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:
22-Mar-2016 to 17-Jun-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

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)
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:
- Name of test material (as cited in study report): di-C16-C18 (evennumbered) alkyl tripropylenetetramine
- Substance type: Viscous liquid
- Physical state: Liquid
- Storage condition of test material: At room temperature container flushed with nitrogen
- Batch/Lot number: B1; batch inspection lot no.:890000394200
- Expiration date of the lot/batch: 22 October 2017
- Purity: 100% (UVCB substance)
- pH: 8.5-10.5 (1% solution)
- specific gravity: 0.83 at 60°C
Specific details on test material used for the study:
-Name of test material (as cited in study report): di-C16-C18 (evennumbered) alkyl tripropylenetetramine
-Substance type: Viscous liquid
-Physical state: liquid
-Purity: Purity/Composition 100% (UVCB substance)
-Batch/Lot number: B1; batch inspection lot no.: 890000394200
-Expiration date of the lot/batch: 22 October 2017

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: males 34.1 ± 1.8 g and females 26.9 ± 1.3 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- 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): 21.2 - 22.2°C
- Humidity (%): 37 - 102%
- Air changes (per hr): 15
- 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: The test item was stable in corn oil for at least 5 hours and suspension could be obtained. Corn oil has been accepted and approved by authorities and international guidelines
- Concentration of test material in vehicle: 32, 62.5, 125 and 250 mg/ml

Duration of treatment / exposure:
Treatment:
Two treatments were performed, administered at a 24-hour interval.
Frequency of treatment:
Twice
Doses / concentrations
Remarks:
Doses / Concentrations:
males 62.5, 125 and 250 mg/kg bw
females 32, 62.5 and 125 mg/kg bw

Basis:
analytical 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: ip
- 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 cover-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 item is considered positive in the micronucleus test if:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative in the micronucleus test if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.
Statistics:
ToxRat Professional v 3.2.1 was used for statistical analysis of the data.

Results and discussion

Test resultsopen allclose all
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: not clastogenic or aneugenic
Key result
Sex:
female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: not clastogenic or aneugenic
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
One male and one female were dosed via intraperitoneal injection with 2000 mg/kg body weight. The animals died within 4 hours after the first dosing. One male and one female dosed with 1000 and 500 mg/kg body weight and one female dosed with 250 mg/kg body weight died within 21 hours after the first dosing. Subsequently, three males and three females were dosed with 250 and 125 mg/kg body weight, respectively. The male animals showed the following toxic signs after dosing: ataxia, lethargy, rough coat, closed eyes and a hunched posture. The female animals showed the following toxic signs after dosing: lethargy, closed eyes and a hunched posture.

RESULTS OF DEFINITIVE STUDY

- Clinical signs of toxicity in test animals:
Male animals treated with 250 mg/kg body weight showed the following toxic signs after dosing: lethargy, ataxia, rough coat, hunched posture and eyes closed. Male and female animals treated with 125 mg/kg body weight showed the following toxic signs after dosing: lethargy, rough coat, hunched posture and eyes closed. Male animals treated with 62.5 mg/kg body weight showed the following toxic signs after dosing: lethargy, hunched posture and rough coat. Female animals dosed with 62.5 mg/kg body weight had hunched posture and rough coat.

No treatment related clinical signs or mortality were noted in any female animal treated with 32 mg/kg body weight. In addition, no treatment related clinical signs or mortality were noted in the control animals receiving vehicle or cyclophosphamide.

- Induction of micronuclei (for Micronucleus assay):
No biologically relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the test item.
- Ratio of PCE/NCE (for Micronucleus assay):
The groups treated with test item concentrations of 62.5 and 32 mg/kg body weight (females) showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicates a lack of toxic effects of this test item on the erythropoiesis. The groups treated with 250, 125 and 62.5 mg/kg body weight (males) and 125 mg/kg body weight (females) and cyclophosphamide 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 intraperitoneal injection .

Applicant's summary and conclusion

Conclusions:
di-C16-C18 (evennumbered) alkyl tripropylenetetramine is not clastogenic or aneugenic in the bone marrow micronucleus test of male and female mice up to a dose of 250 mg/kg body weight and 125 mg/kg body weight, respectively
Executive summary:

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of test item treated animals compared to the vehicle treated animals. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the 95% control limits of the distribution of the historical negative control database.

 

Cyclophosphamide, the positive control item, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database. Hence, all criteria for an acceptable assay were met.