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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three GLP-compliant in vitro tests with „2-propanol and 2-butanol production, distn. residues“, a bacterial reverse mutation assay (Ames test), a mammalian chromosome aberration test, and a mammalian cell gene mutation test, were conducted.

The reported data of the bacterial reverse mutation assay show that under the experimental conditions applied, the substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In the mammalian chromosome aberration test, the substance did not induce structural chromosome aberrations in this test in Chinese Hamster lung cells, both with and without metabolic activation.

In the mammalian cell gene mutation test, the substance did not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells (L5178Y TK+/- 3.7.2 C mouse lymphoma cell line) used.

Hence, in all three different in vitro tests, the gene toxicity of „2-propanol and 2-butanol production, distn. residues“ was evaluated "negative" and the substance is regarded as not mutagenic. Further in vivo tests are not required.

Link to relevant study records

Reference

Reference 1

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

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

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

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 / 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 / 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.

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

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

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

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

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

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

no

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.

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

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 13, 2010 - August 12, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study under GLP with full documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH Guidance S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests For Pharmaceuticals, 1996

Qualifier:

according to guideline

Guideline:

other: ICH Guidance S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals, 1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

tk+/- (thymidine kinase) gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: three types of RPMI 1640 medium (RPMI 5, RPMI 10 and RPMI 20) according to guideline
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: no

Additional strain / cell type characteristics:

other: TK+/- 3.7.2 C

Metabolic activation:

with and without

Metabolic activation system:

cofactor-supplemented post-mitochondrial fraction (S9) (Phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver)

Test concentrations with justification for top dose:

Preliminary test:
1 - 500 µg/mL (with and without metabolic activation, 3h)
1 - 500 µg/mL (without metabolic activation, 24 h)
1000 - 5000 µg/mL (with and without metabolic activation, 3h)
100 - 400 µg/mL (without metabolic activation, 24h)

Main Mutation Assay I
375 - 4000 µg/mL (without metabolic activation, 3h)
375 - 4500 µg/mL (with metabolic activation, 3 h)

Main Mutation Assay II:
12.5 - 200 µg/mL (without metabolic activation, 24 h)
375 - 4000 µg/mL (with metabolic activation, 3 h)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (10 μL/mL) for 24-h treatments; RPMI 5 Medium (the treatment medium) for 3-h treatments
- Justification for choice of solvent/vehicle: preliminary solubility test

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

used in the absence of metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

used as positive control substance for activation study

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: N/A
- Exposure duration: 3 h or 24 h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A

SELECTION AGENT (mutation assays): TFT
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): N/A

NUMBER OF REPLICATIONS: Duplicate cultures were used for each treatment.

NUMBER OF CELLS EVALUATED: N/A

DETERMINATION OF CYTOTOXICITY
- Method: harmonised relative survival; relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: N/A
- Determination of endoreplication: N/A
- Other:

OTHER:
A preliminary Solubility Test was performed for selection of the appropriate vehicle and treatment concentrations to be used in the Dose-Range Finding Test.

Evaluation criteria:

Mutagenicity criteria according to Moore et al. were used (M.M. Moore, M. Honma, J. Clements, G. Bolcsfoldi, B. Burlinson, M. Cifone, J. Clarke, R. Delongchamp, R. Durward, M. Fellows, B. Gollapudi, S. Hou, P. Jenkinson, M. Lloyd , J. Majeska, B. Myhr, M. O’Donovan, T. Omori, C. Riach, R. San, L.F. Jr. Stankowski, A.K. Thakur, F. Van Goethem, S. Wakuri and I. Yoshimura. Mouse Lymphoma Thymidine Kinase Gene Mutation Assay: Follow-up Meeting of the International Workshop on Genotoxicity Testing - Aberdeen, Scotland, 2003 – Assay acceptance criteria, positive controls, and data evaluation. Environ. Mol. Mutagen. 47, 1 – 5 (2006)).

Statistics:

The heterogeneity of the obtained data was tested. The statistical significance of mutant frequencies (total wells with clones) was carried out using Dunnett’s Test, using TOXSTAT statistical software. The positive control data were compared with the respective vehicle control or untreated control data with 2 Sample t-Test, using TOXSTAT statistical software. The data were checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the 24-hour treatment 100% toxicity was detected at the highest examined level of 500 μg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The cytotoxicity results of the evaluated Assay 1 did not fulfil the assay acceptance criterion in so far as the chosen concentration range did not produce 80 - 90% toxicity in presence of exogenous metabolic activation (S9).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The obtained toxicity was ~100% directly after the treatment at the highest examined concentration level, at 200 μg/mL. Based on the harmonised RS, 82% (in the 80 - 90% toxicity assay acceptance range) toxicity was observed at 150 μg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the Assay 2 there were one or two concentration levels where the obtained mutation frequencies were statistically significantly higher than the mutation frequencies of the corresponding vehicle control (Dunnett’s Test, α = 0.05). However, the changes of the mutation frequencies were not dose-related, and the GEF criterion for positive call was not attained in any case. Thus, the obtained statistical significances were regarded as not biologically relevant.

Remarks on result:

other: preliminary test

Conclusions:

Under the conditions of this study, the test item „2-propanol and 2-butanol production, distn. residues“ did not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells (L5178Y TK+/- 3.7.2 C mouse lymphoma cell line) used.

Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of the „2-propanol and 2-butanol production, distn. residues“ to cause gene mutation and/or chromosome damage. Treatments were carried out for 3 hours with and without metabolic activation (±S9 Mix) and for 24 hours without metabolic activation (-S9 Mix).

Due to its solubility properties, the test item was administered directly and the RPMI 5 medium was applied as the “vehicle control” at the 3-hour treatments (±S9 Mix) and was dissolved (mixed with) in DMSO for the lower concentrations of the 24-hour treatments (-S9 Mix).

The concentrations applied in the Assay 1 and Assay 2 were chosen according to the solubility and cytotoxicity results of the pre-experiments.

The following concentrations were investigated in the Assay 1:

3-hour treatment, in absence of exogenous metabolic activation (-S9): 375; 750; 1500; 3000; 3500 and 4000 μg/mL.

3-hour treatment, in presence of exogenous metabolic activation (+S9): 375; 750; 1500; 3000; 4000 and 4500 μg/mL.

The cytotoxicity results of the evaluated Assay 1 did not fulfil the assay acceptance criterion in so far as the chosen concentration range did not produce 80 - 90% toxicity in presence of exogenous metabolic activation (S9). Therefore the Assay 1 (in presence of S9) was completed with investigation of a modified concentration range.

24-hour treatment, in absence of exogenous metabolic activation (-S9): 12.5; 25; 50; 100; 150 and 200 μg/mL.

3-hour treatment, in presence of exogenous metabolic activation (+S9): 375; 750; 1500; 3000; 3500 and 4000 μg/mL

The cytotoxicity results of the evaluated Assay 1 and Assay 2 fulfilled the assay acceptance criterion of the study regarding the degree of the cytotoxicity at the highest examined concentration and regarding to the number of test concentrations.

In the performed mutation assays the cell cultures were treated with a range of the test item concentrations. After the treatment the cell cultures were washed, re-suspended, the cell densities determined and adjusted to 2x10E5/mL. The cells were transferred to flasks for growth through the expression period (for approximately 2 days) or diluted to be plated for survival. At the end of the expression period cells were allowed to grow and form colonies for approximately 2 weeks in culturing plates with and without selective agent (TFT) for determination of mutations and viability. The performed assays fulfilled the validity criteria in connection with the negative control and positive control treatments.

In the performed Assay 1 the obtained mutation frequencies (neither in presence nor in absence of exogenous metabolic activation) did not show dose-related tendencies, remained far below the relevant GEF thresholds for positive call, and were not statistically significantly different from that of the corresponding vehicle controls (Dunnett’s Test, α = 0.05).

In the Assay 2 there were one or two concentration levels where the obtained mutation frequencies were statistically significantly higher than the mutation frequencies of the corresponding vehicle control (Dunnett’s Test, α = 0.05). However, the changes of the mutation frequencies were not dose-related, and the GEF criterion for positive call was not attained in any case. Thus, the obtained statistical significances were regarded as not biologically relevant.

Under the conditions of this study, the test item „2-propanol and 2-butanol production, distn. residues“ did not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells (L5178Y TK+/- 3.7.2 C mouse lymphoma cell line) used.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09. June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study under GLP with full documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for gene toxicity assays with low background aberrations. These cells were chosen because of their small number of chromosomes (diploid number, 2n = 22) and because of the high proliferation rates (doubling time 12 14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male). This cell line was purchased from ECACC (European Collection of Cells Cultures). The cell stocks were kept in a freezer at -80 ±10 °C. Checking for mycoplasma infections was carried out. Trypsin-EDTA (0.25% Trypsin, 1 mM EDTA x 4 Na) solution was used for cell detachment to subculture. The laboratory cultures were maintained in 75 cm2 plastic flasks at 37 ±0.5 °C in an incubator with a humidified atmosphere, set at 5% CO2. The V79 cells for this study was grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine (2 mM) and 1% of Antibiotic-antimycotic solution (containing 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphoptericin-B) and heat-inactivated bovine serum (final concentration 10%). During the 3 and 20 hours treatments with test item, negative (solvent -) and positive controls, the serum content was reduced to 5%.

Additional strain / cell type characteristics:

other: Lot. No.: 05/F/013

Metabolic activation:

with and without

Metabolic activation system:

Metabolic activation of substances achieved by supplementing the cell cultures with liver microsome preparations (S9 mix).

Test concentrations with justification for top dose:

Experiment A with 3/20 h treatment/sampling time
Without: 78.1, 156.3, 312.5 and 468.8 µg/mL
with S9 mix: 78.1, 156.3, 312.5 , 625.0 and 937.5 µg/mL

Experiment B with 20/28 h treatment/sampling time
without S9 mix: 78.1, 156.3, 312.5 and 468.8 µg/mL

Experiment B with 3/28 h treatment/sampling time
with S9 mix: 78.1, 156.3, 312.5 , 625.0 and 937.5 µg/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

no

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Dissolved in DME medium; final concentration: 0.4 and 1.0 µL/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-Nitrosodimethylamine

Remarks:

N-Nitrosodimethylamine, a clastogen that requires metabolic transformation by microsomal enzymes, was dissolved DME medium and was used as a positive control substance for activation study at a final concentration of 1.0 µL/mL

Details on test system and experimental conditions:

Component of Media:
Name: DME
Supplier: Sigma-Aldrich GmbH
Lot No.: RNBB 2470
Expiry: January, 2011
Storage condition: In refrigerator (2-8 °C)

Name: Foetal Bovine Serum
Lot No.: 069K8410
Appearance: liquid
Supplier: Sigma-Aldrich GmbH
Expiration date: June, 2013
Storage condition: In freezer at (-18 ±3 °C).
Name: Antibiotic-antimycotic
Lot No.: 0809M00718
Appearance: liquid
Supplier: Sigma-Aldrich GmbH
Expiration date: August, 2011
Storage condition: In freezer at (-18 ±3 °C)
Test System:
V79: Chinese hamster lung male
Lot. No.: 05/F/013
Supplier: ECACC (European Collection of Cells Cultures)

Evaluation criteria:

The Chromosome Aberration Assay is considered acceptable if it meets the following criteria:
- the number of aberrations found in the negative and/or solvent controls falls within the range of historical laboratory control data.
- the positive control items produces biologically relevant increases in the number of cells with structural chromosome aberrations.
The test item is regarded as non-clastogenic if:
- the number of metaphases with structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data
- and/or no significant increase in the number of metaphases with structural chromosome aberration is observed
A test item is classified as clastogenic if it meets the following criteria:
- increase in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (above the range of our historical control data)
- the increase is reproducible between replicate cultures and between tests (when the treatment conditions are the same)
- the increase is statistically significant

Both, biological and statistical significance should be considered together.

Statistics:

For statistical analysis, Fisher exact test was utilised. The parameters evaluated for statistical analysis were as follows: Number of cells with aberration (with and without gaps).

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

<50 % survival at the highest tested concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A clear solution was obtained up to a concentration of 50 mg/mL. There was no precipitation in the medium at any concentration tested.

Conclusions:

„2-Propanol and 2-butanol production, distn. residues“ was tested up to cytotoxic concentrations, both with and without metabolic activation, and did not induce structural chromosome aberrations in this test in Chinese Hamster lung cells. Therefore, the substance is considered as not clastogenic in this system.

Executive summary:

The test item,„2-propanol and 2-butanol production, distn. residues“ was tested in a Chromosome Aberration Assay in V79 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using S9 mix). In two independent experiments (both run in duplicate) at least 200 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below, ranging from little to maximum (< 50% survival) toxicity:

Experiment A with 3/20 h treatment/sampling time

Without: 78.1, 156.3, 312.5 and 468.8* µg/mL

with S9 mix: 78.1, 156.3, 312.5,625.0 and 937.5* µg/mL

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 78.1, 156.3, 312.5 and 468.8* µg/mL

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 78.1, 156.3, 312.5,625.0 and 937.5* µg/mL

* This concentration was tested but not evaluated because the lower concentrations were evaluated.

 

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and concurrent solvent control groups and no dose-response relationships were noted.

In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent controls, up to cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 28 hours following treatment start. Further, a 3-hour treatment up to cytotoxic concentrations in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations.

In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. The observed chromosome aberration rates were within the ranges of historical control data. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

The validity of the test was shown using ethyl methanesulphonate (0.4 and 1.0 µL/mL) and N-nitrosodimethylamine (1.0 µL/mL) as concurrent positive controls.

„2-Propanol and 2-butanol production, distn. residues“ as tested, both with and without metabolic activation, did not induce structural chromosome aberrations in this test in Chinese Hamster lung cells. Therefore, the substance is considered as not clastogenic in this system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The genetic toxicity of „2-propanol and 2-butanol production, distn. residues“ has been assessed in 3 GLP compliant in vitro studies, including a bacterial reverse mutation assay, a mammalian chromosome aberration test and a mammalian cell gene mutation test. Negative results were reported in all studies in the presence and/or absence of metabolic activation. Therefore, the substance is considered non-mutagenic and not subject to classification for mutagenicity according to CLP (Regulation EC No 1272/2008).