Reaction Products of C4 alcohols and C4 alkenes obtained as by-products from the manufacturing of butan-2-ol by sulfuric acid esterification and hydrolysis of butene

Genetic toxicity: in vitro

Administrative data

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:

The experimental phases of the study were performed between 19 January 2010 and 17 May 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

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 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese New Chemical Substance Law (METI).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Products of C4 alcohols and C4 alkenes obtained as by-products from the manufacturing of butan-2-ol by sulfuric acid esterification and hydrolysis of butene

EC Number:

701-242-6

Molecular formula:

A complex and variable combination of C4 hydrocarbons, 2,2'-oxybisbutane and butan-2-ol.

IUPAC Name:

Reaction Products of C4 alcohols and C4 alkenes obtained as by-products from the manufacturing of butan-2-ol by sulfuric acid esterification and hydrolysis of butene

Method

Target gene:

Not applicable.

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitabilityThe volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Experiment 1:
4 (20)h without-S9 mix: 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 µg/ml
4 (20)h with-S9 mix: 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 µg/ml

Experiment 2:
4 (20)h with-S9 mix: 0, 19.53, 39.06, 78.13, 156.25, 234.38, 312.5 µg/ml
24-hour without S9-mix: 0, 19.53, 39.06, 78.13, 156.25, 234.38, 312.5 µg/ml

Vehicle / solvent:

-Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Acetone was selected as the solvent because the test material was readily soluble in it at the required concentrations.
-A correction for the purity of the test material was not required when the test material formulations were prepared.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

(CP) In the presence of S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

(MMC) In the absence of S9

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in medium


DURATION
- Preincubation period:
48 hrs

- Exposure duration:
Experiment 1 - 4 hrs with and without S9. Experiment 2 - 24 hrs without S9, 4 hrs with S9.

- Expression time (cells in growth medium):
20 hrs for 4 hrs exposure.

- Selection time (if incubation with a selection agent):
Not applicable.

- Fixation time (start of exposure up to fixation or harvest of cells):
24 hrs.


SELECTION AGENT (mutation assays):
No selection agent.

SPINDLE INHIBITOR (cytogenetic assays):
Demecolcine

STAIN (for cytogenetic assays):
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.


NUMBER OF REPLICATIONS:
Duplicate cultures


NUMBER OF CELLS EVALUATED:
100/culture


DETERMINATION OF CYTOTOXICITY
- Method:
mitotic index - A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

-Scoring of Chromosome Damage:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there was approximately 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

OTHER EXAMINATIONS:
- Determination of polyploidy:
Frequency of polyploid cells


OTHER:
None.

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Results and discussion

Test results

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Refer to information on results and attached tables.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS
Preliminary Toxicity Test (Cell Growth Inhibition Test)

The mitotic index data are presented in Appendix 1 (5) and (6). The dose range for the Preliminary Toxicity Test was 19.53 to 5000 µg/ml. The maximum dose was based on the maximum recommended dose level. A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 2500 µg/ml, in the 4(20)-hour and 24-hour exposure groups in the absence of metabolic activation (S9), and at and above 625 µg/ml in the 4(20)-hour exposure group in the presence of S9. The precipitate was observed to be greasy/oily in appearance at and above 2500 µg/ml. Haemolysis was observed in the blood cultures for all exposure groups at and above 39.06 µg/ml.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 5000 µg/ml in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure was 156.25 µg/ml. The mitotic index data are presented in Table 1. The test material induced some evidence of toxicity, particularly in the 24-hour exposure group.

The selection of the maximum dose level was initially based on the maximum recommended dose level for the 4(20)-hour exposure groups in Experiment 1. However, due to the toxicity seen in the initial first experiment in both the absence and presence of S9, where no metaphases suitable for scoring were observed at and above 625 and 312.5 µg/ml respectively, the selection of dose levels was based on toxicity in both experiments.

Chromosome Aberration Test – Experiment 1
The dose levels of the controls and the test material are given in the following table:
Group Final concentration of Reaction Products of C4 alcohols and C4 alkenes (µg/ml)

4(20)-hour without S9 0*, 19.53*, 39.06*, 78.13*, 156.25*, 312.5, 625, 1250, 2500, MMC 0.4*
4(20)-hour with S9 0*, 19.53*, 39.06*, 78.13*, 156.25*, 312.5, 625, 1250, 2500, CP 5*

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the initially performed Experiment 1 and that there were metaphases suitable for scoring present up to 156.25 µg/ml in the absence and presence of metabolic activation (S9). A greasy/oily precipitate of the test material was observed at the end of exposure, at 2500 µg/ml, in both of the exposure groups.
The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in Form 1, Appendix 2. These data show that 57% mitotic inhibition was achieved at 156.25 µg/ml in the absence of S9. In the presence of S9 no reduction in mitotic index was observed in dose levels with metaphases present for scoring.

The maximum dose level selected for metaphase analysis in both exposure groups was limited to 156.25 µg/ml; the maximum dose level with metaphases suitable for scoring.

The chromosome aberration data are given in Form 1, Appendix 2. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test material did not induce any statistically significant increases in the frequency of cells with aberrations in either the absence or presence of metabolic activation.

The polyploid cell frequency data are given in Form 1, Appendix 2. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Chromosome Aberration Test - Experiment 2

The dose levels of the controls and the test material are given in the table below:
Group Final concentration of Reaction Products of C4 alcohols and C4 alkenes (µg/ml)

24-hour without S9 0*, 19.53, 39.06*, 78.13*, 156.25*, 234.38*, 312.5, MMC 0.2*
4(20)-hour with S9 0*, 19.53*, 39.06*, 78.13*, 156.25, 234.38, 312.5, CP 5*

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present up to 234.38 µg/ml in the absence of S9, and 156.25 µg/ml in the presence of S9. No precipitate of the test material was observed at the end of exposure in either exposure group.
The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in Form 2, Appendix 2. These data show that 66% mitotic inhibition was achieved at 234.38 µg/ml in the absence of S9. In the presence of S9 dose-related mitotic inhibition was also observed with 49% achieved at 78.13 µg/ml and 68% at 156.25 µg/ml.

The selection of the maximum dose level for metaphase analysis was based on toxicity for both exposure groups, and was 234.38 µg/ml in the absence of S9 and 78.13 µg/ml in the presence of S9.

The chromosome aberration data are given in Form 2, Appendix 2. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. Due to toxicity and poor slide quality it was only possible to score 45 metaphase cells for the positive control ‘B’ culture. This was considered not to affect the purpose or integrity of the study.

The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of metabolic activation.

The polyploid cell frequency data are given in Form 2, Appendix 2. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Any other information on results incl. tables

Please see attached Appendix 1 Report of Results of Chromosomal Aberration Test in Cultured Mammalian Cells and Appendix 2 Results of Chromosome Aberration Test 2259-0037a.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative

The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Introduction. 

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008 and is acceptable to the Japanese New Chemical Substance Law (METI).

Methods. 

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, ie. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

Results. 

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material was toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included dose levels that induced approximately 50% mitotic inhibition.

Conclusion. 

The test material was considered to be non-clastogenic to human lymphocytes in vitro.