Reaction mass of 2-ethyl-2-[[3-(2-methylaziridin-1-yl)propionyl]methyl]propane-1,3-diyl bis(2-methylaziridine-1-propionate) and 2,2-bis({[3-(2-methylaziridin-1-yl)propanoyl]oxy}methyl)butyl 3-[2,2-bis({[3-(2-methylaziridin-1-yl)propanoyl]oxy}methyl)butoxy]propanoate

Genetic toxicity: in vivo

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:

6 June to 12 July 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Performed according to OECD test guidelines and GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

2-({3-[2,2-bis({[3-(2-methylaziridin-1-yl)propanoyl]oxy}methyl)butoxy]-3-oxopropoxy}methyl)-2-({[3-(2-methylaziridin-1-yl)propanoyl]oxy}methyl)butyl 3-(2-methylaziridin-1-yl)propanoate; 2-ethyl-6-(2-methylaziridin-1-yl)-2-({[3-(2-methylaziridin-1-yl)propanoyl]oxy}methyl)-4-oxohexyl 3-(2-methylaziridin-1-yl)propanoate

EC Number:

939-180-9

Molecular formula:

Not relevant - Multiconstituent substance

IUPAC Name:

2-({3-[2,2-bis({[3-(2-methylaziridin-1-yl)propanoyl]oxy}methyl)butoxy]-3-oxopropoxy}methyl)-2-({[3-(2-methylaziridin-1-yl)propanoyl]oxy}methyl)butyl 3-(2-methylaziridin-1-yl)propanoate; 2-ethyl-6-(2-methylaziridin-1-yl)-2-({[3-(2-methylaziridin-1-yl)propanoyl]oxy}methyl)-4-oxohexyl 3-(2-methylaziridin-1-yl)propanoate

Details on test material:

- Name of test material (as cited in study report): Standard CX-100
- Physical state: clear colourless sllightly viscous liquid
- Batch: 310F-0699
- Purity: 99%
- Expiration date of the lot/batch: 30 January 2012
- Storage condition of test material: At room temperature in the dark

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6-8 weeks old
- Weight at study initiation: males 34-39 g, females 25-29 g
- Assigned to test groups randomly: yes
- Fasting period before study: not reported
- Housing: 5 animals/cage in polycarbonate cages, contining sterilised sawdust as bedding material.
- Diet (ad libitum): Pelleted rodent diet (SM F/M-Z from SSNIFF Spezialdiaten GmbH, Soest, Germany)
- Water (ad libitum): tap water
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1-22.1
- Humidity (%): 41-75
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: physiological saline

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: test substance was suspended in physiological saline. These were dosed within approx 2 hours after preparation.

Duration of treatment / exposure:

not applicable

Frequency of treatment:

once

Post exposure period:

24 or 48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
25, 50, 100 mg/kg bw
Basis:
other: injected

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration: ip
- Doses / concentrations: 40 mg/kg bw

Examinations

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: An intraperitoneal injection of a maximum tolerated, an intermediate and a low dose of substance was given to the mice. The route of administration was chosen to maximise the chance of the substance reaching the target tissue. First a dose range finding study was done.

TREATMENT AND SAMPLING TIMES: Three dose levels were used at the first sampling time. At the second sampling time only the highest dose was used. First sampling time was 24 h after treatment and second sampling time was 48 after treatment.

DETAILS OF SLIDE PREPARATION: Bone marrow suspension was placed on a slide, which was air dried, fixed for 5 min in 100% methanol and air dried overnight. Two slides/animal were prepared. They were stained, based on Giemsa.

METHOD OF ANALYSIS: All slides were randomly coded before examination. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. 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.

OTHER:

Evaluation criteria:

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 historical control data range.
b) 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.
c) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range.

Equivocal results should be clarified by further testing using modification of experimental conditions.
A test substance is considered positive in the micronucleus test if:
- it induced a biologically as well as a statistically significant 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 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:

Statistical significance was determined with the Wilcoxon Rank Sum Test, one-sided, p < 0.05.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

at 100 mg/kg bw, male animals which were sampled at 48 hours, showed a minor decrease in the ratio of polychromatic to normochromatic erythrocytes

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 100 mg/kg bw in one male and one female
- Clinical signs of toxicity in test animals: Within one hour after dosing the animals showed the following toxic signs: hunched posture and rough coat (female). Within 4 hours after dosing the male animal had a rough coat, hunched posture and closed eyes. The female animal remained its hunched posture. After 20 and 44 hours male animals were lethargic, had a hunched posture, a rough coat and closed eyes. The female animals had a hunched posture and a rough coat.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no
- Ratio of PCE/NCE (for Micronucleus assay): minor decrease after 48 hrs
- Appropriateness of dose levels and route: ip route of exposure maximises the chance of the test substance reaching the target tissue. Minimal toxicity was observed by a decrease in PCE/NCE and some clinical signs at the highest dose only.
- Statistical evaluation: Student's t'test, p<0.02

Any other information on results incl. tables

Main test:

In the 100 mg/kg bw group: During the first two hours after dosing all animals had a hunched posture. Four males and two females also had a rough coat. Within 19 hours after dosing all animals still had a hunched posture. All males and two female animals also had a rough coat. After 43 hours the animals had a hunched posture. One male animal also had a rough coat.

In the 50 and 25 mg/kg bw group no clinical signs of toxicity or mortality was observed.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
CX100 showed no evidence of a mutagenic potential, when administered intraperitoneally to mice, in the in vivo micronucleus test.

Executive summary:

An additional study was performed in 2011, according to OECD474. This study was performed because in time the production process of the substance has changed significantly and in addition due to the unknown genetic toxicity status of the substance more information was required for CLP classification and REACH registration (which molecular weight substance would be preferable).

Mice were dosed intraperitoneally once (25, 50 and 100 mg/kg bw). At 100 mg/kg bw no evidence of a mutagenic potential was shown, toxicity was shown by some clinical signs (hunched posture, rough coat, closed eyes), while a decrease in the ratio of polychromatic to normochromatic erythrocytes confirmed exposure of the target tissue.