose
Subacute study: Once a day for each of five consecutive days

Post exposure period:

Acute study: 6 h, 24h and 48h
Subacute study: 6h

Remarks:

Doses / Concentrations:
50, 500 and 5000 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

5 males

Control animals:

yes, concurrent vehicle

Positive control(s):

Triethylene Melamine
- Doses / concentrations: 0.3 mg/kg

Tissues and cell types examined:

The chromosomes of each bonemarrow cell were counted and only diploid cells were analyzed. They were scored for chromatid gaps and breaks, chromosome gaps and breaks, reunions, cells with greater than ten aberrations, polyploidy, pulverization, and any other chromosomal aberrations which were observed. Fifty metaphase spreads were scored per animal . Mitotic indices were obtained by counting at least 500 cells and the ratio of the number of cells in mitosis/the number of cells observed was expressed as the mitotic index.

Details of tissue and slide preparation:

The slides were stained using a 5% Giemsa solution (Giemsa buffer pH 7.2) for 20 minutes, rinsed in acetone, 1:1 acetone:xylene, and placed in fresh xylene for 30 minutes. The slides were then mounted using Permount (Fisher Scientific) and 24 x 50 mm coverglasses. The coverglasses were selected to be 0.17 mm±0.005 mm in thickness by use of a coverglass micrometer.

Evaluation criteria:

None stated

Statistics:

None stated

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Acute study:
The mitotic index in treatment groups and negative controls was similar. No increase in the number of aberrations was reported in the treated groups at all time points compared to untreated controls.
The positive control showed a lower mitotic index and a high incidence of chromosomal abnormalities

Subacute study:
The mitotic index in treatment groups and negative controls was similar. No increase in the number of aberrations was reported in the treated groups compared to untreated controls.
No data on positive controls are available.

No report on bodyweights, clinical findings or pathology

Conclusions:

Interpretation of results (migrated information): negative
The test substance produced no detectable significant increase in the number of aberrations in bone marrow metaphase chromosomes of rats administered orally at the dosage levels employed in this study.
Reliability of this study can be demonstrated as 2 (reliable with restrictions) due to limited data available on methods and results. Study predates approved guidelines and GLP.

Executive summary:

This in vivo chromosome aberration study was conducted using rats. In acute study, animals were killed 6 hours, 24 hours and 48 hours after administration. In subacute study, five doses 24 hours apart, animals were killed 6 hours after last dose. Four hours af ter the last compound administration, and two hours prior to killing, each animal was given 4 mg/kg of colcemid. Bonemarrow cells were harvested and the chromosomes of each cell were counted and only diploid cells were analyzed (50 metaphases per treatment).

The test substance produced no detectable significant increase in the number of aberrations in bone marrow metaphase chromosomes of rats administered orally at the dosage levels employed in this study.

Reliability of this study can be demonstrated as 2 (reliable with restrictions) due to limited data available on methods and results. Study predates approved guidelines and GLP.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

The test substance does not induce mutations in the assays with Salmonella typhimurium and Escherichia coli with and without metabolic activation (Ishidate et al. 1984, Prival et al. 1991)

The test substance did induce chromosomal damage in in vitro studies on chromosomal aberrations, sister chromatid exchange and formation of micronuclei with and without metabolic activation. The studies included both Chinese Hamster cells as well as human lymphocytes (Fabrizio 1974, Ishidate et al. 1977 and 1984, Abe et al. 1977, Mpountoukas et al. 2008, Zengin et al. 2011). For some of these assays, however, it was not clear whether effects were found at concentrations that exhibited cytotoxicity, because of the limited data that were available in the older publicatons. More recent publications (Mpountoukas et al 2008 and Zengin et. al. 2011) confirm the positive results of the older studies.

The test substance showed to be negative in a dominant lethal study, in an in vivo chromosome aberration test and a host mediated assay (Fabrizio 1974)

Based on the information in this dossier, it is concluded that the test substance, although capable of inducing chromosomal damage in vitro, is not mutagenic or clastogenic in vivo. This is further supported by the negative outcome of carcinogenicity studies in both rats and mice.

Summary of in vitro mutagenetic studies

Method/ Guideline	Test system (Organism, strain)	Concentrations tested (giver range)	Results		Remarks give information on cytotoxicity and other	Reference
			+ S9	- S9
Reverse mutation assays in bacteria (all studies are non-GLP)
Reverse mutation assay, sim. to OECD 471	S. typh TA 1535, 1537, 98, 100, 92 and 94	Maximum concentration = 3.0 mg/plate	negative	negative	The test substance dissolved in distilled water	Ishidate M., Jr, Sofuni T., Yoshikawa K., Hayashi M., Nohmi T. , Sawada M. and Matsuoka A. Fd Chem. Toxic., 22(8), 623-636
Reverse mutation assay, sim. to OECD 471	S. typh TA 1535, 1537, 98, 100, 1538, and E. coli WP2	0.033, 0.1, 0.33, 1.0, 3.3 and 10 mg/plate.	negative	negative	The test substance dissolved in0.067M potassium or sodium phosphate buffer (pH 7.0)	Prival Michael J., Simmon Vincent F. and Mortelmans Kristien E., Mutation Res., 260, 321-329
Chromosome aberration tests in mammalian cells in vitro (all studies are non-GLP)
Chromosome aberration test	Human embryonic lung cultures	2.0, 20.0, 200.0 mg/mL	N/A	negative	The test substance dissolved in 0.85% saline.	Fabrizio D.P.A., National Technical Information Service, PB 245453
Chromosome aberration test	Chinese hamster fibroblast cell line (CHL)	Max. 2.00 mg/mL	N/A	Positive	The test substance dissolved in Physiological saline.	Ishidate M., Jr. and Odashima S., Mutation Research, 48 (1977) 337-354
Chromosome aberration test,Sim. to OECD 473	Chinese hamster fibroblast cell line (CHL)	Maximum concentration = 2 mg/plate	N/A	Positive	The test substance dissolved in Physiological saline	Ishidate M., Jr, Sofuni T., Yoshikawa K., Hayashi M., Nohmi T. , Sawada M. and Matsuoka A., Fd Chem. Toxic., 22(8), 623-636
Chromosome aberration test, Sim. to OECD 473	Human peripheral lymphocytes	6.25, 12.5, 25, 50 and 100 µg/mL	N/A	Positive	The test substance dissolved in distilled water	Zengin N., Yüzbasıog˘lu D.,ünal F., Yılmaz S., Aksoy H., Food and Chemical Toxicology 49 (2011) 763-769
Chromosome aberration test	Chinese hamster cell line (Don)	0.001, 0.002, 0.005 and 0.01 M/plate	N/A	ambiguous	The test substance dissolved in HBSS	Abe, S. & Sasaki, M., J. Nat. Cancer Inst. 58, 1635-1641
Chromosome aberration test, Sim. to OECD 487	Human peripheral lymphocytes	6.25, 12.5, 25, 50 and 100 µg/mL	N/A	Positive	The test substance dissolved in distilled water	Zengin N., Yüzbasıog˘lu D.,ünal F., Yılmaz S., Aksoy H., Food and Chemical Toxicology 49 (2011) 763-769
Sister chromosome exchange assays in mammalian cells (all studies are non-GLP)
SCE	human peripheral blood cells	0.02, 0.2, 2, 4 and 8 mM	N/A	Positive	The test substance	Mpountoukas P., Vantarakis A., Sivridis E. and Lialiaris T., Food and Chemical Toxicology 46 (2008) 2390–2393
SCE, Sim. to OECD 479	Human peripheral lymphocytes	6.25, 12.5, 25, 50 and 100 µg/mL	N/A	positive	The test substance dissolved in distilled water	Zengin N., Yüzbasıog˘lu D.,ünal F., Yılmaz S., Aksoy H., Food and Chemical Toxicology 49 (2011) 763-769
SCE	Chinese hamster cell line (Don)	0.001, 0.002, 0.005 and 0.01 M/plate	N/A	ambiguous	The test substance dissolved in HBSS	Abe, S. & Sasaki, M., J. Nat. Cancer Inst. 58, 1635-1641
DNA damage and/or repair	Isolated human lymphocytes	6.25, 12.5, 25, 50 and 100 µg/mL	N/A	Positive	The test substance dissolved in distilled water	Zengin N., Yüzbasıog˘lu D.,ünal F., Yılmaz S., Aksoy H., Food and Chemical Toxicology 49 (2011) 763-769

Summary of in vivo mutagenetic studies

Method/ Guideline	Species, Strain, Sex, No/group	Route and Frequency of application	Sampling times	Dose levels	Results give dose, sampling time and result +/-/+	Remarks	Reference
Chromosome aberration assay, Sim. to OECD 475	Rat, Sprague-Dawley, male, 5/group	Gavage a) single dose b) 5 d	No information given	50, 500 and 5000 mg/kg	negative	The test substance dissolved in 0.85% saline	Fabrizio D.P.A., National Technical Information Service, PB 245453
Dominant lethal assay, Sim. to OECD 478	Rat, Sprague-Dawley, male/female, Ten males and twenty females/group	Gavage a) single dose b) 1 dose per day for 5 d	No information given	50, 500 and 5000 mg/kg	negative	The test substance dissolved in saline	Fabrizio D.P.A., National Technical Information Service, PB 245453
Host mediated assay	Mouse, ICR, male, 10/group	Gavage a) single dose b) 1 dose per day for 5 d	No information given	50, 500 and 5000 mg/kg	negative	The test substance dissolved in saline	Fabrizio D.P.A., National Technical Information Service, PB 245453

Justification for selection of genetic toxicity endpoint

This is an in-vivo study which is conducted according to a reliable method compared to OECD 475 using rats.

Justification for classification or non-classification

Tests in vivo on germ cells (as in the dominant lethal assay) are negative. Therefore there is no need for classification. In principle in vivo tests will overrule the results of in vitro tests, as even for an in vitro mutagen it might not be possible to reach the target site in the organism and exhibit its effect on site.

Under classification the following will be included: Based on the data available sodium benzoate does not need to be classified for genotoxicity (DSD and CLP).