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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test, 4 strains, with and without S9 Hamster mix (Prival), negative

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: read across from analogue substance
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
performed on analogue substance
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
yes
Principles of method if other than guideline:
Prival method
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with
Metabolic activation system:
S9 liver fractions of Syrian Golden hamster, untreated
Test concentrations with justification for top dose:
Pre-test concentrations: 1, 3, 10, 33, 100, 333, 1000, 5000 µg/plate
Toxicity was observed (reduced count of revertants per plate) at concentration of 5000 µ/plate, both with and without S9 mix, in both TA 98 and TA 100 strains.
Based on the pre-test results, the following experimental concentrations were selected: 10, 100, 333.3, 1000 and 5000 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
other: 4-nitro-o-phenylene-diamine
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Details on test system and experimental conditions:
PRE-TEST FOR TOXICITY:
In order to select suitable concentrations of test item for the experimental study which produced some toxicity at the highest dose, the count of revertants per plate at 1, 3, 10, 33, 100, 333, 1000 and 5000 µg/plate of test item was compared to count of revertants of a negative control, solvent control and three positive controls (4-NOPD, 50 µg; sodium azide, 10 µg; 2-aminoantracene, 10 µg). Toxicity by reduced count of revertants was observed at the highest dose, 5000 µg/plate. Therefore, the experimental concentrations selected were 10, 100, 333.3, 1000 and 5000 µg/plate.

PREPARATIONS:
- Bacterial suspensions: ampoules of the four strains were thawed and 0.5 ml bacterial suspensions were transferred to 250 ml erlenmeyer flasks containing 20 ml nutrient medium (8 g/l Difco Nutrient Broth and 5 g/l NaCl). The bacterial culture was incubated in a shaking water bath for 6 hours at 37 °C.
- Agar plates: each petri dish was filled with 20 ml of agar medium (1.5 % Vogel-Bonner-Glucose-Minimal-Agar)
- Overlay agar: 6 g/l Difco Bacto Agar, 6 g/l NaCl, 10.48 mg/l L-histidine x HCl x H2O, 12.2 mg/l biotin
- S9: S9 liver microsomal fraction was obtained from the liver of 8 to 12 week old male Wistar rats which received a single i.p. injection of 500 mg/kg bw Aroclor 1254 in olive oil 5 days previously. After cervical dislocation, the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate was diluted 1:3 in KCl and centrifuged cold at 9000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2 or 5 ml and stored at -70 °C. Small numbers of the ampoules were kept at -20 °C for only several weeks before use. The standardisation of the protein content was made using the analysis kit of Bio-Rad Laboratories. The protein concentration in the S9 preparation was 20 to 45 mg/ml.
- S9 mix: S9 was mixed with S9 cofactor solution in a ratio 3:7 to yield the following concentrations: 8 mM MgCl2; 33 mM KCl; 5 mM glucose-6-phosphate; 5 mM NADP; 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

METHOD:
i) Each test strain was tested both with and without metabolic activation, at all 5 test item concentrations, with an appropriate positive control, and with both a negative control and a solvent, in triplicate. The following materials were mixed in a test tube and poured onto the pre-prepared minimal agar plates:
- 100 µl test item at selected dose level (10, 100, 333, 1000 or 5000 µl), solvent (negative control), or reference mutagen solution (positive control)
- 500 µl S9 mix ("with metabolic activtion") or S9 mix substitution buffer ("without metabolic activation")
- 100 µl bacterial suspension (TA 1535, TA 1537, TA 98 or TA 100)
- 2000 µl overlay agar, pre-prepared
ii) Plates were allowed to solidify, then were incubated upside down for 72 hours at 37 °C in the dark.
iii) Colonies were counted using the BIOTRAN III counter (BIOTRONIK, 6000 Frankfurt, DE)
Rationale for test conditions:
OECD 471 is the most widely used in vitro assay to test in vitro gene mutation in bacteria. It is known that azo compounds can be reduced to free aromatic amines following oral ingestion in mammals. Many aromatic amines have been demonstrated to be mutagenic, and in order to completely evaluate the mutagenic potential of an azo compound, the mutagenicity of any constituent aromatic amines must also be evaluated (Prival and Mitchell, 1982). This study was therefore performed using conditions in which reduction of the compound could occur in the presence of a modified metabolic activation system (the so-called Prival methodology described by Prival and Mitchell (Prival and Mitchell, 1982)).
Evaluation criteria:
A test item is considered as positive to mutagenicity if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced. A test item producing neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
A test item is considered a mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537 and TA 98 it is at least three times higher as compared to the spontaneous reversion rate. A dose-dependent increase in the number of revertants is regarded as an indication ofpossibly existing mutagenic potential of the test article regardless of whether the highest dose induced the described enhancement factors or not.
Statistics:
No statistical evaluation was performed
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
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:
cytotoxicity
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
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
MAIN STUDY
- Toxic effects, evidenced by a reduction in the number of spontaneous revertants, occurred in some test groups with and without metabolic activation at the highest investigated dose. The plates incubated with the test article showed normal background growth of up to 5000 µg/plate with and without S9 mix in all strains used.
- Up to the highest investigated dose, no significant dose-depandant increase in revertant colony numbers was obtained in all Salmonella typhimurium strains used. The presence of liver microsomal activation did not influance thesa findings.
- Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. During the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used.
Conclusions:
The substance was tested for in vitro gene mutation in bacteria following OECD 471 with the Prival modification. Under the experimental conditions the substance did not show any mutagenic potential
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

Classification for mutagenicity under Regulation 1272/2008 is warranted for substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans. The classification in Category 2 is based on:

— positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from:

— somatic cell mutagenicity tests in vivo, in mammals; or

— other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays.

Based on the results of the in vitro gene mutation in bacteria no classification for mutagenicity is foreseen for the target substance