BMS 233110-01

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial gene mutation study

A bacterial gene mutation study was carried out for BMS 233110-01. The bacterial strains were exposed to concentrations of 33.3 to 5000 μg substance per plate, both with and without metabolic activation. A weak positive increase in the mean number of revertants per plate was observed with TA98 strain in the presence of S-9 mix. While this increase was relatively weak, it did meet the minimum criteria for a positive response and was shown to be reproducible. No positive increases were observed in any of the remaining tester strain/activation conditions combinations

In Vitro Gene Mutation

A mouse lymphoma assay test in L5178Y cells was carried out for BMS 233110-01, according to Directive 88/302/EEC, Part B, OECD Guideline No.476. Three tests were carried out with different exposure concentrations (3h), both with and without metabolic activation. The results of the assay indicate that under the conditions of this study, 233110-01 produced a positive increase in the mean number of revertants per plate with tester strain TA98 (3.1-fold in the initial assay (and 2.8-fold in the confirmatory assay) in the presence of S9 mix. It can be concluded that the test substance is not mutagenic in this test system in the absence of S-9 but is weakly mutagenic at highly toxic doses in the presence of S-9.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4th - 22nd May 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identity of test material same as for substance defined in section 1

Species / strain / cell type:

bacteria, other: TA98, TA100, TA1535, TA1537, E Coli WP2uvrA

Metabolic activation system:

Aroclor 1254 induced rat liver S-9 fraction

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 33.3 ... 5000 µg/plate
Concentration range in the main test (without metabolic activation): 33.3 ... 5000 µg/plate

Vehicle / solvent:

Solvent: DMSO

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

other: no data

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 µg/plate)

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

( <=5000 µg/plate)

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

<=5000 µg/plate)

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations: There was no indication of toxicity in either experiments.There was also no observed increase in revertant strains except for weak response in the presence of S-9 in TA-98 in both experiments

Conclusions:

positive with metabolic activation
negative without metabolic activation

The results of the salmonella-escherichia coli / Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, BMS 233110-01 caused a positive increase in the mean number of revertants per plate with tester strain TA98 (3.1-fold in the inital assay and 2.8-fold in the confirmatory assay) in the presence of S9 mix. No positive increases were observed with any of the remaining tester strain/activation conditions combinations.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11th June 1999 - 20th December 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identity of test material same as for substance defined in section 1

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Mouse lymphoma L5178Y cells, tk locus

Metabolic activation system:

Aroclor 1254 induced rat liver post mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 6.25 ... 87.5 µg/ml
Concentration range in the main test (with metabolic activation): 50 ... 150 µg/ml
Concentration range in the main test (with metabolic activation): 50 ... 162.5 µg/ml
Concentration range in the main test (without metabolic activation): 25 ... 300 µg/ml
Concentration range in the main test (without metabolic activation): 150 ... 350 µg/ml

Vehicle / solvent:

DMSO

Details on test system and experimental conditions:

Exposure period (with metabolic activation): 3 hours
Exposure period (without metabolic activation): 3 hours

Expression time:
The expression period was 2 days

Selection time:
The selection period was 8 - 10 days at 37°C

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

( 62.5 µg/ml)

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

( 250 µg/ml)

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

( 150 µg/ml)

Vehicle controls validity:

other:

Remarks:

main test

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

( 300 µg/ml)

Vehicle controls validity:

other: main test

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations: A significant, but weak, result was seen in the presence of S-9 at dose levels producing toxicity to the culture. Toxicity was apparent at the higher dose concentrations tested.

Conclusions:

positive with metabolic activation
negative without metabolic activation

It can be concluded that the test substance is not mutagenic in this test system in the absence of S-9 but is weakly mutagenic at highly toxic doses in the presence of S-9.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo micronucleus assay

An in vivo micronucleus assay was carried out on BMS 233110-01 according to EEC Directive 92/69/EEC, Method B12 and OECD Guidelines for testing of Chemicals No. 474 "Mammalian Erythrocyte Micronucleus Test". The test was carried out on male albino, CD-1 (ICR)BR mice. The material was administered orally to groups of 7 rats at doses of 200, 400 and 800 mg/kg.

There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test substance and it can be concluded that the substance is non genotoxic.

In vivo/In Vitro Unscheduled DNA Synthesis in Rat Liver

An in vivo/in vitro DNA repair (UDS) study was conducted in rats in accordance with OECD Guideline for testing of Chemicals No 486 "Unscheduled DNA synthesis (UDS) with Mammalian Liver Cells In Vivo found that when tested at concentrations of 360 mg/kg and 900 mg/kg there was no NNG values greater that 1.5 or were any more than 1.3% of hepatoocyte cells found in repair at either dose. IT can be concluded that the test substance did not induce UDS under experimental conditions.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9th November 1998 - 1st March 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

other: micronucleus assay

Specific details on test material used for the study:

Identity of test material same as for substance defined in section 1

Species:

mouse

Strain:

other: Crl:CD-1(ICR)BR

Sex:

male

Route of administration:

oral: unspecified

Vehicle:

Carboxymethylcellulose solution

No. of animals per sex per dose:

Male: 200 mg/kg; No. of animals: 7; Sacrifice time: 24 hours
Male: 400 mg/kg; No. of animals: 7; Sacrifice time: 24 hours
Male: 800 mg/kg; No. of animals: 7; Sacrifice time: 24 hours
Male: 800 mg/kg; No. of animals: 7; Sacrifice time: 48 hours

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

up to 800 mg/kg

Toxicity:

yes

Remarks:

at 1000 and 2000 mg/kg

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Doses producing toxicity:
In a range finding study, male and female mice were treated by the oral and intraperitoneal routes. The IP route gave deaths at 500 and 1000 mg/kg, although an absence of a clear dose related response suggested problems with this method of administration. The IP route was not used in the main study.

Oral administration caused 1/4 deaths in males at 1000 mg/kg and 7/13 deaths in males at 2000 mg/kg. There was no mortality with females. Only males were used in the main study.

Observations:
There was no mortality in the main study, but at 800 mg/kg, clinical signs included hunched posture, lethargy and ptosis.

There were no indications of a positive response.

Systemic toxicity inidcated that absorption took place. Subsequent toxicity testing on rats demonstrated systemic effects following oral adminstration.

Conclusions:

Negative.

BMS 233110-01 is considered to be non-genotoxic under the conditions of the test

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

20 Dec 2000 and 01 March 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Version / remarks:

(July 1997).

Principles of method if other than guideline:

BMS 233110-01 was tested using an in vivo/in vitro assay for its ability to cause repairable DNA damage in cultured primary rat hepatocytes. Repair was measured as unscheduled DNA synthesis (UDS) by the uptake of radio-labelled thymidine assayed autoradiographically.
This assay is a useful in vivo evaluation for DNA damage and repair. Although it gives no direct information about the number or type of DNA lesions or about the consequences of repair, it does detect repair of the type of DNA lesions (adducts) caused by chemical carcinogens, many of which are thought to act by interaction with macromolecules within the cell, particularly DNA.

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

BMS 233110-01, batch munber 9E02259, was a white powder. It was received from the Sponsor on 14 June 1999 and was stored at room temperature, under nitrogen and dessicated in the dark. Purity was stated as 92.4% "as is". The expiry date of the test article was stated to be 27 May 2000.

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

An excess number of Male Wistar rats were obtained from Charles River (UK) Ltd, Margate, UK. They were housed in groups of no more than three animals in polypropylene cageswith wire mesh lids and solid floors. Bottled mains tap water and laboratory diet were provided ad libitum.
These are routinely monitored and are not !mown to contain any biological or chemical entity which might interfere with the test system.
Animal holding rooms were designed to be illuminated continuously by fluorescent light for 12 hours out of each 24 hour cycle and to receive at least 15 fresh air changes per hour.
Prior to each main study experiment, 20 male rats were ear-tagged and allocated to groups of five using a system of computer generated random numbers. Group weights were checked within 24 hours prior to treatment to ensure individual group weights differed from the mean by no more than 5%.

Route of administration:

oral: gavage

Vehicle:

Dosing preparations and dilutions were made using arachis oil. The test article preparations were protected from light, stored at room temperature in the dark and used within 3 1/2 hours of initial formulation

Details on exposure:

Animals were weighed before dosing and the volume of vehicle, test article preparation or positive control solution to be administered was calculated based on adose volume of 10 mL/kg. Animals were not starved prior to dosing. All treatments were given via oral gavage to maximise exposure of the test article to the target organ

Duration of treatment / exposure:

Range-finder
An initial range finding study was performed using groups of male rats treated with the test article at suitable doses. Animals were dosed once with the test article via oral gavage. Observations were made over a 4 day period. Clinical signs of toxicity and body weights over this period were recorded and a maximum acceptable dose determined based on these data. This was used as the highest dose level in the main study.

Main study
In the main study, groups of five male rats were treated. To enable a manageable number of animals to be processed at any one time, dosing was carried out on two occasions approximately 2 hours apart, treating three then two animals from each dose group on each occasion. animals were killed a nominal 12-14 hours and 2-4 hours after dosing, respectively

Frequency of treatment:

Range-finder
Animals were dosed once with the test article via oral gavage.

Main study
In the main study, groups of five male rats were treated. To enable a manageable number of animals to be processed at any one time, dosing was carried out on two occasions approximately 2 hours apart, treating three then two animals from each dose group on each occasion

Post exposure period:

Observations were made over a 4 day period. Clinical signs of toxicity and body weights over this period were recorded and a maximum acceptable dose determined based on these data. This was used as the highest dose level in the main study.

Dose / conc.:

360 mg/kg bw/day

Remarks:

Main Study

Dose / conc.:

900 mg/kg bw/day

Remarks:

Main Study

No. of animals per sex per dose:

Range Finder: 9 animals
Main Study: 40 animals

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive Control:
2-Acetamidofluorene (2-AAF) 75 mg/kg in corn oill for the 12-14 hour expirement.
Dimethylnitrosamine (DMN) 10 mg/kg in purified water for the 2-4 hour expirement
Both positive control compounds were administered at a dose volume of 10 mL/kg

Tissues and cell types examined:

Hepatocytes were prepared from three of the five animals in each dose group.

Details of tissue and slide preparation:

Animals were anaesthetised by placing in a gas chamber, supplied from a Halothane gas generator, and were maintained under deep anaesthesia to prevent recovery. The
abdominal surface of each animal was then rinsed with 70% (v/v) ethanol, a 'V' shaped incision made from the centre of the lower abdomen to the rib cage, and the skin and
muscle removed to reveal the abdominal cavity. A cotton tie was placed loosely around the hepatic portal vein. The vein was then cannulated using an appropriate catheter, the
inner needle removed, and the ligature tightened. The inferior vena cava was then clamped superior to the kidney. Cannulation of the superior vena cava was carried out
via the right atrium, following cutting of the diaphragm and removal of the rib cage.
The hepatic portal cannula was connected to a supply of calcium free Buffer 1 (lSO mM NaCl, 3.73 mM NaHC03, 4.84 mM NaiHP04, 4.97 mM KCl, 1.24 mM KHf04 0.62 mM MgS04, 0.62 mM MgCl2, 10 μg/mL Phenol red indicator) which had been gassed for at least S-10 minutes with S% C02 in air (v/v), and pumped at approximately 40 mL/min. The vena cava cannula was connected to a waste line and the liver washed free ofblood, using 400 mL Buffer 1, helped by gentle massaging if required. The liver was then perfused with Buffer 2 (142 mM NaCl, 24 mM NaHC03, 4.37 mM KC!,
1.24 mM KH2P04, 0.62 mM MgS04, 0.62 mM MgC12, 10 μg/mL Phenol red indicator) which was continually gassed with S% C02 in air (v/v), at 40 mL/min until the reservoir
volume had dropped from 400 mL to approximately 200 mL. Calcium and collagenase (approximately SO mg collagenase dissolved in 1 mL 769 mM CaC12 and 10 mL Buffer 2) were added to the reservoir and when the darker colour of this solution entered the liver, the waste line was placed in the Buffer 2 reservoir so that the perfusate recirculated. The pump speed was reduced to 20 mL/min. When the reticular pattern of the liver had begun to break up and the liver became 'spongy' the perfusion was stopped.
The liver was then cut free and transferred to a sterile plastic vessel with approximately 10 mL of the prewarmed (37°C) Buffer 2. The liver capsule was removed and the hepatocytes carefully tease out using a combing method. The separated hepatocytes were gently washed through lSO μm nylon mesh with Williams E medium-Complete
(WE-C) to a volume of 100 mL. Of this suspension, approximately SO mL was taken and centrifuged at approximately 40 x 'g' for 2-3 minutes. The resulting pellet was resuspended in approximately 40 mL WE-C. This centrifugation and resuspension procedure was repeated twice more and cells resuspended finally in 20 mL WE-C. A sample (0.5 mL) of this suspension was taken, diluted with an equal volume of0.4% (w/v) trypan blue and the proportion of viable cells (those with unstained nuclei) determined using a haemocytometer. The culture was then diluted to provide approximately 1.5 x 105
viable cells/mL.
For all hepatocyte suspensions, 3 mL was added to each well of a six-well multiplate, each containing a 25 mm round plastic Thermanox coverslip and incubated at 37°C in a 5% C02 in air (v/v) atmosphere for at least 90 minutes to allow the cells to attach.

Radiolabelling of hepatocyte cultures
Medium was removed from the cells and the monolayers washed with 2 mL Williams E medium-Incomplete (WE-I) which was then replaced with 2 mL WE-I containing 10 μCi/mL [3H] thymidine. After approximately 4 hours incubation at 37°C in a 5% C02 in air (v/v) atmosphere, the medium was removed and the cells washed with three changes of WE-I containing 0.25 mM thymidine. Cultures were then incubated overnight with 3 mL of the same medium.
To prepare for autoradiography, coverslips were washed with 2 mL phosphate buffered saline (PBS) and the cells fixed with three changes of 2 mL glacial acetic acid:ethanol (1 :3 v/v). The coverslips were then washed four times with purified water, allowed to dry and mounted onto previously labelled microscope slides,. cells side up, with DPX.

Autoradiography
Three of the six slides from each animal were coated in Ilford K2 liquid emulsion using a dipping technique. Each slide was dipped individually into the molten emulsion, ensuring that no air-bubbles were generated. After gelling over ice, for 10 minutes face upwards, the slides were incubated in a light-tight box at room temperature for approximately 90 minutes to let the emulsion dry. The slides were then packed in lighttight boxes containing desiccant, sealed with tape and refrigerated for 14 days. At the end of this time, the emulsion was developed in Kodak D 19 developer and fixed using Ilford Hypam fixer. The cell nuclei and cytoplasm were then stained with Meyers haemalurn/eosin Y. Slides were then dehydrated in ethanol, cleared in xylene and mounted with coverslips for microscopic examination. The spare, duplicate set of slides
were not required.

Evaluation criteria:

The following criteria were used for cell analysis:
1) only cells with normal morphology were scored
2) isolated nuclei with no surrounding cytoplasm were not scored
3) cells without nuclear and/or cytoplasmic graining were not scored
4) cells with unusual staining artefacts were not scored
5) heavily labelled cells in S-phase were not scored
6) all other normal cells, 100 per animal were scored
7) all slides were analysed blind (coded).

The test article would be considered as positive in this assay if, at any dose and at either
time point:
1) the test article yielded group mean NNG values greater than 0 NNG and 20% or more of cells responding (mean NNG values >5)
2) an increase was seen in both NNG and the percentage of cells in repair. Cytoplasmic and nuclear grain count values as well as the concurrent negative control data would be considered in relation to the overall NNG values of cultures from treated animals.
If the test article failed to induce UDS at any dose tested after both 2-4 and 12-14 hours exposure, it would be considered clearly negative in this system.

Statistics:

Treatment of data
The following were calculated for each slide, animal and dose point:
1) the population average NNG and standard deviation (SD)
2) the percent of cells responding or in repair (ie > 5 NNG)
3) the population average cytoplasmic and nuclear grain count

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

2-4 hr sacrifice 360 mg/kg Net nuclear grain count (NNG) -2.6, % cells in repair 0.7; 900 mg/kg NNG -1.6, % cells in repair 1.3. 12-14 hr Sacrifice 360 mg/kg (NNG) -1.9, % cells in repair 1.3. 900 mg/kg NNG -1.5, % cells in repair 1.3

Toxicity:

not examined

Vehicle controls validity:

valid

Remarks:

2-4 hr sacrifice (NNG) -2.6, % cells in repair 0.7. 12-14 hr Sacrifice (NNG) -1.5, % cells in repair

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

2-4 hr DMN sacrifice (NNG) 22.5, % cells in repair 97.7. 12-14 hr Sacrifice AAF (NNG) 15.5, % cells in repair 92.

Additional information on results:

The study would be considered valid ifthe negative control animals had a mean NNG value that did not exceed the upper limit of the historical range. The positive control treatments should have group mean values of five or more NNG with 50% or more cells having NNG counts of five or greater.

Validity of study

Acceptance criteria for the in vivo/in vitro hepatocyte DDS assay confirm that:

1) the group mean net grain count for vehicle-treated animals was less than the upper limit of the historical control range (-1.5 and -2.6 for Experiments 1 and 2 respectively)

2) the positive control chemicals 2-AAF and DMN induced increases in group mean net grain count of five or more (15.5 and 22.5 respectively), and 50% or more of cells (92% and 97.7% respectively) had net grain counts of five or

more. This result showed that the test system was sensitive to two known DNA damaging agents requiring metabolism for their action and that the experiment was valid.

Conclusions:

The data obtained in this study indicate that oral treatment of male rats dosed once with 360 or 900 mg/kg BMS 233110-01 did not result in increased UDS in hepatocytes isolated approximately 12-14 or 2-4 hours after dosing.
It was concluded that BMS 233110-01 did not induce UDS detectable under the experimental conditions employed.

Executive summary:

BMS 233110-01 was tested for its ability to induce unscheduled DNA synthesis (UDS) in the livers of orally dosed male rats using an in vivo/in vitro procedure. The Sponsor supplied information indicating that there is no substantial inter-sex difference in the toxicity of the test article to rat and that the maximum tolerated dose was approximately 500 mg/kg. Accordingly only male animals were tested in this study and in an initial toxicity range-finder study, male Wistar rats (three per group) were dosed once with 500, 700 and 900 mg/kg BMS 233110-01. During a 4 day post-dose observation period, clinical signs including piloerection (700 mg/kg), piloerection, staining to the nose, raised gait and being hunched (900 mg/kg) were observed. Furthermore , 3 days after treatment, one of the animals dosed at 900 mg/kg was found dead. No clinical signs were observed in any animal dosed at 500 mg/kg. In view of the toxic signs and the late onset of the mortality, a dose level of 900 mg/kg was considered to be an appropriate maximum tolerated dose for the UDS study (and therefore the maximum dose for the main experiments). A lower dose level of 360 mg/kg (40% of the top dose) was also selected. Groups of five male rats were treated once with the solvent arachis oil, BMS 233110-01 (at 360 or 900 mg/kg) or the required positive control, by oral gavage, at a dose volume of 10 mL/kg. The positive controls used were 75 mg/kg 2-acetamidofluorene (2-AAF) suspended in com oil (12-14 hour experiment) and 10 mg/kg dimethylnitrosamine (DMN) dissolved in purified water (2-4 hour experiment).

Clinical signs observed in the main study included lethargy, piloerection, eye closure and an unsteady gait (900 mg/kg). Piloerection was also observed in the 360 mg/kg dose group. No clinical signs were observed in any other animal group.

Samples were saved from the main study dosing preparations for analysis of achieved concentration. Results proved to be acceptable. Approximately 12-14 hours (Experiment 1) or 2-4 hours (Experiment 2) after dosing, animals

were killed and their livers perfused with collagenase to provide a primary culture of hepatocytes. Cultures were made from three animals in each dose group and were treated with [3H] thymidine. Six slides from each animal were prepared with fixed hepatocytes and of these, three were dipped in photographic emulsion to prepare autoradiograms. Slides were examined microscopically after development of the emulsion and staining, and the net grain count (NNG), the number of grains present in the nucleus minus the mean number of grains in three equivalent areas of cytoplasm, was determined for each of at least two of the three slides, for each animal and dose group.

Negative (vehicle) control animals gave a group mean NNG value ofless than zero with only 0 to 0.7% cells in repair. Group mean NNG values were increased by 2-AAF and DMN treatment to more than 15 .5 and more than 50% cells were found to be in repair. In this study the vehicle control NNG value was consistent with both published and historical control data, and the system was shown to be sensitive to two known DNA damaging agents requiring

metabolism for their action. The assay was therefore accepted as valid.

Treatment with 360 or 900 mg/kg BMS 233110-01 did not produce a group mean NNG value greater than -1.5 nor were any more than 1.3% cells found in repair at either dose.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the results from the in-vivo studies BMS 233110 -01 is considered to be non-mutagenic