trans-(5RS,6SR)-6-amino-2,2-dimethyl-1,3-dioxepan-5-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:

1986-11-21 to 1987-04-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Principles of method if other than guideline:

preincubation method

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

trans-(5RS,6SR)-6-amino-2,2-dimethyl-1,3-dioxepan-5-ol

EC Number:

419-050-3

EC Name:

trans-(5RS,6SR)-6-amino-2,2-dimethyl-1,3-dioxepan-5-ol

Cas Number:

79944-37-9

Molecular formula:

C7H15NO3

IUPAC Name:

6-amino-2,2-dimethyl-1,3-dioxepan-5-ol

Method

Target gene:

his locus

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 102

Remarks:

or E.coli WP2 uvr, were not tested as recommended by the former guideline

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain 6

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: S9-liver mix
- source of S9: S 9 was obtained from Organon Teknika Co., Oklahoma City, OK, USA
- method of preparation of S9 mix: The mix was derived from male Sprague—Dawley rats pretreated with Aroclor 1254 (Batch No. 08495; protein content 28.4 mg/mL; activity (37°C; pmoles/min/mg protein) ethoxy resorufin, 3846; pentoxy resorufin, 428; benzoxy resorufin, 2007).
- concentration or volume of S9 mix and S9 in the final culture medium: The components of the standard S 9 mix were 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate,
4 mM NADP, 100 mM sodium phosphate, pH 7.4 and S 9 in a concentration of 0.3 mL per mL of mix. The final amount on one plate was 0.5 mL.

Test concentrations with justification for top dose:

tested up to limit concentration: 0.05, 0.10, 0.25, 0.50, 1.00, 2.50, and 5.00 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO; phosphate buffered saline

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

N-dimethylnitrosamine

benzo(a)pyrene

cyclophosphamide

other: anthracene-2-amine, with metabolic acitvation, 5 µg/plate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: one

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): E+06 dilution
- Test substance added as preincubation

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 72 h


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition

Rationale for test conditions:

as decribed in Ames et al. 1975

Evaluation criteria:

A positive response was considered if at least 5 mg/plate or up to a toxic dose had been tested (or the compound formed precipitates in the agar) and if the number of induced revertants compared to the spontaneous was higher than 2-fold. Also a dose dependent increase in the number of revertants was considered to indicate a mutagenic effect.
A toxic effect of the substance on the background lawn of non-revertant bacteria and precipitates in the agar were examined stereomicroscopically.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

TA 1535
Dose/Plate			REVERTANTS PER PLATE						QUOTIENT
			-S9	M	SD	+S9	M	SD	-S9	+S9
DMSO	0.1 mL		17	21	4	12	15	3	1.0	1.0
			24			18
			21			16
Phosphate buffer	0.1 mL		21	26	5	16	14	2	1.3	0.9
			27			12
			31			13
Test item	0.05 mg		19	18	1	11	12	1	0.9	0.8
			18			13
			18			12
	0.10 mg		23	20	3	18	17	2	1.0	1.1
			20			15
			17			18
	0.25 mg		26	19	7	15	13	4	0.9	1.2
			12			16
			20			22
	0.50mg		18	19	1	11	12	2	0.9	0.8
			20			11
			19			14
	1.0 mg		18	20	2	25	18	6	1.0	1.2
			21			14
			22			14
	2.5 mg		17	15	2	17	13	6	0.7	0.9
			15			7
			13			16
	5.0 mg		15	16	1	14	15	1	0.8	1.0
			17			15
			16			16
Anthracen-2-amine	5 µg		17	18	2	155	160	5	0.9	10.5
			18			165
			20			161
Cyclophosphamide	400 µg		60	59	4	676	702	25	2.8	45.8
			54			703
			62			726
Sodium azide	5 µg		613	639	24	27	32	5	30.9	2.1
			642			37
			661			31
TA 100
Dose/Plate			REVERTANTS PER PLATE						QUOTIENT
			-S9	M	SD	+S9	M	SD	-S9	+S9
DMSO	0.1 mL		37	91	3	85	88	3	1.0	1.0
			93			90
			93			90
Phosphate buffer	0.1 mL		96	95	3	132	116	16	1.0	1.3
			97			101
			92			115
Test item	0.05 mg		91	93	6	84	82	7	1.1	0.9
			100			88
			103			75
	0.10 mg		97	94	3	83	95	18	1.0	1.1
			93			86
			92			116
	0.25 mg		92	106	13	84	95	12	1.2	1.1
			100			108
			113			94
	0.50mg		95	101	8	113	104	8	1.1	1.2
			110			97
			98			102
	1.0 mg		83	89	6	97	96	5	1.0	1.1
			95			101
			89			91
	2.5 mg		89	99	11	85	84	5	1.1	1.0
			99			79
			110			89
	5.0 mg		83	85	5	96	92	15	0.9	1.0
			91			105
			82			76
Anthracene-2-amine	5 µg		120	114	6	456	510	65	1.2	5.8
			108			492
			113			582
DMNA	5 µL		117	126	9	1144	1092	69	1.4	12.4
			135			1013
			126			1113
Sodium azide	5 µg		903	888	19	111	115	10	9.8	1.3
			867			126
			893			107
TA 1537
Dose/Plate		REVERTANTS PER PLATE							QUOTIENT
			-S9	M	SD	+S9	M	SD	-S9	+S9
DMSO	0.1 mL		9	9	2	20	24	5	1.0	1.0
			8			29
			11			24
Phosphate buffer	0.1 mL		19	15	4	30	29	3	1.6	1.2
			13			25
			12			31
Test item	0.05 mg		15	11	4	24	24	3	1.1	1.0
			9			27
			8			21
	0.1 mg		12	12	2	13	17	5	1.3	0.7
			14			23
			10			15
	0.25 mg		4	11	7	18	21	4	1.2	0.9
			13			20
			17			25
	0.50mg		21	15	6	27	21	5	1.6	0.9
			12			17
			11			19
	1.0 mg		10	10	2	19	20	1	1.0	0.8
			11			20
			8			20
	2.5 mg		14	14	2	28	25	3	1.5	1.0
			16			22
			12			25
	5.0 mg		15	13	2	28	22	6	1.4	0.9
			13			16
			11			22
Anthracene-2-amine	5 µg		12	13	2	67	70	6	1.4	2.9
			13			77
			15			66
Benzo(a)pyrene	5 µg		10	8	2	73	67	11	0.9	2.8
			8			74
			7			54
9-acridineamine	100 µg		-	-	-	715	727	35	-	29.9
			-			767
			-			700
TA 1538
Dose/Plate			REVERTANTS PER PLATE						QUOTIENT
			-S9	M	SD	+S9	M	SD	-S9	+S9
DMSO	0.1 mL		13	13	1	38	32	6	1.0	1.0
			12			30
			13			27
Phosphate buffer	0.1 mL		16	16	1	24	28	6	1.3	0.9
			16			35
			17			24
Test item	0.05 mg		20	16	4	37	31	8	1.3	1.0
			15			22
			13			35
	0.10 mg		14	14	5	28	26	6	1.1	0.8
			19			31
			9			20
	0.25 mg		14	15	4	32	26	8	1.2	0.8
			19			17
			12			28
	0.50mg		12	15	6	23	25	4	1.2	0.8
			11			22
			21			30
	1.0 mg		10	9	3	28	26	4	0.7	0.8
			6			21
			12			28
	2.5 mg		8	11	4	23	31	7	0.9	1.0
			10			35
			15			19
	5.0 mg		13	11	6	25	24	4	0.9	0.7
			16			27
			5			553
Anthracene-2-amine	5 µg		11	15	5	546	531	32	1.2	16.8
			20			495
			14			81
Benzo(a)pyrene	10 µg		11	14	4	87	85	4	1.1	2.7
			14			88
			18			207
2-nitrofluorene	10 µg		1295	1239	49	209	207	2	97.8	6.5
			1213			205
			1208
TA 98
Dose/Plate			REVERTANTS PER PLATE						QUOTIENT
			-S9	M	SD	+S9	M	SD	-S9	+S9
DMSO	0.1 mL		31	29	3	40	31	10	1.0	1.0
			31			21
			25			32
Phosphate buffer	0.1 mL		43	33	10	40	40	12	1.1	1.3
			34			29
			23			52
Test item	0.05 mg		27	25	3	25	25	4	0.9	0.8
			26			22
			21			29
	0.10 mg		32	26	6	32	28	5	0.9	0.9
			22			20
			23			22
	0.25 mg		30	33	4	28	37	8	1.1	1.2
			31			29
			37			44
	0.50mg		29	28	6	43	32	9	1.0	1.0
			33			25
			21			29
	1.0 mg		21	27	12	33	29	7	0.9	0.9
			41			21
			20			33
	2.5 mg		30	28	5	29	32	3	1.0	1.0
			32			34
			22			33
	5.0 mg		25	22	3	25	27	6	0.8	0.9
			22			22
			19			33
Anthracene-2-amine	5 µg		31	25	8	506	485	23	0.9	15.7
			27			460
			16			490
Benzo(a)pyrene	10 µg		26	23	2	155	158	11	0.8	5.1
			22			171
			22			149
2-nitrofluorene	10 µg		1394	1327	65	213	222	11	45.7	7.2
			1321			234
			1265			219

Applicant's summary and conclusion

Conclusions:

None of the five tester strains TA 1535, TA 100, TA 1537, TA 1538 and TA 98 showed increased reversion to prototrophy in assays with Aminodioxepan at the concentrations tested between 0.05 and 5 mg/plate, either in the absence or presence of S 9 mix.

Executive summary:

The test item was examined for mutagenic activity in five histidine-dependent strains of Salmonella typhimurium (TA 1535 and TA 100 for detection of base-pair substitutions, TA 1537, TA 1538 and TA 98 for detection of frameshift mutations) using the modified Ames test (preincubation for 60 min at 37°C). The studies, which were conducted in the presence and absence of an activating system derived from Aroclor 1254 induced rat liver (S9 mix), employed a range of Aminodioxepan concentrations from 0.05 to 5 mg per plate.

Negative controls and positive controls with known mutagens (9-acridinamine, anthracene-2-amine, benzo(a)pyrene, cyclophosphamide, 2-nitrofluorene, N-nitrosodimethylamine, sodium azide) produced the expected numbers of revertant colonies. None of the five tester strains showed increased reversion to prototrophy with Aminodioxepan at the concentrations tested, either in the absence or presence of S 9 mix. Growth inhibition of the background lawn was not observed. There were no precipitates in the agar.

Evaluation of the data does not indicate that Aminodioxepan is a mutagen in the Ames Salmonella/microsome test using the preincubation modification.