Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 April 2015 to 28 September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This testing was performed for the purpose of chemical notification in China. Therefore no testing proposal was included.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
2014
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
other: in vivo micronucleus teste

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): EnvaMul™200
- Substance type: UVCB

Test animals

Species:
mouse
Strain:
NMRI
Details on species / strain selection:
recommended by international guidelines
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 7 weeks
- Weight at study initiation: 31-40 g
- Assigned to test groups randomly: yes
- Fasting period: withheld 3 - 4 hours prior to dosing until 3 - 4 hours after administration
- Housing:
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH,
Soest, Germany) ad libitum)
- Water : tap water ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 - 22.6°C
- Humidity (%): 38-73%
- Air changes (per hr): ca 10/h
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: solubility/stability of the substance
- Amount of vehicle (if gavage or dermal): 10 mL
Details on exposure:
dosing solutions prepared on day of treatment (doses based on the outcome of a pre-test in 3 males and 3 females at 2000 mg/kg bw)
Duration of treatment / exposure:
2 days
Frequency of treatment:
once at 0 and 24 h
Post exposure period:
24 hour after last dose
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
5 males/ dose + additional 18 animals at 2000 mg/kg for blood sampling at 0.5, 1, 2, 4, 6 and 8 hours after the second dosing
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide;
- Justification for choice of positive control(s): according to the guideline
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/kg bw

Examinations

Tissues and cell types examined:
bone marrow for normo- and poly-chromated erithocytes

Details of tissue and slide preparation:
Bone marrow was sampled 48 hours after the first dosing from both femurs, flushed withetal calf serum and the cell suspension was centrifuged at 216 g for 5 min. Smears were prepared after removal of the supernatant (one drop left behind) and suspended on a clean slide that was coloured by Giemsa based staining (Wright-stain-procedure)
Evaluation criteria:
A test substance is considered positive in the micronucleus test if:
It induces a biologically as well as a statistically significant (Dunnett’s test, one-sided, p < 0.05)
increase in the frequency of micronucleated polychromatic erythrocytes and the number of
micronucleated polychromatic erythrocytes in the animals should be above the historical control
data range.

A test substance is considered negative in the micronucleus test if:
None of the tested concentrations show a statistically significant (Dunnett’s test, one-sided,
p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes and the number
of micronucleated polychromatic erythrocytes in the animals should be within the historical control
data range.
Statistics:
ToxRat Professional version 3.0

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
One animal dosed with 1000 mg/kg test substance died immediately after the second dosing due to a technical error while dosing and one animal dosed with 500 mg/kg showed lethargy and a rough coat after the second dosing but recovered within 23 hours after second dosing. No treatment-related clinical signs or mortality were noted in the other animals.

Analyzed blood concentrations of the substance (from the 2000 mg/kg bw additional mice) were above the upper limit of quantification as validated in the analytical method (> 10000 ng/L).

Any other information on results incl. tables

 

GroupTreatment

Dose (mg/kg bw 

Number of micronucleatedpolychromatic erythrocytes (mean ± S.D.)(1,2

Ratio polychromatic/ normochromatic erythrocytes

(mean ± S.D.)(1,2

Vehicle control

0 

3.6 ±0.9

 0.95 ±0.07

Test substance

2000

4.0 ±1.0

 0.90 ±0.08

Test substance

1000 

4.5 ±1.7

 0.94 ±0.05

Test substance

500 

3.4 ±1.1

 0.91 ±0.06

CP

40 

38.2 ±  6.8(4)

 0.54 ±0.12

Vehicle control = Corn oil

CP = Cyclophosphamide.

(1) Four to five animals per treatment group. 

(2) At least 4000 polychromatic erythrocytes were evaluated with a maximum deviation of 5%.

(3) The ratio was determined from at least the first 1000 erythrocytes counted.

(4) Significantly different from corresponding control group (Studentsttest, P = 0.01).

 

Applicant's summary and conclusion

Conclusions:
The substance is not clastogenic in the bone marrow micronucleus test of mice up to a dose of 2000 mg/kg
Executive summary:

In a study according to OECD 474 5 male mice/groupwere dosed twice via oral gavage with vehicle (corn oil), the substance at 2000, 1000 and 500 mg /kg bw or with 40 mg/kg cyclophosphamide (dosed only once). One animal dosed at 1000 mg/kg bw died and one at 500 mg/kg bw showed lethargy and a rough coat after the second dosing but recovered within 23 hours. No treatment related clinical signs or mortality were noted in the other animals. Blood samples collected at 0.5, 1, 2, 4, 6 and 8 hours after dosing from three satellite animals (dosed at 2000 mg/kg bw) per time point treated with 2000 mg showed concentrations of the substance above the upper limit of quantification (> 10000 ng/L), which confirms exposure.

Bone marrow was sampled 48 hours after the first dosing. No biological relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the substance compared to the vehicle treated animals. No toxicity on erythropoiesis of the substance was observed (expressed as the ratio of polychromatic to normochromatic erythrocytes).

The substance is not clastogenic in the bone marrow micronucleus test of mice up to a dose of 2000 mg/kg.