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:
From 30 Aug, 2012 to 14 Oct, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
mouse
Strain:
other: Hsd :ICR (CD-I)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Frederick, USA
- Date of receival: 30 August, 2012 (dose range finding) and 20 September, 2012 (definitive micronucleus study)
- Age at study initiation: 6-7 weeks
- Weight at study initiation: DRF: 29.1-33.2 g (males) or 25.9-26.9 g (females); Definitive study: 30.7-37.0 g (males)
- Number of animals: Dose range finding: 20/sex, definitive study: 35/sex
- Assigned to test groups randomly: Yes, animals randomized using a computer program
- Housing: Animals were housed up to five/micro-barrier cage which were placed on the racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system
- Randomization: Animals were assigned to groups using a randomization procedure based on equalization of group mean body weights. At the time of randomization, the weight variation of animals will not exceed ±20% of the mean weight.
- Bedding: Heat treated hardwood chips were used
- Diet (e.g. ad libitum): Certified laboratory rodent chow (Harlen 2018C certified global rodent diet), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 5 to 11 d (mice were observed each day for signs of illness and other conditions of poor health.)

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3°F
- Humidity: 50 ± 20%
- Air changes: 10/h
- Photoperiod: 12 h dark/12 h light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil
- Lot/batch no. (if required): MKBD6671
- CAS no.: 8001-30-7
- Expiration date: 24 september, 2013
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance dose formulations were prepared fresh for each phase of the study prior to use in dose administration. All formulations for the dose range finding (DRF) assay (0.1, 1, 10, 100, and 200 mg/mL) and all formulations for the definitive micronucleus assay (50, 100, and 200 mg/mL) were prepared as follows:
An appropriate amount of the test substance was combined with an appropriate volume, 80% of the target volume, of the vehicle. Each formulation was vortexed for 2 min and stirred for 10 min using a magnetic stir bar/plate. Remaining volume of the vehicle was added to reach the final targeted volume and each formulation was stirred for an additional 10 min. All formulations appeared as yellow solutions. All formulations were stirred using a magnetic stir bar/plate continuously prior to and during dose administration.

Positive control dose formulation:
An aqueous dosing formulation of cyclophosphamide (CP) at a concentration of 5 mg/mL was prepared fresh on the day of dose administration. An appropriate amount of CP was dissolved in an appropriate volume of sterile water for injection (B. Braun Medical, CAS number: 7732-18-5, lot number: J1L003 expiration date: September 2013). The formulation was vortexed for 2 min. The accuracy of preparation and stability of the CP formulation was demonstrated by acceptable results that met the criteria for a valid test.
Duration of treatment / exposure:
24 or 48 h
Frequency of treatment:
Single administration
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1, 10, 100, 1000 and 2000 mg/kg bw
Basis:
nominal conc.
(for dose range-finding study)
Remarks:
Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:
nominal conc.
(for definitive micronucleus study)
No. of animals per sex per dose:
-Range-finding study: 5 male and 5 female mice at 2000 mg/kg and two male mice each exposed to the test substance at 1, 10, 100 or 1000 mg/kg bw
-Main study: Five male mice/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide monohydrate (CP)
- CAS number: 6055-19-2
- Lot number: SLBC0666V
- Expiration date: 31 March 2015, was obtained from Sigma-Aldrich.

Examinations

Tissues and cell types examined:
Femurs were removed for marrow extraction and the prepared slides were examined for polychromatic erythrocytes (PCEs), normochromatic erythrocytes (NCEs) and total erythrocytes.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dose selection was based on a preliminary dose range-finding test conducted on 5 male and 5 female mice at 2000 mg/kg bw and two male mice each to the test substance at 1, 10, 100 or 1000 mg/kg bw. No mortality and/or toxic signs were recorded after 2 d of exposure.

TREATMENT AND SAMPLING TIMES: 5 mice per treatment were euthanized by CO2 asphyxiation verified by toe pinch reflex at 24 or 48 h after exposure. Immediately following euthanasia. The femurs were exposed, cut just above the knee and the bone marrow was aspirated into a syringe containing fetal bovine serum.The bone marrow cells were transferred to a labeled centrifuge tube containing approximately 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation at about 100 x g for about five minutes and the supernatant was drawn off, leaving a small amount of
serum with the remaining cell pellet

DETAILS OF SLIDE PREPARATION: Bone marrow cells extracted, preparations spread on slides and air-dried. The slides were fixed in methanol. One set of slides was stained with a nucleic acid-specific stain acridine orange and was used in microscopic evaluation. Second set was packaged for storage until finalization for the report. s

METHOD OF ANALYSIS: Slides were scanned to determine the frequency of micronuclei in 2,000 polychromatic erythrocytes (PCEs) per animal. In addition, at least 1,000 total erythrocytes (PCEs + NCEs) were scored per animal to determine the proportion of PCEs as an index of bone marrow cytotoxicity. PCE proportions <20% of vehicle control value were considered excessively cytotoxic and the animal data were excluded from evaluation.
Evaluation criteria:
Criteria for a Valid Test
The micronucleated polychromatic erythrocytes (mnPCE) frequency of the vehicle controls must be within the historical vehicle control range and the positive control must induce a significant increase (p≤0.05) in mnPCE frequency as compared to concurrent vehicle control.

Evaluation of Test Results
Once the criteria for a valid assay were met, the results were evaluated as follows:
- The test substance is considered to be positive if it induces a significant increase in mnPCE frequency (p≤0.05) at any dose level or sampling time compared to the concurrent vehicle control.

- The test substance is considered to be negative if no significant increase in mnPCE frequency is observed (p> 0.05) compared to the concurrent vehicle control.

Mice were observed for clinical signs of toxicity prior to and following each dose administration and at least once daily thereafter. Body weights were recorded at least once on each day of dosing.
Statistics:
The frequency of mnPCEs and the proportion of PCEs to total erythrocytes were determined for each animal and treatment group. Statistical significance (p≤0.05) was determined using the binomial distribution (Kastenbaum-Bowman tables).

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Piloerection was noted in one of the mice at 500 and 1000 mg/kg bw and in all mice at 2000 mg/kg bw following dose administration.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
DRF study:
No mortality was observed in any of the treatment groups. All mice at 1, 10, and 100 mg/kg bw appeared normal during the study period. Piloerection was noted in male mice at 1000 and 2000 mg/kg bw, while this clinical sign was noted transiently in female mice. No appreciable reductions to the mean group body weights were observed.

Definitive Study:
- Clinical Signs
No mortality was observed in any of the treatment groups. All mice in the control groups, and in the test substance groups at 500 mg/kg bw, appeared normal. Piloerection was noted at 1000 and 2000 mg/kg bw.
- Bone Marrow Evaluation
No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes in the test substance groups relative to the respective vehicle control groups were observed, suggesting that the erythropoiesis was not inhibited.
- No statistically significant increase in the incidence of mnPCE in test substance groups relative to the respective vehicle control groups was observed at 24 or 48 h.
CP, the positive control, induced a statistically significant increase in the incidence of mnPCEs (p≤ 0.05, Kastenbaum-Bowman Tables). The number of mnPCEs in the vehicle control groups did not exceed the historical vehicle control range. Based upon this, all criteria for a valid test were met as specified in the protocol.

- Frequency of micronuclei in polychromatic erythrocytes and PCE:NCE ratio: See table 1

Any other information on results incl. tables

Results: Refer to the attached background material for details on results.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the mouse micronucleus assay, di-TMPTTA was negative (not clastogenic).
Executive summary:

An in vivo bone marrow micronucleus test was performed with di-TMPTTA according to OECD Guideline 474, in compliance with GLP. The study was conducted in two phases: a dose range-finding assay and a definitive micronucleus assay. In both phases of the study, test and/or control mice were administered at a dose volume of 10 mL/kg bw by a single oral gavage. The dose range-finding assay was performed exposing two males/group at 1, 10, 100, 1,000 mg/kg bw, while five male and five females were exposed to 2,000 mg/kg bw. No mortality was observed in any of the treatment groups. All mice at 1, 10 and 100 mg/kg bw appeared normal during the study period. Piloerection was noted in males at 1,000 and 2,000 mg/kg bw, while this clinical sign was noted transiently in females. No appreciable reductions in mean group body weights were observed. In the definitive micronucleus assay, mice were dosed either with the control (vehicle or positive) or with the test substance at 500, 1,000 or 2,000 mg/kg bw. No mortality was observed in any of the treatment groups. All mice in the control groups and in the treated groups at 500 mg/kg bw appeared normal. Piloerection was noted at 1,000 and 2,000 mg/kg bw. No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes in the treated groups relative to the respective vehicle control groups were observed, suggesting that the test substance did not inhibit erythropoiesis. No statistically significant increase in the incidence of micronucleated polychromatic erythrocyte cells in treated groups relative to the respective vehicle control groups was observed at 24 or 48 h after dose administration. Under the conditions of the mouse micronucleus assay, the test substance was negative (not clastogenic) (Kulkarni, 2013).