Registration Dossier

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:
16-Sep-2013 to 22-Oct-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Substance type: Clear slightly yellow very viscous liquid
- Purity: 100% Mono constituent substance
- Storage condition of test material: In refrigerator (2-8°C) in the dark



Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: 34.8 ± 1.3 g (range 33 – 38 g).
- Assigned to test groups randomly: yes
- Fasting period before study: Feed was withheld 3 - 4 h prior to dosing until administration
- Housing: In groups of 5 animals per sex per cage in polycarbonate cages containing sterilised sawdust as bedding material. Paper bedding was provided as cage-enrichment
- Diet (e.g. ad libitum): free access
- Water (e.g. ad libitum): free access
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 - 23.0 °C
- Humidity (%): 38 - 87%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline was suspended in propylene glycol . The specific gravity of propylene glycol is 1.036 g/ml. m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline concentrations were vortexed and treated with ultra-sonic waves to obtain a homogeneous suspension.

- Justification for choice of solvent/vehicle: A homogeneous suspension could be obtained in propylene glycol and propylene glycol is accepted and approved by authorities and international guidelines

- Concentration of test material in vehicle: 43.8, 87.5 and 175 mg/ml
- Amount of vehicle (if gavage or dermal): The dosing volume was 10 ml/kg body weight
Duration of treatment / exposure:
Treatment:
Solvent, positive control, low and mid dose level: 24 hours
Highest dose level: 24 and 48 hours

Frequency of treatment:
Once
Doses / concentrations
Remarks:
Doses / Concentrations:
438, 875, and 1750 mg/kg body weight
Basis:
nominal conc.
No. of animals per sex per dose:
At least five animals per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration: Oral
- Doses / concentrations: 40 mg/kg body weight

Examinations

Tissues and cell types examined:
Bone marrow smears
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
-The dose level selected should be ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.

DETAILS OF SLIDE PREPARATION:
- The smears are air-dried, fixed in methanol and stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer, allowed to air-dry and vover-slipped using mounting medium.

METHOD OF ANALYSIS:
- 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.
Evaluation criteria:
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) 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 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
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1750 and 2000 mg/kg BW
- Clinical signs of toxicity in test animals:
One male and one female were dosed with 2000 mg test substance per kilogram body weight. The female had a hunched posture, showed ataxia and was lethargic within 4 hours after dosing. The female died within 19 hours after dosing. The male animal had a hunched posture within 4 hours after dosing. The animal recovered from the treatment within 19 hours after dosing. Two additional males were dosed with 2000 mg/kg body weight. One of the animals died within 20 hours after dosing. Three males and and three females dosed with 1750 mg/kg body weight showed no treatment related clinical signs after dosing


RESULTS OF DEFINITIVE STUDY
- Dose range: 438, 875 and 1750 mg/kg BW
- Clinical signs of toxicity in test animals:
No treatment related clinical signs or mortality were noted in animals dosed with 438 and 875 mg
m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline/kg body weight, or control animals receiving vehicle or cyclophosphamide.

The following clinical observations were made in the groups treated with 1750 mg
m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline/kg body weight:

During the first 2 hours after treatment none of the animals showed treatment related clinical signs. Within 19 hours after dosing 3 animals had died, a third animal showed a hunched posture and was lethargic, a fourth animal was lethargic, showed ventral recumbency and had no reaction to a stimulus. This animal died within 23 hours after dosing. After replacement of the dead animals 4 animals were available for the 24 hour sampling time and 5 animals were available for the 48 hour sampling time.

The other animals treated with 1750 mg m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline/kg body weight showed no treatment related clinical signs or mortality.

- Induction of micronuclei (for Micronucleus assay):
No increase in the mean frequency of micronucleated polychromatic erythrocytes above the historical control data range (0 – 5) was observed in the bone marrow of animals treated with 438 and 875 mg m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline/kg body weight. Two animals treated with 438 mg/kg body weight had a high incidence of 6 and 7 micronucleated polychromatic erythrocytes which is above the historical control data range.

m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline induced a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes compared to the corresponding vehicle control group in animals treated with 1750 mg/kg body weight at both sampling times.
One out of four animals dosed with 1750 mg/kg body weight sampled after 24 hours showed an increase above the historical control data range (0 – 5) and 3 out of 5 animals at the 48 hours sampling time.

- Ratio of PCE/NCE (for Micronucleus assay):
The animals treated with 1750 mg m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline/kg body weight and the animals of the positive control group showed a decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

- Appropriateness of dose levels and route: Adequate evidence of test material toxicity was demonstrated via the oral route administration.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline is not clastogenic or aneugenic in the bone marrow micronucleus test in male mice up to a dose of 875 mg/kg
Executive summary:

Micronucleated polychromatic erythrocytes

No increase in the mean frequency of micronucleated polychromatic erythrocytes above the historical control data range (0 – 5) was observed in the bone marrow of animals treated with 438 and 875 mg m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline/kg body weight. Two animals treated with

438 mg/kg body weight had a high incidence of 6 and 7 micronucleated polychromatic erythrocytes which is above the historical control data range.

 

m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline induced a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes compared to the corresponding vehicle control group in animals treated with 1750 mg/kg body weight at both sampling times. One out of four animals dosed with 1750 mg/kg body weight sampled after 24 hours showed an increase above the historical control data range (0 – 5) and 3 out of 5 animals at the 48 hours sampling time.

Polychromatic to normochromatic ratio

The animals treated with 438 and 875 mg m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline/kg body weight showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects on the erythropoiesis.

 

The animals treated with 1750 mg m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline/kg body weight and the animals of the positive control group showed a decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

Discussion and conclusion

A high level of mortality was observed in the animals dosed with 1750 mg/kg body weight in the main study (4 out of 13 animals died), which did not reflect the data obtained in the dose range finding study where no treatment related clinical signs or mortality was observed.

 

According to the guidelines, a substance should be tested up to the maximum of toxicity, which is defined as the dose producing signs of toxicity such that higher dose levels, based on the same dosing regimen, would be expected to produce lethality. When using this definition, the high dose of 1750 mg/kg where lethality was observed, is considered too high for evaluation of genotoxicity in this assay.

 

Due to excessive toxicity at 1750 mg/kg, the micronucleus scoring at this dose level has no toxicological value.