Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Remarks:
source of read across study record
Adequacy of study:
key study
Study period:
From April 25 to May 11, 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted May 26, 1983
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Similar Substance 01
IUPAC Name:
Similar Substance 01

Method

Species / strain
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Regular checking: regular checking of the properties of the strains with regard to membrane permeability and ampicillin resistance as well as normal spontaneous mutation rates is performed in testing laboratory according to Ames et al.
- Storage: the strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.
- Precultures: from the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. The nutrient medium contains per litre: 8 g Difco Nutrient Broth, 5 g NaCl. The bacterial culture was incubated in a shaking water bath for 6 hours at 37 °C.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal activation system
Test concentrations with justification for top dose:
10.0, 33.3, 100.0, 333.3, 1000.0 and 5000.0 µg/plate
Vehicle / solvent:
- Vehicle: aqua dest, for test item and sodium azide. DMSO for 4-NOPD and 2-AA.
- Justification for choice of vehicle: the vehicle was chosen because of its solubility.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
other: 4-nitro-o-phenylene-diamine // 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation, two independent experiments.

AGAR
- Selective Agar: 2.0 % Vogel-Bonner-Glucose-Minimal-Agar was used as selective agar. Each petri dish was filled with 20 ml of this nutrient medium. Sterilizations were performed at 121 °C in an autoclave.
- Overlay Agar: the overlay agar contains per litre 6.0 g Difco Bacto Agar, 6.0 g NaCl, 10.5 mg L-histidine x HCl x H2O and 12.2 mg biotin. Sterilizations were performed at 121 °C in an autoclave.

NUMBER OF REPLICATIONS: each concentration, including the controls, was tested in triplicate.

EXPERIMENTAL PERFORMANCE
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 control, negative control, 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 (cf. test system, pre-culture of the strains);
2000 µl overlay agar
After solidification the plates were incubated upside down for 72 hours at 37 °C in the dark.

DETERMINATION OF CYTOTOXICITY
To evaluate the toxicity of the test article a pre-study was performed with strains TA 98 and TA 100. 8 concentrations were tested for toxicity and mutation induction with each 3 plates.
The experimental conditions in this pre-experiment were the same as described for the experiment.
Toxicity of the test article may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
S9 preparation
The S9 liver microsomal fraction was obtained from the liver of 8 - 12 weeks old male Wistar rats, strain WU (weight ca. 150 - 200 g) which received a single i.p. injection of 500 mg/kg b.w. 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, diluted 1:3 in RC1 was centrifuged cold at 9,000 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 are kept at -20 “C for only several weeks before use.
The protein concentration in the S9 preparation is usually between 20 and 45 mg/ml.

S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution in a ratio 3:7. The composition of the cofactor solution was concentrated to yield the following concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 5 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.
Evaluation criteria:
A test article is considered as positive 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 article 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 article is considered as 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.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
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
Additional information on results:
Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings.

CYTOTOXICITY
Toxic effects, evidenced by a reduction in the number of revertants, occurred in the strains TA 1535 at 5000.0 µg/plate (exp. I; without S9 mix), in TA 1537 at 1000.0 and 5000.0 µg/plate (exp. I; without S9 mix), and in TA 98 at 1000.0 µg/plate (exp.II; with S9 mix), and 5000.0 µg/plate (exp.I, and II; with S9 mix).
The plates incubated with the test article showed normal back-ground growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

CONTROLS
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Applicant's summary and conclusion

Conclusions:
The test item is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.
Executive summary:

The study was performed to investigate the potential of test item to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100.

The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 10.0, 33.3, 100.0, 333.3, 1000.0 and 5000.0 µg/plate.

Toxic effects, evidenced by a reduction in the number of revertants, occurred in the strains TA 1535 at 5000.0 µg/plate (exp. I; without S9 mix), in TA 1537 at 1000.0 and 5000.0 µg/plate (exp. I; without S9 mix), and in TA 98 at 1000.0 µg/plate (exp.II; with S9 mix), and 5000.0 µg/plate (exp.I, and II; with S9 mix).

The plates incubated with the test article showed normal back-ground growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Conclusion

It can be stated that 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.

Therefore, test item is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.