Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992-10-30 to 1993-02-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with fatty acids, C16-18 (even numbered) and C18 unsatd., Me sulfates (salts)
Cas Number:
1079184-43-2
Molecular formula:
n.a. (UVCB)
IUPAC Name:
1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with fatty acids, C16-18 (even numbered) and C18 unsatd., Me sulfates (salts)
Details on test material:
- Name of test material (as cited in study report): N,N-dimethyl-2-(stearoyloxy)-N-[2-(stearoyloxy)ethyl]ethanaminium chloride in 10% aqueous dispersion.
- Molecular formula (if other than submission substance): Not available
- Molecular weight (if other than submission substance): Not available
- Smiles notation (if other than submission substance): Not available
- InChl (if other than submission substance): Not available
- Structural formula attached as image file (if other than submission substance): Not available
- Substance type: Not available
- Physical state: liquid

Test animals

Species:
rat
Strain:
other: Crl CD BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation: 4 weeks
- Weight at study initiation: 180-210 g (males) and 162-191 g (females)
- Fasting period before study: None
- Housing: individually in wire-mesh cages (for the first two days of the conditioning period, animals were housed 3/cage to allow for acclimation to the automatic watering system).
- Diet (e.g. ad libitum): ground Certified Rodent Chow #5002, Purina Mills, Inc., St. Louis, MO ad libitum
- Water (e.g. ad libitum): ad libitum, water was analyzed on a quarterly basis for the presence of heavy metals, pesticides and other contaminants
- Acclimation period: 15 days


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 73.2 +/- 1.0
- Humidity (%): 47.9 +/- 5.8
- Air changes (per hr): Not available
- Photoperiod (hrs dark / hrs light): 12 hours dark/light


IN-LIFE DATES: From: 1992-10-21 To: 1993-02-04

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was prepared in oral dosing suspensions at concentrations of 0.01, 0.1 and 5.0 % to provide dosage levels of 1, 10, and 500 mg/kg/bw/day.

Dosing suspensions of the test substance required modification of the vehicle in order to produce a consistent pH of 2.5. According to the Sponsor, the stock test preparations were pH 2.15. Since the stock suspensions were to be diluted with the pH Control (pH 2.5) to produce the 5 % dosing suspension, minor adjustment of the pH Control (to slightly increase pH with small amounts of 1 N sodium hydroxide) were necessary to insure a final pH of 2.5 for the dosing suspensions. Pre-test experiments were performed with the stock test article suspensions and the pH Control to determine the necessary adjustment.

Following adjustment of the pH Control for use as a diluent, this modified pH Control was mixed with the stock test substance suspensions to produce a 5 % dosing suspension of test substance (high dose) with a pH of approx. 2.5. The exact proportions of stock test substance suspension to modified pH Control varied with variation of test substance in the stock test substance suspension supplied by the Sponsor. The mid-and low-dose suspensions (0.1 and 0.01 %) were then produced via serial dilution of the 5 % dosing suspension with unmodified pH Control. Each phase of the blending and serial dilutions utilized double-rinsed polypropylene equipment. Each suspension was mixed with a magnetic stirring device for a minimum of 15 minutes between steps. The pH of the suspensions was monitored throughout the mixing period.

Final dilutions permitted once daily administration at a rate of 10 ml/kg. Fresh suspensions were prepared as needed (approx. bi-weekly) and stored at ambient temperature. Test substance suspensions were agitated with magnetic mixers for a minimum of 10 minutes each day immediately prior to dosing.


DIET PREPARATION
- Rate of preparation of diet (frequency): Not available
- Mixing appropriate amounts with (Type of food): Not available
- Storage temperature of food: Not available


VEHICLE: deionized water
- Justification for use and choice of vehicle (if other than water): Not available
- Concentration in vehicle: Not available
- Amount of vehicle (if gavage): Not available
- Lot/batch no. (if required): Not available
- Purity: Not available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the test substance were collected on study days 25, 46, 77, and 92 for stability analysis; and on study days 25, 49, and 77 for concentration analysis. Duplicate samples were taken on days 25, 46, 63, 77, and 92 for microbiological characterization. All samples were delivered for analysis to the Sponsor. Stability and concentration samples were sent frozen and microbiological characterization samples were sent at ambient temperatures. All sampling was conducted immediately after active agitation of the suspensions.

pH TESTING: Dosing suspenstions and the pH Control solution were tested for conformance with pH requirements (pH 2.5 +/- 0.3) each day prior to dosing.

STABILITY ANALYSIS: These samples were collected prior to changeover to fresh test substance and dosing suspensions. They represent material and preparations in use for the preceding, approximate bi-weekly period. Samples were shipped frozen.

CONCENTRATION ANALYSIS: Samples of all dosing preparations and controls were taken on study days 1, 26, 49 and 77 for concentration analysis by the Sponsor. These samples were collected from freshly produced dosing suspensions prepared from recently received test material. Samples were shipped frozen.

MICROBIOLOGICAL CHARACTERIZATION: Samples of the dosing preparations and controls were taken on study days 25, 46, 63, 77, and 92 for microbiological characterization by the Sponsor. Similar to stability samples, those taken for microbiological characterization represented test substance and preparations in use for the proceeding, approximate bi-weekly period. These samples were shipped at ambient temperatures.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily, 7 days/week, for 13 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
500 mg/kg/bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
10 mg/kg/bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1 mg/kg/bw/day
Basis:
actual ingested
No. of animals per sex per dose:
15 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Oral gavage was used to ensure delivery of a controlled and consistent dose of the test substance. Dosage levels were selected based on data generated from a 14-day (range-finding) toxicity study in rats (IRDC 191-1542), and the Sponsor's experience with subchronic
studies for structurally similar compounds. The volume administered to each rat (10 ml/kg) was adjusted based on the most recent body weight.
- Rationale for animal assignment (if not random): Prior to randomization into study groups, the animals were weighed, sexed and examined for evidence of disease and other physical abnormalities. After eliminating animals based on these criteria, animals found to be acceptable for study use
were randomized utilizing a block randomization procedure in which animals were stratified by body weight. Homogeneity of group variance by
body weight was used as the criterion for acceptance, at which time the randomization was accepted and permanent animal numbers were assigned.
- Rationale for selecting satellite groups: NA
- Post-exposure recovery period in satellite groups: NA
- Section schedule rationale (if not random): NA
Positive control:
No data

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes (mortality)
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Rats were observed twice daily for signs of overt toxicity at the times of the mortality/morbidity checks. Detailed clinical examinations were also performed at least weekly and included evaluations of appearance and condition, behaviour and activity, excretory function, respiration, orifices, eyes and palpable masses.


BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were obtained during the pretest period and weekly during the study and prior to termination.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
-Individual food consumption was calculated weekly during the study.


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:N/A


OPHTHALMOSCOPIC EXAMINATION: Yes (performed by a veterinary ophthalmologist using a Keeler Indirect Ophthalmoscope of the cornea, sclera, iris and fundus)
- Time schedule for examinations: during acclimation period and prior to terminal sacrifice
- Dose groups that were examined: all animals


HAEMATOLOGY: Yes
- Time schedule for collection of blood: taken at study termination
- Anaesthetic used for blood collection: Yes (CO2). Samples obtained via cardiac puncture
- Animals fasted: Yes, overnight. Animals had free access to water prior to blood collection. Urine was collected during the fasting period.
- How many animals: 10 animals/sex/group at termination. Studies from 6 animals scheduled for coagulation testing clotted prior to analysis. Blood samples were collected from 6 additional animals for coagulation testing in order to complete the 10/animals/sex/group protocol requirement.
- Parameters examined: leukocyte count, erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelets, differential leukocyte count, prothrombin time (PT), activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: taken at study termination.
- Animals fasted: Yes, overnight. Animals had free access to water prior to blood collection. Urine was collected during the fasting period.
- How many animals: 10 animals/sex/group at termination. Studies from 6 animals scheduled for coagulation testing clotted prior to analysis. Blood samples were collected from 6 additional animals for coagulation testing in order to complete the 10/animals/sex/group protocol requirement.
- Parameters examined: sodium, potassium, chloride, calcium, inorganic phosphorous, alkaline phosphatase, total bilirubin, gamma glutamyl transpeptidase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen, creatinine, total protein, albumin, cholesterol, glucose.


URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected during the fasting period before blood was drawn.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters examined: specific gravity and volume.


NEUROBEHAVIOURAL EXAMINATION: No



OTHER: Not available
Sacrifice and pathology:
All animals received a complete postmortem examination under the direct supervision of a pathologist. All animals were necropsied in a replicate order, and a necropsy body weight was obtained immediately following euthanization, but before exsanguination. This weight was used to determine the organ to body weight ratios. All animals were euthanized by methoxyfluorane anesthesis.

GROSS PATHOLOGY: Yes
After a thorough external examination, the skin was reflected from ventral midline incision and any abnormalities were identified and correlated with antemortem findings. The abdominal, thoracic and cranial cavities were examined for abnormalities and the organs removed, examined and, where required by protocol, placed in phosphate-buffered neutral formalin. All macroscopic abnormalities were recorded on the Pathology Record sheet.

Organ weights were determined for the following tissues for all animals and appropriate weight ratios calculated (absolute and relative to body and brain weights). Paired organs were weighed together. Adrenal, brain with stem, kidney, liver, ovary, testis.

HISTOPATHOLOGY: Yes
Representative samples of protocol designated organs and tissues were processed and embedded in paraffin for the preparation and microscopic examination of stained sections for all animals in the pH Control and 500 mg/kg/bw/day dosage groups.

A four-step grading system of trace, mild, moderate, and severe was used to define gradable lesions for comparison between dosage groups. Representative samples of the following tissues were collected and examined microscopically: adrenal, aorta, bone marrow smear, brain, exoribital lacrimal gland, eye including optic nerve, femur, GI tract, gonads, gross lesions, heart, kidney, liver, lung with bronchi, lymph nodes, mammary gland (females), pancreas, pitituary, prostate and seminal vesicle, salivary gland, sciatic nerve, skeletal muscle (thigh), skin, spinal cord, spleen, sternum (with bone marrow), thymus, thyroid, parathyroid, trachea, urinary bladder, uterus and cervix, vagina.
Other examinations:
Not available
Statistics:
Body weight, food consumption, clinical pathology laboratory, and organ weights (absolute and relative) were analyzed using analysis of variance (one-way classification) and Bartlett's test for homogenecity of variance. Treatment groups were compared to the pH Control group, by sex, using appropriate t-statistic (for equal or unequal variance) as described by Steal and Torrie. Dunnett's multiple comparison tables or pairwise comparisons with a Bonferroni correction were used to determine the significance of differences. Non parametric analyses were conducted as appropriate by transforming the data into ranks prior to analysis as described by Conover and Iman. All statistical analyses were performed with P less than or equal to 0.05 and P less than or equal to 0.01 used as levels of significance.

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 specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
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:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: All rats survived to study termination. No test substance-related signs of overt toxicity were observed during the study. Clinical examinations of the animals did not produce any evidence of test substance-related effects.


BODY WEIGHT AND WEIGHT GAIN: No significant differences in body weights or body weight increases occurred over the course of the study.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No significant differences in food consumption occurred over the course of the study in any of the groups.


FOOD EFFICIENCY: Not available


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not available

OPHTHALMOSCOPIC EXAMINATION: No test substance-related ophthalmoscopic abnormalities were detected; the observations noted were representative of pathology that would be expected for this group of rats considering age, sex and strain.


HAEMATOLOGY: Mean MCV values were statistically significantly lower (p less than or equal to 0.05) in male rats in the water control, 10 mg/kg/bw/day, and 500 mg/kg bw/day treatment groups as compared to the pH Control group. The mean MCV values in these groups were within +/- 2 standard deviations of this laboratory's historical control mean value for this parameter for rats of this strain, age, and sex. The mean MCV values in these groups also fall within published reference ranges for rats of this strain, age, and sex. Because of these considerations, because this change was not present in both sexes, and because this change also occurred in the water control group, this haematological change, although statistically significant, was not considered to be toxicologically significant or test article-related.


CLINICAL CHEMISTRY: (Blood Chemistry) Mean urea nitrogen values were statistically significantly lower (p less than or equal to 0.01) in male rats in the 500 mg/kg bw/day treatment group as compared to the pH Control group. The mean urea nitrogen value in this group was within +/- 2 standard deviations of the testing laboratory's historical control mean value for this paramater for rats of this strain, age, and sex. Because of the small magnitude of the change, because this change was not present in both sexes, and because no other clinical, biochemical, or pathologic alterations correlating with decreased urea nitrogen were observed, this serum biochemical change, although statistically significant, was not considered to be toxicologically significant or test substance-related.


URINALYSIS: There were no changes that were considered to be toxicologically significant or test substance-related.


NEUROBEHAVIOUR: Not available


ORGAN WEIGHTS: There were no test substance-related changes in the mean absolute weights or mean relative organ weight ratios for the brain, adrenal, kidney, liver and testis of male and female rats.

There was a statistically significant decrease in the mean ovary/body weight ratio for female rats in the 500 mg/kg/bw/day group when compared with the pH Control. This ratio change was, at least in part, due to the variability in the mean body weights with the pH Control being the lowest of all the female groups and the 500 mg/kg bw/day group being the highest of the female groups on the study. There were no statistically significant changes in the mean absolute ovary weight and the ovary/body or the ovary/brain weight ratios when the 1, 10 and 500 mg/kg bw/day treatment groups were compared with the water control or when the same parameters of the ovary from the water control group were compared to the pH Control. The mean absolute ovary weight of the 500 mg/kg bw/day treatment group falls within the range of mean absolute ovarian weight fo 13-week CD rat studies at the testing laboratory. For these reasons, the decreased mean ovary/body weight ratio observed in this study was considered not to be biologically relevant and not test substance-related.


GROSS PATHOLOGY: There were no test substance-related macroscopic findings in either male or female rats necropsied 13 weeks following oral intubation with Water or pH Control material or with 1, 10 or 500 mg/kg bw/day of test article.

No rats died during the duration of the study. A few macroscopic findings were noted at the terminal necropsy. The majority of these macroscopic findings were confirmed microscopically. These findings were considered to be incidental and usual for rats of this age and strain and not test article-related.


HISTOPATHOLOGY: NON-NEOPLASTIC: There were no test substance-related microscopic findings in either male or female rats necropsied following 13 weeks oral intubation.

All macroscopic observations (except cystic ovary, # 41229) correlated with microscopic findings. There were several microscopic findings. These findings were considered to be incidental and usual for rats of this strain and age. The incidence of a given lesion was small and/or occurred with equivalent frequency in treated animals versus controls. There was no evidence of any infectious disease present in any of the rats that would adversely affect the results of the study.

The tissues available for microscopic examination were of satisfactory quality and quantity to adequately evaluate this study. Relevant in-life and necropsy data were available to the study pathologist and were considered in interpretation of the pathology findings. All reference to pathology interpretations in the final report were consistent.


HISTOPATHOLOGY: NEOPLASTIC (if applicable): Not available


HISTORICAL CONTROL DATA (if applicable): Not available


OTHER FINDINGS: Not available

Effect levels

Dose descriptor:
NOAEL
Effect level:
> 500 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: There were no significant adverse test substance-related effects in this study for any of the parameters measured.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Statistically significant lower mean MCV values were noted between the 10 mg/kg bw/day, 500 mg/kg bw/day males and the water control male group when compared to the pH control water male group. These differences were not considered biological or test substance related as there were within + or - two standard deviations of the laboratory historical control mean values for rats of this strain, age and sex. In addition, they were within the standard published reference ranges.

The blood urea nitrogen values of the 500 mg/kg bw/day group was significantly lower than the pH control group in male rats. This differences were not considered test substance related as they were within + or - two standard deviatiosn of the laboratory historcal control mean values for rats of this strain, age and sex.

The ovary/body weight ratios of the 500 mg/kg bw/day female rats were signficant lower than the pH control females. The differences in the ovary/body weight ratios were not considered test substance related because of the variability in the mean body weights of pH control group, lowest of all female groups. In addition, there were no test related difference observed in the mean absolute ovary weight of the 500 mg/kg bw/day group in comparison to the other test or control groups and the mean ovary weight also fall with

within + or - two standard deviations of the laboratory historical control mean values for rats of this strain, age and sex.

Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected.

Applicant's summary and conclusion

Conclusions:
The test substance was administered orally via gavage daily to rats at dosage levels of 1, 10, and 500 mg/kg bw/day for a period of 13 weeks. No biologically significant adverse effects were observed in any test group. The NOAEL was determined to be the high dose level of 500 mg/kg bw/day.
Executive summary:

In a subchronic toxicity study comparable to OECD guideline 408, MDEA-Esterquat C16-18 and C18 unsatd.  (10 % a.i.) was administered to 15 Charles River CD rats / sex/ dose by gavage at dose levels of 1, 10 and 500 mg/kg bw/ day for a period of 13- weeks. One control group received the vehicle, deionized water, and a second control group received pH-adjusted, deionized water (pH 2.5). The regimen for both control groups was identical to treatment groups.

The following parameters were monitored during the study: clinical observations (detailed, weekly; mortality, morbidity, and overt signs of toxicity, twice a day); body weights (weekly); food consumption (weekly); clinical pathology (haematology, blood chemistry, and urinalysis; at termination); opothalmoscopic examinations (once pre-test and prior to sacrifice); macroscopic pathologic examination; absolute and relative organ weights; microscopic pathology.

There were no changes in any of these parameters that were considered to be toxicologically significant or test-substance related.

Reproductive parameters:

Absolute and relative organ weights of ovary and testis and histopathology of reproductive organs (gonads, mammary gland (females only), prostate and seminal vesicle, uterus with cervix and vagina) revealed no test substance related findings.

There is no evidence for specific target organ toxicity in this study.

The no effect level (NOEL) for this study is the high dose level of 500 mg/kg bw/day of the test article.

This subchronic toxicity study in rats is acceptable and satisfies to a large extent the guideline requirement for a subchronic oral study, with exception of highest tested dose. 500 mg/kg bw/day was tested as highest dose instead of 1000 mg/kg/bw/day as recommended by the actual OECD guideline 408.