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Toxicological information

Genetic toxicity: in vitro

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Administrative data

in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27. Mar. - 07. Jul. 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
according to guideline
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
solid: crystalline
Specific details on test material used for the study:
- Source and lot/batch No.of test material: 160805
- Expiration date of the lot/batch: 05. Aug. 2018
- Purity test date: not stated

not applicable

- Storage condition of test material: Room temperature 20 ± 5 °C, Keep away from humidity
- Stability under test conditions: assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: tested for solubility, assumed stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: assumed non reactive



Target gene:
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Source of cells: CLS (Eppelheim, Germany)
- Suitability of cells:
The V79 cell line has been used successfully in in vitro experiments for many years because of its sensitivity to chemical mutagens. Especially the high proliferation rate (doubling time 12 – 16 h in stock cultures) and a high cloning efficiency of untreated cells both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22 (Bradley et al., 1981). The cells were purchased by CLS (Eppelheim, Germany) and were sold under the name V79-4. The modal chromosome number was analysed and confirmed by the supplier of the cells.
- Cell cycle length, doubling time or proliferation index: doubling time 12 – 16 h in stock cultures
- Sex, age and number of blood donors if applicable: not applicable
- Whether whole blood or separated lymphocytes were used if applicable: not applicable
- Number of passages if applicable: not stated
- Methods for maintenance in cell culture if applicable: not stated
- Modal number of chromosomes: 22
- Normal (negative control) cell cycle time: not stated

- Type and identity of media including CO2 concentration if applicable: cultivated in DMEM complete culture medium with 5 % HS in cell culture flasks at 37.0 ± 1.5 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat Liver S9
Test concentrations with justification for top dose:
0.03, 0.06, 0.13, 0.25, 0.5, 1, 2 mg/mL in experiment
According to the results of the pre-test, 6 concentrations were chosen for experiment I and II and tested with and without metabolic activation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMEM
- Justification for choice of solvent/vehicle: A solubility test for the determination of a suitable solvent for the test item was performed in a non-GLP with cell culture medium (DMEM) and dimethyl sulfoxide. DMEM was chosen as solvent, because this vehicle has no effects on the viability of cells at the used concentration, does not show genetic toxicity and the test item was completely soluble.
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
Details on test system and experimental conditions:
- Cell density at seeding (if applicable): 1 * 10E6 cells per 10 cm culture dish and 500 cells (for the determination of the cytotoxicity)
per 6 cm culture dish

- Preincubation period: 23 h and 45 min (experiment I) and 24 h (experiment II)
- Exposure duration: 4h / 24 h
- Expression time (cells in growth medium): 168 h
- Selection time (if incubation with a selection agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): 6-Thioguanin

SPINDLE INHIBITOR (cytogenetic assays): not applicable

STAIN (for cytogenetic assays): not applicable


METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: 0.1 % Löffler’s methylene blue solution in 0.01 % KOH solution


NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): not applicable


- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: (absolute and relative)

Rationale for test conditions:
according to Guideline
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be
clearly positive if, in any of the experimental conditions examined:
 at least one of the test concentrations exhibits a statistically significant increase
compared with the concurrent negative control,
 the increase is concentration-related when evaluated with an appropriate trend test,
 any of the results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce gene
mutations in cultured mammalian cells in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly
negative if, in all experimental conditions examined:
 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
 all results are inside the distribution of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian
cells in this test system.
However, in a case by case evaluation this decision depends on the level of the corresponding
solvent control data. If there is by chance a low spontaneous mutation rate within
the laboratories historical control data range, a concentration-related increase of the mutations
within this range has to be discussed. The variability of the mutation rates of solvent
controls within all experiments of this study is also taken into consideration.
In cases when the response is neither clearly negative nor clearly positive as described
above, or in order to assist in establishing the biological relevance of a result, the data
should be evaluated by expert judgement and/or further investigations.
none performed

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
- Effects of pH: see "Any other information on results incl. tables"
- Effects of osmolality: see "Any other information on results incl. tables"
- Evaporation from medium: unlikely
- Water solubility: substance is very water soluble
- Precipitation: not observed
- Definition of acceptable cells for analysis: no individual cells observed
- Other confounding effects: none identified

In the pre-test, 7 concentrations of the test item (for nominal concentrations see Table 7-a)
were used and tested with and without metabolic activation. The exposure time was 4 hours and the exposure date 13. Apr. 2017.
Table 7-a Test Item Concentrations in the Pre-test
Nominal concentrations of
test solutions (mg/mL) 20 10 5 2.5 1.3 0.6 0.3
Resulting nominal concentrations
in experiment (mg/mL) 2 1 0.5 0.25 0.13 0.06 0.03
Determination of Survival by Cloning Efficiency
500 cells were exposed to each concentration of the test item for 4 hours with and without
metabolic activation (duplicate cultures per concentration level). Following treatment, the
cells were washed twice with PBS Dulbecco (2.5 % HS). After an expression period of 7 d
the cells were stained with methylene blue. Afterwards the colonies were counted and the
absolute and the relative cloning efficiency values were calculated.
Results and Conclusion
No cytotoxicity or precipitation was observed in the treatments with and without metabolic
In conclusion it can be stated that the test item TREHALOSE, ANHYDROUS did not induce
a cytotoxic effect in the approach with and without metabolic activation in the pre-test
following a treatment period of 4 h.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: 100 - 461, 261, 66.6, -
- Negative (solvent/vehicle) historical control data: 4 - 35, 16, 10, - (DMEM), 2-39, 14, 8.9, - (DMSO)

Any other information on results incl. tables

Osmolality and pH Values

The osmolality and the pH values of the solvent controls, the positive controls as well as

the test item concentrations (in DMEM medium with 5 % horse serum (HS)) that were

used in the pre-test were determined to exclude a negative influence on the assay by

those parameters. The osmolality was determined using an osmometer and the pH value

was determined using a pH meter. The results are summarized in the following table.

Table 6-a Osmolality and pH values

Sample Osmolality in mosm/kg H2O pH-Value

DMEM (5 % HS) + DMEM 333 7.830

DMEM (5 % HS) + DMSO 408 7.909

DMEM (5 % HS) + EMS (60 mg/mL) 334 7.845

DMEM (5 % HS) + DMBA (0.3 mg/mL) 407 7.832

DMEM (5 % HS) + Test Item 20 mg/mL 337 7.878

DMEM (5 % HS) + Test Item 10 mg/mL 340 7.961

DMEM (5 % HS) + Test Item 5 mg/mL 334 7.924

DMEM (5 % HS) + Test Item 2.5 mg/mL 332 7.879

DMEM (5 % HS) + Test Item 1.3 mg/mL 331 7.923

DMEM (5 % HS) + Test Item 0.6 mg/mL 331 7.920

DMEM (5 % HS) + Test Item 0.3 mg/mL 331 7.865

None of the tested positive controls or test item concentrations provoked a critical change

of the osmolality and the pH value in comparison to the solvent controls. Therefore, a negative influence of these parameters on the assay can be excluded.

Applicant's summary and conclusion

In conclusion, it can be stated that under the experimental conditions of this study
TREHALOSE, ANHYDROUS did not induce gene mutations at the HPRT locus in V79
cells in the absence and presence of metabolic activation.
Therefore, the test item TREHALOSE, ANHYDROUS is considered to be “non-mutagenic
under the conditions of the HPRT assay”.
Executive summary:

This study was performed to investigate the potential of TREHALOSE, ANHYDROUS to

induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in

Chinese Hamster cells (V79).

The assay comprised a pre-test and two independent valid experiments (experiment I and

II). Due to a technical error, the first experiment I was terminated and repeated. The collected

data of this invalid experiment is not included in this final report but will be archived

with the raw data. The pre-test was done to detect a potential cytotoxic effect of the test

item. Based on the results of this test the concentrations for the main experiments were


The first valid main experiment (experiment I) was performed with and without metabolic

activation (liver S9 mix from male rats, treated with Aroclor 1254) and a treatment period of

4 hours. The second experiment (experiment II) was performed with a treatment period of

24 hours without metabolic activation.

The highest nominal concentration (2 mg/mL) applied was chosen with regard to the solubility

of the test item in organic solvents and aqueous media and the cytotoxicity results.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in

mutant colonies and thus, showed enough sensitivity of the testing procedure and the activity

of the metabolic activation system.

No substantial and reproducible dose dependent increase in mutant colony numbers was

observed in both experiments up to the maximal concentration of the test item.