bis(diethylamino)-N,N-diethylmethaniminium chloride

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD 407 (1995); GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Forty seven male and 47 female Crl:CD(SD)IGS BR rats were received in good health from Charles River Laboratories, Inc., Raleigh, North Carolina. The animals were approximately 36 days old at receipt. Each animal was examined by a qualified technician on the day of receipt and weighed one day later. Each animal was uniquely identified by a metal ear tag displaying the permanent identification number. All animals were housed for a 14-day acclimation/pretest period. During this period, each animal was observed twice daily for mortality and changes in general appearance or behavior and food consumption and body weights were recorded. All animals were housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board. Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the study, except during the period of fasting prior to blood collection when food, but not water, was withheld. All animals were housed throughout the acclimation period and during the study in an environmentally controlled room. Actual mean daily temperature ranged from 70.3°F to 70.6°F (21.3°C to 21.5°C) and mean daily relative humidity ranged from 38.9% to 49.1% during the study. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The 12-hour light/12-hour dark photoperiod was interrupted as necessary to allow for the performance of protocol-specified activities. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Test article formulations were prepared as weight/volume mixtures at 25, 100 and 250 mg/ml. The test article formulations were adjusted for the 58.5% active ingredient in the supplied aqueous test solution. The appropriate amount of the test article for each formulation was weighed into a tared, calibrated storage container. A sufficient volume of vehicle (deionized water, prepared on site) was added to each container. The formulations were mixed until uniform using a magnetic stirrer. Vehicle was then added to each container to bring the formulations to the calibration mark. The test article formulations were prepared weekly as single formulations for each dosage level, divided into aliquots for daily dispensation and stored refrigerated. The test article formulations were stirred continuously throughout the preparation, sampling and dose administration procedures.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to the initiation of dose administration, duplicate samples (1 mL each) for homogeneity determination were collected from the top, middle and bottom strata of the 25 and 250 mg/kg/day dosing formulations. In addition, aliquots suitable for one day of dosing were stored refrigerated for 8 days. Duplicate samples (1 mL each) for stability/resuspension determinations were collected from the top and bottom strata of the 25 and 250 mg/kg/day dosing suspensions following refrigerated storage for 8 days. Samples (1 mL each) for concentration analyses were collected from the first and third formulations from all dose groups (including the control group). A high performance liquid chromatography method using ultraviolet detection at a wavelength of 215 nm was used for the determination of test substance concentration in dose formulations. The test article formulations were found to be homogeneous, contained the concentrations of test article specified in the protocol and were stable for at least 8 days (i.e. the analyzed concentrations were within 15% of the target dose concentration). In addition, no test article was found in the vehicle control samples analyzed.

Duration of treatment / exposure:

a minimum of 28 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 25, 100 and 250 mg/kg/day
Basis:

No. of animals per sex per dose:

5/sex at 0 and 250 mg/kg/day for a 14-day recovery period

Control animals:

yes

Details on study design:

Doses were selected based on a series of studies designed to determine the single- and repeated-dose toxicity of the test substance. In addition, these studies examined the potential effect of fasting on the single-dose toxicity. Based on these studies, doses greater than 300 mg/kg/day were considered to be lethal and a dose equal to 300 mg/kg/day was considered too high for a 28-day study based on the loss of body weight after 5 days. Therefore, the high dose of 250 mg/kg/day was selected for the present study.

For the main study, the test substance was administered orally by gavage at 0, 25, 100 or 250 mg/kg/day once daily for a minimum of 28 consecutive days to Crl:CD(SD)IGS BR rats. The vehicle was deionized water and the dose volume was 5 mL/kg for all groups. Groups 1 and 4 each consisted of 13 animals/sex and Groups 2 and 3 each consisted of 8 animals/sex. Individual doses were based on the most recently recorded body weights. Study day 0 and study week 0 were the first day and week of dosing, respectively. Following 28 consecutive days of dose administration, 8 rats/sex/group were euthanized (primary necropsy; study week 4); the remaining 5 rats/sex in the control and high dose groups were euthanized following a 14-day recovery period (recovery necropsy; study week 6).

Examinations

Observations and examinations performed and frequency:

Observations and examinations performed and frequency: All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed twice daily, at the time of dosing and at approximately 1 to 2 hours following dose administration. During the recovery period animals were observed once daily. Detailed physical examinations were performed weekly beginning prior to randomization and concluding on the day of necropsy. Individual body weights and food consumption were recorded weekly. Functional observational battery (FOB) (observations included: home cage; handling; open field; sensory; neuromuscular and physiological) and locomotor activity (MA) data were recorded for all animals prior to the initiation of dose administration and near the end of the dosing period (prior to the daily dose; study week 3). Hematology, serum chemistry and urinalysis parameters (see lists below) were evaluated in all study animals from the primary necropsy (study week 4). The animals were fasted overnight prior to blood collection. Blood was collected at the time of necropsy via the vena cava. Blood was collected into tubes containing EDTA (hematology) or sodium citrate (coagulation tests) or no anticoagulant (serum chemistry). Urine samples were collected overnight using metabolism cages prior to the day blood samples were collected.

Sacrifice and pathology:

A complete necropsy was conducted on all animals. Animals were euthanized at the scheduled necropsies by isoflurane anesthesia and exsanguination. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal and pelvic cavities, including viscera. Select tissues and organs were collected and/or weighed and placed in 10% neutral-buffered formalin (except as noted) (see lists below). After fixation, the specified tissues were trimmed and processed into paraffin blocks, sectioned at 4 to 8 microns, mounted on glass microscope slides and stained with hematoxylin and eosin. Microscopic examination was performed on all tissues listed below from all animals in the control and 250 mg/kg/day groups at the scheduled primary necropsy and all animals found dead. Gross lesions were examined from all animals found dead and those sacrificed at the scheduled primary and recovery necropsies. Microscopic examination was performed at the laboratory.

Statistics:

All statistical tests were performed using appropriate computing devices or programs. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test article-treated group to the control group by sex. Body weight, body weight change, food consumption, clinical pathology, organ weight (absolute and relative) and applicable functional observational battery and locomotor activity data were subjected to a parametric one-way analysis of variance (ANOVA) to determine intergroup differences. If the ANOVA revealed statistically significant (p is less than 0.05) intergroup variance, Dunnett's test was used to compare the test article-treated groups to the control group. Functional observational battery parameters that yielded scalar or descriptive data were analyzed using Fisher’s Exact Test. Clinical pathology values for white blood cell types that occur at a low incidence (i.e., monocytes, eosinophils and basophils) were not subjected to statistical analysis.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

There were no test article-related effects observed throughout the study on survival, detailed physical or clinical observations, or food consumption although one control group male was found dead on study day 24 due to acute inflammation of the kidneys. Because this male was in the control group and not administered test article, the death was not considered test article-related. There were no treatment-related effects on motor activity or home cage, handling, sensory, neuromuscular, and physiological observations.

There were no test article-related effects on body weights. Mean body weight gains were significantly higher in the 100 mg/kg/day group males and significantly lower in the 250 mg/kg/day group females during study days 13 to 20 when compared to the control group. Mean cumulative body weight gains were significantly higher in the 250 mg/kg/day recovery group males when compared for study days 0 to 41. These differences were not observed in a clear dose-related manner, occurred in only 1 week of the dosing period, did not affect cumulative weight gain during the dosing phase of the study, and were not consistent between the sexes and therefore, were not considered test article-related. The apparent increase in weight gain for the recovery animals was considered to be due to the selection of a subpopulation of the animals after the dosing period and not a response to the termination of dosing.

There were no test article-related effects on open field observations at the study week 3 evaluation. Time to first step for the 100 and 250 mg/kg/day group females were both significantly lower and defecation and number of rears for the 100 mg/kg/day group females was significantly higher when compared to the control group. These differences were slight, not observed in a dose-related manner and/or were similar to pretest observations and therefore, were not considered test article-related.

There were no treatment-related effects on hematology or serum chemistry. Females in the
100 mg/kg/day group had a significantly lower mean serum potassium level when compared to controls. This change was not observed in a dose-related manner and therefore, was not attributed to test article administration.

There were no test article-related effects on urinalysis parameters. Significantly higher mean urine specific gravity was noted in the 250 mg/kg/day group males when compared to controls. This slight change was not considered the result of test article administration.

Macroscopic findings noted in the control group male found dead on study day 24 included dark red contents of the jejunum, dilated renal pelvis, enlarged kidneys with white areas, renal calculi, dark red areas of the stomach, distended urinary bladder and distended ureter.

There were no test article-related macroscopic findings in the main study or recovery groups at the scheduled necropsies. All macroscopic changes noted were considered to be spontaneous and/or incidental in nature and unrelated to test article administration.

There were no test article-related effects on organ weights in the main study or recovery animals. Mean relative (to brain weight) heart weights were significantly lower in the 250 mg/kg/day group females at the study day 28 primary necropsy when compared to the control group. This difference was slight and inconsistent between the sexes and therefore, it was not considered test article-related. Mean absolute and/or relative (to final body and brain weights) thymus weights were significantly lower for 250 mg/kg/day recovery group females and significantly higher for 250 mg/kg/day recovery group males at the study day 42 recovery necropsy. Since these differences were only noted at the recovery necropsy and were higher in males and lower in females, they were not considered test article-related.

The cause of death for the one control group male that was found dead on study day 24 was determined to be acute inflammation of the kidneys. Other microscopic findings noted in this animal were cardiomyopathy, bacterial colonies within the kidneys, renal pelvis dilation, histiocytosis of the lymph nodes, acute inflammation of the pancreas and prostate, along with cellular luminal debris of the prostate, congestion of the glandular stomach, necrosis of the thymus and dilated lumen of the ureter. The urinary bladder of this animal was too autolyzed for diagnosis.

There were no test article-related microscopic findings observed in animals euthanized at the primary necropsy (study day 28). Minimal mineralization, characterized by intraluminal lamellated concretions in tubules at the corticomedullary junction, was noted in the kidneys of five females in the 250 mg/kg/day treatment group and one female in the control group. This is a common incidental finding in female rats and was not present in any males; therefore, it was not attributed to test article administration. All other findings observed were consistent with normal background lesions in clinically normal rats of the age and strain used on this study and were considered spontaneous and/or incidental in nature and unrelated to test article administration.

Effect levels

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Maximum dose tested

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Summary of Body Weight Changes (g)

Group:	0 mg/kg/day	25 mg/kg/day	100 mg/kg/day	250 mg/kg/day
Males
Days 13-20
Mean	26.	34.	40.**	34.
S.D.	9.2	7.0	10.8	9.7
N	13	8	8	13
Recovery Males
Days 0-41
Mean	166.	NA	NA	213.*
S.D.	20.0			21.8
N	4			5
Females
Days 13-20
Mean	24.	16.	25.	15.*
S.D.	11.0	6.8	6.3	5.4
N	13	8	8	13
* = Significantly different from the control group at 0.05 using Dunnett’s test ** = Significantly different from the control group at 0.01 using Dunnett’s test ** = Significantly different from the control group at 0.01 using Dunnett’s test N = Number of animals NA = Not applicable

Open Field Observations

Group:	0 mg/kg/day	25 mg/kg/day	100 mg/kg/day	250 mg/kg/day
Females
N	13	8	8	13
Time to First Step (seconds)
Mean	0.6	0.5	0.5*	0.5**
S.D.	0.12	0.12	0.10	0.06
Defecation
Mean	0.0	0.0	0.4*	0.0
S.D.	0.00	0.00	0.74	0.00
Rearing
Mean	8.3	7.9	11.4*	8.0
S.D.	2.14	2.47	3.81	2.38
* = Significantly different from the control group at 0.05 using Dunnett’s test ** = Significantly different from the control group at 0.01 using Dunnett’s test S.D. = Standard deviation N = Number of animals

Serum Chemistry Values

Group:	0 mg/kg/day	25 mg/kg/day	100 mg/kg/day	250 mg/kg/day
Females
Potassium (mEq/L)
Mean	7.16	6.65	5.84*	6.07
S.D.	1.138	0.902	0.718	1.028
N	8	8	8	8
* = Significantly different from the control group at 0.05 using Dunnett’s test S.D. = Standard deviation N = Number of animals

Urine Quantitative Parameters

Group:	0 mg/kg/day	25 mg/kg/day	100 mg/kg/day	250 mg/kg/day
Males
Specific Gravity
Mean	1.029	1.033	1.033	1.050*
S.D.	0.0123	0.0102	0.0159	0.0196
N	8	8	8	8
* = Significantly different from the control group at 0.05 using Dunnett’s test S.D. = Standard deviation N = Number of animals

Organ Weights (g)

Group:	0 mg/kg/day	25 mg/kg/day	100 mg/kg/day	250 mg/kg/day
Recovery Females
Thymus (g)
Mean	0.4889	NA	NA	0.3133*
S.D.	0.1141			0.05178
N	5			5
* = Significantly different from the control group at 0.05 using Dunnett’s test S.D. = Standard deviation N = Number of animals NA = Not applicable

Organ Weights Relative to Final Body Weights (g/100 g)

Group:	0 mg/kg/day	25 mg/kg/day	100 mg/kg/day	250 mg/kg/day
Recovery Females
Thymus
Mean	0.181	NA	NA	0.134**
S.D.	0.0235			0.0166
N	5			5
** = Significantly different from the control group at 0.01 using Dunnett’s test S.D. = Standard deviation N = Number of animals NA = Not applicable

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the no-observed-effect level (NOEL) for oral (gavage) administration of the test substance to rats for 28 consecutive days was 250 mg/kg/day.