Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Holistic evaluation of three independent Ames tests (gene mutation in bacteria), two mouse lymphoma assays (in vitro gene mutation in mammalian cells), and an chromosome aberration test (clastogenicity) results in the conclusion that fumaric acid lacks potential for genetic toxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Guideline-compliant study published in a peer-reviewed journal, no detailed results available
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
not specified
Remarks:
published study
Type of assay:
bacterial reverse mutation assay
Target gene:
No information available.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium, other: TA92, TA94 and TA2637
Metabolic activation:
with and without
Metabolic activation system:
S-9 fraction from liver of Fischer rats pretreated 5 days before with polychlorinated biphenyls
Test concentrations with justification for top dose:
Six different concentrations with a maximum dose of 10 µg/plate fumaric acid
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
not specified
Details on test system and experimental conditions:
Reverse mutation assays were carried out using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98. For some samples, TA2637 was also used. Cells cultured overnight were pre-incubated with both the test sample and the S-9 mix for 20 min at 37 °C before plating. Duplicate plates were used for each of six concentrations.
Evaluation criteria:
The number of revertant colonies (his+) was scored after incubation at 37 °C for 2 days. The result was considered positive if the number of colonies was twice the number in control.
Statistics:
No statistical information available.
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:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
other: S. typhimurium TA92, TA94 and TA2637
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
Interpretation of results (migrated information): Negative with and without metabolic activation
No evidence of mutagenicity was seen when fumaric acid was tested in the Ames assay.
Executive summary:

Fumaric acid was negative in the Salmonella Ames test when tested up to a maximum concentration of 10 µg/plate both with and without metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Guideline-compliant study published in a peer-reviewed journal, but lacks some reporting details
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9 from rats and hamsters
Test concentrations with justification for top dose:
333, 1000, 3333, 6666 and 10000 µg fumaric acid/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
not specified
Details on test system and experimental conditions:
S. typhimurium histidine auxotrophs TA98, TA100, TA1535, TA1537 and TA1538 were cultured overnight in Oxoid nutrient broth no. 2 and incubated until they reached a density of 1.0–2.0E+9 cells/mL. For testing in the absence of S9, 100 µL of the tester strain and 50 µL of the solvent or test chemical were added to 2.5 mL of molten selective top agar at 45 ± 2 °C. When S9 was used, 0.5 mL of S9 mix, 50 µL of tester strain and 50 µL of solvent or test chemical were added to molten selective top agar as described above. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After overlay solidified, plates were incubated for 48 hr at 37 ± 2 °C. Five doses of test chemical, together with solvent and positive controls, were tested in triplicate on each tester strain without metabolic activation and also with metabolic activation by induced rat and hamster liver S9 preparations.
Evaluation criteria:
The doses tested were selected based on levels of cytotoxicity observed in preliminary studies using strain TA100. For a test article to be considered positive, it had to induce at least a doubling (TA98, TA100 and TA1535) in the mean number of revertants per plate of at least one tester strain. This increase in mean revertants per plate had to be accompanied by a dose response to increasing concentrations of test chemical. If the study showed a dose response with a less than 3-fold increase on TA1537 or TA1538, the response had to be confirmed in a repeat experiment.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Positive controls validity:
valid

Results of Ames Salmonella evaluation

Fumaric acid (µg/plate)  

 

Solvent control (DMSO)

 333

 1000

 3333

 6666

10000

 Positive control

TA98 Result                    
no S9 Negative 11 ± 5 15 ± 2 14 ± 5 10 ± 2 15 ± 3 11 ± 4 359 ± 42
rat S9 Negative 16 ± 6 21 ± 2 19 ± 3 19 ± 3 15 ± 6 0 ± 0 894 ± 39
hamster S9 Negative 27 ± 9 18 ± 4 22 ± 4 24 ± 1 17 ± 3 9 ± 9 696 ± 23
TA100 Result                    
no S9 Negative 115 ± 17 124 ± 17  123 ± 7  110 ± 28  128 ± 15  72 ± 15  1106 ± 37 
rat S9 Negative 113 ± 4 104 ± 14  130 ± 31  124 ± 7  100 ± 14  1 ± 2  1868 ± 34 
hamster S9 Negative 127 ± 14 133 ± 15 136 ± 13 133 ± 16 49 ± 121 0 ± 0 1285 ± 33
 TA102 Result                    
no S9 Negative 224 ± 9 233 ± 10 242 ± 39 247 ± 11 235 ± 117 228 ± 16 1384 ± 327
rat S9 Negative 264 ± 4 246 ± 26 227 ± 11  303 ± 15  314 ± 35  185 ± 7  1550 ± 44
hamster S9 Negative 286 ± 16 285 ± 35 290 ± 18 262 ± 28 253 ± 45 0 ± 0 1287 ± 87
TA1535 Result                    
no S9 Negative  31 ± 4 30 ± 2 21 ± 8 37 ± 5 34 ± 1 6 ± 2 899 ± 17
rat S9 Negative  32 ± 8 25 ± 4 26 ± 8 23 ± 10 18 ± 7 2 ± 3 147 ± 9
hamster S9 Negative  24 ± 3 27 ± 4 28 ± 8 23 ± 6 11 ± 2 0 ± 0 139 ± 12
TA1537 Result                    
no S9 Negative 5 ± 2 3 ± 1 5 ± 2 7 ± 3 5 ± 4 6 ± 2 1648 ± 348
rat S9 Negative  6 ± 1 6 ± 3 8 ± 2 6 ± 1 6 ± 4 0 ± 0 134 ± 12
hamster S9 Negative 9 ± 1 10 ± 2  6 ± 1 7 ± 2 4 ± 1 0 ± 0 178 ± 56
TA1538 Result                    
no S9 Negative 15 ± 8 21 ± 6 19 ± 2 19 ± 5 25 ± 6 11 ± 5 1227 ± 48
rat S9 Negative 29 ± 6 24 ± 7 21 ± 1 26 ± 1 23 ± 5 1 ± 2 944 ± 22
hamster S9 Negative 30 ± 4 27 ± 4 29 ± 2 23 ± 6 17 ± 3 0 ± 0 693 ± 47
Conclusions:
Interpretation of results: Negative
No evidence of mutagenicity was seen when fumaric acid was tested in the Ames assay.
Executive summary:

Fumaric acid was non-mutagenic in the bacterial reverse mutation assay (Ames test) using S. typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Guideline-compliant study published in a peer-reviewed journal
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
The order of use of 10% and 30% S-9 was reversed. Initial testing was in strains TA98 and TA100.
Principles of method if other than guideline:
Some 300 materials were used to assess the inter- and intra-lab variability of the Ames test. Fumaric acid was included as one of the test materials. The methods, as indicated by NTP, were complaint with applicable test guidelines.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Standard Ames test parameters e.g. histidine dependency
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 97
Metabolic activation:
with and without
Metabolic activation system:
S-9 fractions of Aroclor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers
Test concentrations with justification for top dose:
Initially tested at half-log dose intervals up to a dose that elicited toxicity.
Vehicle / solvent:
The solvent of choice was distilled water, followed by dimethyl sulfoxide, 95 % ethanol and acetone.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: without activation: sodium azide, 9-aminoacridine and 4-nitro-o-phenylenediamine; with activation: 2-aminoanthracene for all strains
Details on test system and experimental conditions:
A preincubation assay was performed. The test chemical (0.05 mL), Salmonella culture (0.01 mL), and S-9 mix or buffer (0.50 mL) were incubated at 37 °C, without shaking, for 20 mins. The top agar was added and contents of tubes were mixed and poured onto the surface of petri dishes containing Vogel-Bonner medium. The histidine-independent colonies were counted following two days incubation at 37 °C.
Protocol variations included:
1) Testing in strains TA97, TA98, TA100, and TA 1535, with some additional testing in strain TA1537; 10 % S9 was used.
2) The first test of a chemical was without activation and with 10 % S9. Test was repeated for positive results. For negative results, test was repeated without S9 and with 30 % S9.
3) The order of use of 10 % and 30 % S9 was reversed.
4) Initial testing was in strains TA98 and TA100 without activation and with 30 % rat and hamster S9. If positive in the two strains, the test was repeated. If negative, other strains were tested with 30 % and 10 % S9. Occasionally, 5 % S9 was also used in protocol variations.
Evaluation criteria:
Evaluations were made at both the individual trial and overall chemical levels. Individual trials were judged mutagenic, weakly mutagenic, questionable revertants or nonmutagenic, depending on the magnitude of the increase of his+ revertants, and the shape of the dose-response. A chemical was judged mutagenic or weakly mutagenic if it produced a reproducible dose-related response over the solvent control in replicate trials. A chemical was judged questionable if the results of individual trials were not reproducible, did not meet the criteria for weakly mutagenic, or if single doses produced increased his+ revertants in repeat trials. Chemicals were judged nonmutagenic if they did not meet the criteria for a mutagenic or questionable response. A chemical was not designated nonmutagenic unless it had been tested in strains TA98, TA100, TA1535, and TA97 and/or TA1537, without activation and with 10 % and 30 % rat and hamster S9.
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:
not specified

Strain: TA100

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

30% HLI
(Negative)

10% RLI
(Negative)

30% RLI
(Negative)

Protocol

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

113

6.1

81

3.6

107

6.4

82

8.7

114

2.9

100

3.2

33     

102

6.3

75

3.5

98

6.6

83

1.9

121

3.6

97

3.6

100     

105

5.1

90

5.5

85

8.5

81

10

116

4.9

118

4.8

333     

110

5.5

88

2.9

81

3.5

73

2.2

111

6.4

109

5.5

1000     

111

3.8

72

4.9

89

7.4

66

6.8

117

7.3

90

10

2000     

104s

6.1

74s

4

83s

2.7

72s

3.9

110s

4.6

93s

6.6

Positive Control

1047

41.9

743

34.4

1158

14.6

1206

36.4

1580

37.2

818

13.2

Strain: TA1535

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

30% HLI
(Negative)

10% RLI
(Negative)

30% RLI
(Negative)

Protocol

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

43

6.2

30

2.3

21

3.2

11

0.7

29

2.4

21

0.7

33     

46

5.5

29

2.2

15

3.8

11

0.6

31

3.2

12

1.7

100     

43

9.5

31

5

16

2.2

8

1.2

30

2.8

16

3.2

333     

46

3.6

28

1.8

15

0.9

13

4

35

3.8

21

4.2

1000     

42

3.3

33

5

17

2.6

12

1.2

32

1.8

16

1.5

2000     

41

3.2

26

3.1

13

4

12s

0.6

26s

4.5

7s

1.3

Positive Control

881

11.7

948

19.4

77

1.5

210

12.5

119

4.6

118

7.5

Strain: TA97

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

30% HLI
(Negative)

10% RLI
(Negative)

30% RLI
(Negative)

Protocol

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

115

6.4

81

1.5

128

7

137

7.5

137

8.2

177

8.3

33     

108

8.6

83

1.5

125

4.2

137

5.9

141

5.4

179

12.1

100     

107

7.3

93

8.2

120

7.7

152

7.3

124

3.8

171

0.3

333     

99

5.1

87

5.8

116

3.2

148

3.2

131

5.2

166

2.7

1000     

108

6.7

72s

1.5

124

1.3

127

10.8

151

4.5

160

4.2

2000     

119

3.9

88s

6.7

126s

5.4

137s

8.6

125s

8.3

151s

3.7

Positive Control

519

17.4

557

7.4

694

5.3

976

39

1044

12.5

554

13.5

Strain: TA98

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

30% HLI
(Negative)

10% RLI
(Negative)

30% RLI
(Negative)

Protocol

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

28

1.2

17

0.9

35

1.2

30

2.1

38

0.6

27

1

33     

23

2.6

17

4.3

33

3.1

29

1.2

35

4.8

25

2.9

100     

34

4.4

18

3.2

36

2.7

33

2

45

1

33

2

333     

31

6

15

2.7

39

2.9

38

3.2

53

5.7

35

2.2

1000     

34

2.7

18

2.6

42

1.7

32

3.4

35

2.3

36

1.7

2000     

33

4.2

16

0.6

35s

4.3

33s

4.3

36s

3.5

27s

2

Positive Control

1498

62.7

1527

67.4

868

38.8

887

28.7

1175

35.7

427

7.9

Abbreviations:
RLI = induced male Sprague Dawley rat liver S9
HLI = induced male Syrian hamster liver S9
s = Slight Toxicity; p = Precipitate; x = Slight Toxicity and Precipitate; T = Toxic; c = Contamination

Conclusions:
Interpretation of results: Negative
No evidence of mutagenicity was seen with fumaric acid under the conditions of this study.
Executive summary:

The mutagenicity of fumaric acid was investigated in an Ames test (plate incorporation assay) using S. typhimurium strains TA97, TA98, TA100, TA1535 and TA1537. Each strain was exposed to the test material in the presence and absence of an exogenous metabolic activation system (Aroclor 1254-induced male rat liver S9 fraction and male Syrian hamster livers). Exposure to fumaric acid did not induce increased numbers of revertant colonies in any strain.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 May 2010 to 19 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
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 / 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 µg/mL. Since a minimum of four concentrations require evaluation, the lowest concentration, 37.5 µg/ml, was discontinued partway through the study. The remaining five concentrations 75 to 1200 µg/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 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.
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:
methymethanesulfonate = positive control 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 1.0E+7 [4 h exposure] or 3.0E+6 [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 penicillin/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 3.0E+5 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 4.0E+3 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: 10.0E+6

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 10.0E+6 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 50–170E+6 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 300E+6 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 10.0E+6 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
Key result
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
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
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 µg/mL = 7.7
- Effects of osmolality: None despite neutralising higher concentrations Osmolarity (mOsm) for solvent controls = 365 and for high dose fumaric acid, 1200 µg/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.
1.0E+7 cells (3.0E+6 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 3.0E+5 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'.

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 106 cells; the range of the groups treated with the test item was from 70 up to 293 mutant colonies per 106 cells not considering the outlier described above. The highest solvent control values exceeded the recommended 50–170 × 106 control range. The data are acceptable however, since the range of approximately 50-200 colonies per 106 cells 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 % (cf. 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.

Conclusions:
Interpretation of results: Negative with and 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:

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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not stated - various studies reported over a period of two decades
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Guideline-compliant study published in a peer-reviewed journal, but lacks some reporting details
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 1984, revised 1997; transposed into OECD guideline 490 in 2016
Deviations:
no
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Fischer's medium for leukemic cells of mice supplemented with 10 % horse serum and 0.02 % pluronic F-68.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 from Sprague-Dawley rats
Test concentrations with justification for top dose:
2856, 3887, 4913, 5943, 6971 and 8000 µg/mL fumaric acid
Vehicle / solvent:
DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Remarks:
3-methylcholanthrene for test with activation
Positive control substance:
methylmethanesulfonate
Details on test system and experimental conditions:
Duplicate cultures of L5178Y TK ± 3.7.C cells at a concentration of 1.2E+75 cells were exposed to the test chemical, positive control or solvent control for 4 hr at 37 ± 1 °C. The cells were washed twice with growth medium and maintained at 37 ± 1 °C for 48 h in log-phase growth to allow recovery and mutant expression. Cells in cultures were adjusted to 3.0E+5/mL at 24 h intervals and then cloned in soft agar medium containing Fischer's medium, 20 % horse serum, 2 mM sodium pyruvate, 0.02 % pluronic F-68, an 0.23% granulated agar. Plates were incubated at 37 ± 1 °C in 5 % CO2 in air for 10-12 days.
Evaluation criteria:
Colonies were counted with an Artek automated colony counter or ProtoCol colony counter. Only colonies larger than approximately 0.2 mm in diameter were counted. Mutant frequencies were expressed as mutants per 10.0E+6 surviving cells. Results from this study were interpreted using a doubling of the mutant frequency over concurrent solvent control as an indication of a positive effect, together with evidence of a dose-related increase. The authors stated that they used new criteria in which a compound is considered positive if one or more dose levels with 10 % or greater total growth exhibits mutant frequencies of greater than 100 mutants per 10.0E+6 of clonable cells over the background level.
Statistics:
No information provided
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Positive responses were reported by the authors at the two highest concentrations, 6971 and 8000 µg/mL without activation, and at 8000 µg/mL with activation. Relative total growth for non-activated cells at 8000 µg/mL was 3 %, i.e., a 97 % reduction in cell growth. For activated cells, relative total growth was 14 % and 16.5 % at 6971 and 8000 µg/mL, respectively. This corresponds to reductions in cell growth of 86 and 83.5 %, respectively.

Results of the mouse lymphoma assay

Fumaric acid

Non-Activated Cultures S9-Activated Cultures
 Dose (µg/mL)   Average TFT Average VC Mut Freq RTG Dose (µg/mL) Averate TFT Average VC Mut Freq  RTG
2856 35 171 0.4  46  2856 36 118  0.6  54 
     39 181  0.4  49    10 53 0.4  24 
3887 45 169  0.5  41  3887 43 163  0.5  57 
   44 173  0.5  37    57 164  0.7  67 
4913 -- 177  --  30  4913 55 176  0.6  45 
     50  181 0.6  28    50 151  0.7  55 
5943 62 175  0.7  16  5943 93 152  1.2  40 
   74 142  11    88 163  1.1  27 
6971 62 --  --  --  6971 137 152  1.8  14 
   73  169 0.9  13  8000 71 129  1.1  11 
8000 86 89  1.9    139 216  1.3  22 
Solvent control (DMSO) 33 188  0.4    Solvent control (DMSO)  36 138  0.6   
Positive control 386 106  7.3  34  Positive control 146 110  2.6  66 
Conclusions:
Interpretation of results: Negative
In this mouse lymphoma assay, the authors reported positive responses for fumaric acid at concentrations of 8.0 and 6.97 mg/mL without, and 8.0 mg/mL with metabolic activation. This conclusion was based on criteria applicable at the time of publication, considering compounds positive if one or more dose levels with 10 % or greater total growth exhibited mutant frequencies of greater than 100 mutants per 106 of clonable cells over the background level.
Since the publication of this paper, however, the role of cytotoxicity in causing spurious positive responses has received increasing attention. More recent guidance on the interpretation of the mouse lymphoma assay is available from the European Medicines Agency (EMEA/CHMP/ICH/126642/2008). This more recent guidance was developed by the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Expert Working Group and reviewed and accepted by regulatory bodies in accordance with the ICH process. This guidance states that the maximum recommended concentration for the mouse lymphoma assay is 0.5 mg/mL and that it is not necessary to exceed 80 % reduction in relative total growth. Moreover, the recent OECD guideline for the mouse lymphoma assay (OECD test guideline 490, 29 July 2016) specifies that the maximum test concentration should not exceed either the limiting cytotoxicity, precipitation, a concentration of 2 mg/mL (or 10 mM), whichever is the lowest. In the case of this study, all test concentrations exceeded the maximum recommended concentration of 0.5 or 2 mg/mL. In addition, two of three positive responses were reported only at concentrations that exceeded the recommended 80 % reduction relative total growth. The positive response for non-activated cells was observed at a 97 % reduction and the positive responses for activated cells were seen at 86 and 78 % relative inhibition of growth. The positive response seen at 78 % relative growth inhibition could not be replicated and a dose-response relationship was not apparent. This study would not be considered to be a positive study using current ICH and/or OECD 490 criteria.
Executive summary:

In this mouse lymphoma assay, the authors reported positive responses for fumaric acid at concentrations of 8.0 and 6.97 mg/mL without, and 8.0 mg/mL with metabolic activation. This conclusion was based on criteria applicable at the time of publication, considering compounds positive if one or more dose levels with 10 % or greater total growth exhibited mutant frequencies of greater than 100 mutants per 106 of clonable cells over the background level.

Since the publication of this paper, however, the role of cytotoxicity in causing spurious positive responses has received increasing attention. More recent guidance on the interpretation of the mouse lymphoma assay is available from the European Medicines Agency (EMEA/CHMP/ICH/126642/2008). This more recent guidance was developed by the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Expert Working Group and reviewed and accepted by regulatory bodies in accordance with the ICH process. This guidance states that the maximum recommended concentration for the mouse lymphoma assay is 0.5 mg/mL and that it is not necessary to exceed 80 % reduction in relative total growth. Moreover, the recent OECD guideline for the mouse lymphoma assay (OECD test guideline 490, 29 July 2016) specifies that the maximum test concentration should not exceed either the limiting cytotoxicity, precipitation, a concentration of 2 mg/mL (or 10 mM), whichever is the lowest. In the case of this study, all test concentrations exceeded the maximum recommended concentration of 0.5 or 2 mg/mL. In addition, two of three positive responses were reported only at concentrations that exceeded the recommended 80 % reduction relative total growth. The positive response for non-activated cells was observed at a 97 % reduction and the positive responses for activated cells were seen at 86 and 78 % relative inhibition of growth. The positive response seen at 78 % relative growth inhibition could not be replicated and a dose-response relationship was not apparent. This study would not be considered to be a positive study using current ICH and/or OECD 490 criteria.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Guideline-compliant study published in a peer-reviewed journal
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
not specified
Remarks:
published study
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable - chromosome aberration study
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Chinese hamster fibroblast cell line maintained by 4-day passages in MEM supplemented by 10% calf serum
Metabolic activation:
without
Test concentrations with justification for top dose:
Three different doses with a maximum dose of 0.5 mg/mL fumaric acid, corresponding to preliminary data indicating the dose needed for 50 % cell-growth inhibition.
Vehicle / solvent:
Physiological saline
Untreated negative controls:
yes
Remarks:
Chromosome aberration test
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
not specified
Details on test system and experimental conditions:
The modal chromosome number was 25 and the doubling time was approximately 15 hr. Chinese hamster fibroblast cells were exposed to the sample at three different doses for 24 and 48 hr. No metabolic activation was applied. Colcemid was added to cultures 2 hr before harvesting. Cells were fixed and stained and the incidence of polyploidy cells and cells with structural chromosomal aberrations were recorded.
Evaluation criteria:
The results were considered to be negative if the incidence of polyploidy was less than 4.9 %, equivocal if the incidence was between 5.0 and 9.9 %, and positive if it was more than 10.0 %.
Statistics:
No statistical information available.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
Fumaric acid was tested negative for chromosomal aberrations. A dose of 0.5 mg/mL fumaric acid showed an incidence of 0.0 % polyploid cells at 48 hr and an incidence of 1.0 % of cells with structural aberrations at 24 hrs.
Conclusions:
Interpretation of results: Negative without metabolic activation
No evidence of clastogenicity was seen when fumaric acid was tested in the chromosome aberration test.
Executive summary:

Fumaric acid was negative in the chromosome aberration test, with Chinese Hamster Fibroblast cells tested up to a concentration of 0.5 mg/mL without metabolic activation.

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

Genetic toxicity in vivo

Description of key information

Since fumaric acid is negative in in-vitro genetic toxicity tests, in-vivo testing is not required.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The mutagenicity of fumaric acid was investigated using the Ames test in three independent studies comparable to OECD 471. Studies were carried out with S. typhimurium strains TA 92, TA94, TA97, TA98, TA100, TA102, TA1535, TA1537, TA1538 and TA2637 in the presence and absence of metabolic activation. Exposure to fumaric acid at concentrations up to 10000 ug/plate did not induce increased numbers of revertant colonies in any strain.

A chromosome aberration study similar to OECD 473 was carried out using a concentration of up to 0.5 mg/mL fumaric acid. The incidence of polyploid cells at 48 hr and the incidence of cells with structural aberrations at 24 hrs were determined to be 0.0% and 1.0%, respectively, for the highest dose tested.

The mutagenicity of fumaric acid was investigated using the Ames test in three independent studies comparable to OECD 471. Studies were carried out with S. typhimurium strains TA 92, TA94, TA97, TA98, TA100, TA102, TA1535, TA1537, TA1538 and TA2637 in the presence and absence of metabolic activation. Exposure to fumaric acid at concentrations up to 10000 ug/plate did not induce increased numbers of revertant colonies in any strain.

A chromosome aberration study similar to OECD 473 was carried out using a concentration of up to 0.5 mg/mL fumaric acid. The incidence of polyploid cells at 48 hr and the incidence of cells with structural aberrations at 24 hrs were determined to be 0.0% and 1.0%, respectively, for the highest dose tested.

In a mouse lymphoma assay equivalent to OECD 476 (or OECD 490, 29 July 2016), positive responses for fumaric acid were reported for fumaric acid at concentrations of 8.0 and 6.97 mg/mL without, and 8.0 mg/mL with metabolic activation. This conclusion was based on criteria applicable at the time of publication, considering compounds positive if one or more dose levels with 10 % or greater total growth exhibited mutant frequencies of greater than 100 mutants per 106 of clonable cells over the background level.

Since the publication of this paper, however, the role of cytotoxicity in causing spurious positive responses has received increasing attention. More recent guidance on the interpretation of the mouse lymphoma assay is available from the European Medicines Agency (EMEA/CHMP/ICH/126642/2008). This more recent guidance was developed by the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Expert Working Group and reviewed and accepted by regulatory bodies in accordance with the ICH process. This guidance states that the maximum recommended concentration for the mouse lymphoma assay is 0.5 mg/mL and that it is not necessary to exceed 80 % reduction in relative total growth. Moreover, the recent OECD guideline for the mouse lymphoma assay (OECD test guideline 490, 29 July 2016) specifies that the maximum test concentration should not exceed either the limiting cytotoxicity, precipitation, a concentration of 2 mg/mL (or 10 mM), whichever is the lowest. In the case of this study, all test concentrations exceeded the maximum recommended concentration of 0.5 or 2 mg/mL. In addition, two of three positive responses were reported only at concentrations that exceeded the recommended 80 % reduction relative total growth. The positive response for non-activated cells was observed at a 97 % reduction and the positive responses for activated cells were seen at 86 and 78 % relative inhibition of growth. The positive response seen at 78 % relative growth inhibition could not be replicated and a dose-response relationship was not apparent. This study would not be considered to be a positive study using current ICH and/or OECD 490 criteria.

A second mouse lymphoma study conducted in accordance with OECD guideline 476 investigated dose concentrations up to the current limit concentration. The confounding issues resulting from excessive cytotoxicity and reduction in relative total growth were not evident in this assay. No evidence of cytotoxicity or genotoxicity were observed at 1200 µg/mL, equivalent to the limit concentration of 10 mM. The positive controls were validated in the assay and all results confirmed in the replicate assay. Fumaric acid was not mutagenic, either with or without metabolic activation, up to the limit concentration in the mouse lymphoma assay.

Justification for classification or non-classification

The results of the available studies do not trigger classification according to Directive 67/548/EEC. No classification for genotoxicity is required according to Regulation 1272/2008.