bis(diethylamino)-N,N-diethylmethaniminium chloride

Endpoint summary

Description of key information

Under the conditions of this study, the no-observed-effect level (NOEL) for oral (gavage) administration of the test material to rats for 28 consecutive days was 250 mg/kg/day, the highest dose level tested.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 14, 2003 to December 29, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1995

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The animal model, the Crl:CD(SD)IGS BR rat, is recognized as appropriate for subchronic toxicity studies and is a widely used strain for which significant historical control data are available.

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 approximately 36 days old at receipt and approximately 7 weeks old at the initiation of dose administration; body weight values ranged from
196 g to 250 g for males and from 156 g to 195 g for females. 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.

Route of administration:

oral: gavage

Details on route of administration:

The vehicle and test article formulations were administered orally by gastric intubation via a 16-gauge stainless steel gavage dosing cannula.

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.

The dosage volume for all groups was 5 mL/kg. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. Adjusted doses became effective the day after collection of the weekly body weights.

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. More specifically:

- Specificity/selectivity: specificity/selectivity was confirmed when analysis of control formulations revealed that there were no significant peaks at or near the retention time for the test material, which is 3.0 minutes.

- Homogeneity: the RSD for the overall mean concentration was 10% or less at a concentration that was within the acceptable limits (within 85-115% of target concentration).

- Stability: after 8 days the mean concentrations were no less than 90% of the time zero concentrations.

- Confirmation of concentration: the mean concentrations were between 92.9 and 104 percent of target values.

Experimental:
- Instrument: Hewlett-Packard 1100 liquid chromatograph equipped with a UV/VIS detector, autosampler and HP ChemStation data management systems or equivalent.
- Column: Luna C18(2), 75 x 4.6 mm, 5-μm particle size
- Mobile Phase: A: 5mM Heptanesulfonic Acid, B: Acetonitrile (ACN)
- Gradient Program: 70% A : 30% B (0.0 mins and 1.0 mins); 30% A : 70% B (1.1 mins and 4.0 mins); 70% A : 30% B (4.1 mins)
- Flow Rate: 1.0 mL/minute
- Detector: UV at 210 nm
- Injection Volume: 10.0 μL
- Retention Times: Approximately 2.9 minutes for HEG-CL
- Run Time: 6.0 minutes

Duration of treatment / exposure:

a minimum of 28 days, through the day prior to the scheduled primary necropsy

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

8 animals per sex per dose. An additional 5 animals per sex were dosed at 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).

The animals judged suitable for assignment to the study were selected for use in the computerized randomization procedure. A printout containing the animal numbers, corresponding body weights and individual group assignments was generated based on body weight stratification in a block design. The animals then were arranged into groups according to the printout. The control and 250 mg/kg/day groups (Groups 1 and 4, respectively) each consisted of 13 males and 13 females, and the 25 and 100 mg/kg/day groups (Groups 2 and 3, respectively) each consisted of 8 males and 8 females. These animals were then randomized into four study replicates to allow for the reasonable conduct of the functional observational battery and motor activity assessments. Each dose group and sex were approximately equally represented within each study replicate. Individual body weights at randomization were within ± 20% of the mean for each sex.

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). Haematology, 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 (haematology) 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.

Haematology parameters evaluated: Total Leukocyte Count, Erythrocyte Count, Haemoglobin, Haematocrit, Mean Corpuscular Volume, Mean Corpuscular Hemoglobin, Mean Corpuscular Hemoglobin Concentration, Platelet Count, Prothrombin Time, Activated Partial Thromboplastin, Time, Differential Leukocyte Count - Percent and Absolute (Neutrophil, Lymphocyte, Monocyte, Eosinophil, Basophil)

Serum chemistry parameters evaluated: Albumin, Total Protein, Globulin [by calculation], Albumin/Globulin Ratio [by calculation], Total Bilirubin, Urea Nitrogen, Creatinine, Alkaline Phosphatase, Alanine Aminotransferase,, Aspartate Aminotransferase, Gamma Glutamyltransferase, Glucose, Total Cholesterol, Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides

Urinalysis parameters evaluated: Specific Gravity, pH, Urobilinogen, Total Volume, Color, Appearance, Protein, Glucose, Ketones, Bilirubin, Occult Blood, Leukocytes, Nitrites, Microscopy of Sediment

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.

The following tissues and organs were collected: Adrenal glands (2), Bone with marrow, Femur, Sternum, Bone marrow smear (from femur), Brain, Cerebrum level 1, Cerebrum level 2, Cerebellum with medulla/pons, Epididymidesc, Gastrointestinal tract, Esophagus, Stomach, Duodenum, Jejunum, Ileumd, Cecum, Colon, Rectum, Heart, Kidneys (2), Liver (sections of two lobes), Lungs (including bronchi, fixed by inflation with fixative), Lymph nodes, Mandibular (2), Mesenteric, Ovaries with oviducts (2), Peripheral nerve (sciatic), Pituitary, Prostate, Seminal vesicles (2), Spinal cord (cervical, midthoracic, lumbar), Spleen, Testes (2), Thymus, Thyroid [with parathyroids if present (2)], Trachea, Urinary bladder, Uterus with cervix, Vagina, Gross lesions

The following organs were weighed from all animals at the scheduled necropsies: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus

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.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test article-related clinical observations. All observations were noted with similar incidence in the control group, were limited to single animals, were not observed in a dose-related manner and/or were common findings for laboratory rats of this age and strain.

Mortality:

no mortality observed

Description (incidence):

One control group male was found dead on study day 24 due to acute inflammation of the kidneys. All other animals survived to the scheduled necropsies.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test article-related effects on body weights. Mean body weight gains were significantly (p<0.01) higher in the 100 mg/kg/day group males and significantly (p<0.05) 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 (p<0.05) 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.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by test article administration. There were no statistically significant differences when the control and test article-treated groups, including recovery groups, were compared.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Hematology parameters were unaffected by test article administration. There were no statistically significant differences when the control and test article-treated groups were compared.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test article-related effects on serum chemistry parameters. Females in the 100 mg/kg/day group had a significantly (p<0.05) 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.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test article-related effects on urinalysis parameters. Significantly (p<0.05) 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.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- Home cage observations were unaffected by test article administration. There were no statistically significant changes for the test article-treated males and females when compared to the control group at the study week 3 evaluation.
- Handling observations were unaffected by test article administration. There were no statistically significant changes for the test article-treated males and females when compared to the control group at the study week 3 evaluation.
- 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 (p<0.05 or p<0.01) lower and defecation for the 100 mg/kg/day group females was significantly (p<0.05) higher when compared to the control group. These differences were slight, not observed in a dose-related manner and/or were similar to pre-test observations and therefore, were not considered test article-related.
- Sensory observations were unaffected by test article administration. There were no statistically significant changes for the test article-treated males and females when compared to the control group at the study week 3 evaluation.
- Neuromuscular observations were unaffected by test article administration. There were no statistically significant changes for the test article-treated males and females when compared to the control group at the study week 3 evaluation.
- Physiological observations were unaffected by test article administration. There were no statistically significant changes for the test article-treated males and females when compared to the control group at the study week 3 evaluation.
- Mean ambulatory and total motor activity counts were unaffected by test article administration. There were no statistically significant changes for the test article-treated males and females when compared to the control group at the study week 3 evaluation.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

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 (p<0.05) 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 (p<0.01 or p<0.05) lower for 250 mg/kg/day recovery group females and significantly (p<0.01) 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.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic findings noted in the control group male found dead 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.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

One control group male was found dead on study day 24. The cause of death 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. Because this male was in the control group and not administered test article, the death was not considered test article-related.
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.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

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.

Key result

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

Critical effects observed:

no

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

Conclusions:

Under the conditions of this study, the no-observed-effect level (NOEL) for oral (gavage) administration of the test material to rats for 28 consecutive days was 250 mg/kg/day, the highest dose level tested.

Executive summary:

The 28 day oral repeat dose toxicity of the test material to rats was tested in accordance to the standardised guidelines OECD 407, under GLP conditions. 

The test material in the vehicle, deionized water, was administered orally by gavage once daily for a minimum of 28 consecutive days to groups of Sprague-Dawley rats. Dosage levels were 0, 25, 100 and 250 mg/kg/day.

All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily and detailed physical examinations were performed weekly. Individual body weights and food consumption were recorded weekly. Functional observational battery and motor activity data were recorded for all animals prior to the initiation of dose administration and during study week 3. Clinical pathology evaluations (hematology, serum chemistry and urinalysis) were performed on all rats euthanized at the primary necropsy (study day 28). Following 28 days of dose administration, eight rats/sex/group were euthanized; the remaining rats in the control and high dose groups were euthanized following a 14-day nondosing (recovery) period. Complete necropsies were conducted on all animals and selected organs were weighed at the scheduled necropsies. Selected tissues were examined microscopically from all animals in the control and 250 mg/kg/day groups euthanized at the primary necropsy (study day 28).

There were no test material related deaths during the study. There were no test material-related clinical observations or effects on body weights and food consumption. Functional observational battery and motor activity data, hematology, serum chemistry and urinalysis parameters were unaffected by test material administration. There were no test material-related macroscopic or microscopic findings observed or effects on organ weights.

Therefore, under the conditions of this study, the no-observed-effect level (NOEL) for oral (gavage) administration of the test material to rats for 28 consecutive days was 250 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rat

System:

other: NOEL based on highest administered dose due to no adverse effects.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The 28 day oral repeat dose toxicity of the test material to rats was tested in accordance to the standardised guidelines OECD 407, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material in the vehicle, deionized water, was administered orally by gavage once daily for a minimum of 28 consecutive days to groups of Sprague-Dawley rats. Dosage levels were 0, 25, 100 and 250 mg/kg/day.

All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily and detailed physical examinations were performed weekly. Individual body weights and food consumption were recorded weekly. Functional observational battery and motor activity data were recorded for all animals prior to the initiation of dose administration and during study week 3. Clinical pathology evaluations (hematology, serum chemistry and urinalysis) were performed on all rats euthanized at the primary necropsy (study day 28). Following 28 days of dose administration, eight rats/sex/group were euthanized; the remaining rats in the control and high dose groups were euthanized following a 14-day nondosing (recovery) period. Complete necropsies were conducted on all animals and selected organs were weighed at the scheduled necropsies. Selected tissues were examined microscopically from all animals in the control and 250 mg/kg/day groups euthanized at the primary necropsy (study day 28).

There were no test material related deaths during the study. There were no test material-related clinical observations or effects on body weights and food consumption. Functional observational battery and motor activity data, hematology, serum chemistry and urinalysis parameters were unaffected by test material administration. There were no test material-related macroscopic or microscopic findings observed or effects on organ weights.

Therefore, under the conditions of this study, the no-observed-effect level (NOEL) for oral (gavage) administration of the test material to rats for 28 consecutive days was 250 mg/kg/day.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to specific target organ toxicity or repeated dose toxicity.