Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 20 March 1997 to 16 March 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
colorless
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: batch no. 44-0793
- Expiration date of the lot/batch: Not specified
- Purity test date: Not specified
- Date of manufacture: May 1996

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions:about 8 month after the date of manufacture
- Solubility and stability of the test substance in the solvent/vehicle: the test item was soluble in the vehicle Precipitation of the test substance
was found from about 2,500 µg/plate onward.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not specified

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
other: uvrB (S. Typhimurium), uvrA (E. Coli)
Metabolic activation:
with and without
Metabolic activation system:
S9 Fraction from rat liver which were treated intraperitoneally with Aroclor 1254
Test concentrations with justification for top dose:
DOSE RANGE: 15 µg - 5,000 µg/plate (SPT; Salmonella strains) ; 20 µg - 5,000 µg/plate (SPT; E. coli) ; 0.625 µg - 100 µg/plate (PIT; Salmonella strains) ; 4 µg - 2,500 µg/plate (PIT; E. coli)
SPT : Standard plate test ; PIT : Preincubation test
The top dose was chosen as 2500 µg/plate due to the limit of solubility of the test item in vehicle.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Due to limited solubility of the test substance in water, acetone was selected as the vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical data are available
Controls
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
N-ethyl-N-nitro-N-nitrosoguanidine
other: N-methyl-N'-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at seeding (if applicable): 10E9 bacteria/mL

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 to 72 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): revertant colonies were counted after 48 hours to 72 hours exposure period

SELECTION AGENT (mutation assays): not applicable

SPINDLE INHIBITOR (cytogenetic assays): not applicable

STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: triplicates were used per condition

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: individual plate count to determine the nomber of revertant colony

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertants ; clearing or diminution of the background lawn (reduced his- or trp - background growth) ; reduction in titer
- Any supplementary information relevant to cytotoxicity: tested for all test groups both with and without S9 mix in all experiments
Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met:
-A dose-related and reproducible increase in the nuinber of revertant colonies, i.e. about daubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.
A test substance is generally considered nonmutagenic in this test if:
-The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in twa experiments carried out independently of each other.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
for preincubation method condition
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
A bacteriotoxic effect reduced his background growth, decrease in the number of his revertants, reduction in the titer) was observed in the standard plate test depending on the strain and test conditions from about 60 µg - 250 µg/plate onward.
With E. coli only a weak bacteriotoxic effect was observed with S-9 mix only at doses >= 2,500 µg/plate.
In the preincubation assay bacteriotoxicity was observed depending on the strain and test conditions from about 0.625 µg - 6.0 µg/plate onward (Salmonella strains) or from about 4 µg - 20 µg/plate onward (E. coli).

Applicant's summary and conclusion

Conclusions:
According to the results of the study, the test substance did not led to an increase in the number of revertant colonies either without S-9 mix or after adding a metabolizing system in serval experiments carried out independently of each other (standard plate test and preincubation assay). Thus, under the experimental conditions, it is concluded that Tri-N-hexylamine is not a mutagenic agent in a bacterial reverse mutation test in vitro. However, the test item induced bacteriotoxicity effect.
Executive summary:

In this GLP compliant study, the substance Tri-N-hexylamin was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains in a reverse mutation assay according to OECD TG 471/472 method.

TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA strains were used in this study. Two methods were performed, standard plate test, and preincubation method with and without metabolic activation system. For preincubation method, test item and bacteria were incubated 20 minutes. After 48 hours – 72 hours incubation of the test item with bacteria, number of revertant colony and cytotoxicity were determined.

An increase in the number of his or trp revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system. However, bacteriotoxicity occurred under all test conditions.

According to the results of the study, the test substance did not lead to an increase in the number of revertant colonies either without S-9 mix or after adding a metabolizing system in serval experiments carried out independently of each other (standard plate test and preincubation assay). Thus, under the experimental conditions, it is concluded that Tri-N-hexylamin is not a mutagenic agent in a bacterial reverse mutation test in vitro. However, the test item induced bacteriotoxicity effect.