2,2,4,4-tetramethyl-7-oxa-3,20-diazadispiro[5.1.11.2]henicosan-21-one hydrochloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009 -06-22 till 2009-08-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2,4,4-tetramethyl-7-oxa-3,20-diazadispiro[5.1.11.2]henicosan-21-one hydrochloride

EC Number:

277-620-5

EC Name:

2,2,4,4-tetramethyl-7-oxa-3,20-diazadispiro[5.1.11.2]henicosan-21-one hydrochloride

Cas Number:

73833-37-1

Molecular formula:

C22H40N2O2.xClH

IUPAC Name:

2,2,4,4-tetramethyl-7-oxa-3,20-diazadispiro[5.1.11.2]henicosan-21-one hydrochloride

Constituent 2

Reference substance name:

2,2,4,4-tetramethyl-7-oxa-3,20-diaza-dispiro-[5.1.11.2]-heneicosan-21-one hydrochloride

IUPAC Name:

2,2,4,4-tetramethyl-7-oxa-3,20-diaza-dispiro-[5.1.11.2]-heneicosan-21-one hydrochloride

Details on test material:

- Name of test material (as cited in study report): 2,2,4,4-Tetramethyl-7-oxa-3,20-diaza-dispiro-
[5.1.11.2]-heneicosan-21-one hydrochloride
- Physical state: solid/ white
- Analytical purity: 96.6 % (w/w)
- Lot/batch No.: DEF2036584
- Expiration date of the lot/batch: May 07, 2018
- Stability under test conditions: Not indicated
- Storage condition of test material: At room temperature
- Other: None

Method

Species / strain

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
Experiment II
without S9 mix:
Salmonella strains: 0.063; 0.21; 0.63; 2.1; 6.25; 20.83; 62.5; 208.5; and 625 µg/plate
E. coli: 0.21; 0.63; 2.1; 6.25; 20.83; 62.5; 208.5; 625 and 2500 µg/plate
With S9 mix
all strains: 0.21; 0.63; 2.1; 6.25; 20.83; 62.5; 208.5; 625 and 2500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: better than others

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation:
Precipitation of the test item (visible to the unaided eye) was observed in the overlay agar in the test tubes from 2500 µg/plate up to 5000 µg/plate in experiment I and at 2500 µg/plate in experiment II and on the incubated agar plates from 2500 µg/plate up to 5000 µg/plates in experiment I with and without metabolic activation. The undissolved particles had no influence on the data recording.

COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY:

The plates incubated with the test item showed reduced background growth at the following concentrations (µg/plate):
Strain Experiment I Experiment II
without S9 mix with S9 mix without S9 mix with S9 mix
TA 1535 100 - 5000 333 - 5000 20.83 - 625 208.3 - 2500
TA 1537 100 - 5000 333 - 5000 62.5 - 625 208.3 - 2500
TA 98 100 - 5000 333 - 5000 62.5 - 625 208.3 - 2500
TA 100 100 - 5000 333 - 5000 20.83 - 625 208.3 - 2500
WP2 uvrA 1000 - 5000 1000 - 5000 208.3 - 2500 625 - 2500
Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups at the following concentrations (µg/plate):
Strain Experiment I Experiment II
without S9 mix with S9 mix without S9 mix with S9 mix
TA 1535 333 - 5000 333 - 5000 20.83 - 625 625 - 2500
TA 1537 333 - 5000 1000 - 5000 62.5 - 625 208.3 - 2500
TA 98 333 - 5000 333 - 5000 62.5 - 625 625 - 2500
TA 100 333 - 5000 333 - 5000 62.5 - 625 208.3 - 2500
WP2 uvrA 2500 - 5000 1000 - 5000 625 - 2500 2500

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Tabellen

 Summary of Results Pre-Experiment and Experiment I

Study Name: 1268400	Study Code: Harlan -CCR 1268400
Experiment: 1268400 VV Plate	Date Plated: 22/06/2009
Assay Conditions:	Date Counted: 25/06/2009

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO			13 ± 3	12 ± 3	35 ± 4	138 ± 5	55 ± 9
	Untreated			12 ± 4	10 ± 3	33 ± 6	141 ± 18	65 ± 24
	2,2,4,4-tetramethyl-7-oxa-3,20-diazadispiro[5.1.11.2]henicosan-21-one hydrochloride	3 µg		13 ± 1	13 ± 4	30 ± 4	143 ± 10	63 ± 10
		10 µg		13 ± 1	12 ± 6	33 ± 4	138 ± 3	57 ± 7
		33 µg		12 ± 2	14 ± 1	33 ± 10	144 ± 7	60 ± 9
		100 µg		8 ± 1^R	10 ± 4^R	26 ± 6^R	81 ± 3^R	56 ± 7
		333 µg		1 ± 1^M R	1 ± 1^M R	10 ± 3^R	16 ± 3^M R	49 ± 8
		1000 µg		0 ± 0^R	0 ± 0^R	3 ± 1^M R	10 ± 5^M R	36 ± 6^R
		2500 µg		0 ± 0^R P	0 ± 0^P R	0 ± 0^P R M	0 ± 0^P M R	2 ± 1^P M R
		5000 µg		0 ± 0^R P M	0 ± 0^P R M	0 ± 0^P M R	0 ± 0^P M R	1 ± 1^P M R
	NaN3	10 µg		2107 ± 188			1986 ± 24
	4-NOPD	10 µg				321 ± 3
	4-NOPD	50 µg			99 ± 33
	MMS	3.0 µL						1047 ± 11
With Activation	DMSO			19 ± 5	17 ± 2	43 ± 10	167 ± 17	69 ± 4
	Untreated			22 ± 8	18 ± 6	41 ± 7	171 ± 6	64 ± 20
	2,2,4,4-tetramethyl-7-oxa-3,20-diazadispiro[5.1.11.2]henicosan-21-one hydrochloride	3 µg		19 ± 5	16 ± 1	37 ± 1	151 ± 3	69 ± 17
		10 µg		19 ± 3	15 ± 2	39 ± 6	134 ± 7	72 ± 4
		33 µg		30 ± 10	16 ± 4	42 ± 2	142 ± 14	68 ± 5
		100 µg		21 ± 8	15 ± 4	40 ± 3	134 ± 21	69 ± 7
		333 µg		6 ± 3^R	8 ± 3^R	18 ± 4^R	8 ± 2^R M	68 ± 18
		1000 µg		1 ± 1^M R	2 ± 0^M R	3 ± 1^M R	3 ± 1^M R	28 ± 8^R
		2500 µg		0 ± 0^R P	0 ± 0^P R	1 ± 0^P R	0 ± 0^P M R	1 ± 1^P M R
		5000 µg		0 ± 0^R P M	0 ± 0^P R M	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R
	2-AA	2.5 µg		361 ± 39	181 ± 54	1504 ± 65	1786 ± 132
	2-AA	10.0 µg						241 ± 24

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	R M P	Reduced background growth Manual count Precipitate

 Summary of Results Experiment II

Study Name: 1268400	Study Code: Harlan -CCR 1268400
Experiment: 1268400 HV2 Pre	Date Plated: 02/07/2009 / 29/07/2009*
Assay Conditions:	Date Counted: 08/07/2009 / 05/08/2009*

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO			16 ± 4	8 ± 2	31 ± 3	131 ± 18	54 ± 3
	Untreated			12 ± 3	14 ± 7	34 ± 10	145 ± 8	64 ± 9
	2,2,4,4-tetramethyl-7-oxa-3,20-diazadispiro[5.1.11.2]henicosan-21-one hydrochloride	0.063 µg		16 ± 4	10 ± 2	33 ± 7	125 ± 19
		0.21 µg		17 ± 3	13 ± 1	26 ± 6	119 ± 6	48 ± 13
		0.63 µg		15 ± 2	12 ± 3	32 ± 10	107 ± 2	52 ± 8
		2.1 µg		16 ± 4	9 ± 0	30 ± 1	117 ± 9	55 ± 12
		6.25 µg		13 ± 1	13 ± 3	31 ± 2	119 ± 9	52 ± 4
		20.83 µg		7 ± 0^R	10 ± 4	30 ± 4	63 ± 5^R	58 ± 4
		62.5 µg		4 ± 2^M R	3 ± 2^M R	5 ± 1^M R	17 ± 4^M R	54 ± 0
		208.3 µg		0 ± 0^R	0 ± 0^R	0 ± 0^R	0 ± 0^R	28 ± 7^R
		625 µg		0 ± 0^R	0 ± 0^R	0 ± 0^R	0 ± 0^R	23 ± 3^R
		2500 µg						2 ± 2^M R
	NaN3	10 µg		1749 ± 65			1749 ± 122
	4-NOPD	10 µg				452 ± 12
	4-NOPD	50 µg			99 ± 10
	MMS	3.0 µL						493 ± 76
With Activation	DMSO			23 ± 4	17 ± 3	35 ± 1*	138 ± 11	62 ± 5
	Untreated			21 ± 2	21 ± 1	38 ± 5*	129 ± 17	55 ± 2
	2,2,4,4-tetramethyl-7-oxa-3,20-diazadispiro[5.1.11.2]henicosan-21-one hydrochloride	0.21 µg		22 ± 4	19 ± 3	36 ± 2*	128 ± 13	66 ± 2
		0.63 µg		23 ± 3	20 ± 6	40 ± 5*	116 ± 9	56 ± 1
		2.1 µg		22 ± 2	17 ± 3	33 ± 2*	106 ± 10	60 ± 8
		6.25 µg		22 ± 2	17 ± 3	33 ± 3*	116 ± 12	64 ± 8
		20.83 µg		24 ± 4	17 ± 2	37 ± 7*	121 ± 9	64 ± 3
		62.5 µg		20 ± 4	17 ± 1	38 ± 10*	97 ± 9	57 ± 12
		208.3 µg		11 ± 3^R	3 ± 2^M R	20 ± 1^R*	21 ± 4^M R	55 ± 3
		625 µg		3 ± 2^M R	1 ± 1^M R	0 ± 0^M R*	9 ± 6^M R	42 ± 8^R
		2500 µg		1 ± 1^M R	0 ± 0^M R	0 ± 0^M R *	0 ± 0^M R	5 ± 2^M R
	2-AA	2.5 µg		245 ± 12	110 ± 22	1416 ± 220*	1332 ± 53
	2-AA	10.0 µg						349 ± 30

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	R M	Reduced background growth Manual count

* = repeated experiment

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without S9 mix

In conclusion, it can be stated that during the described mutage¬nicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with 2,2,4,4-Tetramethyl-7-oxa-3,20-diaza-dispiro-[5.1.11.2]-heneicosan-21-one hydrochloride at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher muta­tion rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease of induced revertant colonies.