Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
The in vivo micronucleus study was conducted solely to comply with a non-EU national registration requirement, and has been provided here in accordance with REACH, Article 22(1)e.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 17 September 2015, Experimental end date: 04 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
452-330-3
EC Name:
-
Cas Number:
314020-40-1
Molecular formula:
C14H20N2O2
IUPAC Name:
2-(2,6-diethyl-4-methyl-phenyl)propanediamide
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
other: Crl: WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (K) Ltd, Margate, Kent, CT9 4LT, United Kingdom
- Age at study initiation: four to five weeks (dose sighting and range finding), five to six weeks (main study)
- Weight at study initiation: 82 to 123 g for males and 77 to 111 g for females (dose sighting and range finding) and 127 to 170 g (males only, main study)
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing: in groups of up to three, by sex, in grid-floor cages suspended over paper-lined trays
- Diet (ad libitum): pelleted rodent diet, LabDiet 5l0S EURodent Diet, by PMI Nutrition International
- Water (ad libitum): mains tap water
- Acclimation period: 11 days for the dose sighting and range finding phase and five days for the main study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 22 °C
- Humidity (%): 45 to 70%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours darkness

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
The vehicle used in the study was 0.5% w/v carboxymethylcellulose with 0.1% v/v Tween 80, whihc is a common vehicle for oral gavage dosing to rodents.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
A quantity of test item was weighed into a mortar, wetted with a small quantity of vehicle and made into a smooth paste using a pestle. After further addition of vehicle and mixing, the resultant suspension was transferred into a stoppered measuring cylinder. The mortar was thoroughly rinsed out with vehicle and these rinsings were added to the suspension which was then made up to final volume with vehicle and homogenised. Formulations were dispensed into amber glass bottles for dosing.
Formulations were stirred for a minimum of five minutes before the start and until the completion of dosing, to ensure thorough re-suspension and homogeneity.
Duration of treatment / exposure:
Animals were dosed twice, depending on the severity of the clinical signs, approximately 24 hours apart, by oral gavage.
Frequency of treatment:
Two times, approximately 24 hours apart
Post exposure period:
24 hours after first (all groups) and second dose (groups 1 to 4)
Doses / concentrationsopen allclose all
Dose / conc.:
312.5 mg/kg bw/day (nominal)
Dose / conc.:
625 mg/kg bw/day (nominal)
Dose / conc.:
1 250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Six animals per dose group, negative and positive control
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide monohydrate (CPA), the positive Control item, was a 3 mg/mL solution of CPA in UHP water.

Examinations

Tissues and cell types examined:
One femur from each animal was exposed by dissection of the surrounding muscle and connective tissues and the shank of the bone removed. The bone marrow cells from each femur were aspirated using a syringe into labelled centrifuge tubes containing foetal bovine serum.
Details of tissue and slide preparation:
The bone marrow cells were centrifuged, the supernatant withdrawn, and the cells re-suspended in a minimal volume of foetal bovine serum. One drop of cell suspension was placed on each of two slides and spread by drawing an edge of a clean glass microscope slide along from the drop to the end of the slide. All slides were left to air dry and age overnight before fixing for 5 minutes in methanol. Fixed slides were stained for 20 to 30 minutes in 11.5 % (v/v) Giemsa in Sorensen’s buffer pH 6.0, based on the method of Gollapudi and Kamra
Evaluation criteria:
The negative and positive Controls from every experiment were compared with historical ranges of these parameters. The micronucleus test was considered valid because the following criteria were met:
• The incidence of MN-PCE and the PCE/NCE ratio in the negative Control group fell within the historical negative Control range.
• The positive Control item induced a significant increase in the frequency of MN-PCE, and the incidence of MN-PCE and the PCE/NCE ratio in the positive Control group fell within the historical positive Control range.
• At least five animals out of each group were available for analysis.

The test substane was concluded to be neither clastogenic nor aneugenic because the study met the acceptance criteria and no statistically significant increases in the frequency of MN-PCE were seen, relative to negative Controls, in any group given the test substance.
For the test to have been considered positive i.e. the test substance was considered to induce clastogenic/aneugenic damage, the following criteria would have had to be met:
• A statistically significant increase in the frequency of MN-PCE occurred at one or more dose levels.
• The incidence and distribution of MN-PCE in individual animals at the dose level(s) showing statistical significance exceeded Sequani’s historical negative Control data.
• A dose-related increase in the frequency of MN-PCE (where more than two dose levels are analysed) was observed.
Results which only partially satisfy the above criteria would be dealt with on a case-by-case basis. Evidence of a dose-related effect was considered useful but not essential in the evaluation of a positive result.
Biological relevance was taken into account, for example consistency of response within and between dose levels.
A test was considered to be negative if there was neither a dose-response curve nor any group showed statistically significant increases in the frequency of micronucleated PCEs compared to the negative Controls.
Statistics:
The data were analysed in accordance with the UKEMS guidelines. The data analysed were the proportion of MN-PCE and the ratio of polychromatic to normochromatic cells (PCE/NCE).
The proportion of micronucleated PCEs (with respect to the total number of PCEs counted) is considered to be a measure of chromosomal or cell division apparatus damage. The ratio PCE/NCE is considered to provide an estimate of general toxicity of the test item to bone marrow. In this case testing is concerned with a reduction from the negative Control value. The preferred approach for the MN-PCE data is to combine the data within each group and construct a 2x2 contingency table for each treated group with the negative Control. The groups are then compared using a one tailed Fisher Exact test. However, this approach must first be validated by carrying out a test for between animal heterogeneity using a chi square test. If the heterogeneity test is significant at the 1% level then an exact Wilcoxon Rank Sum test is used instead of the Fisher Exact test. The same method is used to compare the positive and negative Controls.
For the PCE/NCE data, the groups given the test substance were compared with the negative Control using one tailed exact Wilcoxon Rank Sum tests.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The only effect observed was yellow stained bedding 24 hours after the second dosing (low dose) or 24 hours after the first and second dosing (mid and high dose).

Any other information on results incl. tables

Table 1: Individual Micronucleus data

Animal number

Group

Dose (mg/kg bw/day)

Total PCE

NCE/1000 cells

MN-PCE

MN-NCE

PCE/NCE

1

1

0

2000

537

3

1

0.86

2

1

0

2000

523

1

0

0.91

3

1

0

2000

450

2

0

1.22

4

1

0

2000

519

3

0

0.93

5

1

0

2000

593

0

0

0.69

6

1

0

2000

620

0

0

0.61

7

2

312.5

2000

536

3

0

0.87

8

2

312.5

2000

528

0

0

0.89

9

2

312.5

2000

579

0

0

0.73

10

2

312.5

2000

506

3

0

0.98

11

2

312.5

2000

529

2

1

0.89

12

2

312.5

2000

640

1

0

0.56

13

3

625

2000

593

1

0

0.69

14

3

625

2000

515

1

0

0.94

15

3

625

2000

497

0

0

1.01

16

3

625

2000

492

0

0

1.03

17

3

625

2000

502

0

1

0.99

18

3

625

2000

518

0

0

0.93

19

4

1250

2000

476

1

0

1.10

20

4

1250

2000

559

1

0

0.79

21

4

1250

2000

473

1

0

1.11

22

4

1250

2000

611

0

0

0.64

23

4

1250

2000

604

1

0

0.66

24

4

1250

2000

605

2

0

0.65

25

5

CPA 15 mg/kg

2000

601

21

2

0.66

26

5

CPA 15 mg/kg

2000

567

17

0

0.76

27

5

CPA 15 mg/kg

2000

560

19

0

0.79

28

5

CPA 15 mg/kg

2000

625

23

0

0.60

29

5

CPA 15 mg/kg

2000

538

25

0

0.86

30

5

CPA 15 mg/kg

2000

649

15

0

0.54

Table 2: Historical control data on rats

Male negative control

 

N

Mean

SD

Range (mean ± SD)

Range (min / max)

PCE

143

2000.0

0.1

1999.9

2000.1

2000

2001

NCE/1000 cells

143

538.0

92.1

445.8

630.1

344

952

MN-PCE/2000 PCE

143

0.9

1.0

-0.2

1.9

0

6

MN-NCE/1000 cells

143

0.1

0.3

-0.2

0.4

0

2

PEC/NCE ratio

143

0.9

0.3

0.6

1.2

0.1

1.9

Male positive control

 

N

Mean

SD

Range (mean ± SD)

Range (min / max)

PCE

123

2000.0

0.5

1999.6

2000.5

2000

2005

NCE/1000 cells

123

594.2

71.6

522.6

665.7

406

809

MN-PCE/2000 PCE

123

24.6

11.2

13.4

35.8

3

70

MN-NCE/1000 cells

123

0.2

0.5

-0.3

0.7

0

2

PEC/NCE ratio

123

0.7

0.2

0.5

0.9

0.2

1.5

Applicant's summary and conclusion

Conclusions:
There was no evidence of clastogenicity or aneugenicity following oral (gavage) administration of the test substance up to the maximum tolerated dose of 1250 mg/kg/day in male rats, as tested in an in vivo micronucleus assay in accordance with OECD TG 474.
Executive summary:

The potential of the test substance to produce clastogenicity or aneugenicity following oral administration by gavage in the male rat was studied under GLP in accordance with OECD TG 474. Female and male rats of the Crl:WI(Han) strain, approximately 4 to 5 weeks old and weighing 77 to 111 g (females) and 82 to 123 g (males), were used in a dose sighting and range finder study conducted with doses of 500, 1250 and 2000 mg/kg bw/day to establish the maximum tolerable dose (MTD). The test substance was administered by oral gavage in a volume of 10 mL/kg bw of a suspension, using 0.5% w/v carboxymethylcellulose with 0.1% v/v Tween 80 as the vehicle. The MTD was determined to be 1250 mg/kg bw/day in female and male rats, and the main study was conducted with male rats only. In the main test, thirty male rats, five to six weeks old and weighing between 160 to 210 g, were arbitrarily assigned to one of three treatment groups, a vehicle control group or a positive control group, so that each group consisted of six individual animals.

In the main study, the test substance was administered at doses of 312.5, 625 or 1250 mg/kg bw/day in 10 mL suspension with the vehicle, animals in the negative control group received the vehicle only, and the positive control substance cyclophosphamide monohydrate was dosed at 15 mg/kg bw/day in 3 mL water. Animals were dosed twice, depending on the severity of the clinical signs, about 24 hours apart, while animals in the positive control group were dosed only once. Animals were killed about 24 hours after the final dose and the bone marrow cells were obtained from one femur taken from each animal. Slides of bone marrow cells were prepared, coded and score following the test guidance.

There were no adverse clinical effects following oral exposure to the test substance, the negative or positive controls in the main test. The micronucleus frequency in male rats exposed to the test substance was not statistically significantly increased. There was no statistically significant reduction in the PCE/NCE ratio in male rats treated with the test substance and there was no evidence of clastogenicity or aneugenicity. Since proof of exposure to the bone marrow was demonstrated in the range finding study, this indicated a lack of toxicity of the test substances to the bone marrow. The animals treated with the positive control substance had statistically significant increases in the number of micronucleated cells compared with the concurrent negative control group, which demonstrated that the test system was capable of detecting a known clastogen.