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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
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to OECD 474, USEPA OPPTS 870.5395 and Method B.12 of Commission Directive EEC.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
(1998)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
(08/06/2000)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
(1997)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Biphenyl FP
- Physical state: white-grey solid
- Analytical purity: 99.6 % ± 0.003% relative standard deviation corrected for water content (0.32% ± 8.3%) by gas chromatography and Karl Fischer coulometric titration with identification by gas chromatography mass spectroscopy and infrared spectroscopy (Lehr et al., 2006)
- Impurities (identity and concentrations): not specified in the report
- Composition of test material, percentage of components: not specified in the report
- Isomers composition: not specified in the report
- Purity test date: not specified in the report
- Lot/batch No.: not specified in the report
- Expiration date of the lot/batch: not specified in the report
- Stability under test conditions: not specified in the report
- Storage condition of test material: not specified in the report

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Kingston, New York)
- Age at study initiation: 9 weeks
- Weight at study initiation: range female day 1: 25.2 - 26.5 g; range male mean weights day 1: 34.1 - 35.0 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: Animals were housed one per cage in stainless steel cages. Cages had wire mesh floors and were suspended above absorbant paper. Cages contained a hanging feeder and a pressure activated lixit value-type watering system
- Diet (e.g. ad libitum): LabDiet Certified Rodent diet #5002 (PMI Nutrition International, St. Louis, Missouri) ad libitium
- Water (e.g. ad libitum): drinking water obtained from the municipal water source ad libitum
- Acclimation period: at least one week prior to the start of the study


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1°C
- Humidity (%): 40 -70 %
- Air changes (per hr): 12-15 times/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: no data for final test
- Amount of vehicle (if gavage or dermal): highest dosing volume allowable for corn oil as a vehicle is 10 mL/kg bw
- Type and concentration of dispersant aid (if powder): not applicable
- Lot/batch no. (if required): no data
- Purity: no data
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing solutions were prepared fresh prior to each dosing. The concentrations of the test material in the dosing solutions were verified using gas chromatography with flame ionization detection (GC/FID) and external standard quantitation


DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data
Duration of treatment / exposure:
Administration of the test material on 2 consecutive days
Frequency of treatment:
Daily for 2 consecutive days
Post exposure period:
24 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg body weight/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
200 mg/kg body weight/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
400 mg/kg body weight/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
800 mg/kg body weight/day
Basis:
nominal conc.
No. of animals per sex per dose:
There were six mice/sex/dose except in the 800 mg/ kg bw/day group where an additional mouse/sex was dosed as a possible replacement in the event of death occurring among the treated animals of this group
Control animals:
yes, concurrent vehicle
Positive control(s):
- Cyclophosphamide monohydrate (CP)
- Justification for choice of positive control(s): no data
- Route of administration: oral gavage
- Doses / concentrations: single administration of 120 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow samples were obtained from both femurs.
(Polychromatic) erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on the results of the range-finding test, groups of male and female mice were administered 0, 200, 400, and 800 mg/kg bw/day of the test material on two consecutive days

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Male and female mice were administered on two consecutive days. Approximately 24 hours after the last dosing, bone marrow samples were collected from all animals

DETAILS OF SLIDE PREPARATION: Wedge smears were prepared on microscope slides using small portions of the cell suspension. The slides were allowed to air dry, fixed in cold methanol and stained with Wright-Giemsa using an automatic slide stainer, (Ames Hema-Tek, Miles Scientific, Naperville, Illinois). All slides were coded, scored, and decoded upon completion to control for bias.


METHOD OF ANALYSIS: Two thousand PCE were examined from each animal and the number of micronucleated polychromatic erythrocytes (MN-PCE) was recorded. Micronuclei were identified as darkly stained bodies with smooth contours and varying shapes such as round, almond, or ring (Schmid, 1976). The ratio PCE to NCE in the bone marrow was determined in the micronucleus test by examining 200 erythrocytes. The ratio was expressed as PCE x 100/PCE+NCE.


Evaluation criteria:
A test material was considered positive in this assay if the following criterion was met: statistically significant increase in MN-PCE frequency at one or more dose levels accompanied by a dose response. A test material was considered negative in this assay if all of the following criteria were met: no statistically significant dose-related increase in MN-PCE compared to the negative control. A result not meeting the criteria for either the positive or the negative response was considered to be equivocal.
Statistics:
The raw data on the counts of MN-PCE for each animal was first transformed by adding one to each count and then taking the natural log of the adjusted number. The transformed MN-PCE data and the data on percent PCE were analyzed separately by a two-way analysis of variance (Winer, 1971). The sex by dose interaction in the two-way analysis was reviewed and if significant, a one-way analysis of variance was performed for each sex. Pairwise comparisons of treated vs. control groups was done, if the dose effect was significant, by Dunnett's t-test, one-sided (upper) for MN-PCE and two-sided for the percent PCE (Winer 1971). Linear dose-related trend tests were performed if any of the pairwise comparisons yielded significant differences. The alpha level at which all tests were conducted was 0.05. The final interpretation of biological significance of the responses was based on both statistical outcome and scientific judgement.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at the highest dose of 800 mg/kg bw/day
Vehicle controls validity:
other: Historical control data available
Negative controls validity:
other: Historical control data available
Positive controls validity:
other: Historical control data available
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: Phase I: 1000 mg/kg bw/day and 2000 mg/kg bw/day; Phase II: 500, 750, and 1000 mg/kg bw/day
- Solubility: Phase I: at the 200 mg/mL concentration, the test material formed a thick suspension that was difficult to administer to the animals. At 100 mg/mL the test material was completely soluble in corn oil. Based upon this information, it was decided to use a split dosing regimen (two administrations within a 2 hour period) to achieve the targed doses. In phase II, the test material was administered as a single bolus dose by oral gavage at a dosing volume of 10 mL/kg bw.
- Clinical signs of toxicity in test animals: Phase I: 2000 mg/kg bw/day: males and females at this dose level, there were significant decreases in body temperature (± 6.8°C in males and 12.8°C females) for 5 or more hours following initial day of dosing; 1000 mg/kg bw/day: 1 male spontaneously died prior to second day of dosing, significant decreases in males and females of body temperature (± 4°C) following first treatment which persisted for 5 hours. Based on these results, it was concluded that the 2000 mg/kg bw/day exceeded the maximum tolerated dose (MTD). Phase II: decreased activity among all female mice, in both sexes there were significant decreases in the body temperature (6-8°C) that persisted for extended periods of time ( = 5 hours) after the first day of dosing; 750 mg/kg bw/day: decreases in body temperature (± 3.5°C in males and 5.8°C in females) 5 hours after the initial dose. 500 mg/kg bw/day dose level: decrease in body temperature of ± 1°C in male and female
- Evidence of cytotoxicity in tissue analyzed: no data
- Harvest times: no data
- Rationale for exposure: based upon the decrease in body temperature observed at the 750 mg/kg bw/day dose level and lethality at the 1000 mg/kg bw/day dose level, dose levels of 0, 200, 400, and 800 mg/kg bw/day biphenyl FP, were chosen for the main micronucleus study (MNT). Because of some indication of differences in toxicity between the sexes, both male and female mice were used in the MNT. The tst material was administered as a single bolus dose on each day of dosing
- High dose with and without activation: not applicable
- Other: no data


RESULTS OF DEFINITIVE STUDY
- There were no significant differences in MN-PCE frequencies between the groups treated with the test material and the negative controls in either male or female mice and all values were within laboratory historical control data. The adequacy of the experimental conditions for the detection of induced micronuclei was ascertained from the observation of a significant increase in the frequencies of micronucleated polychromatic erythrocytes in the positive control group. The percent PCE values observed at 800 mg/kg bw/day biphenyl FP and the positive control dose were significantly lower than the negative control values. See also remarks on results including tables and figures.

Any other information on results incl. tables

Results of concentration analysis:

The analytically determined concentration of the test material in the dosing solutions used for the micronucleus test ranged from 98.96 to 100.7% of the targeted values (The Dow Chemical Company, 2006 - Dose Confirmation (Study 061100; Biphenyl FP)).

Micronucleus assay:

The treatments did not have remarkable effect on the body weight of the animals. There were indications of toxicity upon daily observation during the in-life portion of the micronucleus test at the highest dose of 800 mg/kg bw/day in males and female mice. Decreased activity, crouched posture, and cold to touch were among the observations noted at this dose level. Accidental deaths caused by oral gavage errors occured in a negative control male mouse and a male high dose mouse. At the highest dose of 800 mg/kg bw/day, a significant decrease in body temperature of approximately 6.4°C in males and 8.5°C in females was observed at 2 hours after the initial dose. Decreases in body temperature persisted for > or = 5 hours; however, they recovered prior to the second day of dosing. At the 400 and 200 mg/kg bw/day dose levels, there were minimal to no decreases in body temperature.

Summary of Micronucleated Polychromatic Erythrocytes (MN-PCE) Frequencies and % Polychromatic Erythrocytes (% PCE) - 5 males or females/dose level:

 Dose (mg/kg)   MN-PCE (%) Male  PCE (%) Male  MN-PCE (%) Female PCE (%) Female 
 0*  Mean  0.8  63.0  1.5  67.3
   S.D.  0.8  6.9  0.4  5.2
 200  Mean  1.1  62.8  1.2  65.3
   S.D.  0.7  6.9  1.0  2.5
 400  Mean  1.4  60.0  1.1  63.5
   S.D.  0.8  2.3  0.7  6.1
 800  Mean 1.8  57.4  1.0  58.8
   S.D.  1.0  2.4  0.8  3.8
 CP 120 Mean  53.8  42.4   64.5  50.3
   S.D.  12.0  10.7  14.6  5.0

CP = Cyclophosphamide monohydrate (positive control)

*: Mice were dosed with the vehicle (corn oil)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test material, biphenyl FP, did not induce a significant increase in the frequencies of micronucleated bone marrow polychromatic erythrocytes when given as a single oral dose on two consecutive days to male and female CD-1 mice. Hence, biphenyl FP is considered negative in this test system under the experimental conditions used.