Iron(1+), chloro[dimethyl 9,9-dihydroxy-3-methyl-2,4-di(2-pyridinyl-kN)-7-[(2-pyridinyl-kN)methyl]-3,7-diazabicyclo[3.3.1]nonane-1,5-dicarboxylate-kN3,kN7]-, chloride

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:

From 27 November 2003 to 12 March 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

No certificate of analysis was provided

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,5-dimethyl 3-methyl-9-oxo-2,4-bis(pyridin-2-yl)-7-[(pyridin-2-yl)methyl]-3,7-diazabicyclo[3.3.1]nonane-1,5-dicarboxylate dichloroiron hydrate

EC Number:

610-388-9

Cas Number:

478945-46-9

Molecular formula:

C28H31N5O6FeCl2

IUPAC Name:

1,5-dimethyl 3-methyl-9-oxo-2,4-bis(pyridin-2-yl)-7-[(pyridin-2-yl)methyl]-3,7-diazabicyclo[3.3.1]nonane-1,5-dicarboxylate dichloroiron hydrate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Supplied by the sponsor, sample number S2539801
- Expiration date of the lot/batch: not specified
- Purity test date:not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, in the dark
The characterisation, homogeneity and stability of the test sample were to be assessed in SEAC Study AC030449

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Test article solutions were prepared by dissolving Stainless E-700-2003 in sterile purified water, with the aid of vortexing, immediately prior to assay to give the maximum required treatment solution concentration. This solution was not filter-sterilised and further dilutions were made using purified water. The test article solutions were protected from light and used within approximately 6¼ hours of the initial formulation of the test article.

Method

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: Bacteria were taken from vials of frozen cultures, which had been checked for strain characteristics of histidine dependence, uvrB character, rfa character and resistance to ampicillin (TA98 and TA100) or ampicillin plus tetracycline (TA102).

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was prepared from male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

An initial toxicity Range-Finder Experiment was carried out in strain TA100 only, in the absence and presence of S-9, using final concentrations of Stainless E-700-2003 at 1.6, 8, 40, 200, 1000 and 5000 µg/plate, plus solvent and positive controls. Evidence of toxicity in the form of a marked decrease in revertant numbers (but no concurrent diminution in the background bacterial lawn) was observed following the top two dose treatments in the absence of S-9 and following the top dose treatment in the presence of S-9. This may be indicative of bacteriostasis.

Experiment 1 treatments of all the test strains in the presence of S-9 retained the same test doses as employed for the Range-Finder Experiment. Treatments of all test strains in the absence of S-9 were performed up to a maximum dose of 1000 µg/plate (an estimate of the lower limit of toxicity) using doses of 0.32, 1.6, 8, 40, 200 and 1000 µg/plate.

Experiment 2 treatments of all strains in the absence of S-9 were performed up to 1000 µ g/plate (an estimate of the lower limit of toxicity) using doses of 1.638, 4.096, 10.24, 25.6, 64, 160, 400 and 1000 µg/plate. Treatments of all strains in the presence of S-9 were performed up to 5000 µg/plate using doses of 8.192, 20.48, 51.2, 128, 320, 800, 2000 and 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: Not specified

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene for TA100, TA1535, TA1537, TA102, With metabolic activation ; Glutaraldehyde for TA102 strain, without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) Experiment 1 ; preincubation in experiment 2
- Cell density at seeding (if applicable): not specified

DURATION
- Preincubation period: 1 hour (Experiment 2)
- Exposure duration: 72 hours
- Fixation time (start of exposure up to fixation or harvest of cells): after 72 hours

NUMBER OF REPLICATIONS:triplicate or quintuplicate for negative control

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Colonies were counted electronically using a Seescan Colony Counter (Seescan Plc) or manually where confounding factors such as split agar, bubbles in the agar or a dirty plate affected the accuracy of the automated counter. The background bacterial lawn was inspected for signs of toxicity. Some treatment plates were counted manually for accuracy.

NUMBER OF CELLS EVALUATED: Not specified

DETERMINATION OF CYTOTOXICITY
- Method: Background bacterial lawn was inspected

Evaluation criteria:

The test article was considered to be mutagenic if:

-Dunnett's test gave a significant response (p < 0.01) and the data set(s) showed a significant dose correlation
-The positive responses described above were reproducible.

Statistics:

The m-statistic was calculated to check that the data were Poisson-distributed, and Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was checked by linear regression analysis.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

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

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

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

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

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

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

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

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

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:

RANGE-FINDING/SCREENING STUDIES:
An initial toxicity Range-Finder Experiment was carried out in strain TA100 only, in the absence and presence of S-9, using final concentrations of Stainless E-700-2003 at 1.6, 8, 40, 200, 1000 and 5000 µg/plate, plus solvent and positive controls. Evidence of toxicity in the form of a marked decrease in revertant numbers (but no concurrent diminution in the background bacterial lawn) was observed following the top two dose treatments in the absence of S-9 and following the top dose treatment in the presence of S-9. This may be indicative of bacteriostasis.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Lower Upper
TA98
546.2 1469.2
(293.7) (1614.0)
149.3 383.1
(122.7) (419.0)

TA100
201.7 637.8
(218.3) (745.7)
327.5 2147.8
-(72.6) (2121.9)

TA1535
220.9 605.3
(269.3) (640.0)
43.7 330.6
-(5.8) (303.0)

TA1537
54.1 313.1
-(3.4) (325.7)
48.1 503.2
-(76.7) (447.6)

TA102
144.0 548.9
(48.1) (511.6)
527.1 1497.1
(309.0) (1712.9)

- Negative (solvent/vehicle) historical control data:

Lower Upper
TA98
14.8 34.6
(10.5) (33.1)
20.2 39.6
(13.7) (43.3)

TA100
86.2 141.6
(81.6) (147.6)
88.2 157.8
(71.1) (163.9)

TA1535
13.4 23.8
(9.8) (25.4)
10.0 32.2
(8.7) (31.8)

TA1537
5.6 17.0
(3.4) (16.3)
7.4 20.6
(4.1) (19.9)

TA102
212.0 420.6
(105.9) (447.3)
209.6 461.5
(87.5) (485.1)


ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Background bacterial lawn was inspected

Remarks on result:

other: In experiment 1 and 2

Any other information on results incl. tables

Table1 : Range Finding study

Test strain: TA100 +S-9

 

Treatment (µg/plate)	Revertant  numbers/plate
Solvent - Water	124	143	136	143	126
1.6	157	150	128
8	142	116	136
40	150	128	128
200	130	126	134
1000	119	150	115
5000	20	44	34
Positive	1171	1441	1346

 

Treatment (µg/plate)	Mean (of)	N	Fold Increase	Standard Deviation	Correlation Coefficient		Slope of best fit	Dunnett's t value
Solvent - Water	134.40	5		9.07
1.6	145.00	3	1.08^	15.13	0.46	NS	6.63	0.95	NS
8	131.33	3	0.98	13.61	0.19	NS	-0.66	-0.30	NS
40	135.33	3	1.01	12.70	0.07	NS	-0.05	0.08	NS
200	130.00	3	0.97	4.00	0.23	NS	-0.03	-0.40	NS
1000	128.00	3	0.95	19.16	0.25	NS	-0.01	-0.63	NS
5000	32.67	3	0.24	12.06	0.94	NS	-0.02	-12.70	NS
Positive	1319.33	3	9.82	136.96
	M Statistic = 1.606

 

Key to significance:

 

*  p<0.05  **  p<0.01  ***  p<0.005 NS   notsignificantKeytopostfixes:

^      Maximumincreaseabovecontrol

 

Table2 :Range Finding Study Test strain: TA98 -S-9

 

Treatment(µg/plate)	Revertantnumbers/plate
Solvent - Water	23		27		20		16	22
0.32	28		26		24
1.6	25		18		24
8	16		15		23
40	21		20		21
200	20		28		33
1000	0	M	1	M	2	M
Solvent - DMSO	20		26		24		26	21
Positive	718		908		739

 

Treatment (µg/plate)	Mean (of)	N	Fold Increase	Standard Deviation	Correlation Coefficient		Slope of best fit	Dunnett'st value
Solvent - Water	21.60	5		4.04
0.32	26.00	3	1.20	2.00	0.58	NS	13.75	1.35	NS
1.6	22.33	3	1.03	3.79	0.01	NS	-0.06	0.24	NS
8	18.00	3	0.83	4.36	0.49	NS	-0.65	-1.18	NS
40	20.67	3	0.96	0.58	0.22	NS	-0.06	-0.25	NS
200	27.00	3	1.25^	6.56	0.38	*	0.02	1.56	NS
1000	1.00	3	0.05	1.00	0.81	NS	-0.02	-11.07	NS
Solvent - DMSO	23.40	5		2.79
Positive	788.33	3	33.69	104.16
	M Statistic = 0.746

 

Key to significance:

 

*  p<0.05        **  p<0.01         ***  p<0.005     NS   notsignificantKeytopostfixes:

M        Platecountedmanually

 

^         Maximumincreaseabovecontrol

 

 

Table3 :Summary of mean revertant colonies (-S-9) - Experiment 1

 

Substance	Dose Level µg/plate	TA98	TA100	TA1535	TA1537	TA102
		Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD
Solvent - Water	100 µl	22 ± 4	141 ± 8	14 ± 4	10 ± 2	265 ± 11
Stainless E-700-2003	0.32     1.6     8     40     200     1000	26 ± 2	139 ± 4	17 ± 3	11 ± 4	252 ± 30
		22 ± 4	141 ± 3	13 ± 4	12 ± 6	290 ± 12
		18 ± 4	145 ± 28	19 ± 7	11 ± 3	286 ± 4
		21 ± 1	131 ± 9	17 ± 3	13 ± 1	302 ± 20
		27 ± 7	129 ± 8	18 ± 2 (M)	15 ± 5	252 ± 44
		1 ± 1 (M)	1 ± 1	3 ± 3	0 ± 0 (M)	3 ± 4 (S)
Solvent - DMSO	100 µl	23 ± 3	NT	NT	11 ± 4	NT
Positive controls	Compound     Dose Level     Mean ± SD	2NF	NaN3	NaN3	AAC	GLU
		5 µg	2 µg	2 µg	50 µg	25 µg
		788 ± 104	742 ± 25	598 ± 25	231 ± 24	823 ± 16

 

SD      Standarddeviation

 

2NF    2-Nitrofluorene

NaN3 Sodium azide AAC   9-AminoacridineGLUGlutaraldehyde

 

S        Slight thinningofbackgroundlawn

M       Platescountedmanually

NT     Platesnottreated

 

Table 4:Summary ofmean revertant colonies (+S-9)-Experiment1

 

Substance	Dose Level µg/plate	TA98	TA100	TA1535	TA1537	TA102
		Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD
Solvent - Water	100 µl	28 ± 5	119 ± 13	18 ± 5	15 ± 2	315 ± 23
Stainless E-700-2003	1.6     8     40     200     1000     5000	28 ± 9	141 ± 3	24 ± 5	13 ± 5	271 ± 23
		26 ± 3	133 ± 8	18 ± 3	17 ± 3	289 ± 9
		28 ± 8	128 ± 19	23 ± 7	15 ± 3	307 ± 12
		30 ± 7	127 ± 12	20 ± 12	11 ± 4	324 ± 4
		24 ± 6	145 ± 17	23 ± 6	8 ± 5	225 ± 42
		14 ± 2	9 ± 5	3 ± 2	2 ± 2	69 ± 11
Solvent - DMSO	100 µl	29 ± 6	133 ± 16	23 ± 7	14 ± 5	317 ± 12
Positive controls	Compound     Dose Level     Mean ± SD	B[a]P	AAN	AAN	AAN	AAN
		10 µg	5 µg	5 µg	5 µg	20 µg
		190 ± 11	1445 ± 31	289 ± 35	334 ± 25	1854 ± 106

 

SD      Standarddeviation

 

B[a]P  Benzo[a]pyreneAAN          2-Aminoanthracene

Table5 :Summary of mean revertant colonies (-S-9) - Experiment 2

 

Substance	Dose Level µg/plate	TA98	TA100	TA1535	TA1537	TA102
		Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD
Solvent - Water	100 µl	25 ± 6	149 ± 7	20 ± 2	10 ± 4	435 ± 20
Stainless E-700-2003	1.638     4.096     10.24     25.6     64     160     400     1000	32 ± 7	171 ± 15	16 ± 5	10 ± 4	382 ± 41
		26 ± 2	167 ± 9	25 ± 1	17 ± 1	389 ± 29
		20 ± 8	157 ± 32	18 ± 3	14 ± 10	525 ± 69
		26 ± 8	156 ± 54	19 ± 5	13 ± 6	455 ± 21
		29 ± 16	144 ± 25	14 ± 5	12 ± 3	342 ± 97
		16 ± 5	146 ± 46	22 ± 7	11 ± 4	358 ± 107
		27 ± 8	117 ± 38 (S)	14 ± 2	9 ± 6	370 ± 16
		4 ± 3 (S)	28 ± 10 (S)	3 ± 2 (S)	1 ± 1 (S)	34 ± 27 (S)
Solvent - DMSO	50 µl	23 ± 5	NT	NT	12 ± 5	NT
Positive controls	Compound     Dose Level     Mean ± SD	2NF	NaN3	NaN3	AAC	GLU
		5 µg	2 µg	2 µg	50 µg	25 µg
		1691 ± 443	669 ± 48	679 ± 41	244 ± 50	645 ± 76

SD      Standarddeviation

2NF    2-Nitrofluorene

NaN3 Sodium azide AAC   9-AminoacridineGLUGlutaraldehyde

 

S        Slight thinningofbackgroundlawn

NT     Platesnottreated

 

Table6 :Summary of mean revertant colonies (+S-9)-Experiment2

 

Substance	Dose Level µg/plate	TA98	TA100	TA1535	TA1537	TA102
		Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD
Solvent - Water	100 µl	32 ± 6	163 ± 12	24 ± 8	18 ± 2	435 ± 33
Stainless E-700-2003	8.192     20.48     51.2     128     320     800     2000     5000	29 ± 3	132 ± 19	22 ± 14	19 ± 1	311 ± 40
		30 ± 3	154 ± 13	22 ± 6	10 ± 6	343 ± 13
		23 ± 3	151 ± 9	18 ± 2	14 ± 4	341 ± 25
		31 ± 8	177 ± 9	17 ± 2	12 ± 4	358 ± 41
		22 ± 6	159 ± 20	24 ± 6	9 ± 4	328 ± 6
		29 ± 6	169 ± 7	19 ± 8	13 ± 4	327 ± 34
		27 ± 5	155 ± 36	22 ± 5	13 ± 2	239 ± 40
		22 ± 7	56 ± 12 (S)	13 ± 3	2 ± 1	99 ± 44 (S)
Solvent - DMSO	50 µl	25 ± 9	141 ± 21	21 ± 5	14 ± 4	326 ± 67
Positive controls	Compound     Dose Level     Mean ± SD	B[a]P	AAN	AAN	AAN	AAN
		10 µg	5 µg	5 µg	5 µg	20 µg
		295 ± 41	787 ± 13	83 ± 7	115 ± 4	1403 ± 79

 

SD      Standard deviation

 

B[a]P  Benzo[a]pyreneAAN          2-Aminoanthracene

 

S        Slight thinningofbackgroundlawn

 

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of the study, the Stainless E-700-2003 did not induce mutation in five histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102)
These conditions included treatments at concentrations up to either 5000 µg/plate, or the lower limit of toxicity, in the absence and in the presence of a rat liver metabolic activation system (S-9).

Executive summary:

In this GLP-compliant sutdy, Stainless E-700-2003 was assayed for mutation in five histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments (performed according to OECD Guideline 471 method).

An initial toxicity Range-Finder Experiment was carried out in strain TA100 only, in the absence and presence of S-9, using final concentrations of Stainless E-700-2003 at 1.6, 8, 40, 200, 1000 and 5000 µg/plate, plus solvent and positive controls. Evidence of toxicity was observed following the top two dose treatments in the absence of S-9, and following the top dose treatment in the presence of S-9.

Experiment 1 treatments of all the test strains in the presence of S-9 retained the same test doses as employed for the Range-Finder Experiment. Treatments of all test strains in the absence of S-9 were performed up to a maximum dose of 1000 µg/plate (an estimate of  the  lower  limit  of  toxicity)  using  doses  0.32,  1.6,  8,  40,  200,  1000 µg/plate. Evidence of toxicity was observed following the top dose treatment of all strains in the absence and presence of S-9.

Experiment 2 treatments of all strains in the absence of S-9 were performed using a maximum test dose of 1000 µ g/plate (an estimate of the lower limit of toxicity) and doses of 1.638, 4.096, 10.24, 25.6, 64, 160, 400 and 1000 µ g/plate. Treatments of all strains in the presence of S-9 were performed up to  5000 µ g/plate  using doses  of 8.192, 20.48, 51.2, 128, 320, 800, 2000 and 5000 µ g/plate. In addition, pre-incubation methodology was used for all treatments in the absence and presence of S-9. Evidence of toxicity was observed following the top one or two dose treatments of all strains in the absence and presence of S-9, except strain TA98 in the presence of S-9 where no clear evidence of toxicity was seen.

No statistically significant, dose-related and reproducible increases in revertant numbers were observed following any strain treatments in the absence or presence of metabolic activation, and therefore this study was considered to have provided no evidence of any Stainless E-700-2003 mutagenic activity.

Under the experimental conditions of the study, the Stainless E-700-2003 did not induce mutation in five histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102)

These conditions included treatments at concentrations up to either 5000 µg/plate, or the lower limit of toxicity, in the absence and in the presence of a rat liver metabolic activation system (S-9).