Registration Dossier

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:
Study Initiation Date: 15 June 2015, Experimental Completion Date: 08 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
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:
male
Details on test animals and environmental conditions:
Male Wistar Han rats were received from Harlan, Dublin, VA on 18 June 2015.
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
BioReliance Study No. AE20DP.125M012REACH.BTL 9
and Use of Laboratory Animals adopted by BioReliance. Animals were observed for signs of illness or poor health and 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:
Arachis Oil
Details on exposure:
All dose formulations were administered at a volume of 10 mL/kg by single oral gavage using appropriately sized disposable polypropylene syringes with gastric intubation tubes (needles).
Frequency of treatment:
once
Post exposure period:
24h and 48h
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:

No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Rats treated once with cyclophosphamide monohydrate (CP) at 40 mg/kg, and the bone marrow harvested 24 hours after treatment.

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
Femoral bone marrow was collected at approximately 24 or 48 hours after dose administration, 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 four slides were prepared from each animal, air dried and fixed by dipping in methanol. One set of two slides (including at least five sets of two positive control 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:
A test substance was considered to have induced a positive response if:
a) at least one of the test substance doses exhibited a statistically significant increase when compared with the concurrent vehicle control (p ≤ 0.05), and
b) when multiple doses were examined at a particular sampling time, the increase was dose-related (p ≤ 0.01), and
c) results of the group mean or of the individual animals in at least one group were outside the 95% control limit of the historical negative control data.
A test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met, and there was evidence that the bone marrow was exposed to the test substance (unless intravenous administration was used).
If the response was neither clearly positive nor clearly negative, or in order to assist in establishing the biological relevance of a result, the data were evaluated by expert judgment and/or further investigations. Possible additional work may have included scoring additional cells (where appropriate) or performing an additional experiment that could employ the use of modified experimental conditions. Such additional work would only be carried out following consultation with, and at the request of, the Sponsor.
In some cases, even after further investigations, the data set precluded making a conclusion of positive or negative, at which time the response was concluded to be equivocal. In such cases, the Study Director used sound scientific judgment and reported and described all considerations.
Statistics:
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). Since the variation between groups was 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.
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:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: 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

Day 1

Day 2

Day 3

Males

Pre-Dose

Post-Dose

1Hr

2Hr

Vehicle:Arachisoil

Normal

10/10

10/10

10/10

10/10

5/5

0/10

FAT 41045/A TE

500 mg/kg

Normal

5/5

5/5

5/5

5/5

N/A

0/5

FAT 41045/A TE

1000 mg/kg

Normal *

5/5

5/5

5/5

5/5

N/A

0/5

FAT 41045/A TE

2000 mg/kg

Normal *

10/10

10/10

10/10

10/10

5/5

0/10

*=Redishfeces were observed in this group on Day 2

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

 

Table 2: Individual Body Weight Data (MALES)

Day 1(g)

Day of Euthanasia(g)

Arachisoil-24 Hour

1

177.4

183.1

2

188.1

197.2

3

179.7

188.8

4

161.1

171.5

5

173.7

184.1

MEAN +/-SD

176.0 +/-9.9 

184.9+/-9.4 

Arachisoil-48 Hour

1

183.0

197.2

2

168.3

182.3

3

181.4

195.2

4

183.9

194.6

5

174.8

189.8

MEAN +/-SD

178.3 +/-6.6

191.8 +/-6.0

FAT 41045/A TE-500 mg/kg

1

174.3

184.2

2

166.8

174.5

3

164.8

173.3

4

173.6

179.4

5

179.6

186.6

MEAN +/-SD

171.8+/-6.0

179.6+/-5.8

FAT 41045/A TE- 1000 mg/kg

1

164.9

172.8

2

176.2

185.7

3

177.5

188.1

4

178.1

176.4

5

186.4

193.6

MEAN +/-SD

176.6+/-7.7

183.3+/-8.6


FAT 41045/A TE-2000 mg/kg24 Hour

1

165.8

173.2

2

171.1

176.3

3

176.8

184.3

4

163.5

170.4

5

180.6

186.7

MEAN +/-SD

171.6+/-7.2

178.2+/-7.1

FAT 41045/A TE-2000 mg/kg48Hour

1

185.0

198.1

2

178.4

195.7

3

182.9

196.9

4

192.4

190.3

5

178.1

207.4

MEAN +/-SD

183.4+/-5.9

197.7+/-6.2

SD = Standard deviation 

 

Table 3: Group Mean Body Weight Data

Treatment

Sex

Group Mean Body Weights (g±SD)

% Change¹

Mortality²

Day 1

Day of Euthanasia

Day of Euthanasia

Arachisoil

24 Hour

M±SD

176.0±9.9

184.9±9.4

5.1%

0/5

48 Hour

M±SD

178.3±6.6

191.8±6.0

7.6%

0/5

FAT 41045/A TE-500 mg/kg

M±SD

171.8±6.0

179.6±5.8

4.5%

0/5

FAT 41045/ATE-1000 mg/kg

M±SD

176.6±7.7

183.3±8.6

3.8%

0/5

FAT 41045/ATE-2000 mg/kg

24 Hour

M±SD

171.6±7.2

178.2±7.1

3.8%

0/5

48 Hour

M±SD

183.4±5.9

197.7±6.2

7.8%

0/5

SD = Standard deviation

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

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

 

Table 4: Summary of Bone Marrow Micronucleus Analysis

Treatment

(mg/kg)

Gender

Time(Hrs)

Animals

%PCE

(Mean +/- SD)

Change from

Control (%)

%MnPCE

(Mean +/- SD)

Number of

MnPCE/PCE Scored

Vehicle-0

M

24

5

59.8±3.7

---

0.09±0.03

17/20000

FAT 41045/A TE- 500

M

24

5

60.4±4.8

1

0.08±0.04

15 /20000

FAT 41045/A TE-1000

M

24

5

58.0±1.4

-3

0.11±0.04

21 /20000

FAT 41045/A TE-2000

M

24

5

59.9±1.3

0

0.12±0.04

23 /20000

PC-40

M

24

5

47.5±6.2**

-21

4.19±0.58**

838 /20000

Vehicle0

M

48

5

57.2±4.1

---

0.06±0.03

12 /20000

FAT 41045/A TE-2000

M

48

5

59.4±5.8

4

0.08±0.02

16/20000

SD = Standard deviation ; **p < 0.01, T-Test

24HrsMnPCEMale GLM P-value = 0.403, R-sqr= 16.26%

PCE � polychromatic erythrocytes;MnPCEmicronucleatedpolychromatic erythrocytes

Vehicle =Arachisoil

PC = Scoring positive control slides (fixed and unstained) generated fromBioRelianceStudy No. AE19TL.125M012.BTLwereincluded 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 treatment.

 

Table 5: Induction ofMicronucleatedPolychromatic Erythrocytes in Bone Marrow Collected 24 Hours Post-Dose Administration

 Treatment

 Sex

Animal No.

%PCE

MicronucleatedPCE

(Number/PCE Scored)

 (%MnPCE)

Vehicle-0mg/kg

M

1

61.4

4/4000

0.10

M

2

58.0

3/4000

0.08

M

3

65.6

3/4000

0.08

M

4

56.7

5/4000

0.13

M

5

57.2

2/4000

0.05

FAT 41045/A TE

500 mg/kg

M

6

56.3

2/4000

0.05

M

7

58.9

4/4000

0.10

M

8

68.5

1/4000

0.03

M

9

60.1

5/4000

0.13

M

10

58.0

3/4000

0.08

FAT 41045/A TE

1000 mg/kg

M

11

57.3

4/4000

0.10

M

12

58.6

6/4000

0.15

M

13

57.5

2/4000

0.05

M

14

60.0

3/4000

0.08

M

15

56.4

6/4000

0.15

FAT 41045/A TE

2000 mg/kg

M

16

61.3

3/4000

0.08

M

17

61.3

6/4000

0.15

M

18

59.3

4/4000

0.10

M

19

58.4

7/4000

0.18

M

20

59.4

3/4000

0.08

PC

40 mg/kg

M

CP 74/75

39.8

198/4000

4.95

M

CP 76/77

49.1

138/4000

3.45

M

CP 78/79

46.9

172/4000

4.30

M

CP 80/81

56.8

178/4000

4.45

M

CP 82/83

44.9

152/4000

3.80

PCE � polychromatic erythrocytes;MnPCEmicronucleatedpolychromatic erythrocytes

Vehicle =ArachisOil

PC = Scoring positive control slides (fixed and unstained) generated fromBioRelianceStudy No. AE19TL.125M012.BTLwereincluded 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 treatment.

 

Table 6: Induction ofMicronucleatedPolychromatic Erythrocytes in Bone Marrow Collected 48 Hours Post-Dose Administration

 Treatment

 Sex

Animal No.

%PCE

MicronucleatedPCE

 

(Number/PCE Scored)

(%MnPCE)

Vehicle

0 mg/kg

M

21

62.8

4/4000

0.10

M

22

53.8

2/4000

0.05

M

23

56.0

3/4000

0.08

M

24

53.5

1/4000

0.03

M

25

60.1

2/4000

0.05

FAT 41045/A TE-

2000 mg/kg

M

26

60.0

3/4000

0.08

M

27

58.0

3/4000

0.08

M

28

68.4

4/4000

0.10

M

29

58.0

2/4000

0.05

M

30

52.5

4/4000

0.10

PCE � polychromatic erythrocytes;MnPCEmicronucleatedpolychromatic erythrocytes

Vehicle =ArachisOil

Table7:Historical Control

WistarRat Micronucleus Test Historical Control Data (2008-2014)

Parameter

Ratio of PCE/TotalErythrocytes

Number of MPCE/2000 PCEScored/Animal

Number of MPCE/10000 PCE Scored/Group

Males

Females

Males

Females

Males

Females

Negative Control1

Mean3    

0.53

0.53

0.85

1.23

4.31

6.23

Standard Deviation

0.06

0.04

1.10

1.73

4.15

6.67

Range4   

0.40-0.66

0.42-0.61

0.00-6.00

0.00-7.00

0.00-18.00

0.00-21.00

Positive Control2

Mean3    

0.47

0.45

45.73

39.80

242.00

204.00

Standard Deviation

0.08

0.10

16.55

22.30

82.61

134.29

Range4

0.31-0.69

0.25-0.57

18.00-98.00

12.00-95.00

126.00-439.00

81.00-509.00

1Since no appreciable differences in the induction of MPCEs by different vehicles and solvents (test article carriers) and different routes of administration were observed, this table contains data from carriers and routes of administration widely used during the conduct of contract studies atBioReliance.

Vehicles: water, water soluble vehicles (methylcellulose,carboxymethylcellulose, dextrose), saline, corn oil and other vehicles.

Routes of administration: intraperitoneal (IP), intravenous (IV), oral gavage (PO), subcutaneous (SC).

Bone marrow collection time: 24 and 48 hours post-dose.

2Positive control article: Cyclophosphamide monohydrate (CP); Doses: 40 mg/kg; Route of administration: PO. Bone marrow collection time: 24 hours post-dose.

3Average of the PCE ratio observed out of 1000 erythrocytes scored per animal for the total number of animals used, average of the number of MPCE per 2000 PCE for the total number of animals used; average of number of MPCE/per group (containing 5-7 animals per group) for total number of groups used.   

4Minimum and maximum range of PCE ratio observed out of 1000 erythrocytes scored per animal, the minimum and maximum range of MPCE observed out of 2000 PCE for the total number of animals used, and the minimum and maximum range of MPCE observed out of 10000 PCE for the total number of groups used.

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
All criteria for a valid study were met. The results of the micronucleus assay indicate that FAT 41045/A TE did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes. Therefore, FAT 41045/A TE was concluded to be negative.
Executive summary:

The test substance, FAT 41045/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. Arachis oil was selected as the vehicle. Test and/or control substance formulations were administered at a dose volume of 10 mL/kg by oral gavage.

The definitive assay dose levels tested were 500, 1000 and 2000 mg/kg. Groups 1 and 4 consisted of 10 animals each designated for either 24- or 48-hour bone marrow collection and Groups 2 and 3 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 (4000 PCEs/animal)] were examined microscopically for the presence of micronuclei (micronucleated PCEs; MnPCEs) and statistical analysis of data was performed. The ratio of polychromatic erythrocytes (PCEs) to total erythrocytes (EC) in the test substance groups relative to the vehicle control group was also evaluated to reflect the test substance’s cytotoxicity.

Under the conditions of this study, the administration of FAT 41045/A TE at doses up to and including a dose of 2000 mg/kg was concluded to be negative in the Micronucleus assay.