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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
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Butyl 3-hydroxybutyrate
EC Number:
258-658-1
EC Name:
Butyl 3-hydroxybutyrate
Cas Number:
53605-94-0
Molecular formula:
C8H16O3
IUPAC Name:
butyl 3-hydroxybutanoate
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
Lot/Batch Number: 30705-83-df

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:
not specified
Metabolic activation:
with and without
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene
Details on test system and experimental conditions:
7.1 General Overview:
The selected bacterial strains were combined with the test substance, with or without a metabolic activation system, and plated directly onto minimal medium (plate incorporation method). After a period of incubation, revertant colonies were counted and compared to the number of spontaneous revertants in a negative control culture. For the preincubation method, the bacteria were incubated with the test substance for 20 minutes at 37 ± 1 deg. C with or without metabolic activation.

7.2 Test Strain Identification:
The requirement of exogenous histidine or tryptophan for growth was demonstrated for the S. typhimurium and E. coli strains, respectively. Other phenotypic characteristics were verified by using crystal violet sensitivity and resistance to ampicillin. Spontaneous reversion frequency was in the range expected as established by Toxikon's historical mean values and the published literature. The strains subjected to testing were:

S. typhimurium TA98
S. typhimurium TA 100
S. typhimurium TA 1535
S. typhimurium TA 1537
E. coli WP2 uvrA

7.3 Stock Cultures:
Working stock cultures were grown fresh for the assay. A single colony of bacteria grown on nutrient agar plates was isolated and inoculated in sterile nutrient broth and grown overnight at 37 ± 1 deg. C. Cultures with an absorbance > 1 at 650 nm read against nutrient broth blank were used in the assay.

7.4 The test substance was found to be soluble in DMSO. The study was therefore conducted with DMSO as the vehicle.

7.5 Test Substance Preparation:
Following determination of solubility, the test substance was dissolved in Dimethyl Sulfoxide (DMSO) at appropriate volumes to obtain desired concentrations.

7.6 Negative Control Substance:
The negative control substance was DMSO, without the test substance, and prepared the same way as the test solutions. Each test strain was plated with 100 J.1L of the solvent, both with and without metabolic activation. These negative controls provided reference for background lawns and revertant colony formation.

7. 7.1 Positive Control Substances (Without Metabolic Activation):
For assays performed without metabolic activation, the following strain-specific positive control substances were used.

S. typhimurium TA98 2-Nitrofluorene (1 0 ug/ml)
S. typhimurium TA100 Sodium Azide (100 ug/ml)
S. typhimurium TA1535 Sodium Azide (5 ug/ml)
S. typhimurium TA1537 9-Aminoacridine (800 ug/ml)
E. coli WP2 uvrA 4-Nitroquinoline 1-0xide (100 ug/ml)

7. 7.2 Positive Control Substances (With Metabolic Activation):

S. typhimurium TA98 2-Aminoanthracine (1 0 ug/ml)
S. typhimurium TA100 2-Aminoanthracine (1 0 ug/ml)
S. typhimurium TA1535 2-Aminoanthracine (1 0 ug/ml)
S. typhimurium TA1537 2-Aminoanthracine (1 0 ug/ml)
E. coli WP2 uvrA 2-Aminoanthracine (1 0 ug/ml)

7.8 Application of Test and Control Substances:
All test solutions were applied at a volume of 100 J.1Liplate unless otherwise required based on the appropriate solvent.

7.9 Replication:
All controls and test groups were plated in triplicate.

7.1 0 Non-Activated Assay: Top agar, supplemented with 0.5 mM histidine and 0.5 mM biotin or tryptophan alone, was used as an overlay. The agar was maintained at 42-48 oc until used. The overlay agar mixture was made in sterile tubes and contained:
2 ml of molten top agar
0.1 ml of the appropriate strain of bacteria
0.1 ml of the appropriate concentration of the test substance or control substance
0.5 ml of 0.2 M phosphate buffer pH= 7.4

The tubes were vortexed and then poured onto minimal glucose agar plates. Plates were incubated at 37 ± 1 oc for Approximately 67 hours. After incubation, each plate was checked for uniformity of background lawns and the number of revertant colonies was counted.

7.11 Metabolic Activation Assay:
S9 Microsomal Fraction: The S9 microsomal fraction from Sprague-Dawley rat livers induced with Aroclor® 1254 was purchased from Moltox, Boone, NC, and stored at -80 oc prior to use.

Overlay Agar Preparation with S9 Microsomal Fraction: The overlay agar mixture for testing of metabolic activation contained the S9 fraction of rat liver homogenate in lieu of the phosphate buffer used in the non-activated assay. The S9 activation system was prepared fresh on the day of the assay and kept refrigerated or on ice. The system contained the following per 10 ml:

0.4 M MgCI2/1.65 M KCI 0.20 mL
1 M glucose-6-phosphate 0.05 mL
0.1 M NADP 0.40 mL
0.2 M phosphate buffer pH= 7.4 5.00 mL
USP Sterile Water for Injection 3.35 mL
S9 Fraction 1.00 mL

7.12 Range Finding Study:
Prior to the definitive assay, a range finding study was conducted to determine the cytotoxicity of the test substance and to establish the exposure levels for the definitive assay. Concentrations tested included 5, 1.6, 0.5, 0.18, 0.06 and 0.02 IJL per plate. The test was conducted with S. typhimurium TA98 without metabolic activation.

Evaluation criteria:
EVALUATION CRITERIA
8.1 Statistical Evaluation of the Reverse Mutation Assay: The data obtained from the assay are analyzed by appropriate statistical procedures [e.g. analysis of variance (AN OVA) with Dunnett's Test, using Graph Pad Prism for Windows, version 3.02, Graph Pad Software, San Diego, CA, USA, 2000]. Differences between test and control data are considered to be statistically significant if the probability of the differences being due to chance is equal to or less than 5% (p <; 0.05). The results are also considered for biological relevance.

8.2 Positive Response:
8.2.1 Controls:
The positive control substance assays utilize direct-acting mutagens and a mutagen requiring metabolic biotransformation. All positive controls must exhibit a statistically significant increase in the number of mutants as compared to the corresponding negative control, to demonstrate that the test system is functional with known mutagens. Results for a strain are rejected if the positive control substance does not yield a mutagenic
response.

8.2.2 Test Concentrations:
The test substance is considered to have caused a positive response in the assay if at least one concentration exhibits a reproducible and statistically significant increase (p s: 0.05) in the number of mutants over its concurrent negative control. Alternately, the test substance is considered to have caused a positive response in the assay if a concentration-dependent increase in the number of mutants with r > 0.95 is observed.

8.3 Statistical as well as biological significance are taken into consideration in the evaluation of differences.

8.4 The study and its design employ methodology to minimize uncertainty of measurement and control of bias for data collection and analysis.
Statistics:
The data obtained from the assay are analyzed by appropriate statistical procedures [e.g. analysis of variance (AN OVA) with Dunnett's Test, using Graph Pad Prism for Windows, version 3.02, Graph Pad Software, San Diego, CA, USA, 2000]. Differences between test and control data are considered to be statistically significant if the probability of the differences being due to chance is equal to or less than 5% (p <; 0.05). The results are also considered for biological relevance.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Under the test conditions, n-butyl-3-hydroxybutyrate is non mutagenic in the test species.
Executive summary:

The Salmonella typhimurium ( S. typhimurium) and Escherichia coli (E. coli) Reverse Mutation Assay (Ames Assay) was conducted to evaluate the potential of the test substance, n-butyl-3- hydroxybutyrate, to induce reverse mutations in histidine (his- to his+) and tryptophan (tryp- to tryp+) genes in S. typhimurium and E. coli, respectively.

An initial range finding study was conducted to determine toxicity and select test concentrations for the definitive assay. The test substance was dissolved in Dimethyl Sulfoxide (DMSO) and tested at 5, 1.6, 0.5, 0.18, 0.06 and 0.02 IJL per plate using TA98 strain in the absence of metabolic activation. The results of the range finding assay showed that the test substance was not cytotoxic at 5, 1.6, 0.5, 0.18, 0.06 and 0.02 IJL per plate.

The definitive assay was conducted using 5, 1.6, 0.5, 0.18, 0.06 and 0.02 IJL per plate of the test substance. The definitive assay was conducted with four strains of S. typhimurium and one strain of E. coli in the presence or absence of an exogenous mammalian metabolic activation system using plate incorporation method of exposure. The results of the definitive assay showed that the test substance did not increase the frequency of revertants at any of the test concentrations in any of the strains tested in the presence and absence of metabolic activation. The positive controls exhibited a significant increase in the frequency of revertants as compared to the negative controls in the presence and absence of metabolic activation validating functioning of the assay.

A confirmatory assay was performed in order to verify the results of the definitive reverse mutation assay using preincubation method of exposure. The results of the confirmatory assay were consistent with the definitive assay. Under the test conditions, the test substance is non mutagenic in the test species.

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