Reaction mass of bis[4-(vinyloxy)butyl] hexane-1,6-diylbiscarbamate and 9,18-dioxo-3,8,19-trioxa-10,17-diazapentacos-1-en-25-yl [6-({[4-(vinyloxy)butoxy]carbonyl}amino)hexyl]carbamate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 March 2015 - 06 May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study was performed to fulfill data requirements for a registration in China.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

26 September 2014

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd 06 May 2013

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

420-990-1

EC Name:

-

Cas Number:

146421-65-0

Molecular formula:

C20H36N2O6

IUPAC Name:

4-(ethenyloxy)butyl N-[6-({[4-(ethenyloxy)butoxy]carbonyl}amino)hexyl]carbamate

Constituent 2

Reference substance name:

4-(ethenyloxy)butyl N-{6-[({[6-({[6-({[4-(ethenyloxy)butoxy]carbonyl}amino)hexyl]carbamoyl}oxy)hexyl]oxy}carbonyl)amino]hexyl}carbamate

Cas Number:

1516571-16-6

Molecular formula:

C34H62N4O10

IUPAC Name:

4-(ethenyloxy)butyl N-{6-[({[6-({[6-({[4-(ethenyloxy)butoxy]carbonyl}amino)hexyl]carbamoyl}oxy)hexyl]oxy}carbonyl)amino]hexyl}carbamate

Test material form:

solid: flakes

Details on test material:

- Name of test material (as cited in study report): Uralac P 1920C
- Appearance: Colourless to light yellow flakes

Specific details on test material used for the study:

Purity/composition correction factor: No correction factor required

Test animals

Species:

mouse

Strain:

other: NMRI BR

Details on species / strain selection:

These mice are recommended by international guidelines (e.g. OECD, EC).

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6-7 weeks
- Weight at study initiation (mean per group): 29 - 37 g
- Assigned to test groups randomly: yes
- Housing: group-housed in labelled polycarbonate cages (maximum of 5 animals/cage).
- Diet: Free access to standard pelleted laboratory animal diet (SM RM-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), except 3- 5.5 hours prior to dosing when feed was withheld
- Water: Free access to tap water
- Acclimation period: At least 7 days

Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.6 – 22.2
- Humidity (%): 38 - 50
- Air changes (per hr): appr. 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: propylene glycol
- Amount of vehicle: 10 mL/kg body weight

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was crushed and ground in a mortar with pestle to improve the consistency. URALAC P 1920C concentrations were treated with ultra-sonic waves and heated for approximately 30 to 60 minutes up to a maximum of 80°C to obtain a homogeneous suspension. In the main study the formulation was prepared under nitrogen and the formulation was blended. URALAC P 1920C concentrations were dosed within 4 hours after preparation.

Duration of treatment / exposure:

Test substance and negative control: Two treatments (in two steps each, separated by no more than 2-3 hours) at 0 and 24 hours.
Positive control: Single treatment at start of the experiment (t=0 hours)

Frequency of treatment:

Two treatments at consecutive days. The test substance (and solvent control) was administered as a split dose, i.e., two treatments on the same day separated by no more than 2-3 hours, to facilitate administering a large volume necessary due to limited solubility of the test substance.
Positive control: single oral intubation (40 mg/kg body weight)

Post exposure period:

Test item groups and solvent control group: 24 hours;
Positive control group: 48 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Pre test: 3
Main study: 5 (males only)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide in physiological saline
- Route of administration: Single oral intubation
- Dosis: 40 mg/kg body weight (10 mL/kg body weight)

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Selection of an adequate dose range for the micronucleus main test was based on a dose range finding study. The test procedure and conditions were similar to those applied in the main test. One dose group, comprising 3 males and 3 females received a double dose of URALAC P 1920C with a 24 hour interval. This group was dosed with the highest concentration that was used for the main study (i.e. 2000 mg/kg bw). The observation period after dosing was 3 days. During this period mortality and physical condition were recorded at least once a day.

SAMPLING: Bone marrow was collected by flushing the femurs with fetal calf serum.

DETAILS OF SLIDE PREPARATION: A drop of the bone marrow 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 (Merck, Darmstadt, Germany)/ether (Merck) 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 (Merck) and air-dried overnight. Three slides were prepared per animal. The slides were automatically stained using the "Wright-stain-procedure" (based on Giemsa).

METHOD OF ANALYSIS: The number of micronucleated polychromatic erythrocytes was counted in at least 4000 polychromatic erythrocytes (with a maximum deviation of 5%). The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

Evaluation criteria:

Acceptability of the assay
A micronucleus test is considered acceptable if it meets the following criteria:
a) The incidence of micronucleated polychromatic erythrocytes in the positive control animals should be above the negative historical control data range.
b) The positive control substance induced a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes. The positive control data was analysed by the Students t test (one-sided, p < 0.05) in case of homogeneous variances or by the Welch t test in case of inhomogeneous variances (one-sided, p < 0.05).
c) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range.

Statistics:

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. In case the micronucleus data is not normally distributed the data will be transformed by using the formula y = 1/y. Thereafter the Dunnett’s test will be performed. In case the Dunnett’s test shows that there are statistically significant differences between one or more of the test substance groups and the vehicle control group a Cochran Armitage trend test (p < 0.05) will be performed to test whether there is a significant trend in the induction.
In case the micronucleus data is not normally distributed the data will be transformed by using the formula y = 1/y. Thereafter the Dunnett’s test will be performed. In case the Dunnett’s test shows that there are statistically significant differences between one or more of the test substance groups and the vehicle control group a Cochran Armitage trend test (p < 0.05) will be performed to test whether there is a significant trend in the induction.

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:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: Ataxia and lethargy were observed in all animals within 3 hours after dosing on day 1 and day 2. All animals appeared normal on day 2 (before dosing) and on day 3. No mortality occurred.
- Since there were no differences between sexes in toxicity only male animals were used in the main study.

RESULTS OF DEFINITIVE STUDY
- No mortality occurred. Within 3.5 hour after dosing on the first and second day all animals treated with URALAC P 1920C and the vehicle control animals were lethargic and showed ataxia. Within 20 hours after dosing all animals recovered from the treatment.
- No biologically relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of URALAC P 1920C treated animals compared to the vehicle treated animals. Although a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes was observed at a concentration of 500 mg/kg body weight, the mean and all individual number of micronucleated polychromatic erythrocytes were within the historical control data range and the increase was only observed at the lowest concentration tested, not dose related and therefore not considered biologically relevant.
- The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical vehicle control data range.
- Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes.
- The animals of the groups, which were treated with URALAC P 1920C and the positive control showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test substance on the erythropoiesis.

Applicant's summary and conclusion

Conclusions:

An in vivo micronucleus study with URALAC P 1920C in the mouse (5 males/dose, oral exposure) was conducted according to OECD/EC guidelines and GLP principles. It is concluded that URALAC P 1920C is not clastogenic or aneugenic in the bone marrow micronucleus test.

Executive summary:

An in vivo micronucleus study with URALAC P 1920C in the mouse was conducted according to OECD/EC guidelines and GLP principles. Five males/ dose were exposed via the oral route to 500, 1000 and 2000 mg/kg bw. No biologically relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of URALAC P 1920C treated animals compared to the vehicle treated animals. Positive and negative controls were included and the results validated the study. It is concluded that this test is valid and that URALAC P 1920C is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 48 hours post dosing of male mice up to a dose of 2000 mg/kg bw/day (the maximum recommended dose in accordance with current regulatory guidelines).