3-[(4-aminobenzoyl)amino]propanoic 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:

30 April - 28 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-[(4-aminobenzoyl)amino]propanoic acid

EC Number:

616-017-7

Cas Number:

7377-08-4

Molecular formula:

C10 H12 N2 O3

IUPAC Name:

3-[(4-aminobenzoyl)amino]propanoic acid

Specific details on test material used for the study:

Batch: 12/18

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: MOLTOX, INC., NC 28607, USA (for TA98, TA1535 and TA102) and Xenometrix AG, Switzerland (for TA100 and TA1537)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

31.6, 100, 316, 1000, 2500 and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

A.dest

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation

Controls 2

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

Without metabloic activation

Controls 3

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation

Controls 4

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:
plate incorporation test - EXPERIMENT I; pre-incubation test - EXPERIMENT II

For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
- 100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
- 500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
- 100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
- 2000 µL Overlay agar.

For the pre-incubation method 100 µL of the test item preparation was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
For each strain and dose level, including the controls, three plates were used.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

DURATION
- Preincubation period: 60 minutes at 37°C
- Exposure duration: at least 48 hours at 37°C in the dark

NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity can be detected by a clearing or 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:

The toxicity of the test item was determined with tester strains TA98 and TA100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test).
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.
The test item was tested in the pre-experiment with the following concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Evaluation criteria:

A test item is considered 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 trater strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and TA102 the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS: See Tables 1 and 2

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II.
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with 4-Aminobenzoyl-b-alanine at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.
All criteria of validity were met

Any other information on results incl. tables

Table 1: Results Experiment I (plate incorporation test):

Treatment	Dose/plate	Revertant colonies per plate				Mutation factor
		Without S9		With S9
		Mean	SD	Mean	SD	-S9	+S9
TA98
Water	-	23	2.1	23	2.1	1.0	1.0
DMSO	-	24	2.5	24	1.5	1.0	1.0
Test item	31.6 µg	23	0.6	23	2.5	1.0	1.0
Test item	100 µg	23	2.0	23	1.0	1.0	1.0
Test item	316 µg	24	1.5	24	3.5	1.0	1.0
Test item	1000 µg	25	3.0	24	2.3	1.1	1.0
Test item	2500 µg	26	2.6	23	2.5	1.1	1.0
Test item	5000 µg	25	3.6	24	1.5	1.1	1.0
4-NOPD	10 µg	280	8.1	-	-	11.8	-
2-AA	2.5 µg	-	-	280	8.5	-	11.8
TA100
Water	-	82	1.5	82	3.6	1.0	1.0
DMSO	-	83	2.6	84	4.6	1.0	1.0
Test item	31.6 µg	84	4.6	84	2.5	1.0	1.0
Test item	100 µg	84	2.0	82	2.9	1.0	1.0
Test item	316 µg	83	3.1	85	2.6	1.0	1.0
Test item	1000 µg	84	2.5	83	5.2	1.0	1.0
Test item	2500 µg	86	1.7	84	10.0	1.0	1.0
Test item	5000 µg	89	2.1	85	6.2	1.1	1.0
NaN3	10 µg	457	64.4	-	-	5.5	-
2-AA	2.5 µg	-	-	480	61.5	-	5.7
TA1535
Water	-	21	1.7	21	3.1	1.0	1.0
DMSO	-	21	2.1	20	0.6	1.0	1.0
Test item	31.6 µg	21	2.0	21	3.1	1.0	1.0
Test item	100 µg	21	1.0	21	2.0	1.0	1.0
Test item	316 µg	23	4.4	21	1.5	1.1	1.0
Test item	1000 µg	23	2.5	22	1.2	1.1	1.1
Test item	2500 µg	23	2.1	23	2.9	1.1	1.1
Test item	5000 µg	24	3.8	23	4.2	1.1	1.1
NaN3	10 µg	272	24.5	-	-	12.8	-
2-AA	2.5 µg	-	-	246	14.5	-	12.1
TA1537
Water	-	20	2.9	20	4.2	1.0	1.0
DMSO	-	21	3.5	20	1.5	1.0	1.0
Test item	31.6 µg	21	2.5	21	3.5	1.0	1.0
Test item	100 µg	20	3.5	23	4.5	1.0	1.1
Test item	316 µg	20	2.5	22	5.0	1.0	1.1
Test item	1000 µg	21	3.5	23	2.3	1.0	1.1
Test item	2500 µg	21	2.5	24	4.5	1.0	1.2
Test item	5000 µg	22	3.5	25	3.8	1.0	1.2
4-NOPD	40 µg	144	12.9	-	-	6.8	-
2-AA	2.5 µg	-	-	226	21.7	-	11.1
TA102
Water	-	238	7.9	294	5.0	1.0	1.0
DMSO	-	240	8.6	298	6.2	1.0	1.0
Test item	31.6 µg	240	2.5	295	5.3	1.0	1.0
Test item	100 µg	240	4.0	297	5.7	1.0	1.0
Test item	316 µg	241	10.0	295	11.3	1.0	1.0
Test item	1000 µg	238	3.2	302	4.2	1.0	1.0
Test item	2500 µg	241	7.2	307	4.6	1.0	1.0
Test item	5000 µg	243	3.1	309	5.0	1.0	1.0
MMS	1 µL	1810	99.2	-	-	7.6	-
2-AA	10 µg	-	-	1455	379.3	-	4.9

 

Table 2: Results Experiment II (pre-incubation test):

Treatment	Dose/plate	Revertant colonies per plate				Mutation factor
		Without S9		With S9
		Mean	SD	Mean	SD	-S9	+S9
TA98
Water	-	24	1.0	24	2.3	1.0	1.0
DMSO	-	23	4.0	23	1.5	1.0	1.0
Test item	31.6 µg	23	2.5	23	3.1	1.0	1.0
Test item	100 µg	24	3.5	23	1.5	1.1	1.0
Test item	316 µg	22	2.6	25	2.5	1.0	1.1
Test item	1000 µg	24	1.5	25	1.5	1.0	1.1
Test item	2500 µg	25	2.0	23	1.2	1.1	1.0
Test item	5000 µg	24	1.0	25	4.6	1.0	1.1
4-NOPD	10 µg	225	13.7	-	-	9.8	-
2-AA	2.5 µg	-	-	208	95.6	-	9.2
TA100
Water	-	83	4.9	83	3.6	1.0	1.0
DMSO	-	82	5.7	81	10.7	1.0	1.0
Test item	31.6 µg	82	3.1	81	4.5	1.0	1.0
Test item	100 µg	82	4.4	81	6.7	1.0	1.0
Test item	316 µg	85	4.6	83	1.2	1.0	1.0
Test item	1000 µg	85	4.6	82	1.5	1.0	1.0
Test item	2500 µg	86	6.7	85	8.1	1.0	1.0
Test item	5000 µg	85	7.2	86	4.2	1.0	1.1
NaN3	10 µg	1302	68.6	-	-	15.8	-
2-AA	2.5 µg	-	-	343	20.8	-	4.2
TA1535
Water	-	22	2.1	20	2.0	1.0	0.9
DMSO	-	22	2.9	22	3.1	1.0	1.0
Test item	31.6 µg	21	1.7	22	1.7	0.9	1.0
Test item	100 µg	22	5.7	21	3.8	1.0	1.0
Test item	316 µg	21	1.2	22	3.8	0.9	1.0
Test item	1000 µg	23	2.6	20	1.5	1.0	0.9
Test item	2500 µg	24	3.2	23	5.5	1.1	1.0
Test item	5000 µg	24	4.0	23	5.6	1.1	1.1
NaN3	10 µg	245	7.9	-	-	11.0	-
2-AA	2.5 µg	-	-	249	17.7	-	11.5
TA1537
Water	-	20	2.9	20	0.6	1.0	1.0
DMSO	-	21	2.1	21	3.5	1.0	1.0
Test item	31.6 µg	21	1.0	21	5.0	1.0	1.0
Test item	100 µg	20	3.6	20	1.5	1.0	1.0
Test item	316 µg	21	2.5	22	1.2	1.0	1.1
Test item	1000 µg	21	4.2	23	4.7	1.0	1.1
Test item	2500 µg	22	2.0	22	4.4	1.1	1.1
Test item	5000 µg	23	1.2	22	1.5	1.1	1.1
4-NOPD	40 µg	203	8.7	-	-	9.8	-
2-AA	2.5 µg	-	-	179	11.5	-	8.6
TA102
Water	-	239	8.7	302	5.6	1.0	1.0
DMSO	-	241	1.0	301	6.0	1.0	1.0
Test item	31.6 µg	240	2.6	299	6.0	1.0	1.0
Test item	100 µg	242	9.6	301	2.5	1.0	1.0
Test item	316 µg	241	13.6	303	3.2	1.0	1.0
Test item	1000 µg	246	5.0	304	3.5	1.0	1.0
Test item	2500 µg	244	4.6	304	9.8	1.0	1.0
Test item	5000 µg	245	4.0	304	3.1	1.0	1.0
MMS	1 µL	1290	67.7	-	-	5.4	-
2-AA	10 µg	-	-	1188	48.6	-	3.9

 

Applicant's summary and conclusion

Conclusions:

4-Aminobenzoyl-b-alanine did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, 4-Aminobenzoyl-b-alanine is considered to be non-mutagenic in this bacterial reverse mutation assay.