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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

247-415-5

EC Name:

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

Cas Number:

26021-57-8

Molecular formula:

C8H9NO2

IUPAC Name:

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

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

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

Method

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

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.

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Test results 6

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

Test results 7

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:

Any other information on results incl. tables

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

 

 

Applicant's summary and conclusion

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.