Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 22 Aug, 2012 to 12 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
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Esterification products of acrylic acid and 2,2,2',2'-tetrakis(hydroxymethyl)-3,3'-oxydipropan-1-ol
EC Number:
800-838-4
Cas Number:
1384855-91-7
Molecular formula:
Molecular formula not available for this UVCB.
IUPAC Name:
Esterification products of acrylic acid and 2,2,2',2'-tetrakis(hydroxymethyl)-3,3'-oxydipropan-1-ol
Constituent 2
Chemical structure
Reference substance name:
2-[[3-[(1-oxoallyl)oxy]-2,2-bis[[(1-oxoallyl)oxy]methyl]propoxy]methyl]-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate
EC Number:
249-698-0
EC Name:
2-[[3-[(1-oxoallyl)oxy]-2,2-bis[[(1-oxoallyl)oxy]methyl]propoxy]methyl]-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate
Cas Number:
29570-58-9
Molecular formula:
C28H34O13
IUPAC Name:
2-[[3-[(1-oxoallyl)oxy]-2,2-bis[[(1-oxoallyl)oxy]methyl]propoxy]methyl]-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate
Test material form:
other: Clear colorless liquid
Details on test material:
- Name of test material (as cited in study report): DPHA
- Physical state: Clear colorless liquid
- Analytical purity: 100%
- Storage condition of test material: Room temperature, protected from light
- Receipt/login: 02 August 2012

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan, Frederick, MD
- Age at study initiation: 6 or 7 weeks old
- Weight at study initiation: 31.0 – 35.8 g
- Assigned to test groups randomly: Animals were assigned to these groups using a randomization procedure based on equalization of group mean body weights. Following randomization, animals were identified by sequentially numbered ear tags assigned to each animal during randomization process.
- Housing: Mice were housed up to five per rodent Micro-Barrier cage. Cages were placed on the racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system. The purpose of this system was to supply uninterrupted positive air to each individual rodent Micro-Barrier cage and to capture the effluent air from each cage and re-filter the air (HEPA) prior to introducing the air back into the cage. Heat-treated Sani-Chip hardwood chips were used for bedding to absorb liquids (P.J. Murphy Forest Products, Montville, NJ). Bedding was analyzed by the Manufacturer for any contaminants.
- Diet: Harlan 2018C Certified Global Rodent Diet was provided ad libitum
- Water: Tap water was provided ad libitum
- Acclimation period: 5 or 11 d

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3°F
- Humidity: 50 ± 20%
- Air changes: 10 changes of fresh HEPA-filtered air every hour.
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil
- Concentration of test material in vehicle: 0.1, 1, 10, 100, and 200 mg/mL for dose-range finding study and 50, 100, and 200 mg/mL for definitive micronucleus assay
- Lot/batch no. (if required): MKBD6671
- Purity: Corn oil is characterized as per the Certificate of Analysis on file with the testing facility
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 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.
Duration of treatment / exposure:
24 or 48 h
Frequency of treatment:
Single dose
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1, 10, 100, 1000 and 2000 mg/kg bw
Basis:
nominal conc.
(for dose range-finding assay)
Remarks:
Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:
nominal conc.
(for definitive micronucleus assay)
No. of animals per sex per dose:
Dose range-finding assay: Five male and five female mice were exposed to DPHA at 2000 mg/kg bw and two male mice each were exposed to DPHA at 1, 10, 100 or 1000 mg/kg bw
Definitive micronucleus study: Five male mice/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide monohydrate (CP)
- Route of administration: Injection
- Doses / concentrations: 5 mg/mL (10 to 50 mg/kg bw)

Examinations

Tissues and cell types examined:
Polychromatic erythrocytes (PCE)
Details of tissue and slide preparation:
-Bone marrow collection and slide preparation: At the scheduled bone marrow collection time, five mice per treatment were euthanized by CO2 asphyxiation verified by toe pinch reflex. 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 approx 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation at about 100 x g for about 5 min and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were re-suspended and a small drop of bone marrow suspension was spread onto a clean glass slide. Slides were labeled with harvest date, experiment and animal number using lead pencil. Two slides were prepared from each mouse. The slides were air dried and 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.

-Scoring for micronuclei (bone marrow evaluation): Bone marrow was evaluated by fluorescent microscopy. To control for bias, bone marrow slides were coded using a random number table by an individual not involved with the scoring process. The staining procedure permits the differentiation by color of polychromatic and normochromatic erythrocytes (bright orange PCEs and ghost-like, dark green NCEs, respectively). Slides initially were scanned using medium magnification to locate suitable areas where the cells were well spread and stained. Next, cells were scored using high power oil immersion as follows. The criteria for the identification of micronuclei are those of Schmid (1975). Micronuclei are brightly stained bodies that generally are round and that generally are between 1/20 and 1/5 the size of the PCE. Scoring was based upon the micronucleated cell, not the micronucleus; thus occasional cells with more than one micronucleus are counted as one micronucleated PCE (mnPCE), not two (or more) micronuclei. At least 2000 PCEs/animal were scored for the presence of micronuclei (mnPCEs) whenever possible. In addition, at least 1000 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.
Evaluation criteria:
Criteria for a valid test: The 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. Five animals /group are available for analysis.
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.
-Other criteria may be used in reaching a conclusion about the study results (e.g., magnitude of any increase, dose-dependency, comparison to historical control values, biological significance, etc.).
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:
Transient 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
Positive controls validity:
valid
Additional information on results:
Dose range finding assay: No mortality was observed in any of the treatment groups. All mice at 1, 10, 100 mg/kg bw appeared normal during the study period. Transient piloerection was noted following dose administration at 1000 and 2000 mg/kg bw and persisted in one of the male mice at 2000 mg/kg bw on study Day 2. No appreciable reductions to the mean group body weights were observed.
Definitive micronucleus study:
-Clinical signs: No mortality was observed in any of the treatment groups. All mice in the control groups appeared normal. Transient 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.
-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 test substance did not inhibit erythropoiesis. No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in test substance groups relative to the respective vehicle control groups was observed at 24 or 48 h after dose administration (p > 0.05, Kastenbaum-Bowman Tables).

Any other information on results incl. tables

For data tables, refer to the attached pdf under 'attached background material'.

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the test substance was considered to be non-clastogenic in the mouse micronucleus assay.
Executive summary:

An in vivo bone marrow micronucleus study was conducted to determine the clastogenic potential of the test substance DPHA according to OECD Guideline 474, in compliance with GLP. The study was conducted in two phases: a dose range finding (DRF) assay that evaluated the toxicity of the test article and a definitive micronucleus assay that evaluated test article’s potential to increase the incidence of micronucleated polychromatic erythrocyte cells (mnPCEs). The test article was formulated in corn oil. In the definitive micronucleus assay, corn oil was also used as the vehicle control article and cyclophosphamide monohydrate (CP), at a dose of 50 mg/kg, was used as the positive control article. In both phases of the study, test and/or control articles were administered at a dose volume of 10 mL/kg body weight by a single oral gavage. Animals were observed for signs of toxicity during the course of each phase of the study. In absence of toxicity data for DPHA, a DRF assay was performed exposing two male mice/ group at 1, 10, 100, 1000 mg/kg, while five male and five female mice were exposed to 2000 mg/kg, the maximum regulatory guideline recommended dose level. In absence of mortality or severe signs of clinical signs of toxicity, a dose of 2000 mg/kg was tested as high dose in the definitive micronucleus assay. Two lower doses at 500 and 1000 mg/kg were also tested. Since no differences in the clinical signs of toxicity were observed, only male mice were used in the definitive micronucleus assay. In the definitive micronucleus assay, mice were dosed either with the control (vehicle) or positive articles or with DPHA at 500, 1000, or 2000 mg/kg and were euthanized at the scheduled time points and the femoral bone marrow was collected. Bone marrow smears (slides) were prepared and stained with acridine orange stain (a nucleic acid specific stain). Bone marrow cells [polychromatic erythrocytes (PCEs)] were examined microscopically for the presence of micronuclei (micronucleated PCEs; mnPCEs). A statistical analysis of data was performed using the Kastenbaum-Bowman Tables (binomial distribution, p ≤ 0.05). In addition, the proportion of PCEs to total erythrocytes (PCEs/EC ratio) was also determined as indicator of test article’s cytotoxicity. In the DRF assay, no mortality was observed in any of the treatment groups. All mice at 1, 10, and 100 mg/kg appeared normal during the study period. Transient piloerection was noted following dose administration at 1000 and 2000 mg/kg and persisted in one of the male mice at 2000 mg/kg on study day 2. In the definitive micronucleus assay, no mortality was observed in any of the treatment groups. All mice in the control article groups appeared normal. Transient piloerection was noted in one of the mice at 500 and 1000 mg/kg and in all mice at 2000 mg/kg following dose administration. No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes in the test article groups relative to the respective vehicle control groups were observed, suggesting that the test article did not inhibit erythropoiesis. No statistically significant increase in the incidence of mnPCEs in test article groups relative to the respective vehicle control groups was observed at 24 or 48 hours after dose administration (p >0.05). CP, the positive control, induced a statistically significant increase in the incidence of micronucleated PCEs (p≤ 0.05). The number of micronucleated PCEs 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. Under the study conditions, the test substance was considered to be non-clastogenic in the mouse micronucleus assay (Kulkarni, 2013).