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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:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(2-butoxyethoxy)ethanol
EC Number:
203-961-6
EC Name:
2-(2-butoxyethoxy)ethanol
Cas Number:
112-34-5
Molecular formula:
C8H18O3
IUPAC Name:
2-(2-butoxyethoxy)ethanol
Details on test material:
- Name of test material (as cited in study report): diethylene glycol monobutyl ether, butyl carbitol
- Analytical purity: no data
- Lot/batch No.: S767216
- Other: supplied by Union Carbide Chemical Company (commercial grade material)
Specific details on test material used for the study:
Source: INEOS Oxide
Appearance: Clear, colourless liquid
Batch no: TK309 - 30/3/2017
Purity: 99.5%

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/beta-naphtha flavone induced SD male rat liver S9 fraction. Prepared 21/10/16. Sterile. Final protein content 2g/dL. Sterile cofactors added prior to use and then maintained on ice for duration of study.
Test concentrations with justification for top dose:
Plate incorporation method: 1.5, 5, 15, 50, 150, 500, 1500, 5000 ug/plate. Maximum dose is as recommended in guideline. Done in triplicate
Vehicle / solvent:
- Vehicle: sterile water (supplier Aguettant).
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation): 0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate.
METHOD OF APPLICATION: pre-incubation method: 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates.

DURATION
- Exposure duration: 48 hours at 37C.
- Expression time (cells in growth medium):

NUMBER OF REPLICATIONS: Performed in triplicate
Rationale for test conditions:
According to guideline
Evaluation criteria:
Scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.

A valid positive requires
- A dose-related increase in mutant frequency over the dose range tested.
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Valid statistical significance
- >2x increase in revertant colonies compared to concurrent solvent control for any tester strain

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
Statistical significance was tested using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The acceptability criteria as defined within the study and guideline were all met confirming the study as valid.

Any other information on results incl. tables

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

Applicant's summary and conclusion

Conclusions:
Negative with metabolic activation. Negative without metabolic activation. This substance dose not produce gene mutation in bacteria.
Executive summary:

In a reverse mutation assay in bacteria S typhimurium strains TA98, TA100, TA1535, TA1537 and E coli WP2uvrA there was no evidence of mutation with and without metabolic activation up to the maximum dose which did not cause significant cell toxicity. The substance was considered to be non-mutagenic under the conditions of this test.