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:
2013-02-28 to 2013-04-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD 474 GLP 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:
not applicable
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Test substance: DADPM/DEG-PO
Synonym: Daltolac XR 159
Chemical Name: Reaction mass of 2,2’-Oxydiethanol, propoxylated and Formaldehyde, polymer with benzenamine and 2-methyloxirane
Batch No.: RZBL001781
Description: Clear yellow liquid
Storage Conditions: Room temperature, stored protected from light

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
Test System:
ICR (CD-1) mice were obtained from Harlan, Frederick, MD. At the time of dose administration for each phase of the study, the mice were 6 weeks old. Mouse body weights recorded at randomization were within the following ranges:
Study Phase: 27.4 – 30.9 grams (Males) and 23.7 – 26.8 grams (females)
Definitive Micronucleus Study: 28.9 – 33.3 grams (Males)

Animal Receipt and Acclimation:
Virus antibody-free (VAF) mice were obtained from a supplier that monitored mice for evidence of ectoparasites, endoparasites, pathogenic bacteria, mycoplasmas, and appropriate murine viruses and were acclimated for 5 or 6 days after receipt. At BioReliance, mice were observed each day for signs of illness and other conditions of poor health. All mice were judged to be healthy prior to utilization in the study.

Animal Welfare Provisions and Animal Care:
All procedures involving mice performed at BioReliance follow the specifications recommended in The Guide for the Care and Use of Laboratory Animals adopted by BioReliance.
Animals were housed in an AAALAC-accredited facility with a controlled environment of 50 ± 20% relative humidity and 72 ± 3°F temperature with a 12-hour light/dark cycle. The animal rooms were supplied with at least 10 changes of fresh HEPA-filtered air every hour.

Mice of the same sex 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.
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.
Animals were allowed free access to tap water, which meets U.S. EPA drinking water standards [water source is Washington Suburban Sanitary Commission (WSSC) Potomac Plant]. A certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet) was provided ad libitum.

Randomization:
In each assay, mice were assigned to the appropriate number of treatment groups. Animals were assigned to these groups using a randomization procedure based on equalization of group mean body weights. At the time of randomization, the weight variation of animals did not exceed ±20% from their group mean. Following randomization, animals were identified by sequentially numbered ear tags assigned to each animal during the randomization process. The cage card contained the following information: the animal number, sex, study number, test substance identification, treatment group number, dose level and route of administration. Cage cards were color coded as a function of the treatment group. Raw data records and specimens were also identified by the unique test animal number.

Synopsis:
The study was conducted in two phases: a dose range-finding assay that evaluated the toxicity of the test substance and a definitive micronucleus assay that evaluated the genotoxic potential of the test substance. Based on the absence of mortality in the dose range-finding assay, male mice were exposed to DADPM/DEG-PO at 500, 1000 and 2000 mg/kg in the definitive micronucleus assay. At 24 and 48 hours post dose, bone marrow was collected and microscopically evaluated for the incidence of mnPCEs (Table 1).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Mineral oil
Details on exposure:
Dose Administration Procedure:
All dose formulations were administered at a dose volume of 10 mL/kg by single oral administration using appropriately sized disposable polypropylene syringes with gastric intubation tubes (needles). All mice in the experimental and control groups were weighed immediately before dose administration and the administered volume was based on individual body weight. Mice were observed after dose administration and throughout the course of the study for clinical signs of toxicity.

Definitive Micronucleus Assay
Mineral oil was also used as the negative (vehicle) control and Cyclophosphamide monohydrate (CP), at a dose of 50 mg/kg, was used as the positive control substance in the definitive micronucleus assay. The test or control substances were administered by single oral gavage at a dose volume of 10 mL/kg body weight. Based on the absence of mortality in the dose range-finding assay, 5 male mice in each group were exposed to DADPM/DEG-PO at the OECD guideline recommended high dose of 2000 mg/kg and two lower dose levels, 500 and 1000 mg/kg (Table 1). Animals were observed for clinical signs of toxicity following each dose administration and at least once daily thereafter.
Duration of treatment / exposure:
Dose Range-Finding Assay:
The dose range-finding assay was conducted exposing three mice/sex to DADPM/DEG-PO at a dose of 2000 mg/kg. Mice were observed for clinical signs of toxicity, following each dose administration and at least once daily thereafter. Following the two-day observation period, all surviving animals were euthanized by carbon dioxide inhalation verified by toe pinch reflex, and discarded without further examination.
Frequency of treatment:
A single oral gavage dose.
Post exposure period:
24 and 48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
0
Basis:
other:
Positive control(s):
Cyclophosphamide monohydrate (CP) at a dose of 50 mg/kg

Examinations

Tissues and cell types examined:
At the scheduled time point, animals were euthanized and the femoral bone marrow was collected.
Details of tissue and slide preparation:
At the scheduled bone marrow collection time, five mice per sex 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 approximately 1 mL of fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100xg for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended and a small drop of bone marrow suspension was spread onto a clean glass slide. 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. The second set of slides was stored at BioReliance at room temperature as a backup.
Evaluation criteria:
The bone marrow slides were coded using a random number table by an individual not involved with the scoring process. Bone marrow was evaluated by fluorescent microscopy. 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 a high power oil immersion lens 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.
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 binomial distribution tables (Kastenbaum and Bowman, 1970).

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Dose Range-Finding Assay:
No mortality was observed. Piloerection or diarrhea was noted in male mice, and diarrhea was noted in female mice. No appreciable changes in the mean group body weight were observed during the study period.

Definitive Micronucleus Study:
No mortality was observed in any of the treatment groups. Diarrhea was noted in animals in the vehicle control and the 500, 1000 mg/kg treatment groups. Diarrhea or piloerection was noted in animals in the 2000 mg/kg treatment group. All mice in the positive control group appeared normal during the study period.

The incidence of micronucleated polychromatic erythrocytes per 10,000 polychromatic erythrocytes scored (2000 PCEs/mouse) and the proportion of polychromatic erythrocytes per total erythrocytes are summarized and presented for each treatment group by sacrifice time in Table 2.

• No reductions were observed in the PCEs/EC ratio with DADPM/DEG-PO at any dose level relative to the vehicle control group.
• No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in the test substance-treated groups relative to the respective vehicle control groups was observed at 24 or 48 hours after dose administration (p > 0.05, Kastenbaum-Bowman tables).
• CP, the positive control, induced a statistically significant increase in the incidence of micronucleated PCEs (p = 0.05, Kastenbaum-Bowman tables).
• The incidence of mnPCEs in the vehicle control group at 24 hours post dose was outside the historical control data (0-7 mnPCEs/10,000 PCEs) for vehicle control. However, the incidence of mnPCEs for individual animal was within the range for all animals. Therefore, the Study Director concluded that this out of specification results did not adversely impact the integrity of the data or the validity of the study conclusion.

Any other information on results incl. tables

Table 2: Summary of results

Toxic/Cytotoxic Effects: No appreciable reductions in the PCEs/EC ratio were observed at any dose level relative to the respective vehicle control groups.

Genotoxic Effects: No statistically significant increase in the incidence of mnPCEs was observed at either 24 or 48 hours post dose.

Evidence of Exposure: Piloerection or diarrhea was noted in some animals dosed at 500, 1000 and 2000 mg/kg.

 Sampling time  Controls/Test Substance  Dose (mg/kg)  Sex/No. of Animals/Group     PCE#/1000 Erythrocyte (Mean+/-SD)  Number mnPCE/PCE scored   
 24 hours post-dose  Mineral oil  0  Male  5  0.8 +/- 0.27  8 / 10,000 
 24 hours post-dose  DADPM/DEG-PO  500  Male  5  1.0 +/- 0.35  10  / 10,000
 24 hours post-dose  DADPM/DEG-PO  1000  Male  5  0.8 +/- 0.27  8  / 10,000
 24 hours post-dose  DADPM/DEG-PO  2000  Male  5  1.1 +/- 0.55  11  / 10,000
 24 hours post-dose  Cyclophosphamide  50  Male  5  14.5 +/- 2.57  *145 / 10, 000
               
 48 hours post-dose  Mineral oil  0  Male  5 0.7 +/- 0.27  7 / 10,000 
   DADPM/DEG-PO  2000  Male  5  1.1 +/- 0.89  11  /10,000

* Statistically significant increase relative to the vehicle control, p = 0.05 (Kastenbaum-Bowman tables binomial distribution)                     

# PCE = Polychromatic erythrocytes; b mnPCE = Micronucleated polychromatic erythrocytes 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of the study conducted as described above, a single oral administration of DADPM/DEG-PO at doses up to and including a dose of 2000 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in the bone marrow of male ICR mice. Therefore, DADPM/DEG-PO was concluded to be negative in the mouse micronucleus assay.
Executive summary:

The test substance, DADPM/DEG-PO, was evaluated for in vivo clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (mnPCEs) cells in ICH (CD-1) mouse bone marrow.

The study was conducted in two phases: a dose range-finding assay to evaluate the toxicity of the test substance and a definitive micronucleus assay to evaluate the genotoxic potential of the test substance.

DADPM/DEG-PO was formulated using mineral oil as the vehicle and it was also used as the negative (vehicle) control in the definitive micronucleus assay. Cyclophosphamide monohydrate (CP), at a dose of 50 mg/kg, was used as the positive control substance in the definitive micronucleus assay. In both assays, test or control substances were administered by single oral gavage at a dose volume of 10 mL/kg body weight. Animals were observed for clinical signs of toxicity following each dose administration and at least once daily thereafter.

In the dose range-finding assay, three mice/sex were exposed to DADPM/DEG-PO at the maximum OECD guideline recommended dose level of 2000 mg/kg. No mortality was observed. Piloerection or diarrhea was noted in male mice, and diarrhea was noted in female mice. No appreciable changes in the mean group body weight were observed during the study period.

Due to the absence of mortality at 2000 mg/kg in the dose range-finding assay, the definitive micronucleus assay was conducted exposing the animals to DADPM/DEG-PO at dose levels of 500, 1000 and 2000 mg/kg. The vehicle and positive control substances were tested concurrently. Since no substantial differences in the clinical signs of toxicity between the sexes were observed in the dose range-finding assay, only male mice were used in the definitive micronucleus assay.

At the scheduled time point, animals were euthanized and the femoral bone marrow was collected. Bone marrow smears (slides) were prepared and stained with acridine orange, a nucleic acid specific stain and were examined microscopically for the presence of micronuclei (mnPCEs). A statistical analysis of the data was performed using the Kastenbaum-Bowman tables (binomial distribution, p = 0.05). In addition, the ratio of PCEs to total erythrocytes (PCEs/EC ratio) was also evaluated as an indication of bone marrow toxicity.

The following observations were made in the definitive micronucleus assay:

• No mortality was observed in any of the treatment groups. Diarrhea was noted in animals in the vehicle control and the 500, 1000 mg/kg treatment groups. Diarrhea or piloerection was noted in animals in the 2000 mg/kg treatment group. All mice in the positive control group appeared normal during the study period.

• No reductions were observed in the PCEs/EC ratio with DADPM/DEG-PO at any dose level relative to the vehicle control group.

• No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in the test substance-treated groups relative to the respective vehicle control groups was observed at 24 or 48 hours after dose administration (p > 0.05, Kastenbaum-Bowman tables).

• CP, the positive control, induced a statistically significant increase in the incidence of micronucleated PCEs (p = 0.05, Kastenbaum-Bowman tables).

• The incidence of mnPCEs in the vehicle control group at 24 hours post dose was outside the historical control data (0-7 mnPCEs/10,000 PCEs) for vehicle control. However, the incidence of mnPCEs for individual animal was within the range for all animals. Therefore, the Study Director concluded that this out of specification results did not adversely impact the integrity of the data or the validity of the study conclusion.

Based upon this, all criteria for a valid test were met as specified in the protocol.

Under the conditions of the study as described in this report, a single oral administration of DADPM/DEG-PO at doses up to and including a dose of 2000 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in the bone marrow of male ICR (CD-1) mice. Therefore, DADPM/DEG-PO was concluded to be negative in the mouse micronucleus assay.

This in vivo micronucleus test was requested by the National Institute of Environmental Research (NIER) of Korea for their registration of Daltolac R159.