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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
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Start Date (First Day of Dosing): 30 March 2015, Experimental Completion Date: 15 April 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
None
Specific details on test material used for the study:
None

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
Sprague-Dawley (Hsd:SD) rats were received from Harlan, Frederick, MD on 25 March 2015.

The age at time of initiation and body weights of the rats assigned to the study groups at randomization were within the following ranges:

Sex: Male;

Age at Time of Initiation: 6 weeks;

Body Weight Range at Time of Randomization: 166.6-188.8 grams.

Animal Welfare Provisions, Receipt and Acclimation:
This study was not duplicative or unnecessary. The number of animals, procedures, and design used for this study, were reviewed and approved by the BioReliance Institutional Animal Care and Use Committee. All procedures performed at BioReliance which involved animals, followed the specifications recommended in the most current version of The Guide for the Care and Use of Laboratory Animals adopted by BioReliance. The animals were acclimated for 5 days. Animals were observed daily for signs of illness or poor health. All animals were judged to be healthy prior to use in the study.

Housing:
Animals were housed in a controlled environment at 72 ± 3°F and 50 ± 20% relative humidity with a 12-hour light/dark cycle. The light cycle may have been interrupted for study related activities. The animal rooms were supplied with at least 10 changes of fresh HEPA-filtered air per hour. Animals of the same sex were housed up to five per Micro-Barrier cage. Cages were placed on racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system.

Bedding, Food and Water:
Heat treated hardwood chips were used for bedding to absorb liquids. A certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet) was provided ad libitum. The food was analyzed by the manufacturer for the concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates and specified nutrients. Animals had free access to tap water, which met U.S. EPA drinking water standards [Washington Suburban Sanitary Commission (WSSC) Potomac Plant]. Drinking water was monitored at least annually for levels of specified microorganisms, pesticides, heavy metals, alkalinity and halogens. The results of bedding, food and water analyses are on file at BioReliance. There were no contaminants in the bedding, feed and water that were expected to interfere with the study.

Randomization and Identification:
Animals were assigned to groups using a randomization procedure within Microsoft Excel. At the time of randomization, the weight variation of animals did not exceed ±20% of the mean weight. Following randomization, animals were identified by sequentially numbered ear tags. The cage card contained, at least, the animal number(s), sex, study number, treatment group number, dose level, test substance ID and route of administration. Cage cards were color coded by treatment group. Raw data records and specimens were also identified by the unique animal number.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Deionized water
Details on exposure:
All dose formulations were administered at a volume of 10 mL/kg by oral gavage using appropriately sized disposable polypropylene syringes with gastric intubation tubes (needles). The oral route has been routinely used and is widely-accepted for use in the mammalian bone marrow erythrocyte micronucleus assay. Body weights were recorded prior to the first dose for the purpose of dose volume calculations. Animals were observed prior to, approximately one and two hours after dose administration and daily thereafter for clinical signs of toxicity.
Frequency of treatment:
Once
Post exposure period:
24 or 48 hour
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000, 2000
Basis:
nominal conc.
No. of animals per sex per dose:
Vehicle control: 10 males;
High dose group (2000 mg/kg): 10 males;
Other groups- low (500 mg/kg) and middle dose group (1000 mg/kg) and positve control (Cyclophosphamide 40 mg/kg): 5 males
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide: 40 mg/kg

Examinations

Details of tissue and slide preparation:
Femoral bone marrow was collected at approximately 24 or 48 hours after the final dose, as indicated above. Animals were euthanized by carbon dioxide inhalation. Immediately following euthanasia, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow was transferred to a centrifuge tube containing 2 mL fetal bovine serum, the cells were pelleted by centrifugation, and the supernatant was drawn off leaving a small amount of fetal bovine serum with the pellet. Cells were re-suspended and a small drop of the bone marrow suspension was spread onto a clean glass slide. At least two slides were prepared from each animal, air dried and fixed by dipping in methanol. One set of slides was stained with acridine orange for microscopic evaluation. The other set of slides was kept as backup and will be archived at report finalization. Each slide was identified by the harvest date, study number, and animal number. Slides were coded using a random number table by an individual not involved with the scoring process.
Evaluation criteria:
Criteria for a Valid Test
The MnPCE frequency of the vehicle controls should be consistent with the historical vehicle control range, and must be ≤ 0.4% MnPCEs (Aikihiro et al., 1998), and the positive control must induce significant increase (p≤0.05) in MnPCE frequency as compared to the concurrent vehicle control.
Five animals/group were available for analysis.

Evaluation of Test Results
Once the criteria for a valid assay were met, the results were evaluated. Test substance was considered to be positive if it induced a significant increase in MnPCE frequency (p ≤ 0.05) at any dose level or sampling time compared to the concurrent vehicle control. The test substance was considered to be negative if no significant increase in MnPCE frequency was observed (p >0.05) compared to the concurrent vehicle control. Other criteria may have been used in reaching a conclusion about the study results (e.g., magnitude of any increase, dose-dependency, comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment to clearly report and describe any such considerations.
Statistics:
Kastenbaum-Bowman tables

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Definitive Micronucleus Assay
No mortality occurred at any dose level during the course of the definitive assay. All rats appeared normal throughout the observation period.
Clinical signs are presented in Table 1.

Bone Marrow Analysis
No appreciable reductions in the PCEs/EC ratio in the test substance groups compared to the vehicle control group was observed indicating the test article did not induce cytotoxicity.
No statistically significant increase in the incidence of MnPCEs in the test-substance treated groups was observed relative to the negative control group (p > 0.05, Kastenbaum-Bowman tables). The positive control induced a statistically significant increase in the incidence of MnPCEs (p < 0.05, Kastenbaum-Bowman tables). The number of MnPCEs in the vehicle control groups did not exceed the historical control range.
The incidence of MnPCEs per 10,000 PCEs scored (2000 PCEs/animal) and the proportion of polychromatic erythrocytes per total erythrocytes are summarized and presented for each treatment group by sacrifice time in Table 2. Individual animal data is presented in Table 3 and Table 4.
Based upon this, all criteria for a valid test were met as specified in the protocol. The Common Technical Document (CTD) Summary Table is included in Appendix III.

Any other information on results incl. tables

Table 1: Definitive Assay – Clinical Signs

Treatment

Observation

Number of Animals With Observed Signs/Number of Surviving Animals

Number of Animals Found Dead/Total Number of Animals Dosed

Males

Males

Day 0

Day 1

Day 2

Pre-Dose

Post-Dose

1 Hr

2 Hr

Deionized water

Normal

10/10

10/10

10/10

10/10

5/5

0/10

FAT 40868/A TE

500 mg/kg/day

Normal

5/5

5/5

5/5

5/5

NA

0/5

1000 mg/kg/day

Normal

5/5

5/5

5/5

5/5

NA

0/5

2000 mg/kg/day

Normal

10/10

10/10

10/10

10/10

5/5

0/10

CP

40 mg/kg/day

Normal

5/5

5/5

5/5

5/5

NA

0/5

N/A = No data due to 24 hour bone marrow collection.

Table 2: Summary of Bone Marrow Micronucleus Analysis

Treatment

Sex

Time

(h)

Number of animals

PCE/Total

Erythrocytes

(Mean +/- SD)

Change from

Control (%)

 

Number of

MnPCE/1000 PCE

(Mean +/- SD)

Number of

MnPCE/PCE

scored

Deionized water

M

24

5

0.531±0.00

---

0.1±0.22

1

/

1000

500 mg/kg

M

24

5

0.527±0.00

-1

0.3±0.27

3

/

10000

1000 mg/kg

M

24

5

0.528±0.01

-1

0.2±0.27

2

/

10000

2000 mg/kg

M

24

5

0.535±0.01

1

0.3±0.45

3

/

10000

40 mg/kg

M

24

5

0.548±0.00

3

23.6±1.71

*236

/

10000

Deionized water

M

48

5

0.526±0.01

---

0.2±0.27

2

/

1000

2000 mg/kg

M

48

5

0.527±0.01

0

0.3±0.27

3

/

10000

*Statistical significant increase compared to vehicle control p ≤ 0.05 (Kastenbaum-Bowman Tables)

PCE – polychromatic erythrocytes; MnPCE – micronucleated polychromatic erythrocytes

Table 3: Induction of Micronucleated Polychromatic Erythrocytes in Bone Marrow Collected 24 Hours Post-Dose Administration

Treatment

 

Sex

 

Animal Number

PCE/Total

Erythrocytes

Micronucleated PCE

(Number/PCE scored)

Deionized water

M

1

0.527

0/2000

2

0.526

0/2000

3

0.534

0/2000

4

0.531

0/2000

5

0.535

1/2000

FAT 40868/A TE

500 mg/kg

M

6

0.529

0/2000

7

0.529

1/2000

8

0.531

1/2000

9

0.524

1/2000

10

0.522

0/2000

FAT 40868/A TE

1000 mg/kg

M

11

0.531

0/2000

12

0.527

1/2000

13

0.524

1/2000

14

0.536

0/2000

15

0.521

0/2000

FAT 40868/A TE

2000 mg/kg

M

16

0.541

0/2000

17

0.541

1/2000

18

0.529

0/2000

19

0.537

/22000

20

0.526

0/2000

Cyclophosphamide

40 mg/kg

M

21

0.552

48/2000

22

0.545

46/2000

23

0.553

49/2000

24

0.543

42/2000

25

0.547

51/2000

 

Table 4: Induction of Micronucleated Polychromatic Erythrocytes in Bone Marrow Collected 48 Hours Post-Dose Administration

Treatment

 

Sex

 

Animal Number

PCE/Total

Erythrocytes

Micronucleated PCE

(Number/PCE scored)

Deionized water

M

26

0.528

0/2000

27

0.535

1/2000

28

0.521

1/2000

29

0.517

0/2000

30

0.530

0/2000

FAT 40868/A TE

2000 mg/kg

M

31

0.532

1/2000

32

0.525

1/2000

33

0.519

1/2000

34

0.536

0/2000

35

0.524

0/2000

PCE – polychromatic erythrocytes; MnPCE – micronucleated polychromatic erythrocytes

 

APPENDIX III: Common Technical Document (CTD) Table

Report Title:In VivoMicronucleus Assay in Rats

Test Substance:FAT 40868/A TE

Test for Induction of:

Bone marrow micronuclei

Treatment Schedule:

One dose

BioReliance Study No.:

AE04PT.125M012REACH.BTL

Species/Strain/Sex:

Hsd/SD rats/ males

Sampling Times:

24 & 48 hours post-dose

Age:

6 weeks old

Route of Administration:

Oral gavage

GLP Compliance:

Yes

Cells Evaluated:

Polychromatic erythrocytes (PCE)

Vehicle for the Test Substance Formulation:

Vehicle/Positive control:

Deionized water

Sterile water for injection/Cyclophosphamide (CP)

No. of Cells Analyzed/Animal:

2000 PCE/animal

Feeding Condition:

Ad libitum

Date of Dosing:

30 March 2015

Special Features:

N/A

Toxic/Cytotoxic Effects:

No mortality occurred at any dose level during the course of the definitive assay. All rats appeared normal throughout the observation period.

Genotoxic Effects:

No statistically significant increase in the incidence of MnPCEs in the test-substance treated groups was observed relative to the negative control group (p > 0.05, Kastenbaum-Bowman tables). The positive control induced a statistically significant increase in the incidence of MnPCEs (p < 0.05, Kastenbaum-Bowman tables). The number of MnPCEs in the vehicle control groups did not exceed the historical control range.

Evidence of Exposure:

N/A

 

Sampling Time

Controls/Test Substances

Dose Level mg/kg

Sex/No. of Animals/Group

PCE/ Total Erythrocyte (Mean± SD)

Number MnPCE/PCE scored

24 hrs post-dose

Vehicle**

0

Males/5/group

0.531 ± 0.00

1/10000

FAT 40868/A TE

500

Males/5/group

0.527 ± 0.00

3/10000

1000

Males/5/group

0.528 ± 0.01

2/10000

2000

Males/5/group

0.535 ± 0.01

3/10000

Cyclophosphamide

40

Males/5/group

0.548 ± 0.00

*236/10000

48 hrs post-dose

Vehicle**

0

Males/5/group

0.526 ± 0.01

2/10000

FAT 40868/A TE

2000

Males/5/group

0.527 ± 0.01

3/10000

*Statistically significant increase, p ≤ 0.05 (Kastenbaum-Bowman Tables, binomial distribution)

**Deionized water

PCE: Polychromatic Erythrocytes; MnPCE: Micronucleated Polychromatic Erythrocytes

Applicant's summary and conclusion

Conclusions:
FAT 40868/A TE did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes. Therefore, FAT 40868/A was concluded to be negative.
Executive summary:

The test substance, FAT 40868/A TE, was evaluated for its clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (PCE) cells in rat bone marrow. Deionized water was selected as the vehicle. Test and/or control substance formulations were administered at a dose volume of 10 mL/kg by oral gavage. In the definitive assay, dose levels tested were 500, 1000 or 2000 mg/kg body weight. Groups 1 and 4 consisted of 10 animals each designated for either 24 or 48 hour bone marrow collections and Groups 2, 3 and 5 consisted of 5 animals each designated for 24 hour bone marrow collection. Following scheduled euthanasia times, femoral bone marrow was collected; bone marrow slides were prepared and stained with acridine orange. Bone marrow cells [polychromatic erythrocytes (2000 PCEs/animal)] were examined microscopically for the presence of micronuclei (micronucleated PCEs; MnPCEs) and statistical analysis of data was performed using the Kastenbaum-Bowman Tables (binomial distribution, p ≤ 0.05). The ratio of polychromatic erythrocytes (PCEs) to total erythrocytes (EC) in the test substance groups relative to the vehicle control groups was also evaluated to reflect the test substance’s cytotoxicity. Under the conditions of this study, the administration of FAT 40868/A TE at doses up to and including a dose of 2000 mg/kg was concluded to be negative in the Micronucleus assay.