Carbamic acid, [(1S)-2-[(2-aminoethyl)amino]-1-[(4-ethoxyphenyl)methyl]ethyl]-, phenylmethyl ester, dihydrochloride

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 March to April 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

impurity 1

Reference substance name:

n.a.

Molecular formula:

n.a.

IUPAC Name:

n.a.

impurity 2

impurity 3

Reference substance name:

Carbamic acid [(1S)-,2-[(2-aminoethyl)amino]-1-[(4-ethoxyphenyl)methyl]ethyl]-,phenylmethyl ester, dihydrochloride (9Cl)

IUPAC Name:

Carbamic acid [(1S)-,2-[(2-aminoethyl)amino]-1-[(4-ethoxyphenyl)methyl]ethyl]-,phenylmethyl ester, dihydrochloride (9Cl)

Constituent 1

Reference substance name:

benzyl[(2S)-1-[(2-aminoethyl)amino]-3-(4-ethoxyphenyl)propan-2-yl]carbamate dihydrochlorid

EC Number:

606-946-6

Cas Number:

221640-14-8

Molecular formula:

C21 H29 N3 O3 . 2 Cl H

IUPAC Name:

benzyl[(2S)-1-[(2-aminoethyl)amino]-3-(4-ethoxyphenyl)propan-2-yl]carbamate dihydrochlorid

Test material form:

solid

Method

Target gene:

Histidine gene locus

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not applicable

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male rat liver S9 mix

Test concentrations with justification for top dose:

0, 1.6, 5, 16, 50, 160, 500, 1600, 5000 µg/plate (first test = plate incorporation test; +/-S9 mix, all strains)
0, 1.6 (TA 1537, TA 102), 5, 16, 50, 160, 500 (TA 1535, TA 100, , TA 98; ) µg/plate (repeat test = Preincubation test, +/-S9 mix)

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Remarks:

No solvent control was used since sufficient evidence was available in the literature and from testing laboratory experience, indicating that the solvents used had no influence on the spontaneous mutant counts of the used strains.

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

mitomycin C

other: 4-nitro-1,2-phenylene diamine (TA 1537), 2-aminoanthracene (all strains), cumene hydroperoxide (all strains)

Remarks:

The positive controls sodium azide, nitrofluorene, 4-nitro-1,2-phenylene diamine and mitomycin C were only used without S9 mix; the positive control 2-aminoanthracene was only used with S9 mix.

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): The titel in the plate incorporation experiment ranged from 7.6E+08 to 20.6E+08 per mL and in the preincubation experiment from 4.9E+08 to 20.8E+08 per mL
- Test substance added in medium; in agar (plate incorporation); preincubation

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; other: The toxicity of the substance was assessed by gross appraisal of background growth on the plates for mutant determination. Moreover, a toxic effect of the substance was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the solvent controls.

Rationale for test conditions:

As recommended by the OECD test guideline

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA1535, TA100, TA1537 and TA98 this increase should be about twice that of solvent controls. For TA102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these criteria may be overruled by good scientific judgment. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Statistics:

not specified

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

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 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

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 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

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:

cytotoxicity

Vehicle controls validity:

not examined

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:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not reported
- Precipitation and time of the determination: no precipitates occurred

RANGE-FINDING/SCREENING STUDIES (if applicable): Due to the substance´s toxicity in the plate incorporation tests, doses ranging from 1.6 µg to 500 µg per plate were chosen for the independent repeat test (preincubation) and doses ranging from 1.6 µg to 16 µg per pakte were chosen in addition to the plate incorporation test. The plate first incorporation test was performed with 0, 50, 160, 500, 1600, 5000 µg/plate as recommended by the OECD test guideline.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: yes, please refer to the 'Additional information on results incl. tables' section.

Ames test:
- Signs of toxicity: yes, in the plate incorporation experiment toxicity occurred from 160 µg onwards to the top dose, thus the experiment was repeated with lower concentrations
- Mean number of revertant colonies per plate and standard deviation: yes, please refer to the 'Additional information on results incl. tables' section.



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: yes, please refer to the 'Additional information on results incl. tables' section.
- Negative (solvent/vehicle) historical control data: yes, please refer to the 'Additional information on results incl. tables' section.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1.1: Summary of results from the Salmonella mutagenicity assay (plate incorporation) with Z-Triamin-Dihydrochlorid (mean values of revertants per plate)

Dose (µg per plate)	Without metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
0	8	156	7	17	202
50	7	135	7	24	184
160	7	95	4	17	47
500	0	43	0	7	0
1600	0	0	0	0	0
5000	0	0	0	0	0
Positive control	622*	1222*	76*	1260*	933*
Dose ( µg per plate )	With metabolic activation (liver S9 mix)
	TA 1535	TA 100	TA 1537	TA 98	TA 102
0	11	228	11	32	299
50	9	208	10	29	276
160	8	170	11	29	265
500	5	100	3	12	0
1600	0	0	0	0	0
5000	0	0	0	0	0
Positive control	79*	2083*	136*	1989*	856*

* = mutagenic effect

Table 1.2: Summary of results from the Salmonella mutagenicity assay (plate incorporation) with additional concentrations of Z-Triamin-Dihydrochlorid (mean values of revertants per plate)

 

Dose (µg per plate)	Without metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
0	8	68	5	13	203
1.6	7	70	5	14	182
5	8	81	5	14	168
16	7	78	5	12	198
Positive control	549*	908*	67*	726*	671*
Dose ( µg per plate )	With metabolic activation (liver S9 mix)
	TA 1535	TA 100	TA 1537	TA 98	TA 102
0	9	142	7	24	301
1.6	10	155	6	25	307
5	9	134	6	29	312
16	10	124	6	24	290
Positive control	82*	1204*	58*	1350*	668*

Table 2: Summary of results from the Salmonella mutagenicity assay (Pre-Incubation) with Z-Triamin-Dihydrochlorid

(mean values of revertants per plate)

Dose (µg per plate)	Without metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
0	9	113	6	16	217
1.6	-	-	5	-	235
5	10	124	7	14	237
16	7	120	5	12	209
50	6	93	2	16	184
160	3	25	0	5	0
500	0	0	-	0	-
positive control	1088	1306	73	1257	434
Dose ( µg per plate )	With metabolic activation (liver S9 mix)
	TA 1535	TA 100	TA 1537	TA 98	TA 102
0	8	123	10	22	309
1.6	-	-	-	-	-
5	8	112	9	24	312
16	9	144	9	17	320
50	9	103	8	22	318
160	7	99	7	18	222
500	3	49	0	0	0
Positive control	134	1649	176	1194	550

* = mutagenic effect

The plate incorporation test, employing doses of up to 5000¿g per plate, showed the test item to produce strain-specific bacteriotoxic effects starting already at 160¿g/ plate. Therefore three additional concentrations (1.6, 5 and 16 µg/plate) were applied .The Salmonella/microsome test, using preincubation employing doses of up to 500¿g per plate, showed the test item to produce strain-specific bacteriotoxic effects at 50¿g per plate and above. Substance precipitation did not occur. Evidence of mutagenic activity of the test item was not seen. No biologically relevant increase in the mutant count, in comparison to the solvent controls, was observed in any of the strains tested, without and with S9 mix, in the plate incorporation as well in the preincubation modification, under the experimental conditions applied.

In spite of the low doses used, positive controls increased the mutant counts to well over those of the solvent controls, and thus demonstrated the system's high sensitivity.

Applicant's summary and conclusion

Conclusions:

The mutagenic potential of Z-Triamin-Dihydrochlorid was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471. Doses up to and including 5000 µg/plate were initially used. Due to strain-specific bacteriotoxic effects at concentrations >= 160 µg per plate the concentration range up to and including 500 µg/plate could only partly used for assessment purposes and the concentration range was supplemented by additional concentrations (1.6, 5 and 16 µg/plate). In an independent repeat using doses up to and including 500 µg/plate strain-specific bacteriotoxic effects occured at >=50 µg/plate. In both experiments, the employed positive controls had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding solvent controls. Evidence of mutagenic activity of the test substance was not seen. No biologically relevant increase in the mutant count compared to the corresponding solvent control was observed in any of the strains tested, without and with S9 mix. Therefore, the test item was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/mirosome test.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997), Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 were exposed to Z-Triamine Dihydrochloride in DMSO in concentrations of 0 (control), 50, 160, 500, 1600, and 5000 µg/plate in all strains in the absence and presence of mammalian metabolic activation (rat liver S9 mix). The assay was performed using the plate incorporation method.

The test substance was tested up to cytotoxic concentrations. Cytotoxic effects were noted in all strains from 160 µg/plate with/without metabolic activation on. Therefore a second experiment was conducted using the preincubation method with and without metabolic activation at the following concentrations: 0, 5, 16, 50, 160, 500 µg/plate for the tester srains TA98, TA100, TA1535 and TA1537. TA102 was treated with 0, 1.6, 5, 16, 50, 160, and 500 µg/plate in the presence and absence of S9 liver mix. Again, cytotoxicity occurred from 160 µg/plate onwards. Precipitation was not observed. The positive controls induced the appropriate responses in the corresponding strains. The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.

There was no evidence of an increase in the number of revertant colonies that exceeded twice background in any of the five tester strains (TA98, TA 100, TA102, TA1535or TA1537) examined at dose levels up to 5000 µg/plate in the absence and presence of a metabolic activation source (S9). Therefore, test substance was considered to be non-genotoxic (non-mutagenic) in Salmonella tester strains TA98, TA100, TA102, TA1535 and TA 1537 under the conditions employed (plate incorporation assay and preincubation method).

There was no evidence of induced mutant colonies over background.

Under the conditions of the study, the test substance was negative for mutagenic potential.