Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012-09-28 to 2012-12-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
31 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted July 21, 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): HH-2012-414 solid

Test animals

Species:
mouse
Strain:
other: NMRI BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: males 33.9 ± 1.9 g, females 28.7 ± 2.0 g
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: in groups (maximum 5 animals per sex per cage) in Macrolon cages (type MIII height: 15 cm) containing sterilised sawdust as bedding material
- Diet (e.g. ad libitum): pelleted rodent diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 - 21.5°C
- Humidity (%): 38 - 66%
- Air changes (per hr): app. 15/h
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: not given
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
prepared on the day of administration
- dosing volume: 10 mL/kg bw
Duration of treatment / exposure:
24 h
Frequency of treatment:
1x
Post exposure period:
24 h (+48 h for 2000 mg/kg bw dose group)
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 500, 1000, 2000 mg/kg bw
Basis:

No. of animals per sex per dose:
5 (main test)
3 (pretest)
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: intraperitoneal injection
- Doses / concentrations: 40 mg/kg bw

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: based on results of a dose range finding test

DETAILS OF SLIDE PREPARATION:
- bone marrow was flushed from femurs with approximately 2 mL of fetal calf serum
- cell suspension was collected and centrifuged at 216 g for 5 min
- supernatant was removed with a Pasteur pipette leaving one drop of serum on the pellet, cells were mixed with the remaining serum
- a drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol and cleaned with a tissue
- the drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension; the preparations were air-dried, fixed for 5 min in 100% methanol and air-dried overnight
- two slides were prepared per animal
- staining with Giemsa
METHOD OF ANALYSIS:
- slides were scored at a magnification of 1000 x
- the number of micronucleated polychromatic erythrocytes was counted in at least 2000 polychromatic erythrocytes (with a maximum deviation of 5%)
- the ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time
- micronuclei were only counted in polychromatic erythrocytes
- averages and standard deviations were calculated
Evaluation criteria:
A micronucleus test is considered acceptable if it meets the following criteria:
- The incidence of micronucleated polychromatic erythrocytes in the positive control animals should be above the historical control data range.
- The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
- The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range.

A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately and the number of micronucleated polychromatic erythrocytes in the animals are above the historical control data range.

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes either in the combined data for both sexes or in the data for male or female groups separately and the number of micronucleated polychromatic erythrocytes in the animals are within the historical control data range
Statistics:
Wilcoxon Rank Sum Test, one-sided, p < 0.05

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: hunched posture, rough coat, lethargy

RESULTS OF DEFINITIVE STUDY
- animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality
- 1000 and 2000 mg/kg bw: hunched posture, rough coat, lethargy
- 500 mg/kg bw: hunched posture, rough coat

- Induction of micronuclei (for Micronucleus assay): no increase in the mean frequency of micronucleated polychromatic erythrocytes was observed
- Ratio of PCE/NCE (for Micronucleus assay): no decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test substance on the erythropoiesis
- Appropriateness of dose levels and route:
tested up to maximum recommended dose in accordance with current regulatory guidelines
route of administration (intraperinoneal) was chosen to maximize the chance of the test substance reaching the target tissue

Any other information on results incl. tables

Treatment mg/kg bw

Sampling time [h]

Number of micronucleated polychromatic erythrocytes (mean ± SD)

Ratio polychromatic/ normochromatic erythrocytes (mean ± SD)

males

0 (vehicle)

24

2.2 ±1.3

0.95 ±0.06

2000

24

2.0 ±0.7

0.68 ±0.31

2000

48

1.2 ±0.4

0.70 ±0.20

1000

24

2.0 ±1.4

0.69 ±0.15

500

24

2.2 ±1.3

0.81 ±0.13

Cyclophosphamide, 40 mg/kg bw

48

25.4 ± 4.2*

0.47 ±0.22

females

0 (vehicle)

24

1.8 ±0.8

0.96 ±0.06

2000

24

2.8 ±1.1

0.78 ±0.22

2000

48

0.4 ±0.5

0.79 ±0.14

1000

24

2.8 ±1.1

0.84 ±0.10

500

24

1.4 ±0.9

0.81 ±0.15

Cyclophosphamide, 40 mg/kg bw

48

22.8 ± 5.0*

0.62 ±0.12

* Significantly different from corresponding control group (Wilcoxon Rank Sum Test, P = 0.01).

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
MDEA-Esterquat C16-18 and C18 unsatd. was evaluated for clastogenic potential in the NMRI BR mouse bone marrow micronucleus assay at doses of 0, 500, 1000 and 2000 mg/kg bw. Under the conditions of the study, the test substance did not induce an increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.
Executive summary:

In a NMRI BR mouse bone marrow micronucleus assay according to OECD guideline 474 (adopted July 21, 1997) and EU method B.12 (31 May 2008) 5 animals/sex/dose were treated by intraperitoneal injection with MDEA-Esterquat C16-18 and C18 unsatd. at doses of 0, 500, 1000 and 2000 mg/kg bw.  Bone marrow cells were harvested at 24 h for all dose levels and additionally at 48 h at 2000 mg/kg bw post-treatment.  The vehicle was corn oil.

Hunched posture, rough coat and lethargy were observed at 1000 and 2000 mg/kg bw; hunched posture and rough coat were also present at 500 mg/kg bw.

MDEA-Esterquat C16-18 and C18 unsatd. was tested up to the maximum recommended dose in accordance with current regulatory guidelines. No decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test substance on the erythropoiesis. However the route of exposure was chosen to maximise the chance of the test substance reaching the target tissue.

The positive control induced the appropriate response. 

There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.