L-arabinose

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:

August 19, 2017 - October 19, 2018

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:

1997-07-21

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998-08

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use

Version / remarks:

2012-06

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:

L-arabinose

EC Number:

226-214-6

EC Name:

L-arabinose

Cas Number:

5328-37-0

Molecular formula:

C5H10O5

IUPAC Name:

L-arabinose

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No. of test material: AD16081001
- Expiration date of the batch: 2019-08-09

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: Stable under ambient conditions

Method

Target gene:

his/trp

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

S9 mix of Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver

Test concentrations with justification for top dose:

Selection of the concentrations was done on the basis of a solubility test and a concentration range finding test (Informatory Toxicity Test). The highest dose was 5000 μg test item/plate in absence and in the presence of exogenous metabolic activation (±S9 Mix) in the final treatment mixture under the actual conditions of the test at the start of the experiment for all test strains used. Six concentrations of the test item were tested each separated by approximately √10 factor in the main studies. The test concentrations were: ±S9 Mix: 5000; 1600; 500; 160; 50 and 16 μg/plate.

Vehicle / solvent:

- Solvent used: ultrapure water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene-diamine (NPD) [

Remarks:

TA98: 4 µg/plate, without S9 mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100, TA1535: 2 µg/plate, without S9 mix

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537: 50 µg/plate, without S9 mix

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E.coli WP2 uvrA: 2 µL/plate, without S9 mix

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

all Salmonella strains: 2 µg/plate, with S9 mix

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

E.coli strain: 50 µg/plate, with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar

DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 48 h at 37 °C

SELECTION AGENT : Biotin overlay agar (for Salmonella typhimurium strains), Tryptophan overlay agar (for Escherichia coli strain)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method,other: The viability of each testing culture was determined by plating 0.1 mL of the 10E-5, 10E-6, 10E-7 and 10E-8 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by plating experiments and manual counting.

Evaluation criteria:

The colony numbers on the untreated, vehicle and positive controls and the test item treated plates were determined (counted manually, evaluated by unaided eye), the mean values, standard deviations and the mutation rates were calculated.
Mutation Rate = Mean revertants at the test item (or control*) treatments / mean revertants of vehicle control
* untreated, vehicle or positive control
A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.
Criteria for a Negative Response:
A test item is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

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:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

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:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

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:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

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:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

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:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Summary Table of the Results of the Initial Mutation Test

Concentrations (μg/plate)	TA 98				TA 100				TA 1535				TA 1537				Escherichia coli WP2 uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	19.7	1.09	26.0	1.26	87.7	1.00	91.7	1.01	9.0	0.79	13.3	0.95	8.3	0.93	9.0	1.13	32.7	0.91	46.7	0.85
DMSO Control	16.0	1.00	21.3	1.00	–	–	94.3	1.00	–	–	14.7	1.00	6.0	1.00	7.3	1.00	–	–	43.7	1.00
Ultrapure Water Control	18.0	1.00	20.7	1.00	87.3	1.00	90.7	1.00	11.3	1.00	14.0	1.00	9.0	1.00	8.0	1.00	36.0	1.00	54.7	1.00
5000	28.0	1.56	28.3	1.37	90.0	1.03	110.3	1.22	13.3	1.18	12.0	0.86	6.3	0.70	7.0	0.88	47.0	1.31	54.3	0.99
1600	30.0	1.67	29.3	1.42	79.0	0.90	104.3	1.15	13.0	1.15	11.0	0.79	7.7	0.85	9.0	1.13	46.7	1.30	57.3	1.05
500	21.3	1.19	19.3	0.94	90.0	1.03	108.7	1.20	16.0	1.41	14.0	1.00	10.0	1.11	9.3	1.17	50.0	1.39	55.3	1.01
160	28.3	1.57	23.0	1.11	77.7	0.89	101.3	1.12	13.7	1.21	11.7	0.83	8.0	0.89	7.0	0.88	41.0	1.14	42.0	0.77
50	24.3	1.35	20.0	0.97	73.7	0.84	92.7	1.02	8.7	0.76	13.3	0.95	8.3	0.93	8.0	1.00	40.0	1.11	49.0	0.90
16	23.3	1.30	24.0	1.16	86.3	0.99	96.0	1.06	10.3	0.91	12.0	0.86	10.0	1.11	8.0	1.00	44.3	1.23	43.3	0.79
NPD (4 μg)	357.3	22.33	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2 μg)	–	–	–	–	933.3	10.69	–	–	533.3	47.06	–	–	–	–	–	–	–	–	–	–
9AA (50 μg)	–	–	–	–	–	–	–	–	–	–	–	–	758.0	126.33	–	–	–	–	–	–
MMS (2 μL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	821.3	22.81	–	–
2AA (2 μg)	–	–	1828.0	85.69	–	–	1493.3	15.83	–	–	237.3	16.18	–	–	153.3	20.91	–	–	–	–
2AA (50 μg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	193.7	4.44

MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

Remarks: Ultrapure water was applied as vehicle of the test item and the positive control substances: SAZ and MMS; and the DMSO was applied as vehicle for positive control substances: NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control is given referring to the ultrapure water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.

Table 2: Summary Table of the Results of the Confirmatory Mutation Test

Concentrations (μg/plate)	TA 98				TA 100				TA 1535				TA 1537				Escherichia coli WP2 uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	27.3	1.08	29.0	1.19	92.7	1.06	92.0	0.88	11.0	1.00	11.0	0.92	10.3	1.19	9.0	1.13	17.0	0.46	39.3	0.84
DMSO Control	23.0	1.00	22.7	1.00	–	–	94.3	1.00	–	–	13.0	1.00	7.3	1.00	6.3	1.00	–	–	38.7	1.00
Ultrapure Water Control	25.3	1.00	24.3	1.00	87.3	1.00	104.0	1.00	11.0	1.00	12.0	1.00	8.7	1.00	8.0	1.00	36.7	1.00	46.7	1.00
5000	29.0	1.14	19.3	0.79	83.7	0.96	94.3	0.91	12.3	1.12	9.0	0.75	6.7	0.77	9.0	1.13	39.7	1.08	46.7	1.00
1600	28.3	1.12	22.3	0.92	82.3	0.94	101.0	0.97	9.3	0.85	10.0	0.83	6.7	0.77	9.7	1.21	42.7	1.16	39.3	0.84
500	31.0	1.22	23.0	0.95	81.7	0.94	110.3	1.06	10.7	0.97	8.7	0.72	9.0	1.04	9.7	1.21	39.0	1.06	41.0	0.88
160	25.0	0.99	27.7	1.14	84.3	0.97	98.0	0.94	9.3	0.85	11.0	0.92	7.7	0.88	9.0	1.13	39.3	1.07	41.7	0.89
50	27.3	1.08	17.0	0.70	84.3	0.97	108.0	1.04	8.7	0.79	11.0	0.92	10.0	1.15	7.3	0.92	45.0	1.23	44.0	0.94
16	24.7	0.97	22.7	0.93	87.0	1.00	110.3	1.06	10.7	0.97	9.7	0.81	6.0	0.69	8.7	1.08	36.0	0.98	33.3	0.71
NPD (4 μg)	253.3	11.01	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2 μg)	–	–	–	–	797.3	9.13	–	–	509.3	46.30	–	–	–	–	–	–	–	–	–	–
9AA (50 μg)	–	–	–	–	–	–	–	–	–	–	–	–	528.0	72.00	–	–	–	–	–	–
MMS (2 μL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	741.3	20.22	–	–
2AA (2 μg)	–	–	1532.0	67.59	–	–	2826.7	29.96	–	–	164.0	12.62	–	–	121.7	19.21	–	–	–	–
2AA (50 μg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	243.7	6.30

MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

Remarks: Ultrapure water was applied as vehicle of the test item and the positive control substances: SAZ and MMS; and the DMSO was applied as vehicle for positive control substances: NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control is given referring to the ultrapure water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.

Applicant's summary and conclusion

Conclusions:

In an in vitro bacterial reverse mutation assay (Ames test) according to OECD TG 471, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, L-Arabinose is considered non-mutagenic.

Executive summary:

Five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential of the test item in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test) according to OECD Guideline 471. Each assay was conducted with and without metabolic activation (±S9 Mix). The concentrations, including the controls, were tested in triplicate (positive and negative controls were run concurrently).

In the Initial Mutation Test nearly all of the obtained higher revertant colony numbers (higher than the revertant colony numbers of the vehicle control) remained within the corresponding historical control data ranges. In the case of E. coli WP2 uvrA, at 500 μg/plate (-S9 Mix), the higher revertant colony numbers were above the corresponding historical control data range; however this increase was unique without any tendency and remained within the biological variability range of the applied test system.

All of the obtained increases were far below the biologically relevant threshold for being positive and were considered as reflecting the biological variability of the test system.

In the Initial Mutation Test, inhibitory effect of the test item on bacterial growth was not observed. All of the noticed lower revertant colony numbers (when compared to the revertant colony numbers of the corresponding vehicle control) remained in the range of the biological variability of the applied test system and the background lawn development was not affected in any case. The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, L-Arabinose is considered non-mutagenic in this bacterial reverse mutation assay.

The test item concentrations in experiment II were the same as already tested in the Initial Mutation Test. In the Confirmatory Mutation Test all of the noticed increased revertant colony numbers remained in the corresponding historical control data ranges of the ultrapure water vehicle control, and were without any biological significance. In the Confirmatory Mutation Test inhibitory effect of the test item, similarly to the results of the first experiment was not observed.

In the performed experiments the revertant colony numbers of the untreated and dimethyl sulfoxide (DMSO) control plates in the different experimental phases were slightly higher or lower than the ultrapure water vehicle control plates. The higher or lower revertant counts of these controls remained in the historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains. No substantial increases were observed in revertant colony numbers of any of the five tester strains following treatment with L-Arabinose at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values were observed in both independently performed main experiments; however, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.

The highest revertant colony number increase was observed in the Initial Mutation Test (Plate Incorporation Test), in the case of S. typhimurium TA98, at 1600 μg/plate (-S9 Mix). The mutation rate was: 1.67*. The higher (in comparison with the revertant colony numbers of the vehicle control) revertant colony counts remained in the corresponding ultrapure water historical control data range and far below the genotoxicological threshold for being positive.

* Mutation rate (MR): The mutation rate is the quotient of the mean revertant of test item treatment and the mean revertant of the vehicle control. In the case of Salmonella typhimurium TA98 a biologically relevant increase (positive result) is when the number of reversions is at least three times higher than the reversion rate of the vehicle control (Mutation rate ≥ 3.00).

Signs of cytotoxicity were not observed in either tested strains with and/or without metabolic activation. No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, L-Arabinose is considered non-mutagenic in this bacterial reverse mutation assay.