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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18th July - 24 Aug 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
OJ L 142/240.
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
EPA 712-C-98-226.
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
November 24, 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid
EC Number:
813-120-0
Cas Number:
1262967-45-2
Molecular formula:
C21H36O8 C26H46O8 C31H56O8 C36H66O8 C41H76O8
IUPAC Name:
Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL

- Source and lot/batch No.of test material: JA01YX10
- Expiration date of the lot/batch: April 2012
- Purity test date: 95.8%



RADIOLABELLING INFORMATION (if applicable): N/A




STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL

- Storage condition of test material: Room temperature (ca 20°C), in the dark under nitrogen.
- Stability under test conditions: Not stated

- Solubility and stability of the test substance in the solvent/vehicle: Not stated

- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not stated



TREATMENT OF TEST MATERIAL PRIOR TO TESTING

- Treatment of test material prior to testing: Suspensions of the test substance were prepared in Corn oil obtained from Sigma batch number MKBF6012V.
- Preliminary purification step (if any): N/A

- Final dilution of a dissolved solid, stock liquid or gel: 10 mL/kg/day
- Final preparation of a solid: N/A



FORM AS APPLIED IN THE TEST (if different from that of starting material): N/A


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 UK Limited, Margate, Kent, England, animals
- Age at study initiation: Males and females ca 35 days old. Males and females ca 40 days
- Weight at study initiation: Males weighed between 30.0 g to 30.4 g. Females weighed between 22.4 g to 25.5 g
- Assigned to test groups randomly: yes

- Fasting period before study: Not stated

- Housing: Each group was kept, with the sexes separated, in cages and maintained in a controlled environment
- Diet (e.g. ad libitum): Nestlets
- Water (e.g. ad libitum): tap water ad libitum.
- Acclimation period: 5 days


 ENVIRONMENTAL CONDITIONS

- Temperature (°C): 19 to 23 °C
- Humidity (%): 40 to 70 %
- Air changes (per hr): Not stated

- Photoperiod (hrs dark / hrs light): artificial light for 12 hours per day

IN-LIFE DATES: From: To: 27 July 2011 to 25 August 2011

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Corn oil obtained from Sigma batch number MKBF6012V
Details on exposure:
Preliminary toxicity test:
A dose of 2000 mg/kg/day (200 mg/mL) was administered to 4 animals (2/sex) on two consecutive occasions approximately 24 hours apart followed by 48 hours observation.

The micronucleus test:
Dose levels of 500, 1000 and 2000 mg/kg/day were used once to 5 groups of animals once followed by 24hour observation
Duration of treatment / exposure:
24 hours
Frequency of treatment:
Once
Post exposure period:
Preliminary toxicity test:
48 hours observation.

The micronucleus test:
24hour observation
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Test item
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Test item
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
Test item
Dose / conc.:
12 mg/kg bw/day (nominal)
Remarks:
Mitomycin C
No. of animals per sex per dose:
6 animals per group and 5 for positive control
Control animals:
yes
Positive control(s):
Mitomycin C

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):Femurs were cleaned of all excess tissue and blood and the proximal epiphysis removed from each bone. The bone marrow of both femurs from each animal were flushed out and pooled in a total volume of 3 mL of filtered foetal calf serum by aspiration.

FIXATION AND SLIDE STAINING:
1 Fixed for a minimum of 10 minutes in methanol and allowed to air-dry
2 Rinsed in purified water
3 Stained in acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes
4 Washed in purified water for 5 minutes
5 Rinsed in cold tap water for 2 minutes
6 Stored at room temperature until required
7 Immediately prior to scoring, slides are wet mounted with coverslips using purified water


DETAILS OF SLIDE PREPARATION:
The resulting cell suspensions were centrifuged at 1000 rpm (150 × g) for 5 minutes and the supernatant discarded. The final cell pellet was resuspended in a small volume of foetal calf serum to facilitate smearing in the conventional manner on glass microscope slides

METHOD OF ANALYSIS:
Fluorescence microscopy and 2000 polychromatic erythrocytes per animal were examined for the presence of micronuclei.

OTHER:
The proportion of polychromatic erythrocytes was assessed by examination of a total of at least 1000 erythrocytes per animal and the number of micronucleated normochromatic erythrocytes was recorded.
Evaluation criteria:
The following criteria were applied for assessment of assay acceptability:
1. Each treated and control group should include at least 5 analysable animals.
2. Vehicle control values for micronucleated polychromatic erythrocytes must be consistent
with the laboratory historical vehicle control data.
3. Positive controls must show clear unequivocal positive responses.
Statistics:
The data were received in an Excel document and analysed using SAS 9.1.3 (SAS Institute Inc., 2002) (Jonckheere's and Wilcoxon tests) and StatXact 3 (Cytel 1995) (Linear-by-Linear and Permutation tests).

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No mortalities were observed throughout the duration of the micronucleus test. No clinical signs of toxicity were noted for the vehicle control, positive control and Test item group animals over the duration of the test. Some small incidences of bodyweight loss in all dose groups, including controls, were recorded.

Any other information on results incl. tables

Table 3       Summary of results and statistical analysis

Sampling time after 2nddose

Treatment

Concentration (mg/mL)

Proportion of

PCE (%) #

Incidence MPCE (mean) #

24 Hours

 

Vehicle

-

51.4

1.3

Test item

500

47.0

1.7

Test item

1000

50.0

1.8

Test item

2000

53.2

1.8

Mitomycin Ca

12

51.3

53.6**

Vehicle = Corn Oil

PCE = Polychromatic erythrocytes

MPCE = Number of micronucleated polychromatic erythrocytes observed per 2000 polychromatic erythrocytes examined

a = Positive control dosed once only approximately 24 hours prior to termination at a dose volume of 20 mL/kg/day.

# Occasional apparent errors of ± 1% may occur due to rounding of values for presentation in the table

Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-sided probabilities):

** p < 0.01 (significant), otherwise p > 0.01 (not significant)

Table 4       Results for individual animals - 24 hour sampling time.

Treatment (mg/kg/day)

AnimalNO

Proportion PCE (%)

MPCE

 

PCE

 

NCE

 

MNCE

 

Vehicle

401

402

403

404

405

406

 

50.6

53.2

54.5

49.2

48.3

52.7

1

2

3

0

1

1

515

535

548

500

495

537

503

471

457

516

529

482

0

0

0

0

0

0

Test item (500)

411

451

452

414

415

416

41.5

45.7

53.6

47.9

45.8

47.7

1

1

0

1

5

2

417

457

540

484

467

490

588

544

467

526

553

537

0

0

0

0

0

0

Test item (1000)

421

422

423

424

425

426

51.1

50.4

49.7

50.1

55.8

43.0

1

2

2

1

2

3

514

514

506

510

564

435

492

505

512

507

446

576

0

0

0

0

0

0

Test item (2000)

431

432

433

434

435

436

59.6

59.4

44.5

49.8

55.1

50.6

3

2

3

1

1

1

600

602

452

519

572

513

407

411

563

524

467

501

0

0

0

0

0

0

Mitomycin Ca(12)

441

442

443

444

445

51.5

50.8

52.5

48.6

51.2

58

60

35

40

75

540

508

528

488

516

470

492

477

516

491

0

0

0

0

0

 

Vehicle = Corn Oil

PCE = Polychromatic erythrocytes

MPCE = Number of micronucleated polychromatic erythrocytes observed per 2000 polychromatic erythrocytes examined

NCE = Total number of normochromatic erythrocytes examined for micronuclei

MNCE: Number of micronucleated normochromatic erythrocytes observed

a = Positive control dosed once only approximately 24 hours prior to termination at a dose volume of 20 mL/kg/day.

Table 5       Body weight - Preliminary Toxicity Test

Treatment (mg/kg/day)

 

Animal Number

 

Bodyweights (g)

 

Day after arrival

 

Day 1

Day 2

Day 3

Individual

 

Mean

 

Individual

 

Mean

 

Individual

 

Mean

 

Individual

 

Mean

 

Test item (2000)

      

M -21

30.0

30.2

34.3

 

34.5

33.6#

33.8

35.3

34.7

M-22

30.4

34.6

 

34.0#

34.1

F -27

22.4

24.0

24.2

 

24.6

23.6#

24.4

23.7

24.7

F- 28

25.5

25.0

25.2

 

25.7

# = Denotes weight loss from previous weighing

M Male

F Female

Table 6       Body weight - Micronucleus Test

Treatment (mg/kg/day)

 

Animal Number

 

Bodyweights (g)

 

Day after arrival

 

Day 1

Day 2

Day 3

Individual

 

Mean

 

Individual

 

Mean

 

Individual

 

Mean

 

Individual

 

Mean

 

Vehicle

401

402

403

404

405

406

29.8

32.0

29.6

29.5

30.7

31.4

30.5

31.8

32.2

34.1

32.7

31.8

33.6

32.7

31.8

34.0

33.4#

33.2

31.9

33.1#

 

32.9

31.9

34.7

32.7#

32.7#

31.6#

33.8

 

 

32.9

Test item (500)

 

411

451

452

414

415

416

30.9

31.1

31.3

29.9

28.0

29.5

30.1

33.0

33.2

34.7

31.3

31.0

30.6

32.3

34.5

33.1#

34.8

31.6

31.5

31.3

 

32.8

35.3

33.5

35.0

32.6

32.5

31.8

33.5

Test item (1000)

 

421

422

423

424

425

426

30.5

28.8

33.2

31.8

31.2

30.1

30.9

32.8

31.6

35.6

34.4

34.4

32.4

33.6

32.8

31.9

35.2#

35.0

34.4#

32.6

 

 

33.7

33.0

32.4

35.8

35.0

34.2#

32.7

 

33.9

Test item (2000)

431

432

433

434

435

436

31.1

30.3

31.2

32.1

30.6

29.6

30.8

33.3

33.2

33.7

35.1

32.2

32.0

33.3

33.0#

33.0#

33.7

35.2

31.8#

32.3

 

 

 

33.2

32.5#

33.5

34.0

35.1#

32.4

32.7

 

 

33.4

Mitomycin Ca(12)

 

441

442

443

444

445

30.4

30.5

31.1

32.0

30.5

30.9

-

-

-

-

-

-

31.2

31.7

33.5

32.7

35.5

32.9

31.8

31.5#

33.9

32.8

35.8

33.2

# = Denotes weight loss from previous weighing

M Male

F Female

Applicant's summary and conclusion

Conclusions:
Under the condition of the study, it is concluded that Tetraesters of pentaerythritol with 2- methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid is not clastogenic.
Executive summary:

OECD 474 (2011) - In a study designed to assess the potential induction of micronuclei by Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid in bone marrow cells of CD1 mice, the animal were treated with the test item by oral garage on two occasions approximately 24hr apart.

The preliminary toxicity test demonstrated that a dose of 2000 mg/kg/day, (the standard limit dose for the micronucleus test) administered on two consecutive occasions approximately 24 hours apart was tolerated.  Therefore the main study was conducted at dose levels of 500, 1000 and 2000 mg/kg/day using a dose volume of 10 mL/kg/day.  

The negative control group received the vehicle corn oil (dose volume of 10 mL/kg/day) and the positive control group received Mitomycin C at 12 mg/kg using a dose volume of 20 mL/kg.

Bone marrow smears were obtained from all test groups 24 hours after administration.  One smear from each animal was examined for the presence of micronuclei in 2000 polychromatic erythrocytes. The proportion of polychromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal.

The test item did not induce any statistically significant increases in the frequency of micronucleated polychromatic erythrocytes or decreases in the proportion of polychromatic erythrocytes were observed in all tested concentrations.  Both control control groups produced results within historical control data.

It is concluded that under the condition of the study, the test it is not considered clastogenic.

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