L-arabinose

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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

In an in vitro chromosome aberration assay in the mammalian cell line V79 according to OECD TG 473, the test item did not show clastogenic properties.

In an in vitro mammalian cell forward mutation assay (HPRT) according to OECD TG 476, the test item showed no mutagenic potential.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998-08

Deviations:

no

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

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

Target gene:

his/trp

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

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

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

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

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

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

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.

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

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

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

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

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

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 20, 2018 - April 06, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

July 29, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

May 31, 2008

Deviations:

yes

Remarks:

Negative results were not confirmed as the confirmation of negative results is not required by the most current Guideline (OECD 476, 29 July 2016).

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian gene mutation test

Specific details on test material used for the study:

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

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Target gene:

hprt

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

KI - Sub-line

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures
- Suitability of cells: The system used in this study has been extensively validated.
- Methods for maintenance in cell culture: The cell stocks are kept in liquid nitrogen. Each batch of frozen cells was purged of HPRT mutants and was free for mycoplasma infections, tested by Central Agricultural Office, National Animal Health Institute, Budapest, Hungary; results were fully documented within the raw data file. For each experiment, one or more vials were thawed rapidly, the cells diluted in Ham's F12 medium containing 10 % fetal bovine serum and incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air. When cells were growing well, subcultures were established in an appropriate number of flasks. T

MEDIA USED
- Type and identity of media including CO2 concentration: The CHO KI cells for this study were grown in Ham's F12 medium (F12-10) supplemented with 1 % of Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B) and heat-inactivated bovine serum (final concentration 10 %); humidified atmosphere of 5 % CO2 in air.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: not specified
- Periodically 'cleansed' against high spontaneous background: Yes

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction (S9 mix) of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver.

Test concentrations with justification for top dose:

125, 250, 500, 1000, 2000 µg/mL
The dose selection is based on the results of a per-test on cytotoxicity.

Vehicle / solvent:

- Vehicle used: Ham´s F12 medium
- Justification for choice of vehicle: This vehicle is compatible with the survival of the CHO cells and the S9 activity and was chosen based on the results of the preliminary Solubility Test, and its suitability is confirmed with the available laboratory’s historical database.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;
- Cell density at seeding: 5.0 x 10E6 cells/concentration

DURATION
- Preincubation period: 24 h
- Exposure duration: 5 h
- Selection time: 19 h
- Fixation time (start of exposure up to fixation or harvest of cells): 48 h

SELECTION AGENT: The selection medium (Ham´s F12) for TG resistant mutants contained 3.4 µg/mL of 6-thioguanine (6-TG)

NUMBER OF REPLICATIONS: 1

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After the selection period, the colonies were fixed, stained with Giemsa and counted for mutant selection and cloning efficiency determination.

Evaluation criteria:

Calculation of mutation frequency:
The mutation frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (2x10E6 cells: 2x5 plates at 2 x 10E5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection (viability), and will be expressed as 6-TG resistant mutants per 10E6 clonable cells.

Providing that all acceptability criteria are fulfilled, 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,
• any of the results are outside the distribution of the laboratory historical negative control data (based 95% control limit),
• the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.
Test item is then considered able to induce gene mutations in cultured mammalian cells in this test system.

Providing that all acceptability criteria were fulfilled, the test item is considered clearly negative because:
• none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
• there is no concentration-related increase when evaluated with an appropriate trend test,
• all results are compatible the distribution of the historical negative control data (based 95% control limit).
• The test item is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Statistics:

Statistical analysis was done with SPSS PC+ software for the following data:
• mutant frequency between the negative (solvent) control group and the test item or positive control item treated groups.
• mutant frequency between the laboratory historical negative (solvent) control group and concurrent negative (solvent) control, the test item or positive control item treated groups.
•The data were checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis by Microsoft Excel software.

The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis one-way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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:

not examined

Positive controls validity:

valid

Table 1: Summary of the mutagenesis assay results, 5 h treatment without S9 mix

NON ACTIVATION TEST CONDITION		SURVIVAL TO TREATMENT									REL. POPU- LATION GROWTH (%) OF CONTROL		MUTANT COLONIES DISH NUMBER										TOTAL MUTANT COLONIES		ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
		MEAN COLONY NUMBERS.D.						PERCENT VEH. CONTROL					1		2		3		4		5
Solvent control a		201.7		±		0.58		100			100		0		1		2		2		1		6		100	6.00
Pos. control (EMS 1.0 µL/mL) a		54.0		±		3.61		27			71		193		192		201		198		190		974		71	1371.83**
TEST ITEM
125 µg/mL a		199.7		±		1.53		99			100		0		1		2		0		2		5		100	5.00
250 µg/mL a		198.3		±		1.15		98			99		2		0		1		2		1		6		99	6.06
500 µg/mL a		198.7		±		2.52		99			100		0		3		0		3		0		6		100	6.00
1000 µg/mL a		200.7		±		2.08		100			99		2		2		2		0		0		6		99	6.06
2000 mg/mL a		198.3		±		1.53		98			99		0		0		2		2		1		5		99	5.05
NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT									REL. POPU- LATION GROWTH (%) OF CONTROL		MUTANT COLONIES DISH NUMBER										TOTAL MUTANT COLONIES		ABSOLUTE C.E. %		MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.						PERCENT VEH. CONTROL					1		2		3		4		5
Solvent control b	201.3		±		1.53		100			100		0		1		2		1		2		6		101		5.94
Pos. control (EMS 1.0 µL/mL) b	54.7		±		2.52		27			69		185		189		205		202		196		977		70		1395.71**
TEST ITEM
125 µg/mL b	200.0		±		1.00		99			99		3		0		0		1		2		6		100		6.00
250 µg/mL b	199.3		±		1.15		99			99		0		2		2		0		1		5		100		5.00
500 µg/mL b	199.3		±		2.31		99			99		0		2		0		2		1		5		100		5.00
1000 µg/mL b	200.3		±		0.58		100			100		1		1		1		2		1		6		100		6.00
2000 µg/mL b	198.0		±		1.73		98			99		0		2		2		1		1		6		100		6.00
NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT								REL. POPU- LATION GROWTH (%) OF CONTROL			MUTANT COLONIES DISH NUMBER										TOTAL MUTANT COLONIES		ABSOLUTE C.E. %		MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.						PERCENT VEH. CONTROL					1		2		3		4		5
Solvent control c	203.0		±		2.00		100		100			1		1		1		2		1		6		101		5.94
Pos. control (EMS 1.0 µL/mL) c	52.7		±		1.53		26		66			207		199		189		192		196		983		67		1467.16**
TEST ITEM
125 µg/mL c	201.0		±		3.61		99		99			0		0		2		2		1		5		100		5.00
250 µg/mL c	201.0		±		1.73		99		98			1		1		2		2		0		6		100		6.00
500 µg/mL c	202.0		±		2.00		100		100			2		1		2		2		0		7		101		6.93
1000 µg/mL c	201.3		±		2.08		99		98			1		1		2		2		0		6		99		6.06
2000 µg/mL c	198.0		±		2.65		98		100			2		1		2		1		0		6		101		5.94
NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT								REL. POPU- LATION GROWTH (%) OF CONTROL			MUTANT COLONIES DISH NUMBER										TOTAL MUTANT COLONIES		ABSOLUTE C.E. %		MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.						PERCENT VEH. CONTROL					1		2		3		4		5
Solvent control d	202.7		±		2.08		100		100			0		3		1		2		0		6		101		5.94
Pos. control (EMS 1.0 µL/mL) d	54.0		±		1.00		27		66			197		186		193		199		196		971		67		1449.25**
TEST ITEM
125 µg/mL d	200.0		±		1.00		99		99			1		1		1		1		1		5		100		5.00
250 µg/mL d	200.7		±		0.58		99		99			1		0		2		2		1		6		100		6.00
500 µg/mL d	201.3		±		1.15		99		99			1		2		0		3		0		6		101		5.94
1000 µg/mL d	201.7		±		2.08		100		98			3		2		1		0		0		6		100		6.00
2000 µg/mL d	198.3		±		2.08		98		99			2		1		1		2		1		7		100		7.00
a, b, c, d = corresponding parallel of first culture  abs.C.E. = Absolute Cloning Efficiency EMS = Ethyl methanesulfonate ** = p < 0.01 to the concurrent negative control and to the historical control

Table 2: Summary of the mutagenesis assay results, 5 h treatment with S9 mix

NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT				REL. POPU- LATION GROWTH (%) OF CONTROL			MUTANT COLONIES DISH NUMBER														TOTAL MUTANT COLONIES		ABSOLUTE C.E. %		MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.			PERCENT VEH. CONTROL				1			2			3		4			5
Solvent control a	203.0	±	1.73	100	100			2			1			2		1			0			6		100		6.00
Pos. control (DMBA 20 µg/mL) a	120.3	±	1.53	59	72			101			116			110		119			117			563		73		771.23**
TEST ITEM
125 g/mL a	200.3	±	3.21	99	100			0			2			1		1			2			6		101		5.94
250 g/mL a	199.0	±	3.00	98	100			2			1			1		0			1			5		100		5.00
500 g/mL a	192.7	±	2.31	95	98			3			2			0		0			1			6		98		6.12
1000 g/mL a	189.0	±	1.00	93	99			1			2			1		1			1			6		100		6.00
2000 g/mL a	171.0	±	3.61	84	100			1			0			2		1			2			6		100		6.00
NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT					REL. POPU- LATION GROWTH (%) OF CONTROL			MUTANT COLONIES DISH NUMBER														TOTAL MUTANT COLONIES		ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.			PERCENT VEH. CONTROL					1			2			3			4			5
Solvent control b	204.3	±	1.15	100		100			3			1			1			2			0		7		101	6.93
Pos. control (DMBA 20 µg/mL) b	121.3	±	1.53	59		73			104			117			100			115			92		528		74	713.51**
TEST ITEM
125 g/mL b	198.0	±	1.00	97		99			2			1			1			0			1		5		100	5.00
250 g/mL b	196.3	±	1.53	96		99			2			2			0			2			0		6		100	6.00
500 g/mL b	192.0	±	1.00	94		98			1			0			1			2			1		5		99	5.05
1000 g/mL b	189.3	±	0.58	93		99			1			2			2			0			1		6		100	6.00
2000 g/mL b	169.7	±	0.58	83		99			0			1			2			2			1		6		100	6.00
NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT						REL. POPU- LATION GROWTH (%) OF CONTROL			MUTANT COLONIES DISH NUMBER													TOTAL MUTANT COLONIES		ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.			PERCENT VEH. CONTROL						1			2		3		4			5
Solvent control c	202.3	±	2.52	100			100			2			1		1		1			1			6		100	6.00
Pos. control (DMBA 20 µg/mL) c	122.7	±	1.53	61			74			112			97		91		115			105			520		74	702.70**
TEST ITEM
125 µg/mL c	200.7	±	2.08	99			99			2			2		0		1			1			6		100	6.00
250 µg/mL c	195.0	±	0.00	96			99			1			0		2		2			0			5		99	5.05
500 µg/mL c	190.7	±	2.08	94			100			1			0		2		1			1			5		101	4.95
1000 µg/mL c	191.0	±	1.00	94			100			0			1		2		1			2			6		101	5.94
2000 µg/mL c	167.3	±	1.53	83			100			1			1		1		2			0			5		100	5.00
NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT						REL. POPU- LATION GROWTH (%) OF CONTROL			MUTANT COLONIES DISH NUMBER													TOTAL MUTANT COLONIES		ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.			PERCENT VEH. CONTROL						1		2			3			4		5
Solvent control d	203.0	±	1.00	100			100			2		2			1			1		0			6		101	5.94
Pos. control (DMBA 20 µg/mL) d	124.3	±	1.15	61			73			99		118			101			83		116			517		74	698.65**
TEST ITEM
125 µg/mL d	199.7	±	1.53	98			99			0		2			1			1		1			5		100	5.00
250 µg/mL d	195.3	±	1.15	96			99			0		1			1			2		2			6		100	6.00
500 µg/mL d	192.7	±	1.53	95			100			2		1			1			1		0			5		101	4.95
1000 µg/mL d	190.0	±	0.00	94			99			1		1			1			0		3			6		100	6.00
2000 µg/mL d	167.7	±	2.08	83			99			1		1			1			2		1			6		100	6.00
a, b, c, d = parallel of respective second culture. abs.C.E. = Absolute Cloning Efficiency DMBA= 7,12-Dimethyl benzanthracene ** = p < 0.01 to the concurrent negative control and to the historical control

Conclusions:

In an in vitro mammalian cell forward mutation assay (HPRT) according to OECD guideline 476, the test item showed no mutagenic potential.

Executive summary:

In an in vitro mammalian cell forward mutation assay (HPRT) according to OECD guideline 476 the potential of the test item to induce forward mutation at the hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster ovary (CHO KI) cells was determined. CHO cells were incubated at a cell density of 5.0 x 10E6 cells/plate (duplicates) at concentrations of 125, 250, 500 1000, and 2000 µg test item/mL with or without S9 mix for 5 hours at standard incubation conditions. After 19 h incubation (standard conditions) in selction agent (Ham´s F12 medium containing 3.4 µg/mL of 6-thioguanine), cells were fixed, stained with Giemsa and mutant colonies were counted. No signs of cytotoxicity were observed. Vehicle and positive controls (EMS, without S9 mix; DMBA, with S9 mix) were valid. Incubation with the test item, with or without S9 mix, did not induce statistically and biologically significant increases in mutant frequency over the background (negative solvent control). 

It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with in Chinese hamster ovary cells.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 04 - April 18, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

29-07-2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

14-02-2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

08-1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

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

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures); the V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male).
- Suitability of cells: The V79 cell line is well established in toxicology studies.
- Doubling time: 12 - 14 h
- Methods for maintenance in cell culture: The cell stocks were kept in liquid nitrogen. Checking of mycoplasma infections was carried out before freezing.
Trypsin-EDTA (0.25 % Trypsin, 1 mM EDTA x 4 Na) solution was used for cell detachment to subculture. The laboratory cultures were maintained in 75 cm² plastic flasks at 37 °C in a humidified atmosphere containing 5 % CO2.
- Modal number of chromosomes: diploid number, 2n=22

MEDIA USED
- Type and identity of media including CO2 concentration: The V79 cells for this study were grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine and 1 % of Antibiotic-antimycotic solution (containing 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B) and heat inactivated fetal bovine serum (final concentration 10 %) at 37 °C in a humidified atmosphere containing 5 % CO2.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination:Yes

Cytokinesis block (if used):

colchicine (0.2 µg/mL)

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver

Test concentrations with justification for top dose:

Experiment A:
3-hour treatment, harvest 20 hours from the beginning of treatment: 500, 1000, 2000 µg/mL (with and without S9 mix)
20-hour treatment, harvest 20 hours from the beginning of treatment: 250, 500, 1000, 2000 µg/mL (with and without S9 mix)
20-hour (without S9 mix) and 3-hour (with S9 mix) treatment, harvest 28 hours from the beginning of treatment: 250, 500, 1000, 2000 µg/mL

Experiment B:
In the cytogenetic experiment B the exposure period without metabolic activation was 20 hours. The exposure period with metabolic activation was 3 hours.

Vehicle / solvent:

- Vehicle used: DME medium
- Justification for choice of vehicle: This vehicle is compatible with the survival of the V79 cells and the S9 activity and was chosen based on the results of the preliminary solubility test, and its suitability is confirmed with the available laboratory’s historical database.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DME (Dulbecco’s Modified Eagle’s) medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;
- Cell density at seeding: 5 x 10E5 cells per culture

DURATION
- Exposure duration:
Experiment A: 3 h or 20 h
Experiment B: 20 hours, without S9 mix only or 28 hours, without and with S9 mix
- Expression time (cells in growth medium): 20 h
- Fixation time (start of exposure up to fixation or harvest of cells): 23 or 48 h

NUMBER OF REPLICATIONS: 1

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
cells were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (approx. 10 min. in 3:1 mixture of methanol: acetic-acid until the preparation became plasma free) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies. For control of bias, all slides were coded and scored blind.

NUMBER OF CELLS EVALUATED:
300 metaphase cells containing 2 N ± 2 centromeres were evaluated for structural aberrations from each experimental group


DETERMINATION OF CYTOTOXICITY
- Method: Relative Increase in Cell Counts (RICC)

Treatment of results
–The percentage of cells with structural chromosome aberration(s) was evaluated.
–Different types of structural chromosome aberrations were listed with their numbers and frequencies for experimental and control cultures.
–Gaps were recorded separately and reported but generally not included in the total aberration frequency.
–Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment(s) were recorded.
–Individual culture data were summarised in tabular form.

Rationale for test conditions:

Based on the cell counts in an cytotoxicity pre-test, the Relative Increase in Cell Counts (RICC) was calculated as cytotoxicity parameter. The results obtained were used for the selection of the test item concentrations used in the main cytogenetic experiments (experiments A and B).

Evaluation criteria:

Interpretation of Results

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
–at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
–the increase is dose-related when evaluated with an appropriate trend test,
–any of the results are outside the distribution of the laboratory historical negative control data.

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative because:
–none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
–there is no concentration-related increase when evaluated with an appropriate trend test,
–all results are inside the distribution of the laboratory historical negative control data.

Statistics:

For statistical analysis CHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control.
The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too. The lower and upper 95 % confidence intervals of historical control were calculated with C-chart.
The data were checked for a linear trend in aberration frequency with treatment dose using the adequate regression analysis by Microsoft Excel software.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

500 µg/mL and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Not observed

RANGE-FINDING/SCREENING STUDIES:
Solubility- and dose range finding study (cytotoxicity)

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
see "Any other information on results"

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RICC

Table 1: Mean Percentage of Cells with structural Chromosome Aberrations, Experiment A

Concentration (µg/mL)	S9 mix	Treatment time	Harvesting time	Mean aberrant cells/150 cells
				incl. gaps	excl. gaps
Solvent control (DME medium)	-	3 h	20 h	8	4
L-Arabinose
500 µg/mL	-	3 h	20 h	7	3
1000 µg/mL	-	3 h	20 h	9	4
2000 µg/mL	-	3 h	20 h	8	5
Pos. Control (EMS)	-	3 h	20 h	38**	33**
Solvent control (DME medium)	+	3 h	20 h	6	4
L-Arabinose
500 µg/mL	+	3 h	20 h	7	3
1000 µg/mL	+	3 h	20 h	7	3
2000 µg/mL	+	3 h	20 h	8	4
Pos. Control (Cycl.)	+	3 h	20 h	46**	41**
Positive control (-S9): Ethyl methanesulfonate (1.0 µL/mL) Positive control (+S9): Cyclophosphamide (5.0 µg/mL) **: p<0.01

Table 2: Mean Percentage of Cells with structural Chromosome Aberrations, Experiment B

Concentration (µg/mL)	S9 mix	Treatment time	Harvesting time	Mean aberrant cells/150cells
				incl. gaps	excl. gaps
Solvent control (DME medium)	-	20 h	20 h	6	3
L-Arabinose
250 µg/mL	-	20 h	20 h	7	3
500 µg/mL	-	20 h	20 h	8	4
1000 µg/mL	-	20 h	20 h	7	3
2000 µg/mL	-	20 h	20 h	7	4
Pos. Control (EMS)	-	20 h	20 h	41**	34**
Solvent control (DME medium)	-	20 h	28 h	7	3
L-Arabinose
250 µg/mL	-	20 h	28 h	7	3
500 µg/mL	-	20 h	28 h	8	4
1000 µg/mL	-	20 h	28 h	7	3
2000 µg/mL	-	20 h	28 h	7	4
Pos. Control (EMS)	-	20 h	28 h	42**	36**
Solvent control (DME medium)	+	3 h	28 h	8	4
L-Arabinose
500 µg/mL	+	3 h	28 h	7	3
1000 µg/mL	+	3 h	28 h	8	3
2000 µg/mL	+	3 h	28 h	7	3
Pos. Control (Cycl.)	+	3 h	28 h	45**	39**
Positive control (-S9): Ethyl methanesulfonate (0.4 µL/mL) Positive control (+S9): Cyclophosphamide (5.0 µg/mL) **: p<0.01

Table 3: Historical Control data (without S9 mix)

	number of aberrant cells/ 150 cells
	negative control		positive control (Ethyl methanesulfonate)
	incl. Gaps	excl. Gaps	incl. Gaps	excl. Gaps
Mean	6.26	2.85	40.50	31.70
SD	0.75	0.61	3.51	3.88
Range	4 - 8	2 - 5	35-50	26-39
Lower confidence interval*	4.70	1.59	33.22	23.64
Upper confidence interval*	7.82	4.11	47.78	39.75
n	23	23	23	23
n = number of experiments SD = standard deviation

Table 4: Historical Control data (with S9 mix)

	number of aberrant cells/ 150 cells
	negative control		positive control (Cyclophosphamide)
	incl. Gaps	excl. Gaps	incl. Gaps	excl. Gaps
Mean	6.39	3.02	46.07	39.43
SD	0.83	0.64	2.39	2.65
Range	5-9	2-5	39-51	34-46
Lower confidence interval*	4.66	1.69	41.11	33.95
Upper confidence interval*	8.12	4.35	51.02	44.92
n	23	23	23	23
n = number of experiments SD = standard deviation

Conclusions:

In an in vitro chromosome aberration assay in the mammalian cell line V79 according to OECD 473, the test item did not show clastogenic properties.

Executive summary:

The test item was tested in a chromosome aberration Assay in V79 cells according to OECD TG 473. The test item was dissolved in DME medium and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver). In the two independent experiments of the Chromosome Aberration Assay (Experiments A and B, both run in duplicate) at least 300 well‑spread metaphase cells were analysed at concentrations and incubation/expression intervals given below:

Experiment A with 3/20 h treatment/sampling timewithout and with S9 mix: 500, 1000 and 2000 µg/mL

Experiment B with 20/20 h treatment/sampling timewithout S9 mix: 250, 500, 1000 and 2000 µg/mL

Experiment B with 20/28 h treatment/sampling timewithout S9 mix: 250, 500, 1000 and 2000 µg/mL

Experiment B with 3/28 h treatment/sampling timewith S9 mix: 500, 1000 and 2000 µg/mL

In the performed Chromosome Aberration Assay the concentration levels were chosen mainly based on the maximum recommended concentration. The maximum recommended concentration for lower cytotoxic substances is 2000 µg/mL (based on the updated OECD Guideline 473 (2016)).

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, neither in the absence nor in the presence of metabolic activation, up to the maximum recommended concentration. There were no statistical differences between treatment and concurrent solvent and historical control groups and no dose-response relationships were noted.

In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent and historical controls, up to the maximum recommended concentration without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. Further, a 3-hour treatment up to the maximum recommended concentration in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations.

In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. The observed chromosome aberrations were inside the distribution of the laboratory historical negative control data. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

The number of aberrations found in the solvent controls was compatible with the historical laboratory control data. The concurrent positive controls Ethyl methanesulphonate (0.4 and 1.0 µL/mL) and Cyclophosphamide (5 µg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

The test item assessed up to the maximum recommended concentration without and with mammalian metabolic activation system did not induce structural chromosome aberrations in Chinese Hamster lung cells (V79). Thus, the test item is considered as not clastogenic in this system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

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.

The test item was tested in a chromosome aberration Assay in V79 cells according to OECD guideline 473. The test item was dissolved in DME medium and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver). In the two independent experiments of the Chromosome Aberration Assay (Experiments A and B, both run in duplicate) at least 300 well‑spread metaphase cells were analysed at concentrations and incubation/expression intervals given below:

Experiment A with 3/20 h treatment/sampling time without and with S9 mix: 500, 1000 and 2000 µg/mL

Experiment B with 20/20 h treatment/sampling time without S9 mix: 250, 500, 1000 and 2000 µg/mL

Experiment B with 20/28 h treatment/sampling time without S9 mix: 250, 500, 1000 and 2000 µg/mL

Experiment B with 3/28 h treatment/sampling time with S9 mix: 500, 1000 and 2000 µg/mL

 In the performed Chromosome Aberration Assay the concentration levels were chosen mainly based on the maximum recommended concentration. The maximum recommended concentration for lower cytotoxic substances is 2000 µg/mL (based on the updated OECD Guideline 473 (2016)).

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, neither in the absence nor in the presence of metabolic activation, up to the maximum recommended concentration. There were no statistical differences between treatment and concurrent solvent and historical control groups and no dose-response relationships were noted.

In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent and historical controls, up to the maximum recommended concentration without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. Further, a 3-hour treatment up to the maximum recommended concentration in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations.

In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. The observed chromosome aberrations were inside the distribution of the laboratory historical negative control data. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

The number of aberrations found in the solvent controls was compatible with the historical laboratory control data. The concurrent positive controls Ethyl methanesulphonate (0.4 and 1.0 µL/mL) and Cyclophosphamide (5 µg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

The test item assessed up to the maximum recommended concentration without and with mammalian metabolic activation system did not induce structural chromosome aberrations in Chinese Hamster lung cells (V79). Thus, the test item is considered as not clastogenic in this system.

In an in vitro mammalian cell forward mutation assay (HPRT) according to OECD guideline 476 the potential of the test item to induce forward mutation at the hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster ovary (CHO KI) cells was determined. CHO cells were incubated at a cell density of 5.0 x 10E6 cells/plate (duplicates) at concentrations of 125, 250, 500 1000, and 2000 µg test item/mL with or without S9 mix for 5 hours at standard incubation conditions. After 19 h incubation (standard conditions) in selction agent (Ham´s F12 medium containing 3.4 µg/mL of 6-thioguanine), cells were fixed, stained with Giemsa and mutant colonies were counted. No signs of cytotoxicity were observed. Vehicle and positive controls (EMS, without S9 mix; DMBA, with S9 mix) were valid. Incubation with the test item, with or without S9 mix, did not induce statistically and biologically significant increases in mutant frequency over the background (negative solvent control).

It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with in Chinese hamster ovary cells

In conclusion, the test item did not show mutagenic properties in bacterial or mammalian test systems. Furthermore, the test item did not show clastogenic properties in a mammalian cell system. thus the test item is considered as non-genotoxic.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available information on the test item regarding genetic toxicity are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available experimental information, the test substance is not classified for genetic toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the eleventh time in Commission Regulation (EU) 2018/669.