Trehalose

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Trehalose

EC Number:

202-739-6

EC Name:

Trehalose

Cas Number:

99-20-7

Molecular formula:

C12H22O11

IUPAC Name:

trehalose

Test material form:

solid: crystalline

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:

Test animals

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:

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

1 250 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

2 500 mg/kg bw (total dose)

Doses / concentrations 4

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.

Examinations

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).

Results and discussion

Test results

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.

Applicant's summary and conclusion

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.