Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The following OECD Guideline in vitro genotoxicity studies have been performed:

1). Bacterial reverse mutation test

2). In vitro mammalian cell gene mutation test – Mouse lymphoma assay

3). In vitro micronucleus test

The results of these tests were considered valid and all results were negative and no adverse effects were observed. It can therefore be concluded that 4-aminobenzoyl-b-alanine is not mutagenic.

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Batch: 12/18
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.
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
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
Without metabloic activation
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation
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.
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
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
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
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
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

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

 

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.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 May - 12 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Batch no: 12/18
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Cell bank of Eurofins Munich
- Suitability of cells: V79 cells in vitro have been widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens.
- Methods for maintenance in cell culture if applicable: For the experiments thawed cultures were set up in 75 cm2 cell culture plastic flasks at 37 °C in a 5% carbon dioxide atmosphere (95% air). 5 x 10^5 cells per flask were seeded in 15 mL of MEM (minimum essential medium) supplemented with 10% FBS (fetal bovine serum) and subcultures were made every 3-4 days.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Minimum essential medium (MEM) supplemented with 10% FBS (fetal bovine serum)
- Properly maintained: yes - cells were stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins Munich
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Cytochalasin B (1.5 µg/mL)
Metabolic activation:
with
Metabolic activation system:
Mammalian Microsomal Fraction S9 Homogenate
Test concentrations with justification for top dose:
Experiment I: without and with metabolic activation: 125, 250, 500, 1000, 1500 and 2000 μg/mL

Experiment II: without metabolic activation: 50, 100, 250, 500, 1000, 1500 and 2000 μg/mL

The following concentrations were selected for the microscopic analyses of micronuclei frequencies:
Experiment I with short-term exposure (4 h): without and with metabolic activation: 1000, 1500 and 2000 μg/mL
Experiment II with long-term exposure (24 h): without metabolic activation: 1000, 1500 and 2000 μg/mL

The selection of the concentrations was based on data from the pre-experiment. In the main experiments without and with metabolic activation 2000 μg/mL test item was selected as the highest concentration.
Vehicle / solvent:
- Vehicle/solvent used: Dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 2 mg/mL. Due to the nature of the test item it was not possible to prepare a solution of the test item with cell culture medium.
Untreated negative controls:
yes
Remarks:
Cell culture medium
Negative solvent / vehicle controls:
yes
Remarks:
Cell culture medium with 1% DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
other: Colchicine
Details on test system and experimental conditions:
SEEDING OF THE CULTURES
Three or four day-old stock cultures (in exponential growth), more than 50% confluent, were rinsed with Ca-Mg-free PBS solution prior to the trypsin treatment. Cells subsequently were trypsinised with a solution of 0.2% trypsin in Ca-Mg-free PBS at 37°C for 5 min. By adding complete culture medium the detachment was stopped and a single cell suspension was prepared.

EXPERIMENT I
Exponentially growing V79 cells were seeded into 25 cm2 cell culture flasks (two flasks per test group). Approx. 50 000 cells were seeded per cell culture flask, containing 5 mL complete culture medium (minimum essential medium supplemented with 10% FBS). After an attachment period of approx. 48 h, the complete culture medium was removed and subsequently the test item was added to the treatment medium in appropriate concentrations. The cells were incubated with the test item for 4 h in presence or absence of metabolic activation. At the end of the incubation, the treatment medium was removed and the cells were washed twice with PBS. Subsequently, the cells were incubated in complete culture medium + 1.5 μg/mL cytochalasin B for 20 h at 37 °C.

EXPERIMENT II
If negative or equivocal results are obtained, they were confirmed using continuous treatment (long-term treatment) without metabolic activation. Approx. 50 000 exponentially growing V79 cells were seeded in 25 cm2 cell culture flasks in absence of metabolic activation. After an attachment period of approx. 48 h the test item was added in complete culture medium. 1 h later 1.5 μg/mL cytochalasin B were added and the cells were incubated for 23 h at 37 °C. At the end of the treatment the cell culture medium was removed and the cells were prepared for microscopic analysis.

NUMBER OF CULTURES
Duplicate cultures were performed at each concentration level except for the pre-experiment.

PREPARATION OF THE CULTURES
At the end of the cultivation, the complete culture medium was removed. Subsequently, cells were trypsinated and resuspended in about 9 mL complete culture medium. The cultures were transferred into tubes and incubated with hypotonic solution (0.4% KCl) for some minutes at room temperature.
Prior to this an aliquot of each culture was removed to determine the cell count by a cell counter (ALSystems). After the treatment with the hypotonic solution the cells were fixed with methanol + glacial acetic acid (3+1). The cells were resuspended gently and the suspension was dropped onto clean glass slides. Consecutively, the cells were dried on a heating plate. Finally, the cells were stained with acridine orange solution.

ANALYSIS OF MICRONUCLEI
All slides, including those of positive and negative controls were independently coded before microscopic analysis. For each experimental point, at least 2000 binucleated cells per concentration (1000 binucleated cells per slide) were analysed for micronuclei according to the criteria of Fenech i.e. clearly surrounded by a nuclear membrane, having an area of less than one-third of that of the main nucleus, being located within the cytoplasm of the cell and not linked to the main nucleus via nucleoplasmic bridges. Mononucleated and multinucleated cells and cells with more than six micronuclei were not considered.

ACCEPTABILITY OF THE ASSAY
A mutation assay is considered acceptable if it meets the following criteria:
- The concurrent negative/solvent control is considered acceptable for addition to the laboratory historical negative/solvent control database.
- Concurrent positive controls should induce responses that are compatible with those generated in the laboratory’s historical positive control data base and produce a statistically significant increase compared with the concurrent negative/solvent control.
- Cell proliferation criteria in the negative/solvent control according to OECD 487 should be fulfilled.
- All experimental conditions are tested unless one resulted in positive results.
- Adequate number of cells and concentrations are analysable.
- Criteria for the selection of top concentration are fulfilled.
Evaluation criteria:
A test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- the increase is concentration-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical negative/solvent control data (e.g. Poisson-based 95% control limits).

When all of these criteria are met, the test item is considered able to induce chromosome breaks and/ or gain or loss in this test system.

A test item is considered to be clearly negative if in all experimental conditions examined none of the criteria mentioned above are met.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRE-EXPERIMENT FOR TOXICITY
According to the used guideline the highest recommended concentration is 2000 μg/mL. The test item was dissolved in DMSO. No precipitate of the test item was noted. The highest dose group evaluated in the pre-experiment was 2000 μg/mL. The cytokinesis block proliferation index (CBPI) was used to calculate the cytostasis (cytostasis [%] = 100 - CBPI relative [%]). Cytostasis was used to describe cytotoxicity. The concentrations evaluated in the main experiment were based on the results obtained in the pre-experiment.

CYTOTOXICITY
In experiment I and II with and without metabolic activation no increase of the cytostasis above 30 % was noted.

CLASTOGENICITY / ANEUGENICITY
In experiment I without metabolic activation the mean values of micronucleated cells found after treatment with the test item were 0.80% (1000 µg/mL) and 0.70% (1500 and 2000 µg/mL). The numbers of micronucleated cells were within the historical control limits of the negative and solvent control and did not show a biologically relevant increase compared to the concurrent solvent control.

In experiment I with metabolic activation the mean values of micronucleated cells found after treatment with the test item were 0.60% (1000 µg/mL), 0.85% (1500 µg/mL) and 1.28% (2000 µg/mL). The numbers of micronucleated cells were within the historical control limits of the negative and solvent control and did not show a biologically relevant increase compared to the concurrent solvent control.

In experiment II without metabolic activation the mean values of micronucleated cells found after treatment with the test item were 1.10% (1000 µg/mL), 0.80% (1500 µg/mL) and 1.20% (2000 µg/mL). The numbers of micronucleated cells were within the historical control limits of the negative and solvent control and did not show a biologically relevant increase compared to the concurrent solvent control.

The nonparametric χ² Test was performed to verify the results in both experiments. No statistically significant increase (p< 0.05) of cells with micronuclei was noted in the dose groups of the test item evaluated in experiment I and II without metabolic activation. In experiment I with metabolic activation a statistically significant increase of cells with micronuclei (p = 0.0048) was observed for the test item concentration of 2000 µg/mL. Since the corresponding value of the micronucleus frequency was within the historical control limits of the negative and solvent control the observed increase was considered as not biologically relevant.

The χ² Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions. No statistically significant increase in the frequency of micronucleated cells under the experimental conditions of the study was observed in experiment I and II without metabolic activation. A statistically significant increase in the frequency of micronucleated cells was observed in experiment I with metabolic activation. Since all corresponding values of micronuclei frequencies were within the historical control limits of the negative and solvent control, the increase was considered as not biologically relevant.

MMS (25 µg/mL) and CPA (2.5 µg/mL) were used as clastogenic controls and colchicine as aneugenic controls (0.16 and 2.0 µg/mL). They induced distinct and statistically significant increases of the micronucleus frequency. This demonstrates the validity of the assay.

Table 1: Main Experiment I - CBPI: 4 h treatment (without and with metabolic activation), 24 h fixation period

Dose Group

Concentration

(µg/mL)

CBPI

1/2

CBPI

2/2

Relative cell growth

(%)

Cytostasis

(%)

Precipitate

+/-

Without metabolic activation

C

0

1.58

1.64

98

2

-

S

0

1.61

1.63

100

0

 

4

1000

1.59

1.60

96

4

-

5

1500

1.57

1.54

89

11

-

6

2000

1.61

1.63

99

1

-

MMS

25

1.55

1.52

86

14

-

Colchicine

2.0

1.55

1.54

88

12

-

With metabolic activation

C

0

1.56

1.56

94

6

-

S

0

1.59

1.60

100

0

 

4

1000

1.56

1.54

93

7

-

5

1500

1.55

1.53

91

9

-

6

2000

1.56

1.57

95

5

-

CPA

5.0

1.42

1.37

67

33

-

The CBPI was determined in 500 cells per culture of each test group.

The relative values of the CBPI are related to the negative control.

C:                         Negative Control (Culture medium)

S:                          Solvent Control (DMSO 1% v/v in culture medium)

MMS:                  Methylmethanesulfonate, Positive Control (without metabolic activation)

Colchicine:         Positive Control (without metabolic activation)

CPA:                     Cyclophosphamide, Positive Control (with metabolic activation)

CBPI:                   Cytokinesis Block Proliferation Index, CBPI = ((c1 x 1) + (c2 x 2) + (cx x 3))/n

Relative Cell Growth:      100 x ((CBPI Test conc – 1) / (CBPI control -1))

c1: mononucleate cells

c2: binucleate cells

cx: multinucleate cells

n: total number of cells

CBPI ½:                             Slide 1 of 2

CBPI 2/2:            Slide 2 of 2

Cytostasis [%] = 100- Relative Cell Growth [%]; the cytostasis is defined 0, when the relative cell growth exceeds 100%

 

 

Table 2: Main Experiment I - Micronucleus induction in V79 cells, 4 h treatment, 24 h fixation interval, without metabolic activation

Dose group

Concentration

(µg/mL)

Scored binucleated cells

(total of 2 cultures)

Micronuclei

(total of 2 cultures)

Micronucleated cells frequency

(%, average)

C

0

2000

16

0.80

S

0

4000

27

0.68

4

1000

2000

16

0.80

5

1500

2000

14

0.70

6

2000

2000

14

0.70

MMS

25

3000

66

2.03

Colchicine

2.0

2000

32

1.60

The micronucleated cell frequency was determined where possible in 1000 binucleated cells in each of the two separate cultures per test group. In case of significant difference between both slides (generally factor > 2) additional 1000 binucleated cells of the same concentration were screened to verify this analysis (not considered necessary for the negative control and dose group 4). Only the final count of all analyzed binucleated cells per culture and concentration is given in this table.

 

Table 3: Main Experiment I - Micronucleus induction in V79 cells, 4 h treatment, 24 h fixation interval, with metabolic activation

Dose group

Concentration

(µg/mL)

Scored binucleated cells

(total of 2 cultures)

Micronuclei

(total of 2 cultures)

Micronucleated cells frequency

(%, average)

C

0

2000

11

0.55

S

0

2000

10

0.50

4

1000

2000

12

0.60

5

1500

2000

17

0.85

6

2000

4000

51

1.28

CPA

2.5

2000

58

2.90

The micronucleated cell frequency was determined where possible in 1000 binucleated cells in each of the two separate cultures per test group. In case of significant difference between both slides (generally factor >2) additional 1000 binucleated cells of the same concentration were screened to verify this analysis (not considered necessary for the negative and solvent control). Only the final count of all analyzed binucleated cells per culture and concentration is given in this table.

 

Table 4: Main Experiment II - CBPI: 24 h treatment (without metabolic activation)

Dose Group

Concentration

(µg/mL)

CBPI

1/2

CBPI

2/2

Relative cell growth

(%)

Cytostasis

(%)

Precipitate

+/-

Without metabolic activation

C

0

1.55

1.60

109

0

-

S

0

1.61

1.45

100

0

 

5

1000

1.53

1.42

90

10

-

6

1500

1.53

1.55

102

0

-

7

2000

1.52

1.53

99

1

-

MMS

25

1.45

1.45

85

15

-

Colchicine

0.16

1.36

1.33

65

35

-

 

Table 5: Main Experiment II – Micronucleus induction in V79 cells, 24 h treatment, without metabolic activation

Dose group

Concentration

(µg/mL)

Scored binucleated cells

(total of 2 cultures)

Micronuclei

(total of 2 cultures)

Micronucleated cells frequency

(%, average)

C

0

2000

27

1.35

S

0

2000

20

1.00

5

1000

2000

22

1.10

6

1500

2000

16

0.80

7

2000

2000

24

1.20

MMS

25

2000

105

5.25

Colchicine

0.16

2000

78

3.90

 

Table 6: Summary of Micronuclei Effects: Experiment I without and with metabolic activation

Dose group

Concentration

(µg/mL)

Treatment time

(h)

Fixation interval

(h)

Micronucleated Cells Frequency

(%)

Without metabolic activation

C

0

4

24

0.80

S

0

4

24

0.68

4

1000

4

24

0.80

5

1500

4

24

0.70

6

2000

4

24

0.70

MMS

25

4

24

2.03

Colchicine

2.0

4

24

1.60

With metabolic activation

C

0

4

24

0.55

S

0

4

24

0.50

4

1000

4

24

0.60

5

1500

4

24

0.85

6

2000

4

24

1.28

CPA

2.5

4

24

2.90

 

Table 7: Summary of Micronuclei Effects: Experiment II without metabolic activation

Dose group

Concentration

(µg/mL)

Treatment time

(h)

Fixation interval

(h)

Micronucleated Cells Frequency

(%)

Without metabolic activation

C

0

24

24

1.35

S

0

24

24

1.00

4

1000

24

24

1.10

6

1500

24

24

0.80

7

2000

24

24

1.20

MMS

25

24

24

5.25

Colchicine

0.16

24

24

3.90

 

 

Table 8: Biometry – Trend test

Statistical significance at the 5% level (p < 0.05) was evaluated by the χ² test for trend. The p value was used as a limit in judging for significance levels.

Experiment

Treatment time

(h)

Significance

P value

Exp. I without metabolic activation

4

-

0.7109

Exp. I with metabolic activation

4

+

0.0100

Exp. II without metabolic activation

24

-

0.7545

+:  significant

-: not significant

Statistical significance: statistical significant concentration-related increase in micronucleated cells frequency (χ² test for trend, p < 0.05)

Conclusions:
In conclusion, it can be stated that during the study described and under the experimental conditions reported, 4-Aminobenzoyl-b-alanine did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells.
Therefore, 4-Aminobenzoyl-b-alanine is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Assay.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 September - 30 October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
Batch no: 12/18
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Large stock cultures of the cleansed L5178Y cell line are stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins Munich
- Suitability of cells: Mouse Lymphoma L5178Y cells (clone TK+/- -3.7.2C) have been used successfully in in vitro experiments for many years. These cells are characterised by their high proliferation rate (10 - 12 h doubling time of the Eurofins Munich stock cultures) and their cloning efficiency, usually more than 50%. The cells obtain a near diploid karyotype (40 ± 2 chromosomes).
They are heterozygous at the Thymidine Kinase (TK) locus in order to detect mutation events at the TK- locus.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
The selection of the concentrations used in the main experiment was based on data from the preexperiment. 2000 µg/mL (without and with metabolic activation) was selected as the highest concentrations. The experiment without and with metabolic activation was performed as 4 h short-term exposure assay.
The test item was investigated at the following concentrations:

without metabolic activation:
50, 100, 250, 500, 1000, 1500 and 2000 µg/mL

and with metabolic activation:
50, 100, 250, 500, 1000, 1500 and 2000 µg/mL
Vehicle / solvent:
- Vehicle/solvent used: DMSO;
- Justification for choice of solvent/vehicle: Based on the pre-experiment for solubility
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
1% DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
methylmethanesulfonate
Details on test system and experimental conditions:
CELLS:
Mouse Lymphoma L5178Y cells (clone TK+/- -3.7.2C) have been used successfully in in vitro experiments for many years. These cells are characterised by their high proliferation rate (10 - 12 h doubling time of the Eurofins Munich stock cultures) and their cloning efficiency, usually more than 50%. The cells obtain a near diploid karyotype (40 ± 2 chromosomes). They are heterozygous at the Thymidine Kinase (TK) locus in order to detect mutation events at the TK- locus.
To prevent high backgrounds arising from spontaneous mutation, cells lacking TK can be eliminated by culturing cells in RPMI 1640 supplemented with:
9.0 µg/mL hypoxanthine
15.0 µg/mL thymidine
22.5 µg/mL glycine
0.1 µg/mL methotrexate
The cells are resuspended in medium without methotrexate but with thymidine, hypoxanthine and glycine for 1 - 3 days.
Large stock cultures of the cleansed L5178Y cell line are stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins Munich. This allows the repeated use of the same cell batch in experiments.
Each cell batch is routinely checked for mycoplasma infection.
Thawed stock cultures are maintained in plastic culture flasks in RPMI 1640 complete medium and subcultured three times per week.

MAMMALIAN MICROSOMAL FRACTION S9 HOMOGENATE
An advantage of using in vitro cell cultures is the accurate control of concentration and exposure time of cells to the test item under study. However, due to the limited capacity of cells growing in vitro for metabolic activation of potential mutagens, an exogenous metabolic activation system is necessary. Many substances only develop mutagenic potential when they are metabolized by the mammalian organism. Metabolic activation of substances can be achieved by supplementing the cell cultures with liver microsome preparations (S9 mix). Since the L5178Y cells used in this assay do not have the cytochrome-based P450 metabolic oxidation system, an exogenous metabolic activation system, the S9 microsomal fraction was added. The cells were exposed to the test substance both, in the presence and the absence of metabolic activation.
The S9 liver microsomal fraction was prepared at Eurofins Munich. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and ẞ-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route. The preparation was performed according to Ames et al.
The following quality control determinations were performed:
a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene
b) Sterility Test
A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4 mL and stored
at ≤ -75 °C.
The protein concentration in the S9 preparation (Lot: 280418) was 38 mg/mL

S9 MIX:
The toxicity of the test item was determined in a pre-experiment up to a maximum concentration of 2 mg/mL. Six concentrations [50, 150, 250, 500, 1000 and 2000 µg/mL] were tested without and with metabolic activation. The experimental conditions in these pre-experiment were the same as described below in the paragraph experimental performance. After a 2-day growth period the relative suspension growth (RSG) of the treated cell cultures was calculated according to the method of Clive and Spector.

EXPOSURE CONCENTRATIONS:
The selection of the concentrations used in the main experiment was based on data from the pre-experiment. 2000 µg/mL (without and with metabolic activation) was selected as the highest concentrations. The experiment without and with metabolic activation was performed as 4 h short-term exposure assay.
The test item was investigated at the following concentrations:

without metabolic activation:
50, 100, 250, 500, 1000, 1500 and 2000 µg/mL

and with metabolic activation:
50, 100, 250, 500, 1000, 1500 and 2000 µg/mL
According to OECD Guidelines at least 4 concentrations of the test item were set up in the experiments without and with metabolic activation.

EXPERIMENTAL PERFORMANCE
For a short-term exposure experiment 1 x 10^7cells were suspended in 11 mL RPMI medium with 5% horse serum (25 cm2 flasks) and exposed to designated concentrations of the test item either in the presence or absence of metabolic activation in the mutation experiment. After 4 h the test item was removed by centrifugation (200 x g, 7 min) and the cells were washed twice with PBS. Subsequently the cells were suspended in 30 mL complete culture medium and incubated for an expression and growth period of 2 days in total at 37 °C in 5% CO2/95% humidified air. The cell density was determined each day and adjusted to 3 x 10^5 cells/mL in a total culture volume of 20 mL, if necessary.

After the expression period the cloning efficiency (CE) of the cells was determined by seeding a statistical number of 1.6 cells/well in two 96-well plates. The cells were incubated for at least 7 days at 37 °C in a humidified atmosphere with 5% CO2. Analysis of the results was based on the number of cultures with cell growth (positive wells) and those without cell growth (negative wells) compared to the total number of cultures seeded. Additionally, cultures were seeded in selective medium. Cells from each experimental group were seeded in four 96-well plates at a density of approximately 2000 cells/well in 200 µL selective medium with TFT. The plates were scored after an incubation period of about 12 days at 37 °C in 5% CO2/95% humidified air.

The mutant frequency was calculated by dividing the number of TFT resistant colonies by the number of cells plated for selection, corrected for the plating efficiency of cells from the same culture grown in the absence of TFT. For the microwell method used here the Poisson distribution was used to calculate the plating efficiencies for cells cloned without and with TFT selection. Based on the null hypothesis of the Poisson distribution, the probable number of clones/well (P) is equal to –ln(negative wells/total wells) and the plating efficiency (PE) equals P/(number of cells plated per well). Mutant frequency then was calculated as MF = (PE(cultures in selective medium)/PE(cultures in nonselective medium)). The mutant frequency is usually expressed as “mutants per 106viable cells.

Suspension growth (SG) of the cell cultures reflects the number of times the cell number increases from the starting cell density. When carrying out a short-term treatment (4 h) a 2-day growth period was considered. The relative total growth (RTG) is the product of the relative suspension growth (RSG; calculated by comparing the SG of the dose groups with the SG of the control) and the relative cloning efficiency (RCE) for each culture: RTG = RSG x RCE /100.

The mutant frequencies obtained from the experiments were compared with the Global Evaluation Factor (GEF). To arrive at a GEF, the workgroup (IWGT MLA Workgroup;analyzed distributions of negative/vehicle mutant frequencies of the MLA that they gathered from ten laboratories. The GEF is defined as the mean of the negative/vehicle mutant frequency plus one standard deviation. Applying this definition to the collected data, the GEF arrived to be 126 for the microwell method.
Evaluation criteria:
The test item is considered mutagenic if the following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 106 cells and
- a dose-dependent increase in mutant frequency is detected.

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
Statistical methods might be used as an aid in evaluation of the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative.
Statistics:
The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the solvent/negative controls were used as reference.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRECIPITATION
No precipitation of the test item was noted in the experiments.

TOXICITY
No growth inhibition was observed in the experiment without and with metabolic activation.

MUTAGENICITY
In the main experiment without and with metabolic activation all validity criteria were met. The negative and solvent controls showed mutant frequencies within the acceptance range of 50 - 170 mutants/10^6 cells, according to the IWGT criteria.
The mutant frequencies induced by the test item did not show any biologically relevant increase. The GEF of 126 was not exceeded in any of the dose groups. A statistical analysis displayed that some of the mutant frequencies were significantly increased over those of the solvent controls, but because the corresponding IMF was clearly lower than the GEF of 126, this effect was considered as not biologically relevant.
All mutant frequencies for negative, solvent and positive controls were found within the historical range of the test facility Eurofins Munich.

CLASTOGENICITY
Colony sizing was performed for the highest concentrations of the test item and for the negative and positive controls. An extension of the GEF by the induced mutant frequency in combination with an increased occurrence of small colonies (defined by slow growth and/or morphological alteration of the cell clone) is an indication for potential clastogenic effects and/or chromosomal aberrations. Thus based on the non-mutagenic effects of 4-Aminobenzoyl-b-alanine, an assessment of clastogenicity was not feasible.
The positive controls MMS and B[a]P induced a significant increase in mutant frequency and a biologically significant increase of small colonies (≥40%), thus proving the ability of the test system to indicate potential clastogenic effects.

Table1:  Summary: Main Experiment,withoutandwithmetabolic activation

 

Test Group

Conc.

[µg/mL]

RCEa[%]

RTGb[%]

MFc[mutants/ 106cells]

IMFd[mutants/ 106cells]

GEFeexceeded

Statistical

Significant Increasef

Precipitate

Exp

without

S9

 

C1

0

116.2

117.5

99.2

/

/

-

-

C2

96.0

100.9

/

/

-

S1

0

100.0

100.0

93.6

/

/

/

-

S2

/

/

/

-

2

50

87.3

97.3

127.3

33.8

-

-

-

3

100

92.9

90.9

128.0

34.4

-

+

-

4

250

97.5

107.2

105.8

12.3

-

-

-

5

500

96.0

91.2

107.0

13.4

-

-

-

6

1000

97.5

92.1

109.6

16.0

-

-

-

7

1500

97.5

85.4

143.3

49.7

-

+

-

8

2000

85.9

85.5

124.0

30.4

-

-

-

EMS

300

73.0

66.4

963.1

869.5

+

+

-

MMS

10

52.6

43.1

639.3

545.8

+

+

-

 

Exp

with

S9

 

C1

0

103.2

102.3

98.3

/

/

-

-

C2

100.0

102.1

/

/

-

S1

0

100.0

100.0

95.7

/

/

/

-

S2

/

/

/

-

2

50

122.1

130.1

117.6

21.9

-

-

-

3

100

112.0

105.6

96.3

0.6

-

-

-

4

250

124.4

129.2

96.6

0.9

-

-

-

5

500

108.3

107.2

104.2

8.5

-

-

-

6

1000

104.9

106.6

135.9

40.2

-

-

-

7

1500

85.9

84.9

137.9

42.2

-

-

-

8

2000

82.3

88.5

169.4

73.7

-

+

-

B[a]P

2.5

63.6

36.5

840.8

745.1

+

+

-

C:      Negative Controls
S:        Solvent control (1% DMSO; v/v)
a:      Relative Cloning Efficiency, RCE = [(CEdose group/ CEof corresponding controls) x 100]
           Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)
b:      Relative Total Growth, RTG = (RSG x RCE)/100
c:       Mutant Frequency,
           MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800
d:      Induced Mutant Frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls
e:      Global Evaluation Factor, GEF (126); +: GEF exceeded, -: GEF not exceeded
f:       statistical significant increase in mutant frequency compared to solvent controls (Mann Whitney test, p<0.05).
+: significant; -not significant
EMS:  Ethylmethanesulfonate
MMS:Methylmethanesulfonate
B[a]P:Benzo[a]pyrene

 

Conclusions:
In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item 4-Aminobenzoyl-b-alanine is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

No information available

Additional information

Justification for classification or non-classification

Based on the negative results observed in three valid OECD Guideline in vitro genotoxicity studies, 4-aminobenzoyl-b-alanine is not classified as mutagenic or genotoxic.