Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990-11-19 to 1991-02-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and similar to guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Remarks:
This study was conducted in compliance with Part 792 of 40 CFR (EPA/TSCA Good Laboratory Practice Regulations)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Sample No of test material: 38218-A- Substance type: organic- Physical state: cloudy liquid- Purity: 90 % Triethanol Methyl Ammonium Methyl Sulfate; 10 % Isopropyl alcohol- Expiration date of the lot/batch: August 1992- Storage condition of test material: in a temperature monitored room- Lot/batch: 2005211- Density: 1. 229 g/mL

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Charles River Breeding Laboratories Inc., Kingston, New York 12484, USA- Age at study initiation: approx. 39 weeks old- Weight at study initiation: male: 176.4 g (163.5-188.3); female: 166.5 g (152.6-183.5)- Fasting period before study: no- Housing: individually- Diet: ad libitum, standard laboratory diet (Purina Mills Certified Rodent Mash-type Diet #5002)- Water: ad libitum, tap water (Elizabethtown Water Company, Westfield, N.J., USA)- Acclimation period: 11 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 20.6 - 25.6- Humidity (%): 28 - 70- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:Appropriate amounts of the test material were mixed with tap water to achieve the desired concentrations. Test solution with 1.6 % was prepared by measuring 231.1 mL of the stock solution in an appropriately sized beaker and fill up with 13 liters of tap water. 0.32 % solutions were prepared by diluting 2200 mL of the 1.6 % solution with 11 liters of tap water. 0.096 % solutions were prepared by diluting 660 mL of the 1.6 % solution with 11 liters of tap water.All preparations were mixed thoroughly.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No details on analytical measurements were included in the study report. But homogeneity, stability and confirmation of concentrations during study were analysed. Homogeneity was proven prior to initiation by analysis of mock batches of the low and high concentration solution. Samples of each test solution (dose level) for weeks 1 through 4, 7, 8, and 12 were sent to the sponsor for concentration verification. Stability analysis was performed in the low and high concentration solutions 4, 7 and 14 days after preparation of the testing solutions.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:0.096 % v/v (eq. 115.3 (m) and 139.6 (f) mg/kg bw/d)Basis:nominal in water
Remarks:
Doses / Concentrations:0.32 % v/v (eq. 406.4 (m) and 495.5 (f) mg/kg bw/d)Basis:nominal in water
Remarks:
Doses / Concentrations:1.6 % v/v (eq. 2389.1 (m) and 2703.3 (f) mg/kg bw/d)Basis:nominal in water
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes- Time schedule: daily- Parameters examined: normality, emesis, abnormal stools, other discharges and gross signs of poor health or toxic or pharmacologic effects, e.g. gross abnormalities in general condition, appearance, activity, behaviour, etc.DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: dailyBODY WEIGHT: Yes- Time schedule for examinations: weeklyFOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: YesWATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes- Time schedule for examinations: weeklyOPHTHALMOSCOPIC EXAMINATION: Yes- Time schedule for examinations: prior to test and at termination- Dose groups that were examined: all of themHAEMATOLOGY: Yes- Time schedule for collection of blood: at sacrifice- Anaesthetic used for blood collection: not necessary- Animals fasted: Yes- How many animals: all- Parameters examined: hemoglobin concentration, hematocrit, erythrocyte count, reticulocyte count, platelet count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, prothrombin time, activated partial thromboplastin time, total and differential leukocyte counts, erythrocyte morphologyCLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: at sacrifice- Animals fasted: Yes- How many animals: all- Parameters examined: aspartate aminotransferase (serum glutamic oxaloacetic transaminase), alanine aminotransferase (serum glutamic pyruvictransaminase), alkaline phosphatase, blood urea nitrogen, creatinine, fasting glucose, total protein, albumin, globulin (calculated), A/G ratio (calculated), total bilirubin, sodium, potassium, chloride, calcium, inorganic phosphorus, gamma glutamyl transpeptidaseURINALYSIS: Yes- Time schedule for collection of urine: at sacrifice- Metabolism cages used for collection of urine: No- Animals fasted: Yes- Parameters examined: gross appearance, specific gravity, pH, protein, glucose, ketones, bilirubin, occult blood, 16-hour volume, microscopic analysisNEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, complete gross postmortem examinations were performed on all animals: external surface and all orifices; the external surfaces of the brain and spinal cord; the organs and tissues of the cranial, thoracic, abdominal, and pelvic cavities and neck; and the remainder of the carcass.Organs weighed: adrenals, brain, heart, kidneys, liver, ovaries, spleen, testes with epididymides, thyroid/parathyroids, urinary bladderTissues preserved: adrenal glands, aorta (thoracic), bone marrow (sternum), bone (sternum), brain (medulla/pons, cerebrum and cerebellum), epididymides, esophagus, eyes (with optic nerve), femur, heart, kidneys, lacrimal gland, large intestine (cecum, colon, and rectum), liver (at least 2 lobes), lungs (with mainstem bronchi), lymph node (mesenteric), lymph node (mediastinal), mammary gland, muscle (biceps femoris), nerve (sciatic), ovaries, pancreas, pituitary gland, prostate, salivary gland (mandibulbar), seminal vesicles, skin, small intestine (duodenum, jejunum and ileum), spinal cord (thoracic), spleen, stomach, testes, thymus, thyroid gland (with parathyroids), trachea, urinary bladder, uterus (body/horns) with cervix, gross lesionsHISTOPATHOLOGY: Yes, the following tissues were examined for all animals of the low and high dose group. Target organs identified in the high dose animals were also examined in the remaining animals.Tissues examined: adrenal glands, aorta (thoracic), bone marrow (sternum), bone (sternum), brain (medulla/pons, cerebrum and cerebellum), epididymides, esophagus, heart, kidneys, lacrimal gland, large intestine (cecum, colon, and rectum), liver (at least 2 lobes), lungs (with mainstem bronchi), lymph node (mesenteric), lymph node (mediastinal), nerve (sciatic), ovaries, pancreas, pituitary gland, prostate, salivary gland (mandibulbar), small intestine (duodenum, jejunum and ileum), spinal cord (thoracic), spleen, stomach, testes, thymus, thyroid gland (with parathyroids), trachea, urinary bladder, uterus (body/horns) with cervix, gross lesions
Statistics:
A standard one way ANOVA using the F distribution to assess significance and a Bartlett's test was performed to determine if groups had equal variance. If the variances were unequal, the Kruskal-Wallis test was used, and if differences were indicated a summed rank test (Dunn) was used to determine which treatments differed from control. If the variances were equal, and significant differences among the means were indicated, Dunnett's test was used to determine which means were significantly different from the control. If mean terminal organ weights had unequal variances, the ratios for these organs were analyzed using the Kruskal-Wallis test. A statistical test for trend in the dose levels was also performed. In the parametric case (i.e., equal variance) standard regression techniques with a test for trend and lack of fit were used. In the nonparametric case Jonckheere's test for monotonic trend was used. The test for equal variance (Bartlett's) was conducted at the 1%, two-sided risk level. All other statistical tests were conducted at the 5% and 1%, two-sided risk level. References for these techniques are Snedecor, G.W., and Cochran, W.G., Statistical Methods, 6th edition, Iowa State University Press (1967); Hollander, M. and Wolfe, D.A., Nonparametric Statistical Methods. John Wiley and Sons, New York (1973); Dunnett, C.W., J. Am. Sta. Assn. Val. 50 (1955) and Biometrics. Val. 20 (1964).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITYNo mortality was observed throughout the study period.BODY WEIGHT AND WEIGHT GAINAll animals gained weight throughout the study. However, weights and weight gains for high-dose males were slightly lower than control values and, at study termination, the difference in mean body weights for these two groups was nine percent.FOOD CONSUMPTION AND COMPOUND INTAKEFood consumption values for treated groups were comparable to control values or exhibited normal variability. No consistent differences considered to represent an effect of test material administration were evident.WATER CONSUMPTION AND COMPOUND INTAKEMean weekly water consumption values for high-dose males were statistically significantly higher than mean control values at most intervals from Week 4 throughout study termination (Week 13). Values for high-dose females and for mid- and low-dose animals of both sexes were comparable to control values or exhibited normal variability. No effect of test material was evident in these groups. Mean weekly test substance intake, calculated from water consumption values and based on nominal concentrations are listed in table 1 under "any other information on results incl. tables".OPHTHALMOSCOPIC EXAMINATIONOphthalmoscopic examinations at study termination revealed no ocular abnormalities considered to be related to test material administration.HAEMATOLOGYTerminal values demonstrated no effect of test material administration. Values for control and treated groups were comparable; no statistically significant differences occurred. Differential leukocyte counts were unremarkable.CLINICAL CHEMISTRYAt study termination, values for control and treated groups were considered comparable. No statistically significant differences occurred and no effect of test material administration was evident.URINALYSISUrinary pH values for the high-dose males and females were slightly but statistically significantly lower than values for control animals at study termination. Ranges and mean values are summarized in table 2 under "any other information on results incl. tables".The differences seen for high-dose animals are statistically significant. Other urinalysis values exhibited normal variability. No differences suggestive of an effect of the test material were apparent.ORGAN WEIGHTSThe mean terminal kidney weight for the high-dose males was slightly higher than the control mean even though the mean terminal body weight for this group was slightly lower than the mean control weight. This resulted in a statistically significant elevation, relative to the control value, in the kidney/body weight ratio which is considered suggestive of an effect of test material administration. Similar differences were not evident for females. Other organ weight values for control and treated groups were generally comparable. The only statistically significant difference (elevated liver/brain weight ratio for high-dose females) was slight and no dose-related trend was evident; this was considered unrelated to test material administration.GROSS PATHOLOGY/HISTOPATHOLOGY: NON-NEOPLASTICGross and microscopic postmortem observations were unremarkable. The few abnormalities which were seen occurred sporadically in treated and control animals with comparable incidence and severities and were not considered attributed to the test material.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
ca. 406.4 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No effects were observed in any of the male animals at this dose.
Dose descriptor:
NOEL
Effect level:
ca. 495.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No effects were observed in any of the female animals at this dose.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1 Mean test substance intake

 

Concentration [% v/v]

Mean test substance intake [mg/kg/d]

Males

Females

Group II

0.096

80-190

119-188

Group III

0.32

274-667

395-703

Group IV

1.6

1840-3860

2300-3492

Table 2 Urinary pH values

 

Concentration [% v/v]

Urine pH values – Study Termination

Males

Females

Meana

Range

Meana

Range

Group I

0

8.6

8.0-≥9.0

8.6

8.0-≥9.0

Group II

0.096

8.7

8.0-≥9.0

8.6

8.0-≥9.0

Group III

0.32

8.4

8.0-≥9.0

8.5

8.0-≥9.0

Group IV

1.6

7.8**

6.5-8.5

7.7**

6.5-8.5

aMeans calculated using 9.0 for values≥9.0.

** p≤0.01

Applicant's summary and conclusion