N'-(ethylcarbonimidoyl)-N,N-dimethylpropane-1,3-diamine monohydrochloride

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:

January 2012

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N'-(ethylcarbonimidoyl)-N,N-dimethylpropane-1,3-diamine monohydrochloride

EC Number:

247-361-2

EC Name:

N'-(ethylcarbonimidoyl)-N,N-dimethylpropane-1,3-diamine monohydrochloride

Cas Number:

25952-53-8

Molecular formula:

C8H17N3.ClH

IUPAC Name:

N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide hydrochloride

Test material form:

solid

Specific details on test material used for the study:

Identity: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
Synonyms: EDAC; EDC; EDC HCl; EPCI
CAS number 25952-53-8
Appearance of formulations: Clear colorless solutions (stock in water or DMSO)
Lot number: Y1553T
Description: White powder

Method

Target gene:

Histidine and Tryptophan

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 Fraction of liver homogenate

Vehicle / solvent:

Non-GLP analytical work conducted by the Sponsor demonstrated that EDAC reacts with DMSO forming a reaction product that increased with time; however, EDAC was stable in water. Based on this information, this in vitro bacterial mutagenicity test was conducted using both DMSO and water as vehicle solvents.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

The bacteria were originally supplied by Moltox, NC, USA. Each batch of frozen bacteria was tested for appropriate phenotype characteristics and spontaneous reversion rates; response to diagnostic mutagens is also routinely assessed. The following bacterial strains were employed:
S. typhimurium TA1535 hisG46 rfa uvrB
S. typhimurium TA1537 hisC3076 rfa uvrB
S. typhimurium TA98 hisD3052 rfa uvrB pKM101
S. typhimurium TA100 hisG46 rfa uvrB pKM101
E. coli WP2 trp uvrA

Fresh bacterial cultures were prepared and were in the late-log phase of growth at the time of use. The density of the cultures was confirmed to be ≥ 1000 x 106 bacteria/mL using a bacterial counting chamber before the cultures were used in the assay

Rationale for test conditions:

An initial test, a preliminary and confirmatory assay was carried out, with both vehicles and were performed on the same day using the same bacterial cultures and S9 mix preparations. The confirmatory assay used the same (pre-incubation) method as the preliminary assay,
except a narrower dose interval (2-fold) was employed to confirm results and resolve any issues encountered in the preliminary assay. The same high level stock 50 mg/mL solutions, water and DMSO, were used to prepare all respective lower dose formulations in the preliminry and confirmatory assays.
The supplemental test used the same conditions as the initial test with the exception that only strains TA100 and WP2 uvrA were used. Fresh high level stock 50 mg/mL solutions, water and DMSO, were used to prepare all respective lower dose formulations. The time from preparation of the formulations to completion of the dosing was less than 1 hour (less than 6 hours for the initial assay).

Evaluation criteria:

The mutagenic activity of the test article was assessed by applying the following criteria:
Positive: If treatment with the test article produced a dose-related increase in revertant colony numbers at least twice the concurrent vehicle control levels with bacterial strains TA98, TA100 and WP2 uvrA, (3-fold for TA1535 and TA1537) either in the presence or absence of S9 mix, the test article was considered to show evidence of mutagenic activity in the test system (provided mean value(s) lay outside the historical control range).

Negative: If treatment with the test article did not produce a dose-related increase in revertant colony numbers at least twice the concurrent vehicle controls levels with strains TA98, TA100, and WP2 uvrA (3-fold for strains TA1535 and TA1537) the test article was considered to show no evidence of mutagenic activity in the test system.

Equivocal: If the results obtained failed to satisfy the criteria for a clear “positive” or “negative” response, the results were considered equivocal. It was acceptable to conclude an equivocal response if no clear conclusion could be made. Note that the reproducibility of any apparent effect was taken into account in making any clear conclusion.

For an assay to be considered valid, the mean revertant colony counts of the vehicle controls for each strain had to lie close to or within the current historical control range of the laboratory. All positive control articles (with S9 where required) had to produce increases in revertant colony numbers indicative of a positive response. No invalid assay results were obtained in this study.

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDAC) was mutagenic in the absence of S9 activation when tested in accordance with regulatory guidelines.

Executive summary:

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDAC) was evaluated in a microbial mutagenicity study, using the pre-incubation method, to determine its potential to induce frameshift and/or base-pair substitution mutations in Salmonella typhimurium (histidine⎯), strains TA98, TA100, TA1535 and TA1537, and in Escherichia coli (tryptophan⎯), strain WP2 uvrA. Material safety data information provided to the Sponsor from Toyobo Co., LTD. and Osaka Synthetic Chemical Laboratories, Inc., EDAC vendors, classified EDAC as mutagenic. Sponsor review of an Ames report provided by Toyobo Co., LTD indicated that EDAC, formulated in dimethyl sulfoxide (DMSO), was mutagenic in the presence of S9 activation in both preliminary and confirmatory assays with strains TA100 (3.1-fold) and WP2 uvrA (3.3-fold); elevated responses, not satisfying the 2-fold threshold for a mutagenic response, were observed with these strains in the absence of S9 activation. Non-GLP analytical work conducted by the Sponsor demonstrated that EDAC reacts with DMSO forming a reaction product that increased with time; however, EDAC was stable in water. Based on this information, this in vitro bacterial mutagenicity test was conducted using both DMSO and water as vehicle solvents. Study design included an initial and supplementary trial conducted on different days and each trial consisting of a preliminary and confirmatory assay conducted on the same day. EDAC was dissolved and diluted in sterile water for irrigation USP (water) or DMSO in each of the 5 strains in preliminary and confirmatory assays, with and without exogenous metabolic activation. The metabolic activation system was the S9 fraction of a liver homogenate from rats treated previously with phenobarbital and 5,6-benzoflavone. In each test, the appropriate vehicle and positive controls were included, and cytotoxicity was evaluated as a partial or complete absence of the background lawn or a dose-related reduction in revertant colony counts. In the initial preliminary assays, EDAC was evaluated at 9 concentrations, formulated in water and DMSO, separately, ranging from 5.0 to 5000 µg/plate in triplicate cultures of each strain. The initial confirmatory assays evaluated EDAC, using the same method, at concentrations ranging from 19.5 to 5000 µg/plate. Dosing was performed up to nearly 6 hours following the preparation of the formulations in the initial test. In each assay, the highest 5 concentrations, minimally, of EDAC were assessed. A supplemental test was performed to evaluate if the results obtained in the initial test were influenced by the length of time that had elapsed between preparation of the dosing solutions and dosing completion. In the supplemental test, dosing was performed in less than 1 hour following the preparation of the formulations, EDAC in water and DMSO, individually. The supplemental test was conducted in an analogous manner as the initial test with preliminary and confirmatory assays conducted on the same day but only using strains TA100 and WP2 uvrA. EDAC was tested at 8 concentrations, in triplicate cultures, formulated in water and DMSO, separately, ranging from 15.8 to 5000 µg/plate and 39 to 5000 µg/plate for the preliminary and confirmatory assays, respectively. Precipitate was not observed for any strain at any concentration in the initial test or the supplemental test. Generally, EDAC formulated in DMSO was more toxic than when formulated in water. In the initial assay, toxicity was limited to strains TA1535 and TA1537 when EDAC was formulated in water; toxicity expanded to all Salmonella strains, in the absence of S9 activation, when EDAC was formulated in DMSO. In contrast, toxicity in the supplemental assay was only observed with strain TA100 in the absence of S9 activation, regardless of vehicle. In the initial test, when compared to the vehicle controls, substantial dose related increases in the mean revertant counts, exceeding the 2-fold threshold for a mutagenic response, were obtained with WP2 uvrA when EDAC was formulated in water (2.7-fold). These increases were observed in both the preliminary and confirmatory assays, in the absence of S9 activation. In the supplemental test, preliminary and confirmatory assays, substantial dose related increases in the mean revertant counts were obtained with WP2 uvrA, in the absence of S9 activation, when EDAC was formulated in water (3.6-fold) or DMSO (6.4-fold). Increases in the mean revertant counts, satisfying the mutagenic threshold, were obtained with TA100 when EDAC was formulated in DMSO in the absence of S9 activation (2-fold) only in the confirmatory assay in the supplemental test. As expected, there were significant increases in the histidine+ /tryptophan+ revertant counts in the cultures treated with the positive control articles. In conclusion, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDAC) was mutagenic in the absence of S9 activation when tested in accordance with regulatory guidelines.