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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 July 2012 to 07 August 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

other company data

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

yes

Remarks:

No OECD test guidelines was available for the Comet at the time the test was conducted however, these assays were conducted based on recommended protocols published in the literature

GLP compliance:

no

Type of assay:

mammalian comet 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

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD

Sex:

male

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Sterile Water

Duration of treatment / exposure:

once daily for 30-days, except on Day 15 for the 800/600/450 mg/kg/day dose group

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

150 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

450 mg/kg bw/day

Remarks:

was reduced from 800 mg/kg on day 8 to 600 mg/kg and subsequently 450 mg/kg on day 16 due to toxicity effects

No. of animals per sex per dose:

6 per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

10 mg/kg Cyclophosphamide Days 1, 2, and 27; 10 mg/kg ethyl-N-nitrosourea Days 1, 2, and 3; and 2.5 mg/kg methyl-N-nitrosourea Day 30 (2-4 h prior to sacrifice).

Examinations

Details of tissue and slide preparation:

A portion of the liver (left lobe) was removed, rinsed, and minced in mincing solution to create a single cell suspension. A cell count from the tissue suspension was determined and adjusted to 1.5 x 104 cells/mL prior to preparing slides. Trevigen slides were labeled with the following coded information: study number, date of slide preparation, animal number, tissue, and slide replicate. Three slides were prepared for each animal by diluting the cell suspension 10-fold into low melting point agarose gel. The remaining solution was spread upon 2 wells on each slide. After cooling the gels in a refrigerated environment, the slides were immersed overnight in lysis solution under refrigerated conditions. After lysis treatment, the slides were rinsed with neutralization buffer to remove residual detergents and salts prior to moving them to electrophoresis chamber(s) filled with cold alkaline buffer (pH > 13). Slides are then stored for
40 minutes to allow DNA to unwind, prior to electrophoresis for 40 minutes at 0.7 V/cm (378 mA). Slides were then neutralized, rinsed in water, and fixed in ethanol. After drying, slides were immersed in DNA stain and air-dried prior to scoring

Evaluation criteria:

EDAC was considered positive in this assay if the mean MN-RET value at any dose level was:
1) Statistically significant.
2) The group mean exceeded the upper limit (mean + 2 standard deviations) of the current historical control database.
3) The response was > 3-fold over the concurrent vehicle control
EDAC was considered negative if all of the above criteria were not met.

Statistics:

For both assays, the primary objective of the statistical analyses was to evaluate mean differences in the parameters between the EDAC-treated groups (Groups 2-5) and thepositive
control group versus the vehicle control-group (Group 1), separately on each study day. For the comet assay, an additional objective was to evaluate the data for presence of a linear trend. For each assessment, homogeneity of group variances was assessed at the 10% significance level If significant heterogeneity of variance was observed (p0.10), or if there was observed non-normality, an appropriate transformation of data was utilized. If a log transformation was utilized, an offset of 0.1 was used to account for frequencies of 0, if needed.
If appropriate, a one-way analysis of variance (ANOVA) model was utilized to analyze each parameter; t-tests were computed in the context of the ANOVA model to compare the mean of the vehicle-control group to the mean of each EDAC-treated group on each study day. The Dunnett multiple-comparison t-test procedure was utilized for the comparisons on each study day.
Two-sided Dunnett t-tests were used for the comet assay endpoints. Because differences in variance were expected between the endpoints for the vehicle-control and positive-control groups, a Welch t-test3
For the comet endpoints, a linear regression model using log mean and median %tail intensities was used to assess the presence of a linear trend, at the 5% significance level. Analyses were performed using SAS version 9.1.2. was utilized to compare the vehicle control and positive control group means. The test was performed at the 5% significance level.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

High dose (800 mg/kg, Group 5) was reduced on Day 8 to 600 mg/kg (Group 4) and then to 450 mg/kg (Group 7) on Day 16. Animals in this cohort were not dosed on Day 15.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Throughout the study animals in Group 4 (800 mg/kg), 5 (600mg/kg), and 7 (450 mg/kg) were euthanized in response to labored and noisy respiration coupled with salvation, indicating that doses of 450 mg/kg/day and higher were not tolerated. On Day 26, an animal in the 300 mg/kg/day dose group was euthanized due to similar observations. However, this dose level was well tolerated by the other animals in this dose group. Hence, despite this mortality, the 300 mg/kg/day dose group was considered the maximally tolerated dose (MTD) in this 30-day oral repeat dose study. Other sporadic clinical observations at 300 mg/kg included decreased activity, chromodacryorrhea, chromorhinorrhea, scant feces, and red-stained haircoat. One animal in the 300 mg/kg/day dose group had yellow soiling around the perianal cavity while another animal had a torn nail. All of these clinical signs, along with absent feces, were also observed in the 800/600/450 dose group(s). In all treatment groups, including vehicle and positive controls, animals were observed to have thinning of the haircoat around their forefeet/hands, which was attributed to the feeding apparatus. Hence, the only reproducible adverse clinical observation noted in doses 300 mg/kg/day was salvation coupled with labored and noisy respiration. Animals in the positive control group showed no consistent treatment related clinical signs. Incidental clinical signs included focal scabbing on the shoulders of one animal with concomitant observationof a transient dorsal mass, which were likely due to the animal rubbing against the caging

The results of the DNA damage evaluation in liver met the acceptance criteria

Applicant's summary and conclusion

Conclusions:

EDAC was not genotoxic in rat liver cells when tested up to, and above, the MTD of 300 mg/kg/day

Executive summary:

An investigative non-GLP in vivo study was conducted to evaluate the relevance of the in vitro genotoxicity response. A 1-month rat study was designed to assess clastogenicity, aneuploidy, and base-pair mutation potential in a single in vivo study. The Comet assay in the rat is recommended by international regulatory agencies as the appropriate tests to determine the in vivo genotoxic potential of a compound. The comet assay in liver assesses the potential of EDAC or a metabolite to induce deoxyribonucleic acid (DNA) damage. No OECD test guidelines are available for the Comet assays at the time of testing; however, this assay was conducted based on recommended protocols published in the literature.

EDAC was administered orally by gavage for 30 consecutive days to groups of 6 males at dose levels of 150, 300, 600, and 800 mg/kg. EDAC was not well tolerated and on Day 9 the dose administered to the 4 surviving animals from the high dose group (800 mg/kg/day) was reduced to 600 mg/kg/day.  Due to continued poor tolerability and a scheduled blood collection, the 6 remaining animals in the 800/600 mg/kg group were not dosed on Day 15.

Dosing resumed on Day16 at a reduced dose of 450 mg/kg for the remainder of the study (14 Days). Hence, doses at termination were 150, 300, and 800/600/450 mg/kg/day. An additional 6 males served as the vehicle control group and were administered sterile water for injection (orally by gavage) on the same schedule. Three positive control agents (10 mg/kg ENU, 10 mg/kg CP, and 2.5 mg/kg MNU) were intermittently administered to an additional 6 males to demonstrate biological sensitivity to the genotoxic endpoints evaluated

 

Dose-related clinical observations were salivation coupled with labored and noisy respiration. At doses > 300mg/kg/day (maximum tolerated dose (MTD)), respiratory distress was the cause for euthanasia. Respiratory distress was likely due to aspiration of stomach contents. In those animals necropsied following euthanasia, there were no signs of dosing error, but the stomachs of these animals were bloated with gases and fluid. Based on the clinical observations, the MTD was 300 mg/kg.

There were no statistically significant increases in DNA damage, as measured by mean or median% Comet tail fluorescence intensities (%TI) for any EDAC treatment group when compared with the vehicle control group. The positive control treatments induced statistically significant increases in %TI measured in liver cells measured by the alkaline comet assay (Day 30).The reagent, EDAC, was not genotoxic following 1-month administration to male rats at doses up to, and exceeding, MTD (300 mg/kg).