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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:
9 November 1989 to 17 February 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Test Substance: MARK HP-10
Lot No.: 103
Concentration: 100% Active Ingredient
Description: White powder
Dates Received: 21 September 1989 (Containers 1 - 24); 28 November 1989 (Container 25)
Analysis: The identity, strength, purity and composition and stability, synthesis, fabrication, and/or derivation of the test substance have been documented by the sponsor.
Storage: Room temperature under N2 seal
Sampling: An archival sample of approximately 10 grams of test substance is stored in the Archives of Bio/dynamics, Inc.
Disposition: All remaining containers of the test substance will be returned to the sponsor.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Test Animals: Rats
Strain: CD® (Sprague-Dawley derived)
Justification For Animal Selection: Standard laboratory rat available in good supply.
Number of Animals: Received: 199 total (99 males, 100 females); Placed on Test: 160 total (80 males, 80 females)
Supplier: Charles River Breeding Laboratories, Inc., Kingston, New York 12484
Date Received: 26 October 1989
Age at Receipt: Approximately 4 weeks old.
Age at Initiation of Treatment: Approximately 6 weeks old.
Weight at Initiation of Treatment (grams): Mean Range
Males: 207 192 - 223
Females: 165 151 - 180
Acclimation Period: Animals were acclimated for 2 weeks (26 October to 8 November 1989). All animals were examined by the staff veterinarian during the acclimation period.
Selection: More animals than required for the study were purchased and equilibrated. Animals considered unsuitable for the study on the basis of pre-test physical examinations, outlying body weight or ophthalmoscopic examination were eliminated prior to random selection for group assignment.
Protocol Deviation: Extra animals were transferred to the Bio/dynamics, Inc. in-house colony.
Group Assignment: Animals considered suitable for study were distributed into 4 groups of
20 animals per sex by a computerized random sort program so that body weight means for each group were comparable. Groups were assigned to control and dose levels randomly.
Animal Identification: Each rat was identified with a metal ear tag bearing its unique Bio/dynamics, Inc. animal number. If the tag was lost, it was replaced and/or the rat was identified with a tail tattoo. In addition, each cage was provided with a cage card which was color coded for dose level identification and contained the project number, animal number, sex and dose-group information.

Husbandry:
Housing: Animals were doubly housed in elevated stainless steel wire mesh cages during the first week of the acclimation period and individually housed thereafter.
Food: ad libitum; standard laboratory diet (Purina Certified Rodent Chow® Brand Animal Diet #5002). Fresh food presented weekly.
Analysis of Feed: Analysis of each feed lot used during this study was performed by the Ralston-Purina® Company prior to receipt at Bio/dynamics, Inc. Results are maintained on file at Bio/dynamics.
Water: ad libitum; by automated watering system (Elizabethtown Water Company).
Analysis of Water: Water analysis was provided by Elizabethtown Water Company, Westfield,
New Jersey. Results are maintained on file at Bio/dynamics.
Contaminants: There are no known contaminants in the feed or water which are expected to be capable of interfering with the results of this study.

Environmental Conditions: 12 hour light/dark cycle (7 AM to 7 PM) via automatic timer; temperature and humidity monitored and recorded twice daily.
Temperature: Desired: 67 - 76° F; Actual: 68- 75° F
Humidity: Desired: 30 - 70%; Actual: 3- 55%
Deviations from the desired humidity range were not considered to have had an adverse effect on the results of this study.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Appropriate amounts of test substance were mixed with standard laboratory diet weekly. Control animals received standard laboratory diet. Prepared diets were stored at room temperature through Day 6 and stored frozen thereafter.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
SCOPE: This method describes the analytical procedure for the analysis of Mark HP-10 by liquid chromatography. The limit of detection is 300 ng injected (30 ul of a 10.0 ug/ml standard).

EQUIPMENT
-120 ml and 250 ml amber glass bottles
-Analytical balances, Mettler H33AR and Mettler P1210 or the equivalent
-Disposable glass pipets and labeling tape
-15 ml test tubes and rack
-A reciprocal or orbital shaker
-Sorval GLC-28 centrifuge or the equivalent
-10.0, 25.0 and 100 ml Class A volumetric flasks
-1.0, 5.0, 10.0 and 20.0 ml Class A volumetric pipets
-A liquid chromatograph equipped with a Waters 510 pump, a Water 481 LC spectrophotometer, a WISP 710 B autoinjector, a Waters 730 data module and a Lichrosorb 10 Diol column or the equivalent for any component

STANDARD PREPARATION: Approximately 100 mg of Mark HP-10 test material is weighed out and transferred to a 100 ml volumetric flask. This flask is diluted to volume with hexane for an approximate stock standard concentration of 1,000 ug/ml. This standard must be sonicated for
2.0 minutes to facilitate mixing.
Dilutions for injection are prepared as follows:
-20.0 ml of a 1000 ug/ml std. diluted to 100 ml for a 200 ug/ml std.
-10.0 ml of a 1000 ug/ml std. diluted to 100 ml for a 100 ug/ml std.
-5.0 ml of a 1000 ug/ml std. diluted to 100 ml for a 50.0 ug/ml std.
-1.0 ml of a 1000 ug/ml std. diluted to 100 ml for a 10.0 ug/ml std.
Dilutions for injection are also brought to volume with hexane.
Further standard dilutions may be prepared provided they are within the linear range. All standards are stored in the freezer between analyses and prepared fresh on a monthly basis.

SAMPLE PREPARATION: Weigh out 10 grams of diet into a 250 ml amber glass bottle. Dietary levels of Mark HP-10 at or greater than 50,000 ppm require only 5 grams of diet to be weighed out.
Fortify control samples with an appropriate standard volume and concentration. If dietary levels are over 500 ppm, then the test material should be weighed and added to the diet and shaken by hand for 10 seconds.
Add a total of 200 ml of hexane to samples and fortifications (include standard volume used to fortify if necessary) and shake for 1 hour.
Centrifuge approximately 10 ml of each sample and fortification at 2500 rpm for 5 minutes.
Perform the following dilutions for the specified levels:
Control: Diluted the same as the lowest level on test.
(300 - 3590 ppm): Undiluted
(3600 - 35900 ppm): 1.0 ml diluted to 10.0 ml
(36000 - 89900 ppm): 1.0 ml diluted to 25.0 ml
Dilutions are prepared with hexane and are stored in the freezer between analyses. For diet storage refer to stability data.

LIQUID CHROMATOGRAPHIC ANALYSIS: A standard curve for the analyses is constructed by injecting at least three different concentrations at the initiation of the chromatographic run, a standard after every fourth sample and at the end of the run. The standard response is plotted at the peak area of Mark HP-10 versus the concentration of Mark HP-10 in the standard. A standard correlation coefficient is then calculated by linear regression and the sample concentrations are obtained using this coefficient.

LIQUID CHROMATOGRAPHIC CONDITIONS
Instrument: Waters 510 Pump, Waters 481 LC Spectrophotometer, WISP 710 B Autoinjectior, Waters 730 Data Module
Column: Lichrosorb 1 0 Diol , 25 cm x 4.6 mm
Mobile phase: Hexane
Flow rate: 1.0 m1/minute
Column temperature: Ambient
Chart speed: 0.5 cm/minute
Sample size: 30 ul
AUFS: 0. 02
Attenuation: 23
Analytical wavelength: 275 nm
Duration of treatment / exposure:
98 to 100 days, depending on day of necropsy.
Frequency of treatment:
Daily (7 days per week) through the day prior to necropsy.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 500, 5000, 50000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
(20/sex/group) at dose levels of 500, 5,000, and 50,000 ppm for at least 90 days. Control animals (20/sex) received standard laboratory diet.
Control animals:
yes, plain diet
Details on study design:
Species and strain of test animal, method and route of test substance administration and dose levels were determined by the sponsor.
Positive control:
Positive control not used in this study.

Examinations

Observations and examinations performed and frequency:
For Mortality and Gross Signs of Toxicologic or Pharmacologic Effects: Twice daily, once in the morning and once in the afternoon.
Detailed Physical Examination for Signs of Local or Systemic Toxicity, Pharmacologic Effects and Palpation for Tissue Masses: Pre-test and weekly thereafter.
Ophthalmoscopic Examination: Pre-test: 3 November 1989; Termination: 8 February 1990
Body Weight: Twice pre-test, weekly during treatment and terminally (after fasting).
Food Consumption: Weekly, beginning one week prior to treatment.
Feed Efficiency: Pre-test and weekly through 14 weeks. Calculated from body weight and food consumption data.
Test Substance Intake: Calculated from food consumption data and based on nominal concentrations.
Water Consumption: Pre-test and weekly thereafter.
Protocol Deviation: Because of scheduling error, water consumption measurements were not performed pre-test or during Weeks 1 through 13. Measurements were performed during Week 14 based on a three day interval.
Laboratory Studies: Blood was obtained via venipuncture of the orbital sinus (retrobulbar venous plexus) under light ether anesthesia. Rats were fasted overnight prior to blood collections. Urinalysis was performed on unfasted rats.
Number of Animals: Performed on all animals at study termination.
Parameter Evaluated
Hematology: hemoglobin concentration; hematocrit; erythrocyte count; platelet count; total and differential; leukocyte counts.
Termination: 15 to 17 February 1990
Clinical Chemistry: aspartate aminotransferase; alanine aminotransferase; alkaline phosphatase; plasma cholinesterase; blood urea nitrogen; fasting glucose; cholesterol; total protein; albumin; globulin (calculated); A/G ratio (calculated); creatinine; total bilirubin; sodium; potassium; sodium/potassium ratio; chloride; calcium; inorganic phosphorus; gamma glutamyl transpeptidase.
Termination: 15 to 17 February 1990
Urinalysis: gross appearance; pH; protein; glucose; ketones; occult blood; urobilinogen; 16-hour volume; microscopic analysis.
Termination: 1, 2, 5, 6 and 7 February 1990
Protocol Deviation: Urinalysis was not performed the week prior to sacrifice because the study was extended.
Sacrifice and pathology:
Postmortem:
Animals Found Dead, Killed in a Moribund Condition or Killed at Terminal Sacrifice: Complete gross postmortem examinations were performed on all animals. External surface, all orifices, the cranial cavity, carcass, the external surface of the brain, the thoracic, abdominal and pelvic cavities and their associated tissues and organs, the neck with its associated tissues and organs and the carcass were examined for all animals. Animals were fasted prior to scheduled sacrifices.
Necropsy: 15, 16 and 17 February 1990
Number of Animals: 158 animals total
Sacrifice Method: Exsanguination under ether anesthesia.
Organs Weighed and Organ/Body Weight and Organ/Brain Weight Ratios Calculated: The following organs were weighed for all animals killed at terminal sacrifice. Paired organs were weighed together.
Adrenals; brain; heart; kidneys; lungs; pituitary; testes; thyroid/parathyroids (weighed post fixation); liver; ovaries; thymus; spleen
Tissues Preserved: All tissues listed below and on the following page were taken and preserved from all animals.
adrenals (2); aorta (abdominal); bone (femur); bone marrow (sternum); bone marrow smear (rib, costochondral junction); brain; ear with tag; epididymides (2); esophagus; exorbital lacrimal glands (2); eyes and contiguous Harderian glands and optic nerve (2); heart; intestine; cecum; colon; duodenum; ileum; jejunum; rectum; kidneys (2); larynx; liver; lungs (with mainstem bronchi and trachea); lymph nodes (mesenteric and mediastinal); mammary gland (right inguinal taken with skin); nasal turbinates; ovaries and Fallopian tubes (2); pancreas; peripheral nerve (sciatic - taken with biceps femoris); pituitary; preputial glands; prostate; salivary gland (submandibular) (2); seminal vesicles; skeletal muscle (biceps femoris); spinal cord (cervical and lumbar) (2); spleen; sternum; stomach; testes (2); thymus; thyroid/parathyroids (2); tongue; trachea; ureter (2); urinary bladder; uterus (corpus and cervix uteri); uterine horns; vagina; Zymbals glands; gross lesions.
Number in parentheses indicates number of organs/sections preserved.
Tissues Examined Histopathologically: All tissues listed below were examined for all animals in the control and high dose groups (Groups I and IV, respectively) and for the animal found dead in Group III. In addition, all gross lesions were examined for all animals.
Adrenals; aorta; bone marrow; brain (frontal cortex, basal ganglia, parietal cortex, thalamus, cerebellum and pons); esophagus; eyes; heart; intestine, large and small (duodenum and cecum); kidneys (2); liver (section of two major lobes); lungs (with mainstem bronchi); lymph nodes (mesenteric); mammary gland; ovaries (2); pancreas; pituitary; prostate; salivary gland; sciatic nerve (with biceps femoris); spinal cord (cervica and lumbar); spleen; sternum; stomach; testes; thymus; thyroid/parathyroids (2); trachea; urinary bladder; uterus; gross lesions.
Number in parentheses indicates number of organs/sections examined.
Other examinations:
Not specified in the study report.
Statistics:
Statistical Analysis: Body weight, food consumption, water consumption, hematology and clinical chemistry parameters, organ weights, organ/body weight and organ/brain weight ratios were analyzed. Mean values of all dose groups were compared to control values at each time interval. Statistically significant differences from control are indicated on mean tables of appendices.

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:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
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:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Metabolism and Analytical Chemistry: Analysis was performed on the dietary mixtures of MARK HP-10. The admixtures were stable and homogeneous. The weekly analyses showed that the diets were within acceptable tolerances throughout the study.
Mortality: All female rats survived the duration of the study. In males, one rat in the 5,000 ppm dose group died during Week 12 and one rat in the 50,000 ppm dose group was killed moribund during Week 13. The cause of death could not be determined from the results of gross post-mortem examinations or histopathological evaluations performed on these animals. Since these deaths occurred only in one sex and not in a dose-related pattern, they were not considered to be treatment related.
Physical Observations: There were few remarkable physical observations seen. The few abnormalities seen were of the type commonly observed in laboratory rats and were not treatment related.
Ophthalmoscopic Examinations: At termination of the study few abnormalities were noted. The abnormalities that were observed are commonly seen in laboratory rats and were not treatment related.
Body Weights: There were no statistically significant differences in mean body weight values between controls and any of the treatment groups.
Food Consumption: The mean food consumption values for high-dose (50,000 ppm) animals were frequently statistically significantly increased compared to controls. This is a test substance effect which is not considered to be a manifestation of toxicity. Administration of the test substance at 500 and 5,000 ppm did not affect the food consumption of the animals in those groups. There were no consistent patterns of differences that would be indicative of a test substance effect in food utilization values.
Test Substance Intake: Mean weekly intake of test substance, based on nominal dietary concentrations, ranged as follows:
Group: II III IV
ppm: 500 5000 50000
Test Substance Intake (mg/kg/day):
Males: 26 - 61 264 - 588 2739 - 6301
Females: 33 - 56 321 - 594 3435 – 5960
Water Consumption: There were no statistically significant differences in liquid consumption values between the control group and any of the treated groups.

Clinical Laboratory Studies:
Hematology: There were no statistically significant differences between groups in any of the hematology parameters measured.
Clinical Chemistry: The only statistically significant effect in clinical chemistry parameters was a increase in serum chloride values for males in the 5000 ppm dose group. This effect is not dose or treatment related.
Urinalysis: There were no apparent treatment-related trends in any urinalysis parameter measured.
Terminal Organ and Body Weights and Organ/Body Weight and Organ/Brain Weight Ratios: The only statistically significant findings were an increase in absolute testes weights of the 500 and 50,000 ppm dose group males. The testes weights in the 5,000 ppm dose group were not statistically significantly different from controls. The relative organ weight ratios were not statistically significantly different from controls, however. Histopathological evaluation revealed no indication of a treatment-related effect on the testes. Overall, these findings did not indicate a treatment-related toxicological effect on the testes.
Pathology: There were no consistent treatment-related alterations noted in the tissues evaluated by histopathology.

Effect levels

Dose descriptor:
NOEL
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on a statistically significant increase in food consumption in the 50,000 ppm dose group, the no effect level in this study was determined to be 5,000 ppm of MARK HP-10 when administered via dietary admixture to SpragueDawley® rats for a period of three months (Males: at least 263.7 mg/kg/day; Females: at least 320.5 mg/kg/day).
Executive summary:

This study was designed to assess the toxicity of MARK HP-10 when administered orally, via dietary admixture to 120 Sprague-Dawley CD® rats (20/sex/group) at dose levels of 500, 5,000, and 50,000 ppm for at least 90 days. Control animals (20/sex) received standard laboratory diet. The study design was based on FDA Redbook Guidelines (Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives used in Food, 1982, Appendix II, pg. 19-29) as well as the OECD Guidelines for Testing of Chemicals, Section 4: Health Effects, May 12, 1981, 408.

Physical observations and body weight and food consumption measurements were performed on all animals pre-test and weekly thereafter. Water consumption measurements were performed for all animals at two intervals. Ophthalmoscopic examinations were performed for all animals pre-test and at study termination. Hematology, clinical chemistry, and urinalyses parameters were evaluated for all animals at study termination.

After at least 90 days of treatment, all survivors were sacrificed, selected organs were weighed and organ/body and organ/brain weight ratios calculated. Complete gross postmortem examinations were conducted on all animals. Histopathological evaluation of selected tissues were conducted on all animals in the control and high-dose groups. In addition, gross lesions were examined for all animals.

Analytical results indicated that the dietary admixture of MARK HP-10 was stable, homogeneous and within acceptable ranges, for all doses, throughout the study.

All females survived the duration of the study. Two male animals died but these deaths were not considered to be treatment related. The physical observations indicated no gross treatment-related effects on the health of the animals in this study.

There were no treatment-related effects on the eyes, body weights, water consumption, hematology, urinalysis or histopathology of the animals in this study.

There was a statistically significant increase in food consumption in the 50,000 ppm dose group but no consistent pattern of difference in food utilization values.

There was a non-treatment-related increase in serum chloride values for the males in the 5,000 ppm dose group.

There was a statistically significant increase in testes weights but not organ/body weight or organ/brain weight ratios in the 500 and 50,000 ppm dose groups but not in the 5,000 ppm dose group. This was not considered a toxic effect.

Since there was a statistically significant increase in food consumption at the 50,000 ppm dose level, the no effect level of MARK HP-10 administered via dietary admixture for 3 months to Sprague-Dawley® rats was determined to be 5,000 ppm (Males: at least 263.7 mg/kg/day; Females: at least 320.5 mg/kg/day).