DL-malic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

19 May 2010 to 19 July 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant study with no deviations, conducted by a reliable laboratory

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

Conducted in accordance with current test guidelines

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fumaric acid

EC Number:

203-743-0

EC Name:

Fumaric acid

Cas Number:

110-17-8

IUPAC Name:

but-2-enedioic acid

Constituent 2

Reference substance name:

trans-butendiacid

IUPAC Name:

trans-butendiacid

Details on test material:

- Name of test material (as cited in study report): Fumaric acid (trans-butendiacid or Fumarsaure)
- Molecular formula (if other than submission substance): C4H4O4
- Smiles notation (if other than submission substance): C(=C/C(=O)O)\C(=O)O
- InChl (if other than submission substance): 1/C4H4O4/c5-3(6)1-2-4(7)8/h1-2H,(H,5,6)(H,7,8)/b2-1+
- Physical state: white crystalline powder
- Analytical purity: 99.7%
- Impurities (identity and concentrations): not stated
- Composition of test material, percentage of components: 99.7% fumaricacid
- Purity test date: 22 October 2009
- Lot/batch No.: LPFS6A4011 for analysis, also used for inhalation test.
- Expiration date of the lot/batch: 30 June 2010
- Stability under test conditions: stable
- Storage condition of test material: refrigerated
- Other: solubility in water = 5 g/L at20 °C
For the mouse lymphoma assay fumaric acid was dissolved in DMSO butthe stability in this solvent was not determined in the assay.

Method

Target gene:

Cells deficient in thymidine kinase (TK) due to the mutation TK+/- to TK-/- are resistant to cytotoxic effects of the pyrimidine analogue trifluorthymidine (TFT). TK proficient cells are sensitive to TFT (which causes inhibition of cellular metabolism and halts further cell division), so mutant cells can proliferate in the presence of TFT but normal cells, containing thymidine kinase, cannot.
A similar effect can be detected if mutations arise at the HPRT locus for hypoxanthine-guanine phosphoribosyl transferase or XPRT - a transgene of xanthine-guanine phosphoribosyl transferase.

This in vitro test is an assay for the detection of forward gene mutations at the autosomal thymidine kinase (TK) locus of heterozygous L5178Y/TK+/- cells to TK-/- mutants. In addition, evidence has been obtained that small TK-/- colonies may result from chromosomal damage to the TK locus and adjacent genes (1). Gene and chromosome mutations are considered as an initial step in the carcinogenic process (2).
The L5178Y/TK+/- cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional TK enzyme.
TK catalyses the conversion of TFT (Trifluorothymidine) to its cytostatic and cytotoxic tri-fluorothymidine-monophosphate derivative. Therefore, cells deficient in TK due to a forward mutation are resistant to TFT. These cells are able to proliferate in the presence of TFT whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 48 hours.

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Complete culture medium - RPMI 1640 medium supplemented with 15 % horse serum (3 % HS during 4 hour treatment), 1% of 100 U/100 µg/mL Penicillin/Streptomycin, 220 µg/mL Sodium-Pyruvate, and 0.5 – 0.75 % Amphotericin used as antifungal.
Details of all other media used during the course of the assay are given in the study report

- Properly maintained: yes

- Periodically checked for Mycoplasma contamination: yes
Large stocks of the cleansed L5178Y cell line are stored in liquid nitrogen in the cell bank of Harlan CCR allowing the repeated use of the same cell culture batch in many experiments. Before freezing, each batch is screened for mycoplasma contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells

Additional strain / cell type characteristics:

other: thymidine kinase deficiency

Metabolic activation:

with and without

Metabolic activation system:

S9 from rats

Test concentrations with justification for top dose:

In all four experimental conditions (Experiment I with or without metabolic activation) and Experiment II (4 or 24 hours with or without metabolic activation) the same dose concentrations were selected - 37.5, 75, 150, 300, 600 and 1200 ug/mL. Since a minimum of four concentrations require evaluation, the lowest concentration, 37.5 ug/ml, was discontinued partway through the study. The remaining five concentrations 75 to 1200 ug/ml were evaluated in each case.
The highest concentration should be 10 mM, but not higher than 5 mg/mL, unless limited by the solubility or toxicity of the test item.
RSG (Relative Suspension Growth) or RTG (Relative Total Growth) values (main experiment) below 50 % are considered toxic. In case of toxic effects, the highest test item concentration of the main experiment should reduce the RSG or RTG value to approximately 10 - 20 %.
The pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test item concentrations between 9.4 µg/mL and 1200 µg/mL were used. The highest concentration in the pre-experiment was chosen with regard to the purity (min. 99.7 %) and the molecular weight (116.07 g/mol) of the test item. Isolated minor toxic effects occurred at 18.8 and 300 µg/mL in the presence of metabolic activation. In the pre-experiment and in the main experiments the pH at the two highest concentrations of 600 and 1200 µg/mL was neutralised with 2N NaOH. There was no relevant shift of osmolarity of the medium even at the maximum concentration of the test item. activation following 4 hours treatment
The maximum concentration was again 1200 µg/mL equal to a molar concentration of approximately 10 mM.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:The solvent was chosen according to its solubility properties and its relative non-toxicity to the cells. The final concentration of DMSO in the culture medium was 0.5 % v/v.

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

cyclophosphamide for test with activation

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: for test without activation

Details on test system and experimental conditions:

Prior to testing the number of spontaneous mutants is reduced by growing cells in supplemented RPMI 160-HAT medium (supplementedwith hypoxanthine, aminopterin and thymidine). The cells are then allowed to recover for two days in RPMI 1640 medium supplemented with hypoxanthine and thymidine only, cells are then returned to complete culture medium. Stocks of cleansed cell ilines are then maintained in liquid nitrogen storage.

Thawed cells are subcultured twice prior to treatment and incubated at 37±1°C in a humidified CO2 enriched ambient air mix.

For the mutation experiments 1x10 E7 [4h exposure] or 3x10 E6 [24 h exposure] cells/flask were suspended in 10 ml RPMI medium with 3 % horse serum ( or 15 % horse serum during 24 h exposure) and exposed to various concentrations of fumaric acid (37.5, 75, 150, 300, 600 or 1200 µg/mL) in the presence or absence of metabolic activation.

After 4 h (or 24 h in the second experiment) the test material was removed by centrifugation and the cells were washed twice with "saline G". The cells were resuspended in 30 ml complete culture medium and incubated for an expression and growth period of 48 h.

Complete culture medium - RPMI 1640 medium (GIBCO, invitrogen) supplemented with 15 % horse serum (HS, GIBCO, invitrogen) (3 % HS during 4 hour treatment), 1% of 100 U/100 µg/mL Penicil¬lin/Streptomycin, 220 µg/mL Sodium-Pyruvate, and 0.5 – 0.75 % Amphotericin used as antifungal
Selective medium - RPMI 1640, complete culture medium, supplemented by addition of 5 µg/mL TFT.

The cell density was determined each day and adjusted to 3 x 10 E5 cells/ml. The relative suspension growth (RSG) of the treated cell cultures was calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold-increase in cell number
.
After the expression period the cultures were selected. Cells from each experimental group were seeded into 2 microtiter plates so that each well contained approximately 4 x 10³ cells in selective medium. The viability (cloning efficiency) was determined by seeding about 2 cells per well into microtiter plates (selective medium without TFT). The plates were incubated at 37±1.5 °C in 4.5 % CO2/95.5 % water saturated air for 10 - 15 days. Then the plates were evaluated.

METHOD OF APPLICATION: in medium
DURATION

- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 10-15 days

SELECTION AGENT (mutation assays): RPMI 1640, complete culture medium, supplemented by addition of 5 µg/mL TFT.

NUMBER OF REPLICATIONS: 2 per experiment

NUMBER OF CELLS EVALUATED: 10 E6

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - no indications that less than 50% survival occurred in parallel cultures

Evaluation criteria:

Colonies were counted with an automated colony counter. Mutant frequencies were expressed as mutants per 10 E6 surviving cells.

The criteria for acceptance of assay validity were:
1 .All plates, from either the cloning efficiency or the TFT resistance-testing portion of the experiment are analysable.
2. The absolute cloning efficiency at the time of mutant selection (CE) of the solvent controls is 65 – 120 %.
3. The total suspension growth of the solvent control calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold-increase in cell number is 8 to 32. Following 24 h treatment the total suspension growth is 32 – 180.
4. The range of the solvent control mutant frequency is in the range of 50 – 170 x 10 E6 cells.
5. The positive controls (MMS and CPA) should yield an absolute increase in total MF, that is an increase above spontaneous background MF (an induced MF [IMF]) of at least 300 x 10 E6 cells. At least 40 % of the induced mutation frequency (IMF) should be reflected in the small colony MF. Alternatively, the positive controls should induce at least 150 small colonies.
6.The upper limit of cytotoxicity observed in the positive control culture should be the same as for the experimental cultures (I.e. the relative total growth – RTG- should be greater than 10 % of the concurrent selective control group).

Results evaluation
Test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10 E6 cells above the corresponding solvent control.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is reproducible if it occurs in both parallel cultures. In the evaluation of results the historical variability of the mutation rates in the solvent controls are also taken into consideration. Results of test groups are generally rejected if the relative total growth is less than 10 % of the vehicle control

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT 11 statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the probability p-value is below 0.05.

experimental group p-value
experiment I, culture I without S9 mix 0.621
experiment I, culture II without S9 mix 0.476
experiment I, culture I with S9 mix 0.156
experiment I, culture II with S9 mix 0.104
experiment II, culture I without S9 mix 0.226
experiment II, culture II without S9 mix 0.720
experiment II, culture I with S9 mix 0.676
experiment II, culture II with S9 mix 0.036

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The two highest concentrations were neutralised with 2M NaOH but this did not result in any shift in osmolarity in the medium. pH of solvent control medium = 7.56 and for high dose fumaric acid ,1200 ug/mL - 7.7
- Effects of osmolality: None despite neutralising higher concentrations Osmolarity (mOsm) for solvent controls = 365 and for high dose fumaric acid, 1200 ug/mL = 360

RANGE-FINDING/SCREENING STUDIES:
A pre-test was performed in order to determine the concentration range of the mutagenicity experiments. Both, pH value and osmolarity were determined at the maximal concentration of the test item and in the solvent control without metabolic activation.
1x10 E7 cells (3x10 E6 cells at the beginning of 24 h treatment) were exposed to each concentration of the test item for 4 and 24 hours without and 4 hours with metabolic activation. During the 4 h treatment period the serum concentration was reduced from 15 % to 3 %. Following treatment the cells were washed twice by centrifugation (425 g, 10 min) and resuspended in "saline G". Subsequently the cells were resuspended in 30 mL complete culture medium for a 2-day growth period. The cell density was determined immediately after treatment and at each day of the growth period and adjusted to 3x10 E5 cells/mL, if necessary. The relative suspension growth (RSG) of the treated cell cultures was calculated at the end of the growth period

COMPARISON WITH HISTORICAL CONTROL DATA:
Historical data presented in study report and used for comparison where appropriate in evaluation of results.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

No relevant cytotoxic effects, indicated by a relative total growth of less than 50 % of survival in both parallel cultures, were observed up to the maximum concentration with and without metabolic activation, following 4 and 24 hours of treatment.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in the first experiment up to the maximum concentration with and without metabolic activation. In the second experiment the threshold of 126 above the corresponding solvent control count and the historical range of solvent controls was exceeded in culture II at an intermediate concentration of 300 µg/mL. However, no comparable increase was noted in the parallel culture under identical conditions or in both parallel cultures at any other, even higher concentration. Furthermore, there was no dose dependent trend as indicated by the lacking statistical significance. The isolated increase of the mutation frequency was consequently judged as an irrelevant outlier.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT statistics software. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely determined in the second culture of the second experiment with metabolic activation. Since the mutation frequency did not exceeded the threshold as indicated above, the statistical result is considered as biologically irrelevant fluctuation.

In this study the range of the solvent controls was from 127 up to 204 mutant colonies per 10⁶cells; the range of the groups treated with the test item was from 70 up to 293 mutant colonies per 10⁶cells not considering the outlier described above. The highest solvent control values exceeded the recommended 50 – 170 x 10⁶control range. The data are acceptable however, since the range of approximately 50-200 colonies per 10⁶cells recommended by the IWGT in 2003 (11) was covered. The cloning efficiency 2 of the solvent control of the second culture of the first experiment with metabolic activation exceeded the upper limit of 120% (c.f. table 9, column 8). The data are acceptable since the parallel culture remained within the recommended range and the absolute values of the cloning efficiency 2 were used to calculate the mutation frequency.

MMS (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and CPA (4.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies. In the first experiment the positive controls also showed a distinct shift towards small colonies. In the second experiment however, most induced colonies were large ones. The criterion of at least 150 induced small colonies was generally not met in the second experiment although the positive controls showed a substantial increase of total colonies. The substantial increase of the total number of induced colonies showed that the test is sensitive and valid but based on the low number of induced small colonies the second experiment can not distinguish clastogenic effects from point mutations. Even MMS which is known to primarily induce clastogenic effects produced mostly large colonies in the second experiment. Since the test item was clearly non-mutagenic this inability to distinguish clastogenic effects and point mutations has no impact on this study.

Applicant's summary and conclusion

Conclusions:

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

Under the experimental conditions reported fumaric acid did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Executive summary:

Read across to malic acid is considered valid as metabolism of fumaric acid to malic acid is a key part of the Krebbs Cycle

The assay was performed in two independent experiments, using two parallel cultures each. The first experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

The highest concentration (1200 µg/mL) was chosen with regard to the molecular weight of the test item corresponding to a molar concentration of about 10 mM - the limit concentration for this assay.

No substantial and reproducible dose dependent increase in mutant colony numbers was observed in either experiment. No relevant shift in the ratio of small versus large colonies was observed up to the maximum concentration applied.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

Under the experimental conditions reported fumaric acid did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.