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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

study conducted similar to OECD test guideline 471, result: negative (plate incorporation)

study conducted similar to OECD test guideline 471, result: negative (preincubation)

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:

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

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

Target gene:

his locus

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 / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

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

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.

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

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

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

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

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

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986-05-30 to 1986-07-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Principles of method if other than guideline:

plate incorporation method

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his locus

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 102

Remarks:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: S 9 was obtained from Litton Bionetics, Charleston, SC
- method of preparation of S9 mix: derived from male Sprague—Dawley rats pretreated with Aroclor 1254 (Batch No. 06585; protein content 31.9 mg/mL; activity (37°C; pmoles/min /mg protein) ethoxy resorufin (P448), 2383; pentoxy resorufin, 351; benzoxy resorufin, 848).
- concentration or volume of S9 mix and S9 in the final culture medium: The components of the standard S9 mix were 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 4 mM NADP, 100 mM sodium phosphate, pH 7.4 and S9 in a concentration of 0.1 mL per mL of mix. 0.5 mL of the mix were used for one plate.

Test concentrations with justification for top dose:

0.1, 0.25, 0.5, 1.0, 2.5, 5.0 mg/plate

Vehicle / solvent:

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

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

benzo(a)pyrene

cyclophosphamide

other: anthracene-2-amine, with metabolic activation 2 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 in agar (plate incorporation)

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 by Ames et al.

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.

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

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

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

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

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

TA 1535
Dose/Plate			REVERTANTS PER PLATE						QUOTIENT
			-S9	M	SD	+S9	M	SD	-S9	+S9
DMSO	0.1 mL		30	23	8	18	18	4	1.0	1.0
			25			22
			15			15
Phosphate buffer	0.1 mL		26	28	5	28	25	4	1.2	1.3
			34			21
			24			25
Test item	0.10 mg		21	23	3	11	13	4	1.0	0.7
			22			18
			26			10
	0.25 mg		16	21	5	9	12	4	0.9	0.7
			21			12
			25			16
	0.50mg		30	24	6	20	15	8	1.0	0.8
			23			6
			18			20
	1.0 mg		26	23	5	7	12	4	1.0	0.6
			25			13
			18			15
	2.5 mg		31	27	5	14	18	4	1.2	1.0
			29			18
			21			21
	5.0 mg		29	25	4	18	16	3	1.1	0.9
			22			16
			23			13
Anthracen-2-amine	2 µg		23	24	2	153	160	6	1.0	8.7
			26			163
			23			163
Cyclophosphamide	400 µg		62	63	2	206	185	23	2.7	10.1
			63			189
			65			161
Sodium azide	5 µg		713	707	6	234	245	13	30.3	13.4
			705			259
			702			242
TA 100
Dose/Plate			REVERTANTS PER PLATE						QUOTIENT
			-S9	M	SD	+S9	M	SD	-S9	+S9
DMSO	0.1 mL		107	100	7	94	97	3	1.0	1.0
			99			100
			93			96
Phosphate buffer	0.1 mL		103	105	2	95	102	12	1.1	1.1
			107			116
			105			95
Test item	0.10 mg		90	89	2	95	101	11	0.9	1.0
			91			114
			87			95
	0.25 mg		84	92	9	101	94	9	0.9	1.0
			92			97
			101			83
	0.50mg		105	94	10	88	92	5	0.9	1.0
			91			97
			86			91
	1.0 mg		116	101	15	93	91	14	1.0	0.9
			87			104
			100			76
	2.5 mg		106	115	13	100	100	6	1.2	1.0
			130			94
			110			106
	5.0 mg		116	119	3	106	105	3	1.2	1.1
			121			107
			121			102
Anthracene-2-amine	2 µg		107	113	5	1433	1421	76	1.1	14.7
			115			1490
			116			1339
Benzo(a)pyrene	5 µg		106	97	8	600	573	36	1.0	5.9
			92			587
			93			532
Sodium azide	5 µg		968	1004	56	361	356	5	10.1	3.7
			1068			357
			975			351
TA 1537
Dose/Plate		REVERTANTS PER PLATE							QUOTIENT
			-S9	M	SD	+S9	M	SD	-S9	+S9
DMSO	0.1 mL		19	17	3	17	20	3	1.0	1.0
			14			22
			18			21
Test item	0.1 mg		15	17	3	28	22	6	1.0	1.1
			15			17
			20			22
	0.25 mg		17	17	2	25	21	4	1.0	1.1
			15			18
			18			20
	0.50mg		18	17	3	27	22	5	1.0	1.1
			14			18
			20			20
	1.0 mg		15	16	4	23	23	2	1.0	1.2
			21			21
			13			25
	2.5 mg		14	18	4	24	21	3	1.1	1.0
			20			18
			21			20
	5.0 mg		17	17	3	24	22	2	1.0	1.1
			14			23
			19			20
Anthracene-2-amine	2 µg		10	14	5	110	123	13	0.8	6.1
			14			123
			19			135
Benzo(a)pyrene	5 µg		14	16	2	84	86	9	1.0	4.3
			18			78
			17			95
9-acidineamine	100 µg		1065	1030	96	-	-	-	60.6	-
			1104			-
			922			-
TA 1538
Dose/Plate			REVERTANTS PER PLATE						QUOTIENT
			-S9	M	SD	+S9	M	SD	-S9	+S9
DMSO	0.1 mL		19	18	4	31	31	1	1.0	1.0
			22			31
			14			30
Test item	0.10 mg		12	15	3	30	30	4	0.8	1.0
			15			34
			18			27
	0.25 mg		11	12	3	27	28	7	0.7	0.9
			10			35
			15			22
	0.50mg		16	15	2	30	33	3	0.8	1.1
			16			36
			12			34
	1.0 mg		19	22	4	27	30	6	1.2	1.0
			27			36
			20			26
	2.5 mg		17	16	5	35	39	5	0.9	1.3
			10			38
			19			44
	5.0 mg		17	15	5	43	36	7	0.8	1.2
			10			29
			19			36
Anthracene-2-amine	2 µg		17	19	3	1558	1468	82	1.1	47.9
			22			1399
			19			1446
Benzo(a)pyrene	10 µg		12	16	4	133	138	5	0.9	4.5
			20			143
			15			139
2-nitrofluorene	10 µg		1474	1493	21	435	457	24	81.4	14.9
			1489			453
			1516			482
TA 98
Dose/Plate			REVERTANTS PER PLATE						QUOTIENT
			-S9	M	SD	+S9	M	SD	-S9	+S9
DMSO	0.1 mL		21	19	7	33	40	8	1.l0	1.0
			25			38
			11			48
Test item	0.10 mg		22	21	4	42	36	6	1.1	0.9
			17			36
			24			30
	0.25 mg		14	22	7	41	33	12	1.1	0.8
			23			39
			28			20
	0.50mg		22	18	4	33	33	2	0.9	0.8
			14			32
			17			35
	1.0 mg		23	24	3	33	36	3	1.3	0.9
			22			37
			28			38
	2.5 mg		20	22	7	33	36	4	1.2	0.9
			17			34
			30			40
	5.0 mg		23	25	2	32	31	3	1.3	0.8
			27			28
			24			33
Anthracene-2-amine	2 µg		19	20	2	1110	1168	77	1.0	20.5
			22			1139
			18			1256
Benzo(a)pyrene	10 µg		21	20	3	276	282	17	1.0	7.1
			16			269
			22			301
2-nitrofluorene	10 µg		1207	1174	55	440	422	16	61.8	10.6
			1111			411
			1205			415

Conclusions:

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

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 direct plate incorporation procedure developed by Ames et al.

The studies, which were conducted in the absence and presence of an activating system derived from Aroclor 1254 induced rat liver (S 9 mix), employed a range of Aminodioxepan concentrations from 0.1 to 5 mg per plate.

Negative controls and positive controls with known mutagens (9-acridinamine, anthracene-2-amine, benzo(a)pyrene, cyclophosphamide, 2-nitrofluorene, sodium azide) produced the expected numbers of revertant colonies. None of the five tester strains showed increased reversion to prototrophy with test item at the concentrations tested either in the presence or absence 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

study conducted similar to OECD test guideline 474, result: negative

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 1993 to July 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

26 May 1983

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse was chosen as test animal since a wide range of experience with regard to micronucleus tests exists and since the mouse has been recommended by national and international authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzheim, FRG
- Age at study initiation: adult, 8-9 weeks
- Weight at study initiation: 25-35 and 25-29 g for males and females, respectively
- Fasting period before study: 17-21 h
- Housing: housed individually in Macrolon type ll cages containing wood-chip bedding.
- Diet (e.g. ad libitum): ad libitum, (Altromin®R; Altromin, Lage, FRG)
- Water (e.g. ad libitum): ad libitum, mildly acidified demineralized water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23
- Humidity (%): 46-50
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used:physiological saline
- Concentration of test material in vehicle: 50, 100 and 200 mg/mL

Duration of treatment / exposure:

treated once and killed after 24 and 48 h

Frequency of treatment:

once

Dose / conc.:

2 000 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

500 mg/kg bw (total dose)

No. of animals per sex per dose:

10-13 per treatement and 10 in the negative control group and 5 in the positive control group

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): Cyclophosphamide, a known mutagen, was used as a positive control.
- Route of administration: intraperitoneal
- Doses / concentrations: 30 mg/kg bw

Tissues and cell types examined:

bone marrow from femurs

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: lt was assumed from an acute toxicity study in male mice that 2 g/kg would be a dose level at which toxic effects might be noted (at 2g/kg all animals showed moderate apathy).

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
5 males and 5 females from each of the negative control and the treatment groups were killed by carbon dioxide asphyxiation ca. 24 or 48 hours after treatment (the positive control animals were killed 24 hours after treatment) and both femurs were dissected out from each animal.

DETAILS OF SLIDE PREPARATION:
The bone marrow was flushed/aspirated into fetal calf serum. The resulting cell suspensions were centrifuged and smears were prepared from drops of the cell pellets which had been resuspended in a few drops of serum. The slides were air-dried and stained using May-Gruenwald and Giemsa solutions.

METHOD OF ANALYSIS:
The slides were coded and analysed "blind" in random order. The stained smears were examined using oil immersion high power magnification in regions where cells were well-spread and stained. The slides were examined for the incidence of micronucleated cells per 2000 polychromatic (POE) and 1000 normochromatic (NCE) erythrocytes per animal. The ratio of polychromatic to normochromatic erythrocytes was calculated on the basis of 1000 NCE scored.

Statistics:

The statistical analysis was conducted for each of the following variables:
p1: proportion of micronucleated PCE
p2: proportion of micronucleated NOE
p3: ratio of PCE/NCE
For investigation of treatment differences the variables p1 and p2- were arc sin transformed. The analyses were conducted separately for the sample times. Regarding the first sample time one-sided t-tests were performed to assess the difference between positive and negative controls with pooled values for both sexes; the positive control group was then excluded from further analysis. Thereafter in a two-factorial ANOVA (factors "sex", treatment") for each sample time it was investigated as to whether any treatment effect was present. In case of significant interactions, comparisons between the control and each of the treatment groups were conducted separately for each sex. Where no significant interactions occurred but a global treatment effect, comparisons were performed with values pooled for both sexes. The pair-wise comparisons were performed with one-sided t-tests (increase of p1 and D2, decrease of p3), using the error estimate of the ANOVA table. The test levels were always or = 0.05 (least significant differences test-LSD). In addition to the non-transformed individual data the arithmetic means and standard deviations were included.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

in the high dose group

Vehicle controls validity:

valid

Positive controls validity:

valid

After application of the high dose only 1 male and 2 females out of 13 males and 13 females survived the first day. Therefore, only two dose groups could be evaluated. Signs of slight toxicity could be observed in 4 out of 10 males and 4 out of 10 females in the mid dose group.
Regarding micronucleated PCE and NCE counts as well as the ratio POE/NCE, there were no biologically relevant or statistically significant differences (p > 0.05) at any sample time. The positive control group showed a significant increase in micronucleated POE and NCE counts (p < 0.05).

 

MICRONUCLEUS ASSAY RESULTS

MICRONUCLEI (IN PER MILL) SCORED PER TWO THOUSAND POLYCHROMATIC AND ONE THOUSAND ERYTHROCYTES WITH RATIO PCE/NCE

Negative Control 10 mL/kg bw
24 h post application				48 h post application
No.	PCE (M)	NCE (M)	Ratio PCE/NCE	No.	PCE (M)	NCE (M)	Ratio PCE/NCE
Male				Male
1	1.00	1.00	0.97	6	1.00	1.00	0.92
2	0.50	1.00	0.96	7	0.00	1.00	1.14
3	1.00	1.00	0.96	8	1.00	1.00	1.12
4	0.50	0.00	0.98	9	1.50	1.00	1.02
5	1.50	1.00	0.90	10	1.50	1.00	1.11
MV=	0.90	0.80	0.95	MV=	1.00	1.00	1.06
SD=	0.42	0.45	0.03	SD=	0.61	0.00	0.09
N=	5	5	5	N=	5	5	5
Female				Female
1	0.50	0.00	0.95	6	1.00	2.00	0.94
2	1.00	1.00	1.05	7	0.50	0.00	1.02
3	1.00	1.00	0.87	8	0.50	1.00	1.06
4	0.50	0.00	0.95	9	1.00	1.00	1.01
5	0.50	1.00	1.10	10	1.00	0.00	0.88
MV=	0.70	0.60	0.98	MV=	0.80	0.80	0.98
SD=	0.27	0.55	0.09	SD=	0.27	0.84	0.07
N=	5	5	5	N=	5	5	5
Male + Female				Male + Female
MV=	0.80	0.70	0.97	MV=	0.90	0.90	1.02
SD=	0.35	0.48	0.07	SD=	0.46	0.57	0.09
N=	10	10	10	N=	10	10	10
0.5 g/kg bw
24 h post application				48 h post application
No.	PCE (M)	NCE (M)	Ratio PCE/NCE	No.	PCE (M)	NCE (M)	Ratio PCE/NCE
Male				Male
1	1.00	2.00	0.96	6	1.00	1.00	1.07
2	0.50	0.00	0.96	7	0.50	1.00	1.06
3	0.50	0.00	1.15	8	0.50	0.00	1.14
4	1.00	1.00	1.00	9	1.00	1.00	1.04
5	1.50	1.00	1.09	10	1.00	0.00	0.92
MV=	0.90	0.80	1.03	MV=	0.80	0.60	1.05
SD=	0.42	0.84	0.08	SD=	0.27	0.55	0.08
N=	5	5	5	N=	5	5	5
Female				Female
1	0.50	2.00	0.98	6	1.00	1.00	1.10
2	1.00	0.00	1.00	7	0.50	0.00	0.91
3	0.50	1.00	0.94	8	0.50	1.00	1.06
4	1.00	1.00	0.96	9	0.50	1.00	0.94
5	0.50	0.00	0.90	10	0.50	1.00	1.13
MV=	0.70	0.80	0.96	MV=	0.60	0.80	1.03
SD=	0.27	0.84	0.04	SD=	0.22	0.45	0.10
N=	5	5	5	N=	5	5	5
Male + Female				Male + Female
MV=	0.80	0.80	0.99	MV=	0.70	0.70	1.04
SD=	0.35	0.79	0.07	SD=	0.26	0.48	0.09
N=	10	10	10	N=	10	10	10
1.0 g/kg bw
24 h post application				48 h post application
No.	PCE (M)	NCE (M)	Ratio PCE/NCE	No.	PCE (M)	NCE (M)	Ratio PCE/NCE
Male				Male
1	1.00	0.00	0.96	6	1.00	1.00	1.10
2	1.00	1.00	1.03	7	0.50	1.00	1.04
3	1.00	1.00	1.06	8	0.50	0.00	0.97
4	1.00	0.00	0.95	9	1.00	1.00	0.99
5	1.00	1.00	0.83	10	1.00	0.00	0.92
MV=	1.00	0.60	0.96	MV=	0.80	0.60	1.00
SD=	0.00	0.55	0.09	SD=	0.27	0.55	0.07
N=	5	5	5	N=	5	5	5
Female				Female
1	0.50	0.00	1.00	6	1.00	2.00	1.06
2	0.50	1.00	1.05	7	0.50	1.00	1.10
3	0.50	1.00	1.03	8	1.00	1.00	1.05
4	1.00	1.00	0.90	9	0.50	0.00	1.07
5	0.50	1.00	1.01	10	0.50	0.00	1.07
MV=	0.60	0.80	1.00	MV=	0.70	0.80	1.07
SD=	0.22	0.45	0.06	SD=	0.27	0.84	0.02
N=	5	5	5	N=	5	5	5
Male + Female				Male + Female
MV=	0.80	0.70	0.98	MV=	0.75	0.70	1.04
SD=	0.26	0.48	0.07	SD=	0.26	0.67	0.06
N=	10	10	10	N=	10	10	10
Cyclophosphamide 30 mg /kg bw
24 h post application
No.	PCE (M)	NCE (M)	Ratio PCE/NCE
Male
1	9.50	2.00	0.95
2	11.00	1.00	0.95
3	13.50	2.00	0.89
4	15.00	1.00	1.00
5	15.00	1.00	0.96
MV=	12.80	1.40	0.95
SD=	2.46	0.55	0.04
N=	5	5	5
Female
1	10.00	2.00	0.98
2	7.50	1.00	0.89
3	10.50	3.00	0.87
4	11.00	1.00	0.81
5	9.50	1.00	0.96
MV=	9.70	1.60	0.90
SD=	1.35	0.89	0.07
N=	5	5	5
Male + Female
MV=
SD=
N=	10	10	10
PCB = POLYCHROMATIC ERYTHROCYTES NCE = NORMOCHROMATIC ERYTHROCYTES PCE(M) = MICRONUCLEATED POLYCHROMATIC ERYTHROCYTES (IN PER MILL) NCE(H) = MICRONUCLEATED NORMOCHROMATIC ERYTHROCYTES (IN PER HILL) MV: MEAN VALUE SD: STANDARD DEVIATION N: NUMBER 0F ANIMALS PER GROUP

 

Conclusions:

Evaluation of the data showed that Aminodioxepan has no evidence of mutagenic potential, when administered intraperitoneally up to the toxic dose level of 1.0 g/kg in the mouse micronucleus test.

Executive summary:

In a NMRI mouse bone marrow micronucleus assay equivalent to OECD test guideline 474, (5 animals/sex/dose) were treated intraperitoneally with Aminodioxepan (100 % a.i.) at doses of 0, 500, 1000 and 2000 mg/kg bw. Bone marrow cells were harvested at 24 and 48 h post-treatment.  The vehicle was physiological saline.

There were signs of toxicity present during the study. After application of the high dose only 1 male and 2 females out of 13 males and 13 females survived the first day. Therefore, only two dose groups could be evaluated. Signs of slight toxicity could be observed in 4 out of 10 males and 4 out of 10 females in the mid dose group.

Regarding micronucleated PCE and NCE counts as well as the ratio POE/NCE, there were no biologically relevant or statistically significant differences (p > 0.05) at any sample time. The positive control group showed a significant increase in micronucleated POE and NCE counts (p < 0.05). Aminodioxepan was tested at an adequate dose (based on an acute toxicity study in mice). There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

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 direct plate incorporation procedure developed by Ames et al.

The studies, which were conducted in the absence and presence of an activating system derived from Aroclor 1254 induced rat liver (S 9 mix), employed a range of Aminodioxepan concentrations from 0.1 to 5 mg per plate.

Negative controls and positive controls with known mutagens (9-acridinamine, anthracene-2-amine, benzo(a)pyrene, cyclophosphamide, 2-nitrofluorene, sodium azide) produced the expected numbers of revertant colonies. None of the five tester strains showed increased reversion to prototrophy with test item at the concentrations tested either in the presence or absence 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.

 

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.

 

In a NMRI mouse bone marrow micronucleus assay equivalent to OECD test guideline 474, (5 animals/sex/dose) were treated intraperitoneally with Aminodioxepan (100 % a.i.) at doses of 0, 500, 1000 and 2000 mg/kg bw. Bone marrow cells were harvested at 24 and 48 h post-treatment.  The vehicle was physiological saline.

There were signs of toxicity present during the study. After application of the high dose only 1 male and 2 females out of 13 males and 13 females survived the first day. Therefore, only two dose groups could be evaluated. Signs of slight toxicity could be observed in 4 out of 10 males and 4 out of 10 females in the mid dose group.

Regarding micronucleated PCE and NCE counts as well as the ratio POE/NCE, there were no biologically relevant or statistically significant differences (p > 0.05) at any sample time. The positive control group showed a significant increase in micronucleated POE and NCE counts (p < 0.05). Aminodioxepan was tested at an adequate dose (based on an acute toxicity study in mice). There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.

Justification for classification or non-classification

Based on the available data, Aminodioxepan does not need to be classified for genotoxicity according to Regulation (EC) No. 1272/2008 (CLP).