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EC number: 205-181-1 | CAS number: 135-16-0
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017-09-07 to 2017-12-08
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted July 21, 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- N-[4-[[(2-amino-1,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid
- EC Number:
- 205-181-1
- EC Name:
- N-[4-[[(2-amino-1,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid
- Cas Number:
- 135-16-0
- Molecular formula:
- C19H23N7O6
- IUPAC Name:
- (2S)-2-[(4-{[(2-amino-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl)methyl]amino}phenyl)formamido]pentanedioic acid
- Test material form:
- solid
Constituent 1
Method
- Target gene:
- S. typhimurium: his operon
E. choli: trp operon
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate.
The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used:water
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 1) 4-nitro-o-phenylene-diamine 2) 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium; in agar (plate incorporation) for experiment 1; preincubation for experiment 2
- Cell density at seeding (if applicable): approx. 1E09 cells/mL
DURATION
- Preincubation period: 60 min at 37°C
- Exposure duration: at least 48h at 37°C in the dark
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: other: Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control. - Rationale for test conditions:
- According to OECD test guideline
- Evaluation criteria:
- Evaluation of Cytotoxicity
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "N" or “B”, respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
Evaluation of Mutagenicity
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and E. coli WP2 uvrA the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher as compared to the reversion rate of the solvent control. - Statistics:
- According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary. A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- experiment 1 & 2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- experiment 1& 2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Remarks:
- experiment 1& 2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Remarks:
- experiment 1 & 2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Remarks:
- experiment 1& 2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed
RANGE-FINDING/SCREENING STUDIES: The toxicity of the test item was determined with tester strains TA 98 and TA 100 in a preexperiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The test item was tested in the pre-experiment at the following concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate. Toxicity was detected at 316 µg/plate (TA 98 without met. act.) and 1000 µg/plate (TA 100 without met. act.) and at 5000 µg/plate for both strains with met. act..
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: See table 1 under "any other information on results"
- Negative (solvent/vehicle) historical control data: See table 2 under "any other information on results"
Any other information on results incl. tables
Table 1:
Historical Laboratory Control Data of the Positive Control (in 2014 - 2016) without S9 (-S9)
|
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2 uvrA |
Substance Conc./plate |
4-NOPD 10 µg |
NaN310 µg |
NaN310 µg |
4-NOPD 40 µg |
MMS 1 µL |
Mean |
430.7 |
612.1 |
792.0 |
94.5 |
524.2 |
SD |
155.5 |
220.0 |
299.5 |
22.7 |
113.5 |
Min |
141 |
132 |
38 |
35 |
208 |
Max |
1830 |
1423 |
1854 |
273 |
918 |
RSD [%] |
36.1 |
35.9 |
37.8 |
24.0 |
21.7 |
n |
971 |
1188 |
931 |
929 |
229 |
Historical Laboratory Control Data of the Positive Control (in 2014 - 2016) with S9 (+S9)
|
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2 uvrA |
Substance Conc./plate |
2-AA 2.5 µg |
2-AA 2.5 µg |
2-AA 2.5 µg |
2-AA 2.5 µg |
2-AA 10 µg |
Mean |
1880.5 |
1727.7 |
133.9 |
234.1 |
227.0 |
SD |
708.5 |
522.0 |
134.9 |
101.4 |
83.7 |
Min |
70 |
169 |
22 |
26 |
84 |
Max |
3606 |
3132 |
1954 |
682 |
451 |
RSD [%] |
37.7 |
30.2 |
100.8 |
43.3 |
36.9 |
n |
966 |
1184 |
927 |
925 |
230 |
Table 2:
Historical Laboratory Control Data of the Negative Control (in 2014 - 2016) without S9 (-S9)
|
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2 uvrA |
Mean |
24.2 |
90.7 |
13.8 |
8.2 |
44.4 |
SD |
6.7 |
15.6 |
6.7 |
2.9 |
6.9 |
Min |
11 |
49 |
4 |
3 |
32 |
Max |
58 |
155 |
41 |
35 |
66 |
RSD [%] |
27.7 |
17.2 |
48.6 |
35.3 |
15.5 |
n |
972 |
1191 |
929 |
931 |
231 |
Historical Laboratory Control Data of the Negative Control (in 2014 - 2016) with S9 (+S9)
|
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2 uvrA |
Mean |
29.0 |
96.4 |
10.5 |
8.3 |
48.3 |
SD |
6.8 |
14.1 |
4.5 |
3.1 |
8.8 |
Min |
15 |
62 |
3.0 |
3 |
30 |
Max |
59 |
160 |
38 |
36 |
78 |
RSD [%] |
23.4 |
14.6 |
42.7 |
37.4 |
18.3 |
n |
967 |
1189 |
925 |
926 |
231 |
S9: metabolic activation
Mean: mean of revertants/plate
Min.: minimum of revertants/plate
Max.: maximum of revertants/plate
SD: Standard Deviation RSD: Relative Standard Deviation
n: Number of control values
Table 3: Number of revertants per plate (mean of 3 plates) Experiment 1
|
[TA98] |
[TA100] |
[TA1535] |
[TA1537] |
[WP2uvrA] |
||||||||||
Conc. |
— MA |
+ MA |
Cytotoxic |
— MA |
+ MA |
Cytotoxic |
— MA |
+ MA |
Cytotoxic |
— MA |
+ MA |
Cytotoxic |
— MA |
+ MA |
Cytotoxic |
0* |
23 |
40 |
no |
73 |
111 |
no |
20 |
22 |
no |
32 |
28 |
no |
48 |
62 |
no |
3.16 |
33 |
34 |
no |
91 |
112 |
no |
23 |
21 |
no |
33 |
27 |
no |
50 |
54 |
no |
10.0 |
31 |
37 |
no |
90 |
106 |
no |
20 |
21 |
no |
27 |
25 |
no |
52 |
49 |
no |
31.6 |
28 |
41 |
no |
78 |
109 |
no |
22 |
17 |
no |
30 |
31 |
no |
54 |
51 |
no |
100.0 |
33 |
30 |
no |
76 |
94 |
no |
21 |
23 |
no |
25 |
26 |
no |
59 |
46 |
no |
316 |
37 |
30 |
yes |
46 |
114 |
no |
21 |
23 |
no |
30 |
28 |
no |
64 |
59 |
no |
1000.0 |
4 |
24 |
yes |
2 |
104 |
yes |
3 |
16 |
yes |
23 |
27 |
no |
48 |
35 |
no |
2500 |
2 |
50 |
yes |
0 |
151 |
yes |
0 |
18 |
yes |
10 |
8 |
yes |
52 |
29 |
yes |
5000 |
0 |
28 |
yes |
0 |
133 |
yes |
0 |
1 |
yes |
0 |
0 |
yes |
15 |
9 |
yes |
Positive control |
1132 |
2958 |
|
604 |
2223 |
|
930 |
317 |
|
167 |
231 |
|
596 |
171 |
|
*solvent control with .water
Table 4: Number of revertants per plate (mean of ... plates) Experiment 2
|
[TA98] |
[TA100] |
[TA1535] |
[TA1537] |
[WP2uvrA] |
||||||||||
Conc. |
— MA |
+ MA |
Cytotoxic |
— MA |
+ MA |
Cytotoxic |
— MA |
+ MA |
Cytotoxic |
— MA |
+ MA |
Cytotoxic |
— MA |
+ MA |
Cytotoxic |
0* |
24 |
26 |
no |
99 |
82 |
no |
13 |
13 |
no |
21 |
29 |
no |
60 |
60 |
no |
3.16 |
22 |
29 |
no |
113 |
89 |
no |
14 |
12 |
no |
29 |
31 |
no |
53 |
55 |
no |
10.0 |
28 |
30 |
no |
112 |
81 |
no |
10 |
16 |
no |
29 |
28 |
no |
64 |
63 |
no |
31.6 |
22 |
25 |
no |
102 |
100 |
no |
11 |
13 |
no |
29 |
30 |
no |
58 |
65 |
no |
100.0 |
28 |
23 |
no |
109 |
75 |
no |
11 |
15 |
no |
26 |
26 |
no |
58 |
67 |
no |
316 |
20 |
31 |
no |
104 |
99 |
no |
10 |
13 |
no |
29 |
23 |
no |
60 |
64 |
no |
1000.0 |
11 |
27 |
yes |
63 |
81 |
no |
5 |
6 |
yes |
28 |
26 |
no |
56 |
55 |
no |
2500 |
3 |
32 |
yes |
17 |
133 |
yes |
1 |
8 |
yes |
26 |
28 |
no |
63 |
51 |
no |
5000 |
0 |
6 |
yes |
0 |
42 |
yes |
0 |
4 |
yes |
0 |
0 |
yes |
5 |
22 |
yes |
Positive control |
347 |
978 |
|
474 |
757 |
|
934 |
188 |
|
207 |
110 |
|
653 |
150 |
|
*solvent control with .water
Applicant's summary and conclusion
- Conclusions:
- The present study was conducted according to OECD guideline 471 (1997). Four Salmonella thyphimurium strains and one Escherichia coli strain were incubated with Tetrahydrofolic acid at the following concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate for at least 48h at 37°C. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Tetrahydrofolic acid did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, Tetrahydrofolic acid is considered to be non-mutagenic in this bacterial reverse mutation assay.
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