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Genetic toxicity in vitro

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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 28 July 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
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
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. QA statement)
Remarks:
Date of GLP inspection 15-09-2009. Date of signature on GLP form 26-11-2009.
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 calf 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 in house 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 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 8.10 8.10 8.15 8.13 8.03 7.73 7.29 6.65 6.46
mOsm 302 305 - - 302 300 - 302 303 - 305
- 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 and positive controls were used in parallel with the test material. Solvent treatment groups were used as the negative controls. Ethyl methane
sulphonate (EMS) Sigma Batch Numbers 142314732109252 and 0001423147 were used in Experiment 1 and Experiment 2 respectively at 500 and
750 µg/ml as the positive control in cultures without S9. Dimethyl benzanthracene (DMBA) Sigma Batch Number 105K1312 at 0.5 and 1 µg/ml was
used as the positive control in cultures with S9. All positive controls were dissolved in dimethyl sulphoxide and dosed at 1%.
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 20 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 levels of test material used were 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 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 a minimum of five 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 both in the presence and absence of S9 metabolic activation in Experiment 1. In Experiment 2 the dose range was modified to 27.5 to 440 µg/ml for both exposure groups.
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 106 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 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 subcultured and maintained at 2 x 106 cells/225 cm2 flask on days 2 to 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 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 37°C in an incubator with a humidified atmosphere of 5% CO2 in air, then fixed with methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each flask.
The percentage of viability 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.
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 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.
Precipitate was not seen in the cultures at the beginning or end of the exposure period.

Mutagenicity Test - Experiment 1
The dose levels of the controls and the test material are given in the table below:
Group Final concentration of n-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*

The Day 0 and Day 7 cloning efficiencies are presented in the attached Table 2 and Table 3. The vehicle control cultures demonstrated acceptable
values for cloning efficiencies confirming the experiment was valid. 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 10E-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 n-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*
The Day 0 and Day 7 cloning efficiencies are presented in the attached Tables 4 and 5. 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. Although the day 7 cloning efficiencies of the vehicle control groups of the with S9 exposure group of Experiment 1 and the without S9 exposure group of Experiment 2 were marginally less than 70% they were considered to be acceptable.

The mutation frequency counts and mean mutation frequency per survivor per 10E6 cells values are presented in attached Tables 4 and 5. In the absence and
presence of metabolic activation there were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10E-6.
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.

All tables are attached.
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.

The test material demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

Conclusion.

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

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions (only tests without metabolic activation, no duplicates tested for individual exposure times, only 100 metaphases scored, limited reporting)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
: only tests without metabolic activation, no testing of duplicates, only 100 metaphases scored, limited reporting
Principles of method if other than guideline:
No guideline stated but chromosome aberration tests were performed according to Ishidate and Odashima (1977) Mutat Res 48, 337
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Chinese hamster lung fibroblast cell line (CHL)
Details on mammalian cell type (if applicable):
- Type and identity of media: Minimum Essential Medium (Gibco) supplemented by 10% calf serum
Metabolic activation:
without
Test concentrations with justification for top dose:
3 different concentrations up to 1 mg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: physiological saline
- Justification for choice of solvent/vehicle: no data
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 and 48 hr
- Expression time (cells in growth medium): 24 and 48 hr
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.2µg/mL, 2 hr before cell harvesting)
STAIN (for cytogenetic assays): Giemsa solution (1.5%, at pH 6.8; E. Merck; for 12 - 15 min)

NUMBER OF REPLICATIONS: 1.5 and 3 (cell doubling time 15 hr)

NUMBER OF CELLS EVALUATED: 100 metaphases were evaluated

DETERMINATION OF CYTOTOXICITY
- Method: cytotoxicity was determined in a preliminary test evaluating the dose for 50% cell-growth inhibition (cell-densitometer measurements). This was selected as maximum test dose.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no data
- Other: structural chromosomal aberrations as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others
Evaluation criteria:
In untreated and solvent-treated cells, the incidence of aberrations was usually less than 3.0%
Results were considered to be
- negative if the incidence was less than 4.9%
- equivocal if the incidence was between 5.0 and 9.9%
- positive if the incidence was more than 10.0%

For the quantitative evaluation of the clastogenic potential of the positive samples, the D20 was calculated, which is the dose (mg/mL) at which structural aberrations (including gaps) were detected in 20% of the metaphases observed.
Species / strain:
other: Chinese hamster lung fibroblast (CHL)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at maximum dose tested 50% cell-growth inhibition
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: yes, a dose of 1 mg/mL (maxium dose applied in main test) was found to inhibit cell-growth by 50 %
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation

Butyric acid was found to be negative in a chromosome aberration test without metabolic activation at concentrations up to 1 mg/mL. It was shown to be non-clastogenic to CHL cells in vitro.
Executive summary:

In a mammalian cell cytogenetics assay (Chromosome aberration test in Vitro), Chinese hamster fibroblast cell cultures were exposed to butyric acid in physiological saline at 3 graduate concentrations up to 1 mg/mL for 24 and 48 hours in a chromosome aberration study in the absence of mammalian metabolic activation.

 

Butyric acid was tested up to cytotoxic concentrations (50% cell-growth inhibition). Data on positive controls are not reported. There was no evidence for a concentration related response of chromosomal aberrations induced over background. The test substance was shown to be non-clastogenic to CHL cells in vitro (Ishidate, 1984).

 

This study is classified as reliable with restrictions due to deviations from the OECD test guideline 473 (only tests without metabolic activation, no duplicates tested for individual exposure times, only 100 metaphases scored, limited reporting). The study results are assessed as valid.

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:
comparable to guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
: only 4strains of S. thyphimurium tested
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
his
Species / strain / cell type:
S. typhimurium, other: TA97, TA 98, TA 100, TA1535
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from livers of induced male Sprague Dawley rats and induced male Syrian hamsters (10% and 30% each)
Test concentrations with justification for top dose:
0, 100, 333, 1000, 3333, 10000 µg/plate, for TA 100 in addition 5000, 6667, and 7500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without metabolic activation: 9-aminoacridine (TA97), sodium azide (TA 100, TA 1535), 2-nitrofluorene (TA 98); with metabolic activation: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 2 days
- Expression time (cells in growth medium): 2 days at 37°C

DETERMINATION OF CYTOTOXICITY
- Method: no data
Evaluation criteria:
Number of mutant colonies of positive controls must be significantly increased over the spontaneous control numbers for the test to be considered valid.
Numbers of mutant colonies of test plates have to be significantly increased over the mutant colony numbers of negative control plates.
Statistics:
Mean and standard error of the mean of replicate plates were calculated
Key result
Species / strain:
S. typhimurium, other: TA97, TA 98, TA 100, TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
observed at concentrations above 5000 µg/plate in some strains
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Tables presenting the revertant counts for all tests are appended under attached background material.

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Butyric acid was not mutagenic in Salmonella typhimurium strains TA 97, TA 98, TA100 and TA1535 with and without metabolic activation.
According to OECD 471 butyric acid is not mutagenic to Salmonella typhimurium.
Executive summary:

In a reverse gene mutation assay in bacteria, strains of S. typhimurium (TA 97, TA 98, TA 100, and TA 1535) were exposed to butyric acid at concentrations of 0, 100, 333, 1000, 3333, and 10000 µg/plate in the presence and absence of mammalian metabolic activation (S9 mix from induced rabbit and rat liver) with 20 min preincubation before plating.

  

Butyric acid was tested up to the limit concentration of 10000 µg/plate. With the highest concentration cytotoxicity was observed. The positive controls induced the appropriate responses in the corresponding strains. Butyric acid did not increase the number of revertants in any of the test strains. There was no evidence of induced mutant colonies over background (NTP, 1991).

  

This study is classified as acceptable. It was performed according to OECD test guideline 471 with minor restrictions (only 4 strains of S. thyphimurium tested).

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 n-butanol to butyric acid, information on toxicokinetics for the metabolization of n-butanol in the liver where it undergoes rapid oxidation. n-butyraldehyde and subsequently n-butyric acid is formed. Thus n-butanol is a precursor for the asessed substance n-butyric acid.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical n-butanol was investiagated as a pure compound without significant impurities. The target substance 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

For butyric acid, no study concerning genetic toxicity in vivo could be located. As substitute, data for n-butanol will be used based on following reasons. After administration, butanol will rapidly be metabolized in vivo to butyraldehyde by alcohol dehydrogenases and subsequently to butyric acid by aldehyde dehydrogenases. In the course of metabolic transformation of butanol, butyric acid is generated rapidly and predominantly as intermediary metabolite. Thus, it is justified to use butanol as supporting substance in the evaluation of the systemic in vivo effects of butyric acid.

The genetic toxicity in vivo of n-butanol has been evaluated in one valid study of high reliability (GLP study) which is used as key study (BASF 1998 (Mouse Micronucleus Tests, key study)). In a mouse bone marrow micronucleus test, 5 NMRI-mice per sex/dose were treated orally with n-butanol dissolved in olive oil at doses of 0, 500, 1000, and 2000 mg/kg bw. Animals were sacrificed and bone marrow cells of the two femora were prepared 24 and 48 post treatment. 2,000 poly­chromatic erythrocytes were evaluated per animal. Negative and positive controls displayed the appropriate responses. The administration of the test substance led to evident signs of toxicity in the highest dose group of 2,000 mg/kg body weight.

There was no significant increase in the frequency of micronucleated polychromatic erythrocytesin bone marrow after any dose and any treatment time. Under the experimental conditions of the test, n-butanol had no chromosome damaging (clastogenic) effect nor were there any indication of an impairment of chromosome distribution in the course of mitosis(BASF, 1998).

Justification for Transfer of result from the supporting substance to butyric acid

Based on the results for n-butanol (BASF, 2000), butyric acid is assessed not to exhibit a chromosome damaging (clastogenic) effect in mice in vivo.
Genetic toxicity in vitro Butyric acid was found not to be mutagenic in two independent in-vitro reverse gene mutation assays in bacteria according to Ames with and without metabolic activation (NTP, 1991; Ishidate, 1984). In addition, butyric acid did not show genotoxic potential in vitro in mammalian cells (CHL) without metabolic activation in a Chromosomal Aberration test (Ishidate, 1984). Genetic toxicity in vivo No data for butyric acid could be located. Data for n-butanol are used as supporting substance. Supporting substance: n-butanol In a valid in vivo mouse micronucleus test, n-butanol did not increase the rate of micronuclei in polychromatic and normochromatic erythrocytes at the three doses tested (BASF, 1998). Endpoint Conclusion: No adverse effect observed (negative)
Reason / purpose for cross-reference:
read-across source
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH, WIGA, Sulzfeld, Germany
- Age at study initiation: 5-8 weeks
- Weight at study initiation: 26.9 g (mean)
- Assigned to test groups randomly: yes
- Housing: during acclimation period: in groups of 5, separated by sex, Makrolon cages, type MIII; during test: individually, Makrolon cages, type MI
- Diet: standardized pelleted feed (Kliba Haltungsdiät, Klingentalmühle AG, Kaiseraugst, Switzerland); ad libitum
- Water: drinking water; ad libitum
- Acclimation period: about 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- 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: due to the limited solubility of the test item in water; olive oil had been demonstrated to be suitable in the MN-assay and historical control data are available
- Concentration of test material in vehicle: 5 g / 100 mL; 10 g / 100 mL; 20 g / 100 mL
- Amount of vehicle: 10 ml/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- solution of the test substance in the vehicle was prepared immediately before dosing
Duration of treatment / exposure:
- 24 h (all dose groups and control group)
- 48 h (highest dose group and control group)
Frequency of treatment:
- single application
Post exposure period:
- none
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide (CPP; 2 males, 3 females) and vincristine sulphate (VCR; 3 males, 2 females)
- Justification for choice of positive control(s): CPP causes clastogenicity; VCR is a known spindle poison
- Route of administration: CPP: orally; VCR: intraperitoneally
- Doses: 20 mg CPP/kg bw ; 0.15 mg VCR/kg bw (both dissolved in purified water; application volume: 10 ml/kg bw)
- Exposure duration: 24 h
Tissues and cell types examined:
- polychromatic erythrocytes and normocytes isolated from the bone marrow of the femora
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- testing up to the highest recommended dose in OECD TG 474


TREATMENT AND SAMPLING TIMES:
- sampling of bone marrow of the two femora of each animal 24 or 48 hours after dosing


DETAILS OF SLIDE PREPARATION:
- bone marrow smear was prepared on microscopic slides and dried in the air
- STAINING:
- 5 minutes modified May Grünwald solution (Wrights solution): eosin, methylene blue
- rinsed in purified water and finally stained for 15 minutes in Giemsa solution
- embedded in Corbit Balsam


METHOD OF ANALYSIS:
In general, 2,000 polychromatic erythrocytes (PCE) from each of the male and female animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCE) which occur are also scored. The following parameters are recorded:
• Number of polychromatic erythrocytes.
• Number of polychromatic erythrocytes containing micronuclei.
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome-breaking (clastogenic) effect or of a spindle activity of the substance tested.
• Number of normochromatic erythrocytes.
• Number of normochromatic erythrocytes containing micronuclei.
The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals shows the situation before test substance administration and may serve as a control value. A substance-induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice intervals.
• Ratio of polychromatic to normochromatic erythrocytes.
An alteration of this ratio indicates that the test substance actually reached the target.
Individual animals with pathological bone marrow depression may be identified and excluded from the evaluation.
• Number of small micronuclei (d < D/4) and of large micronuclei (d >/= D/4) (d = diameter of micronucleus, D = cell diameter).
The size of micronuclei may give an indication an the possible mode of action of the test substance, i.e. a clastogenic or a spindle poison effect.
Slides were coded before microscopic analysis.

Acceptance criteria:
The mouse micronucleus test is to be considered valid if the following criteria are met:
The quality of the slides allowed the identification and evaluation of a sufficient number of analyzable cells, i.e. >/= 2,000 polychromatic erythrocytes per animal.
• The proportion of cells with micronuclei in negative control animals was within the normal range of the historical control data.
• The two positive control chemicals induced a significant increase in the number of cells containing small and large micronuclei.


Evaluation criteria:
The test chemical is to be considered positive in this assay if the following criteria are met:
A dose-related and significant increase in the number of micronucleated polychromatic erythrocytes at the 24 hour interval.
• The proportion of cells containing micronuclei exceeded both the values of the concurrent negative control 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:
The statistical evaluation of the data was carried out using the program system MUKERN (BASF AG).
The number of micronuclei in polychromatic erythrocytes was analyzed.
A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians. Here, the relative frequencies of cells with micronuclei of each animal were used. If the results of this test were significant, labels (* for p
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
: in the mid and high dose group (1000 and 2000 mg/kg bw)
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- pretest for determination of acute oral toxicity
- Dose range: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: abdominal position, irregular respiration, staggering, squatting posture and narcotic like state
- no mortality,
- Rationale for exposure: recommended highest dose by OECD TG 474


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: none
- Ratio of PCE/NCE (for Micronucleus assay): see below
- Statistical evaluation: no statistical significant increase in micronuclei was observed
- no clinical signs of toxicity in the animals of the vehicle control, 500 mg/kg bw and positive control group;
- 1000 mg/kg bw: piloerection after about 30 minutes until sacrifice of the animals;
- 2000 mg/kg bw: animals were in poor condition: abdominal position, irregular respiration, staggering, squatting posture, narcotic like state
-

The single oral administration of olive oil in a volume of 10 mL/kg body weight led to 1.5% polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 0.8% after the 48-hour sacrifice interval.

After the single administration of the highest dose of 2,000 mg/kg body weight, 0.7% polychromatic erythrocytes containing micronuclei were found after 24 hours and 0.6% after 48 hours.

In the two lower dose groups, rates of micronuclei of about 0.4% (1,000 mg/kg group) and 0.6% (500 mg/kg group) were detected after a sacrifice interval of 24 hours in each case.

An 11.6% increase in polychromatic erythrocytes was observed in animals treated with 20 mg/kg cyclophosphamide, the positive control agent. This expected increase consisted mainly of small micronuclei indicative of clastogenicity.

Vincristine, a spindle poison agent, produed a 60.9%increase of micronuclei in polychromatic erythrocytes. A significant portion of this increase,

7.7% was attributable to large micronuclei.

The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals.

Thus, the test substance, n-Butanol, did not lead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d >/= D/4) did not deviate from the vehicle control value at any of the sacrifice intervals.

No inhibition of erythropoiesis induced by the treatment of mice with n-Butanol was detected; the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups.

Table 1:Polychromatic and normochromatic erythocytes (males and females)

 

Interval: 24 hours

Interval: 48 hours

 

Total No. of

MN (o/oo) in

Total No. of

MN (o/oo) in

 

PCEs

NCEs

PCEs

NCEs

PCEs

NCEs

PCEs

NCEs

Vehicle Olive Oil

20,000

7942

 1.5

0.3

20,000

7963

0.8

0.6

 

 

 

 

 

 

 

 

 

 500 mg/kg

20,000

6912

 0.6

0.0

 

 

 

 

1000 mg/kg

20,000

8570

 0.4

0.1

 

 

 

 

2000 mg/kg

20,000

7959

 0.7

0.4

20,000

8995

0.6

0.3

 

 

 

 

 

 

 

 

 

20 mg/kg CPP

10,000

4610

11.6**

0.4

 

 

 

 

0.15 mg/kg VCR

10,000

4721

60.9**

0.0

 

 

 

 

Wilcoxon test (one-sided): *: p </= 0.05, ** p </= 0.01

Pairwise comparison of each dose group with the vehicle control group.

Table 2:Polychromatic erythrocytes: differentiation between small and large micronuclei (males and females)

 

Interval: 24 hours

Interval: 48 hours

 

Total No. of PCEs

Cells (o/oo) with

Total No. of PCEs

Cells (o/oo) with

 

 

MN.d<D/4

MN.d=/>D/4

 

MN.d<D/4

MN.d=/>D/4

Vehicle Olive Oil

20,000

 1.5

0.0

20,000

0.8

0.0

 

 

 

 

 

 

 

 500 mg/kg

20,000

 0.6

0.0

 

 

 

1000 mg/kg

20,000

 0.3

0.1

 

 

 

2000 mg/kg

20,000

 0.7

0.0

20,000

0.5

0.1

 

 

 

 

 

 

 

20 mg/kg CPP

10,000

11.5**

0.1

 

 

 

0.15 mg/kg VCR

10,000

53.2**

7.7**

 

 

 

Wilcoxon test (one-sided): *: p </= 0.05, **: p </= 0.01

Pairwise comparison of each dose group with the vehicle control group.

Conclusions:
Interpretation of results (migrated information): negative
Butan-1-ol did not induce micronuclei in erythrocytes of mice after single oral application up to the maximum tolerable dose of 2000 mg/kg bw. Under the experimental conditions of this assay butan-1-ol has no chromosome damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis.
Executive summary:

The substance n-Butanol was tested for clastogenicity and for the ability to have spindle poison effects in NMRI mice using the micronucleus test method. For this purpose, the test substance, dissolved in olive oil, was administered once orally to male and female animals at dose levels of 500 mg/kg, 1,000 mg/kg and 2,000 mg/kg body weight in a volume of 10 mL/kg body weight in each case.

As a negative control, male and female mice were administered merely the vehicle, olive oil, by the same route, which gave frequencies of micronucleated polychromatic erythrocytes within the historical control range.

Both of the positive control chemicals, i.e. cyclo­phosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.

Animals which were administered the vehicle or the positive control substances cyclophosphamide or vincristine did not show any clinical signs of toxicity.

The administration of the test substance led to evident signs of toxicity in the highest dose groupof 2,000 mg/kg body weight.

The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2,000.mg/kg body weight and in the vehicle controls. In the test groups of 500 mg/kg and 1,000 mg/kg bodyweight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2,000 poly­chromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2,000 polychromatic erythrocytes were also registered. In addition, the number of normochromatic erythrocytes with micronuclei and the ratio of polychromatic to normochromatic erythrocytes were determined.

According to the results of the present study, the single oral administration of n-Butanol did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always in the same range as that of the negative control in all dose groups and at all sacrifice intervals (Engelhardt and Hoffmann, 1998).

No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.

Thus, under the experimental conditions chosen here,the test substance n-Butanol does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis. This study is reliable without restrictions (RL1).

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

Additional information

Genetic toxicity in vitro

 

Three key studies have been identified to assess the genetic toxicity potential of butyric acid. In all studies, no evidence was found for a mutagenic/genotoxic potential of butyric acid.

 

NTP 1991

 

In a reverse gene mutation assay in bacteria, strains of S. typhimurium (TA 97, TA 98, TA 100, and TA 1535) were exposed to butyric acid at concentrations of 0, 100, 333, 1000, 3333, and 10000 µg/plate in the presence and absence of mammalian metabolic activation (S9 mix from induced rabbit and rat liver) with 20 min preincubation before plating.

 

Butyric acid was tested up to the limit concentration of 10,000 µg/plate. With the highest concentration, cytotoxicity was observed. The positive controls induced the appropriate responses. Butyric acid did not increase the number of revertants in any of the test strains. There was no evidence of induced mutant colonies over background (NTP, 1991).

 

Ishidata 1984 (Ames test)

 

In a reverse gene mutation assay in bacteria according to Ames (1975), strains of Salmonella typhimurium (TA92, TA94, TA98, TA100, TA1535, and TA1537) were exposed to butyric acid (99.9%) in DMSO at 6 graduate concentrations up to 10,000 µg/plate (preincubation assay) in the presence and absence of mammalian metabolic activation (S9 mix from PCB induced male rat liver).

 

Butyric acid did not show cytotoxicity at 10,000 µg/plate. Data on positive controls are not reported. Butyric acid did not increase the number of revertants in any of the test strains. There was no evidence of induced mutant colonies over background (Ishidata, 1984).

 

 

Ishidata 1984 (Chromosome Aberration test)

 

In a mammalian cell cytogenetics assay, Chinese hamster fibroblast cell cultures were exposed to butyric acid in physiological saline at 3 graduate concentrations up to 1 mg/mL for 24 and 48 hours in a chromosome aberration study in the absence of mammalian metabolic activation.

 

Butyric acid was tested up to cytotoxic concentrations (50% cell-growth inhibition). Data on positive controls are not reported. There was no evidence for a concentration related response of chromosomal aberrations induced over background. The test substance was shown to be non-clastogenic to CHL cells in vitro (Ishidate, 1984).

 

Harlan 2010 (CHO HPRT Test)

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 demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

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

Genetic toxicity in vivo

 

For butyric acid, no study concerning genetic toxicity in vivo could be located. As substitute, data for n-butanol will be used based on following reasons.

 

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

 

Supporting substance: n-butanol

 

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

 

BASF 1998 (Mouse Micronucleus Tests, key study)

 

In a mouse bone marrow micronucleus test, 5 NMRI-mice per sex/dose were treated orally with n-butanol dissolved in olive oil at doses of 0, 500, 1000, and 2000 mg/kg bw. Animals were sacrificed and bone marrow cells of the two femora were prepared 24 and 48 post treatment. 2,000 poly­chromatic erythrocytes were evaluated per animal.

 

Negative and positive controls displayed the appropriate responses. The administration of the test substance led to evident signs of toxicity in the highest dose group of 2,000 mg/kg body weight.

 

There wasno significant increase in the frequency of micronucleated polychromatic erythrocytesin bone marrow after any dose and any treatment time. Under the experimental conditions of the test, n-butanol had no chromosome damaging (clastogenic) effect nor were there any indication of an impairment of chromosome distribution in the course of mitosis(BASF, 1998).

 

Transfer of result from the supporting substance to butyric acid

 

Based on the results for n-butanol (BASF, 2000), butyric acid is assessed not to exhibit a chromosome damaging (clastogenic) effect in mice in vivo.


Short description of key information:
Genetic toxicity in vitro
Butyric acid was found not to be mutagenic in two independent in-vitro reverse gene mutation assays in bacteria according to Ames with and without metabolic activation (NTP, 1991; Ishidate, 1984).
In addition, butyric acid did not show genotoxic potential in vitro in mammalian cells (CHL) without metabolic activation in a Chromosomal Aberration test (Ishidate, 1984).
Genetic toxicity in vivo
No data for butyric acid could be located. Data for n-butanol are used as supporting substance.
Supporting substance: n-butanol
In a valid in vivo mouse micronucleus test, n-butanol did not increase the rate of micronuclei in polychromatic and normochromatic erythrocytes at the three doses tested (BASF, 1998).

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 n-butyric acid in vitro and

with butan-1-ol in vivo it is concluded that n-butyric acid has not to be classified for

genotoxicity according to Regulation (EC) No 1272/2008.