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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 18, 1987 to June 21, 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPP 82-1 (90-Day Oral Toxicity)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
This is formulation of 49.2% of the test substance in water.
Specific details on test material used for the study:
- Name of test material (as cited in study report): Alkyl dimethyl benzyl ammonium hloride
- Physical state: Pale yellow viscous liquid
- Analytical purity: 80.51 and 79.7% w/w active substance in aqueous/ethanol solution
- Lot/batch No.: Lot # 6158-59-60 (batch 1) and Lot # SC132-65 (batch 2)
- Stability under test conditions: The test substance is hydrolytically and photolytically stable under the conditions of this study and has been shown to be stable in aqueous, alcohol and alcohol/aqueous solutions for extended periods, e.g. at least five years under standard laboratory conditions.
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Test animals
- Source: Sprague-Dawley CD rats were obtained from Charles River Breeding Laboratories, Portage, MI, USA.
- Age at study initiation: 7 weeks
- Weight at study initiation: 221.6-250.9 g (males); 147.7-174.4 g (females)
- Housing: Animals were housed one animal/side of divided stainless steel cages (solid sides with wire mesh floors) mounted in a stainless steel Maxi-Rack (Hazleton Systems, Inc., Aberdeen, MD).
- Diet: Ground Purina Certified Rodent Chow # 5002 (Ralstor Purina Co., St. Louis, MO), ad libitum
- Water: Tap water, ad libitum. Water was provided by an automatic watering system with demand control valves mounted on each cage rack
- Acclimation period: 1 week

Environmental conditions:
- Temperature: 22 ± 3°C
- Humidity: 40-70%
- Photoperiod: 12h light/12h dark

In-life dates: From: 18 June 1987 to: 22 September 1987

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: Ground Purina Certified Rodent Chow # 5002
Details on oral exposure:
Diet preparation: Test diets were prepared by direct addition of the test substance to ground rodent feed. A concentrated premix was prepared to ensure maximal loss of the ethanol (approximately 12% by weight) from the test substance during the original mixing time of 1h. Test diets were prepared by appropriate dilutions of the concentrated premix or higher diet concentrations.
- Rate of preparation of diet (frequency): Fresh diet was prepared and offered to the animals each week.
- Mixing appropriate amounts with (Type of food): Ground Purina Certified Rodent Chow # 5002
- Storage temperature of food: Diets were stored in polypropylene containers at room temperature.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Experimental diets were analyzed for stability, homogeneity and concentration of test substance in the diet by using liquid chromatography.
- Homogeneity studies performed on samples from all dietary concentrations indicated that test substance was uniformly distributed in the diet.
- Stability studies were conducted on diets (8000 and 100 ppm) stored at room temperature in closed polypropylene containers and in glass jars. These analyses indicated that the test substance was stable in the diets for at least 21 d under both storage conditions for all dose levels.
- Concentration verification analyses revealed that the test substance concentrations were 90.7 to 111.8% of nominal for all 5 concentrations.
Duration of treatment / exposure:
95 and 96 days for males and females, respectively.
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 500, 1000, 4000 or 8000 ppm (equivalent to 0, 6, 31 and 62 mg/kg bw/day (males) and 0, 8, 38 and 77 mg/kg bw/day (females). [Due to mortality in the 4000 and 8000 ppm treatment groups, the corresponding daily intakes could not be calculated.]
Basis: nominal in diet
No. of animals per sex per dose:
15
Control animals:
yes, plain diet
Details on study design:
- Rationale for animal assignment: Animals were assigned to test groups, based on body weight, by a computer generated, weight stratified randomization procedure. Only rats with body weights within ± 20% of the population mean for each sex were used in the study.

Assignment of animals: The animals were assigned into following groups in the study:
- Group 1(Diet control): Plain diet
- Group 2: 100 ppm test substance in diet
- Group 3: 500 ppm test substance in diet
- Group 4: 1000 ppm test substance in diet
- Group 5: 4000 ppm test substance in diet
- Group 5: 8000 ppm test substance in diet


Examinations

Observations and examinations performed and frequency:
MORTALITY and CLINICAL SIGNS: Yes
- Time schedule: Twice daily

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed once a week for detailed clinical observations, and six days a week for overt clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight data were collected weekly for all animals.

FOOD 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: Yes, food consumption data were collected weekly for all animals.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmic examinations were performed prior to treatment and prior to final sacrifice.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Just prior to sacrifice at study termination
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: Yes, overnight
- How many animals: Blood samples were collected from 10 animals/sex/group (or from the surviving animals in the 4000 ppm group) for haematology analyses.
- Parameters checked: Hemoglobin, hematocrit, erythrocyte count, erythrocyte indices, platelet count, total leukocyte count, differential leukocyte count, reticulocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Just prior to sacrifice at study termination
- Animals fasted: Yes, overnight
- How many animals: Blood samples were collected from 10 animals/sex/group for clinical chemistry
- Parameters checked: AST (SGPT), ALT (SGOT), creatinine, alkaline phosphatase, gamma glutamyl transpeptidase, glucose, urea nitrogen, total protein, albumin, globulin, A/G ratio, total bilirubin, direct bilirubin, indirect bilirubin, calcium, phosphorous, sodium, potassium, chloride

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
SACRIFICE: Animals were anesthetized with methoxyflurane and exsanguinated by severing the brachial vessel.
GROSS PATHOLOGY: Yes, all animals (surviving) were subject to gross necropsy.
HISTOPATHOLOGY: Yes, histopathologic examinations were performed on selected tissues from 10 randomly selected animals/group from the control and 1000 ppm dose groups. The 1000 ppm group was selected for complete examination because it was the highest dose group without significant mortality.
- Tissues collected for histopathology: Gross lesions, brain, eyes, pituitary, salivary gland, heart, aorta, thymic region, thyroid, lungs, adrenals, trachea, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, spinal cord, pancreas, liver, kidneys, urinary bladder, testes, prostate, epididymis, ovaries, uterus, spleen, lymph nodes, sciatic nerves, sternum
Other examinations:
ORGAN WEIGHT: Prior to sacrifice, body weights were obtained to allow expression of relative organ weights. The liver, spleen, kidney, heart, brain, adrenals, testes and ovaries were weighed for all animals.
Statistics:
- Parametric variables were compared between the dose and control groups using Levene’s test for homogeneity of variances, by analysis of variance and by pooled variance t-tests.
- Non-parametric data were analyzed by the Kruskal-Wallis test or by the Wilcoxon rank sum test as modified by Mann-Whitney.
- Frequency data were compared using Fisher’s exact tests.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All animals in the 8000 ppm group died. For the 4000 ppm group 12/15 males and 11/15 females died or were sacrificed in a moribund condition.

Mortality:
mortality observed, treatment-related
Description (incidence):
Treatment-related clinical findings were restricted to animals from the 4000 and 8000 ppm groups. The observations were of two types, general cachexia and loose faeces. Findings for the surviving animals were similar to those that died.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Decrease in body weight was observed for the 4000 and 8000 ppm dose group surviving the first week of the study. A slight but significant decrease was also noticed in the males of the 1000 ppm dose group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Decrease in food consumption was observed for the 4000 and 8000 ppm dose group surviving the first week of the study. A slight decrease in the food consumption was also noticed in the males of the 1000 ppm dose group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related finding at any treatment level was observed.
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment related change was observed for males or females from any treatment group (4000 ppm or lower).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No treatment related effect up to 1000 ppm. Significant increases in ALT and phosphorus were observed for 3 surviving males of the 4000 ppm group.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
No treatment related effect up to 1000 ppm was observed.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No treatment related effect up to 1000 ppm was observed. Gross lesions related to the treatment were restricted to the animals that died in the 8000 and 4000 ppm group and to a lesser degree in the animals that survived the 4000 ppm group. The findings were fecal staining of perineal area, emaciation, colour changes of the organs, ileus consisting of distended fluid and gas-filled viscera.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathologic changes were observed for animals in the 4000 and 8000 ppm dose group and consisted of congestion of various organs or tissues, mucosal cell degeneration of the villus tips of small intestine and cecum, submucosal edema of stomach, splenic contraction and hepatocellular atrophy.
Histopathological findings: neoplastic:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
31 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: the dose of 500 ppm is equivalent to 31 mg/kg bw/day for males and 38 mg/kg bw/day for females
Key result
Dose descriptor:
NOEL
Effect level:
38 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: the dose of 500 ppm is equivalent to 31 mg/kg bw/day for males and 38 mg/kg bw/day for females

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
62 mg/kg bw/day (nominal)
System:
other: GI tract, hepatobiliary and spleen (males)
Organ:
stomach
intestine
ileum
colon
liver
spleen
Treatment related:
yes
Dose response relationship:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
77 mg/kg bw/day (nominal)
System:
other: GI tract, hepatobiliary and spleen (females)
Organ:
stomach
intestine
ileum
colon
liver
spleen
Treatment related:
yes
Dose response relationship:
yes

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the rat NOAEL of the test substance in rats was considered to be 500 ppm in the diet, i.e., equivalent to 31 mg/kg bw/day for males and 38 mg/kg bw/day for females.
Executive summary:

A study was conducted to determine the repeated dose oral toxicity of the test substance (ca. 80% purity) according to OECD Guideline 408 and US EPA OPP 82 -1, in compliance with GLP. This sub-chronic oral toxicity study (90 d) was realised in Sprague-Dawley rats. The rats were administered daily dietary levels of 0, 100, 500, 1000, 4000 and 8000 ppm test substance, equivalent to 0, 6, 31 and 62 mg/kg bw/d (i.e. 0, 4.8, 25 and 50 mg a.i./kg bw/d) (males) and 0, 8, 38 and 77 mg/kg bw/d (i.e. 0, 6.4, 30 and 62 mg a.i./kg bw/d) (females) for 95 and 96 days, respectively. Daily intakes at 4000 and 8000 ppm could not be calculated due to high mortality. The animals were observed for mortality, clinical signs, body weight, food consumption, hematology and clinical chemistry at termination. Gross and histopathological examinations were also performed. A slight trend in reduction of body weight and food consumption in males at 1000 ppm was observed. Besides this, there were no treatment-related findings at 1000 ppm or lower. There was 80 and 100% mortality at 4000 and 8000 ppm, respectively. Clinical signs of toxicity (general cachexia and loose faeces), decreased food consumption and body weights, gross necropsy findings (including increased amounts of liquids or semi-solid material within the stomach, jejunum, ileum and cecum) were also observed at 4000 and 8000 ppm. The cause of morbidity and death was assumed to be shock secondary to fluid and/or ionic shifts in the gastro-intestinal tract. Under the study conditions, the NOAEL was considered to be 500 ppm in the diet, equivalent to 31 mg/kg bw/d (i.e. 25 mg a.i./kg bw/d) for males and 38 mg/kg bw/d (i.e. 30 mg a.i./kg bw/d) for females (Van Miller, 1988).