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Diss Factsheets

Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 30, 2005 to March 31, 2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
: see overall remarks section for details.
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
478-130-6
EC Name:
-
Cas Number:
50940-49-3
Molecular formula:
C9H12O6
IUPAC Name:
2-(acryloyloxy)ethyl hydrogen succinate
Details on test material:
Test article ID: MAES
Test article description: Clear, viscous liquid.
Lot No: C513708-A
Storage conditions: Ambient temperature (temperature range was 19 to 26°C
Purity: 94%

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (Wilmington, MA).
- Age at study initiation: 8 weeks.
- Weight at study initiation: 24 - 35 g.
- Assigned to test groups randomly: Yes
- Fasting period before study: No.
- Housing: Mice were group-housed, in polycarbonate static cages containing Sani Chip screened soft wood as bedding. Up to five males or five females ot various treatment groups occupied a single cage for the dose range finding assay. Up to five males or five females of the same treatment group occupied a single cage for the definitive and repeat assays.
- Diet (e.g. ad libitum): A commercial diet was available to the mice ad libitum.
- Water (e.g. ad libitum): Tap water was available to the mice ad libitum.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22ºC
- Humidity (%):
Dose range finding assay,67-83%
Definitive assay, 64-84%
Repeat assay, 56-76%

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil
- Justification for choice of solvent/vehicle: Chosen by sponsor.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Fresh test article concentrations were prepared each treatment day, one to two hours before treatment initiation. As the test article was very viscous and a higher density than the vehicle, it settled to the bottom of the tube. Therefore, the test article concentrations were mixed by vortexing, shaking, or pipetting, immediately before making dilutions or preparing doses. Positive control chemical was prepared fresh each day one to two hours before initiating treatment. All solutions were kept at room temperature during use, and were discarded after use.

Test article was prepared at 0.01, 0.05, 0.1, 0.5, 1, 5 and 10 ml/kg bw/day for the dose range finding.
Test article was prepared at 0.125, 0.25, 0.5 and 1 ml/kg bw/day for the definitive assay and repeat assay.

TREATMENT VOLUME:
The volume of each test article concentration administered to each animal (for the dose range finding, the definitive assay and the repeat assay) was 10 ml/kg/ bw/day, resulting in the doses specified above.




Duration of treatment / exposure:
Three consecutive days
Frequency of treatment:
Treatment consisted of daily administration of the test article concentrations and controls for three consecutive days at 24 ± 2 hour intervals.
Post exposure period:
30 ± 6 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
Definitive and repeat assays: 0, 0.125, 0.25, 0.5, 1 ml/kg bw.
Basis:
nominal conc.
No. of animals per sex per dose:
Repeat Assay: Five males and five females were assigned to each treatment group.
Control animals:
yes, concurrent vehicle
Positive control(s):
Methyl methanesulfonate (MMS)
- Justification for choice of positive control(s): Historical data confirms the substance to be a positive control.
- Route of administration: Intraperitoneal injection.
- Doses / concentrations: Animals were treated with a dose of 50 mg/kg bw/day.

Examinations

Tissues and cell types examined:
The objective of this Mouse Micronucleus (MN) Assay was to evaluate the ability of four concentrations of a test article to induce cytogenetic damage (i.e., micronuclei) in mouse erythrocyte precursor cells.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Dose Range Finding Assay:
Seven test article treatment groups, plus a vehicle control (0, 0.01, 0.05, 0.1, 0.5, 1, 5, and 10 ml/kg bw), were included for the dose range finding assay, with 2 males and 2 females in each treatment group.

Mice were observed, on each day of treatment, immediately after treatment (within 5 minutes), and at the following intervals after treatment: 10 to 30 m, approximately 1 h, approximately 2 h, approximately 4 h, and approximately 6 h.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Definitive and repeat assay:
Treatment consisted of daily administration of the test article concentrations and controls for three consecutive days at 24 ± 2 hour intervals.
Observation of mice after treatment:
Definitive and repeat assay: mice were observed, on each day of treatment, immediately after treatment (within 5 minutes), and 10 to 30 m after treatment. Animals that exhibited signs of reaction to dose within the 10 to 30 m time period were observed approximately 1 h, approximately 2 h, approximately 4 h, and approximately 6 h after treatment.

DETAILS OF SLIDE PREPARATION:
Blood Collection:
Blood samples were collected into a heparin solution from a tail vein of each mouse approximately 25 hours after the last treatment. Mice were euthanized after blood collection.

Blood Fixation:
The blood samples were fixed into tubes containing ultracold methanol within three hours of collection. The fixed blood samples were stored at -75°C to -90 °C until analysis (at least 4 days after fixation).

Flow Cytometric Analysis:
Blood Cell Preparation:
The fixed blood samples were washed out of fixative with 12 ml ice-cold, Hanks' Balanced Salt Solution (HBSS) and isolated by centrifugation. The resulting cell pellets were stored at 4 ± 4°C until staining.

Staining for Identification of Cell Populations:
Blood cells were stained for flow cytometric (FCM) analysis on the day they were washed out of fixative. Twenty microliter aliquots of each cell pellet were added to tubes containing 80 µl antibody and RNase solution (prepared as follows: 10 µl anti-CD71-FITC, 5 µl anti-CD61-PE, and 1 mg RNase per ml HBSS). This solution prepared cells for analysis by labeling RETs with anti-CD71-FITC, labeling platelets with anti-CD61-PE, and degrading cellular RNA. Cells were incubated in this solution for 30 minutes at 4 ± 4°C and 30 minutes at room temperature. They were then stored at 4 ± 4°C until the addition of 1 to 2 ml ice-cold propidium iodide staining solution (1.25 µg/ml), which was used to label DNA. Cells were then analyzed by flow cytometry.

METHOD OF ANALYSIS:
Analysis of Blood Samples:
Each stained blood sample was quantitatively analyzed by high speed FCM. The stained cells were moved at a high velocity past a laser set to provide 488 nm excitation. The fluorescence from each cell was collected by photomultiplier tubes and the data sent to an online computer.
Using the staining procedure described above, the propidium iodide-stained DNA of the micronuclei emitted a red fluorescence and the anti-CD71-FITC antibody emitted a high green fluorescent signal. Platelets were excluded based on their yellow fluorescence. All remaining blood samples were discarded upon successful FCM analysis.

Methods for Measurement of Bone Marrow Toxicity:
Toxicity is indicated by an 80% reduction in % RET, or doses that cause severe distress (symptoms that do not improve and will likely lead to death) or death. Due to the unexpected deaths in the negative control group, severe distress or death cannot be used as indications of toxicity for the definitive assay.







Evaluation criteria:
Criteria for a Valid Assay:
The criteria for determining whether an assay is valid were met and are:
- The average % MN-RET for the vehicle control group was between 0.10% and 0.4%.
- The average % MN-RET for the positive control group (MMS) was at least 1.0%

Criteria for a Positive Test:
Biological relevance of the data is considered along with the statistical analysis. A test article is considered positive if the assay is valid, and if any of the following conditions is met:
- The ANOVA test for effect of treatment on % MN-RET results in a p value <0.05 for treatment (for parametric tests).
- The regression analysis results in a p value <0.025 (for parametric tests).
- The Kruskall-Wallis test results in a p value <0.05 (for non-parametric tests).

A positive result indicates that the test article induces chromosomal damage or damage to the mitotic apparatus in mouse erythroblasts that results in MN formation.

Significant bone marrow toxicity is indicated by an 80% decrease in the percentage of RET when compared to vehicle control.
Statistics:
Although the definitive assay met criteria for a valid assay, the assay was repeated due to unexpected deaths in the negative control group. For this reason, statistical analysis is only provided for data from the repeat assay.

The data for the repeat assay met all criteria for a valid assay. Due to the fact that there were only two females and four males (in the 1 ml/kg bw treatment group) available for blood collection after three days of treatment, this treatment group is being excluded from the statistical analysis. A test for normality (ShapiroWilk W-Test) was used to determine whether the negative % MN-RET data were normally distributed. A value of p = 0.0107 indicated that the data was not normally distributed, and non-parametric tests were performed.

A Wilcoxon Two Sample test was performed to determine if sex affected % MN-RET. The value of p = 0.0901 indicated that sex did not have a significant effect. A Kruskall-Wallis test was used to determine if treatment affected % MN-RET. The value of p = 0.3803 indicated that treatment did not have a significant
effect.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
(Reaction to dose and death).
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
See Table 1 (Dose Range FInding Assay Individual Animal Data) in attached background material for results.

Only 1 animal of 4 died in the 1 ml/kg bw/day dose group. The remaining three animals in this treatment group appeared to be healthy at the completion of the dose range finding assay. 1 ml/kg bw/day was therefore chosen as the high dose group in the definitive assay.


RESULTS OF DEFINITIVE STUDY
See Tables 2, 3, 4, 5 and 6 (attached background material) for results.

Originally, three test article treatment groups, plus a vehicle control (0, 0.25, 0.5, and 1 ml/kg bw), and a positive control, were included for the definitive assay. The top dose was chosen based on the results from the dose range finding assay, where only one animal died of the four animals treated. Since the remaining 3 animals in this treatment group appeared to be healthy at the completion of the dose range finding assay, low mortality was expected in the definitive assay. Even so, 8 males and 8 females were assigned to the high dose treatment group. Five males and five females were assigned to each remaining treatment group.

Before the second treatment, 6 of the 16 high dose animals had either been euthanized because of severe distress or died. It was the decision of the study director to discontinue this dose, in order to prevent additional animals in this treatment group from dying before the end of the study. Therefore, 0.5 ml/kg bw was the top dose. All animals from the 1 ml/kg bw top dose were euthanized, and a new low dose, 0.125 ml/kg bw was chosen.

During the course of the definitive assay, 2 additional males and 2 additional females were added to the vehicle control treatment group, due to unexpected animal deaths. One additional male was added to the 0.5 ml/kg bw treatment group to compensate for an animal death.

Only 4 females in the negative control treatment group were available for bleeding and flow cytometric analysis. Due to the unexplained deaths in the negative control group, the assay was repeated. One of the negative control females (animal 61) was sent for necropsy and histopathology after euthanasia. These non-GLP reports indicated that a procedural error may have been the cause of death (foreign body in the lung).

REPEAT ASSAY:
See Tables 7, 8, 9, 10 and 11 (attached background material) for results.

Four test article treatment groups, plus a vehicle control (0, 0.125, 0.25, 0.5, and 1 ml/kg bw), and a positive control, were included for the repeat assay. The top dose was chosen based on the results from the dose range finding assay, where only one animal died of the four animals treated. Since the remaining 3 animals in this treatment group appeared to be healthy at the completion of the dose range finding assay, low mortality was expected in the repeat assay. Even so, a fourth treatment group, at a lower dose (0.125 ml/kg bw) was added. Five males and five females were assigned to each treatment group.

During the course of the repeat assay, 1 additional female was added to the 1 ml/kg bw treatment group to compensate for an animal death (due to a procedural error). This replacement animal was treated in a similar manner to the original animals assigned to the group


No significant bone marrow toxicity was observed for animals in any group.



Any other information on results incl. tables

Discussion of Results:

Given the information provided on the test article purity and stability both neat and in combination with the vehicle and the procedures used to prepare the dosing solutions, it is reasonable to expect that the actual amount of test article administered to the animals was consistent with the concentrations of the dosing solutions used in the study. However, the study outcome could not be fully evaluated due to the lack of concentration data on the actual dosing solutions administered to the animals. However, in this experiment, the mean MN-RET frequency for all test article doses was not significantly higher than that of the negative control. Since there was no bone marrow toxicity (reduction in the % RET), there is no indication that the test article or its metabolites reached the bone marrow. However, there was toxicity, as evident by animal deaths and adverse reactions to dose.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the experimental conditions of this assay, test article does not induce micronuclei in CD-1 mice.
Executive summary:

The objective of this Mouse Micronucleus (MN) Assay was to evaluate the ability of four concentrations of a test article to induce cytogenetic damage (i.e., micronuclei) in mouse erythrocyte precursor cells. The study was carried out based on OECD and International Conference for Harmonisation (ICH) testing guidelines.

A dose range finding assay was performed to determine the appropriate doses for the definitive assay. The definitive assay was performed, but due to unexplained deaths in the negative control group, the assay was not considered scientifically valid. A repeat assay was performed, and data for both the definitive and repeat assays are provided in this report. However, only data for the repeat assay was used to evaluate the genotoxic potential of this test article.

For the repeat assay, male and female mice were treated via oral gavage with four concentrations of test article on three consecutive days at 24 ± 2 hour intervals. Signs of toxicity (reaction to dose and death) were observed. Peripheral blood samples were collected 30 ± 6 hours following the last administration.

Flow cytometric analysis of 20,000 reticulocytes (RETs) per blood sample was carried out and the frequency of RETs was determined and used to provide an indication of bone marrow toxicity. Treatment with test article up to 1.0 ml/kg body weight/day did not cause a significant reduction to the RET frequency, indicating a lack of bone marrow toxicity.

The frequency of micronucleated reticulocytes (MN-RETs) was determined to provide an indication of genotoxic potential. Statistical analysis of the data indicates that no significant increase was observed when data from test article groups was compared to data from the negative control group.

Under the experimental conditions of this assay, test article does not induce micronuclei in CD-1 mice.