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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
December 4th, 2012 to July, 17th 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)
Qualifier:
according to
Guideline:
other: ICH Guideline S2 (R1)
GLP compliance:
yes
Type of assay:
other: mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Crl:CD (SD) rats chosen as the test system as there is a large amount of historical biological data available for this species and strain. Its sensitivity to chemical-induced toxicity has been shown to be sufficient for detecting the potential toxicity of compounds.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh.
- Age at study initiation: 7 weeks old.
- Weight at study initiation: definitive study: range-finding test of initial dosing: 202-248 g (males), 152-175 g (females) range-finding phase after the study restart: 201-224 g (males); 147-198 g (females); definitive test:189-237 g (males); 152-186 g (females).
- Assigned to test groups randomly: each group (Groups 1-4) consisted of 3 animals/sex during initial dosing and study restart in order to allow an evaluation of toxicity while accounting for potential individual animal variability in response. Then 6 animals/sex/group during the study start in the definitive phase in order to ensure that there were 5 suriving analysable animals/sex/ concentration as per the OECD Guideline 474.
- Housing: upon arrival, the animals were housed individually in clean, stainless-steel, wire-mesh cages suspended above cage-board. Enrichment devices provided for environmental enrichment and to maintain their oral health and were sanitised weekly.
- Diet: basal certified Rodent LabDiet 5002 (PMI Nutrition International, LLC).
- Water: ad libitum, reverse-osmosis treated (on-site) drinking water delivered by an automatic watering system throughout the day.
- Acclimation: minimum 7 days. During this period, each animal was observed twice for mortality and changes in general appearance or behaviour.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C.
- Humidity: 50 ± 20 %.
- Air changes: 10 per hr.
- Lighting: fluorescent
- Photoperiod: 12 hrs dark / 12hrs light.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Vehicle(s)/solvent(s) used:
(1) polyethylene glycol 400 (PEG 400)
- Lot/batch no.: 2AC0722
(2) Glycerol Formal
- Lot/batch no.: YOYLK
Details on exposure:
INITIAL DOSING:RANGE-FINDING TEST
- Preparation of solutions: PEG 400, was dispensed on the day of dosing for administration to the vehicle control group (Group 1) and for preparation of the test substance formulations during the initial dosing. Due to viscosity, the vehicle could not be sterile-filtered so it was transferred into a sterile container and capped with a septum; these procedures were performed within a laminar flow hood using sterilised glassware and utensils. The vehicle was mixed throughout the preparation procedure. The test substance formulations were prepared on the days of dose administration as single formulations and stored at room temperature until use, were sterile-filtered into sterile containers and capped with a septum. Due to viscosity, the Group 4 (2000 mg/kg/day) test substance formulations could not be sterile-filtered; these formulations were transferred into a sterile container and capped with a septum.
- Route of administration: intraperitoneal injection.
- Treatment: the vehicle and test substance formulations were administered once followed by 2 days of observation prior to scheduled euthanasia (study day 3).
- Dosage levels: 500, 1000, 2000 mg/kg/day corresponding to test substance concentrations 50, 100 and 200 mg/ml.
- Dose volume: 10 ml/kg.
- Control: vehicle group (PEG 400)
- Due to mortality noted in all groups (including the control group) after a single dose administration, dosing was suspended and the surviving animals were observed for 2 days. All survivng animals were euthanised and discarded without necropsy on study day 3.

STUDY RESTART: RANGE-FINDING PHASE
- Preparation of solutions: glycerol formal, was dispensed daily for administration to the vehicle control group (Group 1) and for preparation of the test substance formulations after study restart. The vehicle was too viscose to be sterile-filtered so it was instead transferred to a sterile container and capped with a septum under a laminar hood and using sterilised glassware and utensils. The vehicle was mixed throughout the preparation and sampling procedure. The test substance formulations were prepared on the days of dose administration as single formulations and stored at room temperature until use.
- Route of administration: intraperitoneal injection.
- Treatment: the vehicle and test substance formulations were administered once daily for 3 concecutive days, through the period prior to scheduled euthanasia (study day 3).
- Dosage levels: 500, 1000, 2000 mg/kg/day corresponding to test substance concentrations 250, 500 and 1000 mg/ml.
- Dose volume: 2 ml/kg for test and vehicle group.
- Controls: vehicle group (glycerol formal).

STUDY RESTART: DEFINITIVE STUDY
- Preparation of solutions: in vehicle glycerol formal.
- Route of administration: intraperitoneal injection.
- Treatment: the vehicle and test substance formulations were administered once daily for 3 concecutive days, through the period prior to scheduled euthanasia (study day 3).
- Dosage levels: 500, 1000, 2000 mg/kg/day corresponding to test substance concentrations 250, 500 and 1000 mg/ml.
- Selection of doses: the highest dose selected was the limit test level as specified in the OECD Guideline 474, the 1000 mg/kg/day was selected to be half of the high dose and the 500 mg/kg/day was selected to be half of the mid dose. Findings in the range-finding phase were generally limited to the 2000 mg/kg/day group and included observations of hypoactivity/decreased activity and a single death shortly after the second dose. All other animals survived until study termination. It was expected that the high dose would produce chemical-specific effects but incidents of mortality would be limites and allow for interpretation of the data.
- Dose volume: 2 ml/kg for test and vehicle group.
Frequency of treatment:
Initial dosing - one single dose
After study restart (range finding and definitive phase)- once daily for 3 consecutive days
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
Range finding phase initial dosing and after study restart (Groups 1-4): 3 males and 3 females
Definitive phase after study restart (Groups 1-5): 6 males and 6 females
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide monohydrate (CPS)
- Lot no.: SLBC0666V.
- Purity: 100 %.
- Route of administration: oral.
- Frequency of treatment: single oral dose on study day 2, prior to the scheduled euthanasia.
- Dose: 60 mg/kg/day.
- Dose volume: 10 ml/kg.

Examinations

Details of tissue and slide preparation:
PARAMETERS EVALUATED
- Mortality and moribundity: all animals were observed twice daily, once in the morning and once in the afternoon. Moribund animals received a final detailed examination and had final body weights collected prior to euthanasia.
- Clinical examinations: performed at the time of dose administration and approximately 1-2 hours following dosing. In addition, all animals were observed once daily on nondosing days during the initial range-finding phase. Detailed physical examinations were performed on all animals 3 days of receipt, at randomization, on study day 0 and prior to the scheduled euthanasia (study day 3).
- Body weights: individual body weights were recorded within 3 days of receipt, at randomization, on study day 0 and on the day of the scheduled euthanasia (study day 3).
- Food consumption: in the definitive phase, individual food weights were recorded from the start of the pretest period to the time of randomization and from study day 0 to the day of the scheduled euthanasia. Food consumption was calculated as g/animal/day.
- Anatomic pathology-Macroscopic examination (range-finding phase): a complete necropsy was conducted on all animals found dead. Examination of the external surface, all orifices and the cranial, thoracis, abdominal and pelvic cavities, including viscera. Carcasses were discarded without tissue collection.

MICRONUCLEUS EVALUATION (DEFINITVE PHASE)
-Collection of bone marrow: collected from the first 5 of 6 animals/sex/group at the time of euthanasia from the right femur of animals euthanised by inhalation of carbone oxide. Bone marrow was aspirated or flushed 2 to 3 times from the right femur into a centrifuge tube using a syringe containing heat inactivated fetal bovine serum (HI FBS). The bone marrow was centrifuged and all but approx. 0.25 ml (or a volume approximately twice that of the cell pellet) of HI FBS was decanted, and the pellet was resuspended in the remaining HI FBS.
- Slide preparation: bone marrow smears were prepared by placing approximately 1 drop of cell suspension onto a minimum of 2 clean microscope slides. The slides were air dried, fixed in 100 % methanol for approx. 20 minutes and allowed to air dry a second time. Prior to analysis, the coded slides were stained with acridine orange (A/O) staining solution and placed in numerical order using the animal numbers. Two separate evaluations were made for each slide 1) a total of 1000 erythrocytes (both polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) per animal were counted and the PCE:total erythrocytes (TE) ratio was determined and 2) the number of micronuclated PCEs from a total of 2000 PCEs was scored per animal.
Statistics:
All statistical tests were performed using WTDMS unless otherwise stated. Analysis conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1 % and 5 %, comparing each test substance-treated group to the vehicle control group by sex. Body weight, body weight change, food consumption data and the percentages of micronucleated cells in PCEs and in the ratio of PCEs to TEs for the test substance-treated and vehicle control group were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the vehicle group. The positive control data were evaluated using the 2-sample t-test and compared to the vehicle control group. Statistical significance was assessed at a 95 % confidence level (p ≤ 0.05).

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Definitive study
Additional information on results:
MICRONUCLEUS EVALUATION
- Significant decrease in the mean percentage of micronucleated polychromatic erythrocytes (%MN-PCEs) was noted at the 500 and 1000 mg/kg/day dose levels in male rats when compared to the vehicle control group.
- The substance did not induce an increase in the mean number of MN-PCEs compared to the vehicle control group.
- No bone marrow cytotoxicity (decreases in the ratio of polychromatic to total erythrocytes, PCE:TE ratio) was noted in any test substance-treated group.
- Group mean values for both % MN-PCEs and PCE:TE ratios for the vehicle and positive control groups were within the respective historical control ranges.

INITIAL DOSING: RANGE FINDING PHASE
- Survival: mortality was noted in all groups including the control group on study day 1 or 2 following a single dose administration on study day 0. 3/6 animals in the 2000 mg/kg/day, 500 mg/kg/day and control group; all animals in the 1000 mg/kg/day group were found dead.
- Clinical observations: decreased activity, prostration, impaired muscle contraction, hypoactivity, intermittent convulsions or tremors, decreased respiration rate, body cool to touch, blue extremities, clear discharge from eyes, partial closure of eyes, mucoid or soft faeces, diarrhea, rectal mucous exudate, yellow/brown/red material on various body surfaces were noted in all groups.
- Body weight: no statistically significant differences found between the control and test substance-treated groups.
- Anatomic pathology: in the control group: dark red contents noted in the stomach for 2 females, yellow matting of skin and small spleen for 2 females; at 500 mg/kg/day clear fluid observed in abdominal cavity for 1 male and 1 female; at 1000 mg/kg/day clear fluid in the uterus and a mass on the abdominal soft tissue noted for 1 female; at 2000 mg/kg/day dark red areas were noted on the stomach of one male and on the thymus of one female.

STUDY RESTART: RANGE-FINDING PHASE
- Survival: test substance-related mortality was noted at 2000 mg/kg/day.
- Clinical observations: decreased activity and impaired muscle coordination on study days 0, 1 and 2 and reddened ears on study days 1 and 2 in the 2000 mg/kg/day male and female group, approximately 1-2 hours following dose administration. Hypoactivity was noted for male on study day 0.
- Body weight: unaffected by test substance and no statistically significant differences found between the control and test substance-treated groups.
- Anatomic pathology: for the 2000 mg/kg/day group male found dead on study day 1, a white area was noted on the left kidney at the time of necropsy.

STUDY RESTART: DEFINITIVE PHASE
- Survival: all animals survived to the scheduled euthanasia on study day 3.
- Clinical observations: decreased activity, reddened left ear (males only) and impaired muscle coordination were noted in the 2000 mg/kg/day group males and females approx. 1-2 hours following dose administration on study days 0, 1 and 2. Clinical observations of flushed extremities for three animals (1 male, 2 females) were noted in the 2000 mg/kg/day group on study day 0.
- Body weight: unaffected by test substance and no statistically significant differences found between the control and test substance-treated groups.
- Food consumption unaffected by test substance administration and no statistically significant differences found between the control and test substance- treated groups

ANALYSIS OF DOSING FORMULATIONS
The analysed formulations were found to contain 104 % to 106 % of the test substance.

Applicant's summary and conclusion

Conclusions:
Negative response for bone marrow cytotoxicity and clastogenicity.
Executive summary:

The aim of the study was to determine the potential for toxicity of the test material to induce micronuclei in polychromatic erythrocytes in rat bone marrow following 3 consecutive days of treatment administered by intraperitoneal injection. The study was performed according to ICH guideline S2 (R1) and the OECD Guideline 474. The animals were acclimatised for 7 days. The range-finding phase was initially conducted using PEG 400 as a vehicle, however the vehicle was not tolerated and mortality was seen following a single dose (50 % male and 7 % females) in all groups (including the control) and thus dosing was suspended. Glycerol Formal was chosen as the new vehicle and the study was restarted. In the range-finding phase, the test substance in the new vehicle, was injected peritoneally once daily for 3 consecutive days at dosage levels 500, 1000 and 2000 mg/kg/day for Groups 2-4 respectively, each consisting of 3 animals/sex. All surviving animals were euthanised and discarded without necropsy, 18-24 hours after the last dose administration (study day 3). In the definitive phase, test substance was administered in the same dosage levels and same frequency to Groups 2-4 and a concurrent vehicle group (Group 1) received the vehicle on a comparable regime. A positive control group (Group 5) received a single oral dose of 60 mg/kg/day of cyclophosphamide on study day 2. All animals were observed twice daily for mortality and morbidity. Mortality, body weight loss and several clinical observations seen when PEG 400 was used as the vehicle.

During the range-finding phase following study restart with Glycerol Formal, mortality was noted in the test substance-treated group and 1 male in the 2000 mg/kg/day group was found dead. There were no test substance-related effects on body weight. During the definitive phase, all animals survived until scheduled euthanasia on study day 3. Decreased activity, reddened left ear (males only) and impaired muscle coordination seen in the 2000 mg/kg/day male and female group. Clinical observations of flushed extremities noted for 3 animals in the 2000mg/kg/day group.

The test substance did not increase the mean number of micronucleated polychromatic erythrocytes compared to the vehicle control group and no bone marrow cytotoxicity noted in the test substance-treated group. The test substance met the criteria for a negative response for bone marrow cytotoxicity and clastogenicity under the conditions of the assay.