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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 Starting Date (first day of data collection): 05 October 2015 ; Experimental Start Date (first day test substance administered to test system): 12 October 2015 and Experimental Completion Date: 30 October 2015.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Wistar Han female rats were received from ENVIGO RMS, Dublin, VA on 05 October 2015 (definitive assay).
- Age at study initiation: 6 weeks
- Weight at study initiation: 112.7- 138.5 grams
- Acclimation period: 7 days

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 (ENVIGO 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.


Justification for the test system: This species has been routinely used as an animal model of choice for the mammalian bone marrow erythrocyte micronucleus assay. This strain was an outbred strain that maximizes genetic heterogeneity and therefore tends to eliminate strain-specific response to the test substance.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
The vehicle used in the assay was deionized (DI) water obtained from BioReliance.
Details on exposure:
Preparation of Test Substance Dose Formulations:
Dose formulations were prepared prior to dose administration as follows:
An appropriate amount of test substance was weighed into a suitable sized amber vial with a PTFE stir bar. An appropriate amount of vehicle was added and mixed magnetically until homogenous. The dose formulations thus prepared appeared to be red solutions and were stored at room temperature until use.
Residual dose formulations were discarded after use.

Dose Formulation Collection and Analysis.
Analyses to determine the concentration, uniformity and stability of the test substance dose formulations were not performed.

Duration of treatment / exposure:
Not applicable
Frequency of treatment:
One single application
Post exposure period:
Not applicable
Doses / concentrations
Remarks:
Doses / Concentrations:
0/500/1000/2000 mg/kg body weight
Basis:
nominal conc.
No. of animals per sex per dose:
- At 0 mg/kg bw: 10 animals.
- At 500 mg/kg bw: 5 animals
- At 1000 mg/kg bw: 5 animals
- At 2000 mg/kg bw: 10 animals
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive Control
For positive control, slides (fixed and unstained) generated from BioReliance Study Number AE36SK.125M012.BTL, were included to verify scoring. These slides were generated from rats treated once with cyclophosphamide monohydrate (CP) at 40 mg/kg and the bone marrow harvested 24 hours after the treatment.
The stability of the vehicle and positive control components and their mixtures was demonstrated by acceptable results that met the criteria for a valid test.

Examinations

Tissues and cell types examined:
Bone Marrow Collection
Femoral bone marrow was collected at approximately 24 or 48 hours after the final dose, as indicated. 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.
Details of tissue and slide preparation:
Preparation of Micronucleus Slides
The bone marrow was transferred to a centrifuge tube containing 2 mL fetal bovine serum, the cells were pelleted by centrifugation, and the supernatant 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 four slides were prepared from each animal, air dried and fixed by dipping in methanol. One set of two slides (including at least 5 sets of two positive control slides) were stained with acridine orange for microscopic evaluation. The other set of slides was kept as backup. Each slide was identified by the harvest date, study number, and animal number (or slide number for positive control slides). Slides were coded using a random number table by an individual not involved with the scoring process


Scoring of Micronucleus Slides
Slides were evaluated by fluorescent microscopy. The staining procedure permits the differentiation by color of polychromatic and normochromatic erythrocytes (bright orange PCEs and ghost like, dark green NCEs, respectively).
The criteria for the identification of micronuclei are those of Schmid (1975). Micronuclei are brightly stained bodies that generally are round and that generally are between 1/20 and 1/5 the size of the PCE. The frequency of micronucleated cells was recorded with cells containing one or more micronuclei counted as one micronucleated PCE (MnPCE).
At least 4000 PCEs/animal were scored for the presence of micronuclei (MnPCEs) whenever possible. In addition, at least 500 total erythrocytes (PCEs + NCEs) were scored per animal to determine bone marrow cytotoxicity.
Stained slides will be discarded prior to report finalization.
Evaluation criteria:
Evaluation of Test Results
The test substance was considered to have induced a positive response if
• at least one of the test substance doses exhibits a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
• when multiple doses are examined at a particular sampling time, the increase is dose-related (p ≤ 0.01), and
• results of the group mean or of the individual animals in at least one group are outside the 95% control limit of the historical negative control data.
The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met and there is evidence that the bone marrow was exposed to the test substance (unless intravenous administration was used).
Statistics:
Statistical analysis was performed on the micronucleus frequency (MnPCE%) and PCE% using the animal as the unit. The mean and standard deviation of MnPCE% and PCE% were presented for each treatment group.
The use of parametric or non-parametric statistical methods in evaluation of data was based on the variation between groups. The group variances for micronucleus frequency for the vehicle and test substance groups at the respective sampling time were compared using Levene’s test (significant level of p  0.05). If the variation between groups is found not to be significant, a parametric one-way ANOVA was performed followed by a Dunnett’s post-hoc analysis to compare each dose group to the concurrent vehicle control. If Levene’s test indicates heterogeneous group variances (p  0.05), a non-parametric statistical method (Kruskal-Wallis test and/or Mann-Whitney test) may be used in evaluation of the data.
A linear regression analysis was conducted to assess dose responsiveness in the test substance treated groups (p 0.01).
A pair-wise comparison (Student’s T-test) was used to compare the positive control group to the concurrent vehicle control group.

Results and discussion

Test results
Sex:
female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Micronucleus Assay
No mortality occurred at any dose level during the course of the definitive assay. All animals appeared normal throughout the observation period.

Bone Marrow Analysis
The scoring results and a statistical analysis of data indicated the following:
• No appreciable reductions in the PCEs/EC ratio in the test substance groups compared to the vehicle control group were observed indicating the test 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 (ANOVA, p > 0.05, Dunnett’s post hoc analysis).
• The positive control, CP, induced a statistically significant increase in the incidence of MnPCEs (Student’s t test, p ≤ 0.05).
• The number of MnPCEs in the vehicle control groups did not exceed the historical control range (Appendix I). Based upon this, all criteria for a valid test were met as specified in the protocol.
Based upon this, all criteria for a valid test were met as specified in the protocol.

Any other information on results incl. tables

Micronucleus Assay – Clinical Signs of Toxicity:

Treatment

mg/kg/day

Observation

Number of Animals With Observed Signs/Number of Surviving Animals

Number of Animals Found Dead/Total Number of Animals Dosed

Females

Day 1

Day 2

Day 3

Females

Pre-Dose

Post-Dose

1 Hr

2 Hr

Deionized water

Normal

10/10

10/10

10/10

10/10

5/5

0/10

FAT-40870/A TE

500

Normal

5/5

5/5

5/5

5/5

N/A

0/5

1000

Normal

5/5

5/5

5/5

5/5

N/A

0/5

2000

Normal

10/10

10/10

10/10

10/10

5/5

0/10

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

Micronucleus assay- Mean group body weight.

 Treatment

 

Group Mean Body Weights (g ± SD)

% Change ¹

 

mg/kg/day

 Sex

Day 1

Day of Euthanasia

Day of Euthanasia

Mortality ²

Deionized water

24 Hour

F

144.7

147.0

1.6%

0/5

±6.9

±5.6

48 Hour

F

136.1

140.7

3.4%

0/5

±7.6

±9.7

FAT-40870/A TE

500

F

136.1

138.7

1.9%

0/5

±3.9

±2.9

1000

F

142.7

143.0

0.2%

0/5

±6.0

±4.7

2000

F

135.0

138.4

2.5%

0/5

24 Hour

±2.1

±2.2

F

139.4

144.6

3.7%

0/5

48 Hour

±8.0

±7.0

   

SD = Standard deviation, F = Females

 1 % Change =[(Post-treatment weight - Pretreatment weight) x 100]/Pretreatment weight

 2 Reported as number of animals found dead after dose administration/total number tested.

Summary of Bone Marrow Micronucleus Analysis.

Treatment

Time

%PCE

Change from

% MnPCE

Number of

mg/kg

Gender

(Hrs)

Animals

(Mean +/- SD)

Control (%)

(Mean +/- SD)

MnPCE/PCE Scored

Deionized water

F

24

5

52.8

±

0.9

---

0.13

±

0.08

25

/20000

 

 

 

 

 

 

FAT 40870/A TE

500

F

24

5

53.3

±

2.3

1

0.09

±

0.01

17

/20000

 

 

 

 

 

 

1000

F

24

5

51.2

±

2.3

-3

0.08

±

0.02

16

/20000

 

 

 

 

 

 

2000

F

24

5

51.8

±

1.1

-2

0.13

±

0.02

26

/20000

 

 

 

 

 

 

CP, 40

(Slide Scoring Control)

M

24

5

46.5

±

2.2**

-12

4.72

±

0.29**

943

/20000

 

 

 

 

 

 

Deionized water

F

48

5

52.0

±

0.4

---

0.10

±

0.08

20

/20000

 

 

 

 

 

 

FAT 40870/A TE

2000

F

48

5

51.4

±

1.5

-1

0.12

±

0.03

23

/20000

 

 

 

 

 

 

  *p < 0.05 or **p < 0.01, One-Way ANOVA with Post-Hoc analysis or T-Test, F – Females, M - Males

24 Hrs MnPCE GLM P-value = 0.216, R-sqr = 23.70%

 

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

Treatment 

 

Animal No.

%PCE

Micronucleated PCE

 

mg/kg

Sex

(Number/PCE Scored)

(%MnPCE)

Deionized water

F

1

51.8

 

3

/4000

0.08

2

52.8

 

3

/4000

0.08

3

52.8

 

2

/4000

0.05

4

52.6

 

7

/4000

0.18

5

54.2

 

10

/4000

0.25

 

 

 

FAT 40870/A TE

F

6

57.2

 

3

/4000

0.08

500

7

53.2

 

4

/4000

0.10

8

51.6

 

4

/4000

0.10

9

51.4

 

3

/4000

0.08

10

53.2

 

3

/4000

0.08

 

 

 

FAT 40870/A TE

F

11

53.4

 

3

/4000

0.08

1000

12

47.4

 

4

/4000

0.10

13

51.6

 

2

/4000

0.05

14

51.2

 

4

/4000

0.10

15

52.6

 

3

/4000

0.08

 

 

 

FAT 40870/A TE

F

16

51.2

 

4

/4000

0.10

2000

17

51.0

 

5

/4000

0.13

18

53.6

 

6

/4000

0.15

19

51.4

 

5

/4000

0.13

20

51.8

 

6

/4000

0.15

 

 

 

CP

M

CP31/32

44.6

 

180

/4000

4.50

40

CP33/34

49.4

 

198

/4000

4.95

CP35/36

45.0

 

203

/4000

5.08

CP37/38

48.4

 

175

/4000

4.38

CP39/40

45.2

 

187

/4000

4.68

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

Treatment 

 

Animal No.

%PCE

Micronucleated PCE

 

 

 

 

mg/kg

Sex

 

 

(Number/PCE Scored)

(%MnPCE)

 

 

 

Deionized water

F

21

52.0

 

2

/4000

0.05

 

 

 

 

22

52.6

 

9

/4000

0.23

 

 

 

23

51.8

 

2

/4000

0.05

 

 

 

24

51.6

 

2

/4000

0.05

 

 

 

25

52.0

 

5

/4000

0.13

 

 

 

 

 

 

 

 

 

FAT 40870/A TE

 

F

26

52.6

 

4

/4000

0.10

2000

 

 

27

52.4

 

6

/4000

0.15

 

 

 

28

50.2

 

3

/4000

0.08

 

 

 

29

49.4

 

6

/4000

0.15

 

 

 

30

52.2

 

4

/4000

0.10

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of the assay described in this report, FAT 40870/A TE was concluded to be negative for the induction of MnPCEs in the In Vivo Mammalian Erythrocyte Micronucleus Assay in Rats.
Executive summary:

The test substance, FAT 40870/A TE, was evaluated for its clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (PCE) cells in female rat bone marrow. Deionized water was used as the vehicle. Test and/or control substance formulations were administered at a dose volume of 10 mL/kg via oral gavage.

In the definitive assay, the dose levels tested were 500, 1000, and 2000 mg/kg based on the toxicity information provided by the Sponsor.

No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes scored (PCEs/EC ratio) was observed in the test substance groups compared to the vehicle control group, indicating the test did not induce cytotoxicity. No statistically significant increase in the incidence of micronucleated PCEs (MnPCEs) in the test substance treated groups was observed relative to the vehicle control groups. The positive control induced a statistically significant increase in the incidence of MnPCEs. The number of MnPCEs in the vehicle control groups did not exceed the historical control range.  

 

Under the conditions of this study, the administration of FAT 40870/A TE at doses up to and including a dose of 2000 mg/kg was concluded to be negative in theIn VivoMammalian Erythrocyte Micronucleus Assay in female rats.