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Genetic toxicity: in vitro

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Reference substance name:
n-butyric acid
IUPAC Name:
n-butyric acid
Constituent 2
Chemical structure
Reference substance name:
Butyric acid
EC Number:
203-532-3
EC Name:
Butyric acid
Cas Number:
107-92-6
Molecular formula:
C4H8O2
IUPAC Name:
butyric acid
Details on test material:
Sponsor's identification: n-butyric acid
Description : Clear colourless liquid
CAS No : 107-92-6
Batch number : 50000023747
Date received : 08 February 2010
Expiry Date : 08 February 2011
Storage conditions: Approximately 4°C in the dark

Method

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
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.
Controlsopen allclose all
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"

Results and discussion

Test results
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'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.