3,4-dihydro-2H-1,4-benzoxazin-6-ol

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Nov 2004 to 26 May 2005

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Test material

Test material information

Constituent 1

Reference substance name:

3,4-dihydro-2H-1,4-benzoxazin-6-ol

EC Number:

247-415-5

EC Name:

3,4-dihydro-2H-1,4-benzoxazin-6-ol

Cas Number:

26021-57-8

Molecular formula:

C8H9NO2

IUPAC Name:

3,4-dihydro-2H-1,4-benzoxazin-6-ol

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 0508918
- Expiration date of the lot/batch: September 2005
- Purity test date : 98.3%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (>0 to 10°C) protected from light and with nitrogen gas
- Stability under storage conditions:
- Stability under test conditions: Stability of Hydroxybenzomorpholine (A025) in 0.5% carboxymethylcellulose over a range of 1-200 mg/mL was confirmed in CIT study number 26976 AHS, when protected from light and under nitrogen atmosphere for a maximum time period of 6 hours.
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: The stability, homogeneity, and/or concentration of the dosing preparations were not analyzed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article was supplied as a brown crystalline powder. Prior to preparation, the vehicle was degassed by sonication for at least 15 minutes and then saturated with nitrogen gas, and kept under nitrogen atmosphere for at least 15 minutes prior to dosing. The highest dosing solution was prepared by adding a weighed amount of test article to a measured volume of 0.5% carboxymethylcellulose and mixing well. Lower doses were prepared by dilution of the highest dosing solution with the vehicle. Brown opaque homogeneous suspensions were obtained over the entire target concentration range of approximately 37.5 to 200 mg/mL. The dosing solutions were prepared within approximately 2.5 hours of dosing and were held at room temperature, protected from light and under nitrogen atmosphere.

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Remarks:

Crl:CD® (SD)IGS BR rats

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animals purchased from Charles River Laboratories, Raleigh, North Carolina, were used for the assay.
- Age at study initiation: The animals were approximately 9 weeks of age at the initiation of dosing.
- Weight at study initiation: Animals were weighed prior to dosing and were dosed based upon the individual animal weights. The animals were dosed on 02 December 2004
(2000 mg/kg) or 03 December 2004 (1000 and 1500 mg/kg) and ranged in weight from 287 to 330 grams for the males, and 195 to 236 grams for the females.
- Fasting period before study: not specified
- Housing: Animals were housed up to 2 per cage during acclimation and singly after randomization in suspended stainless-steel cages measuring 24.2 cm x 22.0 cm x 17.3 cm (DxWxH)
- Diet (e.g. ad libitum): PMI Certified Rodent Dietâ 5002, and tap water were supplied ad libitum. The feed was analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates, and specified nutrients.
- Water (e.g. ad libitum): The water was analyzed on a retrospective basis for specified microorganisms, pesticides, heavy metals, alkalinity, and
halogens.
- Acclimation period: Animals were acclimated for at least 5 days prior to the initiation of dosing. They were identified by eartag after computer-generated random assignment to treatment groups according to Covance-Vienna SOPs. Treatment groups were identified by cage label. . Animals were anesthetized prior to surgery to obtain the hepatocytes (Ketamine:Xylazine at approximately 100 mg/kg:13.4 mg/kg by intraperitoneal injection) and exsanguinated during the procedure.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 4°C (64 to 79°F). Temperature was recorded at least once daily.
- Humidity (%): humidity was recorded at least once daily. 55 ± 15%
The lighting controls were set to maintain a 12-hour light/12-hour dark cycle (lights on
- Air changes (per hr): The air handling controls were set for ten or greater air changes/hour in the study room.
- Photoperiod (hrs dark / hrs light): The lighting controls were set to maintain a 12-hour light/12-hour dark cycle (lights on approximately 0600 to 1800 hours), which was interrupted during animal dosing of the 14- to 16-hour timepoint.

IN-LIFE DATES:
The animals were dosed on 02 December 2004 (2000 mg/kg) or 03 December 2004 (1000 and 1500 mg/kg)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: The vehicle control was 0.5% carboxymethylcellulose (CMC, from Sigma Chemical Co., Lot No. 052K0117; and distilled water from Crystal Springs, Lot Nos. Btld 06/21/04 and Btld. 10/15/04).
- Justification for choice of solvent/vehicle: no
- Concentration of test material in vehicle: The vehicle control animals were dosed with the same lot of 0.5% carboxymethylcellulose used to dilute the test article and dosed by the same route as, and concurrently with, the test article in amounts equal to the maximum volume of dosing formulations administered to the experimental animals.
- Amount of vehicle (if gavage or dermal): The dosing volume was 10 mL/kg. Four control rats at each timepoint in the UDS study were treated by oral gavage (no control articles were used in the dose range finding assay).
- Type and concentration of dispersant aid (if powder): Vehicle control hepatocytes were subjected to all of the manipulations used for the hepatocytes derived from test article-treated animals.

An acute dosing regimen (single administration) was used, and the route of administration for test article and the vehicle control groups was oral gavage. The dosing volume was kept constant at 10 mL/kg. The positive control was prepared fresh for each timepoint and administered by IP injection at a dosing volume of 1 mL/kg. Delivery volumes were calculated on the basis of the most recent animal weight. The animals were observed for toxic signs and mortality within 1 hour of dosing and just prior to perfusion for hepatocyte collection.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The highest dose selected for the UDS assay was 1500 mg/kg, based on the results of the dose range finding assay. Two additional dose levels were selected, using dilutions of the highest dose. The test article was therefore tested at 375, 750, and 1500 mg/kg. Because females were found in the dose range finding study to be more sensitive to acute toxicity than the males, only females were treated in the UDS assay

Duration of treatment / exposure:

Two timepoints for UDS analysis were employed, one at 2 to 4 hours after administration of a single dose of the test article and another at approximately 14 to 16.5 hours after administration of a single dose of the test article

Frequency of treatment:

single administration

Post exposure period:

cell collection and culture of hepatocytes

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 500 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

750 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

375 mg/kg bw (total dose)

No. of animals per sex per dose:

4 or 6

Control animals:

yes, concurrent vehicle

Positive control(s):

N-dimethylnitrosamine
- Justification for choice of positive control(s): The positive control article, N-dimethylnitrosamine (DMN: CAS No. 62-75-9, Sigma Chemical Co., Lot No. 062K1506), is known to induce UDS in rat hepatocytes in vivo and was included in the UDS assay.
- Route of administration: DMN was dissolved in sterile deionized water and administered at a dosing volume of about 1 mL/kg. DMN was administered at approximately 10 mg/kg and 15 mg/kg for the 2- to 4-hour and 14- to 16-hour timepoints, respectively. The positive control was prepared fresh for each timepoint and administered by intraperitoneal injection to four rats per timepoint.
- Doses / concentrations: Two positive control groups, dosed intraperitoneally with dimethylnitrosamine (DMN) were included. The dose was 10 mg DMN/kg for the 2 to 4 hour
harvest and 15 mg DMN/kg for the 14 to 16 hour harvest

Examinations

Tissues and cell types examined:

nuclear labeling of cultured hepatocytes for detection of unscheduled DNA synthesis

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Dose Range Finding Study
A dose range finding study was performed with a high dose of 2000 mg/kg of the test article in 0.5% carboxymethylcellulose. A single group of three males and three females was dosed initially at 2000 mg/kg, then two additional groups consisting of three males and three females each were dosed at 1000 or 1500 mg/kg. Each rat was treated by oral gavage at a dosing volume of 10 mL/kg.
Body weights were taken prior to dosing. The animals were dosed on 02 December 2004 (2000 mg/kg) or 03 December 2004 (1000 and 1500 mg/kg) and ranged in weight from 287 to 330 grams for the males, and 195 to 236 grams for the females. The weight variation of the animals did not exceed ± 20% of the mean weight. All three groups of animals were observed immediately after dosing and within approximately 1 hour after dosing, and daily thereafter. The animals dosed at 2000 mg/kg were also observed approximately 5 hours after dosing. All surviving animals were euthanized by CO2 inhalation followed by penetration of the thorax 2 days after receiving a single dose.
The daily observations of toxic symptoms and/or mortality data were used to select doses for the subsequent UDS assay.

Dose Selection
The highest dose selected for the UDS assay was 1500 mg/kg, based on the results of the dose range finding assay. Two additional dose levels were selected, using dilutions of the highest dose. The test article was therefore tested at 375, 750, and 1500 mg/kg. Because females were found in the dose range finding study to be more sensitive to acute toxicity than the males, only females were treated in the UDS assay.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
UDS Study
Dosing and Sacrifice Timepoints
Two timepoints for UDS analysis were employed, one at 2 to 4 hours after administration of a single dose of the test article and another at approximately 14 to 16.5 hours after administration of a single dose of the test article. The group of animals for each analysis was dosed on different dates, independent of the ordering of the timepoint.
For the 2- to 4-hour timepoint (dosing date of 21 December 2004), the animals ranged in weight from 185 to 215 grams. For the 14- to 16-hour timepoint (dosing date of 27 December 2004), the weight range of the animals used was 206 to 248 grams. At the initiation of dosing, the weight variation of animals did not exceed ±20% of the mean weight at each timepoint.
An acute dosing regimen (single administration) was used, and the route of administration for test article and the vehicle control groups was oral gavage. The dosing volume was kept constant at 10 mL/kg. The positive control was prepared fresh for each timepoint and administered by IP injection at a dosing volume of 1 mL/kg. Delivery volumes were calculated on the basis of the most recent animal weight. The animals were observed for toxic signs and mortality within 1 hour of dosing and just prior to perfusion for hepatocyte collection.

DETAILS OF SLIDE PREPARATION:
Cell Collection and Culture
This assay was based on the procedures described by Butterworth et al. (1987). The hepatocytes were obtained by perfusion of livers from 4 animals per group in situ with HBSS/EGTA followed by WMEC. The hepatocytes were obtained by mechanical dispersion of excised liver tissue in a sterile culture dish containing WMEC. The suspended tissues and cells were allowed to settle to remove cell clumps and debris prior to collection. The collected cell suspension was centrifuged and the cell pellet resuspended in WME+. After obtaining a viable cell count, a series of culture dishes was inoculated with approximately 0.5 x 106 viable cells in 3 mL of WME+, where possible. Culture dishes that were used for the UDS assay contained plastic coverslips. Dishes used to assess attachment efficiency had no coverslips. Cultures were
identified with the animal eartag number.
An attachment period of 1.5 to 2 hours at 35 to 38°C in an atmosphere of 4 to 6% CO2 in air was used to establish the cell cultures as monolayers. Unattached cells were then removed, the cultures washed twice, and labeling was initiated by refeeding the cultures with 2.5 mL of WME-treat. Three of the replicate cultures from each animal were used for the UDS assay, and one culture was used to assess cell attachment. Any remaining cultures were kept for analysis in the event of technical problems.
Attachment efficiency, an estimate of the number and viability of cells attaching to the dishes, was determined for one culture from each animal using trypan blue dye exclusion and in situ analysis.
After a labeling period of about 4 hours, the labeled cell cultures were washed twice, refed with WMEI containing 0.25 mM thymidine, and returned to the incubator for 16 to 20 hours.
Termination
The nuclei were swollen by addition of 1% sodium citrate to the cultures (containing cell monolayers) for 8-12 minutes. Next, the cells were fixed in acetic acid:ethanol (1:3) and dried at least overnight. The coverslips were mounted on glass slides, dipped in an emulsion of Kodak NTB and water, and air-dried. The emulsion-coated slides were stored for 6-10 days at >0-10°C in light-tight boxes containing a desiccant. The emulsions were developed in Kodak D19, fixed with Kodak Rapid Fixer, and stained with a modified hematoxylin and eosin procedure.

METHOD OF ANALYSIS:
After autoradiography, all slides were reviewed for quality before analysis. The quality of the autoradiography, the number and distribution of cells on the slides, and cellular morphology were considered in the evaluation. Three treatment groups from each timepoint were analyzed for nuclear labeling. Three animals from the vehicle, positive control and test article dose groups were analyzed, beginning with the lowest numbered animal having cells acceptable for analysis.
The cells were examined microscopically at approximately 1500x magnification under oil immersion and the field was displayed on the video screen of an automatic counter. Only normally-appearing nuclei were scored, and any occasional nuclei blackened by grains too numerous to count were excluded as cells in which replicative DNA synthesis occurred rather than repair synthesis. UDS was measured by counting nuclear grains and subtracting the average number of grains in three nuclear-sized areas adjacent to each nucleus (cytoplasmic count). This value is referred to as the net nuclear grain count. The coverslips were coded to prevent bias in grain counting.
The net nuclear grain count was routinely determined for 50 randomly selected cells on duplicate or triplicate coverslips (150 total nuclei) for each animal. The average mean net nuclear grain count (± standard deviation) was determined from the duplicate or triplicate coverslips (150 total nuclei) for each animal and averaged for each treatment condition.

Evaluation criteria:

An assay normally is considered acceptable for evaluation of the test results only if all of the criteria listed below are satisfied. This listing may not encompass all test situations, thus the study director must exercise scientific judgment in modifying the criteria or considering other causes that might affect reliability and acceptance

Results and discussion

Test results

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

squinted eyes, lacrimation, hypoactivity, sensitivity to touch and brown urine

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

not determinable

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: Three rats/sex/dose group were dosed by oral gavage with the test article in 0.5% carboxymethylcellulose at 1000, 1500 and 2000 mg/kg
- Clinical signs of toxicity in test animals: Clinical signs included hypoactivity, squinted eyes, labored respiration, flattened posture, brown oral discharge, lacrimation, lateral right recumbency, ataxia, brown nasal discharge, yellow oral discharge and tremors. One out of three females dosed at 1000 mg/kg and one of three females dosed at 1500 mg/kg was found dead one day after dosing.
Two females dosed at 2000 mg/kg were found dead approximately 8 hours after dosing. Because females were found in the dose range finding study to be more sensitive to acute
toxicity than the males, only females were treated in the UDS assay.


RESULTS OF DEFINITIVE STUDY
- Dose range: the test article was administered at 375, 750, and 1500 mg/kg in 0.5% carboxymethylcellulose, based on the results of the dose range finding study
- The test material was administered by oral gavage in volumes of 10 mL/kg to four to six female rats per dose group at each timepoint. Four animals from each group were perfused and hepatocytes were seeded for attachment. UDS was determined from three animals per group.
-observation : Animal observations from the UDS assay are given in Tables 3 and 6. At the 2- to 4-hour timepoint (Table 3), clinical signs observed in the high dose group prior to perfusion included squinted eyes, lacrimation, hypoactivity, sensitive to touch and/or brown urine. Two animals in the low dose group (375 mg/kg) were observed with slight hypoactivity and squinted eyes after dosing. All other animals were normal.
At the 14- to 16-hour timepoint (Table 6), all animals were normal after dosing with the exception of hypoactivity observed in all animals dosed at 375 mg/kg, and slight hypoactivity in one animal dosed at 1500 mg/kg. Prior to perfusion, animals treated at 1500 mg/kg were observed with hypoactivity/slight hypoactivity, brown urine, labored breathing, ataxia and/or chromodachorrhea.
For the early UDS timepoint, perfusions were initiated 2.6 to 3.0 hours after dose administration. The hepatocytes ranged in viability (determined by trypan blue dye exclusion) from 72.5% to 89.6% of the total cells collected in the perfusate (Table 4). The attachment efficiency varied from 48.5% to 83.1%, and the viability of the attached cells was good, ranging from 86.3% to 98.7%.
For the 14- to 16-hour timepoint, perfusions were initiated 16.0 to 16.5 hours after dose administration (see Protocol Deviations). The hepatocytes ranged in viability from 62.7% to
89.0% of the total cells collected in the perfusate (Table 7). The attachment efficiency varied from 0.8% to 89.8%, and the viability of the attached cells was good, ranging from 92.6% to 100.0%.
All three test article treatment groups (375, 750, and 1500 mg/kg) were analyzed for nuclear labeling at both timepoints.
The UDS data for both timepoints is summarized in Table 1.
For the 2- to 4-hour timepoint, the mean net nuclear grain count for the vehicle control animals was 0.30, and the average percent of cells containing five or more net nuclear grains was 1.33%.
None of the treatment groups yielded a positive mean net nuclear grain count, and the highest percent cells with > 5 grains was 10.44%, below the criterion for a positive response. Thus, no evidence for UDS was obtained at the early timepoint of 2 to 4 hours after treatment of the animals. (Individual UDS slide data for each animal are shown in Table 5.)
For the 14- to 16-hour timepoint, the mean net nuclear grain count for the vehicle control animals was 0.63, and the average percent of cells containing five or more net nuclear grains was 3.11% (Table 1). None of the treatment groups yielded a positive mean net nuclear grain count, and the highest percent cells with > 5 grains was only 2.22%, well below the criterion for a positive response. Thus, no evidence for UDS was obtained at the timepoint of 14 to 16.5 hours after treatment of the animals. (Individual UDS slide data for each animal are shown in Table 8.)
The DMN positive control induced large increases in nuclear labeling as measured by the mean number of net nuclear grain counts as well as the mean percentage of cells with ³ 5 net nuclear grains.
The vehicle control results were well within the acceptable criteria for this study. The DMN positive control treatments induced large increases in nuclear labeling that clearly exceeded both criteria used to indicate UDS. Since the positive control animals were responsive, the test results were considered to provide conclusive evidence for the lack of UDS induction by the test article.

Any other information on results incl. tables

 

DATA TABLES

 

 

TABLE 1: SUMMARY OF UDS SLIDE DATA

 

Treatment	Dose (mg/kg)	Na	Time (hr)		Mean NuclearGrains^b +/-SD	MeanNetNuclear Grains^c +/-SD	Mean Cytoplasmic Grains^d +/-SD	Mean % Cells With>5NNG^e +/-SD
Vehicle Control	0	3	2-4	Mean	2.90	0.30	2.60	1.33
				+SD	0.52	0.31	0.81	0.67
	0	3	14-16	Mean	4.03	0.63	3.40	3.11
				+SD	1.03	0.56	0.98	5.39
Positive Control	10	3	2-4	Mean	16.05	13.52	2.53	93.11
				+SD	2.34	2.53	0.21	3.08
	15	3	14-16	Mean	16.19	12.72	3.48	97.55
				+SD	1.85	2.42	0.64	2.34
Hydroxybenzomorpholine(A025)	375	3	2-4	Mean	3.56	-0.22	3.79	6.67
				+SD	0.91	0.38	0.63	4.81
		3	14-16	Mean	3.26	-0.15	3.42	1.33
				+SD	0.62	0.05	0.63	0.67
	750	2	2-4	Mean	4.20	0.19	4.01	3.67
				+SD	1.15	0.75	1.67	2.85
		3	14-16	Mean	3.59	-0.22	3.81	1.11
				+SD	0.44	0.33	0.68	1.39
	1500	3	2-4	Mean	6.25	0.26	5.99	10.44
				+SD	1.73	0.35	1.40	6.40
		3	14-16	Mean	3.56	0.08	3.49	2.22

Notes:

^aNumberofanimalsanalyzed.

^bAverage nuclear graincount.

^cAverage of net nuclear grain count with standard deviation (SD) between coverslips. Net nuclear grains (NNG) = Nuclear grain count - Average cytoplasmic grain count.

^dAverage of cytoplasmic grain count.

^eAverage percentage of cells with greater than or equal to 5 net nuclear grains.

Vehicle control article = 0.5% carboxymethylcellulose, 10 mL/kg.; Positive control article =Dimethylnitrosamine, 1 mL/kg. Criteria for a positive response:

2-4 hr timepoint - mean net nuclear grain counts³3.30 or nuclei containing³5 NNG³11.33%. 14-16 hr timepoint - mean net nuclear grain counts³3.63 or nuclei containing³5 NNG³13.11%.

 

TABLE 2. ANIMAL OBSERVATIONS, DOSE RANGEFINDING STUDY

Trial Initiation Date: 02 December 2004 (2000mg/kg)

03 December (1000 and 1500mg/kg)

Animal Number	Body Weight (g)	Immediately After Dosing	Approximately 1 Hour After Dosing	Approximately 5 Hours After Dosing	Day1After Dosing	Day2After Dosing
Test Article
MALES
1000 mg/kg
7721	309	Normal	2,3,4,7	Not performed	Normal	Normal
7722	287	Normal	2,3,4,6	Not performed	Normal	Normal
7723	296	Normal	2,3,4,7	Not performed	Normal	Normal
1500 mg/kg
7724	330	Normal	2,3,4,6,7	Not performed	Normal	Normal
7725	305	Normal	2,3,4	Not performed	Normal	Normal
7726	307	Normal	2,3,4	Not performed	1,12	1,9,12
2000 mg/kg
7718	312	1	2,3,4,5	1,4	Normal	Normal
7719	315	1	2,3,4,5	1,3,4	9	9
7720	319	1	2,3,4,5	1,3,12	9,10,11	Normal
FEMALES
1000 mg/kg
7733	202	Normal	2,3,4,5,7	Not performed	13	-
7734	236	Normal	2,3,4,5,7	Not performed	1,9	Normal
7735	226	Normal	2,3,4	Not performed	Normal	Normal
1500 mg/kg
7736	226	Normal	2,3,4	Not performed	13	-
7737	201	Normal	2,3,4	Not performed	1	1
7738	225	Normal	2,3,4	Not performed	Normal	Normal
2000 mg/kg
7730	226	Normal	2,3,4,5	2,3,4,6,7,8^a	-	-
7731	223	Normal	2,3,4,5	2,3,4	Normal	Normal
7732	195	Normal	2,3,4,5	2,3,4,5^a	-	-

 

1 = slightly hypoactive 2 = hypoactive

3 = squinted eyes

4 = labored respiration 5 = flattened posture

6 = oral discharge – brown 7 = lacrimation

8 = recumbent – lateral, right 9 = ataxic

10 = nasal discharge -brown11 = oral discharge - yellow 12 = tremors

13 = found dead

^aAnimalfound dead approximately 8 hours after dosing

Test Article =Hydroxybenzomorpholine(A025), 10 mL/kg.

 

TABLE 3. ANIMAL OBSERVATIONS, 2- TO 4-HOUR TIMEPOINT

Trial Initiation Date: 21 December 2004

Animal Number	After Dosing		Before Perfusion
Vehicle Control 8032	Normal		Normal
8033	Normal		Normal
8034	Normal		Normal
8035	Normal		Normal
Positive Control 8028	Normal		Normal
8029	Normal		Normal
8030	Normal		Normal
8031	Normal		Normal
Test Article 375 mg/kg 8036	1,2		Normal
8037	1,2		Normal
8038	Normal		Normal
8039	Normal		Normal
750 mg/kg 8040	Normal		Normal
8041 8042* 8043*	NormalNormalNormal		NormalNormalNormal
8044	Normal		Normal
8045	Normal		Normal
1500 mg/kg 8046	Normal		2,3,4,6
8047*	Normal		2,3,4
8048	Normal		2,3,4,6
8049	Normal		1,2,3,6
8050	Normal		2,3,4,5
8051*	Normal		1,2,3
1 = slightly hypoactive		4 = hypoactive
2 = squinted eyes 3 = lacrimation		5 = sensitive to touch 6 = brown urine

Vehicle control = 0.5% carboxymethylcellulose, 10 mL/kg. Positive control =Dimethylnitrosamine, 10 mg/kg (1 mL/kg). Test Article =Hydroxybenzomorpholine(A025), 10 mL/kg.

*Extra animals sacrificed prior to perfusion.

 

TABLE 4. SUMMARY OF CULTURE DATA, 2- TO 4-HOUR TIMEPOINT

 

Trial Initiation Date: 21 December 2004

	Body Weight	Attachment Efficiency	Attachment Viability	Perfusion Viability
Test Condition	(g)	(%)	(%)	(%)
Vehicle Control
8032	209	75.7	93.0	82.6
8033	206	62.6	86.3	78.6
8034	211	57.5	93.6	82.3
8035	185	76.9	91.2	81.5
Positive Control
8028	210	67.5	93.6	86.1
8029	207	68.0	90.9	79.9
8030	205	61.0	89.6	77.8
8031	202	83.1	96.8	85.6
Test Article
375 mg/kg
8036	213	56.6	95.5	81.1
8037^a	192	71.1	96.9	76.5
8038	191	60.5	98.7	72.5
8039	201	56.5	92.6	76.7
750 mg/kg
8040	204	55.6	98.2	79.5
8041	195	54.6	95.9	82.0
8044	202	57.5	97.8	78.9
8045	203	64.9	97.2	82.1
1500 mg/kg
8046	185	63.7	94.7	89.6
8048	192	52.6	95.6	80.5
8049	212	48.5	97.7	88.3
8050	195	52.7	96.8	79.0

Vehicle control = 0.5% carboxymethylcellulose, 10 mL/kg. Positive control =Dimethylnitrosamine, 10 mg/kg (1 mL/kg). Test Article =Hydroxybenzomorpholine(A025), 10 mL/kg.^aMuchdebris and many red blood cells.

 

TABLE 5. INDIVIDUAL UDS SLIDE DATA, 2- to 4-HOUR TIMEPOINT

 

Initiation of Dosing: 21 December 2004

 

Slide Code		Animal Number^a	Mean NuclearGrains^b	Mean Net NuclearGrains^c	Mean CytoplasmicGrains^d	% Cells with =5NNG^e
VehicleControlDose:0 mg/kg
8.0		8032B	2.06	0.26	1.80	0.00
14.0		8032E	3.94	0.94	3.00	4.00
18.0		8032F	2.68	0.04	2.64	0.00
		Mean	2.89	0.41	2.48	1.33
		Standard Deviation	0.96	0.47	0.62	2.31
2.0		8034A	2.08	-0.08	2.16	4.00
7.0		8034B	2.16	0.32	1.84	0.00
10.0		8030D	2.92	1.34	1.58	2.00
		Mean	2.39	0.53	1.86	2.00
		Standard Deviation	0.46	0.73	0.29	2.00
5.0		8035B	2.46	0.92	1.54	2.00
9.0		8035C	4.76	-0.98	5.74	0.00
15.0		8035E	3.04	-0.08	3.12	0.00
		MEAN	3.42	-0.05	3.47	0.67
		Standard Deviation	1.20	0.95	2.12	1.15
		Mean (Group)	2.90	0.30	2.60	1.33
		Standard Deviation	0.52	0.31	0.81	0.67

 

Positive Control Dose: 10 mg/kg

1.0	8028A	16.20	13.62	2.58	96.00
4.0	8028B	13.72	11.20	2.52	98.00
17.0	8028F	12.94	10.32	2.62	80.00
	MEAN	14.29	11.71	2.57	91.33
	Standard Deviation	1.70	1.71	0.05	9.87
6.0	8029B	12.08	9.86	2.22	90.00
11.0	8029D	25.29	22.88	2.41	100.00
13.0	8029E	18.74	16.48	2.26	100.00
	MEAN	18.70	16.41	2.30	96.67
	Standard Deviation	6.61	6.51	0.10	5.77
3.0	8030A	14.44	12.32	2.12	92.00
12.0	8034C	10.72	9.08	1.64	84.00
16.0	8030F	20.30	15.92	4.38	98.00
	MEAN	15.15	12.44	2.71	91.33
	Standard Deviation	4.83	3.42	1.46	7.02
	MEAN	16.05	13.52	2.53	93.11
	Standard Deviation	2.34	2.53	0.21	3.08

                                      (Group)                                      

 

 

TABLE 5 cont. INDIVIDUAL UDS SLIDE DATA, 2- to 4-HOUR TIMEPOINT

 

 

Slide Code	AnimalNumber^a	Mean NuclearGrains^b	Mean Net NuclearGrains^c	Mean CytoplasmicGrains^d	% Cells with=5NNG^e
Test Article Dose: 375mg/kg
32.0	8036C	2.82	-0.64	3.46	0.00
33.0	8036B	2.18	-0.84	3.02	2.00
40.0	8036E	2.72	-0.46	3.18	2.00
	MEAN	2.57	-0.65	3.22	1.33
	Standard Deviation	0.34	0.19	0.22	1.15
28.0	8037C	3.48	-0.60	4.08	2.00
34.0	8037B	2.58	-0.18	2.76	0.00
38.0	8037A	5.20	1.04	4.16	22.00
	MEAN	3.75	0.09	3.67	8.00
	Standard Deviation	1.33	0.85	0.79	12.17
31.0	8039C	4.06	-1.38	5.44	0.00
35.0	8039B	1.96	-1.14	3.10	2.00
39.0	8039A	7.06	2.18	4.88	30.00
	MEAN	4.36	-0.11	4.47	10.67
	Standard Deviation	2.56	1.99	1.22	16.77
	MEAN	3.56	-0.22	3.79	6.67
	Standard Deviation (Group)	0.91	0.38	0.63	4.81

 

Test Article Dose: 750mg/kg

 

24.0	8040E	3.40	0.28	3.12	2.00
25.0	8040F	2.84	-0.14	2.98	0.00
26.0	8040A	3.30	0.02	3.28	0.00
	MEAN	3.18	0.05	3.13	0.67
	Standard Deviation	0.30	0.21	0.15	1.15
20.0	8041A	4.05	0.99	3.07	2.67
21.0	8041D	3.88	1.02	2.86	10.00
	MEAN	3.96	1.00	2.96	6.34
	Standard Deviation	0.12	0.02	0.15	5.18
19.0	8045A	3.70	-0.88	4.58	0.00
22.0	8045D	3.98	0.34	3.64	10.00
23.0	8045E	8.66	-0.92	9.58	2.00
	MEAN	5.45	-0.49	5.93	4.00
	Standard Deviation	2.79	0.72	3.19	5.29
	MEAN	4.20	0.19	4.01	3.67
	Standard Deviation (Group)	1.15	0.75	1.67	2.85

 

 

TABLE 5 cont. INDIVIDUAL UDS SLIDE DATA, 2- to 4-HOUR TIMEPOINT

 

 

 

Slide Code	AnimalNumber^a	Mean NuclearGrains^b	Mean Net NuclearGrains^c	Mean CytoplasmicGrains^d	% Cells with=5NNG^e
Test Article Dose: 1500 mg/kg
27.0	8046C	7.34	0.04	7.30	2.00
36.0	8046B	3.60	0.04	3.56	2.00
37.0	8046A	7.24	0.22	7.02	10.00
	MEAN	6.06	0.10	5.96	4.67
	Standard Deviation	2.13	0.10	2.08	4.62
29.0	8048C	4.42	-0.84	5.26	2.00
41.0	8048E	4.86	0.26	4.60	12.00
42.0	8048D	4.60	0.64	3.96	14.00
	MEAN	4.63	0.02	4.61	9.33
	Standard Deviation	0.22	0.77	0.65	6.43
30.0	8049C	6.12	-1.86	7.98	0.00
43.0	8049D	8.86	1.86	7.00	24.00
44.0	8049F	9.24	2.00	7.24	28.00
	MEAN	8.07	0.67	7.41	17.33
	Standard Deviation	1.70	2.19	0.51	15.14
	MEAN	6.25	0.26	5.99	10.44
	Standard Deviation (Group)	1.73	0.35	1.40	6.40

^aThree animals per dose level were analyzed (except animal 8041).

^bAverage nuclear grain counts.

^cAverage of net nuclear grain counts with standard deviation (SD) between coverslips. Net nuclear grains (NNG) = Nuclear grain count - Average cytoplasmic grain count.

^dAverage of cytoplasmic grain counts.

^eAverage percentage of cells with greater than or equal to 5 net nuclear grains. Vehicle control article = 0.5% carboxymethylcellulose, 10 mL/kg.

Positive control article =Dimethylnitrosamine, 1 mL/kg. Test Article =Hydroxybenzomorpholine(A025), 10 mL/kg.

 

TABLE 6. ANIMAL OBSERVATIONS, 14- TO 16-HOUR TIMEPOINT

Trial Initiation Date: 27 December 2004

Animal Number	After Dosing	Before Perfusion
Vehicle Control 8168	Normal	Normal
8169	Normal	Normal
8170	Normal	Normal
8171	Normal	Normal
Positive Control 8164	Normal	Normal
8165	Normal	Normal
8166	Normal	Normal
8167	Normal	Normal
Test Article 375 mg/kg 8172	3	Normal
8173	3	Normal
8174	3	Normal
8175	3	Normal
750 mg/kg 8176	Normal	Normal
8177	Normal	Normal
8178	Normal	Normal
8179	Normal	Normal
8180*	Normal	Normal
8181*	Normal	Normal
1500 mg/kg 8182	3	2,3
8183	Normal	1,2,4,5
8184	Normal	2,3
8185	Normal	2,3
8186*	Normal	2,3,6
8187*	Normal	1,2,4,5

1 =hypoactive2 =brownurine

3 =slightly hypoactive4 = labored breathing 5 =ataxic

6 =chromodacyorrhea

Vehicle control = 0.5% carboxymethylcellulose, 10 mL/kg. Positive control =Dimethylnitrosamine, 15 mg/kg (1 mL/kg). Test Article =Hydroxybenzomorpholine(A025), 10 mL/kg.

*Extra animals sacrificed prior to perfusion.

 

TABLE 7. SUMMARY OF CULTURE DATA,14- TO 16-HOUR TIMEPOINT

 

Trial Initiation Date: 27 December 2004

	Body Weight	Attachment Efficiency	Attachment Viability	Perfusion Viability
Test Condition	(g)	(%)	(%)	(%)
Vehicle Control
8168	230	69.2	93.3	78.1
8169	206	83.0	94.8	84.9
8170	206	83.1	97.6	79.0
8171	232	45.6	96.2	83.6
Positive Control
8164	228	74.9	96.1	77.7
8165	222	89.8	97.4	84.3
8166	231	86.9	98.5	80.3
8167	227	64.6	92.8	79.6
Test Article
375 mg/kg 8172^a	242	85.7	94.0	80.4
8173	214	71.4	93.3	78.3
8174	233	69.0	95.2	80.7
8175	235	45.2	93.4	70.3
750 mg/kg
8176	214	76.5	97.7	76.9
8177	224	77.9	96.3	81.2
8178	219	48.7	98.3	72.7
8179	228	28.8	95.9	78.4
1500 mg/kg 8182^b	231	18.1	97.2	62.7
8183^b	236	0.8	100.0	62.9
8184	248	60.0	97.1	81.5
8185^c	218	65.5	92.6	89.0

Vehicle control = 0.5% carboxymethylcellulose, 10 mL/kg. Positive control =Dimethylnitrosamine, 15 mg/kg (1 mL/kg). Test Article =Hydroxybenzomorpholine(A025), 10 mL/kg.^aHeavydebris on monolayer.

^bSparsegrowth.

^cManyred blood cells.

 

TABLE 8. INDIVIDUAL UDS SLIDE DATA, 14- to 16-HOUR TIMEPOINT

 

Initiation of dosing: 27 December 2004

 

Slide Code	AnimalNumber^a	Mean NuclearGrains^b	Mean Net NuclearGrains^c	Mean CytoplasmicGrains^d	% Cells with=5NNG^e
Vehicle Control Dose: 0 mg/kg
3.0	8168A	2.82	0.60	2.22	0.00
4.0	8168C	3.24	0.30	2.94	0.00
12.0	8168B	2.48	0.46	2.02	0.00
	MEAN	2.85	0.45	2.39	0.00
	Standard Deviation	0.38	0.15	0.48	0.00
5.0	8169C	3.18	0.54	2.64	0.00
6.0	8169E	6.32	2.54	3.78	22.00
17.0	8169F	4.62	0.70	3.92	6.00
	MEAN	4.71	1.26	3.45	9.33
	Standard Deviation	1.57	1.11	0.70	11.37
1.0	8170A	4.08	0.66	3.42	0.00
7.0	8170E	5.04	-0.70	5.74	0.00
8.0	8170D	4.48	0.58	3.90	0.00
	MEAN	4.53	0.18	4.35	0.00
	Standard Deviation	0.48	0.76	1.22	0.00
	MEAN	4.03	0.63	3.40	3.11
	Standard Deviation (Group)	1.03	0.56	0.98	5.39

 

Positive Control Dose: 15 mg/kg

2.0	8164A	12.96	9.30	3.66	92.00
13.0	8164B	16.74	11.08	5.66	100.00
14.0	8164D	12.72	10.10	2.62	94.00
	MEAN	14.14	10.16	3.98	95.33
	Standard Deviation	2.25	0.89	1.55	4.16
15.0	8165A	15.20	11.44	3.76	98.00
16.0	8165F	16.78	13.24	3.54	100.00
18.0	8165C	18.10	14.34	3.76	94.00
	MEAN	16.69	13.01	3.69	97.33
	Standard Deviation	1.45	1.46	0.13	3.06
9.0	8167D	16.54	14.06	2.48	100.00
10.0	8167A	18.44	15.38	3.06	100.00
11.0	8167E	18.24	15.50	2.74	100.00
	MEAN	17.74	14.98	2.76	100.00
	Standard Deviation	1.04	0.80	0.29	0.00
	MEAN	16.19	12.72	3.48	97.55
	Standard Deviation	1.85	2.42	0.64	2.34

                                      (Group)                                      

 

 

Test Article Dose: 375mg/kg

19.0	8172A	3.34	0.24	3.10	2.00
30.1	8172B	2.92	-0.06	2.98	0.00
36.0	8172E	4.10	-0.82	4.92	2.00
	MEAN	3.45	-0.21	3.67	1.33
	Standard Deviation	0.60	0.55	1.09	1.15
20.0	8173A	6.14	0.68	5.46	6.00
31.1	8173B	2.78	0.06	2.72	0.00
45.0	8173F	2.38	-1.10	3.48	0.00
	MEAN	3.77	-0.12	3.89	2.00
	Standard Deviation	2.07	0.90	1.41	3.46
34.0	8174C	2.30	-0.04	2.34	0.00
35.0	8174E	2.44	0.24	2.20	0.00
44.0	8174F	2.98	-0.60	3.58	2.00
	MEAN	2.57	-0.13	2.71	0.67
	Standard Deviation	0.36	0.43	0.76	1.15
	MEAN	3.26	-0.15	3.42	1.33
	Standard Deviation (Group)	0.62	0.05	0.63	0.67

 

Test Article Dose: 750mg/kg

23.0	8176D	3.12	0.44	2.68	0.00
29.0	8176B	3.58	0.26	3.32	0.00
37.0	8176E	4.94	-0.88	5.82	0.00
	MEAN	3.88	-0.06	3.94	0.00
	Standard Deviation	0.95	0.72	1.66	0.00
28.0	8177B	3.80	0.32	3.48	4.00
38.0	8177E	4.68	-1.24	5.92	0.00
43.0	8177F	2.96	-0.88	3.84	4.00
	MEAN	3.81	-0.60	4.41	2.67
	Standard Deviation	0.86	0.82	1.32	2.31
21.0	8178A	3.16	0.46	2.70	0.00
22.0	8178D	2.92	0.12	2.80	0.00
42.0	8178F	3.18	-0.54	3.72	2.00
	MEAN	3.09	0.01	3.07	0.67
	Standard Deviation	0.14	0.51	0.56	1.15
	MEAN	3.59	-0.22	3.81	1.11
	Standard Deviation (Group)	0.44	0.33	0.68	1.39

Test Article Dose: 1500 mg/kg

24.0	8182D	2.74	0.32	2.42	0.00
27.0	8182B	3.04	0.52	2.52	0.00
33.0	8182C	2.70	0.20	2.50	0.00
	MEAN	2.83	0.35	2.48	0.00
	Standard Deviation	0.19	0.16	0.05	0.00
32.0	8184C	3.64	-0.08	3.72	0.00
39.0	8184E	4.80	-0.66	5.46	6.00
41.0	8184F	4.30	-0.32	4.62	6.00
	MEAN	4.25	-0.35	4.60	4.00
	Standard Deviation	0.58	0.29	0.87	3.46
25.0	8185D	3.44	0.30	3.14	4.00
26.0	8185B	3.02	-0.26	3.28	2.00
40.0	8185E	4.36	0.64	3.72	2.00
	MEAN	3.61	0.23	3.38	2.67
	Standard Deviation	0.69	0.45	0.30	1.15
	MEAN	3.56	0.08	3.49	2.22
	Standard Deviation (Group)	0.71	0.37	1.06	2.04

 

^aThree animals per dose level were analyzed.

^bAverage nuclear grain counts.

^cAverage of net nuclear grain counts with standard deviation (SD) between coverslips. Net nuclear grains (NNG) = Nuclear grain count - Average cytoplasmic grain count.

^dAverage of cytoplasmic grain counts.

^eAverage percentage of cells with greater than or equal to 5 net nuclear grains. Vehicle control article = 0.5% carboxymethylcellulose, 10 mL/kg.

Positive control article =Dimethylnitrosamine, 1 mL/kg. Test Article =Hydroxybenzomorpholine(A025), 10 mL/kg.

 

Comments on the Data

Various models of calculators, computers, and computer programs were used to analyze data in this study. Because different models round off or truncate numbers differently, values in some tables (e.g., means, standard deviations, or individual values) may differ slightly from those in other tables, from individually calculated data, or from statistical analysis data. Neither the integrity nor the interpretation of the data was affected by these differences.

 

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:

Hydroxybenzomorpholine (A025) did not cause any dose-related changes in the degree of nuclear labeling of cultured hepatocytes after treatment of female rats at doses of 375, 750 and 1500 mg/kg (Maximum Tolerated Dose), whether assayed at 2 to 4 hours after treatment or at 14 to 16.5 hours. Therefore, Hydroxybenzomorpholine (A025) was evaluated as negative in the in vivo/in vitro assay for unscheduled DNA synthesis (UDS) in the livers of female Crl:CDÒ(SD)IGS BR rats under the conditions of this study.

Executive summary:

Hydroxybenzomorpholine was investigated for the induction of unscheduled DNA synthesis (UDS) in hepatocytes of rats. Rats were treated in vivo. The highest dose selected for this UDS assay was 1500 mg/kg bw based on the results of a dose range finding study. In this dose range

finding study 2 out of 3 females died about 8 h after dosing in the 2000 mg/kg bw group whereas 1 out of 3 females treated with 1500 mg/kg bw died one day after dosing. Because females were found to be more sensitive to acute toxicity than the males, only females were used in the main

experiment. Next to 1500 mg/kg bw, 2 additional dose levels were selected using dilutions of the highest dose.

Hepatocytes for UDS analysis were collected at 2 - 4 h and 14 - 16.5 h after administration of hydroxybenzomorpholine. All animals from each group were perfused for the collection of hepatocytes and establishment of cultures. After attachment of the cultures they were labelled for

4 h with 10 μCi/ml 3H-thymidine. Evaluation of autoradiography was done after 6-10 days exposure.

UDS was measured by counting nuclear grains and substracting the average number of grains in 3 nuclear sized areas adjacent to each nucleus; this value is referred to as nuclear grain count.

The nuclear labelling, measured as the mean net nuclear grain count or the percent of nuclei with five or more net nuclear grains, is used to determine if a response has occurred.

Unscheduled synthesis was determined in 50 randomly selected hepatocytes per dose. Negative and positive controls were in accordance with the OECD guideline.

Results

At the 2-4 h time point clinical signs observed in the high dose group prior top perfusion were squinted eyes, lacrimation, hypoactivity, sensitivity to touch and brown urine. At the 14-16.5 time point all animals were normal after dosing with the exception of hypoactivity in the low dose group and slight hypoactivity in the high dose group. Prior to perfusion animals treated with the high dose (1500 mg/kg bw) were observed with (slight) hypoactivity, brown urine, laboured breathing, ataxia and/or chromodacryorrhea.

Both for the 2-4 h and the 14-16.5 time point after treatment none of the individual groups showed an increased mean net nuclear grain count as compared with the untreated control. Also the number of cells with 5 or more nuclear grains per cells never reached the necessary criterion

of 10% above the percentage found for the untreated control. Thus, no evidence for UDS was obtained in any treatment group at both time points.

Conclusion

Under the experimental conditions used hydroxybenzomorpholine did not induce unscheduled DNA synthesis and, consequently, is not genotoxic in rats in the in vivo UDS test.