Registration Dossier

Administrative data

Key value for chemical safety assessment

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:
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 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).