Biphenyl-4,4'-diol

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06-NOV-2014 to 14-SEP-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This GLP-compliant study was conducted according to standardised guidelines (i.e., OECD 474, EU B.14 and EPA OTS 798.5395).

Cross-reference

Reason / purpose for cross-reference:

exposure-related information

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Reason / purpose for cross-reference:

exposure-related information

Objective of study:

bioaccessibility (or bioavailability)

Qualifier:

according to guideline

Guideline:

other: OECD 474

Version / remarks:

The study was conducted according to the following test guideline:
- OECD Guideline for the Testing of Chemicals No. 474, 29 Jul 2016, “Mammalian Erythrocyte Micronucleus Test”; Section: Target tissue exposure.

Deviations:

no

Principles of method if other than guideline:

In a previously performed Micronucleus study in NMRI mice (BASF Project No.26M0276/13M335) the absence of a clastogenic and aneugenic potential of the test substance Biphenyl-4,4’-diol after oral administration was demonstrated.
To fulfill the recommendations of the current OECD Guideline No. 474 an additional bioavailability study was performed to demonstrate the occurrence of the test substance in the plasma samples and to prove the exposure of the bone marrow of the animals.
Definite test substance concentrations in the samples were determined.

GLP compliance:

yes

Specific details on test material used for the study:

Biphenyl-4,4’-diol, CAS 92-88-6

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

Age: 5 -8 weeks

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 5 – 8 weeks
- Weight at study initiation: 25.0 ± 0.82 g (mean ± standard deviation)
- Housing: Polycarbonate cages, type II; single housing
- Diet (e.g. ad libitum): Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): Drinking water from bottles was available ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Photoperiod: 12 hours light from 6 am to 6 pm; 12 hours dark from 6 pm to 6 am

IN-LIFE DATES:
- Arrival in the testing facility: 22 Nov 2016

Route of administration:

oral: gavage

Vehicle:

other: Due to the limited solubility in water, DMSO was selected as vehicle (suitable in the micronucleus test, with available historical data). To achieve a higher volume (DMSO used up to 4 mL/kg bw) the DMSO solution was filled up to 10 mL/kg bw corn oil.

Details on exposure:

All animals were given 2000 mg/kg body weight (maximum recommended dose level) or 10 mL/kg body weight of a test substance preparation with a concentration of 200 mg/mL.

Duration and frequency of treatment / exposure:

The animals were treated once orally (gavage) with a volume of 10 mL/kg body weight of the test substance. The animals were sacrificed 4 hours after the treatment.

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Control animals:

other:

Details on study design:

PREPARATION OF THE DOSING PREPARATION:
The substance to be administered per kg body weight was dissolved in DMSO (2 parts of total intended volume) and then emulsified in corn oil (3 parts of total intended volume).
To achieve the homogeneity of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and shaken thoroughly.
To keep the test substance homogeneously in the vehicle, the test substance preparation was constantly stirred.
The test substance preparation was prepared about 30 minutes before the beginning of administration.

ANALYSIS OF THE SUBSTANCE PREPARATIONS:
In the previously performed Micronucleus study in NMRI mice (reference: 26M0276/13M335) the stability of the test substance in the vehicle DMSO/corn oil over the application period was confirmed indirectly by recovery rates in the range of about 100% of the
nominal concentration in the test substance preparations.
In the current study the test substance concentration in the vehicle for the administered dose were determined analytically (Control procedure No. 13/0276_02-01). The homogeneity of the test substance preparation was confirmed indirectly based on these data.
For the determination of the test substance concentration in the vehicle, 6 samples (including three retain samples) were taken from the test substance preparation after treatment of the last animal (approximately 1 hour) and then were kept deep-frozen.
Retain samples were stored at the Laboratory for Cellular Methods (at – 20°C). Analysis of these samples was performed due to inadequate recovery in the original samples after agreement by the Study Director. Analysis are documented and justified in both the raw data and report. Retain samples are described by the suffix “R” in the report. Following finalization of the report, all analytical samples, including retain samples, will be discarded.
The analyses were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.

DOSING:
At the beginning of the study, the animals were weighed and the substance to be administered or the amount of volume was related to the specific weight of the individual animals.
NMRI mice of both sexes, two animals per sex, were administered a dose of 2000 mg/kg body weight once orally (gavage) with a volume of 10 mL/kg body weight of the test substance.

CLINICAL EXAMNATIONS:
After treatment up to the time of sacrifice, the animals were examined for any clinically evident signs of toxicity several times.

SAMPLING OF BLOOD AND SACRIFICE:
Blood was taken from vena facialis 2 hours after administration (about 100 μL per animal; sample code B).
Blood was collected with EDTA (ethylendiamintetraacetic acid) as anticoagulant in ready-to-use tubes (Microvette® tubes, Sarstedt, Germany). The samples were centrifuged for 2 minutes at 20000 x g at 4°C. The supernatant, plasma, was put into a second tube (1.5 mL Eppendorf tube) without additive, transferred to the Analytical Chemistry Laboratory of the test facility and was kept frozen at about -80°C prior to analysis.
Four hours after administration, the animals were sacrificed by decapitation under isoflurane anesthesia. Again, blood samples were taken (about 500 μL per animal; sample code C).

ANALYSIS OF PLASMA SAMPLES
The test substance batch used in this study was used as reference item for plasma analysis.
In order to have plasma control samples of untreated animals (blank samples), from all animals blood was taken from vena facialis the day before test substance administration (about 100 µL per animal) under isoflurane anesthesia. These samples were processed as those from treated animals.
The analyses were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE (Control procedure No. 13/0276_03-01).

Type:

other: Exposure of the target tissue (bone marrow cells)

Results:

The results of the plasma anlysis demonstrated the exposure of the target cells.

Key result

Test no.:

#1

Toxicokinetic parameters:

C(time): 2h after administration

Key result

Test no.:

#2

Toxicokinetic parameters:

C(time): 4h after administration

Metabolites identified:

not specified

Results of clinical examinations:

The single oral administration of the test substance did not lead to clinical signs of toxicity which was in accordance with the observations in the previous Micronucleus study.

Results of the analytical investigation of dosing preparations:

The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of 90% - 110% of the nominal concentration.

Based on the recovery rates it has to be considered that the test substance was stable at room temperature in the vehicle DMSO/corn oil at least over a period of 40 minutes.

Plasma analysis:

The bioavailability of the test substance Biphenyl-4,4’-diol in mouse plasma samples was clearly demonstrated 2 and 4 hours after oral administration of 2000 mg/kg body weight in either male and female NMRI mice. The plasma level was in a concentration range of 293.67 to 1113.34 ng/mL depending on the animal and time point. The blank plasma samples taken from the same animals about 24 hours before test substance administration confirmed the absence of test substance as expected.

Sample ID	Sex	Time point	Concentration in plasma (ng/mL)
1A	Male	Before application (Blank sample)	< 99.6
2A	Male		< 99.6
3A	Female		< 99.6
4A	Female		< 99.6
1B	Male	2 hours after application	431.4
2B	Male		1113.3
3B	Female		303.0
4B	Female		435.5
1C	Male	4 hours after application	293.7
2C	Male		659.9
3C	Female		316.4
4C	Female		495.3

< 99.6 ng/mL: not detected, = Limit Of Quantification (LOQ)

Conclusions:

Based on the previously performed Micronucleus study in NMRI mice (BASF Project No. 26M0276/13M335), 2000 mg/kg body weight Biphenyl-4,4’-diol was administered orally to male and female NMRI mice. No clinical signs of toxicity were observed until sacrifice. The bioavailability of the test substance in blood after oral administration was clearly demonstrated by analysis of plasma samples taken 2 and 4 hours after administration.
It has to be concluded that due to the confirmation of bioavailability in plasma in the recent study, the bone marrow of the animals of the previously performed Micronucleus study in NMRI mice (BASF Project No. 26M0276/13M335) was clearly exposed to the test substance Biphenyl-4,4’-diol.

Executive summary:

In a previously performed Micronucleus study in NMRI mice (BASF Project No. 26M0276/13M335) the absence of a clastogenic and aneugenic potential of the test substance Biphenyl-4,4’-diol after oral administration was demonstrated. To fulfill the recommendations of the current OECD Guideline No. 474 an additional bioavailability study was performed which demonstrates the occurrence of the test substance in the plasma samples. Definite test substance concentrations in the samples were determined.

NMRI mice of both sexes, two animals per sex, were given 2000 mg/kg body weight once orally (gavage) with a volume of 10 mL/kg body weight of the test substance. The animals were sacrificed 4 hours after the treatment.

At the beginning of the study, the animals were weighed and the substance to be administered was related to the specific weight of the individual animals.

Blood was taken from vena facialis 2 hours after administration (about 100 μL per animal). Four hours after administration, the animals were sacrificed by decapitation under isoflurane anesthesia. Again, blood samples were taken, i.e. 4 hours after administration (about 500 μL per animal). In order to have plasma control samples of untreated animals (blank samples), from all animals blood was taken from vena facialis the day before test substance administration (about 100 μL per animal) under isoflurane anesthesia. These samples were processed as those from treated animals.

Blood was collected with EDTA as anticoagulant. The samples were centrifuged for 2 minutes at 20000 x g at 4°C. The supernatant, plasma, was transferred to the Analytical Chemistry Laboratory of the test facility and was kept frozen at about -80°C prior to analysis.

The single oral administration of the test substance did not lead to clinical signs of toxicity which was in accordance with the observations in the previous Micronucleus study

The plasma level was in a concentration range of 293.67 to 1113.34 ng/mL depending on the animal and time point.

The blank plasma samples taken from the same animals about 24 hours before test substance administration confirmed the absence of test substance as expected.

These additional data confirm the exposure of the bone marrow of the animals in the above mentioned Micronucleus study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian bone marrow chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Biphenyl-4,4'-diol

EC Number:

202-200-5

EC Name:

Biphenyl-4,4'-diol

Cas Number:

92-88-6

Molecular formula:

C12H10O2

IUPAC Name:

[1,1'-biphenyl]-4,4'-diol

Test material form:

solid

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 5-8 weeks
- Weight at study initiation: 28.26 g (mean)
- Assigned to test groups randomly: yes, according to a randomization plan prepared with an appropriate computer program
- Fasting period before study: no data available
- Housing: single housing in polycarbonate cages, type II
- Diet: standardized pelleted feed (Maus/Ratte Haltung "GLP",Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: muncipal water from bottles, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity: 30-70%
- Air changes: no data available
- Photoperiod: 12 hrs dark / 12 hrs light (light from 6 am to 6 pm; dark from 6 pm to 6 am)

IN-LIFE DATES: From 10-NOV-2014 to 11- NOV-2014 (all test substance concentrations, vehicle, positive control) and From 10-NOV-2014 to 12- NOV-2014 (highest test substance concentration, vehicle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: DMSO and corn oil (final ratio 2:3)
- Justification for choice of solvent/vehicle: the choice was done based on preliminary solubility testing in which DMSO was the most suitable vehicle. It was the only one tested with which a homogeneous suspension was obtained at the highest required dose. Using the vehicles DMSO and corn oil subsequently a homogeneous suspension of the test substance was obtained. Therefore, a mixture of DMSO and corn oil (ratio 2:3) was selected as vehicle, which was demonstrated to be suitable in the mouse micronucleus test and for which historical control data were available.
- Concentration of test material in vehicle: 50, 100, and 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL
- Lot/batch no., purity: no data available

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
In the two lower dose groups the substance to be administered was dissolved in DMSO (125 mg/mL or 250 mg/mL) and then emulsified in corn oil (ratio 2:3, each). Besides, in the top dose a suspension in DMSO (500 mg/mL) was obtained which was subsequently suspended in corn oil (ratio 2:3).
To achieve homogeneity of the test substance in the vehicle, the test substance preparation of the high dose in DMSO was stirred with an ultraturrax and treated with ultrasonic waves. Then, it was shaken thoroughly to obtain a homogeneous suspension in corn oil. The preparations of the two lower dose groups were shaken thoroughly to obtain solutions in DMSO and emulsions in corn oil, each.
All test substance formulations were prepared immediately before administration.

For the determination of the test substance concentrations in the vehicle, 6 samples of each dose (including three retain samples) were taken from the test substance preparations after treatment of the last animal (approximately 1 hour) and then were kept deep-frozen. The determination of the concentrations in the vehicle, three samples per dose, was carried out by means of high performance liquid chromatography (HPLC). The homogeneity of the samples and the stability of the test substance in the vehicle were confirmed indirectly based on these data.
The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e., were in a range of 90%-110% of the nominal concentration, or above. Based on the recovery rates it had to be considered that the test substance was stable at room temperature in the vehicle DMSO/corn oil at least over a period of 40 minutes.


EXPOSURE OF TARGET CELLS
> To fulfill the recommendations of the current OECD Guideline No. 474 an additional bioavailability study (BASF - Project No.: 99M0276/13M370) was performed to demonstrate the occurrence of the test substance in the plasma samples and to prove the exposure of the bone marrow of the animals. Mice of both sexes were given orally 2000 mg/kg body weight of the test substance. Blood was taken from vena facialis 2 hours after administration (about 100 μL per animal). Four hours after administration, the animals were sacrificed by decapitation. gain, blood samples were taken (about 500 μL per animal). Blood was collected with EDTA, centrifuged for 2 minutes at 20000 x g at 4°C. The supernatant, plasma was kept frozen at about -80°C prior to analysis.
Definite test substance concentrations in the samples were determined.
> Results: No clinical signs of toxicity were observed until sacrifice. The plasma level was in a concentration range of 293.67 to 1113.34 ng/mL depending on the animal and time point. The bioavailability of the test substance in blood after oral administration was clearly demonstrated
For more details, see study described in the endpoint "Toxicokinetics", from BASF, study number 99M0276/13M370; author: Dr. M. Schulz.

Duration of treatment / exposure:

Single oral exposure

Frequency of treatment:

Single oral administration

Post exposure period:

Animals were sacrificed 24 and 48 hours after oral administration.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 per group

Control animals:

other: DMSO/corn oil (ration 2:3)

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): routinely used in the experimental laboratory
- Route of administration: oral
- Doses / concentrations: 20 mg/kg or 2 mg/mL in deionized water

Examinations

Tissues and cell types examined:

Bone marrow of the two femora

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The doses were selected in accordance with the requirements set forth in the test guidelines and based on the results of a preliminary range finding test. The pretest was performed following the method described for the main experiment. The test substance was administered by gavage (orally) to NMRI mice of both sexes (3/dose and sex). Then, the animals were examined for clinically evident signs of toxicity several times. About 48 hours after administration the surviving animals were sacrificed by isoflurane anaesthesia followed by cervical dislocation.
Based on the data of the pretest 2000 mg/kg bw was selected as the highest dose in the present cytogenetic study. 1000 mg/kg and 500 mg/kg body weight were administered as further doses.

TREATMENT AND SAMPLING TIMES:
Animals were sacrificed 24 and 48 hours after administration in the highest dose group and in the vehicle controls. In the medium and low dose groups and in the positive control group, the 24-hour sacrifice interval was only investigated.
All animals were anaesthetized with isoflurane. Subsequently, they were sacrificed either by decapitation in the course of the blood sampling or by cervical dislocation (only positive control group). Then, the two femora were prepared by dissection and all soft tissues were removed. After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with foetal calf serum (FCS with EDTA) which was pre-heated up to 37°C (about 2 mL/femur).

DETAILS OF SLIDE PREPARATION:
The slides were stained with eosin and methylene blue for about 5 minutes. After briefly rinsing in deionized water, the preparations were soaked in deionized water for about 2-3 minutes. Subsequently, the slides were stained with Giemsa solution for about 15 minutes. After rinsing twice in deionized water and clarifying in xylene, the preparations were mounted in Corbit-Balsam.

METHOD OF ANALYSIS:
In general, 2000 polychromatic erythrocytes (PCEs) were evaluated for the occurrence of micronuclei from each animal of every test group, so in total 10000 PCEs were scored per test group. The normocytes erythrocytes (i.e., normocytes; NCEs) with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded. The following parameters were recorded:
- Number of PCEs
- Number of PCEs containing micronuclei
- Number of NCEs
- Number of NCEs containing micronuclei
- Ratio of PCEs to NCEs
- Number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4) [d = diameter of micronucleus, D = cell diameter]

Slides were coded before microscopic analysis.

Evaluation criteria:

ACCEPTANCE CRITERIA:
The mouse micronucleus test was considered valid if the following criteria were met:
- The quality of the slides must allow the evaluation of a sufficient number of analysable cells; i.e., ≥ 2000 PCEs per animal and a clear differentiation between PCEs and NCEs.
- The ratio of PCEs/NCEs in the concurrent vehicle control animals had to be within the normal range for the animal strain selected.
- The number of cells containing micronuclei in vehicle control animals had to be within the range of the historical vehicle control data for PCEs.
- The positive control substance had to induce a distinct increase in the number of PCEs containing small and/or large micronuclei

ASSESSMENT CRITERIA:
A finding was considered positive if the following criteria were met:
- A statistically significant and dose-related increase in the number of PCEs containing micronuclei.
- The number of PCEs containing micronuclei had to exceed both the concurrent vehicle control value and the range of the historical vehicle control data.

A test substance was considered negative if the following criteria were met:
- The number of cells containing micronuclei in the dose groups was not statistically significant increased above the concurrent vehicle control value and was within the range of the historical vehicle control data.

Statistics:

The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there were statistically significant differences between the untreated control group and the treated dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test. Statistical significances were identified as follows:
* p ≤ 0.05
** p ≤ 0.01
However, both biological relevance and statistical significance were considered together.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Solubility: no data available
- Clinical signs of toxicity in test animals: the recommended highest oral dose of 2000 mg/kg bw was survived by all animals showing weak signs of toxicity. However, there were no distinct differences in clinical observations between male and female animals.
- Evidence of cytotoxicity in tissue analysed: not applicable (i.e., acute oral toxicity study)
- Rationale for exposure: according to OECD guideline 474
- Harvest times: About 48 hours after administration the surviving animals were sacrificed by isoflurane anaesthesia followed by cervical dislocation.

RESULTS OF DEFINITIVE STUDY (see in Table 1 below)
- Induction of micronuclei: the single oral administration of the vehicle DMSO/corn oil led to 1.3‰ PCEs containing micronuclei after the 24-hour sacrifice interval or to 1.8‰ after the 48-hour sacrifice interval, respectively.
After the single administration of the highest dose of 2000 mg/kg bw, 1.5‰ PCEs containing micronuclei were found after 24 and 48 hours, each. In the two lower dose groups, rates of micronuclei of 1.7‰ (1000 mg/kg group) and 1.2‰ (500 mg/kg group) were detected at a sacrifice interval of 24 hours in each case.
The positive control substance, cyclophosphamide, led to a statistically significant increase (17.8‰) in the number of PCEs containing exclusively small micronuclei, as expected.
The number of NCEs containing micronuclei did not differ to any appreciable extent in the vehicle control group or in the various dose groups at any of the
sacrifice intervals.
- Ratio of PCE/NCE: at 24-hour sacrifice interval a slight inhibition of erythropoiesis determined from the ratio of PCE/NCE was detected at all doses (500, 1000 and 2000 mg/kg bw) after test substance administration (87%, 89% and 87% of control, respectively).
- Statistical evaluation: there were thus no statistical significances or biologically relevant differences in the frequency of erythrocytes containing micronuclei either between the vehicle control groups and the three dose groups (500 mg/kg, 1000 mg/kg and 2000 mg/kg) or between the two sacrifice intervals (24 and 48 hours). The number of NCEs or PCEs containing small micronuclei (d < D/4) or large micronuclei (d ≥ D/4) did not deviate from the vehicle control values at any of the sacrifice intervals and was within the historical vehicle control data range
- Other: the administration of the test substance led to distinct clinical signs of toxicity in the top dose: piloerection, hunched posture and/or reduced general condition at several observations.

Any other information on results incl. tables

Table 1: Summary table – Induction of micronuclei in bone marrow cells

Test group	Sacrifice interval [hrs]	Animal No.	Micronuclei in PCE		PCEs per 2000 erythrocytes^c
			Total^a [‰]	Large MN^b [‰]
Vehicle control DMSO/corn oil	24	5	1.3	0.0	1437
Test substance 500 mg/kg bw	24	5	1.2	0.0	1254
Test substance 1000 mg/kg bw	24	5	1.7	0.1	1280
Test substance 2000 mg/kg bw	24	5	1.5	0.1	1256
Positive control cyclophosphamide 20 mg/kg bw	24	5	17.8**	0.0	1430
Vehicle control corn oil	48	5	1.8	0.0	1357
Test substance 2000 mg/kg bw	48	5	1.5	0.2	1301

PCE = polychromatic erythrocytes

NCE = normochromatic erythrocytes

bw = body weight

^a = sum of small and large micronuclei

^b = large micronuclei (indication for spindle poison effect)

^c = calculated number of PCEs per 2000 erythrocytes (PCE + NCE) when scoring a sample of 10000 PCE per test group

* = p ≤ 0.05

** = p ≤ 0.01

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions applied, the test substance had no chromosome-damaging (clastogenic) effect nor did it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells of NMRI mice in vivo.

Executive summary:

The test substance was assessed for its potential to induce chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method, according to OECD guideline 474 and in compliance with GLP.

 

The test substance, dissolved or suspended in DMSO and subsequently emulsified or suspended in corn oil, was administered once orally to male animals at dose levels of 500 mg/kg, 1000 mg/kg and 2000 mg/kg body weight (bw) in a volume of 10 mL/kg body weight in each case. The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2000 mg/kg body weight and in the vehicle controls. In the test groups of 1000 mg/kg and 500 mg/kg body weight and in the positive control group, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded.

 

As vehicle control, male mice were administered merely the vehicle, DMSO/corn oil (ratio 2:3), by the same route and in the same volume as the animals of the dose groups, which gave frequencies of micronucleated polychromatic erythrocytes within the historical vehicle control data range. The positive control substance cyclophosphamide led to the expected increase in the rate of polychromatic erythrocytes containing only small micronuclei.

 

A slight inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes (slightly below 90% of control, each) was detected after test substance administration at all doses at 24-hour sacrifice interval. According to the results of the present study, the single oral administration of the test item did not lead to any biologically relevant increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was close to the range of the concurrent vehicle controls in all dose groups and at all sacrifice intervals and within the range of the historical vehicle control data.

In an additional study, the exposure of the bone marrow was confirmed by the analysis of the test substance in plasma after oral dosing of animals (see study described in the section "Toxicokinetics", from BASF, study number 99M0276/13M370, author:   Dr. M. Schulz).

Thus, under the experimental conditions of this study, the test substance did not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo.