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Diss Factsheets

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:
from 1 August 2011 to 23 february 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
methyl 5-(dimethylamino)-2-methyl-5-oxopentanoate
EC Number:
700-204-6
Cas Number:
1174627-68-9
Molecular formula:
C9H17NO3
IUPAC Name:
methyl 5-(dimethylamino)-2-methyl-5-oxopentanoate
Details on test material:
Name in study report: Rhodiasolv Polarclean

Test animals

Species:
mouse
Strain:
ICR
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: approximately 6-10 weeks
- Weight at study initiation: 22-30g
- Fasting period before study: not applicable
- Housing: pre-mating: groups of up to 7
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 50 +/- 20
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: from 16August 2011 to 3 November 2011

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Distilled water
Duration of treatment / exposure:
Single exposure
Frequency of treatment:
Once
Post exposure period:
24 or 48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
7 males
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide, intraperitoneal route, 50 mg/kg

Examinations

Tissues and cell types examined:
Bone marrow
Evaluation criteria:
The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored.
A positive mutagenic response would be demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to the vehicle control group.
A positive response for bone marrow toxicity would be demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the vehicle control group.
Statistics:
All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a (x 1) transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Hunched posture, ptosis and ataxia at and above 1000 mg/kg in 24 and 48-h groups
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

The group mean data from the micronucleus study are summarised in Table 3. The individual data from each group are summarised in Tables 4-9.

 

Table 3: Micronucleus Test - Summary of Group Mean Data

Treatment group

Number of PCE with Micronuclei per 2000 PCE

PCE/NCE ratio

Group mean

SD

Group mean

SD

1

Vehicle control (Distilled water)

10 ml/kg

24-hour sampling time

1.1

1.5

0.98

0.30

2

Positive control (Cyclophosphamide) 50 mg/kg

24-hour sampling time

38.4***

20.8

0.97

0.23

3

Rhodiasolv Polarclean

2000 mg/kg

48-hour sampling time

0.7

0.8

0.79

0.24

4

Rhodiasolv Polarclean

2000 mg/kg

24-hour sampling time

0.7

0.8

0.66

0.24

5

Rhodiasolv Polarclean

1000 mg/kg

24-hour sampling time

0.7

0.8

0.94

0.27

6

Rhodiasolv Polarclean

500 mg/kg

24-hour sampling time

0.4

0.5

1.03

0.33

PCE = Polychromatic erythrocytes

NCE = Normochromatic erythrocytes

SD = Standared deviation

*** = P < 0.001

Table 4: Micronucleus Test - Individual Data and Group Means and Standard Deviations: Vehicle Control (10 ml/kg): 24-Hour Sampling Time

Treatment group

Animal number

BW (g) when dosed

PCE

NCE

PCE/NCE ratio

Number scored

PCE+MN

%PCE+MN

Number scored

NCE+MN

1.

Vehicle control

10 ml/kg

24-h sampling time

1

26

2000

1

0.05

435

1

1.30

2

22

2000

2

0.10

691

0

0.45

3

24

2000

1

0.05

465

0

1.15

4

24

2000

4

0.20

488

0

1.05

5

24

2000

1

0.05

446

0

1.24

6

25

2000

0

0.00

554

0

0.81

7

28

2000

0

0.00

541

0

0.85

Group mean

24.7

2000

1.3

0.06

517

0.1

0.98

SD

1.9

0

1.4

0.07

89

0.4

0.30

BW = bodyweight

 

Table 5: Micronucleus Test - Individual Data and Group Means and Standard Deviations: Cyclophosphamide (50 mg/kg): 24-Hour Sampling Time

Treatment group

Animal number

BW (g) when dosed

PCE

NCE

PCE/NCE ratio

Number scored

PCE+MN

%PCE+MN

Number scored

NCE+MN

1.

Vehicle control

10 ml/kg

24-h sampling time

8

27

2000

45

2.25

449

1

1.23

9

22

2000

49

2.45

528

2

0.89

10

26

2000

18

0.90

467

0

1.14

11

26

2000

64

3.20

614

0

0.63

12

29

2000

16

0.80

508

0

0.97

Group mean

26.0

2000

38.4

1.92

513

0.6

0.97

SD

2.5

0

20.8

1.04

65

0.9

0.23

 

Table 6: Micronucleus Test - Individual Data and Group Means and Standard Deviations: Test item (2000 mg/kg): 48-Hour Sampling Time

Treatment group

Animal number

BW (g) when dosed

PCE

NCE

PCE/NCE ratio

Number scored

PCE+MN

%PCE+MN

Number scored

NCE+MN

1.

Vehicle control

10 ml/kg

24-h sampling time

13

27

2000

1

0.05

596

0

0.68

14

28

2000

0

0.00

551

0

0.81

15

29

2000

1

0.05

703

0

0.42

16

27

2000

0

0.00

514

0

0.95

17

24

2000

2

0.10

463

0

1.16

18

28

2000

1

0.05

627

0

0.59

19

27

2000

0

0.00

526

0

0.90

Group mean

27.1

2000

0.7

0.04

569

0.0

0.79

SD

1.6

0

0.8

0.04

80

0.0

0.24

 

Table 7: Micronucleus Test - Individual Data and Group Means and Standard Deviations: Test item (2000 mg/kg): 24-Hour Sampling Time

Treatment group

Animal number

BW (g) when dosed

PCE

NCE

PCE/NCE ratio

Number scored

PCE+MN

%PCE+MN

Number scored

NCE+MN

1.

Vehicle control

10 ml/kg

24-h sampling time

20

30

2000

0

0.00

653

0

0.53

21

24

2000

1

0.05

688

2

0.45

22

25

2000

1

0.05

497

1

1.01

23

26

2000

0

0.00

680

0

0.47

24

24

2000

1

0.05

649

1

0.54

25

30

2000

0

0.00

613

0

0.63

26

25

2000

2

0.10

505

0

0.98

Group mean

26.3

2000

0.7

0.04

612

0.6

0.66

SD

2.6

0

0.8

0.04

80

0.8

0.24

 

Table 8: Micronucleus Test - Individual Data and Group Means and Standard Deviations: Test item (1000 mg/kg): 24-Hour Sampling Time

Treatment group

Animal number

BW (g) when dosed

PCE

NCE

PCE/NCE ratio

Number scored

PCE+MN

%PCE+MN

Number scored

NCE+MN

1.

Vehicle control

10 ml/kg

24-h sampling time

27

23

2000

1

0.05

628

1

0.59

28

25

2000

0

0.00

553

0

0.81

29

26

2000

0

0.00

415

0

1.41

30

26

2000

2

0.10

533

2

0.88

31

29

2000

1

0.05

524

0

0.91

32

24

2000

0

0.00

554

0

0.81

33

26

2000

1

0.05

466

0

1.15

Group mean

25.5

2000

0.7

0.04

525

0.4

0.94

SD

1.9

0

0.8

0.04

68

0.8

0.27

 

Table 9: Micronucleus Test - Individual Data and Group Means and Standard Deviations: Test item (500 mg/kg): 24-Hour Sampling Time

Treatment group

Animal number

BW (g) when dosed

PCE

NCE

PCE/NCE ratio

Number scored

PCE+MN

%PCE+MN

Number scored

NCE+MN

1.

Vehicle control

10 ml/kg

24-h sampling time

34

25

2000

0

0.00

602

0

0.66

35

22

2000

0

0.00

546

0

0.83

36

26

2000

0

0.00

524

0

0.91

37

26

2000

0

0.00

408

0

1.45

38

26

2000

1

0.05

521

0

0.92

39

28

2000

1

0.05

391

0

1.56

40

29

2000

1

0.05

522

0

0.92

Group mean

26.0

2000

0.4

0.02

502

0.0

1.03

SD

2.2

0

0.5

0.03

76

0.0

0.33

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Rhodiasolv Polarclean was considered to be non-genotoxic under the conditions of the test.
Executive summary:

The potential of Rhodiasolv Polarclean to produce damage to chromosomes or aneuploidy was investigated in GLP-compliant study according to OECD Guideline 474 (1997).

The micronucleus test was conducted using the intraperitoneal route in groups of seven mice (males) at the maximum tolerated dose (MTD) of 2000 mg/kg and with 1000 and 500 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Additional groups of mice were given a single intraperitoneal dose of distilled water (7 male mice) or dosed orally with cyclophosphamide (5 male mice), to serve as vehicle and positive controls respectively. Vehicle and positive control animals were killed after 24 hours.

There were no premature deaths seen in any of the dose groups in the main test. Clinical signs consisting of hunched posture, ptosis and ataxia were observed in animals dosed with the test item at and above 1000 mg/kg in both the 24 and 48-hour dose groups Animals dosed at 2000 mg/kg in both the 24 and 48-hour dose groups exhibited decreased respiratory rate, laboured respiration and loss of righting reflex up to 10 minutes after dosing. The observation of the clinical signs was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved. No statistically significant decreases in the PCE/NCE ratio were observed in any of the test item dose groups when compared to the vehicle control group. There were no statistically significant increases in the frequency of micronucleated PCE in any test item dose groups. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Rhodiasolv Polarclean was considered to be non-genotoxic under the conditions of the test.