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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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

The test substance induced toxicologically significant increases in the mutant frequency at the TK +/- locus in Mouse Lymphoma L5178Y cells following four hours exposure and is therefore considered to be mutagenic under the conditions of the test. These increases were observed in the absence and presence of metabolic activation and were predominantly due to small colony formation indicating a clastogenic response.

Link to relevant study records

Reference

Reference 1

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)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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

Target gene:

Histidine and Tryptophan

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.

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.

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine Kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
Cells were obtained from MRC Cell Mutation unit at the University of Sussex, Brighton, UK. These were routinely cultured in medium at 37°C with 5% CO2 in air. The cells have a regeneration time of ca. 12 hours and were subcultured accordingly. Master stocks of cells were tested and found to be free of mycoplasma.

CELL CLEANSING:
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. For the following 24 hours the cells were cultured in THG medium before being returned to R10 medium.

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix from the livers of male Sprague-Dawley rats treated with phenobarbital/Beta-naphthoflavone

Test concentrations with justification for top dose:

Preliminary toxicity test: 7.5, 15, 30, 60, 120, 240, 480, 960 and 1920 µg/mL.
Experiment 1 (4 hours with and without S9 mix): 0.94, 1.88, 3.75, 7.5, 11.25, 15, 22.5 and 30 µg/mL.
Experiment 2 (4 hours without S9 mix): 1.25, 2.5, 5, 7.5, 10, 12.5, 15 and 17.5 µg/mL
Experiment 2 (4 hours with S9 mix): 7.5, 10, 12.5, 15, 17.5, 20, 22.5 and 25 µg/mL.

Results from the preliminary test were used to set the dose levels for the main experiments (maximum dose level should produce 10 to 20% survival).

Vehicle / solvent:

The test substance was completely soluble in R0 medium and accordingly was used as supplied. The addition of the substance at the highest possible dose (1920 µg/mL) did not lead to any pH or osmolality effects.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Experiment 1:
Several days before the start of the experiment, an exponentially growing stock culture of cells was setup so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1x10^6 cells/mL in 10 mL aliquots in medium. Treatments were performed in duplicate both with and without metabolic activation at 8 dose levels. To each universal was added S9 mix if required, treatment solutions and sufficient medium. These vessels were incubated at 37°C for 4 hours with continuous shaking.

Experiment 1:
As with experiment 1, exponentially growing cultures were obtained and treated similarly although dose levels varied.

Measurements of survival, viability and mutant frequency:
At the end of the treatment periods, the cells were washed using medium and resuspended at a cell density of 2x10^5 cells/mL. These cultures were incubated at 37°C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days by counting and diluting. On Day 2 of the experiments, the cells were counted diluted and plated for mutant frequency (2000 cells/well) in selective mediumcontaining TFT. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium. The daily counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the viability data a Relative Total Grwoth (RTG) value.

Plate Scoring:
Plates were scored using a magnifying mirror box after 10 to fourteen days incubation at 37°C with 5% CO2 in air. The number of positive wells (those with colonies) were recorded together with the total number of scorable wells. The numbers of small and large colonies seen in the TFT mutation plates were also recorded. Colonies are scored manually by eye using qualitative judgement. Large colonies are define as those covering 1/4 to 3/4 of the surface area of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally more than two cells thick. MTT solution was added to assist in the scoring of TFT mutant colonies.

Calculation of Percentage Relative Suspension Growth (%RSG):
Suspension growth, % RSG, day 2 viability (%V), RTG and the mutation frequency were calculated as per the relevant OECD guideline.

Rationale for test conditions:

As per OECD guidelines with doses selected based on the results of a preliminary test.

Evaluation criteria:

The normal range for mutant frequency per survivor is 50 to 170 x10^-6 for the TK+/- locus in L5178Y cells at the testing laboratory with vehicle control results usually within this range.

Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.

The optimum toxicity is approximately 20% survival but no less than 10% survival. RTG values are usually the primary factor to designate the level of toxicity achieved by the test item for any individual dose level. h

For a test item to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutation frequency (IMF) over the concurrant vehicle mutant frequency value. A Global Evaluation Factor (GEF) of 126x10^-6 was used as a benchmark for positive responses. Toxicological significance of the data itself was also considered.

Statistics:

Analysed using a dedicated computer programme, Mutant 240C by York Electronic Research.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Experiment 1:
Evidence of marked dose-related toxicity following exposure to the test substance (with and without S9 mix) was observed as indicated by %RSG and RTG values. There was some evidence of reductions in viability (%V) with and without S9 mix indicating residual toxicity had occured. Optimum levels of toxicity were achieved both with and without S9 mix. Control and positive controls groups provided adequate results. The test substance induced statistically significant dose related (linear-trend) increases in the mutant frequency both with and without S9 mix. Statistically significant increases in mutant frequency were also observed at an individual dose level, the upper surviving dose level, both with and without S9 mix. The MF values observed were higher than the acceptable range for controls, with the GEF also exceeded in the absence of metabolic activation and approached in the presence of metabolic activation. However, neither of the individual dose levels showed MF significantly above the respective controls showed any marked increases in absolute numbers of mutant colonies. therefore it was considered appropriate to perform an additional experiment with a modified dose range. No precipitate of the test substance was observed at any dose. The slight increases in the proportion of small colonies in both exposure groups indicated that some clastogenic activity was involved in the response.

Experiment 2:
As observed previously, there was evidence of marked toxicity following exposure to the test substance both with and without S9 mix. Optimum levels of toxicity had been achieved in the presence of S9 mix, and near optimum levels had been achieved in the absence of S9 mix. Evidence of dose related reductions in viability were also observed indicating that some residual toxicity had occured. Control and positive controls groups provided adequate results. The test substance induced statistically significant dose related (linear-trend) increases in the mutant frequency both with and without S9 mix. Statistically significant increases in mutant frequency were also observed at individual dose levels both with and without S9 mix. The MF values observed were markedly higher than the acceptable range for controls and the GEF was exceeded. There was also evidence of increases in absolute numbers of mutant colonies and a shift towards small colony formation, indicative of clastogenic activity. The reproducible responses observed were therefore considered to be of toxicological significance.

Experiment 1

Treatment (µg/mL)	4 hours (-) S9			Treatment (µg/mL)	4 hours (+) S9
	%RSG	RTG	MF^a		%RSG	RTG	MF^a
0	100	1.00	138.18	0	100	1.00	123.19
0.94^b	92	-	-	0.94^b	103	-	-
1.88	87	0.86	134	1.88^b	88	-	-
3.75	90	1.03	127	3.75	101	0.99	104.88
7.5	66	0.69	152.74	7.5	95	0.99	132.28
11.25	33	0.24	197.23	11.25	88	0.81	126.16
15	21	0.11	307.65*	15	90	0.92	110.78
22.5^C	7	0.02	730.57	22.5	31	0.18	248.15*
30^b	4	-	-	30^C	15	0.03	729.64
Linear trend		***		Linear trend		*
EMS (400)	63	0.45	1169.69	CP (2)	60	0.32	1458.95

 

Experiment 2

Treatment (µg/mL)	4 hours (-) S9			Treatment (µg/mL)	4 hours (+) S9
	%RSG	RTG	MF^a		%RSG	RTG	MF^a
0	100	1.00	145.33	0	100	1.00	167.29
1.25^b	88	-	-	7.5^b	84	-	-
2.5	85	0.84	186.33	10	81	0.80	195.12
5	74	0.62	218.62*	12.5	73	0.72	222.36
7.5	61	0.53	225.15*	15	59	0.56	194.35
10	35	0.24	336.24*	17.5	46	0.34	244.39*
12.5^C	19	0.04	396.41	20	38	0.30	281.31*
15^C	12	0.02	826.69	22.5	24	0.15	460.57*
17.5^b	9	-	-	25^C	13	0.01	609.89
Linear trend		***		Linear trend		***
EMS (400)	51	0.28	1524.73	CP (2)	39	0.20	1177.42

a – 5 TFT resistant mutants /10⁶viable cells 2 days after treatment

b – Not plated for viability or 5 TFT resistance

c – Treatment excluded from test statistics due to toxicity

* – p< 0.05

*** – p< 0.001

Conclusions:

The test substance induced toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells following four hours exposure and is therefore considered to be mutagenic under the conditions of the test. These increases were observed in the absence and presence of metabolic activation and were predominantly due to small colony formation indicating a clastogenic response.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

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 in the rat was conducted. The rodent micronucleus test, Comet assay and Erythrocyte Mutation Frequency (PIG-A) was evaluated and recommended by international regulatory agencies as the appropriate tests to determine the in vivo genotoxic potential of a compound.

 

There were no statistically significant increases in the frequency of mutation in any EDAC treatment group when compared with the corresponding vehicle control group in the 3 individual studies. The reagent, EDAC, was not genotoxic following 1-month administration to male rats at doses up to, and exceeding, MTD (300 mg/kg).

Link to relevant study records

Reference

Reference 1

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

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

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

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD

Sex:

male

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

Dose / conc.:

150 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

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).

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.

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

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).

Reference 2

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

no

Type of assay:

mammalian erythrocyte micronucleus test

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

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl;CD

Sex:

male

Details on test animals or test system and environmental conditions:

Forty-five male Sprague Dawley Crl:CD rats were received from Charles River Laboratories on 28-Jun-2012 and were approximately 6 weeks of age upon arrival. Rats were selected for inclusion in the study based upon acceptable clinical condition, body weight, and basal Pig-a gene mutation frequency. Six animals were randomly assigned to 1 of 6 groups using a computer-generated stratified randomization procedure to achieve approximately equal group mean body weights. On Day 1, study animals were approximately 7 weeks of age and weighed between 203.6 and 226.9 grams. Animals were individually identified by an implantable microchip and each animal enclosure was identified with the animal's unique number.
Upon receipt and throughout the study, animals were individually housed in appropriately sized stainless-steel wire-bottom cages. Animals arrived and were conditions to this environment for 7 days prior to pre-study activities on 05-Jul-2012 (Day -4). Animals were provided food (Harlan Diet #2018C: Certified 18% Protein Rodent Diet) and purified chlorinated tap water ad libitum. In addition, animals were provided with an enriched environment as part of the animal enrichment program

Route of administration:

oral: gavage

Vehicle:

Sterile Water

Duration of treatment / exposure:

30 days

Post exposure period:

Viability checks were made at least once daily starting on the first day of dosing. During the dosing period, each animal was observed at least two times daily, predose, and 2 to 3 hours after dosing for changes in condition and behavior. Each animal was observed on the day of scheduled necropsy.
On Day 30 blood was collected the micronucleus analyses. Animals received their final treatment of vehicle, EDAC, or positive control article (MNU) 2 to 3 hours prior to anesthetization with isoflurane and exsanguination

Dose / conc.:

150 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

450 mg/kg bw/day

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.

No. of animals per sex per dose:

6

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).

Tissues and cell types examined:

On Days 4 and 30, approximately 24 hours after the last dose, 0.3 to 0.5 mL of peripheral blood from the tail vein from all animals was collected into K2 EDTA coated tubes. The tubes were inverted several times to ensure mixing and were diluted and fixed in duplicate within 24 hours. The fixed blood samples were stored in a freezer set to maintain ≤ -70°C until analysis or disposition

Evaluation criteria:

At a procedurally convenient time, the frequency of micronucleated reticulocytes (MN-RET), micronucleated normochromatic reticulocytes (MN-NCE), and percent RET were measured from the peripheral blood samples using a BD FACSCanto II™ flow cytometer. Remaining samples were used for re-analysis (Day 4) or were discarded after data acceptance.
The percent MN-RET was determined for each animal by scoring up to 20,000 RET per animal from the dose groups. The number of RET acquired for samples collected from the positive control group animals varied due to compound toxicity.

EDAC was considered positive in this assay if the mean MN-RET value at any dose level was:
1) Statistically significant at any one timepoint.
2) The MN-RET frequency in any test-article group exceeded the upper limit (mean + 2 standard deviations) of the current historical database.
3) Is at least 3-fold greater than the concurrent negative-control mean value. EDAC was considered negative if all of the above criteria were not met.

Statistics:

The micronucleus data were evaluated using appropriate descriptive and statistical methods. Specifically, the means and standard deviations were calculated for each of the treatment groups. The following parameters were subject to statistical analysis: % RET, % MN-RET, and % MN- NCE. A positive or negative response was determined based upon a comparison of the observed test article responses with concurrent vehicle-control values. Statistical comparisons between EDAC treatments and vehicle control were performed using Dunnett's multiple comparison t-test procedure in the context of a one-way analysis of variance model. Appropriate weighted regression methods were used to evaluate the presence of a linear trend with dose at each timepoint. For the micronucleus positive control article, CP, a "t" test was performed with the vehicle-control animals serving as the controls. Any value exceeding the 5% (P ≤ 0.05) level of probability was deemed statistically significant.

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

The results of this peripheral blood micronucleus evaluation met the acceptance criteria outlined in the study protocol. There were no statistically significant treatment related effects observed for %RET at Day 4 or 30. The high dose 800 mg/kg had elevated %RETs compared to the vehicle control on Day 4 with larger dose-related elevation by Day 30, suggestive of toxicity followed by stimulation erythropoiesis. In the literature, excessive toxicity followed by stimulation of erythropoiesis has resulted in the induction of micronuclei. However, in this study, at doses that exceeded MTD for both the Day 4 and Day 30 micronucleus assessments, elevations in micronucleus frequency were not observed. There were no statistically significant increases in %MN-RET in any EDAC treatment group when compared with the corresponding vehicle control groups at either sampling time. On Day 4, at 0, 150, 300, 600, and 800 mg/kg/day, mean %MN-RET frequencies were 0.13%, 0.08%, 0.10%, 0.12%, and 0.11%, respectively. On Day 30, at 0, 150, 300, and 800/600/450 mg/kg/day, mean %MN-RET frequencies were 0.10%, 0.10%, 0.13%, and 0.11%, respectively.

 

The positive control treatments induced statistically significant decreases in %RET at both sampling times, which is indicative of bone marrow exposure and hematopoietic toxicity (82 and 77% decreases on Days 4 and 30, as compared to corresponding VC groups, respectively). The positive control treatments induced statistically significant increases in %MN-RET on Days 4 and 30. There was a statistically significant but not biologically meaningful increase in the %MN-NCE in the positive control treatments. The increases observed (Group mean %MN-NCE 0.03 and 0.02 on Days 4 and 30, respectively), were within the historical control range of this laboratory.

Conclusions:

In conclusion, EDAC was not genotoxic in the in vivo rat peripheral blood micronucleus assay when tested up to, and above, the maximium tolerated dose 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 rodent micronucleus test and the Comet assay in the rat are recommended by international regulatory agencies as the appropriate tests to determine the in vivo genotoxic potential of a compound. The micronculeus test assesses the potential of EDAC to act as a clastogen or anuegen,

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 the frequency of micronucleated reticulocytes (%MN-RET) in any EDAC treatment group when compared with the corresponding vehicle control group. The reagent, EDAC, was not genotoxic following 1-month administration to male rats at doses up to, and exceeding, MTD (300 mg/kg).

Reference 3

Endpoint:

genetic toxicity in vivo, other

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:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline available

Principles of method if other than guideline:

The phosphatidylinositol glycan, Class A (Pig-a) mutation assay was included to assess the potential of EDAC to induce mutagenicity in vivo

GLP compliance:

no

Type of assay:

other:

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

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD

Sex:

male

Details on test animals or test system and environmental conditions:

Forty-five male Sprague Dawley Crl:CD rats were received from Charles River Laboratories on 28-Jun-2012 and were approximately 6 weeks of age upon arrival. Rats were selected for inclusion in the study based upon acceptable clinical condition, body weight, and basal Pig-a gene mutation frequency. Six animals were randomly assigned to 1 of 6 groups using a computer-generated stratified randomization procedure to achieve approximately equal group mean body weights. On Day 1, study animals were approximately 7 weeks of age and weighed between 203.6 and 226.9 grams. Animals were individually identified by an implantable microchip and each animal enclosure was identified with the animal's unique number.
Upon receipt and throughout the study, animals were individually housed in appropriately sized stainless-steel wire-bottom cages. Animals arrived and were conditions to this environment for 7 days prior to pre-study activities on 05-Jul-2012 (Day -4). Animals were provided food (Harlan Diet #2018C: Certified 18% Protein Rodent Diet) and purified chlorinated tap water ad libitum. In addition, animals were provided with an enriched environment as part of the animal enrichment program

Route of administration:

oral: gavage

Vehicle:

Sterile water

Duration of treatment / exposure:

30 days

Post exposure period:

Viability checks were made at least once daily starting on the first day of dosing. During the dosing period, each animal was observed at least two times daily, predose, and 2 to 3 hours after dosing for changes in condition and behavior. Each animal was observed on the day of scheduled necropsy.
On Day 30 blood was collected the micronucleus analyses. Animals received their final treatment of vehicle, EDAC, or positive control article (MNU) 2 to 3 hours prior to anesthetization with isoflurane and exsanguination

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

150 mg/kg bw/day

Dose / conc.:

450 mg/kg bw/day

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.

No. of animals per sex per dose:

6

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).

Tissues and cell types examined:

Blood samples (0.2 to 0.3 mL) were collected from the tail vein in tubes of appropriate size containing K2EDTA on Days -4, 15, and 30.

Details of tissue and slide preparation:

Blood was processed according to the instruction manual provided with the prototype rat Pig-a MutaFlow® kit from Litron Laboratories (v120209). Briefly, 80 µL of peripheral blood from each animal sampled was diluted with anticoagulant solution, layered on Lympholyte Solution, and centrifuged at 800 x g for 20 minutes. The leukodepleted blood was labeled with phycoerythrin (PE)-conjugated antibodies against CD61 and CD59, and paramagnetic particles were added to the samples to bind the PE molecules. A small aliquot of the sample was stained with DNA stain containing a homogenous suspension of counting beads and analyzed via flow cytometry. These analyses represent "pre- column" data. The remaining sample volume was added to a magnetic column to deplete platelets (CD61-PE+) and Pig-a wild type (CD59-PE+) cells. The eluate was concentrated, stained with DNA stain plus counting beads, and subjected to flow cytometric analysis. These analyses represent "post-column" data. The counting beads provided a common denominator between pre- and post-column data, which permited determination of mutant frequencies for both RET and RBC.

Evaluation criteria:

1) The response is statistically significant at any one timepoint.
2) The group mean exceeds the mutation frequencies in our historical control database.
3) The response is biologically relevant based on the experienced judgment of the Study Director.

The test article was considered negative if all of the above criteria were not met.

Statistics:

The frequencies of %RET, mutant RET, and mutant RBC was determined via flow cytometry. An instrument calibration standard (ICS) sample, which contains an approximate 50% mixture of labeled and unlabeled cells (the latter mimicking the mutant phenotype), was used to verify gating logic. After setting compensation and gates based upon the ICS data acquisition settings remained constant throughout the analysis of all experimental samples for a given day.
Mutation frequency was determined for each surviving animal at each time point by acquiring data in the following fashion:
1. Pre-column: Data were acquired until at least 1,000 counting beads were acquired.
2. Post-column: Data were acquired until the sample volume was nearly depleted (approximately 4 minutes for each sample).

For each animal, the following parameters were calculated. Quadrant abbreviations are defined as UL = upper left, UR = upper right, LL = lower left, and LR = lower right.
1. Calculations from pre-column acquisitions:
a) %RETs = (UL + UR) I (UL + UR + LL + LR) x 100
b) RBC to counting bead ratio = (UL + UR + LL + LR) I Beads c) RET to counting bead ratio = (UL + UR) I Beads

2. Calculations from post-column data acquisition, for specifics, refer to the MutaFlow®
instruction manual:
a) Mutant RET to bead ratio = UL I Beads
b) Mutant RBC to bead ratio = (UL + LL) I Beads
c) Total RBC Equivalents, based on dilution factors d) Total RET Equivalents, based on dilution factors
e) Number of mutant RBC and mutant RET per 106 cells
The group means and standard deviations for %RET, mutant RBC, and mutant RET were calculated for each of the test- and control-article treatment groups.

Key result

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

The results of this erythrocyte Pig-a gene mutation evaluation met the acceptance criteria outlined in the study protocol and are summarized in the Biostatistics Memorandum (Appendix 4). The individual animal data are listed in Appendix 5. Historical vehicle and positive control data from previous studies conducted in this laboratory are shown in Appendix 6. On Days 15 and 30 there were no statistically significant treatment related effects observed for %RET. On Day 30 %RET increased in a dose-dependent fashion, with a 20% increase observed for the 800/600/450 mg/kg/day dose group, as compared to the corresponding vehicle control group. However, this was driven by one animal that had 7.12 %RET, and omission of this animal brought the group mean to within interanimal variability of all EDAC treatment groups on Day 30 (4.49% compared to %RET range of 3.65% to 5.67%). There were no statistically significant increases in Pig-a gene mutation, measured by CD59NEG phenotype, in erythrocytes and reticulocytes, on either day of sampling. On Day 15, at 0, 150, 300, and 800/600 mg/kg/day, mean Pig-a gene mutation frequencies in reticulocytes was 0.653, 0.416, 0.260, and 0.287 x 10-6 mutants, respectively, and in total erythrocytes was 0.239, 0.359, 0.241, and 0.435 x 10-6   mutants, respectively. On Day 30, at 0, 150, 300, and 800/600/450 mg/kg/day, mean Pig-a gene mutation frequencies in reticulocytes was 1.083, 1.801, 0.363, and 0.589 x 10-6 mutants, respectively, and in total erythrocytes was 0.348, 0.733, 0.298, and 0.419 x 10-6 mutants, respectively.

By Day 30, the positive control treatments induced statistically significant decreases in %RET (1.57% compared to the corresponding VC group mean of 4.26%). The positive control treatments induced statistically significant increases in Pig-a mutant reticulocytes (65.2 and 61.2 x 10-6 mutants) and erythrocytes (20.7 and 37.58 x 10-6 mutants) on Days 15 and 30, respectively.

Conclusions:

The reagent, EDAC, was not genotoxic following 1-month administration to male rats at doses up to, and exceeding, MTD (300 mg/kg).

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 phosphatidylinositol glycan, Class A (Pig-a) mutation assay was included to assess the potential of EDAC to induce mutagenicity in vivo. No OECD test guideline is available for thr Pig-a mutation assay; however, this assays wasconducted based on recommended protocols published in the literature.The Pig-a assay design used has been shown to have sufficient power to detect a 1.7x increase in mutation frequency with 80% probability. Recently, the Pig-a mutation assay has gained increased international attention as a tool to investigate the potential of a compound to induce in vivo somatic cell mutation. The Pig-a gene is one of several genes that are involved in glycosylphosphatidyl inositol (GPI) anchor synthesis, but the Pig-a gene is the only gene that resides on the X-chromosome. Because it resides on the X-chromosome, only a single mutation is required to prevent expression of GPI anchors. Therefore, the lack of GPI-linked proteins on cell membranes (CD59-negative phenotype [CD59Neg]) acts as a reporter of Pig-a mutation. Measurement of CD59Neg phenotype for reticulocytes (RETs) and erythrocytes (RBCs) in peripheral blood by flow cytometry provides a fast-non-invasive method to measure mutation frequency in large numbers of cells. Useful characteristics of the Pig-a mutation assay include the accumulation of mutant phenotype cells with repeat dosing, a low background mutation frequency, and a large dynamic response range.

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 Pig-a gene mutation, measured by CD59NEG phenotype, in RBCs or RETs (Table 2-2). The reagent, EDAC, was not genotoxic following 1-month administration to male rats at doses up to, and exceeding, MTD (300 mg/kg).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)