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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test substance : LAB 3085
Batch number : HW 11/10/00
I.P.L. Registration number : 010109
Physical form : white cristalline powder
Purity : 99.5% (molar purity)
Storage : at +4°C, protected from light and moisture
Solvent used : distilled water for injectable preparations, Fresenius #4807

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
The two strains TA 1535 and TA 100 show a base pair substitution in the hiis G gene (G-C in place of A-T). These two strains can be used to detect products which cause reverse base pair substitution.
The strain TA 1537 has, in the his C gene, a loop of several cytosines which give rise to a frameshift mutation. The strain TA 98 contains, in the his D gene, a sequence of four base pairs of cytosine-guanine (CG) which also give rise to a frameshift mutation.
The four strains also carry two other types of mutation : they are deficient in DNA repair mechanism (uvr B-) and show a change in the structure of cell-wall lipopolysaccharides (LPS) (rfa-). These two mutations make the bacteria more sensitive to genotoxic agents and more permeable to cell penetration by large molecules.
The R factor (present in strains TA 98 and TA 100), which is a pKM 101 ampicillin resistance plasmid, increases sensitivity to mutagenic agents via error-prone repair mechanisms (Maron and Ames, 1983).
Species / strain / cell type:
other: WP2 pKM101
Details on mammalian cell type (if applicable):
Bacterial strains obtained from CROFTON-SLEIGH C, kept in the laboratory and preserved in liquid nitrogen :
WP2 pKM101:trp- presence of plasmid (R factor: pKM 101 ampicilline-resistant plasmid)
This Escherichia coli strains show the same type of mutation in the trp gene: the TAA ochre non-sens mutation. Moreover, they are deficient in DNA excision repair (uvrA-). That mutation makes the bacteria more sensitive to genotoxic agents such as UV which causes lesions in DNA.
The R factor increases sensitivity to mutagenic agents via error-prone repair mechanisms.
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Details on mammalian cell type (if applicable):
Bacterial strains obtained from CROFTON-SLEIGH C, kept in the laboratory and preserved in liquid nitrogen :
WP2 uvrA.pKM101: trp- ; uvrA- mutation presence of plasmid pKM 101 (R factor : pKM 101 ampicilline- resistant plasmid)
This Escherichia coli strains show the same type of mutation in the trp gene: the TAA ochre non-sens mutation. Moreover, they are deficient in DNA excision repair (uvrA-). That mutation makes the bacteria more sensitive to genotoxic agents such as UV which causes lesions in DNA.
The R factor increases sensitivity to mutagenic agents via error-prone repair mechanisms.
Metabolic activation:
with and without
Metabolic activation system:
hepatic microsomes from rat livers induced by Aroclor 1254- Incubation period 48 hours
Test concentrations with justification for top dose:
0, 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water

Controlsopen allclose all
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation for TA1535 and TA100
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation for TA 1537
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without metabolic activation for TA 98
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation for WP2pKM101
Positive controls:
yes
Positive control substance:
other: 2-athramine
Remarks:
with metabolic activation for TA 1535, TA1537, TA98 and TA100
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation for WP2pKM101 and WP2 uvrA pKM101
Positive controls:
yes
Positive control substance:
other: Potassium dichromate
Remarks:
without metabolic activation for WP2 uvrA pKM101
Details on test system and experimental conditions:
After 48 hours of incubation at 37°C, the prototrophic mutant colonies that have developed in the plates are counted.
Evaluation criteria:
The results are expressed as the mean of mutants per plate and, for each concentration of the test product.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Species / strain:
other: E. coli WP2 pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test compound LAB 3085 provided by ROQUETTE induced no mutagenic activity in the four Salmonella typhimurium and the two Escherichia coli strains tested.
Executive summary:

MUTAGENICITY TEST ON BACTERIA

(Salmonella typhimurium his- and Escherichia coli trp-)

USING B.N.AMES'S TECHNIQUE

This study was carried out in compliance with good laboratory practice.

Methods

Strains used: S.typhimurium: TA 1535, TA 1537, TA 98, TA 100

E.coli: WP2(pkM101) and WP2uvrA(pKM101)

Preliminary test of toxic activity: Carried out on 6 strains - Incubation period: 48 hours

Sterility test: Normal

Mutagenicity test: Carried out both with and without metabolic activation using hepatic microsomes from rat livers induced by Aroclor 1254 -Incubation period: 48 hours

Number of assays: 2 (the second assay with metabolic activation was performed according to pre-incubation method)

Initial solution: Distilled water

Limiting factor for maximum dose: Maximum dose according to OECD procedures

Doses used in main test: expressed as µg/plate

Without S9 mix

 Strain     TA1535 TA1537     TA98     TA100     WP2(pKM101)     WP2uvrA(pKM101)    
 Assay  1
Doses µg/plate                  0  0  0  0  0  0  0  0  0  0  0  0
 50  50  50  50  50  50  50  50  50  50  50  50
 150  150  150  150  150  150  150  150  150  150  150  150
 500  500  500  500  500  500  500  500  500  500  500  500
 1500  1500  1500  1500  1500  1500  1500  1500  1500  1500  1500  1500
 5000  5000  5000  5000  5000  5000  5000  5000  5000  5000  5000  5000

With S9 mix

 Strain     TA1535 TA1537     TA98     TA100     WP2(pKM101)     WP2uvrA(pKM101)    
 Assay  1 2 * 2*  2*  2*  2*  2 *
Doses µg/plate                  0  0  0  0  0  0  0  0  0  0  0  0
 50  50  50  50  50  50  50  50  50  50  50  50
 150  150  150  150  150  150  150  150  150  150  150  150
 500  500  500  500  500  500  500  500  500  500  500  500
 1500  1500  1500  1500  1500  1500  1500  1500  1500  1500  1500  1500
 5000  5000  5000  5000  5000  5000  5000  5000  5000  5000  5000  5000

* assay with pre-incubation

Results:

The test compound LAB 3085 provided by ROQUETTE induced no mutagenic activity in the four Salmonella typhimurium and the two Escherichia coli strains tested.