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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Hydroxybenzomorpholine produced gene mutations in bacteria in the presence of S9 metabolic activation but not in mammalian cells on the hprt locus of mouse lymphoma cells. Negative results were also found in an in vitro chromosome aberration test (CHO cells) up to the limit of cytotoxicity.

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:

12 Dec 2003 to 19 Jan 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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 (the expiry date specified in the Study plan (September 2004)
was reviewed in the final analytical certificate)
- Purity test date: 98.3%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at +4°C, protected from light and under nitrogen gas
- Stability under storage conditions: not specified
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: The test item dosage forms were prepared extemporaneously under nitrogen atmosphere and were stored at room temperature, protected from light (using an aluminium foil) and under nitrogen atmosphere until treatment, for a maximum period of 4 hours according to stability results obtained in CIT/Study No. 26976 AHS.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in the vehicle at a concentration of 50 mg/mL for the preliminary toxicity test and both mutagenicity experiments.
- Preliminary purification step (if any): no

Target gene:

Each strain derived from Salmonella typhimurium LT 2 contains one mutation in the histidine operon, resulting in a requirement for histidine.
In addition, to increase their sensitivity to mutagenic items, further mutations have been added:
• the rfa mutation causes partial loss of the lipopolysaccharide barrier that coats the surface of the bacteria and increases permeability to large molecules that do not penetrate the normal bacteria cell wall,
• the uvrB mutation is a deletion of a gene coding for the DNA excision repair system, which renders the bacteria unable to use this repair mechanism to remove the damaged DNA,
• the addition of the plasmid pKM 101 to strains TA 98, TA 100 and TA 102 enhances their sensitivity of detection to some mutagens,
• in case of TA 102 strain, the histidine mutation is located on the multicopy plasmid pAQ1.
The TA 1535, TA 100 and TA 102 strains are reverted by base-pair substitution mutagens and the TA 1537 and TA 98 strains by frameshift mutagens. In addition, the TA 102 strain detects oxidative mutagens.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: five strains of Salmonella typhimurium (a): TA 1535, TA 1537, TA 98, TA 100 and TA 102 were supplied by B.N. Ames' Laboratory (University of California, Berkeley, USA)
- Suitability of cells: They are stored in a cryoprotective medium (1 mL nutrient broth and 0.09 mL dimethylsulfoxide) in liquid nitrogen.
- Normal cell cycle time (negative control): yes

Cultures : the day before treatment, cultures will be inoculated from frozen permanents: a scrape will be taken under sterile conditions and put into approximately 6 mL of nutrient broth. The nutrient broth will then be placed under agittaion in an incubator at 37°C for about 14 hours, to produce bacterial suspensions.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : The S9 mix consists of induced enzymatic systems contained in rat liver post-mitochondrial fraction (S9 fraction) and the cofactors necessary for their function. S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route. Each batch of S9 is tested and validated by Moltox for its ability to activate benzo(a)pyrene and 2-anthramine (also known as 2-amino anthracene) to mutagenic intermediates.
The S9 fraction was preserved in sterile tubes at -80°C, until use.
- method of preparation of S9 mix: The S9 mix was prepared at + 4°C immediately before use and maintained at this temperature until added to the overlay agar.
- concentration or volume of S9 mix and S9 in the final culture medium : 38.5 mg/mL
The composition of S9 mix was as follows:
Ingredient
Glucose-6-phosphate: 5 mM
NADP: 4 mM
KCl 33: mM
MgCl2: 8 mM
Sodium phosphate buffer pH 7.4: 100 mM
S9 fraction, batch No. 1727, protein concentrations: 38.5 mg/mL 10% (v/v)
water: to volume

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: The test item was freely soluble in the vehicle (DMSO) at 50 mg/mL.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-anthramine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 3: a preliminary test and 2 independent experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk : The preliminary test, all experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the preincubation method.
The direct plate incorporation method was performed as follows: test item solution (0.1 mL), S9 mix when required or phosphate buffer pH 7.4 (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: The preincubation method was performed as follows: test item solution (0.1 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C, under shaking, before adding the overlay agar and pouring onto the surface of a minimum agar plate.
After 48 to 72 hours of incubation at 37°C, revertants were scored with an automatic counter.
- Exposure duration/duration of treatment: after 48 to 72 h of incubation at 37°C, revertants will be scored with an automatic counter. Manual counting may be performed as needed.

Rationale for test conditions:

Treatment of results
In each experiment, for each strain and for each experimental point, the number of revertants per plate was scored. The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test item/mutants obtained in the presence of the vehicle), are presented in tabular form.
Acceptance criteria
This study is considered valid if the following criteria are fully met:
• the number of revertants in the vehicle controls is consistent with the historical data of the testing facility (appendix 2),
• the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with the historical data of the testing facility.

Evaluation criteria:

A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

not determined

Remarks:

slight increase in the number of revertants but within the range of historical control and without reproducibility. coloration of agar observed

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

not determined

Remarks:

slight increase in the number of revertants but within the range of historical control and without reproducibility. coloration of the agar observed

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

dose-related and reproducible increase in the number of revertant colonies (up to 7.3-fold the vehicle control mean value).coloration of the agar observed

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

coloration of the agar observed

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

coloration of the agar observed

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

coloration of the agar observed

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The direct plate incorporation method was performed as follows: test item solution (0.1 mL), S9 mix when required or phosphate buffer pH 7.4 (0.5 mL)

RANGE-FINDING/SCREENING STUDIES (if applicable): To assess the toxicity of the test item to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA 98, TA 100 and TA 102 strains, with and without S9 mix. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test item was freely soluble in the vehicle (DMSO) at 50 mg/mL.
Consequently, with a treatment volume of 100 μL/plate, the dose-levels were 10, 100, 500, 1000, 2500 and 5000 μg/plate.
A coloration of agar was noted in the Petri plates when scoring the revertants at dose-levels ≥ 2500 μg/plate.
A moderate precipitate was sometimes observed in the Petri plates when scoring the revertants at 5000 μg/plate.
No noteworthy toxicity was observed at any dose-level.

STUDY RESULTS
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria.
The study was therefore considered valid.
Since the test item was freely soluble and non toxic, the highest dose-level selected for the main test was 5000 μg/plate, according to the criteria specified in the international guidelines.
The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000 μg/plate, for all the strains in both experiments.
No precipitate was observed in the Petri plates when scoring the revertants at any dose-level. Except for a marked toxicity noted in the TA 98 strain without S9 mix at 5000 μg/plate, no toxicity was observed towards all the strains used, with and without S9 mix.
Some slight increases in the number of revertants were noted in the TA 1535 and TA 98 strains without S9 mix. However, since the numbers of revertants remained within the range of our historical control values and since these slight increases were not reproducible, they were considered not to be biologically relevant.
A dose-related and reproducible increase in the number of revertant colonies (up to 7.3-fold the vehicle control mean value) was observed in the TA 98 strain, with S9 mix.

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures
o Number of cells plated in selective and non-selective medium
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency
o When using the thymidine kinase gene on L5178Y cells: colony sizing for the negative and positive controls and if the test chemical is positive, and related mutant frequency. For the MLA, the GEF evaluation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:

Table 1: Preliminary toxicity test Direct plate incorporation method

 

Revertant colony numbers per plate using strains TA 98, TA 100, TA 102

 

Strain	Compound	Dose level per plate	S9 mix	Mean revertant colony counts	SD	Ratio treated/solvent	Individual revertant colony counts
TA 98	DMSO		-	22	-		22
	test item	10 µg	-	25	-	1.1	25
		100 µg	-	29	-	1.3	29
		500 µg	-	26	-	1.2	26
		1000 µg	-	23	-	1.0	23
		2500 µg	-	25	-	1.1	25
		5000 µg	-	38	-	1.7	38Mp+ Co
	DMSO		+	43	-		43
	test item	10 µg	+	24	-	0.6	24
		100 µg	+	46	-	1.1	46
		500 µg	+	103	-	2.4	103
		1000 µg	+	145	-	3.4	145
		2500 µg	+	320	-	7.4	320 Co
		5000 µg	+	232	-	5.4	232Mp+ Co
TA 100	DMSO		-	168	-		168
	test item	10 µg	-	132	-	0.8	132
		100 µg	-	174	-	1.0	174
		500 µg	-	166	-	1.0	166
		1000 µg	-	165	-	1.0	165
		2500 µg	-	183	-	1.1	183
		5000 µg	-	254	-	1.5	254 Co +Mp
	DMSO		+	159	-		159
	test item	10 µg	+	102	-	0.6	102
		100 µg	+	138	-	0.9	138
		500 µg	+	152	-	1.0	152
		1000 µg	+	210	-	1.3	210
		2500 µg	+	205	-	1.3	205
		5000 µg	+	181	-	1.1	181 Co
TA 102	DMSO		-	440	-		440
	test item	10 µg	-	432	-	1.0	432
		100 µg	-	405	-	0.9	405
		500 µg	-	486	-	1.1	486
		1000 µg	-	561	-	1.3	561
		2500 µg	-	496	-	1.1	496
		5000 µg	-	396	-	0.9	396Mp+Co
	DMSO		+	333	-		333
	test item	10 µg	+	564	-	1.7	564
		100 µg	+	254	-	0.8	254
		500 µg	+	321	-	1.0	321
		1000 µg	+	263	-	0.8	263
		2500 µg	+	325	-	1.0	325
		5000 µg	+	274	-	0.8	274Mp+ Co

 

Table 2: First experiment

Direct plate incorporation method

 

Revertant colony numbers per plate using strains TA 1535, TA 1537, TA 98, TA 100, TA 102

 

Strain	Compound	Dose level per plate	S9 mix	Mean revertant colony counts	SD	Ratio treated/solvent	Individual revertant colony counts
TA 1535		DMSO				-		13		3				11,17,12
		test item		312.5µg		-		17		8		1.3		11,26,14
			625 µg		-		19		1		1.4		19,18,20
			1250 µg		-		29		10		2.2		19,30,38
	2500 µg		-	45	3	3.4	47Co,46Co,41Co
	5000 µg		-	34	2	2.6	35Co,36Co,32Co
	NAN3	1 µg	-	649	39	48.7	683,658,606
	DMSO		+	14	4		10,14,17
	test item	312.5µg	+	7	1	0.5	8,7,6
		625 µg	+	15	3	1.1	18,14,12
		1250 µg	+	18	5	1.3	13,23,17
		2500 µg	+	25	7	1.8	31,18,26
		5000 µg	+	17	3	1.3	19Co,19Co,14Co
	2AM	2 µg	+	285	21	20.8	267,279,308
TA 1537	DMSO		-	7	4		11,7,4
	test item	312.5µg	-	6	1	0.9	7,5,7
		625 µg	-	5	3	0.7	8,5,2
		1250 µg	-	9	3	1.2	12,7,8
		2500 µg	-	7	4	0.9	2Co, 10Co, 8Co
		5000 µg	-	6	1	0.8	5Co,7 Co, 5Co
	9AA	50 µg	-	602	305	82.1	303,913,590
	DMSO		+	10	3		8,13,8
	test item	312.5µg	+	13	5	1.3	10,10,19
		625 µg	+	14	2	1.4	12,16,13
		1250 µg	+	14	3	1.4	11,17,14
		2500 µg	+	15	2	1.6	13,17,16
		5000 µg	+	8	2	0.8	7Co,6Co,10Co
	2AM	2 µg	+	102	10	10.5	91,110,104
TA 98	DMSO		-	17	6		24,13,14
	test item	312.5µg	-	20	0	1.2	20,20,20
		625 µg	-	15	3	0.9	16,12,18
		1250 µg	-	26	7	1.5	29,31,18
		2500 µg	-	23	7	1.3	31Co,17Co,20Co
		5000 µg	-	2	2	0.1	0Co+St, 4Co+St, 2Co+St
	2NF	0.5 µg	-	346	1	20.4	347,345,346
	DMSO		+	27	4		24,32,26
	test item	312.5µg	+	57	5	2.1	52,57,62
		625 µg	+	90	2	3.3	89,92,90
		1250 µg	+	97	11	3.5	108,86,97
		2500 µg	+	200	28	7.3	231,192,176
		5000 µg	+	92	44	3.4	69Co, 65Co,143Co
	2AM	2 µg	+	926	114	33.9	808,1036,934
TA 100	DMSO		-	131	8		133,137,122
	test item	312.5µg	-	146	11	1.1	134,153,152
		625 µg	-	148	16	1.1	129,159,155
		1250 µg	-	159	23	1.2	149,143,186
		2500 µg	-	160	6	1.2	159Co,166Co,155Co
		5000 µg	-	145	20	1.1	133Co,133Co,168Co
	NAN3	1µg	-	751	46	5.7	726,723,804
	DMSO		+	105	8		98,104,113
	test item	312.5µg	+	117	19	1.1	95,129,126
		625 µg	+	115	22	1.1	104,141,101
		1250 µg	+	113	8	1.1	110,108,122
		2500 µg	+	96	13	0.9	84,95,109
		5000 µg	+	103	21	1.0	79Co,114Co,116Co
	2AM	2 µg	+	490	38	4.7	460,533,477
TA 102	DMSO		-	367	26		387,337,376
	test item	312.5µg	-	360	4	1.0	362,355,363
		625 µg	-	346	58	0.9	304,322,412
		1250 µg	-	376	53	1.0	426,321,382
		2500 µg	-	341	33	0.9	364Co,355Co,303Co
		5000 µg	-	354	21	1.0	377Co,349Co, 337Co
	MMC	0.5 µg	-	2106	109	5.7	2149,2188,1982
	DMSO		+	350	61		383,387,279
	test item	312.5µg	+	437	87	1.3	485,490,337
		625 µg	+	393	65	1.1	449,407,322
		1250 µg	+	468	62	1.3	418,448,537
		2500 µg	+	405	23	1.2	405,383,428
		5000 µg	+	402	15	1.1	418Co,399Co,388Co
	2AM	10 µg	+	1642	267	4.7	1910,1640,1376

 

SD: Standard deviation

-       : Absence of S9

 + :Presence of S9

Co :coloration of agar

St :strong toxicity

 

 

Table 3: Second experiment

Direct plate incorporation method (without S9 mix) and preincubation method (with S9 mix)

 

Revertant colony numbers per plate using strains TA 1535, TA 1537, TA 98, TA 100, TA 102

 

Strain	Compound	Dose level per plate	S9 mix	Mean revertant colony counts	SD	Ratio treated/solvent	Individual revertant colony counts
TA 1535	DMSO		-	15	1		14,16,16
	test item	312.5µg	-	17	4	1.1	22,14,16
		625 µg	-	11	6	0.7	11,17,5
		1250 µg	-	18	3	1.2	20,20,14
		2500 µg	-	34	4	2.2	34Co,30Co,37Co
		5000 µg	-	32	10	2.1	42Co,22Co,31Co
	NAN3	1 µg	-	463	12	30.2	450,466,473
	DMSO		+	14	3		10,16,16
	test item	312.5µg	+	11	3	0.8	7,13,13
		625 µg	+	14	3	1.0	12,12,17
		1250 µg	+	18	5	1.3	12,22,19
		2500 µg	+	22	8	1.6	14,24,29
		5000 µg	+	39	9	2.8	40Co,47Co,30Co
	2AM	2 µg	+	153	17	10.9	171,138,150
TA 1537	DMSO		-	6	2		5,5,8
	test item	312.5µg	-	5	4	0.8	8,1,5
		625 µg	-	7	4	1.2	6,4,11
		1250 µg	-	6	1	1.1	6,6,7
		2500 µg	-	7	3	1.2	5Co,6Co,11Co
		5000 µg	-	12	6	1.9	5Co,17Co,13Co
	9AA	50 µg	-	476	123	79.4	511,578,340
	DMSO		+	7	3		10,4,6
	test item	312.5µg	+	12	6	1.8	6,17,13
		625 µg	+	10	3	1.3	7,12,12
		1250 µg	+	7	1	1.1	7,8,7
		2500 µg	+	13	7	2.0	7,13,20
		5000 µg	+	13	5	1.9	14Co,17Co,7Co
	2AM	2 µg	+	121	10	18.2	114,133,117
TA 98	DMSO		-	10	6		5,7,17
	test item	312.5µg	-	8	4	0.9	12,8,5
		625 µg	-	12	5	1.2	11,7,17
		1250 µg	-	20	5	2.1	25,19,16
		2500 µg	-	19	5	2.0	14Co,24Co,19Co
		5000 µg	-	6	3	0.6	2Co+St, 7Co+St,8Co+St
	2NF	0.5 µg	-	274	34	28.4	281,237,305
	DMSO		+	23	3		25,24,20
	test item	312.5µg	+	43	11	1.9	31,46,52
		625 µg	+	62	2	2.7	60,63,63
		1250 µg	+	130	23	5.6	145,141,103
		2500 µg	+	124	19	5.4	103,128,140
		5000 µg	+	76	7	3.3	73Co,71Co,84Co
	2AM	2 µg	+	1750	76.1	76.1	1801,1880,1568
TA 100	DMSO		-	159	19		172,168,138
	test item	312.5µg	-	165	11	1.0	162,177,156
		625 µg	-	165	6	1.0	164,172,160
		1250 µg	-	160	12	1.0	163,170,146
		2500 µg	-	221	12	1.4	223Co,232Co,208Co
		5000 µg	-	182	10	1.1	170Co, 187Co,189Co
	NAN3	1µg	-	430	11	2.7	419,440,432
	DMSO		+	187	28		171,171,219
	test item	312.5µg	+	146	5	0.8	150,147,140
		625 µg	+	163	36	0.9	121,187,181
		1250 µg	+	157	27	0.8	186,133,153
		2500 µg	+	183	7	1.0	176,184,190
		5000 µg	+	189	38	1.0	152Co,187Co,228Co
	2AM	2 µg	+	1028	102	5.5	1146,963,976
TA 102	DMSO		-	352	30		320,355,380
	test item	312.5µg	-	289	31	0.8	317,255,295
		625 µg	-	287	69	0.8	222,281,359
		1250 µg	-	268	13	0.8	283,263,259
		2500 µg	-	298	20	0.8	309Co;311Co,275Co
		5000 µg	-	239	18	0.7	218Co,252Co,247Co
	MMC	0.5 µg	-	1166	102	3.3	1260,1058,1180
	DMSO		+	352	36		371,375,310
	test item	312.5µg	+	340	17	1.0	326,335,359
		625 µg	+	372	19	1.1	387,378,350
		1250 µg	+	337	43	1.0	328,384,299
		2500 µg	+	522	34	1.5	561,502,502
		5000 µg	+	423	49	1.2	393Co,396Co,480Co
	2AM	10 µg	+	1858	3	5.3	1860,1860,1854

 

SD: Standard deviation

-       : Absence of S9

 + :Presence of S9

Co :coloration of agar

St :strong toxicity

 

 

Conclusions:

Under our experimental conditions, the test item Hydroxybenzomorpholine (A025) (batch No. 0508918) showed mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium TA 98 strain, with metabolic activation (S9 mix).

Executive summary:

Hydroxybenzomorpholine was investigated for the induction of gene mutations in Salmonella typhimurium (Ames test). Liver S9 fraction from Aroclor 1254-induced rats was used as exogenous metabolic activation system. Test concentrations were based on the level of toxicity in a preliminary toxicity test with strains TA98, TA100 and TA102. Toxicity was evaluated on the basis of a reduction in the number of revertant colonies and/or thinning of the bacterial background lawn. Since hydroxybenzomorpholine was freely soluble and non toxic in this preliminary toxicity test, it was tested up to the prescribed maximum concentration of 5000

μg/plate. The preliminary toxicity test, experiment 1 and experiment 2 without S9 were performed with the direct plate incorporation method, experiment 2 with S9 according the preincubation method. Negative and positive controls were in accordance with the OECD guideline.

Results

Precipitation of hydroxybenzomorpholine was not observed. Marked toxicity was only seen in the TA98 strain without S9 at 5000 μg/plate. Toxicity was not noted towards all the other strains used without or with S9. A coloration of agar was noted at dose levels of 2500 μg/plate and above without S9 and at 5000 μg/plate with S9.

In experiment 1 without S9 a slight increase in the number of revertants was seen in the TA1535 strain. This increase was considered not biologically relevant since the number of revertants remained within the range of the historical control and the increase could not be confirmed in the second experiment.

A more or less dose related and reproducible increase was found in the number of revertants in strain TA 98 with S9.

Conclusion

Under the experimental conditions used hydroxybenzomorpholine was genotoxic (mutagenic) in the gene mutation tests in bacteria in strain TA98 in the presence of S9 metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1983

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Op.90
- Expiration date of the lot/batch: 12 April 1990
- Purity test date: >99.9%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under storage conditions: room temperature
- Stability under test conditions: soluble in DMSO and good stability in DMSO
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Imexine OV was dissolved in DMSO immediately before use at a concentration of 5000 µg/mL when dosed at 1% v/v into tissue culture medium. 5000 µg/mL is the maximum advisable concentration used in this test system due to the suggested artefacual increases in chromosomal aberrations associated with high ionic strenghts and osmolality

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: strain K1-BH4 obtained form BIBRA and stored in polypropylene ampoules at -196 °C in 90% foetal calf serum and 10% dimethylsulphoxide
- Normal cell cycle time (negative control): the cells were routinely grown and subcultured in Hams F12 medium (Imperial) supplemented with 5% foetal calf serum (Gibco) at 37°C in a humid atmosphere containing 5% carbon dioxide in 175 cm² plastic tissue culture flasks

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Rat Sprague Dawley (11 males, 6-8 weeks, 170-220g), albino outbred. Olac Limited, shaws farm, Blackthorn, Bicester , Oxon
- method of preparation of S9 mix : mixed function oxidase systems in the rat liver were stimulated following a single i/p injection of Aroclor 1254 (diluted in Arachis oil to 200 mg/mL) at a dosage of 500 mg/kg. On the fifth day of induction, following an overnight starvation, the rats were killed and their livers asepticaly removed.
Preparation of homogenate S-9: All steps were at 0-4°C using sterile solutions and glassware. The livers were placed in beakers containing 0.15 M KCl. After weighing, livers were transferred to a beaker containing 3 mL of 0.15 M KCl per 1g of liver and were homogenised in a Janke and Kunkel "Ultra-Turrax" homogeniser. This homogenate was centrifuged for 10 minutes at 9000 x "g" and the supernatant divided into aliquots. These were stored at -80°C and tested before use, with the carcinogen, 7, 12-dimethylbenzen-(a)anthracene.
Each mL S9 mix contained:
- S-9 fraction: 0.1 mL
- 0.4 M Mg Cl2: 0.02 mL
- 0.2 M Na2HPO4: (pH 7.4): 0.5 mL
- 1.0 M glucose-6-phosphate: 0.005 mL
- 1.0 M NADP: 0.04 mL
- distilled water: 0.335 mL
All the above solutions were mixed and then filter-sterilised (apart from the S9 fraction which was added after filter-sterilisation of the other S9- mix components)

Test concentrations with justification for top dose:

In the dose range finding study, doses tested 250, 500, 1000, 2000, 5000 mg/kg. 400 mg/kg was selected as the maximum tolerated dose level for use in the micronucleus study

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: arachis oil B.P. Safepharm Co/374.
- description: clear, straw-coloured oily liquid
- container: plastic screw-top bottle
- storage conditions: room temperature
- Justification for choice of solvent/vehicle: no data
- Justification for percentage of solvent in the final culture medium: no data
The identification and stability of the vehicle control were not determined

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

ANIMALS AND ANIMAL HUSBRANDY
Sufficient male and female albino CD1 strain mice were supplied by Charles River (UK) Ltd., Manston, Kent. At the start of the main study the males weighed 23-
30 g, and the females 21-28g, and were approximately five to eight weeks old. After a minimum acclimatisation period of five days the animals were selected at random and given a unique number within the study by ear punching and a number written on a colour coded cage card.
The animals housed in groups of up to five by sex in sold-floor polypropylene cages with sawdust bedding. With the exception of a 2-4 hour fast immediately before dosing and for approximately two hours after dosing, free access to mains drinking water and food (Rat and Mouse Expanded Diet N°1, Special Diet Services Limited, Witham, Essex, U.K.) was allowed throughout the study.
The animal room was maintained at a temperature of 17-21°C and relative humidity of 30-38%. The rate of air exchange was approximately 15 changes per hour and the lighting was controlled by a time switch to give 12 hours light and 12 hours darkness.

RANGE-FINDING TOXICITY STUDY
Range-finding studies were performed to determine a suitable dose level for the micronucleus study. The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of cytotoxicity ou to a maximum recommended dose of 5000 mg/kg.

MICRONUCLEUS STUDY
All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable.

- slide preparation:
immediately following sacrifice (i.e. 24, 48 or 72 hours following dosing), one femur was dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, and stained in May-Grünwald/Giemsa.

- evaluation of slides:
Stained bone marrow smears were examined at random using light microscopy at x 1000 magnification. The incidence of micronucleated cells per 1000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally x circular in shape, although occasionally they may be oval of half-moon shaped, and have a sharp contour with even sytaining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 polychromatic erythrocytes were counted; these cells were also scored for incidence of micronuclei.
The ratio of normochromatic to polychrmatic erythrocytes was calculated together with appropriate group mean values for males and females separately and combined.

Evaluation criteria:

A comparison was made between the number of micronucleated polychromatic erthrocytes occuring in each of the three test material groups and the number occuring in the corresponding vehicle control groups.
A positive mutagenic response is demonstrated when a statistically significant increase in the number of micronucleated polychromatic erthrocytes is observed for either the 24, 48 or 72 - hour kill times. A positive response for bone marrow toxicity is demonstrated when the dose group mean normochromatic to polchromatic ratio is shown to be statstical significat from the concurrent vehicle control group.
If the above criteria are not demonstrated, then the test material is condidered to be non-mutagenic under the conditions of the test.

Statistics:

if necessary, and where possible, all data were statistically analysed using appropriate statistical methods as recommended by the UKEMS sub-committee on guidelines for mutagenicity testing, report part III (1989).

Key result

Species / strain:

other: mouse bone marrow cells

Remarks:

erythrocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

400 mg/kg

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH:
- Data on osmolality:
- Possibility of evaporation from medium:
- Water solubility:
- Precipitation and time of the determination:
- Definition of acceptable cells for analysis:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES (if applicable):

STUDY RESULTS
- Concurrent vehicle negative and positive control data

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any
- Any other criteria: e.g. GEF for MLA

Ames test:
- Signs of toxicity
- Individual plate counts
- Mean number of revertant colonies per plate and standard deviation

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For lymphocytres in primary cultures: mitotic index (MI)
o For cell lines: relative population doubling (RPD), relative Increase in cell count (RICC), number of cells treated and cells harvested for each culture, information on cell cycle length, doubling time or proliferation index.
- Genotoxicity results (for both cell lines and lymphocytes)
o Definition for chromosome aberrations, including gaps
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excludling gaps
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes) if seen

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
o In the case of the cytokinesis-block method: CBPI or RI; distribution of mono-, bi- and multi-nucleated cells
o When cytokinesis block is not used: RICC, RPD or PD, as well as the number of cells treated and of cells harvested for each culture
o Other observations when applicable (complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells)

- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture and defining whether from binucleated or mononucleated cells, where appropriate

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures
o Number of cells plated in selective and non-selective medium
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency
o When using the thymidine kinase gene on L5178Y cells: colony sizing for the negative and positive controls and if the test chemical is positive, and related mutant frequency. For the MLA, the GEF evaluation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:

Range-finding toxicity study

DOSE LEVEL mg/kg	SEX	NUMBER OF ANIMALS TREATED	DEATHS ON DAY 0	DEATHS ON DAY 1	DEATHS ON DAY 2	DEATHS ON DAY 3	TOTAL DEATHS
5000	MALE	2	2	-	-	-	4/4
	FEMALE	2	1	1	-	-
2000	MALE	2	1	0	0	0	2/4
	FEMALE	2	1	0	0	0
1000	MALE	2	0	0	0	0	2/4
	FEMALE	2	2	-	-	-
500	MALE	7	0	0	0	0	1/14
	FEMALE	7	0	0	1	0
250	MALE	2	0	0	0	0	0/4
	FEMALE	2	0	0	0	0

 

-       Clinical signs: were observed in all of theImexineOV treatment groups, with the exception of the 250 mg/kg group, the number of different signs observed and their persistence was clearly dose-related. He clinical signs observed included: hunched posture, ataxia, lethargy, ptosis, laboured respiration, loss of righting reflex, occasional bod tremors and stains around the eyes. 400 mg/kg was selected as the maximum tolerated dose level for use in the micronucleus study.

 

-       Mortality: there was only one premature death in the groups of animals dosed withImexineOV, this animal was from the 48-hour group females. Many of the animals dosed withImexineOV showed lethargic signs one hour after dosing, and ptosis in some animals at 24, 48 and 72 hours post dosing.

 

-       Evaluation of bone marrow slides:

A summary of the results of the micronucleus study is given in Table 1.

Individual and group mean data are presented in Tables 2 to 8.

 There was no significant increase in the frequency ofmicronucleatedPCE’s in any of the test material dose groups when compared to their concurrent vehicle control group.

The positive control group showed a marked increase in the incidence ofmicronucleatedpolychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

The test material,ImexineOV, was found not to produce micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

Table 1: summary of groupmeandata (males and females combined)

TREATMENT GROUP	NUMLBER OF PCE WITH MICRONUCLEI PER 1000 PCE		NUMLBER OF PCE WITH MICRONUCLEI PER 1000 PCE		NCE/PCE RATIO
	GROUP MEAN	SD	GROUP MEAN	SD	GROUP MEAN	SD
VEHICLE CONROL 72-hour sampling time	0.9	1.0	0.4	1.1	0.78	0.23
VEHICLE CONROL 48-hour sampling time	0.5	0.7	0.4	1.1	0.92	0.26
VEHICLE CONROL 24-hour sampling time	0.3	0.5	0.2	0.5	0.91	0.17
IMEXINE OV 400 mg/kg72 hoursampling time	1.6	1.7	0.5	0.8	1.74**	0.79
IMEXINE OV 400 mg/kg48 hoursampling time	0.4	0.7	0.2	0.5	0.95	0.19
IMEXINE OV 400 mg/kg24 hoursampling time	0.9	1.0	0.4	1.2	0.71	0.23
POSITIVE CONTROL24 hoursampling time	18.9	11.3	1.2	1.3	1.17	0.36

 

PCE = polychromatic erythrocytes

NCE =normochromaticerythrocytes

SD = standard deviation

** = significantly different from concurrent vehicle control group (t-test; p<0.01)

 Table2 :individual and group means and standard deviations

IMEXINE OV

 - doselevel :0 mg/kg

- sample time 72 hours

ANIMAL NUMBER AND SEX	TOT CELLS SCORED (PCE+NCE)	POLYCHROMATIC ERTHROCYTES (PCE)		NORMOCHROMATIC ERYTHROCYTES (NCE)			NCE PCE RATION
		SCORED	+veMN	SCORED	+veMN	/1000 NCE
1M	1804	1000	2.0	804	0.0	0.0	0.80
2M	2041	1000	2.0	1041	0.0	0.0	1.04
3M	1932	1000	0.0	932	0.0	0.0	0.93
4M	1681	1000	2.0	681	0.0	0.0	0.68
5M	1552	1000	0.0	552	0.0	0.0	0.55
male
Mean	1802	1000	1.2	802	0.0	0.0	0.80
SD	194	0	1.1	194	0.0	0.0	0.19
6F	1571	1000	1.0	571	2.0	3.5	0.57
7F	1930	1000	0.0	931	0.0	0.0	0.93
8F	1423	1000	0.0	423	0.0	0.0	0.42
9F	1716	1000	0.0	716	0.0	0.0	0.72
10F	2154	1000	2.0	1154	0.0	0.0	1.15
female
Mean	1759	1000	0.6	759	0.4	0.7	0.76
SD	290	0	0.9	290	0.9	1.6	0.29
group
Mean	1781	1000	0.9	781	0.2	0.4	0.78
SD	234	0	1.0	234	0.6	1.1	0.23

SD :standard deviation

Table3 :individual and group means and standard deviations

IMEXINE OV

 - doselevel :0 mg/kg

- sample time 48 hours

ANIMAL NUMBER AND SEX	TOT CELLS SCORED (PCE+NCE)	POLYCHROMATIC ERTHROCYTES (PCE)		NORMOCHROMATIC ERYTHROCYTES (NCE)			NCE PCE RATION
		SCORED	+veMN	SCORED	+veMN	/1000 NCE
11M	2491	1000	1.0	1491	1.0	0.7	1.49
12M	1718	1000	0.0	718	0.0	0.0	0.72
13M	2087	1000	2.0	1087	0.0	0.0	1.09
14M	1981	1000	1.0	981	0.0	0.0	0.98
15M	1858	1000	1.0	858	3.0	3.5	0.86
male
Mean	2027	1000	1.0	1027	0.8	0.8	1.03
SD	294	0	0.7	294	1.3	1.5	0.29
16F	1508	1000	0.0	508	0.0	0.0	0.51
17F	1807	1000	0.0	807	0.0	0.0	0.81
18F	1946	1000	0.0	946	0.0	0.0	0.95
19F	1878	1000	0.0	878	0.0	0.0	0.88
20F	1917	1000	0.0	917	0.0	0.0	0.92
female
Mean	1811	1000	0.0	811	0.0	0.0	0.81
SD	177	0	0.0	177	0.0	0.0	0.18
group
Mean	1919	1000	0.5	919	0.4	0.4	0.92
SD	255	0	0.7	255	1.0	1.0	0.26

SD :standard deviation

 

Table4 :individual and group means and standard deviations

IMEXINE OV

 - doselevel :0 mg/kg

- sample time 24 hours

ANIMAL NUMBER AND SEX	TOT CELLS SCORED (PCE+NCE)	POLYCHROMATIC ERTHROCYTES (PCE)		NORMOCHROMATIC ERYTHROCYTES (NCE)			NCE PCE RATION
		SCORED	+veMN	SCORED	+veMN	/1000 NCE
21M	1865	1000	1.0	864	0.0	0.0	0.86
22M	2087	1000	0.0	1087	0.0	0.0	1.09
23M	1708	1000	0.0	708	0.0	0.0	0.71
24M	1670	1000	1.0	670	1.0	1.5	0.67
25M	1889	1000	0.0	889	0.0	0.0	0.89
male
Mean	1844	1000	0.4	844	0.2	0.3	0.84
SD	166	0	0.5	166	0.4	0.7	0.17
26F	1934	1000	0.0	934	1.0	1.1	0.93
27F	1985	1000	0.0	985	0.0	0.0	0.99
28F	2002	1000	0.0	1002	0.0	0.0	1.00
29F	2203	1000	1.0	1203	0.0	0.0	1.20
30F	1732	1000	0.0	732	0.0	0.0	0.73
female
Mean	1971	1000	0.2	971	0.2	0.2	0.97
SD	168	0	0.4	168	0.4	0.5	0.17
group
Mean	1907	1000	0.3	907	0.2	0.3	0.91
SD	171	0	0.5	171	0.4	0.5	0.17

SD :standard deviation

Table5 :individual and group means and standard deviations

IMEXINE OV

 - doselevel :400 mg/kg

- sample time 72 hours

ANIMAL NUMBER AND SEX	TOT CELLS SCORED (PCE+NCE)	POLYCHROMATIC ERTHROCYTES (PCE)		NORMOCHROMATIC ERYTHROCYTES (NCE)			NCE PCE RATION
		SCORED	+veMN	SCORED	+veMN	/1000 NCE
31M	2568	1000	0.0	1568	1.0	0.6	1.57
32M	4127	1000	2.0	3127	1.0	0.3	3.13
33M	1999	1000	0.0	999	0.0	0.0	1.00
34M	2088	1000	0.0	1088	0.0	0.0	1.09
35M	2518	1000	3.0	1518	1.0	0.7	1.52
male
Mean	2660	1000	1.0	1660	0.6	0.3	1.66
SD	858	0	1.4	858	0.5	0.3	0.86
36F	4123	1000	2.0	3123	1.0	0.3	3.12
37F	2441	1000	5.0	1441	4.0	2.8	1.44
38F	2420	1000	3.0	1420	1.0	0.7	1.42
39F	2857	1000	0.0	1857	0.0	0.0	1.86
40F	2302	1000	1.0	1302	0.0	0.0	1.30
female
Mean	2829	1000	2.2	1829	1.2	0.8	1.83
SD	753	0	1.9	753	1.6	1.2	0.75
group
Mean	2744	1000	1.6	1744	0.9	0.5	1.74
SD	766	0	1.7	766	1.2	0.8	0.77

SD :standard deviation

Table6 :individual and group means and standard deviations

IMEXINE OV

 - doselevel :400 mg/kg

- sample time 48 hours

ANIMAL NUMBER AND SEX	TOT CELLS SCORED (PCE+NCE)	POLYCHROMATIC ERTHROCYTES (PCE)		NORMOCHROMATIC ERYTHROCYTES (NCE)			NCE PCE RATION
		SCORED	+veMN	SCORED	+veMN	/1000 NCE
41M	1954	1000	0.0	954	0.0	0.0	0.95
42M	2174	1000	0.0	1174	0.0	0.0	1.17
43M	1782	1000	1.0	782	0.0	0.0	0.78
44M	1728	1000	0.0	728	1.0	1.4	0.73
45M	1895	1000	1.0	895	0.0	0.0	0.9
male
Mean	1907	1000	0.4	907	0.2	0.3	0.91
SD	174	0	0.5	174	0.4	0.6	0.17
46F	1951	1000	0.0	951	0.0	0.0	0.95
47F	-	-	-	-	-	-	-
48F	1974	1000	2.0	974	0.0	0.0	0.97
49F	2317	1000	0.0	1317	0.0	0.0	1.32
50F	1811	1000	0.0	811	0.0	0.0	0.81
female
Mean	2013	1000	0.5	1013	0.0	0.0	1.01
SD	215	0	1.0	215	0.0	0.0	0.21
group
Mean	1954	1000	0.4	954	0.1	0.2	0.95
SD	189	0	0.7	189	0.3	0.5	0.19

SD :standard deviation

Table7 :individual and group means and standard deviations

IMEXINE OV

 - doselevel :400 mg/kg

- sample time 24 hours

ANIMAL NUMBER AND SEX	TOT CELLS SCORED (PCE+NCE)	POLYCHROMATIC ERTHROCYTES (PCE)		NORMOCHROMATIC ERYTHROCYTES (NCE)			NCE PCE RATION
		SCORED	+veMN	SCORED	+veMN	/1000 NCE
51M	2168	1000	1.0	1168	0.0	0.0	1.17
52M	1381	1000	2.0	381	0.0	0.0	0.38
53M	1890	1000	0.0	890	0.0	0.0	0.89
54M	1675	1000	0.0	675	0.0	0.0	0.68
55M	1815	1000	1.0	815	3.0	3.7	0.82
male
Mean	1786	1000	0.8	786	0.6	0.7	0.79
SD	289	0	0.8	289	1.3	1.6	0.29
56F	1598	1000	1.0	598	0.0	0.0	0.60
57F	1859	1000	3.0	859	0.0	0.0	0.86
58F	1540	1000	0.0	540	0.0	0.0	0.54
59F	1574	1000	0.0	574	0.0	0.0	0.57
60F	1550	1000	1.0	550	0.0	0.0	0.55
female
Mean	1624	1000	1.0	624	0.0	0.0	0.62
SD	133	0	1.2	133	0.0	0.0	0.13
group
Mean	1705	1000	0.9	705	0.3	0.4	0.71
SD	229	0	1.0	229	0.9	1.2	0.23

SD :standard deviation

Table8 :individual and group means and standard deviations

cyclophosphamide

 - doselevel :50 mg/kg

- sample time 24 hours

ANIMAL NUMBER AND SEX	TOT CELLS SCORED (PCE+NCE)	POLYCHROMATIC ERTHROCYTES (PCE)		NORMOCHROMATIC ERYTHROCYTES (NCE)			NCE PCE RATION
		SCORED	+veMN	SCORED	+veMN	/1000 NCE
61M	1954	1000	10.0	954	0.0	0.0	0.95
62M	2456	1000	19.0	1456	1.0	0.7	1.46
63M	1982	1000	29.0	982	0.0	0.0	0.98
64M	1698	1000	41.0	698	3.0	4.3	0.70
65M	1991	1000	32.0	991	2.0	2.0	0.99
male
Mean	2016	1000	26.2	1016	1.2	1.4	1.02
SD	274	0	12.0	274	1.3	1.8	0.27
66F	2642	1000	11.0	1642	2.0	1.2	1.64
67F	2430	1000	13.0	1430	2.0	1.4	1.43
68F	2366	1000	17.0	1368	0.0	0.0	1.37
69F	1646	1000	8.0	646	1.0	1.5	0.65
70F	2504	1000	9.0	1504	2.0	1.3	1.50
female
Mean	2318	1000	11.6	1318	1.4	1.1	1.32
SD	389	0	3.6	389	0.9	0.6	0.39
group
Mean	2167	1000	18.9	1167	1.3	1.2	1.17
SD	355	0	11.3	355	1.1	1.3	0.36

SD :standard deviation

 

 

Conclusions:

Imexine OV was considered to be non-genotoxic under the conditions of the test

Executive summary:

Hydroxybenzomorpholine has been investigated in the absence and presence of metabolic activation for the induction of chromosomal aberrations in CHO cells. From a range of concentrations up to prescribed maximum concentration of 5000 μg/ml, the dose level causing a

decrease in mitotic index of 50% of the solvent control value was used as the highest dose level.

In the absence of S9 cells were treated for 21 h and immediately harvested; in the presence of S9 cells were treated for 4 h and harvested 21 h after the start of treatment. Two hours before harvest, each culture was treated with colchicine solution (final concentration 0.25 μg/ml) to

block cells at metaphase of mitosis. Liver S9 fraction from Aroclor 1254-induced rats was used as exogenous metabolic activation system. Toxicity was determined by measuring the decrease in the mitotic index. Chromosome (metaphase) preparations were stained with 10% Giemsa and examined microscopically for chromosomal aberrations. Negative and positive controls were in accordance with the OECD draft guideline.

Results

Hydroxybenzomorpholine induced sufficient toxicity as the mitotic index after the highest dose was reduced with about 50% compared to the negative control both in the absence or presence of S9.

In both the absence and the presence of a metabolic activation, hydroxybenzomorpholine (Imexine OV) did not cause a biological relevant and dose dependent increase in cells with chromosome aberrations.

Conclusion

Under the experimental conditions used hydroxybenzomorpholine did not show evidence for a genotoxic (clastogenic) activity in CHO cells in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 dec 2004 to 27 June 2005

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

21 July 1997

Deviations:

not specified

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

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: sept 2005
- Purity test date: 98.3%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerated under nitrogen, at approximately 1-10°C in the dark
- Stability & storage under test conditions: Prior to preparation of all test article solutions, the solvent was saturated with nitrogen gas (kept under nitrogen atmosphere for 15 minutes). Formulations of Hydroxybenzomorpholine (A025) at 0.1 to 500 mg/mL in DMSO were stable for up to 4 hours following formulation when stored at room temperature, away from light and under inert gas atmosphere
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Preliminary solubility data indicated that Hydroxybenzomorpholine (A025) was soluble in sterile anhydrous analytical grade dimethyl sulphoxide (DMSO), with the aid of vortex mixing, up to at least 169.6 mg/mL. The solubility limit in tissue culture medium was in the region of 1696 μg/mL. A maximum test dose of 1510 μg/mL (equivalent to 10 mM: molecular weight of 151.16 supplied by Sponsor) was selected for the cytotoxicity range-finding experiment

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test article solutions were prepared by dissolving Hydroxybenzomorpholine (A025) in DMSO (with the aid of vortex mixing) immediately prior to assay to give the required concentration. Further dilutions were made using nitrogen flushed DMSO. The test article solutions were protected from light and used within 2½ hours of initial formulation of the test article



INFORMATION ON NANOMATERIALS
- Chemical Composition:
- Density:
- Particle size & distribution:
- Specific surface area:
- Isoelectric point:
- Dissolution (rate):

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added:
- other information:

Target gene:

only four mammalian cell lines have been sufficiently well validated for use in in vitro mutation assays (mouse lymphoma L5178Y, CHO, V79 and TK6 cells). Similarly, only three genetic loci have been suggested as suitable: hprt (6-thioguanine resistance), tk (trifluorothymidine resistance) and Na+/K+ cell
membrane ATPase (ouabain resistance). The use of cells in suspension culture is preferable because cell numbers are not restricted by problems of metabolic
co-operation and therefore the mouse lymphoma L5178Y system is statistically more sensitive than standard CHO and V79 systems. Further, a fluctuation protocol has been developed for use with mouse lymphoma L5178Y cells which has been well validated. Such a procedure affords a more accurate determination of mutagenic potential than conventional plate assays.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y TK +/- mouse lymphoma cells were stored as frozen stocks in liquid nitrogen, the original cultures were obtained from Dr Donald Clive, Burroughs Wellcome Co
- Suitability of cells: Each batch of frozen cells was purged of TK- mutants, checked for spontaneous mutant frequency and that it was mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified
atmosphere of 5% v/v CO2 in air. When the cells were growing well, subcultures were
established in an appropriate number of flasks.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was prepared from male Sprague Dawley rats, induced with Aroclor 1254. The S-9 was obtained from Molecular Toxicology Incorporated, USA.
The batches of MolToxTM S-9 were stored frozen at –80ºC prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities). The quality control statements relating to the batches of S-9 preparation were provided i the report
- method of preparation of S9 mix : Glucose-6-phosphate (180 mg/mL), NADP (25 mg/mL), 150 mM KCl and rat liver S-9 were mixed in the ratio 1:1:1:2. For all cultures treated in the presence of S-9, a 1 mL aliquot of the mix was added to each cell culture (19 mL) to give a total of 20 mL. Cultures treated in the absence of S-9 received 1 mL 150 mM KCl

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Negative controls comprised treatments with the solvent DMSO diluted 100-fold in the treatment medium.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

GROWTH MEDIA
Three types of RPMI 1640 medium were prepared as follows:
Final concentration in
RPMI A:
- Horse serum (heat inactivated): 0% v/v
- Penicillin: 100 units/mL
- Streptomycin: 100 µg/mL
- Amphotericin B: 2.5 µg/mL
- Pluronic: 0.5 mg/mL

RPMI 10:
- Horse serum (heat inactivated): 10% v/v
- Penicillin: 100 units/mL
- Streptomycin: 100 µg/mL
- Amphotericin B: 2.5 µg/mL
- Pluronic: 0.5 mg/mL

RPMI 20:
- Horse serum (heat inactivated): 20% v/v
- Penicillin: 100 units/mL
- Streptomycin: 100 µg/mL
- Amphotericin B: 2.5 µg/mL
- Pluronic: /

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 2 replicates in 2 independent experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density: After 3 hours rocking at 37ºC, tubes were centrifuged at 200 x 'g' for 5 minutes, the cells washed with tissue culture medium and resuspended further in 20 mL RPMI 10/tube. Cell densities were determined using a Coulter counter and, the concentrations adjusted to 2 x 10E5/mL. Cells were transferred to flasks for growth through the expression period or were diluted to be plated for survival as described: Following adjustment of the cultures to 2 x 10E5 cells/mL after treatment, samples from these were diluted to 8 cells/mL as follows:
- initial cell concentration (A) : 2x10E5 /mL - Dilution A : 1mL, 9.9 medium mL
- Intermediate cell concentration (B) : 2x10E3 /mL - Dilution B: 0.2 mL, 50 medium RPMI 20
- Final cell concentration (C): 8/mL
Using an eight-channel pipette, 0.2 mL of concentration C of each culture was placed into each well of two 96-well microtitre plates (192 wells, at an average of 1.6 cells per well). The plates were incubated at 37ºC in a humidified incubator gassed with 5% v/v CO2 in air until scorable (7 to 8 days). Wells containing viable clones were identified by eye using background illumination and counted.

- Treatment : At least 10E7 cells in a volume of 18.8 mL of RPMI 5 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were placed in each of a series of sterile disposable 50 mL centrifuge tubes. Solvent, test article or positive control solution (0.2 mL) was added. 1 mL of S-9 mix or 150 mM KCl were added for treament with or witout S9 mix.
Each treatment, in the absence or presence of S-9, was performed in duplicate cultures (single cultures only used for positive control treatments).


FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): Cultures were maintained in flasks for a period of 7 days during which the HPRTmutation would be expressed. Sub-culturing was performed as required with the aim of not exceeding 1 x 10E6 cells per mL and, where possible, retaining at least 6 x 10E6 cells/flask. From observations on recovery and growth of the cultures during the expression period, the following cultures were selected to be plated for viability and 6-thioguanine (6TG) resistance (mutation assessment):
Experiment 1 (µg/mL): without S9: 0, 25,50, 100, 150, 200, 250, 300,350, 400, 500. With S9: 0, 50,100,200, 400, 600, 800, 1000, 1250, 1510, positive control NQO without S9: NQO 0.10, 0.15, with S9 BP2, BP3
Experiment 2 (µg/mL): without S9: 0,50, 75, 100, 150, 200, 250 With S9: 0, 50,100,200, 300, 375, 450, positive control NQO without S9: NQO 0.10, 0.15, with S9 BP2, BP3

- 6-thioguanine (6TG) resistance: At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 10E5/mL. Acceptable cultures usually
yield at least 80 mL of cells at this concentration and 6TG (9 x 10-3 M) was diluted 100-fold into these suspensions to give a final concentration of 15 μg/mL.
Using an eight-channel pipette, 0.2 mL of each suspens ion was placed into each well of four 96-well microtitre plates (384 wells at 2 x 104 cells per well). Plates
were incubated at 37ºC in a humidified incubator gassed with 5% v/v CO2 in air until scorable (11 to 12 days) and wells containing clones were identified as above
and counted.

Determination of survival or viability
From the zero term of the Poisson distribution the probable number of clones/well (P) on microtitre plates in which there are EW empty wells (without clones) out of a total
of TW wells is given by:
P = -ln (EW/TW)
The plating efficiency (PE) in any given culture is therefore:
PE = P/No of cells plated per well and as an average of 1.6 cells per well were plated on all survival and viability plates,
PE = P/1.6
The percentage relative survival (%RS) in each test culture was therefore determined by comparing plating efficiencies in test and control cultures thus:
% RS = [PE (test)/PE (control)] x 100
To take into account any loss of cells during the 3 hour treatment period, percentagerelative survival values for each dose of test article were adjusted as follows:

Adjusted %RS = %RS x (Post-treatment cell concentration for dose/ Post-treatment cell concentration for solvent control)
All percentage relative survival (%RS) values were adjusted as described above.

Determination of mutant frequency
It is usual to express mutant frequency (MF) as "mutants per 10E6 viable cells". In order to calculate this, the plating efficiencies of both mutant and viable cells in the
same culture were calculated,
MF = [PE (mutant)/PE (viable)] x 10E6
From the formulae given and with the knowledge that 2 x 10E4 cells were plated/well
for mutation to 6-thioguanine resistance,
PE (mutant) = P (mutant)/2 x 10E4
PE (viable) = P (viable)/1.6
where, in each case, P = -1n (EW/TW)
Therefore,
MF = [P (mutant)/2 x 10E4] x [1.6/P (viable)] x 10E6
= {-ln [EW/TW (mutant)]/-ln [EW/TW (viable)]} x 80

Acceptance criteria
The assay was considered valid if the following criteria were met:
1. the mutant frequencies in the negative (solvent) control cultures fell within the normal range (not more than three times the historical mean value)
2. at least one concentration of each of the positive control chemicals induced a clear increase in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value).

Evaluation criteria:

Evaluation criteria
The test substance was considered to be mutagenic if:
1. the assay was valid
2. the mutant frequency at one or more doses was significantly greater than that of the negative control
3. there was a significant dose-relationship as indicated by the linear trend analysis
4. the effects described above were reproducible.

Statistics:

Assessment of statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. Thus the control log mutant frequency (LMF) was compared with the LMF from each treatment dose, and secondly the data were checked for a linear trend in mutant frequency with treatment dose. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Toxicity
In the cytotoxicity range-finding experiment, six doses of Hydroxybenzomorpholine (A025) were tested in the absence and presence of S-9, separated by two fold intervals
and ranging from 47.19 to 1510 μg/mL (equivalent to 10 mM at the highest dose tested). Extreme or complete toxicity (<10% relative survival) was observed at the highest three doses tested in the absence of S-9 (377.5-1510 μg/mL). The highest doses to give >10% relative survival was 188.8 mg/mL in the absence of S-9 and 1510 mg/mL in the presence of S-9, which yielded 41% and 11% relative survival,
respectively. The raw plate counts and relative survival values are shown in Table 1.

Accordingly, in Experiment 1, ten doses of Hydroxybenzomorpholine (A025), ranging from 25 to 500 μg/mL were tested in the absence of S-9 and nine doses, ranging from 50 to 1510 μg/mL, were tested in the presence of S-9. All doses in both the absence and presence of S-9 were scored to determine viability and 6TG resistance. However, the highest two doses tested in the absence of S-9 (400 and
500 μg/mL) were later excluded from final test statistics, due to excessive toxicity. Furthermore, five of the highest six doses tested in the presence of S-9 (400, 800-1510 μg/mL) were later excluded from analysis. Of these five doses, two (400 and 1250 mg/mL) were discarded due to excessive heterogeneity and three (800, 1000 and 1510 mg/mL) were discarded due to toxicity, yielding 16%, 10% and 13% relative survival, respectively. The highest doses considered suitable for mutation assessment were the lowest concentrations to give 10%-20% relative survival, as required by the test guidelines. The highest doses selected were 350 μg/mL in the absence of S-9 and 600 μg/mL in the presence of S-9, which yielded 15% and 17% relative survival, respectively (see Tables 2 and 3).

In Experiment 2 ten doses, ranging from 50 to 500 μg/mL, were tested in the absence of S-9 and eleven doses, ranging from 50 to 1510 μg/mL, were tested in the presence of S-9. Seven days after treatment, the highest four doses tested in the absence of S-9 (300-500 μg/mL) and the highest five doses tested in the presence of S-9 (600-1510 μg/mL) were not selected to determine viability and 6TG resistance due to
toxicity, based primarily on Day 0 relative survival values. All other doses were selected. The highest doses considered suitable for mutation assessment were the lowest concentrations to give 10%-20% relative survival, as required by the test guidelines. These were 200 μg/mL in the absence of S-9 and 300 μg/mL in the presence of S-9, both of which yielded 18% relative survival (see Tables 2 and 3).

Mutation
A summary of the results for Experiments 1 and 2 is shown in Table 3.
For each experiment, the individual plate counts observed (on two plates for survival or viability and on four plates for 6TG resistance for each culture) are shown on the first page of each appendix. A summary of individual replicate results is shown on the second page of each appendix. The heterogeneity factors for the survival, viability and mutant plates are shown on the third page of each appendix, in addition to the comparison of negative and positive controls with historical means. Finally, the % relative survival values, the mutant frequencies and the statistical analyses are shown on the fourth page of each appendix. The acceptance criteria were met and the study was therefore accepted as valid.
In the absence of S-9, when tested up to toxic doses in Experiment 1, no statistically significant increases in mutant frequency were observed following treatment with Hydroxybenzomorpholine (A025) at any dose level tested. When tested up to toxic doses in Experiment 2, a statistically significant increase in mutant frequency was observed at one intermediate dose level (100 μg/mL). However, all mutant frequency values were within the historical negative solvent control range (upper limit 16.17 mutants per 106 viable cells; see Appendix 6) and no increases were observed at two higher dose levels analysed (up to 200 mg/mL). Moreover, the statistically significant increase observed at 100 μg/mL in Experiment 2 was set against a concurrent solvent control value of 3.48 mutants per 106 viable cells, which was lower than the historical solvent control mean value based on the last 20 experiments . A weak linear trend was obtained, but there appeared no real
evidence of a dose-related increase in mutant frequency over the entire dose range.
Overall, the isolated increase in mutant frequency in Experiment 2 was not doserelated and was not reproduced between experiments under the same experimental conditions. It was therefore considered a chance event of no biological relevance.

In the presence of S-9, when tested up to toxic doses in Experiment 1, a statistically significant increase in mutant frequency was observed at 600 μg/mL, the highest dose analysed, which was associated with 17% relative survival. A weak linear trend was also observed. When tested up to toxic doses in Experiment 2, no statistically significant increases in mutant frequency were observed at any dose level tested.
The mutant frequency observed at 600 mg/mL in Experiment 1 (where a statistically significant increase was observed) was 11.48 mutants per 10E6 viable cells. This value was similar to the upper limit of the historical negative control range (11.37 mutants per 10E6 viable cells; see Appendix 6), calculated as mean mutant frequency± 2 standard deviations, over the last 20 experiments. Moreover, one of the
protocol acceptance criteria states that mutant frequencies in solvent control cultures should not exceed three times the historical mean value. This value was 6.78 mutants per 10E6 viable cells at the time of this study, therefore a solvent control mutant frequency of up to 20.34 mutants per 10E6 viable cells would have been considered acceptable in this experiment. Moreover, the statistically significant
increase observed at 600 mg/mL in Experiment 1 was set against a concurrent solvent control value of 4.79 mutants per 10E6 viable cells, which was lower than the historical solvent control mean value based on the last 20 experiments. The lowest doses to yield 10%-20% relative survival in the presence of S-9 were 600 and 300 mg/mL in Experiments 1 and 2, respectively. As such, the dose giving a
significant increase in mutant frequency in Experiment 1 (600 mg/mL), which yielded 17% relative survival in Experiment 1, was associated with extreme toxicity (<10% relative sur vival) in Experiment 2. There was therefore a slight toxicity shift between experiments (although there appeared to be a toxicity “plateau” in Experiment 1 between 400 and 1510 mg/mL).
Overall, it is clear that the single increase in mutant frequency in Experiment 1 in the presence of S-9 was associated with a concentration with marked cytotoxicity and was not reproduced between experiments.

Table 1

 

Hydroxybenzomorpholine (A025):Raw plate counts and adjusted relative survival in the cytotoxicity range-finder

 

Treatment (µg/mL)	In the absence of S-9					In the presence of S-9
	Day0cells x10^5	Survival § (Day 0)	%CE (Day 0)	%S	%RS	Day0cells x10^5	Survival § (Day 0)	%CE (Day 0)	%S	%RS
0	4.90	86	141.36	141.36	100.00	4.47	81	116.02	116.02	100.00
47.19	4.66	81	116.02	110.34	78.05	4.16	77	101.24	94.22	81.21
94.38	4.47	72	86.64	79.04	55.91	4.04	65	70.65	63.85	55.04
188.8	4.36	62	64.87	57.72	40.84	3.93	60	61.30	53.90	46.45
377.5	4.02	13	9.09	7.46	5.28	4.00	35	28.34	25.36	21.86
755	3.53	0	0.00	0.00	0.00	3.99	23	17.12	15.28	13.17
1510	3.26	0	0.00	0.00	0.00	3.95	19	13.78	12.18	10.50

 §            Positivewellsperplate.96wellsscoredunlessotherwisestatedeg52/95

1.6 cells/well plated for survival.

 

 

Table 2

Hydroxybenzomorpholine(A025): summary of cytotoxicity data

 

Experiment 1 –S-9			Experiment 1 +S-9			Experiment 2 –S-9			Experiment 2 +S-9
Treatment (mg/mL)		%RS	Treatment (mg/mL)		%RS	Treatment (mg/mL)		%RS	%RS		MF§
0		100.00	0		100.00	0		100.00	0		100.00
25		108.93	50		95.77	50		62.28	50		74.66
50		88.03	100		74.51	75		56.48	100		59.48
100		78.36	200		40.21	100		47.12	200		28.85
150		46.16	400		21.52	150		31.92	300		18.11
200		41.46	600		16.55	200		18.00	375	X	14.80
250		29.23	800	X	16.19	250	X	8.70	450	X	8.23
300		20.58	1000	X	9.86	300	X	9.27	600	X	7.75
350		15.19	1250	X	12.73	350	X	3.52	800	X	5.04
400	X	7.26	1510	X	13.41	400	X	3.24	1000	X	4.49
500	X	1.92				500	X	0.73	1250	X	1.57
									1510	X	NE

 X           Too toxic to beanalysedfor viability and 6-TG resistance

 

Table 3

Hydroxybenzomorpholine(A025): summary of results

Experiment1

Treatment (µg/mL)	-S-9		Treatment (µg/mL)		+S-9
	%RS	MF§			%RS	MF	§
0	100.00	5.10	0		100.00	4.79
25	108.93	5.25            NS	50		95.77	8.50               NS
50	88.03	6.37            NS	100		74.51	5.57               NS
100	78.36	6.55            NS	200		40.21	6.15               NS
150	46.16	10.42           NS	400	$$	21.52	(10.84)
200	41.46	7.51            NS	600		16.55	11.48                *
250	29.23	7.39            NS
300	20.58	8.99            NS
350	15.19	6.84            NS
Linear trend		NS		Linear trend		*
NQO			BP
0.1	85.51	39.86	2		56.68	56.64
0.15	72.18	48.36	3		34.31	116.09

 

Experiment2

Treatment (µg/mL)	-S-9		Treatment (µg/mL)	+S-9
	%RS	MF§		%RS	MF§
0	100.00	3.48	0	100.00	4.33
50	62.28	9.17           NS	50	74.66	8.43                NS
75	56.48	6.11          NS	100	59.48	8.59                NS
100	47.12	15.75!             *	200	28.85	8.21                NS
150	31.92	9.31           NS	300	18.11	6.18                NS
200	18.00	7.92           NS
Linear trend		*	Linear trend		NS
NQO			BP
0.1	84.33	43.59	2	48.44	90.89
0.15	83.76	68.34	3	33.11	112.43

 

§     6-TG resistant mutants/10⁶viable cells7days aftertreatment

$    Not evaluated (due totoxicity)

%RS  Percent relative survival adjusted by post treatment cell counts

NE  Not evaluated

$$    Treatment excluded from analysis due to unacceptably high heterogeneity

Data in parentheses indicates marked heterogeneity observed

! based on one replicate only

X     Treatment excluded from final test statistics due to toxicity

NS   Not significant

*                   Comparison of each treatment with control :Dunnett 's test (one-sided), significant at 5% level

*, **, ***       Test for linear trend :?² (one-sided), significant at 5%, 1% and 0.1% level respectively

 

 

Conclusions:

It is concluded that when tested up to toxic doses, Hydroxybenzomorpholine (A025) did not show reproducible evidence of inducing mutation at the hprt locus of L5178Y mouse lymphoma cells in two independent experiments in the absence or presence of S-9. Under the conditions of the test, Hydroxybenzomorpholine (A025) is therefore considered not mutagenic in the absence and presence of S-9 in this test system.

Executive summary:

Hydroxybenzomorpholine was assayed for gene mutations at the hprt locus of mouse lymphoma cells both in the absence and presence of S9 metabolic activation. Test concentrations were based on the results of a cytotoxicity range-finding experiment measuring relative survival. In the main test, cells were treated for 3 h followed by an expression period of 7 days to fix the DNA damage into a stable hprt mutation. Liver S9 fraction from Arachlor 1254-induced rats was used as exogenous metabolic activation system. Toxicity was measured as percentage relative survival of the treated cultures relative to the survival of the solvent control cultures. Negative and positive controls were in accordance with the OECD guideline.

Results

The test meets to all acceptance criteria and, consequently, the study was considered valid.

In both experiments in the absence and presence of S9 the appropriate level of toxicity (10 -20% survival after the highest dose) was reached pointing to sufficient exposure of the cells. In the absence of S9 no increase in mutant frequency was seen in the first experiment. In the second experiment a statistical significant increase was found at an intermediate concentration.

As this result appeared not reproducible and remained within the historical control values this increase was considered not biological relevant.

In the presence of S9 in the first experiment a statistical significant increase in mutant frequency was found at the highest dose evaluated. This mutant frequency was similar to the upper limit of the historical control value, indicating that the increase was minimal whereas significance may be due to a low concurrent control value. In the second experiment no increases in mutant frequencies were found. At the highest dose (which is lower than the highest dose in experiment 1) an almost similar level of toxicity was found but no increase in mutant frequency. The increase in mutant frequency at the highest dose in experiment 1 was, therefore, considered as

not biological relevant.

Conclusion

Under the experimental conditions used, hydroxybenzomorpholine was considered not mutagenic in the gene mutation test with mouse lymphoma cells at the hprt locus.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In two in vivo bone marrow micronucleus tests performed up to lethal doses in mice and in rats, hydroxybenzomorpholine did not produce any increased micronucleus frequency. Finally, hydroxybenzomorpholine was non-genotoxic in an in vivo test for the evaluation of DNA damage and repair (UDS test in rats) that was conducted up to the MTD.

Overall, the genotoxicity program on hydroxybenzomorpholine investigated both endpoints of genotoxicity: gene mutations and chromosome aberrations. As hydroxybenzomorpholine did not produce gene mutations and chromosome aberrations in mammalian cells in vitro and in vivo, and as the positive result in bacteria was covered by a negative in vivo UDS test, hydroxybenzomorpholine is considered not genotoxic.

Link to relevant study records

Reference

Reference 1

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)

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

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.

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)

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

Dose / conc.:

1 500 mg/kg bw (total dose)

Dose / conc.:

750 mg/kg bw (total dose)

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

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

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.

 

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.

 

 

 

 

 

 

 

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.

Reference 2

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 december 1990 to 25 February 1991

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1981

Deviations:

not specified

GLP compliance:

yes

Type of assay:

mammalian bone marrow chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: E70 CXB
- date received: 13 November 1990
- Purity test date: not provided

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not specified
- Stability under storage conditions: not specified
- Stability under test conditions: the test material was freshly prepared as required as a suspension at the appropriate concentration in arachis oil B.P.. The identification and stability of the test material and the preparations were note determined.
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: the identification ans stability of the test material and the preparations were not determined.

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

CD1 strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK)
- Age at study initiation: 5 to 8 weeks old
- Weight at study initiation: males : 23 - 30 g, females: 21- 28g
- Fasting period before study: 2-4 hour fast immediately before dosing and for approximately 2 hours after dosing
- Housing: in groups of up to five by sex in solid-floor polypropylene cages with sawdust bedding
- Diet (e.g. ad libitum) and water: free access to mains drinking water and food
- Acclimation period and assigned to test groups randomly:: after a minimum acclimatisation period of five days the animals were selected at random and given a unique number within the study by ear punching and a number written on a colour coded cage card.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-21 °C
- Humidity (%): 30-38%
- Air changes (per hr): the rate of air exchange was approximately 15 changes per hour
- Photoperiod (hrs dark / hrs light): the lighting was controlled by a time switch to give 12 hours light and 12 hours darkness.

IN-LIFE DATES: From: To: not specified

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle: not specified
- Concentration of test material in vehicle: for the dose range finding: 5000, 2000, 1000, 500, 250 mg/kg. For the main study : 400 mg/kg
- Amount of vehicle (if gavage or dermal): 10 mL/kg
- Lot/batch no. (if required): Co/374
- Purity: not specified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: dose level were administered by gavage using a metal cannula attached to a graduated syringue. The volume aadministered to each animal was calculated according to its fasted bodyweight at the time of dosing.

Duration of treatment / exposure:

dose range-finding study: animals were dosed once only at the appropriate dose level by gavage using metal cannula attached to a graduated syringe
micronucleus study: the study was performed using one dose level at three kill times of 24, 48 and 72 hours after dosing

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide monohydrate
- Justification for choice of positive control(s): not specified
- Route of administration: gavage
- Doses / concentrations: the positive control material was freshly prepared as required as a solution at the appropriate concentration in distilled water

Tissues and cell types examined:

micronucleated cells of bone marrow

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 24,48 or 72 hours following dosing
DETAILS OF SLIDE PREPARATION: immediately following sacrifice (i.e. 24,48 or 72 hours following dosing) one femur was dissected from each animal, aspirated with foetal calf serum and bone marow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, and stained in May-Grünwald/Giemsa

METHOD OF ANALYSIS: stained bone marrow smears were examined at random using light microscopy at x1000 magnification. the incidence of micronucleated cells per 1000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or hlaf-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 polychromatic eythrocytes were counted; these cells were also scored for incidence of micronuclei.
The ratio of normochromatic to polychromatic erthrocytes was calculated together with appropriate group mean values for males and females separately and combined.

Evaluation criteria:

A comparison was made between the number of micronucleated plochromatic erythrocytes occuring in each of the three test material groups and the number occuring in the corresponding vehicle control groups.
A positive mutagenic response is demonstrated when a statistically significant increase in the number of micronucleated polychromatic erythrocytes is observed for either the 24, 48 or 72-hour kill times.
If the above criteria are not demonstrated, then the test material is considered to be non-mutagenic under the conditions of the test.
A positive response for bone-marrow toxicity is demonstrated when dose group mean normochromatic to polychromatic ratio is shown to be statistically significant from the concurrent vehicle control group

Statistics:

If necessary, and where possible, all data were statistically analysed using appropriate statistical methodes as recommened by the UKEMS sub-committee on guidelines for mutagenicit testing, report, part III (1989)

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 5000, 2000, 1000, 500, 250
- Clinical signs of toxicity in test animals: clinical sings were observed in all of the test substance treatment groups, with the exception of the 250 mg/kg group, the numbr of different signs observed and their persistance was clearly dose related. the clinical signs observed included: hunched posture, ataxia, lethary, ptosis, laboured respiration, loss of righting reflex, occasional body tremors and stains around the eyes. 400 mg/kg was selected as the maximum tolerated dose level fo use in the micronucleux study.


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): there was only one premature death in the groups of animals dosed with test substance, this animal was from the 48 hours group females. Many of the animals dosed with the test substance showed lethargic signs one hour after dosing, and ptosis in some animals at 24, 48 and 72 hours post dosing.
- Ratio of PCE/NCE (for Micronucleus assay): there was a significant increase in the frequency of micronucleated NCE's in any of the test material dose groups when compared to their concurrent vehicle control group

A summary of the results of the micronucleus study is given in Table 1.

Individual and group mean data are presented in Tables 2 to 8.

There was no significant increase in the frequency ofmicronucleatedPCE’s in any of the test material dose groups when compared to their concurrent vehicle control group.

There was a significant change in the NCE/PCE ratio in the test material 400 mg/kg 72-hour dose group when compared to its concurrent vehicle control group.

The positive control group showed a marked increase in the incidence ofmicronucleatedpolychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

The test material, was found not to produce micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

 

Table 1: summary of groupmeandata (males and females combined)

 

Treatment group	Number of PCE with micronuclei per 1000 PCE Group Mean	Number of PCE with micronuclei per 1000 PCE SD	Number of PCE with micronuclei per 1000 NCE Group Mean	Number of PCE with micronuclei per 1000 NCE SD	NCE/PCE ratio Group Mean	NCE/PCE ratio SD
Vehicle control 72 hour sampling time	0.9	1.0	0.4	1.1	0.78	0.23
Vehicle control 48 hour sampling time	0.5	0.7	0.4	1.1	0.92	0.26
Vehicle control 24 hour sampling time	0.3	0.5	0.2	0.5	0.91	0.17
Test substance 400 mg/kg 72 hour sampling time	1.6	1.7	0.5	0.8	1.74**	0.79
Test substance 400 mg/kg 48 hour sampling time	0.4	0.7	0.2	0.5	0.95	0.19
Test substance 400 mg/kg 24 hour sampling time	0.9	1.0	0.4	1.2	0.71	0.23
Positive control24 hoursampling time	18.9	11.3	1.2	1.3	1.17	0.36

 

PCE = polychromatic erythrocytes

NCE =normochromaticerythrocytes

SD = standard deviation

** = significantly different from concurrent vehicle control group, (t-test; p<0.01)

 

Table 2: micronucleus study – individual and group means and standard deviations

Doselevel :0 mg/kg

Sampletime :72 hour

 

Animal number and sex	Total cells scored (PCE+NCE)	Polychromatic erythrocytes (PCE) scored	Polychromatic erythrocytes (PCE) +veMN	Normochromatic erythrocytes (NCE) scored	Normochromatic erythrocytes (NCE) +veMN	Normochromatic erythrocytes (NCE) /1000 NCE	NCE/PCE ratio
1M	1804	1000	2.0	804	0.0	0.0	0.8
2M	2041	1000	2.0	1041	0.0	0.0	1.04
3M	1932	1000	0.0	932	0.0	0.0	0.93
4M	1681	1000	2.0	681	0.0	0.0	0.68
5M	1552	1000	0.0	552	0.0	0.0	0.55
Male
Mean	1802	1000	1.2	802	0.0	0.0	0.8
SD	194	0	1.1	194	0.0	0.0	0.19
6 F	1571	1000	1.0	571	2.0	3.5	0.57
7 F	1931	1000	0.0	931	0.0	0.0	0.93
8F	1423	1000	0.0	423	0.0	0.0	0.42
9F	1716	1000	0.0	716	0.0	0.0	0.72
10 F	2154	1000	2.0	1154	0.0	0.0	1.15
Female
Mean	1759	1000	0.6	759	0.4	0.7	0.76
SD	290	0	0.9	290	0.9	1.6	0.29
Group
Mean	1781	1000	0.9	781	0.2	0.4	0.78
SD	234	0	1.0	234	0.6	1.1	0.23

SD :standard deviation

 

 Table 3: micronucleus study – individual and group means and standard deviations

Doselevel :0 mg/kg

Sampletime :48 hour

 

Animal number and sex	Total cells scored (PCE+NCE)	Polychromatic erythrocytes (PCE) scored	Polychromatic erythrocytes (PCE) +veMN	Normochromatic erythrocytes (NCE) scored	Normochromatic erythrocytes (NCE) +veMN	Normochromatic erythrocytes (NCE) /1000 NCE	NCE/PCE ratio
11M	2491	1000	1.0	1491	1.0	1.49
12M	1718	1000	0.0	718	0.0	0.72
13M	2087	1000	2.0	1087	0.0	1.09
14M	1981	1000	1.0	981	0.0	0.98
15M	1858	1000	1.0	858	3.0	0.86
Male
Mean	2027	1000	1.0	1027	0.8	1.03
SD	294	0	0.7	294	1.3	0.29
16 F	1508	1000	0.0	508	0.0	0.51
17 F	1807	1000	0.0	807	0.0	0.81
18F	1946	1000	0.0	946	0.0	0.95
19F	1878	1000	0.0	878	0.0	0.88
20 F	1917	1000	0.0	917	0.0	0.92
Female
Mean	1811	1000	0.0	811	0.0	0.81
SD	177	0	0.0	177	0.0	0.18
Group
Mean	1919	1000	0.5	919	0.4	0.92
SD	255	0	0.7	255	1.1	0.26

SD :standard deviation

Table 4: micronucleus study – individual and group means and standard deviations

 

Doselevel :0 mg/kg

Sampletime :24 hour

 

Animal number and sex	Total cells scored (PCE+NCE)	Polychromatic erythrocytes (PCE) scored	Polychromatic erythrocytes (PCE) +veMN	Normochromatic erythrocytes (NCE) scored	Normochromatic erythrocytes (NCE) +veMN	Normochromatic erythrocytes (NCE) /1000 NCE	NCE/PCE ratio
21M	1864	1000	1.0	864	0.0	0.0	0.86
22M	2087	1000	0.0	1087	0.0	0.0	1.09
23M	1708	1000	0.0	708	0.0	0.0	0.71
24M	1670	1000	1.0	670	1.0	1.5	0.67
25M	1889	1000	0.0	889	0.0	0.0	0.89
Male
Mean	1844	1000	0.4	844	0.2	0.3	0.84
SD	166	0	0.5	166	0.4	0.7	0.17
26 F	1934	1000	0.0	934	1.0	1.1	0.93
27 F	1985	1000	0.0	985	0.0	0.0	0.99
28F	2002	1000	0.0	1002	0.0	0.0	1.00
29F	2203	1000	1.0	1203	0.0	0.0	1.20
30 F	1732	1000	0.0	732	0.0	0.0	0.73
Female
Mean	1971	1000	0.2	971	0.2	0.2	0.97
SD	168	0	0.4	168	0.4	0.5	0.17
Group
Mean	1907	1000	0.3	907	0.2	0.3	0.91
SD	171	0	0.5	171	0.4	0.5	0.17

SD :standard deviation

Table 5: micronucleus study – individual and group means and standard deviations

Doselevel :400 mg/kg

Sampletime :72 hour

 

Animal number and sex	Total cells scored (PCE+NCE)	Polychromatic erythrocytes (PCE) scored	Polychromatic erythrocytes (PCE) +veMN	Normochromatic erythrocytes (NCE) scored	Normochromatic erythrocytes (NCE) +veMN	Normochromatic erythrocytes (NCE) /1000 NCE	NCE/PCE ratio
31M	2568	1000	0.0	1568	1.0	0.6	1.57
32M	4127	1000	2.0	3127	1.0	0.3	3.13
33M	1999	1000	0.0	999	0.0	0.0	1.00
34M	2088	1000	0.0	1088	0.0	0.0	1.09
35M	2518	1000	3.0	1518	1.0	0.7	1.52
Male
Mean	2660	1000	1.0	1660	0.6	0.3	1.66
SD	858	0	1.4	858	0.5	0.3	0.86
36 F	4123	1000	2.0	3123	1.0	0.3	3.12
37 F	2441	1000	5.0	1441	4.0	2.8	1.44
38F	2420	1000	3.0	1420	1.0	0.7	1.42
39F	2857	1000	0.0	1857	0.0	0.0	1.86
40 F	2302	1000	1.0	1302	0.0	0.0	1.30
Female
Mean	2829	1000	2.2	1829	1.2	0.8	1.83
SD	753	0	1.9	753	1.6	1.2	0.75
Group
Mean	2744	1000	1.6	1744	0.9	0.5	1.74
SD	766	0	1.7	766	1.2	0.8	0.77

SD :standard deviation

Table 6: micronucleus study – individual and group means and standard deviations

Doselevel :400 mg/kg

Sampletime :48 hour

 

Animal number and sex	Total cells scored (PCE+NCE)	Polychromatic erythrocytes (PCE) scored	Polychromatic erythrocytes (PCE) +veMN	Normochromatic erythrocytes (NCE) scored	Normochromatic erythrocytes (NCE) +veMN	Normochromatic erythrocytes (NCE) /1000 NCE	NCE/PCE ratio
41M	1954	1000	0.0	954	0.0	0.0	0.95
42M	2174	1000	0.0	1174	0.0	0.0	1.17
43M	1782	1000	1.0	782	0.0	0.0	0.78
44M	1728	1000	0.0	728	1.0	1.4	0.73
45M	1895	1000	1.0	895	0.0	0.0	0.9
Male
Mean	1907	1000	0.4	907	0.2	0.3	0.91
SD	174	0	0.5	174	0.4	0.6	0.17
46 F	1951	1000	0.0	951	0.0	0.0	0.95
47 F	-	-	-	-	-	-	-
48F	1974	1000	2.0	974	0.0	0.0	0.97
49F	2317	1000	0.0	1317	0.0	0.0	1.32
50 F	1811	1000	0.0	811	0.0	0.0	0.81
Female
Mean	2013	1000	0.5	1013	0.0	0.0	1.01
SD	215	0	1.0	215	0.0	0.0	0.21
Group
Mean	1954	1000	0.4	954	0.1	0.2	0.95
SD	189	0	0.7	189	0.3	0.5	0.19

SD :standard deviation

-       : animal died prematurely

 

Table 7: micronucleus study – individual and group means and standard deviations

Doselevel :400 mg/kg

Sampletime :24 hour

 

Animal number and sex	Total cells scored (PCE+NCE)	Polychromatic erythrocytes (PCE) scored	Polychromatic erythrocytes (PCE) +veMN	Normochromatic erythrocytes (NCE) scored	Normochromatic erythrocytes (NCE) +veMN	Normochromatic erythrocytes (NCE) /1000 NCE	NCE/PCE ratio
51M	2168	1000	1.0	1168	0.0	0.0	1.17
52M	1381	1000	2.0	381	0.0	0.0	0.38
53M	1890	1000	0.0	890	0.0	0.0	0.89
54M	1675	1000	0.0	675	0.0	0.0	0.68
55M	1815	1000	1.0	815	3.0	3.7	0.82
Male
Mean	1786	1000	0.8	786	0.6	0.7	0.79
SD	289	0	0.8	289	1.3	1.6	0.29
56 F	1598	1000	1.0	598	0.0	0.0	0.60
57 F	1859	1000	3.0	859	0.0	0.0	0.86
58F	1540	1000	0.0	540	0.0	0.0	0.54
59F	1574	1000	0.0	574	0.0	0.0	0.57
60 F	1550	1000	1.0	550	0.0	0.0	0.55
Female
Mean	1624	1000	1.0	624	0.0	0.0	0.62
SD	133	0	1.2	133	0.0	0.0	0.13
Group
Mean	1705	1000	0.9	705	0.3	0.4	0.71
SD	229	0	1.0	229	0.9	1.2	0.23

SD :standard deviation

Table 8: micronucleus study – individual and group means and standard deviations

Cyclophosphamide

Doselevel :50 mg/kg

Sampletime :24 hour

 

Animal number and sex	Total cells scored (PCE+NCE)	Polychromatic erythrocytes (PCE) scored	Polychromatic erythrocytes (PCE) +veMN	Normochromatic erythrocytes (NCE) scored	Normochromatic erythrocytes (NCE) +veMN	Normochromatic erythrocytes (NCE) /1000 NCE	NCE/PCE ratio
61M	1954	1000	10.0	954	0.0	0.0	0.95
62M	2456	1000	19.0	1456	1.0	0.7	1.46
63M	1982	1000	29.0	982	0.0	0.0	0.98
64M	1698	1000	41.0	698	3.0	4.3	0.7
65M	1991	1000	32.0	991	2.0	2.0	0.99
Male
Mean	2016	1000	26.2	1016	1.2	1.4	1.02
SD	274	0	12.0	274	1.3	1.8	0.27
66 F	2642	1000	11.0	1642	2.0	1.2	1.64
67 F	2430	1000	13.0	1430	2.0	1.4	1.43
68F	2368	1000	17.0	1368	0.0	0.0	1.37
69F	1646	1000	8.0	646	1.0	1.5	0.65
70 F	2504	1000	9.0	1504	2.0	1.3	1.50
Female
Mean	2318	1000	11.6	1318	1.4	1.1	1.32
SD	389	0	3.6	389	0.9	0.6	0.39
Group
Mean	2167	1000	18.9	1167	1.3	1.2	1.17
SD	355	0	11.3	355	1.1	1.3	0.36

SD :standard deviation

 

Conclusions:

the test substance was considered to be non-genotoxic under the conditions of the test

Executive summary:

Hydroxybenzomorpholine has been investigated for the induction of micronuclei in bone marrow cells of mice. The test concentration was based on the result of a range finding toxicity study in which mice were exposed to a range of concentrations; 400 mg/kg was selected as the maximum tolerated dose level. In the main experiment mice were exposed by gavage to a single dose of 400 mg/kg bw. Bone marrow cells were collected 24, 48 and 72 h after dosing. Toxicity and thus exposure of the target cells was determined by measuring the ratio between normochromatic to polychromatic erythrocytes (PCE/NCE ratio). Moreover, all animals were observed daily for signs of overt toxicity and death. Bone marrow preparations were stained and examined microscopically for the PCE/NCE ratio and micronuclei. Negative and positive controls were in accordance with the OECD guideline.

Results

One female mouse from the 48 h group died. The ratio PCE/NCE substantially changed in the 72h group as compared to the untreated controls at 72 h indicating that hydroxybenzomorpholine did have cytotoxic properties in the bone marrow and consequently must have been biologically

available. Moreover, many of the treated mice showed lethargic signs immediately after dosing and ptosis at 24, 48 and 72 h after dosing indicating to systemic toxicity and confirming exposure to hydroxybenzomorpholine.

Biological relevant increases in the number of micronucleated PCEs compared to the concurrent vehicle controls were not found following treatment with hydroxybenzomorpholine at any time point.

Under the experimental conditions used hydroxybenzomorpholine did not induce micronuclei in bone marrow cells of treated mice and, consequently, hydroxybenzomorpholine was not genotoxic (clastogenic and/or aneugenic) in bone marrow cells of mice.

Reference 3

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 dec 2003 to 6 June 2005

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

21 july 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian bone marrow chromosome aberration test

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:
sept 2005
- Purity test date:
98.3%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: storage conditions: at +4°C, protected from light and under nitrogen gas
- Stability under test conditions: The test item dosage forms were prepared extemporaneously under nitrogen atmosphere and were stored at room temperature, protected from light (using aluminium foil) and under nitrogen atmosphere until treatment. They were used within 6 hours of preparation, according to known stability results (CIT/Study No. 26976 AHS).
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: The test item was ground to a fine powder using a mortar and pestle, suspended in the vehicle in order to achieve the concentrations of 50, 100 and 200 mg/mL and then homogenized using a magnetic stirrer. Using a treatment volume of 10 mL/kg, the target dose-levels were 500, 1000 and 2000 mg/kg/day, respectively.
The vehicle was 0.5% carboxymethylcellulose, batch No. 101K0185 (Sigma, Saint-Quentin-Fallavier, France) in purified water (CIT, Millipore).

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Sprague-Dawley rats, Ico: OFA-SD (IOPS Caw).
Reason for this choice: rodent species generally accepted by regulatory authorities for this type of study.
Breeder: Charles River Laboratories France, L'Arbresle, France.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Breeder: Charles River Laboratories France, L'Arbresle, France.
- Age at study initiation: on the day of treatment, the animals were approximately 6 weeks old
- Weight at study initiation: not specified
- Assigned to test groups randomly: Acclimation: at least 5 days before the day of treatment. Constitution of groups: upon arrival, the animals were randomly allocated to the groups by sex. Subsequently, each group was assigned to a different treatment group. Identification: individual tail marking upon treatment.
- Fasting period before study: not sepcified
- Housing: The housing conditions (temperature, relative humidity and ventilation) and corresponding instrumentation and equipment were verified and calibrated at regular intervals.
The animals were housed by groups in polycarbonate cages. Each cage contained autoclaved sawdust (SICSA, Alfortville, France).
Sawdust is analyzed by the supplier for composition and contaminant levels.
- Diet (e.g. ad libitum): All animals had free access to A04 C pelleted maintenance diet (SAFE, Epinay-sur-Orge,France).
Each batch of food is analyzed by the supplier for composition and contaminant levels.
- water: Drinking water filtered by a FG Millipore membrane (0.22 micron) was provided ad libitum.
Bacteriological and chemical analysis of water are performed regularly by external laboratories, These analyses include the detection of possible contaminants (pesticides, heavy metals and nitrosamines).
No contaminants were known to have been present in the diet, drinking water or bedding material at levels which could be expected to interfere with or prejudice the outcome of the
study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 30 to 70%,
- Air changes (per hr): 12 cycles/hour of filtered non-recycled fresh air
- Photoperiod (hrs dark / hrs light): 12 h/12 h (07:00 - 19:00),

IN-LIFE DATES:
- 20 April 2004 for the preliminary toxicity test
- From: 16 June 2004 To: 1 June 2004, for first cytogenetic test
- From: 5 January 2005 To: 7 January 2005 , for second cytogenetic test

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: The vehicle was 0.5% carboxymethylcellulose, batch No. 101K0185 (Sigma, Saint-Quentin-Fallavier, France) in purified water (CIT, Millipore). The positive control was cyclophosphamide (CPA), batch Nos. 3B257L, 0D203A and 2B249G (Laboratoire Asta Médica, Mérignac, France) dissolved in distilled water at a concentration of 1.5 mg/mL. The preparation was made immediately before use or was stored at -20°C and thawed immediately before use
- Concentration of test material in vehicle: The test item was ground to a fine powder using a mortar and pestle, suspended in the vehicle in order to achieve the concentrations of 50, 100 and 200 mg/mL and then homogenized using a magnetic stirrer.
- Amount of vehicle (if gavage or dermal): Using a treatment volume of 10 mL/kg, the target dose-levels were 500, 1000 and 2000 mg/kg/day, respectively

For the positive control (CPA):
⋅ Route: oral,
⋅ Frequency: one treatment,
⋅ Volume: 10 mL/kg.
The quantity of each item administered to each animal was adjusted according to the most recently recorded body weight.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the vehicle and the test item:
⋅ Route: oral (gavage), was selected to provide an exaggerated model of the normal exposure in human by cutaneous application,
⋅ Frequency: two treatments separated by 24 hours,
⋅ Volume: 10 mL/kg.

For the positive control (CPA):
⋅ Route: oral,
⋅ Frequency: one treatment,
⋅ Volume: 10 mL/kg.
The quantity of each item administered to each animal was adjusted according to the most recently recorded body weight.

Duration of treatment / exposure:

Blood samples for the determination of plasma levels of the test item were taken at the following times:
⋅ 0.5, 1 and 4 hours following the last treatment, for the high-dose treated group (three males and three females per time point, one or two time-points per animal).
Venous blood (approximately 1 mL) was taken from the orbital sinus of the animals under light isoflurane anesthesia, into a tube containing lithium heparinate.
After the blood sampling, the animals were killed by carbon dioxide asphyxiation and discarded without necropsy.
The blood was centrifuged (10 min at 4000 rpm, at +4°C), then 52 μL of 0.1% ascorbic acid per milliliter of plasma was added to the samples and the plasma was kept frozen in individual tubes at -20°C until analysis.

Frequency of treatment:

one treatment

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

5, 6 or 8

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration: oral
- Doses / concentrations: 15 mg/kg bw/day

Tissues and cell types examined:

microscopic examination of the bone marrow smears and scoring of micronucleus

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Blood samples for the determination of plasma levels of the test item were taken at the following times:
⋅ 0.5, 1 and 4 hours following the last treatment, for the high-dose treated group (three males and three females per time point, one or two time-points per animal).
Venous blood (approximately 1 mL) was taken from the orbital sinus of the animals under light isoflurane anesthesia, into a tube containing lithium heparinate.
After the blood sampling, the animals were killed by carbon dioxide asphyxiation and discarded without necropsy.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The blood was centrifuged (10 min at 4000 rpm, at +4°C), then 52 μL of 0.1% ascorbic acid per milliliter of plasma was added to the samples and the plasma was kept frozen in individual tubes at -20°C until analysis.

DETAILS OF SLIDE PREPARATION:
At the time of sacrifice, all the animals were killed by CO2 inhalation in excess. The femurs were removed and bone marrow was flushed and suspended in fetal calf serum. The separation of anucleated erythrocytic cells from other myeloic cells was carried out using a cellulose column. This elution step enables the production of slides containing only polychromatic and normochromatic erythrocytes without any nucleated cells or mast cell granules. After centrifugation of the eluate containing the cells, the supernatant was removed and the cells in the sediment were resuspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa.
The slides were coded so that the scorer was unaware of the treatment group of the slide under evaluation ("blind" scoring).

METHOD OF ANALYSIS:
For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

Evaluation criteria:

For a result to be considered positive, a statistically significant increase in the frequency of MPE must be demonstrated when compared to the concurrent vehicle control group. Reference to historical data, or other considerations of biological relevance were also taken into account in the evaluation of data obtained.

Statistics:

When there was no significant within-group heterogeneity, using the heterogeneity chi-square test value (Lovell et al., 1989) (e), the frequencies of MPE in each treated group was compared with those in the concurrent vehicle control groups by using a 2 x 2 contingency table to determine the χ2 value (Lovell et al., 1989) (e).
When there was significant within-group heterogeneity, then that group was compared with the control group using a non-parametric analysis, the Mann-Whitney test (Schwartz, 1969) (f).
The student "t" test was used for the PE/NE ratio comparison.
Probability values of p ≤ 0.05 were considered as significant.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable

Additional information on results:

RESULTS OF FIRST EXPERIMENT
Plasma levels of the test item
The results of the plasma levels of the test item for animals given 2000 mg/kg:
Following the treatment, mean ± SD test item plasma levels were as follows:
⋅ in males, 182 ± 35, 69.1 ± 66.5 and 5.77 ± 9.47 μg/mL, at the 0.5, 1 and 4-hour sampling times, respectively,
⋅ in females, 121 ± 104, 169 ± 49 and 55.7 ± 94.6 μg/mL, at the 0.5, 1 and 4-hour sampling times, respectively.

Preliminary toxicity test
In order to determine the highest dose-level, a preliminary test was performed on a group of six animals (three males and three females). Clinical signs and any mortality were recorded for a period of 48 hours. At the end of this period, the animals were killed by CO2 inhalation in excess.
In order to select the top dose-level for the cytogenetic study, 2000 mg/kg was administered to three males and three females.
No mortality was noted in the animals. Piloerection, half-closed eyes, hypoactivity and dyspnea were noted in all animals on day 1 only.
The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines. Since non-severely toxic effects were observed, the top dose-level selected was 2000 mg/kg. The two other selected dose-levels were 500 and 1000 mg/kg.

Main cytogenetic test
No mortality and no clinical signs were noted in the animals of either sex given 500, 1000 or 2000 mg/kg.
For all treated males as well as for females from the low and intermediate treated groups, the mean values of MPE as well as the PE/NE ratio in the groups treated with the test item, were equivalent to those of the vehicle groups at both harvest times .
A slightly high frequency of micronucleated polychromatic erythrocytes (7.1 MPE/1000 PE), even though not statistically significant, was noted in the female high-dose group (2000 mg/kg) at the 24-hour sampling time. The frequency of MPE in the other female groups, including vehicle control group, was also higher than the values usually obtained in this test
(1.0-1.8 MPE/1000 PE obtained for vehicle control females which received two consecutive oral administrations).
Therefore, in order to check the reliability of the slightly high value observed in the main test at the 24-hour sampling time, a confirmatory test was performed in females at the highest dose of 2000 mg/kg.

Confirmatory cytogenetic test
No mortality and no clinical signs were noted in the animals given 2000 mg/kg.
No statistically significant increase in the frequency of MPE was noted with the test item in the confirmatory experiment. However, the frequency of MPE at 2000 mg/kg
(2.7 MPE/1000 PE versus 1.4 for the vehicle control) was still slightly above historical data for the vehicle control group. Therefore, at the request of the Sponsor, in order to check the
reliability of these slightly high values, a second experiment was performed using a modified treatment schedule (two treatments separated by a 24-hour interval and harvest of the bone marrow cells 24 hours following the second treatment).

RESULTS OF SECOND EXPERIMENT
Plasma levels of the test item
The results of the plasma levels of the test item for animals given 2000 mg/kg:

Following the treatment, mean ± SD test item plasma levels were as follows:
- in males, 152 ± 117, 55.3 ± 77.8 and 3.65 ± 2.12 µg/mL, at the 0.5, 1 and 4-hour sampling times, respectively,
- in females, 82 ± 54, 25.4 ± 15.9 and 7.61 ± 7.61 µg/mL, at the 0.5, 1 and 4-hour sampling times, respectively.

These results demonstrated that bone marrow cells were effectively exposed to the test item.

Preliminary experiment
In order to select the top dose-level for the cytogenetic study, 2000 mg/kg/day was administered twice, to three males and three females. The interval between each administration was 24 hours. No mortality was noted in the animals. The animals showed piloerection and hypoactivity after the first treatment. Piloerection, hypoactivity and dyspnea were noted after the second treatment.
The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines. Since non-severely toxic effects were observed, the top dose-level selected was 2000 mg/kg/day. The two other selected dose-levels were 500 and 1000 mg/kg/day.

Cytogenetic test
No clinical signs and no mortality were observed in the animals of either sex given 500 mg/kg/day.
No mortality was noted in the animals receiving 1000 mg/kg/day, however piloerection was observed in all animals 24 hours after the second treatment.
At 2000 mg/kg/day, 1/5 females was found dead 2 hours after the second treatment and 1/3 of the supplementary females was found dead 24 hours the second treatment. Hypoactivity was noted in all animals after the first treatment, and piloerection was observed in all surviving animals after the second treatment.
For both males and females, the mean values of MPE as well as the PE/NE ratio in the groups treated with the test item, were equivalent to those of the vehicle groups.


Considering all data generated in this study, since no statistically significant increase in the frequency of MPE was noted either in the first experiment, or in the second experiment using a modified treatment schedule, the slightly high MPE frequency values noted in the first experiment were not considered as relevant.

Throughout the study, Cyclophosphamide induced a highly statistically significant increase (p < 0.001) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid

Table 1: Results of the preliminary toxicity test of the first experiment

 

Doses          Time          Animal number                            Clinical signs

mg/kg		Males	Females
2000	30 min	01-02-03	04-05-06	Half-closed eyes,
				hypoactivity, dyspnea
	2 h	01-02-03	04-05-06	Hypoactivity, piloerection,
				dyspnea
	5 h	01-02-03	04-05-06	Hypoactivity, piloerection
	6 h	01-02-03	04-05-06	None
	24 h	01-02-03	04-05-06	None
	48 h	01-02-03	04-05-06	None
h :hour

Table 2: Results of the first cytogenetic test: clinical signs

 

 

Doses mg/kg	Time	Animal number Males		Females	Clinical signs
0 *	2 h 5 h	89-90-91-92-93	06-07-08-09-10		None None
	24 h	89-90-91-92-93	06-07-08-09-10		None
0 **	2 h 5 h	94-95-96-97-98	16-17-18-19-20		NoneNone
	24 h	94-95-96-97-98	16-17-18-19-20		None
	48 h	94-95-96-97-98	16-17-18-19-20		None
500 *	2 h 5 h	99-100-101-102-103	26-27-28-89-90		NoneNone
	24 h	99-100-101-102-103	26-27-28-89-90		None
1000 *	2 h 5 h	104-105-106-107-108	36-37-38-39-40		NoneNone
	24 h	104-105-106-107-108	36-37-38-39-40		None
2000 *	2 h 5 h	109-110-111-112-113	46-47-48-49-50		NoneNone
	24 h	109-110-111-112-113	46-47-48-49-50		None
2000 **	2 h 5 h	114-115-116-117-118	56-57-58-59-60		NoneNone
	24 h	114-115-116-117-118	56-57-58-59-60		None
	48 h	114-115-116-117-118	56-57-58-59-60		None
2000 ** (1)	2 h		74-75-76		None
	5h	124 -125 -126			None

	24 h	124-125-126	74-75-76	None
	48 h	124-125-126	74-75-76	None
CPA *	2 h 5 h	119-120-121-122-123	66-67-68-69-70	NoneNone
	24 h	119-120-121-122-123	66-67-68-69-70	None

0      : vehiclecontrol

*      : 24-hour harvesttime

**    : 48-hour harvesttime

⁽¹⁾     : supplementaryanimals

h      :hour

CPA :cyclophosphamide

Table 3: Results of the confirmatory cytogenetic test: clinical signs

 

 

Doses mg/kg	Time	Animal number Females	Clinical signs
0	2 h	133-134-135-136-137	None
	24 h	133-134-135-136-137	None
2000	2 h	138-139-140-141-142	None
	24 h	138-139-140-141-142	None
CPA	2 h	143-144-145-146-147	None
	24 h	143-144-145-146-147	None

0: vehiclecontrol h:hour

CPA :cyclophosphamide

Table 4: Results of the preliminary toxicity test of the second experiment

 

Doses         Time                Animal number              Clinical signs

mg/kg/day		Males	Females
2000 *	2 h	07-08-09	10-11-12	Hypoactivity, piloerection
	5 h	07-08-09	10-11-12	Piloerection Hypoactivity, piloerection
	24 h	07-08-09	10-11-12	None
2000 **	2 h	07		Hypoactivity, piloerection
		08-09	10-11-12	Hypoactivity, piloerection, dyspnea Hypoactivity, piloerection
	6 h	07-08-09	10-11-12	Hypoactivity, piloerection
	24 h	07-08-09	10-11-12	Piloerection

*       : firsttreatment

** :second treatment h : hour

Table 5: Results of the second cytogenetic test: clinical signs

 

 

Doses mg/kg/day	Time	Animal Males	number Females	Clinical signs
0 *	2 h	148-149-150-151-152	153-154-155-156-157	None
	24 h	148-149-150-151-152	153-154-155-156-157	None
0 **	2 h	148-149-150-151-152	153-154-155-156-157	None
	24 h	148-149-150-151-152	153-154-155-156-157	None
500 *	2 h	158-159-160-161-162	163-164-165-166-167	None
	24 h	158-159-160-161-162	163-164-165-166-167	None
500 **	2 h	158-159-160-161-162	163-164-165-166-167	None
	24 h	158-159-160-161-162	163-164-165-166-167	None
1000 *	2 h	168-169-170-171-172	173-174-175-176-177	None
	24 h	168-169-170-171-172	173-174-175-176-177	None
1000 **	2 h	168-169-170-171-172	173-174-175-176-177	None
	24 h	168-169-170-171-172	173-174-175-176-177	Piloerection
2000 *	2 h	178-179-180-181-182	183-184-185-186-187	Hypoactivity
	24 h	178-179-180-181-182	183   184-185-186-187	None Ocular secretion,rhinorrea None
2000 **	2 h		183	Mortality
		178-179-180-181-182	184-185-186-187	Piloerection
	24 h	178-179-180-181-182	184-185-186-187	Piloerection
2000 *^(1)	2 h	198-199-200	201-202-203	Hypoactivity
	24 h	198-199-200	201-202-203	None
2000 **^(1)	2 h	198-199-200	201-202	Piloerection
	24 h	198-199-200	203   201-202	Piloerection, dyspnea,sedation Piloerection
			203	Mortality
CPA	2 h	188-189-190-191-192	193-194-195-196-197	None
	24 h	188-189-190-191-192	193-194-195-196-197	None

0             : vehiclecontrol

*             : firsttreatment

**    : second treatment

⁽¹⁾     : supplementaryanimals

h      :hour

CPA :cyclophosphamide

Table 6: Results of the first cytogenetic test: data summary

 

Doses     MPE/1000PE          PE/NEratio          Timeof

Sex	Group	(mg/kg)	mean	(sd)		mean	(sd)	sacrifice (hours)
Male
	Vehicle	-	1.2	(0.8)		0.5	(0.1)
		500	2.7	(1.3)		0.7	(0.3)	24
	Test item	1000	0.9	(1.2)		0.6	(0.1)
		2000	1.9	(2.1)		1.0	(0.4)
	Cyclophosphamide	15	34.0	(6.5)	***	0.5	(0.1)
Female
	Vehicle	-	4.9	(2.5)		0.9	(0.3)
		500	4.8	(2.3)		0.6	(0.3)
	Test item	1000	3.9	(1.9)		0.8	(0.3)	24
		2000	7.1	(2.6)		0.7	(0.2)
	Cyclophosphamide	15	32.2	(5.2)	***	0.7	(0.2)
Male
	Vehicle	-	2.7	(1.1)		0.6	(0.1)
								48
	Test item	2000	2.0	(1.0)		0.5	(0.1)
Female
	Vehicle	-	2.9	(1.6)		1.3	(0.4)
	Test item	2000	2.6	(1.3)		0.9	(0.2)	48

 

 

5 animals per group

Doses frequency: one administration

 

Route: oral

Vehicle: 0.5% carboxymethylcellulose

 

 

MPE:Micronucleated Polychromatic Erythrocytes PE: Polychromatic Erythrocytes

NE:Normochromatic Erythrocytessd: standard deviation

 

Statisticaltestsused:                                    *** p <0.001The 2 x 2 contingency table for MPE Student 's"t" test for PE/NEratio

Mann-Whitney test

Table 7: Results of the first cytogenetic test: individual values

 

24 hours after treatment

Vehicle                                                        Test substance (500mg/kg)

   sex   slideMPE/2000PE   PE    NE Ratio           sex   slideMPE/2000PE      PE    NERatio

Male	76	4	376	624	0.6		Male	98	7	344	656	0.5
	91	2	356	644	0.6			86	9	541	459	1.2
	89	2	327	673	0.5			100	4	453	547	0.8
	103	4	387	613	0.6			85	3	330	670	0.5
	72	0	278	722	0.4			99	4	364	636	0.6
mean		2.40			0.53		mean		5.40			0.72
sd		1.67			0.10		sd		2.51			0.29
Female	40	10	566	434	1.3		Female	49	13	312	688	0.5
	13	9	456	544	0.8			37	5	496	504	1.0
	29	10	449	551	0.8			26	5	393	607	0.6
	54	17	374	626	0.6			45	15	349	651	0.5
	07	3	431	569	0.8			21	10	200	800	0.3
mean		9.80			0.86		mean		9.60			0.57
sd		4.97			0.26		sd		4.56			0.27

Test item(1000mg/kg)                                                   Test item (2000mg/kg)

   sex   slideMPE/2000PE   PE    NE Ratio           sex   slideMPE/2000PE      PE    NERatio

Male	83	0	362	638	0.6		Male	97	10	498	502	1.0
	71	1	358	642	0.6			101	6	323	677	0.5
	92	1	373	627	0.6			80	0	586	414	1.4
	87	6	378	622	0.6			73	1	415	585	0.7
	82	1	450	550	0.8			78	2	562	438	1.3
mean		1.80			0.63		mean		3.80			0.98
sd		2.39			0.11		sd		4.15			0.39
Female	33	6	426	574	0.7		Female	10	12	332	668	0.5
	42	12	429	571	0.8			65	20	431	569	0.8
	64	10	562	438	1.3			18	12	468	532	0.9
	04	2	266	734	0.4			53	19	366	634	0.6
	59	9	453	547	0.8			35	8	409	591	0.7
mean		7.80			0.79		mean		14.20			0.68
sd		3.90			0.33		sd		5.12			0.15

Cyclophosphamide (15 mg/kg)

   sex   slideMPE/2000PE   PE    NERatio

Male	74	56	321	679	0.5
	84	87	272	728	0.4
	105	72	330	670	0.5
	94	56	354	646	0.5
	104	69	393	607	0.6
mean		68.00			0.51	Route: oral
sd		12.90			0.10	Vehicle: 0.5% carboxymethylcellulose
Female	46	56	408	592	0.7
	22	65	501	499	1.0
	70	52	409	591	0.7	MPE:MicronucleatedPolychromatic Erythrocytes
	67	74	423	577	0.7	PE: Polychromatic Erythrocytes
	11	75	349	651	0.5	NE:NormochromaticErythrocytes
mean		64.40			0.73	sd: standard deviation
sd		10.36			0.17

Table 7 (continued)

 

48 hours after treatment

Vehicle                                                           Test item (2000mg/kg)

   sex  slideMPE/2000PE   PE    NE Ratio         sex    slideMPE/2000PE   PE    NERatio

Male	88	6	326	674	0.5		Male	77	5	263	737	0.4
	79	8	317	683	0.5			95	1	295	705	0.4
	93	2	396	604	0.7			90	5	301	699	0.4
	75	5	441	559	0.8			102	6	376	624	0.6
	96	6	316	684	0.5			81	3	338	662	0.5
mean		5.40			0.57		mean		4.00			0.46
sd		2.19			0.15		sd		2.00			0.09
Female	52	7	551	449	1.2		Female	30	4	485	515	0.9
	36	2	482	518	0.9			05	5	413	587	0.7
	41	3	504	496	1.0			44	9	465	535	0.9
	60	7	620	380	1.6			17	6	523	477	1.1
	50	10	636	364	1.7			28	2	523	477	1.1
mean		5.80			1.31		mean		5.20			0.94
sd		3.27			0.36		sd		2.59			0.17

 

 

Route: oral

Vehicle: 0.5% carboxymethylcellulose

 

 

MPE:MicronucleatedPolychromatic Erythrocytes PE: Polychromatic Erythrocytes

NE:NormochromaticErythrocytessd: standard deviation

Table 8: Results of the confirmatory cytogenetic test: data summary

 

 

Second experiment

Doses     MPE/1000PE                     PE/NE ratio                Time of sacrifice

Group (mg/kg)	mean	(sd)	mean	(sd)	after the last administration
Vehicle                0	1.4	(1.0)	0.7	(0.5)
Testitem            2000	2.7	(1.0)	0.4	(0.1)
Cyclophosphamide      15	22.4	(10.3)***	0.5	(0.2)	24 h
Five females per group

 

Doses frequency: one administration Route: oral

Vehicle: 0.5% carboxymethylcellulose

 

MPE:MicronucleatedPolychromatic Erythrocytes PE: Polychromatic Erythrocytes

NE:NormochromaticErythrocytessd: standard deviation

 

Statisticaltestsused:                                      *** p <0.001For MPE: 2 x 2 contingencytable

Student's "t" test for PE/NE ratio

Table 9: Results of the confirmatory cytogenetic test: individual values

 

Vehicle                                                          Test item (2000mg/kg)

sex	slide	MPE/2000PE	PE	NE	Ratio		sex	slide	MPE/2000PE	PE	NE	Ratio
Female	135	2	601	399	1.5		Female	143	8	364	636	0.6
	139	1	368	632	0.6			137	6	230	770	0.3
	145	3	374	626	0.6			134	6	310	690	0.4
	142	2	159	841	0.2			141	4	310	690	0.4
	136	6	337	663	0.5			147	3	218	782	0.3
mean		2.80			0.68		mean		5.40			0.41
sd		1.92			0.49		sd		1.95			0.12

 

Cyclophosphamide (15 mg/kg)

sex	slide	MPE/2000PE	PE	NE	Ratio
Female	138	74	479	521	0.9
	146	38	338	662	0.5
	140	47	325	675	0.5
	144	48	247	753	0.3
	133	17	284	716	0.4
mean		44.80			0.53
sd		20.54			0.23

 

Route: oral

Vehicle: 0.5% carboxymethylcellulose

 

MPE:MicronucleatedPolychromatic Erythrocytes PE: Polychromatic Erythrocytes

NE:NormochromaticErythrocytessd: standard deviation

Table 10: Results of the second cytogenetic test: data summary

(mg/kg/day) 

          MPE/1000PE                         PE/NE ratio                                                         Time of sacrifice Group                                                                                                               after thelast

      mean         (sd)                      mean        (sd)                            administration

 

Males Vehicle	-	1.7	(1.5)		0.5	(0.2)
	500	1.7	(1.4)		0.5	(0.2)	24 h
Test item	1000	2.1	(1.6)		0.7	(0.6)
	2000	1.8	(1.2)		0.8	(0.4)
Cyclophosphamide	15 mg/kg	41.1	(8.7)	***	0.6	(0.2)
Females Vehicle	-	2.5	(2.0)		0.8	(0.4)
	500	1.8	(1.4)		0.9	(0.5)
Test item	1000	1.7	(1.0)		0.5	(0.2)	24 h
	2000	2.9	(0.7)		0.5	(0.2)
Cyclophosphamide	15 mg/kg	28.0	(8.9)	***	0.6	(0.3)

   

Five animals per group

(1) Dose frequency:

- vehicle and test item: oral route, two administrations separated by a 24-hourinterval

- cyclophosphamide: oral route, one administration Vehicle: 0.5 %carboxymethylcellulose

MPE:MicronucleatedPolychromatic Erythrocytes PE: Polychromatic Erythrocytes

NE:NormochromaticErythrocytes

 

statistical testsused :

X ² test for MPE

Student's "t" test forPE/NEratio                           *** p <0.001

Table 11: Results of the second cytogenetic test: individual values

 

Vehicle                                                                      Test item (500mg/kg/day)     sex  slide MPE/2000PE   PE    NE Ratio          sex  slide MPE/2000PE   PE       NERatio
Male	161	2	330	670	0.5		Male	196	8	351	649	0.5
	151	8	422	578	0.7			150	3	446	554	0.8
	182	1	398	602	0.7			166	0	227	773	0.3
	167	1	216	784	0.3			172	3	247	753	0.3
	173	5	361	639	0.6			154	3	306	694	0.4
mean		3.40			0.54		mean		3.40			0.48
sd		3.05			0.18		sd		2.88			0.21
Female	159	6	466	534	0.9		Female	165	7	326	674	0.5
	181	1	215	785	0.3			158	6	330	670	0.5
	197	11	569	431	1.3			174	1	400	600	0.7
	175	2	467	533	0.9			189	3	636	364	1.7
	190	5	404	596	0.7			168	1	511	489	1.0
mean		5.00			0.80		mean		3.60			0.89
sd		3.94			0.38		sd		2.79			0.53
Test item(1000mg/kg/day)                                      Test item (2000mg/kg/day)     sex  slide MPE/2000PE   PE    NE Ratio          sex  slide MPE/2000PE   PE       NERatio
Male	183	1	383	617	0.6		Male	195	3	431	569	0.8
	176	9	641	359	1.8			185	2	431	569	0.8
	160	4	275	725	0.4			149	5	361	639	0.6
	153	5	301	699	0.4			194	1	312	688	0.5
	164	2	347	653	0.5			171	7	581	419	1.4
mean		4.20			0.75		mean		3.60			0.78
sd		3.11			0.59		sd		2.41			0.36
Female	148	1	321	679	0.5		Female	179	6	292	708	0.4
	180	3	205	795	0.3			155	6	449	551	0.8
	184	6	379	621	0.6			193	5	335	665	0.5
	157	5	273	727	0.4			187	4	301	699	0.4
	188	2	409	591	0.7			169	8	319	681	0.5
mean		3.40			0.48		mean		5.80			0.53
sd		2.07			0.17		sd		1.48			0.17
Cyclophosphamide (15 mg/kg)     sex   slide  MPE/2000PE   PE     NERatio
Male	177	66	299	701	0.4
	162	99	313	687	0.5	Route: oral
	152	80	406	594	0.7	Vehicle: 0.5 % carboxymethylcellulose
	178	65	347	653	0.5	Vehicleandtestitem:twoadministrationsseparatedbya24-hourinterval
	163	101	451	549	0.8	Cyclophosphamide: one administration
mean		82.20			0.58
sd		17.31			0.17
Female	186	33	230	770	0.3
	191	68	487	513	0.9
	156	73	389	611	0.6	MPE:MicronucleatedPolychromatic Erythrocytes
	192	65	269	731	0.4	PE: Polychromatic Erythrocytes
	170	41	344	656	0.5	NE:NormochromaticErythrocytes
mean		56.00			0.56	sd: standard deviation
sd		17.80			0.26

 

Conclusions:

Under our experimental conditions, the test item Hydroxybenzomorpholine (A025) (batch No. 0508918) did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells either after a single oral administration, or after two oral administrations at a 24-hour interval, at the dose-levels of 500, 1000 and 2000 mg/kg.

Executive summary:

Hydroxybenzomorpholine has been investigated for the induction of micronuclei in bone marrow cells of rats.

Test concentrations were based on a preliminary toxicity test in which clinical signs and mortality was recorded for a period of 48 h. In the first experiment rats were exposed by gavage to single doses of 0, 500, 1000 and 2000 mg/kg bw hydroxybenzomorpholine. Bone marrow cells were collected 24 h or 48 h (highest dose and vehicle control only) after dosing. Additional rats were used in a confirmatory test (highest dose, vehicle control and positive control). Satellite rats allocated for determination of plasma level of hydroxybenzomorpholine (determined 0.5, 1 and 4 h after treatment) were incorporated.

In the second experiment rats were exposed by gavage to two doses 24 h apart of 0, 500, 1000 and 2000 mg/kg bw hydroxybenzomorpholine. Bone marrow cells were only collected 24 h after the second dose. Again satellite rats allocated for determination of plasma level of hydroxybenzomorpholine (determined 0.5, 1 and 4 h after treatment) were incorporated.

Toxicity and thus exposure of the target cells was determined by measuring the ratio between polychromatic and normochromatic erythrocytes (PCE/NCE ratio). Moreover, all animals were observed daily for clinical signs and mortality. Bone marrow preparations were stained and

examined microscopically for the PCE/NCE ratio and micronuclei. Negative and positive controls were in accordance with the OECD guideline.

Results

The results of the measurement of plasma levels in experiment 1 clearly demonstrated that animals were systemically exposed to hydroxybenzomorpholine. However, the PCE/NCE ratios of the treated groups were equivalent with those from the untreated controls. No mortality and no clinical signs were observed in the rats of either sex. Exclusively, in the female high dose group a slightly higher (but not statistically significant) frequency of micronucleated erythrocytes was found. In the additional confirmatory groups again a slightly higher frequency in micronucleated erythrocytes (again not statistically significant but slightly above historical data) was found.

In experiment 2 measurement of plasma levels again demonstrated the systemic exposure of the animals. The PCE/NCE ratios of all treated groups were equivalent with those from the untreated controls. No mortality and no clinical signs were observed in the rats of the low dose group. Rats

receiving the mid dose showed piloerection after the second treatment. At the high dose 1 of the 5 females was found dead 2 h after the second treatment and 1 out of 3 of the supplementary (back up) females 24 h after the second treatment. All rats showed hypoactivity after the first

treatment and pilo-erection was observed in all surviving rats after the second treatment. A biological relevant increase in micronucleated erythrocytes was not found in any of the groups treated with hydroxybenzomorpholine.

Since the findings in the high dosed females in the first could not be confirmed in the second experiment, the slightly higher frequency in micronucleated erythrocytes in the high dose females was not considered biologically relevant.

Conclusion

Under the experimental conditions used hydroxybenzomorpholine did not induce micronuclei in bone marrow cells of treated rats and, consequently, hydroxybenzomorpholine was not genotoxic (clastogenic and/or aneugenic) in bone marrow cells of rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Considering all the in vitro and in vivo mutagenic or clastogenic studies on somatic cells and CLP crtiteria of the Regulation (EC) No 1272/2008, it could be concluded that there is enough information to conclude on the absence of genotoxicity and no classifcation should apply.