Trehalose

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

different Guideline studies on genetic toxicity available.

No indication of genetic toxicity found.

Link to relevant study records

Reference

Reference 1

Endpoint:

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

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)

Qualifier:

according to guideline

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 160805
- Expiration date of the lot/batch: 05. Aug. 2018
- Purity test date: not stated

RADIOLABELLING INFORMATION (if applicable)
not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- 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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
none

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- 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

MEDIA USED
- 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:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;
- 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

DURATION
- 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

NUMBER OF REPLICATIONS: 2

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

NUMBER OF CELLS EVALUATED: not applicable

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

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: not applicable

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

OTHER EXAMINATIONS:
none

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
test,
 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.

Statistics:

none performed

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- 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

RANGE-FINDING/SCREENING STUDIES:
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
activation.
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)

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.

Conclusions:

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

determined.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994/1995

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:

26 May 1983

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 931129
- Expiration date of the lot/batch: 28 May 1995
- Purity test date: not stated

RADIOLABELLING INFORMATION (if applicable)
not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: assumed stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: assumed stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
none

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:

Target gene:

his-, trp-

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

The highest concentration was 50mg/ml of test substance in the chosen solvent, which provided a final
concentration of 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Prior to commencing testing the solubility of the test substance was assessed at 50 mg/ml in water in which it was found to dissolve. Therefore water was chosen as the solvent forthis study.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation);
- Cell density at seeding (if applicable): not applicable

DURATION
- Preincubation period: none
- Exposure duration: 72 h
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): not applicable

SPINDLE INHIBITOR (cytogenetic assays): not applicable

STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: not applicable

NUMBER OF CELLS EVALUATED: not applicable

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

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: m relative total growth;
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
none

- OTHER:

Rationale for test conditions:

according to Guideline

Evaluation criteria:

If treatment with a test substance produces an increase in revertant colony numbers of at least
twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in
two separate experiments with anybacterial strain either in the presence or absence of S-9
mix, it is considered to show evidence of mutagenic activity in this test system.

If treatment with a test substance does not produce reproducible increases of at least 1.5 times
the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to
show no evidence of mutagenic activity in this test system

Statistics:

No statistical analysis is performed

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle 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

Vehicle 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

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

It is concluded that,when tested in water, trehalose (crystal) shows no evidence of mutagenic activity
in this bacterial system.

Executive summary:

In this in vitro assessment of the mutagenic potential of trehalose (crystal), histidine dependent

auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100）and

a tryptophan dependent mutant of Escherichia coli (WP2 uvrA) were exposed to the test substance,

diluted in water which was also used as a negative control.

Two independent mutation tests were performed, in the presence and absence of liver preparations

from Aroclor 1254-induced rats.

In the preliminary dose range finding study with dose levels of up to 5000 μ.g/plate no toxicity was

observed. A top dose level of 5000 μ.g/plate was chosen for the subsequent mutation study. Other

dose levels used in the mutation assays were: 2500, 1250, 625, 312.5 μ.g/plate.

No evidence of mutagenic activity was seen at any dose level of trehalose (crystal) in either mutation

test.

The concurrent positive control compounds demonstrated the sensitivity of the assay and the

metabolising activity of the liver preparations.

It is concluded that,when tested in water, trehalose (crystal) was not mutagenic in this bacterial

system.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17. June 1997 - 4. August 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 50615
- Expiration date of the lot/batch: not stated
- Purity test date: not stated

RADIOLABELLING INFORMATION (if applicable)
not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from Light
- Stability under test conditions: assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble in water and assumed stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: assumed stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
none

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:

Target gene:

no single target gene, whole genom

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection (A TCC), Rockville, MD
- Suitability of cells: recomended in Guideline
- Cell cycle length, doubling time or proliferation index: not stated
- 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: not stated
- Normal (negative control) cell cycle time: not stated

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: F-12
medium with 2.5% FBS
- Properly maintained: [yes/no] not stated
- Periodically checked for Mycoplasma contamination: [yes/no] not stated
- Periodically checked for karyotype stability: [yes/no) not stated
- Periodically 'cleansed' against high spontaneous background: [yes/no] not stated

Cytokinesis block (if used):

colcemid

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

Cytotox Expt.; 312, 1250, and 5000 μg/mL
Definitive Expts.; 1250, 2500, and 5000 μg/mL
The top dose of 5000 μg/mL in the initial chromosome ·
aberration assay was chosen based upon the results of the
cytotoxicity assay

Vehicle / solvent:

sterile water
common solvent in nature and in test protocol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;
- Cell density at seeding (if applicable): 5 x 10E5 cell per flask

DURATION
- Preincubation period:
- Exposure duration: 3 and 21 hours
- Harvest time: 21 and 45 hours after exposure initiation

SELECTION AGENT (mutation assays): not applicable

SPINDLE INHIBITOR (cytogenetic assays): Colcemid, 2,5 h before harvest

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: two experiments

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
After -2.5 hours in colcemid, the cells were harvested. At harvest, cells were
removed from the surface of the flask using a cell-scraper. The cells with their media
were then transferred to a 15-mL centrifuge tube. This cell suspension was centrifuged,
and the supernatant was aspirated. Then 9 mL of warm hypotonic solution (0.075 M
KCl) was added to each tube using gentle agitation. The cell suspension was incubated
from between 10 to 20 minutes at 37°C. One mL of Carnoy's fixative (3:1, v/v methanol:
glacial acetic acid) was added to each tube, the tubes were vortexed and centrifuged. The
supernatant were aspirated, and the cells resuspended in 5 mL fixative. Each tube sat at
room temperature for at least 5 min followed by centrifugation. After two changes of
fixative, air-dried slides were prepared from all flasks. Upon drying the slides were
evaluated, using a light microscope, for metaphase quality and concentration. Only
scorable slides were stained with Giemsa for evaluation. After staining cells were rinsed
in deionized water, passed through xylene, and coverslips mounted with Permount.

NUMBER OF CELLS EVALUATED:
Mitotic Index: At least 500 cells per culture
Chromosome Aberrations: 100 cells per culture from vehicle and positive control and
top three scorable doses of test article. A minimum of 25
cells instead of 100 cells were scored when 25% of those
evaluated cells were aberrant.
Polyploidy (replicate Assay only): 45 hour harvest only,% polyploidy cells from a minimum
of 100 mitosis.
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): not applicable

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: not stated

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index;
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
none

- OTHER:

Rationale for test conditions:

according to:
Galloway, S. M., A. D. Bloom, M. Resnick, B. H. Margolin, F. Nakamura, P. Archer, and E. Zeiger. 1985. Development of a standard protocol for in vitro cytogenetic testing with Chinese hamster ovary cells: Comparison of results for 22 compounds in two laboratories. Environ. Mutagenesis 7, 1-52.

Hamerton, J. L. 1971. Human Cytogenetics, Vol. 2, Clinical Cytogenetics. Academic Press, New York.

Preston, J. R., W. Au, M.A. Bender, J. G. Brewen, A. V. Carrano, J. A. Heddle, A. F. Mcfee, S. Wolff, and J. S. Wasscom. 1981. Mammalian in vivo and in vitro cytogenetic assays: A report of the U.S. EPA's Gene-Tax Program. Mutat. Res. 87, 143-188.

Savage, J .R .K. 197 5. Classification and relationships of induced chromosomal structural changes. J. Med. Genet. 12, 103-122.

Evaluation criteria:

1. Frequency of cells with structural chromosome
aberrations in positive control significantly (p < 0.05)
elevated above solvent control.
2. Highest concentration evaluated in chromosome
aberration assay within a factor of two of the level that
resulted in a significant effect on the mitotic index.

Criteria for Interpretation:
Positive:
Negative:
(1) Significant increase (p < 0.01) in the frequency of cells
with structural chromosomal damage and (2) increase either
dose-related or reproducible in a replicate chromosome
aberration assay.
Neither criterion for a positive response met.
Inconclusive: Statistically significant elevation in chromosomal
abnormalities not dose-related.

Statistics:

Analysis of data from chromosome aberration assays conducted with and
without MA were performed separately. The following statistics calculated for
each treatment:
• Mitotic index
• Total number of chromosomal aberrations
• Frequency of chromosomal aberrations per cell
• Frequency of aberrant cells
• Number and frequency of aberrations in each category
• Number and frequency of cells with structurally aberrant
chromosomes
• Number and frequency of severely damaged cells (i.e., cells
with 5 or more chromosome aberrations).
• Number and frequency of polyploid cells for 45 hr
treatments with and without MA for negative control and
top treatment concentrations.
• The number of cells with structural chromosomal damage
observed in the test article and positive control treatment
groups statistically compared with those on the concurrent
solvent control group using Fisher's Exact test (significance
level of p < 0.01, I-tailed).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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:

Based on the criteria established in the protocol, it is concluded that Trehalose elicits a
negative response in the CHO chromosomal aberration assay in the absence and presence of
metabolic activation.

Conclusions:

Based on the criteria established in the protocol, it is concluded that Trehalose elicits a negative response in the CHO chromosomal aberration assay in the absence and presence of metabolic activation.

Executive summary:

This study was performed to evaluate the ability of Trehalose to induce chromosomal

aberrations (CA) in Chinese hamster ovary (CHO) cells in the presence and absence ofrat S9

metabolic activation (MA).

In the cytotoxicity assay, CHO cells were exposed for 3 hr to Trehalose at concentrations

of 312, 1250, and 5000 μg/mL in both the absence and presence of MA. Metaphase cells were

harvested at 21 hr after initiation of exposure. No significant decrease in mitotic index or

confluency was observed at any dose level.

Based on the cytotoxicity results, the initial chromosomal aberration study was

performed by exposing CHO cells for 3 hr to Trehalose concentrations of 1250, 2500, and 5000

μglmL in both the absence and presence of MA. Metaphase cells were then harvested at 21 hr

after initiation of exposure. In the absence and presence of MA, no statistically significant

increase in the frequency of cells with structural chromosomal aberrations was observed at any

dose. As expected, the positive controls (methyl methane sulfonate and cyclophosphamide)

produced significant increases in the number of cells with structural chromosomal aberrations.

For the replicate experiment cultures were exposed to Trehalose for 21 hr in the absence

of MA or 3 hr in the presence of MA. Cultures were then harvested at 21 hr and, in the case of

the vehicle control and high dose, at 45 hr. At the 21- and 45-hr harvest times no statistically

significant increases in the frequency of cells with structural chromosomal aberrations were

observed at any dose in the presence or absence of MA. The positive controls produced

significant increases in the number of cells with structural chromosomal aberrations The 45-hr

harvest was also evaluated for frequency of polyploidy at the 5000μg/rnL. No increase in

polyploidy above control values were observed.

Based on the criteria established in the protocol, it is concluded that Trehalose elicits a

negative response in the CHO chromosomal aberration assay in the absence and presence of

metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

one Guideline study present, no effects observed

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9. July 1997 - 12. November 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

mammalian bone marrow chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
50615
- Expiration date of the lot/batch:
not stated
- Purity test date: not stated

RADIOLABELLING INFORMATION (if applicable)
not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
room temperature, protected from Light
- Stability under test conditions: assumed stable
- Solubility and stability of the test substance in the solvent/vehicle:
soluble in water and assumed stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:
assumed stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
none

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:

Species:

mouse

Strain:

Swiss Webster

Details on species / strain selection:

no further details, standard species for Assay

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Hollister CA
- Age at study initiation: Males: 6-7 weeks
Females: 7-8 weels
- Weight at study initiation: Males: 24.1-31.3 g
Females: 21.8-29.3 g
- Assigned to test groups randomly: randomly
- Fasting period before study: not specified
- Housing: no more than 5/cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 66 -72 °F
- Humidity (%): 46-54 %
- Air changes (per hr): not stated
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: To:

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: water;
- Justification for choice of solvent/vehicle: common solvent in nature and for testing
- Concentration of test material in vehicle: depending on dose, 125 / 250 / 500 mg/mL
- Amount of vehicle (if gavage or dermal): not applicable
- Type and concentration of dispersant aid (if powder): not applicable
- Lot/batch no. (if required): not applicable
- Purity:

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose solutions were prepared by diluting weighted amounts of trehalose in sterile water for injection, USP. Dose preparation was mixed with a stir bar for approximately 45 minutes and then sonicated for approximately 5 minutes, before serial dilutions were prepared.

Duration of treatment / exposure:

single dose

Frequency of treatment:

single dose

Post exposure period:

24 or 48 hours

Dose / conc.:

0 mg/kg bw (total dose)

Dose / conc.:

1 250 mg/kg bw (total dose)

Dose / conc.:

2 500 mg/kg bw (total dose)

Dose / conc.:

5 000 mg/kg bw (total dose)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): not stated, but according to guideline
- Route of administration: intraperitoneal
- Doses / concentrations:
The positive control was prepared by adding 25 mg
of cyclophosphamide to sterile water. It was then
brought up to a volume of 10 mL. Dose
formulations were stored at room temperature until
used.

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
not stated
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
1 dosage
DETAILS OF SLIDE PREPARATION:
At 24, or 48 hrs post-treatment, mice were
euthanized with C02 followed by cervical
disolcation. femurs were removed and three bone
marrow slides were prepared, fixed in absolute
methanol and stained with acridine orange.

METHOD OF ANALYSIS:
Two principal parameters were determined using 1
slide/animal: 1) the number of PCE among 200 total
erythrocytes (RBC) per animal, and 2) the number
of micronucleated RNA positive erythrocytes (PCE)
among a total of 2,000 PCE per animal.
Two principal parameters were determined using 1
slide/animal: 1) the number of PCE among 200 total
erythrocytes (RBC) per animal, and 2) the number
of micronucleated RNA positive erythrocytes (PCE)
among a total of 2,000 PCE per animal.
additional requirement that the micronuclei exhibit
the bright yellow fluorescence characteristic of
acridine orange stain. The data from a given slide
were registered directly to an IBM PC data file
during scoring (using the SRI-developed software
program MNSCORE). After analysis, the slides
were decoded and the data summarized by using a
decoding program in an IBM PC (using the SRIdeveloped
software program MNSUM).
OTHER:

Evaluation criteria:

Positive. The test article was considered positive if 1 )there was a statistically
significant (p<0.05) increase in micronucleated PCE, 2) this increase was dose-related,
and 3) the micronucleated PCE frequency was greater than the mean historical
micronucleus vehicle frequency± 2 SD.
Negative. The test article was considered negative if the none of the criteria for a
positive or inconclusive response were met.
Inconclusive. The results were considered inconclusive if a statistically
significant increase in the micronucleated PCE frequency was observed that was either
not dose-related or was not greater the mean historical micronucleus frequency± 2 SD.

Statistics:

Clinical Observations were not evaluated statistically. Body weights were
evaluated by the LABCAT computerized data capture system (Innovative Programming ·
Associates, Inc., Princeton, NJ; module BWT/CO v.4.64). One-way analysis of variance
(ANOVA) followed by Dunnett's test to compare each treated group with the vehicle
control group were performed. The probability level for all null hypothesis rejection was
p<0.05.
For the micronucleus assay, the data were analyzed separately for each gender and
each harvest time. The ratio of micronucleated PCEs to total PCEs and the ratio of PCEs
to total erythrocytes in percentages were calculated for each animal.
The frequency of micronucleated PCEs was statistically analyzed using the
Cochran-Armitage test (using the SRI-developed software COCHRAN) for trend in
binomial proportions, to determine if a significant dose-response relationship was present,
and the normal test for equality of binomial proportions (Kastenbaum and Bowman,
1970) to determine whether values for individual dose groups were statistically different
from those from vehicle controls (using the SRI-developed software package MNSUM).
These tests and their rationale are discussed in the ASTM Standard Guide for the Conduct
of Micronucleus in Mammalian Bone Marrow Erythrocytes and other publications
(ASTM Committee, 1988; Margolin et al., 1983).

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the micronucleus experiment, 10 mice per sex per dose group were treated with
a single i.p. dose of Trehalose at 1250, 2500, or 5000 mg/kg. Five mice per sex per dose.
group were sacrificed approximately 24, or 48 hours after treatment and bone marrow
was evaluated for cytotoxicity and micronucleus formation. A vehicle control group and
a cyclophosphamide positive control group were treated similarly (except for n=4 at 24
hrs in the cyclophosphamide positive control group) and evaluated concurrently with the
Trehalose-treated test groups. No statistically significant increases in micronucleated
PCE frequency was seen in any of the Trehalose-treated animals. As expected, the
frequency of micronucleated PCEs of male mice treated with cyclophosphamide was
statistically significant at the 24- and 48-hr harvest using the binomial proportions test.
The data from this experiment was considered acceptable because the frequency
of micronucleated PCEs in the vehicle control groups was within SRI's normal historical
mean± 2 SD (Appendix D), the administration of the cyclophosphamide positive control
resulted in a statistically significant elevation of micronucleated cells, and there were at
least three surviving animals of each sex in two or more dose groups with a PCE/RBC
ratio greater than or equal to 0.1.

Conclusions:

In conclusion, Trehalose meets the criteria for a negative response in the mouse bone marrow micronucleus assay.

Executive summary:

The genotoxic potential of Trehalose administered by intraperitoneal (ip) injection to

induce micronucleus formation in bone marrow erythrocytes was determined in Swiss-Webster

mice.

In the micronucleus experiment, 10 mice per sex per dose group were treated with a

single i.p. dose of Trehalose at 1250, 2500, or 5000 mg/kg. Five mice per sex per dose group

were sacrificed approximately 24, or 48 hours after treatment and bone marrow was evaluated for

cytotoxicity and micronucleus formation. A vehicle control group and a cyclophosphamide

positive control group were treated similarly and evaluated concurrently with the Trehalosetreated

test groups. There were no early deaths or significant clinical signs. No statistically

significant increases in micronucleated PCE frequency was seen in any of the Trehalose-treated

animals.

In conclusion, Trehalose meets the criteria established in the study protocol for a negative

response in the mouse bone marrow erythrocyte micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

No mode of action identified.

Justification for classification or non-classification

The presented information is conclusive but not sufficient for classification.