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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non mutagenic

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
From April 27 to May 05 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The test was conducted by means of Read Across approach. The reliability of the source study report is 1. Further information was attached at section 13
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
84/449/EWG, B.14, Prival Modification
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidine dependent
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from rat and hamster liver
Test concentrations with justification for top dose:
Preliminary test: 4 to 10000 µg/plateMain test: 20 to 10000 µg/plate
Vehicle / solvent:
At the day of the experiment the test substance was dissolved in Aqua bidest at appropriate concentrations. Two independent experiments were performed for each protocol (Ames, Prival).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
Remarks:
without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
congo red
other: 2-aminoanthracene, benzidine
Remarks:
with S9
Details on test system and experimental conditions:
Preparation and storage of a liver homogenate fraction ("S-9")
Liver preparations were performed from liver of Aroclor induced Sprague Dawley rats and from non pretreated Syrien hamsters. Male Sprague Dawley rats (200 -300 g) receive a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bodyweight) 5 days before sacrifice. Preparation is performed at 0 to 4 °C using cold sterile solution and glassware. The livers from at least 5-6 Sprague Dawley rats or from 5-6 male Syrian golden hamsters (7-8 weeks old) are removed and pooled, washed in 150 mM KCl (approximately 1 ml/g wet livers). The washed livers are cut into small pieces and homogenized in three volumes of KC1. The homogenate is centrifuged at 9000 g for 10 minutes. The supernatant is the S-9 fraction. It is divided into small portions, rapidly frozen and stored at -80 °C for not longer than three months.

Preparation of S-9 Mix
Sufficient S-9 fraction is thawed immediately before each test at room temperature. One or three volumes of S-9 fraction is mixed with nine or seven volumes of the S-9 cofactor solution and kept on ice until used. This preparation is termed S-9 Mix. The concentrations of the different compounds in the S-9 Mix of the rat liver are:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP+
100 mM phosphate buffer
pH 7.4
According to the modification proposed by Prival using 30 minutes preincubation in the presence of 30 % Syrian golden hamster

S-9 Mix The S-9 Mix consists of:
8 mM MgCl2
33 mM KCI
20 mM glucose-6-phosphate
2.8 units/ml glucose-6-phosphate dehydrogenase
4 mM NAPD+
2 mM NADH
2 mM FMN (Riboflavin-5’-phosphate-Na-salz)
100 mM phosphate buffer
pH 7.4

Bacteria
Bacteria are grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No 2 /liter) at 37 °C. The suitable amount of bacteria in the cell suspension is checked by nephelometry. For inoculation, stock cultures which are stored at -80 °C, are used. The compound is tested with the strains Salmonella typhimurium TA 100, TA 1535, TA 1537, and TA 98.

Toxicity experiments and dose range finding
Preliminary toxicity tests were performed with five or four tester strains using three plates per dose to get information on mutagenicity and toxicity for calculation of an appropriate dose range. A reduced rate of spontaneously occuring colonies as well as visible thinning of the bacterial lawn were used as indicator for toxicity. Thinning of the bacterial lawn was controlled microscopically. In combination with the main experiments, toxicity testing was performed as follows: 0.1 ml of the different dilutions of the test compound were thoroughly mixed with 0.1 ml of 10-6 dilution of the overnight culture of TA 100 and plated with histidine and biotin rich top agar (3 plates per dose). The solvent control is compared with the number of colonies per plate in the presence of the test compound. Results are given as a ratio of these values (= surviving fraction).

Mutagenicity test
Two independent experiments which each of the two protocols (Ames, Prival) were performed.
a) - with 10 % rat liver S-9 Mix or buffer and the strains TA 100, TA 1535, TA 1537, and TA 98
- with 30 % rat liver S-9 Mix and the stains TA 100, TA 1535, TA 1537 and TA 98
Top agar is prepared for the Salmonella strains by mixing 100 ml agar (0.6 % agar, 0.6 % NaCl) with 10 ml of a 0.5 mM histidine-biotin solution. The following ingredients are added (in order) to 2 ml of molten top agar at 45 °C:
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound solution 0.5 ml 10 % or 30 % rat liver S-9 Mix or buffer
After mixing, the liquid is poured into a petridish with minimal agar (1.2 % agar, Vogel-Bonner E medium with 2 % glucose). After incubation for 48 to 72 hours at 37 °C in the dark, colonies (his+ revertants) are counted.

b) with 30 % Syrian golden hamster S-9 Mix and preincubation
0.1 ml test solution, 0.1 ml bacterial suspension and 0.5 ml S-9 Mix are incubated at 30 °C for the duration of 30 minutes. Subsequently, 2 ml of soft agar which consists of 100 ml agar (0.6 % agar + 0.6 % NaCl) and 10 ml amino-acid solution (minimal amino-acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) is added. After mixing, the samples are poured on to the Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds. After incubation for 48 to 72 hours at 37 °C in the dark, colonies (his+ revertants) are counted.

Positive controls
Positive control plates were included for each strain. The following substances were used as positive controls.
a) without metabolic activation:
Na-azide: TA 100, TA 1535
9-Aminoacridine: TA 1537
2-Nitrofluorene: TA 98
b) with rat liver S-9 Mix (10 %):
Benzo[a]pyrene: TA 98, TA 100, TA 1535, TA 1537
2-Aminoanthracene: TA 98, TA 100, TA 1535, TA 1537
c) with rat liver S-9 Mix (30 %):
Benzo[a]pyrene: TA 98, TA 100, TA 1535, TA 1537
2-Aminoanthracene: TA 98, TA 100, TA 1535, TA 1537
d) with hamster liver S-9 Mix (30 %):
2-Aminoanthracene: TA 100, TA 1535, TA 1537
Benzidine: TA 98 Congored: TA 98
Evaluation criteria:
No data
Statistics:
No data
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
Positive controls validity:
valid
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
Remarks:
up to 10000 µg/plate
Vehicle controls validity:
valid
Untreated negative 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 nor precipitates, but tested up to recommended limit concentrations
Remarks:
up to 10000 µg/plate
Vehicle controls validity:
valid
Untreated negative 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 nor precipitates, but tested up to recommended limit concentrations
Remarks:
up to 10000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
not specified
Genotoxicity:
other: Not available
Cytotoxicity / choice of top concentrations:
other: Not available
Vehicle controls validity:
other: Not available
Untreated negative controls validity:
other: Not available
True negative controls validity:
other: Not available
Positive controls validity:
other: Not available
Additional information on results:
Sterility checks and control plates
Sterility of S-9 Mix and the test compound were indicated by the absence of contamination on the test material and S-9 Mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies.

Toxicity test
The test compound was tested at doses of 4 to 10000 microgram/plate and proved to be not toxic to the bacterial strains. For mutagenicity testing 10000 microgram/plate was chosen as the highest dose in the main experiments.

Mutagenicity test
Ames-Test:The test compound did not cause a significant increase in the number of revertant colonies with any of the tester strains either in the absence or in the presence of rat S-9 Mix (10 %). No dose dependent effect was obtained.

Prival-Test
In the presence of rat liver S-9 Mix (30 %) and hamster liver S-9 Mix (30 %) using the preincubation method according to Prival the test compound did not show any relevant increases in the number of revertant colonies under the experimental conditions described.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Non mutagenic.
Executive summary:

Method

The test substance was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium, according to the OECD guideline 471.

The mutagenicity studies were conducted in the standard plate test (Ames Test) and in a modified preincubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolizing system derived from rat or hamster liver homogenate. A dose range of 6 different doses from 20 microgram/ plate to 10000 microgram/plate was used.

Results

Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies. The test compound proved to be not toxic to the bacterial strains. 10000 microgram/plate was chosen as top dose level for the mutagenicity study.

a) Ames Test

Mutagenicity: In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with the test substance did not result in relevant increases in the number of revertant colonies.

b) Prival Test

In the presence of hamster liver S-9 using the preincubation method according to Prival the test substance di d not induce a significant increase in the number of revertant colonies, with any of the tester strains.

Conclusions

It can be stated that the test substance is not mutagenic in the standard plate test and in the preincubation test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

GERM CELL MUTAGENICITY

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

Category 1: Substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans. Substances known to induce heritable mutations in the germ cells of humans.

Categoty 2: Substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

Classification for heritable effects in human germ cells is made on the basis of well conducted, sufficiently validated tests as In vitro mutagenicity tests such as these indicated in 3.5.2.3.8:

- in vitro mammalian chromosome aberration test;

- in vitro mammalian cell gene mutation test;

- bacterial reverse mutation tests

The substance did not create gene mutations in the strains of Salmonella typhimurium and E. Coli under the performed test, therefore according to the 3.5. of the CLP Regulation EC n.1272/2008, it cannot be classified as mutagenic for germ cells.