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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 27 - November 9, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian germ cell cytogenetic assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light
- Stability under test conditions: Expected to be stable.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was ground until it passed through a 425 micron sieve.
- Final dilution of a dissolved solid, stock liquid or gel: The test substance was dissolved in distilled water. A separate dilution was prepared for each dose level in order to maintain a constant dose volume.

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

Test animals

Species:
mouse
Strain:
ICR
Details on species / strain selection:
Mice were selected as micronucleated erythrocytes are not readily removed from the blood.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo Laboratories, Inc.
- Age at study initiation: 8 weeks (preliminary test), 7 weeks (main test)
- Weight at study initiation: Preliminary test - 31-39.7 g males, 24.8-28.5 g females; Main test - 36-39 g males, 23-27 g females
- Assigned to test groups randomly: yes
- Fasting period before study: No
- Housing: Plastic solid bottom cages
- Diet: Harlan Teklad Global 16% Protein Rodent Diet #2016, ad libitum
- Water: Filtered tap water ad libitum
- Acclimation period: 5-18 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 42-55
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: October 27, 2015 To: November 19, 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: distilled water
- Concentration of test material in vehicle: Preliminary test - 500 and 2000 mg/kg bw, Main test - 2000 mg/kg bw
- Amount of vehicle (if gavage or dermal): Enough vehicle for constant volume dose of 5 ml.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Prepared on day of dosing. The test substance was ground until it passed through a 425 micron sieve. The test substance was dissolved in distilled water. A separate dilution was prepared for each dose level in order to maintain a constant dose volume.
Duration of treatment / exposure:
Preliminary test: two doses administered at a 24 hr interval
Main test: two doses administered at a 24 hr interval, positive control single dose on Day 2
Frequency of treatment:
Once daily
Post exposure period:
2 days
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day
Remarks:
Preliminary test only
Dose / conc.:
2 000 mg/kg bw/day
Remarks:
Preliminary and main test
No. of animals per sex per dose:
Preliminary test: 3 animals per group per sex
Main test: 5 animals per group per sex
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide monohydrate
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/kg bw/day

Examinations

Tissues and cell types examined:
Blood, erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on preliminary test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Sampling performed 44-48 hrs after final treatment.

DETAILS OF SLIDE PREPARATION: Duplicate samples of 60-120 microliters of blood per sample were collected. The blood was fixed by rapid pipetting in chilled methanol, and stored for at least three days. The samples were centrifuged, the methanol removed, and the cells suspended in shipping buffer. One set of samples was sent to Litron Laboratories, 3500 Winton PI, Rochester, New York 14623, United Sates of America, for analysis. Cells were stained with fluorescent labeled anti-CD71, fluorescent labeled anit-CD61 antibody, and propidium iodide following RNAse treatment.

METHOD OF ANALYSIS: The cells were analyzed by flow cytometry for a minimum of 4000 immature erythrocytes per animals, and DNA content in both immature and mature erythrocytes.

Evaluation criteria:
Micronucleated immature erythrocytes (MIE) values within expected range based on published values and laboratory control values for negative controls. Clear increase in MIE values outside historical control range for negative control animals in the positive controls. A statistically significant dose-related increase in MIE values as compared to the negative control group with at least two individual animals outside the laboratory control range was considered a positive result.
Statistics:
Analysis of variance followed by Bonferroni-corrected multiple comparison test.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500-2000 mg/kg bw/day
- Clinical signs of toxicity in test animals: No
- Evidence of cytotoxicity in tissue analyzed: No
- Harvest times: 44-48 hrs after final exposure

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No significant difference from negative controls.
- Appropriateness of dose levels and route: The route chosen (oral gavage) is the recommended route. The dose levels were chosen based on the preliminary test. The highest dose in the preliminary test (2000 mg/kg bw/day) was chosen based on the lack of toxicity and cytotoxocity.
- Statistical evaluation: No test substance related change in reticulocyte fraction (% RET) was observed. No statistically significant increase in micronucleus frequency (% MN-NCE) was observed.

Applicant's summary and conclusion

Conclusions:
The test substance is not genotoxic with respect to micronucleus induction.
Executive summary:

An In Vivo Mouse Erythrocyte Micronucleus Test (Flow Cytometry) was performed using the test substance Sucrose Palmitate Stearate MDT. A dose of 2000 mg/kg bw was administered to 5 female and 5 male mice on two consecutive days. The mice were sacrificed two days later, and blood samples drawn for analysis of micronuclei. Other groups of mice were used as negative and positive controls. The test substance Sucrose Palmitate Stearate MDT Grade is not genotoxic with respect to micronucleus induction.