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:
22 October 2014 to 18 December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
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:
other: Water white liquid
Details on test material:
Name: NovaSpec base oilBatch/Lot No.: TS13732CAS number: 1472010-43-7Chemical name: Alkenes, C-10-16a-, mixed with (6E)-7,11-dimethyl-3-methylene-116,10-dodecatriene, dimers, tetramers and trimers, hydrogenatedPurity: 100%Manufacture date: 06 June 2014Expiry date: 06 June 2016Description: Water white liquidStorage condition: Controlled room temperature (15-25 °C below 70 RH%)Safety Precautions: Routine safety precautions for unknown materials (lab coat, gloves, safety glasses and face mask) were applied to assure personnel health and safety.No correction for purity of the test item was applied.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Species and strain: Crl:WI rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, from SPF colony. Males and females originated from different units to avoid subsequent brother/sister matings.
Hygienic level: Standard laboratory conditions during the study
Justification of species/strain: The rat is regarded as a suitable species for toxicology and reproduction studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. Crl:WI rats were used for dose range finding study (study code:14/328-220PE).
Number of animals: 48 male, 48 female rats, 12 animals/sex/group, 4 groups (main animals); 10 male and 10 female rats, 5/sex/group (recovery group) and 12 males and 12 females rats in the positive control group for the micronucleus test).
Age of animals: Young adult rats, 10-11 weeks old at starting and 12-13 weeks at mating.
Body weight range: Males: 327 g – 478 g, Females: 207 g - 273 g;
Acclimation period: At least 5 days
Husbandry
Animal health: Only healthy animals were used for the test, as certified by the staff Veterinarian. Females were nulliparous and non-pregnant.
Room number: 508
Cage type: Type II and/or III polypropylene/polycarbonate
Bedding: Lignocel® and GRADE 5 type wooden chips were available to the animals
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.1 – 25.1 °C (target range 22±3°C)
Relative humidity: 31 – 64 % (target range 30-70%)
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were group-housed, up to 5 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting)
.Food and water supply: Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and Maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum, and tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum.

Water quality control analysis is performed once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36, Hungary). The quality control results are retained in the archives at CiToxLAB Hungary Ltd.Food and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Animal identification: Each parental animal (P Generation) was identified by a number unique within the study written with indelible ink on the tail and cross-referenced to the Animal Master File at CiToxLAB Hungary Ltd. The animal number consisted of 4 digits, the first digit being the group number, the second, 0 for the males and 5 for the females, and the last 2, the animal number within the group, as indicated in the Experimental design section. The boxes (cages) were marked by identity cards, with information at least about study code, sex, dose group, cage number and individual animal numbers, date of mating and delivery. Boxes were arranged in such a way that possible effects due to cage placement were minimized.Randomization: All adult animals were sorted according to body weight by computer and divided in to weight ranges. There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that the mean group weights were similar. The grouping was controlled by SPSS/PC software, according to the actual body weight verifying the homogeneity/variability between/within the groups and cages. Males and females were randomized separately.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Name: Poly(ethylene glycol) 400
Lot No.: BCBL5307V/BCBM8497V//BCBK 9981V
Manufacturer: Sigma-Aldrich Co.
Expiry Date: 31 January 2015/31 January 2016/January 2015
Storage: Room temperature
Name: 0.2% Polysorbate 80Lot No.: BCBL9041V/BCBN3690V
Manufacturer: Sigma-Aldrich Co.
Expiry Date: 30 November 2014/31 July 2016
Storage: Room temperature under inert gas
Preparation of the vehicle: For the preparation of 100 g (approximately 100 mL) vehicle, 0.2 g Polysorbate 80 was weighed by analytical balance into 99.8 g PEG 400, and was stirred by a magnetic stirrer.
Details on exposure:
The test item was formulated in the vehicle as a visibly stable homogenous formulation at the appropriate concentrations according to the dose level and volume selected, in the Central Dispensary of CiToxLAB Hungary Ltd. Formulations were prepared for 7 days and kept in refrigerator pending dosage in the first 14 days of the study, and daily afterwards.The positive control material (cyclophosphamide) was dissolved in physiological saline (10 mg/mL) for the treatment. The solution was prepared just before the treatment. The test item solutions were given to assure the same dosing volumes in rat (2 mL/kg bw).No dose formulation analysis was performed from the positive control solutions.
Duration of treatment / exposure:
At least until the first scheduled euthanasia of dams.
Frequency of treatment:
Daily
Post exposure period:
At least 14 days after the first schedules euthanasia of dams
Doses / concentrations
Remarks:
Doses / Concentrations:0, 1000 mg/kg bw/dayBasis:nominal conc.
No. of animals per sex per dose:
5 male and 5 female in the treatment group12 animals/sex/group served as the positive control group for the Mammalian Erythrocyte Micronucleus Test (MNT).
Control animals:
yes
Positive control(s):
12 animals/sex/group served as the positive control group for the Mammalian Erythrocyte Micronucleus Test (MNT). They were mated and femalesallowed to deliver similarly to the Main animals, then treated once with 20 mg/kg bw Cyclophosphamide, administered by intraperitoneal injection(2 mL/kg bw) approximately 24 h prior to scheduled necropsy.

Examinations

Tissues and cell types examined:
Polychromatic erythrocytes (PCEs) were scored per animal
Details of tissue and slide preparation:
At the end of the treatment period, bone marrow slides were prepared from all animals in the vehicle control and the positive control groups. The bone marrow was obtained from the right femurs of the rats immediately after euthanasia and flushed with foetal bovine serum (5 mL). The left femur of Main and Recovery group animals was used for routine histopathology, the left femur of positive control animals was discarded.After vortex mixing, the cell suspension was concentrated by centrifugation and the supernatant was discarded. Smears of the cell pellet were made on four standard microscope slides. Slides were then dried at room temperature until considered to be completely dry. Subsequently the slides were stained as follows:1. Fixed for a minimum of 5 minutes in methanol and allowed to air-dry.2. Stained with 10% Giemsa solution for 20 minutes.3. Rinsed in distilled water.4. Dried at room temperature (at least 12 hours).5. Coated with EZ-mountingPrior to sending to Microptic for microscopic analysis, one slide from each animal was given a code number for blind microscopic analysis. The code labels covered the original animal numbers to ensure that the slides were scored without bias.
Evaluation criteria:
Two thousand Polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The frequency of micronucleated cells was expressed as percent of the micronucleated cells based on the first 2000 PCEs counted in the optic field. The proportion of immature among total (immature + mature) erythrocytes was determined for each animal by also counting a total of at least 1000 cells (immature erythrocytes, PCEs plus mature normochromatic erythrocytes, NCEs). During this process, the number of micronuclei was recorded in mature erythrocytes (NCEs) as well. Criteria for Identification of Micronucleated ErythrocytesA micronucleus is defined in following way:- A bluish mauve strongly coloured uniform round or oval particle in the cell.- The particle should be large enough for the colour to be recognisable, and it should be located inside the cells. Areas with micronucleus-like particles outside the cells should not be used for analysis.- During focusing, the particle should stay uniform in colour/light refraction and shape within a large interval and focus in the same plane as the erythrocyte.- The unit of damage is deemed to be the cell, and therefore cells with two or more micronuclei will be counted as single micronucleated cells. The Micronucelus Test is considered acceptable/valid in the conditions of this study, as it met the following criteria:- the frequencies of micronucleated polychromatic erythrocytes found in the negative and/or solvent controls fell within the range of historical laboratory control data.- the positive control item produced biologically relevant increases in the number of micronucleated polychromatic erythrocytes. - Each treated and control group included at least 5 analysable animals.
Statistics:
Statistical analysis was performed using Kruskal Wallis Non Parametric ANOVA test (level of significance 5%)

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The groups with 1000 mg/kg bw (high dose) were compared with their vehicle control group using Kruskal Wallis test. These gave values of H = 0.014 in the males and H = 0.149 in the females. Both H values are non-significant, giving a negative response. The positive and negative control results were also compared, and gave a value of H = 17.633 (p<0.001) in the males, and H = 17.561 (p<0.001) in the females. The positive control treatment therefore caused a very substantial increase, demonstrating the sensitivity of the test system. The positive and negative control data are considered to give adequate data to confirm the validity of the study.

Any other information on results incl. tables

Table 1: Dose Group – Males – Negative Control

Animal no.

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 NCE+PCE

1001

84

2

310

1002

101

3

291

1003

99

3

360

1004

43

3

356

1005

104

1

382

1006

26

3

305

1007

107

1

460

1008

5

2

372

1009

79

1

217

1010

16

1

432

1011

28

3

308

1012

22

1

380

Mean

 

2.00

347.8

SD

 

0.95

66.0

 

Table 2: Dose Group – Males – High Dose 1000 mg/kg bw 24h

Animal no.

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 NCE+PCE

4001

106

2

361

4002

98

2

395

4003

116

5

370

4004

103

4

295

4005

25

2

336

4006

83

1

451

4007

56

2

425

4008

13

1

384

4009

54

4

391

4010

90

0

208

4011

36

1

369

4012

50

1

380

Mean

 

2.08

363.8

SD

 

1.51

62.9

 

Animal no.

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 NCE+PCE

5001

24

7

213

5002

29

9

316

5003

33

11

481

5004

55

7

305

5005

111

7

200

5006

118

12

297

5007

113

10

215

5008

114

6

388

5009

15

10

221

5010

120

13

239

5011

44

32

395

5012

11

8

269

Mean

 

11.00

294.9

SD

 

6.97

88.0

 

Table 4: Dose Group – Females – Negative Control

Animal no.

Slide code

Micronucleated PCE/2000 PCE

PCE/1000

NCE+PCE

1501

20

3

601

1502

37

2

484

1503

70

1

536

1504

14

2

537

1505

32

1

544

1506

45

2

397

1507

10

0

511

1508

35

2

420

1509

1

3

409

1510

30

2

507

1511

19

5

509

1512

80

2

479

Mean

 

2.08

494.5

SD

 

1.24

60.8

 

Table 5: Dose Group – Females – High Dose 1000 mg/kg bw 48h

Animal no.

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 NCE+PCE

4501

77

4

552

4502

62

1

589

4503

81

2

417

4504

4

1

554

4505

65

0

364

4506

109

2

489

4507

94

1

500

4508

74

0

556

4509

21

6

477

4510

88

3

419

4511

72

1

399

4512

57

5

486

Mean

 

2.17

483.5

SD

 

19.5

71.6

 

Table 6: Dose Group – Females - Cyclophosphamide

Animal no.

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 NCE+PCE

5501

47

30

464

5502

75

52

569

5503

2

42

544

5504

87

35

424

5505

59

36

516

5506

49

101

592

5507

9

27

497

5508

38

162

522

5509

41

34

416

5510

96

11

271

5511

52

61

429

5512

34

37

399

Mean

 

52.33

470.3

SD

 

41.08

89.0

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negativeIn conclusion, no induction of micronuclei in bone marrow erythrocytes was observed following administration of NovaSpec Base Oil to rats at up to and including 1000 mg/kg bw. Thus there was no evidence of any genotoxic activity of the test item under the conditions of this part of the study.
Executive summary:

The objective of this work phase is to assess the potential genotoxic effects of the test item by examining the induction of micronuclei in bone marrow erythrocytes of treated and control animals.

 

In conclusion, no induction of micronuclei in bone marrow erythrocytes was observed following administration of NovaSpec Base Oil to rats at up to and including 1000 mg/kg bw. Thus there was no evidence of any genotoxic activity of the test item under the conditions of this part of the study.