Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 May 2019 - 25 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reference substance 001
Cas Number:
67-47-0
Molecular formula:
C6H6O3
impurity 1
Chemical structure
Reference substance name:
Fructose
EC Number:
200-333-3
EC Name:
Fructose
Cas Number:
57-48-7
Molecular formula:
C6H12O6
IUPAC Name:
D-fructose
impurity 2
Chemical structure
Reference substance name:
1-(furan-2-yl)-2-hydroxyethanone
Cas Number:
17678-19-2
Molecular formula:
C6H6O3
IUPAC Name:
1-(furan-2-yl)-2-hydroxyethanone
impurity 3
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
water
impurity 4
Chemical structure
Reference substance name:
Sodium formate
EC Number:
205-488-0
EC Name:
Sodium formate
Cas Number:
141-53-7
Molecular formula:
CH2O2.Na
IUPAC Name:
sodium formate
impurity 5
Chemical structure
Reference substance name:
Sodium nitrate
EC Number:
231-554-3
EC Name:
Sodium nitrate
Cas Number:
7631-99-4
Molecular formula:
HNO3.Na
IUPAC Name:
sodium nitrate
impurity 6
Chemical structure
Reference substance name:
Glucose
EC Number:
200-075-1
EC Name:
Glucose
Cas Number:
50-99-7
Molecular formula:
C6H12O6
IUPAC Name:
D-glucose
impurity 7
Chemical structure
Reference substance name:
Sodium acetate
EC Number:
204-823-8
EC Name:
Sodium acetate
Cas Number:
127-09-3
Molecular formula:
C2H4O2.Na
IUPAC Name:
sodium acetate
impurity 8
Chemical structure
Reference substance name:
2-furaldehyde
EC Number:
202-627-7
EC Name:
2-furaldehyde
Cas Number:
98-01-1
Molecular formula:
C5H4O2
IUPAC Name:
2-furaldehyde
impurity 9
Chemical structure
Reference substance name:
Sodium 4-oxovalerate
EC Number:
243-378-4
EC Name:
Sodium 4-oxovalerate
Cas Number:
19856-23-6
Molecular formula:
C5H8O3.Na
IUPAC Name:
sodium 4-oxopentanoate
impurity 10
Chemical structure
Reference substance name:
Formaldehyde
EC Number:
200-001-8
EC Name:
Formaldehyde
Cas Number:
50-00-0
Molecular formula:
CH2O
IUPAC Name:
formaldehyde
Test material form:
solid: crystalline
Details on test material:
freeze-dried material
Batch: 1808A-RO-C-I1-FD-Chg#3
Appearance Crystal / brown
Storage: Refrigerator (2 - 8 °C)
Expiring date: 31 December 2019
Specific details on test material used for the study:
- Batch No.: 1808A-RO-C-I1-FD-Chg#3
- Storage conditions: 2-8°C

Method

Target gene:
histidine loci of several strains of Salmonella typhimurium and tryptophan locus of Escherichia coli strain WP2 uvrA
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
10 % (v/v) rat liver S9 Mix
Test concentrations with justification for top dose:
Initial Mutation Test and Confirmatory Mutation Test: 5000, 1600, 500, 160, 50 and 16 µg/plate

For soluble, non-toxic test compounds the maximum test concentration is 5 mg/plate as recommended in the guideline for soluble non-toxic test compounds.

Concentration Range Finding Test (Informatory Toxicity Tests):
Based on the solubility test, the stock solution with a concentration of 50 mg/mL was prepared in ultrapure water (ASTM Type I), and diluted in 6 steps by factor of approximately v10.
The number of revertant colonies and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 1600, 500, 160, 50, 16 and 5 µg/plate of the test item.
In the Informatory Toxicity Test no inhibitory effect of the test item was observed, background lawn development were not affected at any of the tested concentrations. Slight revertant colony number decreases or increases (compared to the number of revertant colonies numbers of the vehicle control) remained within the corresponding historical control data ranges and biological variability range of the applied test system.
No precipitation of the test item was observed on the plates in the bacterial strains at any of the examined concentration levels, in the presence or in the absence of S9 Mix.
Vehicle / solvent:
- Vehicle/solvent used: Ultrapure water (ASTM Type I)
- Justification for choice of solvent/vehicle: In the preliminary solubility and concentration range finding tests, ultrapure water (ASTM Type I) was selected as appropriate vehicle for preparing the test item solutions. This vehicle is compatible with the survival of the bacteria and the S9 activity.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
methylmethanesulfonate
other: 2-aminoanthracene (2AA), 4-Nitro-1,2-phenylenediamine (NPD)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ultrapure water (ASTM Type 1)
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION
Initial mutation test: in agar (plate incorporation); confirmatory test: in agar (plate incorporation) with preincubation
- Bacterial cultures: The frozen bacterial cultures were thawed at room temperature and 200 µL inoculum was used to inoculate each 50 mL of Nutrient Broth No. 2 for the overnight cultures in the assay. The cultures were incubated for approximately 10-13 hours in a 37°C Benchtop Incubator Shaker.

- Molten top agar was prepared and kept at 45°C. Two mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. Conditions were investigated in triplicate. The test item and other components were prepared fresh and added to the overlay (45°C).
The typical content of the tubes:
top agar 2000 µL
vehicle or solution of test item or positive controls 100 µL
overnight culture of test strain 100 µL (containing aprox 10^9 CFU/ml)
phosphate buffer (pH: 7.4) or S9 mix 500 µL
This solution was mixed and poured on the surface of the properly labeled minimal agar plates. For in cubations with metabolic activation, instead of phosphate buffer, 0.5 mL of the S9 Mix was added to each overlay tube.

- Exposure duration: incubated at 37°C for about 48 hours

- Preincubation period (in confirmatory test): Before the overlaying of the test item, the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to allow direct contact between bacteria and the test item (in its vehicle). These tubes were gently mixed and incubated for 20 min at 37ºC in a shaking incubator. After the incubation period, two mL of molten top agar was added to the tubes, and the content was mixed and poured onto minimal glucose agar plates.

NUMBER OF REPLICATIONS: Triplicates

METABOLIC ACTIVATION SYSTEM: Rat Liver S9 Fraction
The S9 fraction of Phenobarbital (PB) and ß-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).

VALIDITY CRITERIA
The tests (Initial and Confirmatory Mutation Tests) are considered valid if:
- All of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene.
- The Salmonella typhimurium TA98 and TA100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- The Escherichia coli WP2 uvrA culture demonstrate the deletion in the uvrA gene.
- The bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- The tester strain culture titers are in the 109 cells/mL order.
- The batch of S9 used in this study shows the appropriate biological activity.
- The reference mutagens show the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective vehicle control.
- There are at least five analyzable concentrations (at each tester strain) (a minimum of three nontoxic dose levels is required to evaluate assay data).

A dose level is considered toxic if
- reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- reduced background lawn development occurs.
- For soluble, non-toxic test compounds the maximum test concentration is 5 mg/plate or 5 µL/plate. F or test compounds that are not soluble at 5 mg/plate or 5 µL/plate and that are not toxic at levels lower than an insoluble level, the highest doses tested is at least one insoluble concentration in the final treatment mixture under the actual conditions of the test at the start of the experiment. Insolubility is assessed as precipitation in the final mixture under the actual test conditions and evident to the unaided eye.
- The test has to be included five analyzable concentrations (where the precipitate does not interfere with the scoring) and a minimum of three non-precipitated dose levels.

Evaluation criteria:
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 rev ersion 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.
Statistics:
Based on the evaluation criteria no statistical analysis was required.

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA98, TA100, TA1535, TA1537, and E. coli WP2 uvrA
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:
- Validity criteria: All criteria for the validity of the performed experiments according to the OECD guideline were met.
- Controls: In the Initial and Confirmatory Mutation Tests the revertant colony numbers of the ultrapur e water (ASTM Type 1) vehicle control plates with and without S9 Mix were in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in both main experimental phases, in all tester strains.
- Results on initial and confirmatory mutation tests: Some increases in the number of revertant colonies were observed. These increases did not show a clear dose-response relationship, and the mutation rate associated to these increases remained below the thresholds established as the criteria for a positive response. The obtained increases were therefore considered as biologically not relevant, being in the range of the biological variability of the applied test system.
The highest number of revertant colonies was observed in the confirmatory mutation test (pre-incubation method) in Salmonella typhimurium TA100 strain at 1600 µg/plate, in the absence of metabolic activation. This value was above the upper value of the corresponding vehicle historical control data ranges. However, since this increase was only observed at one dose level, the increase was not considered to be dose-related. Furthermore, the mutation rate was 1.86, which was well below the 2-fold increase threshold established as the criteria for a positive response in this test. 3.
Based on these results, no significant dose-related increases were observed in the number of revertant colonies in any of the five test strains following treatment with 5-(Hydroxymethyl)furfural at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.
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.
In the confirmatory mutation test slight inhibitory effect of the test item was noticed in Salmonella typhimurium TA98, TA100 and TA1537 strains at 5000 µg/plate, in the absence of exogenous metabolic activation (-S9). The cytotoxicity was indicated by affected background lawn development (reduced background lawn), and decreased revertant colony counts (revertants below the historical control data ranges and/or corresponding vehicle control data ranges).
In the two experiments, no precipitation of the test item was observed on the plates in any of the bacterial strains at any examined concentration level, in the absence or in the presence of S9 Mix.

Applicant's summary and conclusion

Conclusions:
Based on the results obtained under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item 5-(Hydroxymethyl)furfural has no mutagenic activity on the bacterial tester strains under the test conditions used in this study.
Executive summary:

The test item 5-(Hydroxymethyl)furfural was tested with regard to its potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/ß-naphthoflavone-induced rats.

The study included a preliminary solubility test, a preliminary concentration range finding test (informatory toxicity test following the plate incorporation method), an initial mutation test (plate incorporation method), and a confirmatory mutation test (pre-incubation method).

Based on the results of the solubility test and the concentration range finding test, the test item was dissolved in ultrapure water, and the following concentrations were prepared and investigated in the initial and confirmatory mutation tests: 5000; 1600; 500; 160; 50 and 16 µg/plate with and without S9 Mix.

The maximum test concentration was in all strains 5000 µg/plate, as recommended in the guideline for soluble non-toxic compounds.

No precipitation of the test item was observed on the plates in any of the bacterial strains at the examined concentration levels, in the presence and the absence of S9 Mix, throughout the study.

In the initial mutation test, inhibitory effect of the test item on bacterial growth was not observed.

In the confirmatory mutation test, slight inhibitory effect of the test item, indicated by reduced background lawn and decreased revertant colony counts were noticed in Salmonella typhimurium TA98, TA100 and TA1537 strains at 5000 µg/plate, in the absence of S9 Mix.

The number of revertant colonies in the vehicle control (ultrapure water) plates with and without S9 Mix was within the historical control data ranges for the number of spontaneous revertant colonies per strain.

The positive control reference items induced the expected, biological relevant increases (more than 3-fold increase) in revertant colonies and the number of revertants was within the historical control data range per strain, thereby meeting the criteria for a valid positive control in both experimental phases and all tester strains.

No biologically relevant increases were observed in the number of revertant colonies in any of the five tester strains following treatment with 5-(Hydroxymethyl)furfural at any concentration levels, either in the presence or absence of S9 Mix in the performed experiments.

Based on the results obtained under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item 5-(Hydroxymethyl)furfural has no mutagenic activity in the bacterial tester strains under the test conditions used in this study.