2-propanol and 2-butanol production, distn. residues

Genetic toxicity: in vitro

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)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study under GLP with full documentation

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471, "Bacterial Reverse Mutation Test", adopted 21st July, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

May 30, 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

June 1996 (Public draft)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-propanol and 2-butanol production, distn. residues, sample 2010

Molecular formula:

UVCB substance

IUPAC Name:

2-propanol and 2-butanol production, distn. residues, sample 2010

Test material form:

liquid

Details on test material:

- The analysis was performed for the reference sample "Sample 2010" as defined in Section 1.4
- Lot/batch No.: 10013593
- Expiration date of the lot/batch: March 2011
- Storage condition of test material: For several days, the test item was stored in the test facility at room temperature protected from light and kept under inert gas. As soon as a fridge with explosion prevention was available, the test item was stored at 2 – 8 °C. No relevant changes in composition were detected before and after storage at room temperature (GC chromatograms).

Method

Target gene:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100: Main DNA target is GC
E. coli WP2 uvr A: Main DNA target is AT

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: see table in "any other information on materials and methods"

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Remarks:

see table in "any other information on materials and methods"

Metabolic activation:

with and without

Metabolic activation system:

Post mitochondrial supernatant (S9) prepared from livers of phenobarbital/beta-naphthoflavone-induced rats

Test concentrations with justification for top dose:

5000; 1581; 500; 158; 50; 15.8 and 5 µg/plate.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO); DMSO was found to be an appropriate vehicle for solubilising the test item up to 50 mg/mL.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 4-Nitro-o-phenylenediamine

Remarks:

4 µg; Strain Salmonella TA98; non-activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

2 µg; Strain Salmonella TA100 and TA1535; non-activation

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

50 µg; Strain Salmonella TA1537; non-activation

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

2 µl; Strain E.Coli WP2 uvrA; non-activation

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2 µg; all of Salmonella strains; activation

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

50 µg; E.Coli WP2 uvrA; activation

Controls 7

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

no

Controls 8

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

True negative controls:

yes

Positive controls:

no

Details on test system and experimental conditions:

The study included a Preliminary Solubility Test, and a Preliminary Range Finding Test (Informatory Toxicity Test)

METHOD OF APPLICATION: Initial Mutation Test (Plate Incorporation Test) and Confirmatory Mutation Test (Pre-Incubation Test)

A) INITIAL MUTATION TEST (PLATE INCORPORATION TEST):
DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS:
The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate.

B) CONFIRMATORY MUTATION TEST (PRE-INCUBATION TEST):
DURATION
- Preincubation period: 20 minutes at 37 °C
- Exposure duration: 48 h

NUMBER OF REPLICATIONS:
The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate.

Statistics:

The colony numbers on the control, positive control and the test plates were determined (counted manually), the mean values and appropriate standard deviations and mutation rates were calculated.
Mutation rate = (Mean revertants at the test item (or control) treatments)/(Mean revertants of vehicle control)

A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control

According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Criteria for a Negative Response: A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Results and discussion

Test results

Test results 1

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:

cytotoxicity

Remarks:

For concentration levels of 500 µg/plate and higher in S. typhimurium TA98, TA1535 and TA1537. For concentration levels of 158 µg/plate and higher in S. typhimurium TA100.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

For concentration levels of 500 µg/plate and higher.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the Confirmatory Mutation Test the pre-incubation method was applied. This experiment was carried out using Salmonella typhimurium strains (TA98, TA100, TA1535, TA1537) and Escherichia coli WP2 uvrA strain, in presence and absence of metabolic activation (S9 Mix) with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate.
In the Confirmatory Mutation Test the examined concentrations were the same as in the Initial Mutation Test.

In general the pre-incubation procedures have equal or greater sensitivity than the plate incorporation assays and during the pre-incubation procedure the test compound, S9, and bacteria are incubated at higher concentrations than in the standard plate incorporation method. Due to the greater sensitivity the inhibitory effect of the test item (already observed in the performed Informatory Toxicity Test and Initial Mutation Test) manifested stronger in the Confirmatory Mutation Test.
Inhibition was observed in S. typhimurium TA98, TA1535, TA1537 and in E. coli WP2 uvrA down to and including the concentration level of 500 µg/plate, in TA100 down to and including the concentration level of 158 µg/plate, without metabolic activation (±S9 Mix) furthermore in all Salmonella typhimurium strains at the concentrations of 5000 and 1581 µg/plate (+S9 Mix) and in E. coli WP2 uvrA at 5000 µg/plate (+S9 Mix).
In the above cases the lower revertant colony numbers than the revertant colony numbers of the vehicle control plates (mostly below the corresponding historical control data ranges) and/or reduced or slightly reduced background lawn development indicated the inhibitory, toxic effect of the test item. Pinpoint colonies appeared in S. typhimurium TA98, at 5000 and 1581 µg/plate (±S9 Mix) and in E. coli WP2 uvrA at 5000 µg/plate (±S9 Mix).
Bacterial growth was not observed in S. typhimurium TA1535 at 5000 µg/plate, in absence of metabolic activation (±S9 Mix).
The further observed slightly lower (than the revertant colony numbers of the vehicle control plates), not dose dependently appeared revertant colony numbers* were evaluated (similarly to the experimental phases before) as reflecting the biological variability of the applied test system.
* The revertant colony numbers were slightly lower than the revertant colony numbers of the vehicle control plates at 1581 µg/plate in E. coli WP2 uvrA (+S9 Mix); at 500 µg/plate in S. typhimurium TA98 (+S9 Mix); at 158 µg/plate in TA1535 (±S9 Mix); at 50 µg/plate in TA98 (±S9 Mix); and at 5 µg/plate in TA98 (±S9 Mix), in E. coli WP2 uvrA (+S9 Mix).
All of the obtained revertant colony number increases were in the historical control data and biological variability ranges** and far below the thresholds for being positive (Section: 8.0).
** The revertant colony numbers were slightly higher than the revertant colony numbers of the vehicle control plates in the concentration range of 500-5 µg/plate in S. typhimurium TA1535 (+S9 Mix); furthermore at 158 µg/plate in E. coli WP2 uvrA (±S9 Mix), in TA1537 (+S9 Mix); at 50 µg/plate in TA1537 (±S9 Mix); and at 15.8 µg/plate in TA1537 (+S9 Mix).
The experimental results (revertant colony numbers per plate, mutation factors, standard deviations) are summarised in Table 9 (Appendix I), and in Tables 17-21 (Appendix IV).

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

The test item „2-propanol and 2-butanol production, distn. residues“ has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test item „2-propanol and 2-butanol production, distn. residues“ was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay in accordance to OECD 471 and EU B.13/14 guidelines.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of phenobarbital/b-naphthoflavone-induced rats.

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test).

Based on the results of the preliminary Range Finding Test the following concentrations of the test item were prepared and used in the Initial and Confirmatory Mutation Tests:

5000; 1581; 500; 158; 50; 15.8 and 5 µg/plate.

The revertant colony numbers of vehicle control plates with and without S9 Mix were within the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with „2-propanol and 2-butanol production, distn. residues“ at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

The reported data of this mutagenicity assay show (see Appendix I to IV) that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item „2-propanol and 2-butanol production, distn. residues“ has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.