N-[4-[[(2-amino-1,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid

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:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

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

Test material information

Constituent 1

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

Method

Target gene:

S. typhimurium: his operon
E. choli: trp operon

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

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 results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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. [unit]	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
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. [unit]	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
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.