Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Tetrabutane was tested in 3 in vitro genotoxicity assays for bacterial mutagenicity, for mutagenicity in mammalian cells and for cytogenic effects in mammalian cells. As a result, tetrabutane did not induce a mutagenic effect in bacteria and mammlian cells with an without metabolic activation. Furthermore, Tetrabutane did not show any evidence for cytogenetic effects, i.e. increase in the frequency of structural chromosome aberrations in a mammalian cell line.

As a conclusion, reliable bacterial and mammalian cell gene mutation and cytogenetic tests demonstrate that tetrabutane has no in vitro genotoxic or mutagenic potential.

Link to relevant study records

Referenceopen allclose all

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:
28 August to 28 October 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted to appropriate OECD and EC Test Guidelines
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH (1996) Guideline S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. PAB/PCD Notification No. 444.
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. PMSB/ELD Notification No. 554.
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes:
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
liver fraction (S9) from male CD rats
Test concentrations with justification for top dose:
First test
In the absence of S9 mix: 27.86, 46.44, 77.40, 129, 215, 358.34, 597.24, 995.4, 1659 and 2765 μg/mL.
In the presence of S9 mix: 27.86, 46.44, 77.40, 129, 215, 358.34, 597.24, 995.4, 1659 and 2765 μg/mL.

Second test
In the absence of S9 mix: 250, 500, 1000, 1500, 2000, 2500 and 2765 μg/mL.
In the presence of S9 mix: 250, 500, 1000, 1500, 2000, 2500 and 2765 μg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle:
Prior to commencing testing, the solubility of the test substance in solvents compatible with
the test system was assessed. Tetrabutane was found to be miscible in acetone at
276.5 mg/mL (1M) following sonication and incubation at 37°C. On dosing a 276.5 mg/mL
solution at 1% v/v into aqueous tissue culture medium, giving a final concentration of
2765 μg/mL, no precipitate was observed.
Concentrations with high ionic strength and osmolality may cause chromosomal aberrations
(Galloway et al. 1987). Therefore, concentrations greater than 5000 μg/mL or 10 mM are not
used in this test system.
The osmolality and pH of the test substance in medium was tested at 2765 μg/mL; no
fluctuation in osmolality of more than 50 mOsm/kg and no change in pH of more than 1.0
unit was observed compared with the solvent control.
In this case, the highest final concentration used for subsequent testing was 2765 μg/mL
(10 mM). In this study Tetrabutane was added to cultures at 1% v/v (50 μL per 5 mL
culture).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
mitomycin C (without S9), Cyclophosphamide (with S9) Migrated to IUCLID6: Cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk


DURATION
- Preincubation period: 48 hours
- Exposure duration:
The study comprised two independent tests. In the first test, a three hour treatment was used
in both the absence and presence of S9 mix. In the second test, a 21 hour continuous
treatment was used in the absence of S9 mix, and a three hour treatment using an increased
S9 concentration (5% v/v) was used in the presence of S9 mix.

- Fixation time (start of exposure up to fixation or harvest of cells):
First test: three hours after dosing, the cultures were centrifuged at 500g for 5 minutes. The
supernatant removed and the cell pellets resuspended in fresh medium. They were then
incubated for a further 18 hours.
Second test: three hours after dosing, the cultures containing S9 mix were centrifuged. The cell pellets
were rinsed and resuspended in fresh medium. They were then incubated for a further
18 hours. Cultures treated in the absence of S9 mix were incubated continuously for
21 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid®
STAIN (for cytogenetic assays): 10% Giemsa, prepared in buffered water (pH 6.8).

NUMBER OF REPLICATIONS: duplicate


NUMBER OF CELLS EVALUATED: 100 each replicate


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index:
The prepared slides were examined by light microscopy using a low power objective. The
proportion of mitotic cells per 1000 cells in each culture was recorded except for positive
control treated cultures, or cultures where there were no signs of cytotoxicity.

OTHER EXAMINATIONS:
- Determination of polyploidy: Polyploid and endoreduplicated cells were noted when seen.



OTHER:
Evaluation criteria:
An assay is considered to be acceptable if the negative and positive control values lie within
the current historical control range.

The test substance is considered to cause a positive response if the following conditions are
met:
Statistically significant increases (P<0.01) in the frequency of metaphases with aberrant
chromosomes (excluding gaps) are observed at one or more test concentration.

The increases exceed the negative control range of this laboratory, taken at the 99% confidence limit.

The increases are reproducible between replicate cultures.

The increases are not associated with large changes in pH, osmolality of the treatment
medium or extreme toxicity.

Evidence of a concentration-related response is considered to support the conclusion.

A negative response is claimed if no statistically significant increases in the number of
aberrant cells above concurrent control frequencies are observed, at any concentration.

A further evaluation may be carried out if the above criteria for a positive or a negative
response are not met.
Statistics:
The number of aberrant metaphase cells in each treatment group was compared with the
solvent control value using the one-tailed Fisher exact test (Fisher 1973).

A Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test
substance groups. If this is significant at the 1% level, the test is reiterated excluding the
highest dose group - this process continues until the trend test was no longer significant.
Key result
Species / strain:
lymphocytes: Human lymphocytes
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 applicable
Positive controls validity:
valid

First test

Toxicity data

       

Mitotic indices of cultured human lymphocytes treated with Tetrabutane are shown in Tables 2a and 2b (attachment below).

In the absence of S9 mix following three hour treatment, Tetrabutane caused no significant reduction in the mitotic index at 2765mg/mL, compared to the solvent control value. The concentrations selected for the metaphase analysis were 995.4, 1659 and 2765 mg/mL.

In the presence of S9 mix (2% v/v final concentration) following three hour treatment,Tetrabutane caused no reduction in the mitotic index at 2765mg/mL, compared to the solvent control value. The concentrations selected for the metaphase analysis were 995.4, 1659 and 2765 mg/mL.

Metaphase analysis

     

The effects of Tetrabutane on the chromosomes of cultured human lymphocytes are summarised in Table1 (attachment below).

In the absence of S9 mix, Tetrabutane caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations at either 995.4 or 1659mg/mL, when compared with the solvent control. The mean values for the solvent control (acetone) and both Tetrabutane treatment concentration were within the historical control range for this laboratory. However, at 2765mg/mL, a statistical significance (p<0.001: including gaps and p<0.01: excluding gaps) which exceeded the historical solvent control range for this laboratory was observed. As the data suggested no clear concentration-related response, the result was considered biologically non-relevant.

In the presence of S9 mix, Tetrabutane caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any concentration, when compared with the solvent control. All mean values for the solvent control (acetone), and all Tetrabutane treatment concentrations were within the historical control range for this laboratory.

Both positive control compounds, Mitomycin C and Cyclophosphamide, caused statistically significant increases (P<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.

Second test

Toxicity test

        

Mitotic indices of cultured human lymphocytes treated with Tetrabutane are shown in Tables 4a and 4b (attachment below).


In the absence of S9 mix following 21 hour continuous treatment, Tetrabutane caused no reduction in the mitotic index at 2765mg/mL, compared to the solvent control value. The concentrations selected for the metaphase analysis were 2000, 2500 and 2765mg/mL.

In the presence of S9 mix (5% v/v final concentration) following three hour treatment, Tetrabutane caused no reduction in the mitotic index 2765 atmg/mL, compared to the solvent control value. The concentrations selected for the metaphase analysis were 2000, 2500 and 2765mg/mL.

Metaphase analysis

       

The effects of Tetrabutane on the chromosomes of cultured human lymphocytes are summarised in Table1 (attachment below).

In both the absence and the presence of S9 mix, Tetrabutane caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any concentration, when compared with the solvent control.

All mean values for the vehicle control (acetone), were within the historical control range for this laboratory. All mean values for Tetrabutane were within the historical control range for this laboratory, with the exception of 2500mg/mL (excluding gaps) and 2765mg/mL (including and excluding gaps) in the absence of S9 mix only, were values were marginally outside the historical control range for this laboratory.

Both positive control compounds, Mitomycin C and Cyclophosphamide, caused statistically significant increases (P<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.

Polyploid anaylysis

No statistically significant increases in polyploid metaphases were observed during metaphase analysis in either test.

Conclusions:
It is concluded that the test substance Tetrabutane has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.
Executive summary:

A study was performed at the Laboratories of Huntingdon Life Sciences, Eye, on behalf of Evonik Oxeno GmbH,, to investigate the possible clastogenic properties of the test substance Tetrabutane. The study investigated the ability of the test substance (initially at 10 concentrations up to a maximum of 2765 µg/mL) to induce chromosomal aberrations on cultured human lymphocytes from healthy donors in two independent experiments. The investigations were performed with and without rat liver S9 fraction as metabolizing system. The study was conducted to GLP and according to OECD guidelines 473 and EU method B.10. At the highest practical concentration of 2765 µg/mL there was no significant reduction of the mitotic index compared to the vehicle control, in the presence or absence of metabolic activation. The concentrations selected for metaphase analysis in the first test were 995.4, 1659 and 2765 µg/mL and in the second test 2000, 2500 and 2765 µg/mL.

In the first test at 2765 ug/mL, in the absence of S9 mix, a statistical significance (p<0.001: including gaps and p<0.01: excluding gaps) which exceeded the historical solvent control range for this laboratory was observed. As the data suggested no clear concentration-related response, the result was considered biologically non-relevant. Tetrabutane caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations at either 995.4 or 1659 ug/mL. In the presence of S9 mix, Tetrabutane caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any concentration, when compared with the solvent control.

 

In the second test, both in the absence and the presence of S9 mix, Tetrabutane caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any concentration, when compared with the solvent control.

 

It is concluded that the test substance Tetrabutane has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

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:
28 August 2009 to 23 September 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted to appropriate OECD, EU and EPA Test Guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH (1996) Guideline S2A: Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. PAB/PCD Notification No. 444.
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. PMSB/ELD Notification No. 554.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Monitoring Authority (MHRA)
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase locus (TK+/-)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: The follwing media, obtained from a suitable supplier were used:
R0 RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 μg/mL gentamicin.
R10p R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v.
R30p R0, supplemented with 0.02% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 30% v/v.
R10p medium was used for cell culture unless otherwise specified. R20p medium was used for the cloning efficiency plating. This was prepared by
mixing equal volumes of R10p and R30p. Selective medium consisted of R10p containing 4 μg/mL trifluorothymidine (TFT).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Preliminary toxicity test: 5.4, 10.8, 21.6, 43.2, 86.41, 172.81, 345.63, 691.25, 1382.5 and 2765 μg/mL
Mutation tests:
-S9 mix Test 1 (3 hours) 86.41, 172.81*, 345.63, 691.25, 1382.5 and 2765 μg/mL
+S9 mix Test 1 (3 hours) 86.41, 172.81, 345.63, 691.25, 1382.5 and 2765 μg/mL
-S9 mix Test 2 (24 hours) 86.41, 172.81, 345.63, 691.25, 1382.5 and 2765 μg/mL
* - Cultures assessed but not reported due to excessive heterogeneity seen in the viability
plates.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: On the basis of solubility determination from Huntingdon Life Sciences Study BBB0036 (In Vitro
Mammalian Chromosome Aberration Test In Human Lymphocytes). Tetrabutane was found to be soluble in acetone at a maximum concentration of
276.5 mg/mL.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: Main test 1: 3 hours
- Exposure duration: Test 1: 3 hours ; Test 2: 24 hours
Test 1 (3h in the absence and presence of S9 mix)
Test 2 (24h in the absence of S9 mix)
- Expression time (cells in growth medium): Test 1: 48 hours

NUMBER OF REPLICATIONS: 4

NUMBER OF CELLS EVALUATED:
Test 1: 2x10E3/well
Test 2: 2x10E5/ml

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth and relative total growth


Evaluation criteria:
Criteria for assessing mutagenic potential: The following criteria were applied for assessment of individual assay results using data for MF where the RTG normally exceeded 10%:
Definitions: GEF = Global Evaluation Factor. For microwell assays this is 126 x 10-6 (Moore et al., 2006). The assay was considered valid in
accordance with the assay acceptance criteria. The test agent was regarded as negative if: The mean mutant frequency of all test concentrations was
less than the sum of the mean concurrent solvent control mutant frequency and the GEF. If the mutant frequency of any test concentrations exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, a linear trend test was applied: If the linear trend test was negative,
the result was regarded as negative. If the linear trend test was positive, this indicated a positive, biologically relevant response. Where appropriate,
other factors were considered in the interpretation of the results, for example, the reproducibility within and between tests, the overall number of
mutant colonies (as opposed to mutation frequency) and the nature of any concentration-related effect(s). Results that only partially satisfied the
assessment criteria described above were considered on a case-by-case basis. In cases where the results were inconclusive, further testing and/or a
test modification may have been required to better define the assay response.
Statistics:
The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods
described by Robinson et al. (1989).
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No changes in pH were observed of more than 1.0 unit at 276.5 μg/mL.
- Effects of osmolality: no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the solvent (vehicle)
control.
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation:None I
- Other confounding effects: None reported


RANGE-FINDING/SCREENING STUDIES:
Precipitate (observed by eye at the end of treatment) was observed at concentrations of 691.25 μg/mL and greater in the absence of S9 mix
following a 3 hour exposure. No precipitate was observed by eye at the end of exposure to any concentrations tested in the presence of S9 mix
following a 3 hour exposure.

Preliminary test:

Exposure to Tetrabutane at concentrations from 5.4 to 2765 μg/mL in the absence and presence of S9 mix (3 hour exposure) resulted in relative suspension growth (RSG) values from 110 to 91% and from 115 to 76% respectively. No precipitate was observed by eye at the end of exposure to any concentrations tested in the absence of S9 mix for a 24 hour exposure. Exposure to concentrations from 5.4 to 2765 μg/mL resulted in RSG values from 103 to 76%. Concentrations used in the main test were based upon these data.

Results shown in Table 1 attached (pdf).

Main mutation test 1: 3 hour treatment in the absence of S9 mix: Cultures were exposed to Tetrabutane at concentrations from 86.41 to 2765 μg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to Tetrabutane at concentrations from 86.41 to 2765 μg/mL were assessed for determination of mutation frequency. Relative total growth (RTG) values from 121 to 65% were obtained relative to the solvent control. There were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent solvent control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants. Results shown in Tables 2 & 3 attached (pdf). Main mutation test 1: 3 hour treatment in the presence of S9 mix: Cultures were exposed to Tetrabutane at concentrations from 86.41 to 2765 μg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to Tetrabutane at concentrations from 86.41 to 2765 μg/mL were assessed for determination of mutation frequency. RTG values from 143 to 87% were obtained relative to the solvent control. There were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, within acceptable levels of toxicity. The positive control, benzo[a]pyrene, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants. Results shown in Tables 4 and 5 attached (pdf). Main mutation test 2: 24 hour treatment in the absence of S9 mix: Cultures were exposed to Tetrabutane at concentrations from 86.41 to 2765 μg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to Tetrabutane at concentrations from 86.41 to 2765 μg/mL were assessed for determination of mutation frequency. RTG values from 169 to 87% were obtained relative to the solvent control. There were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, within acceptable levels of toxicity. The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants. Results shown in Tables 6 and 7 attached (pdf).

Conclusions:
It was concluded that Tetrabutane did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.
Executive summary:

A study was performed at the Laboratories of Huntingdon Life Sciences, Eye, Suffolk, UK on behalf of Evonik Oxeno GmbH, Germany, to assess the mutagenic potential of the test substance Tetrabutane in an in vitro mammalian cell mutation assay.

The study was conducted under GLP and in accordance with OECD Guideline 476, EU Method B.17 and EPA OPPTS 870.5300. Tetrabutane, was tested for potential mutagenicity in the mouse lymphoma L5178Y cell mutation test. The test involves detection of mutation of mouse lymphoma L5178Y cells using the microtitre® fluctuation technique in 96-well microtitre plates. The study consisted of a preliminary toxicity test and two main tests (which were based on the results of the preliminary test) and comprised of three independent mutagenicity assays. The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix. Following a 3 hour exposure to Tetrabutane at concentrations from 5.4 to 2765 μg/mL, relative suspension growth (RSG) was reduced from 110 to 91% and from 115 to 76% in the absence and presence of S9 mix respectively. Following a 24 hour exposure in the absence of S9 mix RSG was reduced from 103 to 76%. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations up to 10mM as no cytotoxicity was seen in response to Tetrabutane. Following 3 hour treatment in the absence and presence of S9 mix, there were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent solvent control mutant frequency and the global evaluation factor (GEF), within acceptable levels of toxicity. The maximum concentration assessed for mutant frequency in the 3 hour treatment in the absence and presence of S9 mix was 2765 μg/mL. In the absence and presence of S9 mix relative total growth (RTG) was reduced to 65 and 87% respectively. In the 24 hour treatment, the maximum concentration assessed for mutant frequency was 2765 μg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF was observed at concentrations up to 2765 μg/mL, where RTG was reduced to 87%. In all tests the concurrent solvent and positive control were within acceptable ranges. It was concluded that Tetrabutane did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

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:
31 August 2000 to 11 September 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted to appropriate Test Guidelines
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
National (Germany) GLP Monitoring Authority (1997)
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
50, 160, 500, 1600 and 5000 µg/plate. in triplicate both with and without the addition of a metabolising system.
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
Tetrahydrofuran
Positive controls:
yes
Positive control substance:
other: 2-Aminofluorene
Remarks:
with metabolic activation
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
with metabolic activation
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without metabolic activation
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); pre-incubation test
- PLATE INCORPORATION TEST
AM-OO/07
TETRABUTAN, TECHNISCH.
In a sterile tube,
- 0.025 ml of the appropriately diluted test material (or 0.025 ml of the solvent) (or 0.1 ml [0.05 ml for TA 1535] of the strain specific positive control substance)
- 0.5 ml phosphate buffer (or 0.5 ml 89 mix in the experiment with metabolic activation)
- 2 ml of molten trace histidine supplemented top agar at approx. 45°C
- 0.1 ml of the bacterial overnight culture were mixed. Mixing was done in triplicate, for each bacterial strain and for each concentration of the test
material. The mixture was then poured onto the surface of minimal agar plates. These plates were incubated at 37°C for 72 hours and then the
number of revertant colonies was counted.
- PRE-INCUBATION TEST
In a sterile tube, a 0.1 ml aliquot of each one of the bacterial overnight cultures was mixed with a 0.5 ml volume of S9 mix
(for tests with metabolic activation) or phosphate-buffer (for tests without metabolic activation). Then either 50 µl of the positive controls
2-Aminofluorene and 2-Aminoanthracene (+89), 10 µl of the solvent, or 10 ul of the test substance solution were added. The tubes were incubated
at 30°C for 30 min with gentle agitation. At the end of the incubation period, 2 ml of molten top agar was added to each tube, mixed briefly and
poured onto minimal agar plates. These plates were incubated at 37°C for 72 hours and then the number of revertant colonies was counted.

DURATION
- Preincubation period: approx. 18hours
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 18 hours
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): 72 hours


SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):


NUMBER OF REPLICATIONS: 3


NUMBER OF CELLS EVALUATED:


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:


OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:


OTHER:
Evaluation criteria:
For a test compound to be considered positive, it must (in two independent experiments) cause at least a doubling in the mean revertants per plate of at least one tester strain. This increase must be accompanied by a dose response towards increasing concentrations of the test article. A test article
that does not meet these criteria will be called non-mutagenic in bacteria. Single increases in revertant frequencies, which are not dose-related and
not reproducible in two independent tests are considered non-relevant. If however these increases do occur in both tests, this will be taken as an
indication of a mutagenic effect.
Statistics:
No data
Key result
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:
in TA 98 at 1600 and 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
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
Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
Some test results were insignificantly higher (TA 98 [control; -S9; #AM-00/07.1], TA 102 [Mitomycin C, -S9, #AM-00/07.1], TA 98 [control, -S9,
#AM-00/07.2], TA 98 [2-Nitrofluorene, -S9, #AM-00/07.2]) and others were lower than the minimal resp. maximal historical control data values
(TA 1537 [9-Aminoacridine, -S9, #AM00/07.1], TA 102 [2-Aminoanthracene, +S9, #AM-00/07.2], TA 1537 [9-Aminoacridine, -S9,
#AM-00/07.2]). But they were regarded as acceptable.

Toxic effects by treatment with the test substance were shown in both tests by the strain TA 98 in the two highest concentrations (1600 and 5000 µg/plate) in the plate incorporation test and in the highest concentration (5000 µg/plate) in the preincubation test.

Precipitation was regarded by using the highest concentration of 5000 µg/plate in the plate incorporation test and in the preincubation test by using the concentration of 1600 and 5000 µg/plate. With two exceptions all five bacterial strains exhibited a positive mutagenic response with the positive controls tested both with and without metabolic activation by S9 mix. The strain TA 1535 showed a limited response to the presence of the positive control substance 2-Aminofluorene in case of the preincubation test with metabolic activation. But in case of TA 1535 the parallel tested additional positive control substance 2-Aminoanthracene showed enough activity. The strain TA 102 also showed a limited response to the positive control substance 2-Aminoanthracene in both experiments, but in this case 2-Aminofluorene showed enough activity.

Negative (solvent) controls were also tested with each strain, and the mean numbers of spontaneous revertants were considered acceptable.

Conclusions:
TETRABUTAN, TECHNISCH did not induce a mutagenic effect in S. typhimurium with an without metabolic activation. It is therefore not considered to be a bacterial mutagen.
Executive summary:

A study was conducted at the Laboratories of Infracor GmbH on behalf of Oxeno Olefinchemie GmbH to investigate the ability of the test substance TETRABUTAN, TECHNISCH to induce reverse mutations in an in vitro bacterial system. Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 were treated with the test compound by the Ames test plate incorporation as well as by the preincubation method. Dose levels covering the range of 50 to 5000 µg/plate, in triplicate both with and without the addition of a metabolising system (Phenobarbital/β-Naphthoflavone co-induced rat liver S9 mix) were employed. The study was conducted to GLP and to OECD 471 and EU B14 Test Guidelines. With two exceptions all five bacterial strains exhibited a positive mutagenic response with the positive controls tested both with and without metabolic activation by S9 mix. The strain TA 1535 showed a limited response to the presence of the positive control substance 2-Aminofluorene in case of the preincubation test with metabolic activation. But in case of TA 1535 the parallel tested additional positive control substance 2-Aminoanthracene showed enough activity. The strain TA 102 also showed a limited response to the positive control substance 2-Aminoanthracene in both experiments, but in this case 2-Aminofluorene showed enough activity. Solvent controls were also tested with each strain and the mean numbers of spontaneous revertants were in an acceptable range. A reproducible mutagenic activity of the test compound to any of the tester strains TA 98, TA 100, TA 102, TA 1535 or TA 1537 was not observed with and without metabolic activation. It is therefore concluded, that TETRABUTAN, TECHNISCH is not a bacterial mutagen.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

No classification for genetic toxicity is indicated according to the classification, labeling and packaging (CLP) regulation (EC) No 1272/2008.