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Genetic toxicity in vitro

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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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given; comparable to guideline study (deviation: only 4 strains of S. thyphimurium tested)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
: only 4 strains of S. thyphimurium tested
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
his¯
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix prepared from Dawlay rat livers after Aroclor 1254 activation
Test concentrations with justification for top dose:
20, 100, 500, 2500, 5000 ug/plate (Standard plate test)
20, 100, 500, 2500, 5000 ug/plate (Preincubation test)
4, 4, 20, 100, 500, 1000 µg/plate (Preincubation test) TA 1537/TA 98 without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: see details
Details on test system and experimental conditions:
Standard plate test
Test tubes containing 2 ml portions of saft agar kept in a water bath at 45'C, and the remaining companents are added in the following order:
0.1 ml test solution or vehicle
0.1 ml bacterial suspension
0.5 ml S-9 mix (in tests with metabolic activatian) or
0.5 ml phosphate buffer (in tests without metabalic activatian)
After mixing, the samples are poured onto Vogel-Bonner agar plates. After incubation at 37'C for 48 -72 haurs in the dark, the bacterial calonies (his+ revertants) are counted.


Preincubation test
0.1 ml test solution or vehicle, 0.1 ml bacterial Suspension and 0.5 ml S-9 mix are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx.30 seconds.


Both Tests
In each experiment 3 Test plates per dose per control used. After incubation at 37°C for 48 -72 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Posivite Control:
with S-9 mix: 10 µg 2-aminoanthracene (2-AA)(dissolved in DMSO) for the strains TA 100, TA 98, TA 1537and TA 1535

without S-9 mix: 5 µg N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) (dissolved in DMSO) for the strains TA 100 and TA 1535
10 µg 4-nitro-o-phenylendiamine (NOPD) (dissolved in DMSO) for the strain TA 98
100 µg 9-aminoacridine (AAC) chloride monohydrate (dissolved in DMSO) for the strain TA 1537

The Titer was determined and in regularly measurements the strain characteristics were checked. Sterility control performed.
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: Salmonella typhimurium TA1535, TA100, TA1537, TA98
Remarks:
Migrated from field 'Test system'.

Solubility: Complete solubility of the test substance in aqua. dest.

Toxicity: A bacteriotoxic effect was abserved only without S-9 mix in the PIT at doses >= 1000 µg/plate (TA 98) or at >= 2500 µg/plate (TA 100 and TA 1537)

Mutagenicity: An increase in the number of his+ revertants was not observed both in the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system.

The slightly enhanced values due to some single increased colony numbers observed with TA 1537 and TA 98 in the preincubation test did not show any dose dependency and were not confirmed in a 2nd preincubation assay selecting closer doses. The findings are therefore regarded as incidental.

Conclusions:
Interpretation of results (migrated information):
negative

According to the results of the present study, the test substance isobutyric acid is not mutagenic in the Ames Test under the experimental conditions chosen here.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA 1535, and TA1537 of Salmonella typhimurium were exposed to isobutyric acid (purity 99.33%) at concentrations of 0 (controls), 20, 100, 500, 2500, and 5000 µg/plate in the presence and absence of mammalian metabolic activation. Tests were performed as plate incorporation and as preincubation assays.

 

Isobutyric acid was tested up to limit concentration (5000 µg/plate). The positive controls induced the appropriate responses. There was no evidence of induced mutant colonies over background.

 

Isobutyric acid was demonstrated to be not mutagenic to Salmonella typhimurium under the conditions of this test (BASF, 1989).

 

This study is classified as acceptable. It satisfies the requirement of Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data with minor restrictions (only 4 strains tested).

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:
The study was performed between 25 February 2010 and 25 June 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with the following minor deviation. No analysis was carried out to determine the homogeneity, concentration or stability of the test material formulation. This exception is considered not to affect the purpose or integrity of the study or the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
No analysis was carried out to determine the homogeneity, concentration or stability of the test material formulation. This exception is considered not to affect the purpose or integrity of the study.
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No. 440/2008 and the United Kingdom Environmental Mutagen Society (Cole et al, 1990). The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6­TG) as the selective agent.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
To assess the potential mutagenicity of the test material on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster
ovary (CHO) cells.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Properly maintained: yes

- Periodically checked for Mycoplasma contamination:yes

- Periodically checked for karyotype stability: no

- Periodically "cleansed" against high spontaneous background: yes

Cell Line
The Chinese hamster ovary (CHO-K1) cell line was obtained from ECACC, Salisbury, Wiltshire.
Cell Culture
The stocks of cells were stored in liquid nitrogen at approximately -196°C. Cells were routinely cultured in Ham's F12 medium, supplemented with 5%
foetal bovine serum and antibiotics (Penicillin/Streptomycin at 100 units/100 µg per ml) at 37°C with 5% CO2 in humidified air.
Cell Cleansing
Cell stocks spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen down they were cleansed of HPRT- mutants by
culturing in HAT medium for 4 days. This is Ham's F12 growth medium supplemented with Hypoxanthine (13.6 µg/ml, 100 µM), Aminopterin (0.0178
µg/ml, 0.4 µM) and Thymidine (3.85 µg/ml, 16 µM). After 4 days in medium containing HAT, the cells were passaged into HAT-free medium and grown for 4 to 7 days. Bulk frozen stocks of HAT cleansed cells were frozen down, with fresh cultures being recovered from frozen before each experiment.


Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 was prepared from the livers of male rats weighing approximately 250g. These had received three daily oral doses of a mixture of phenobarbitone (80 mg/kg) and beta-naphthoflavone (100 mg/kg), prior to S9 preparation on the fourth day.
Test concentrations with justification for top dose:
The molecular weight of the test material was supplied as 88.10, therefore the maximum recommended dose level was the 10mM concentration, 880
µg/ml. The purity of the test material was greater than 99% and was therefore not accounted for in the formulations. The osmolality did not increase
by more than 50 mOsm when the test material was dosed into media. However the pH did decrease by more than 1 pH unit at the maximum recommended dose of 880 µg/ml and at the intermediate dose of 660 µg/ml, therefore the maximum dose was limited to 440 µg/ml (Scott et al 1991). The pH
and osmolality data can be seen in the following table.
Dose level (µg/ml) 0 3.44 6.88 13.75 27.5 55 110 220 440 660 880
pH 8.12 8.14 8.09 8.10 8.14 8.13 8.02 7.71 7.28 6.68 6.47
mOsm 302 305 - - 302 301 - 303 305 - 310

- = Not determined
The dose range of test material was selected based on the results of a preliminary cytotoxicity test and was 13.75 to 440 µg/ml in both the presence
and absence of metabolic activation in Experiment 1. In Experiment 2 the dose range was modified to 27.5 to 440 µg/ml for both exposure groups.
The maximum dose was limited to 440 µg/ml as a result of a decrease in pH of greater than 1 unit at the maximum recommended dose of 880 µg/ml.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Hams F12 cell culture media
- Justification for choice of solvent/vehicle:The test material formed a solution with the solvent suitable for dosing.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Hams F12
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Dimethyl benzanthracene (DMBA)
Remarks:
With metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Hams F12
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Details on test system and experimental conditions:
Preliminary Cytotoxicity Test
A preliminary cytotoxicity test was performed on cell cultures using a 4-hour exposure time with and without metabolic activation. The cells were
plated out at 3 x 10E6 cells/75 cm2 flask approximately 18 hours before dosing. On dosing, the growth media was removed and replaced with serum
free media (Ham's F12). One flask per dose level was treated with and without S9 metabolic activation, 9 dose levels using halving dilutions and vehicle
controls were dosed. The dose range of test material used was 1.72 to 440 µg/ml. Exposure was for 4 hours at 37°C, after which the cultures were
washed twice with phosphate buffered saline (PBS) before being trypsinised. Cells from each flask were suspended in growth medium; a sample was
removed from each dose group and counted using a Coulter counter. For each culture, 200 cells were plated out into three 25 cm2 flasks with 5 ml of growth medium and incubated for 7 days at approximately 37°C ± 2°C in an incubator with a humidified atmosphere of 5% CO2 in air. The cells were
then fixed and stained and total numbers of colonies in each flask counted to give cloning efficiencies.
Results from the preliminary cytotoxicity test were used to select the test material dose levels for the mutagenicity test.

Mutagenicity Test
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. Cells were seeded at 3 x 10E6/75 cm2 flask for the 4 hour exposure groups and allowed to attach overnight before being exposed to the test or control materials. Duplicate cultures were set up, both in the presence and absence of metabolic activation, with six dose levels of test material, and vehicle and positive controls. Treatment was for 4 hours in serum free media (Ham's F12) at approximately 37°C in an incubator with a humidified atmosphere of 5% CO2 in air. The dose range of test material was 13.75 to 440 µg/ml for both exposure groups of Experiment 1 and 27.5 to
440 µg/ml for Experiment 2.

At the end of the treatment period the flasks were washed twice with PBS, trypsinised and the cells suspended in growth medium. A sample of each
dose group cell suspension was counted using a Coulter counter. Cultures were plated out at 2 x 10E6 cells/flask in a 225 cm2 flask to allow growth and expression of induced mutants, and in triplicate in 25 cm2 flasks at 200 cells/flask for an estimate of cytotoxicity. Cells were grown in growth media and incubated at approximately 37°C in an incubator with a humidified atmosphere of 5% CO2 in air.

Cytotoxicity flasks were incubated for 6 or 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.
During the 7 Day expression period the cultures were sub-cultured and maintained at 2 x 10E6 cells/225 cm2 flask on days 3 or 4 to maintain logarithmic growth. At the end of the expression period the cell monolayers were trypsinised, cell suspensions counted using a Coulter counter and plated out as follows:
i) In triplicate at 200 cells/25 cm2 flask in 5 ml of growth medium to determine cloning efficiency. Flasks were incubated for 6 to 7 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.
ii) At 2 x 10E5 cells/75 cm2 flask (5 replicates per group) in Ham's F12 growth media (5% serum), supplemented with 10 µg/ml 6-Thioguanine (6-TG), to determine mutant frequency. The flasks were incubated for 14 days at approximately 37°C in an incubator with a humidified atmosphere of 5% CO2 in air, then fixed with methanol and stained withGiemsa. Mutant colonies were manually counted and recorded for each flask.

The percentage cloning efficiency and mutation frequency per survivor were calculated for each dose group.
Fixation and staining of all flasks was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.

ASSAY ACCEPTANCE CRITERIA
An assay will normally be considered acceptable for the evaluation of the test results only if all the following criteria are satisfied. The with and without metabolic activation portions of mutation assays are usually performed concurrently, but each portion is, in fact, an independent assay with its own positive and negative controls. Activation or non-activation assays will be repeated independently, as needed, to satisfy the acceptance criteria.
i) The average absolute cloning efficiency of negative controls should be between 70 and 115% with allowances being made for errors in cell counts and dilutions during cloning and assay variables. Assays in the 50 to 70% range may be accepted but will be dependent on the scientific judgement of the
Study Director. All assays below 50% cloning efficiency will be unacceptable.
ii) The background (spontaneous) mutant frequency of the vehicle controls are generally in the range of 0 to 25 x 10E-6. The background values for the with and without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays. Assays with backgrounds greater than 35 x 10-6 will not be used for the evaluation of a test material.
iii) Assays will only be acceptable without positive control data (loss due to contamination or technical error) if the test material clearly shows mutagenic activity. Negative or equivocal mutagenic responses by the test material must have a positive control mutant frequency that is markedly elevated over the concurrent negative control.
iv) Test materials with little or no mutagenic activity, should include an acceptable assay where concentrations of the test material have reduced the clonal survival to approximately 10 to 15% of the average of the negative controls, reached the maximum recommended dose (10 mM or 5 mg/ml) or twice the solubility limit of the test material in culture medium. Where a test material is excessively toxic, with a steep response curve, a concentration that is at least 75% of the toxic dose level should be used. There is no maximum toxicity requirement for test materials that are clearly mutagenic.
v) Mutant frequencies are normally derived from sets of five dishes for mutant colony count and three dishes for viable colony counts. To allow for contamination losses it is acceptable to score a minimum of four mutant selection dishes and two viability dishes.
vi) Five dose levels of test material, in duplicate, in each assay will normally be assessed for mutant frequency. A minimum of four analysed duplicate
dose levels is considered necessary in order to accept a single assay for evaluation of the test material.
Evaluation criteria:
ASSAY ACCEPTANCE CRITERIA
An assay will normally be considered acceptable for the evaluation of the test results only if all the following criteria are satisfied. The with and without metabolic activation portions of mutation assays are usually performed concurrently, but each portion is, in fact, an independent assay with its own positive and negative controls. Activation or non-activation assays will be repeated independently, as needed, to satisfy the acceptance criteria.
Due to the amount of information involved it was not possible to insert all the required information within this section therefore for full details please see ASSAY ACCEPTANCE CRITERIA in the section "Details on test system and conditions"
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
non-mutagenic
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary Cytotoxicity Test
Doses of 1.72 to 440 µg/ml were used in the preliminary cytotoxicity test. The results of the individual flask counts and their analysis are presented in
the attached Table 1. It can be seen that there was no consistent dose-related reduction in the cloning efficiency (CE) either in the presence or absence of S9.
Mutagenicity Test - Experiment 1
The dose levels of the controls and the test material are given in the table below:
Group Final concentration of iso-butyric acid (µg/ml)
4-hour without S9 0*, 13.75*, 27.5*, 55*, 110*, 220*, 440*, EMS 500* and 750*
4-hour with S9 0*, 13.75*, 27.5*, 55*, 110*, 220*, 440*, DMBA 0.5* and 1.0*
The Day 0 and Day 7 cloning efficiencies are presented in the attached Table 2 and Table 3. All of the vehicle controls had acceptable levels for cloning efficiencies.
It can be seen that there was no marked toxicity with the test material when compared to the vehicle controls. The toxicity observed was similar to that seen in the preliminary cytotoxicity test.
All of the vehicle control cultures had mutant frequencies within the expected range. The positive control materials induced significant increases in
mutant frequency. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The mutation frequency counts and mean mutation frequency per survivor values are presented in attached Table 2 and Table 3. There were no
increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10-6 with or without the presence of S9, or exceeded
the expected upper limit for vehicle control cultures.

Mutagenicity Test - Experiment 2
The dose levels of the controls and the test material are given in the table below:
Group Final concentration of iso-butyric acid (µg/ml)
4-hour without S9 0*, 27.5*, 55*, 110*, 220*,330*, 440*, EMS 500* and 750*
4-hour with S9 0*, 27.5*, 55*, 110*, 220*, 330*, 440*, DMBA 0.5* and 1.0*
The Day 0 and Day 7 cloning efficiencies are presented in the attached Tables 4 and 5. All the vehicle controls were considered to have acceptable levels for cloning efficiencies, although the Day 7 vehicle control of the with S9 exposure group of Experiment 2 was marginally less than 70%. It can be seen that, as in
Experiment 1, there was no marked toxicity with the test material when compared to the vehicle controls, and the toxicity observed was similar to that
seen in the preliminary cytotoxicity test.
All of the vehicle control cultures had mutant frequencies within the expected range. The positive control materials induced significant increases in
mutant frequency. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The mutation frequency counts and mean mutation frequency per survivor values are presented in attached Table 4 and Table 5. There were no
increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10-6 with or without the presence of S9, or exceeded
the expected upper limit for vehicle control cultures.
CONCLUSION
The test material did not any induce significant or dose-related increases in mutant frequency per survivor in either the presence or absence of
metabolic activation in either of the two experiments. The test material was therefore considered to be non-mutagenic to CHO cells at the HPRT locus
under the conditions of this test.
EMS = Ethyl methane sulphonate
* = Dose levels plated for mutant frequency
DMBA= Dimethyl benzanthracene
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Due to the nature of the table format’s it is not possible to insert them within this section, therefore please see attached tables 1 to 5 & Appendix 1 Historical Background Data.

Conclusions:
Interpretation of results (migrated information):
negative Non-mutagenic

The test material did not induce any significant or dose-related increases in mutant frequency per survivor in either the presence or absence of
metabolic activation in either of the two experiments. The test material was therefore considered to be non-mutagenic to CHO cells at the HPRT locus
under the conditions of this test.
Executive summary:

Introduction.

The study was conducted to assess the potential mutagenicity of the test material on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells. The protocol used was designed to comply with the OECD Guidelines for Testing of Chemicals No. 476'In Vitro Mammalian Cell Gene Mutation Tests', Commission Regulation (EC) No. 440/2008 and the United Kingdom Environmental Mutagen Society (Cole et al, 1990). The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6­TG) as the selective agent.

Methods.

Chinese hamster ovary (CHO) CHO-K1 cells were treated with the test material at six dose levels, in duplicate, together with vehicle (solvent) and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was 4-hours using modified dose levels.

The dose range of test material was selected based on the results of a preliminary cytotoxicity test and was 13.75 to 440 µg/ml in both the presence and absence of metabolic activation in Experiment 1. In Experiment 2 the dose range was modified to 27.5 to 440 µg/ml for both exposure groups. The maximum dose was limited to 440 µg/ml as a result of a decrease in pH of greater than 1 unit at the maximum recommended dose of 880 µg/ml.

Results.

The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus.

The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.

CONCLUSION

The test material did not induce any significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation in either of the two experiments. The test material was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.

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

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
As working hypothesis for the read-across approch from iso-butanol to iso-butyric acid information on toxicokinetics for isobutanol showing a readily absorbtion from the gastrointestinal as well as from the respiratory tract. Isobutanol is metabolized in the liver and it undergoes rapid oxidation (Hedlund 1969, Saito 1975, Ehrig 1988, Sinclair 1990). Isobutyraldehyde and subsequently isobutyric acid is formed (Saito 1975, Poet and Corley (OPP/ACC) 2004). Because the metabolization takes place rapidly it is scientifically relevant to assume that possible genotoxic effects will depend most likely on the metabolites (isobutyraldehyde and isobutyric acid) than on the original substance isobutanol

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemicals Isobutanol was investiagate as a pure compound without significant impurities. The target substance iso-butyric acid is as well regarded and evaluated as pure compound and mono-constituent substance under REACh Legislation. No significant impurities need to be assessed according to analytical data.

3. ANALOGUE APPROACH JUSTIFICATION

Isobutanol is readily absorbed from the gastrointestinal as well as from the respiratory tract. Maximum blood levels are reached within 15 minutes (rat, respiration; Poet and Corley (OPP/ACC) 2004), 45 to 60 minutes (human, rabbit, oral application; Bilzer 1990, Saito 1975) or 90 minutes (rat, oral application; Gaillard 1965). Elimination from blood is considerably fast as well. In humans, an elimination half-life of 1.45 h was determined (Bilzer 1990). In rabbits, isobutanol could not be detected any more 6 hours after application (Saito 1975).

Isobutanol is metabolized in the liver and it undergoes rapid oxidation (Hedlund 1969, Saito 1975, Ehrig 1988, Sinclair 1990). Isobutyraldehyde and subsequently isobutyric acid are formed (Saito 1975, Poet and Corley (OPP/ACC) 2004) by oxidation with alcohol dehydrogenase and aldehyde dehydrogenase respectively (Hedlund 1969, Saito 1975, Rüdell 1983), Beta-oxidation and decarboxylation of isobutyric acid results in propionyl-coenzyme A (Saito 1975) which can enter the tricarbxylic acid cycle. Accordingly, propionaldehyde, propionic acid, and succinic acid were identified as additional metabolites (Rüdell 1983).

Only minor amounts of applied isobutanol doses are excreted unchanged (rabbit 0.54 %, Saito 1975; rat 0.27%, Gaillard 1965). Small amounts have been found as glucuronides in the urine of rabbits (4.4 %, Kamil 1953).
In expired air, no metabolites (aldehyde, ketone) could be detected (Kamil 1953).
In conclusion, isobutanol is rapidly metabolized to isobutyraldehyde and further on to isobutyric acid with higher levels of isobutyric acid detectable in blood.

Genotoxicity
For isobutyric acid, no study concerning genetic toxicity in vivo could be identified. As substitute, data for isobutanol will be used based on following reason (for more detailed information on toxicokinetics/metabolism and justification of supporting substances see Section 7.1, endpoint summary).
After administration, isobutanol will rapidly be metabolized in vivo to isobutyraldehyde by alcohol dehydrogenases and subsequently to isobutyric acid by aldehyde dehydrogenases. In the course of metabolic transformation of isobutanol, isobutyric acid is generated rapidly and predominantly as intermediary metabolite. Thus, it is justified to use isobutanol as supporting substance in the evaluation of the systemic in vivo effects of isobutyric acid.
The genetic toxicity in vivo of isobutanol has been evaluated in one valid study of high reliability (GLP study) which is used as key study.
In a mouse bone marrow micronucleus test, 5 NMRI-mice per sex/dose were treated orally with isobutanol at doses of 0, 500, 1000, and 2000 mg/kg bw. Bone marrow cells were harvested at 24 and 48 hours post treatment. The test substance was administered orally dissolved in olive oil in single doses.

There were signs of toxicity during the study. Isobutanol was tested at adequate doses. The positive controls induced the appropriate responses.

There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any dose and any treatment time. Under the experimental conditions of the test, isobutanol had no chromosome damaging (clastogenic) effect nor did it lead to any impairment of chromosome distribution in the course of mitosis

Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: mean weight 26 g
- Assigned to test groups randomly: yes, assignment with an appropriate computer program
- Housing: in groups of 5 of the same sex in Makrolon cages, type MIII
- Diet (e.g. ad libitum): standardized pelleted feed (Kliba Haltungsdiät, Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water (e.g. ad libitum): drinking water from bottles, ad libitum
- Acclimation period: 3 - 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 /12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil
- Justification for choice of solvent/vehicle: insolubility of test substance in water, suitability of olive oil in the vivo micronucleus test as demonstrated by historical data
- Concentration of test material in vehicle: 5 g, 10 g, and 20 g/100 mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test substance was dissolved in olive oil immediately before administration. Concentrations were 5 g, 10 g, and 20 g TS/100 mL vehicle.
Duration of treatment / exposure:
Test substance was orally administered in one dose
Frequency of treatment:
single dose
Post exposure period:
24 h (all groups) and 48 h (vehicle controls, 2000 mg/kg group)
Remarks:
Doses / Concentrations:
500, 1000, and 2000 mg/kg bw
Basis:
actual ingested
TS concentrations in vehicle were analytically tested and were found to be in the range of 97 - 105% of the nominal value
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control substances: cyclophosphamide (CPP) and vincristine (VCR)
- Justification for choice of positive control(s): control substances are proven to induce micronuclei by a chromosome breaking effect and by spindle activity respectively.
- Route of administration: CCP oral, VCR intraperitoneal
- Doses / concentrations: 20 mg CPP/kg bw and 0.15 mg VCR/kg bw each dissolve in 10 mL purified water/kg bw.
- Number of animals for positive controls: 5 males and 5 females, subdivided between CPP (2 males/3 females) and VCR (3 males/2 females)
Tissues and cell types examined:
bone marrow, polychromatic erythrocytes with and without micronuclei, normochromatic erythrocytes with and without micronuclei
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: doses were selected on basis of a preliminary experiment. In a pretest for the determination of the acute oral toxicity, all animals (males and females) survived treatment with 2000 mg/kg bw which is set as limit dose according to OECD TG 474. As signs of toxicity only piloerrection was observed. In addition to 2000 mg/kg bw, doses of 1000 and 500 mg/kg bw were selected.

DETAILS OF SLIDE PREPARATION: bone marrow was prepared according to W. Schmidt (see study report for references). Shortly, bone marrow was prepared from the two femora by flushing it out of the diaphysis into a centrifuge tube with fetal calf serum. After mixing and centrifugation, one drop of the resuspended precipitate in FCS was placed onto clean microscopic slides. Smears were produced, dried in the air, and subsequently stained.
Slides were first stained in eosin and methylen blue solution, rinsed in purified water, and finally stained in 7.5% Giemsa solution. After additional rinsing and clarification in xylene, the preparations were mounted using Corbit-Balsam.

METHOD OF ANALYSIS: 2000 polychromatic erythrocytes (PCE) from each animal of every test group was investigated for micronuclei. The normochromatic erythrocates (NCE) were also scored. Slides were coded before microscopic analysis.
The following parameters were recorded:
Number of polychromatic erythrocytes
Number of polychromatic erythrocytes containing micronuclei
Number of monochromatic erythrocytes
Number of monochromatic erythrocytes containing micronuclei
Ratio of polychromatic to normochromatic erythrocytes
Number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4)
Evaluation criteria:
Criteria for positive results in this test:
A dose-related and significant increase in the number of micronucleated polychromatic erythrocytes at any of the intervals.
The proportion of cells containing micronuclei exceeded both the values of the concurrent negative controls range and the negative historical control range.
A test substance is generally considered negative in this test system if:
There was no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies and at any time.
The frequencies of cells containing micronuclei were within the historical control range.
Statistics:
Statistical evaluation of data was carried out using the program system MUKERN (BASF AG).
A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test (Lienert, Verteilungsfreie Methoden in der Biostatistic, Tafelband, Verlag Anton Hain, Meisenheim am Glan, 1975) for the hypothesis of equal medians. Here, the relative frequencies of cells with micronuclei of each animal were used (statistic significance at p ≤ 0.05 and 0.01). The test was performed one-sided.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: single dose 2000 mg/kg bw
- Solubility: no sufficiently soluble in water, soluble in vehicle (olive oil)
- Clinical signs of toxicity in test animals: slight signs of toxicity were observed (piloerection)
- Evidence of cytotoxicity in tissue analyzed: no

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no
- Ratio of PCE/NCE (for Micronucleus assay): ca. 2000/670; for all dose groups in the same range as that of controls
- Appropriateness of dose levels and route: yes

There are no relevant differences in the frequency of erythrocytes containing micronuclei either between the vehicle control and the 3 dose groups or between the two sacrifice intervals (see attached tables taken from the report, file: Mous Micronucleus Study isobutanol results.pdf).

 

Clinical signs were only observed in the 1000 and 2000 mg/kg bw dose groups. Males and females revealed the same clinical signs. For the 1000 mg/kg bw dose group, piloerection was observed for all animals at 30 min and for some animals again at day one. All animals of the 2000 mg/kg bw dose group showed a narcotic-like state at 30 min after dosing but no symptoms from 90 min until day 1. On day 1, piloerection was noticed in all animals which could not be seen any more on day 2.

 

In feed, water and bedding no components were detected which could have interfered with the study.

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of the micronucleus test performed, isobutanol did not lead to any increase in the rate of micronuclei in polychromatic and normochromatic erythrocytes at doses of 500, 1000, and 2000 mg/kg bw..
Executive summary:

In a mouse bone marrow micronucleus test, 5 NMRI-mice per sex/dose were treated orally with isobutanol at doses of 0, 500, 1000, and 2000 mg/kg bw. Bone marrow cells were harvested at 24 and 48 hours post treatment. The test substance was administered orally dissolved in olive oil in single doses.

 

There were signs of toxicity during the study (narcotic-like state shortly after exposure and later (1 day) piloerection in the 2000 mg/kg dose group, piloerection shortly after exposure in the 1000 mg/kg dose group). Isobutanol was tested at adequate doses. The positive controls induced the appropriate responses.

 

There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any dose and any treatment time. Under the experimental conditions of the test, isobutanol had no chromosome damaging (clastogenic) effect nor did it lead to any impairment of chromosome distribution in the course of mitosis (CMA/BASF 2000).

 

This study is classified as acceptable. It satisfies the requirement of EU Method B.12 and OECD Test Guideline 474 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test) for in vivo cytogenetic mutagenicity data.

Based on the results for isobutanol (CMA/BASF, 2000), isobutyric acid is assessed not to exhibit a chromosome damaging (clastogenic) effect in mice in vivo.

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

Additional information

Genetic toxicity in vitro

 

Two studies are used to assess the mutagenic potential of isobutyric acid (BASF AG 1989, Hüls 1984). Both studies are performed according or similar to OECD TG 471. Documentation for the Hüls study is only available as short abstract. Thus, reliability is low. It is used as supporting information for the study of BASF. This study is used as key study. The reliability is 2. In both studies, the result is negative.

 

A third study (Szybalski 1958) does not use a validated standard method. Thus, its reliability is low (RL3). The result of this study also is negative.

 

BASF 1989 (Ames test. key study)

 

In a valid reverse gene mutation assay in bacteria according to OECD TG 471, strains TA98, TA100, TA 1535, and TA1537 of Salmonella typhimurium were exposed to isobutyric acid (purity 99.33%) at concentrations of 0 (controls), 20, 100, 500, 2500, and 5000 µg/plate in the presence and absence of mammalian metabolic activation. Tests were performed as plate incorporation and as preincubation assays.

 

Isobutyric acid was tested up to limit concentration (5000 µg/plate). The positive controls induced the appropriate responses. There was no evidence of induced mutant colonies over background.

 

Isobutyric acid was demonstrated not to be mutagenic to Salmonella typhimurium under the conditions of this test (BASF, 1989).

 

Hüls 1984 (Ames test, supporting information)

 

Isobutyric acid was negative in a duplicate bacterial reverse gene mutation assay (Ames test, plate incorporation and preincubation method) with and without metabolic activation at concentrations from 10 to 5000 µg/plate. Because only an abstract is available, the results of this study are used as supporting information (Hüls AG, 1984).

OXEA, EASTMAN, BASF 2010 (CHO HPRT)

The study according to OECD 476 was conducted to assess the potential mutagenicity of the test material on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells using 6-thioguanine (6­TG) as the selective agent. The test material did not induce any significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation in either of the two experiments. 

The test material was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.

Genetic toxicity in vivo

 

For isobutyric acid, no study concerning genetic toxicity in vivo could be identified. As substitute, data for isobutanol will be used based on following reason (for more detailed information on toxicokinetics/metabolism and justification of supporting substances see Section 7.1, endpoint summary).

After administration, isobutanol will rapidly be metabolized in vivo to isobutyraldehyde by alcohol dehydrogenases and subsequently to isobutyric acid by aldehyde dehydrogenases. In the course of metabolic transformation of isobutanol, isobutyric acid is generated rapidly and predominantly as intermediary metabolite. Thus, it is justified to use isobutanol as supporting substance in the evaluation of the systemic in vivo effects of isobutyric acid.

 

Supporting substance: isobutanol

 

The genetic toxicity in vivo of isobutanol has been evaluated in one valid study of high reliability (GLP study) which is used as key study.

 

CMA/BASF 2000 (Mouse Micronucleus Tests, key study)

 

In a mouse bone marrow micronucleus test, 5 NMRI-mice per sex/dose were treated orally with isobutanol at doses of 0, 500, 1000, and 2000 mg/kg bw. Bone marrow cells were harvested at 24 and 48 hours post treatment. The test substance was administered orally dissolved in olive oil in single doses.

 

There were signs of toxicity during the study. Isobutanol was tested at adequate doses. The positive controls induced the appropriate responses.

 

There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any dose and any treatment time. Under the experimental conditions of the test, isobutanol had no chromosome damaging (clastogenic) effect nor did it lead to any impairment of chromosome distribution in the course of mitosis (CMA/BASF, 2000).

 

Transfer of result from the supporting substance to isobutyric acid

 

Based on the results for isobutanol (CMA/BASF, 2000), isobutyric acid is assessed not to exhibit a chromosome damaging (clastogenic) effect in mice in vivo.


Short description of key information:
Genetic toxicity in vitro
Isobutyric acid was found not to be mutagenic in two in-vitro reverse gene mutation assays in bacteria according to Ames with and without metabolic activation (BASF AG 1989, Hüls 1984).
Isobutyric acid was found not to be mutagenic in a CHO HPRT test (Oxea, Eastman, BASF 2010).
Genetic toxicity in vivo
No data for isobutyric acid could be located. Data for isobutanol are used as supporting substance.
Supporting substance: isobutanol
In a valid mouse micronucleus test, isobutanol did not increase the rate of micronuclei in polychromatic and normochromatic erythrocytes at the three doses tested (CMA/BASF, 2000).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the negative results obtained in all tests for genotoxicity performed with Isobutyric acid in vitro and

with Isobutanol in vivo it is concluded that Isobutyric acid has not to be classified for genotoxicity according to Regulation (EC) No 1272/2008.