Quaternary ammonium compounds, C12-14-alkyl(hydroxyethyl)dimethyl, chlorides

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2000-09-13 to 2001-11-01

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study was conducted according to U.S. EPA OPPTS Health Effects Test Guideline 870.3100 (90-Day Oral Toxicity Study in Rodents), which is comparable to OECD Test Guideline 408. The study was well documented and conducted. For Read across justification refer to IUCLID point 13 Assessment reports.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Principles of method if other than guideline:

N/A

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

C8-C10 alkyl dimethyl hydroxyethyl ammonium chloride

IUPAC Name:

C8-C10 alkyl dimethyl hydroxyethyl ammonium chloride

Details on test material:

- Name of test material (as cited in study report): SS0772.01
- Molecular formula (if other than submission substance): N/A
- Molecular weight (if other than submission substance): N/A
- Smiles notation (if other than submission substance): N/A
- InChl (if other than submission substance): N/A
- Analytical purity: 97.67 %
- Purity test date: 2000-07-28
- Lot/batch No.: SS0772.01
- Expiration date of the lot/batch: 2002-07-01
- Isomers composition: N/A
- Impurities (identity and concentrations): N/A
- Other: The test substance was stored at room temperature under nitrogen.
- Physical state: Pale yellow solid

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan
- Age at study initiation: ~ 6 weeks (41-45 days)
- Weight at study initiation: Males: 178-244 g; Females: 142-198 g
- Fasting period before study: N/A
- Housing: The animals were housed individually in stainless-steel cages.
- Diet (e.g. ad libitum): ad libitum except when animals were fasted
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 9 days- During the acclimation, the animals were examined for abnormalities indicative of health problems, and ophthalmic examination was done, and body weights were recorded for all animals at arrival and at randomization. Expanded Clinical Observations (hand-held and open field observations and elicited behavior tests) and Motor Activity testing were conducted on 10 animals/sex/group prior to initiation of treatment.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): N/A
- Photoperiod (hrs dark / hrs light): 12/12 (interrupted to accommodate study-related procedures)


IN-LIFE DATES: From: 2000-09-14 To: 2001-01-18

Administration / exposure

Route of administration:

oral: feed

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Dietary concentrations were based on the test substance as supplied and were mixed according to the mixing procedure which is maintained in the study records. Dose preparation concentrations (ppm) were calculated using projected body weight and food consumption values. (See diet preparation)

DIET PREPARATION
- Rate of preparation of diet (frequency): ~ weekly
- Mixing appropriate amounts with (Type of food): Certified rodent diet (#5002 meal, Lab Diet by Purina Mills, Inc.)-Each dose level was prepared independently in sequential order of increasing concentration. A specified amount of carrier (feed) was weighed into a labeled Hobart mixing bowl, size A-200. The required amount of test substance was weighed, placed into a labeled beaker and covered until use. The required volume of RO water was measured, transferred to the beaker, and mixed manually with a spatula until all the test substance was dissolved. Approximately 500 g of feed from the A-200 bowl was transferred to a labeled Hobart mixing bowl, size N-50, to prepare a premix. While mixing, the test substance/RO water mixture was added slowly using a syringe, then mixed for at least 2 minutes. Start and stop times for mixing were recorded. In the A-200 bowl, a pocket was formed in the remaining feed. The premix was sifted using a No. 25 sieve and transferred back to the A-200 bowl. Approximately 100 g of clean feed was obtained from the A-200 bowl, transferred to the N-50 bowl, and mixed manually to pick up any diet adhering to the bowl. The sieve was rinsed with clean diet, and the rinses were transferred to the A-200 bowl. The premix was covered with feed from the edge of the bowl and mixed for 10 minutes. Samples were taken as directed by the protocol, and a net prepared diet weight was taken.
- Storage temperature of food: Diets were stored in a freezer set to maintain -10 to -30 degrees C until dispensed into food containers. The test material container head space was flushed with nitrogen prior to return to storage.


VEHICLE
- Justification for use and choice of vehicle (if other than water): N/A
- Concentration in vehicle: N/A
- Amount of vehicle (if gavage): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity was determined for the lowest and highest concentrations mixed for Week 1. Samples (approximately 100 g) taken from the top, middle, and bottom of the mix were sent to the sponsor's designee for analyses. Samples were stored in a freezer set to maintain -10 to -30 degrees C until shipped to the Sponsor's designee. Samples were stored in a freezer until shipped to the designee.

To evaluate the stability of the test substance in the carrier, samples (approximately 100 g each) were taken from the lowest and highest dose concentrations mixed from Week 1. Samples were stored at room temperature for at least 10 days and then stored in a freezer until shipment to designee. The results from the analysis of the middle homogeneity sample from the low- and high-dose concentrations served as the initial stability results.

Samples (approximately 100 g each) were collected from the middle of each diet preparation for Weeks 1 through 4, 8, and 13 for routine analysis. If the samples were not packed on dry ice and shipped to the Sponsor's designee on the same day of sampling, they were stored in a freezer until sent to the Sponsor's designee for analysis. The results from the analysis of the middle homogeneity sample from the low- and high-dose concentrations served as the Week 1 routine analysis results.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Treated food was available ad libitum for at least 13 weeks.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 15, 50, and 75 mg/kg bw
Basis:
nominal in diet

No. of animals per sex per dose:

18-control and high dose groups (six rats/sex were designated as recovery animals); 17- low and mid dose groups (5 rats/sex were designated as recovery animals)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The goal of dose selection was a gradient of toxic effects. Signs of toxicity were expected at the high dose level. The low dose level was anticipated to be a no effect level. The intermediate dose level was selected as an additional dose for the purpose of evaluating any potential toxicological effects.
- Rationale for animal assignment (if not random): Animals were assigned to treatment groups using a computerized blocking procedure designed to achieve body weight balance with respect to treatment group. Prior to assignment to groups, the weight variation of the animals did not exceed +/- 2 standard deviations of the mean body weight for each sex. After assignment to groups, the mean body weight for each group were not statistically different at the 5.0 % probability level. At randomization, the coefficient of variation of the mean weight for each sex and group was less than 10%. Group mean body weights were analyzed using Levene's test for homogeneity of variance at the 5.0 % probability level and were found to be homogeneous.
- Rationale for selecting satellite groups: N/A
- Post-exposure recovery period in satellite groups: 4 weeks
- Section schedule rationale (if not random): N/A

Positive control:

N/A

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations included: The animals were observed twice daily (a.m. and p.m.) for mortality and moribundity. Signs of poor health or abnormal behavior were recorded as they were observed.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to initiation of treatment, on the day of initiation of treatment, and weekly during treatment, each animal was removed from its home cage and examined for, but not limited to, changes in skin, fur, eyes, and mucous membranes; occurrences of secretions and excretions; and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in posture and reactivity to handling and the presence of clonic or tonic movements, sterotypies (e.g., excessive grooming, circling), or bizarre behavior (e.g., self-mutilation, walking backwards) were also recorded. Abnormal findings (ranked/graded, if appropriate) or an indication of normal were recorded. By allowing the animal to walk freely, changes in gait were assessed during the detailed observations. Additional findings were recorded as they were observed.
- Expanded clinical observations for hand held, open field, and elicited behavior tests were conducted on 10 animals/sex/group once prior to treatment and once during Week 13 and on 5 animals/sex/group once during Week 17. Expanded clinical observations for hand held and open field observations were conducted on 10 animals/sex/group weekly during Weeks 1 through 12 and on 5 animals/sex/group during Weeks 14 through 16.
- Expanded clinical observations and motor activity testing were conducted on the same day during Weeks 13 and 17. The technician was unaware of each animal's dose level, and the observations were done on a day other than that scheduled for the weekly detailed observations.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weight data were recorded for all animals at arrival, once for randomization, on the first day of treatment, and weekly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal was recorded weekly and reported in grams: Yes
- Compound intake calculated as [((diet concentration x food consumption)/ food consumption interval x (first body weight of interval + (daily body weight gain x day factor))]: Yes

FOOD EFFICIENCY:
- Food efficiency was calculated as (mean weekly body weight gain/mean weekly food consumption) x 100: Yes

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmic examinations were done by a board certified veterinary ophthalmologist once prior to initiation of treatment and once during Weeks 14 and 18.
- Dose groups that were examined: All groups-The pupils were dilated with a mydriatic agent, and the eyes were examined with an indirect ophthalmoscope. The ophthalmic observations were done on a day other than that scheduled for expanded clinical observations, and animals were examined in random order during Weeks 14 and 18.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected before the terminal and recovery sacrifices (Weeks 14 and 19, respectively). Blood was collected from a jugular vein. The anticoagulant used was potassium EDTA.
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: red blood cell (erythrocyte) count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, white blood cell (leukocyte) count, differential blood cell count (two blood smears for differential blood cell counts were prepared and stained for each animal), blood cell morphology, reticulocyte smear (made but not examined, because there were no group differences in measurements on the erythron or leukon)
-Other: Coagulation-Blood was taken as described above, except sodium citrate was used as the anticoagulant. Prothrombin time and activated partial thromboplastin time were measured.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected before the terminal and recovery sacrifices (Weeks 14 and 19, respectively). Blood was collected from a jugular vein.
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: glucose, urea nitrogen, creatinine, total protein, albumin, globulin, albumin/globulin ratio, total bilirubin, cholesterol, triglycerides, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma glutamyltransferase, calcium, inorganic phosphorus, sodium, potassium, chloride

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected before the terminal and recovery sacrifices (Weeks 14 and 19, respectively). Urine was collected on wet ice.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters examined: specific gravity, pH, protein, glucose, ketones, bilirubin, blood, urobilinogen, volume (approximately 16 hours), microscopic examination of sediment, appearance

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: Motor activity testing was conducted on 10 animals/sex/group once prior to treatment and once during Week 13 and on 5 animals/sex/group once during Week 17.
- Dose groups that were examined: all
- Battery of functions tested: Motor activity - the animals were placed into an automated photocell activity recording device and activity was recorded for 40 minutes; activity counts were recorded at 2-minute intervals.
- Expanded clinical observations and motor activity testing were conducted on the same day during Weeks 13 and 17. The technician was unaware of each animal's dose level, and the observations were done on a day other than that scheduled for the weekly detailed observations.

OTHER: N/A

Sacrifice and pathology:

NECROPSY: After at least 13 weeks of treatment (Week 14, Day 97 and Day 98), 12 animals/sex/group (including 5 animals/sex/group that had expanded clinical observation testing) were fasted overnight and bled for clinical pathology tests from a jugular vein. After at least 13 weeks of treatment followed by a 4 week recovery period, the remaining animals were fasted overnight and bled for clinical pathology tests from a jugular vein (Week 19, Day 127). All animals were euthanatized with carbon dioxide, weighed, exsanguinated, and necropsied. All animals were necropsied in random order and a veterinary pathologist was present at each scheduled necropsy.

GROSS PATHOLOGY: Yes- The necropsy included a macroscopic examination of the external features of the carcass; external body orifices; the abdominal, thoracic, and cranial cavities; organs; and tissues.
- Organ weights: The following organs (when present) were weighed; paired organs were weighed together: adrenal (2), brain, epididymis (2), heart, kidney (2), liver, ovary (2), pituitary, prostate, spleen, testis (2), thymus, thyroid (2) with parathyroid, uterus with cervix. Organ-to-body weight percentages and organ-to-brain weight ratios were calculated.

HISTOPATHOLOGY: Yes-The following tissues (when present) or representative samples were collected and preserved in 10% neutral-buffered formalin, unless otherwise specified: adrenal 2), aorta, brain, cecum, cervix, colon (proximal and distal), duodenum, epididymis (2), esophagus, eye (2), femur with bone marrow (articular surface of the distal end), Harderian gland, head (including pharynx, larynx, and nose - preserved for possible future examination), heart, ileum (including Peyer's Patch), jejunum, kidney (2), lacrimal gland (exorbital), liver, lung with mainstem bronchi, lymph node (mandibular and mesenteric), mammary gland (females only), ovary (2), pancreas, pituitary, prostate, rectum, salivary gland (mandibular) (2), sciatic nerve, seminal vesicle (2), skeletal muscle (thigh), skin, spinal cord (cervical, thoracic, and lumbar), spleen, sternum with bone marrow, stomach (nonglandular and glandular), testis (preserved in Bouin's fixative for sacrificed animals) (2), thymus, thyroid (2) with parathyroid, tissues with macroscopic changes or alterations (i.e. gross lesions), tongue, trachea, urinary bladder, uterus with uterine horns, and vagina.
- Tissues from each animal in the control and high-dose groups were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically by a board-certified pathologist.
- Any observed lesions from any animal in any group, and the rectum, cecum, colon, duodenum, jejunum, and ileum from each animal in the low- and mid-dose groups were also examined microscopically.
- Bone marrow smears from the femur of each animal at the scheduled sacrifices were prepared, stained with Wright's stain, and retained for possible future examination.

Other examinations:

Estrous Cycle- Vaginal smears were taken daily from all females beginning on Day 64 and continuing to the day before the terminal sacrifice (for main study females) and continuing until the day before the recovery sacrifice (for recovery females). Vaginal smears were prepared and examined to evaluate the stage of the estrous cycle. Examinations were done at approximately the same time each day and after completion of the expanded clinical observations.

Male Reproductive Assessment- At the terminal and recovery sacrifices, males were evaluated for reproductive capacity by Pathology Associates, A Charles River Company. At each necropsy, the right vas deferens was removed from each male and transferred to a technician from Pathology Associates, A Charles River Company for evaluation and processing for sperm motility and morphology. The right epididymis was removed from each male, placed on dry ice, and transferred to Pathology Associates, A Charles River Company for assessment of total sperm count. The left epididymis and remaining portion of the right epididymis were collected. Following collection from each male, the epididymides were separated from the testes. The tunic of each testis was nicked at the caudal end to facilitate fixation in Bouin's fixative, and the left and right testes were weighed together. Both epididymides were weighed together as a paired organ and the right epididymis was divided in half by cross sectioning the epididymides in the middle. The head and proximal midsection of the right epididymis was placed in 10% neutral-buffered formalin. The tail and distal midsection of the right epididymis were placed on dry ice for analysis of total sperm count evaluations. After adequate fixation in Bouin's fixative, each testis was sectioned transversely in half, and the cranial pole was sectioned, embedded in paraffin, histologically processed, and stained with hematoxylin and eosin. The remaining testicular tissue was stored in 70% ethyl alcohol. The left epididymis and portions of the right epididymis saved for histology were sectioned longitudinally and processed for histologic evaluation. The remaining portions of the epididymides were stored in 10% neutral-buffered formalin. Testes and epididymides from each male in the control and high-dose groups were examined microscopically by a board-certified veterinary pathologist.

Statistics:

The observed values for body weights, body weight changes, food consumption, motor activity, grip strength, nociceptive reflex, continuous clinical pathology data, and organ weight data were evaluated statistically.

If Levene's test for variance homogeneity was not significant (p>0.05), one way analysis of variance (ANOVA) was performed on the observed values. If Levene's test was significant (p
Expanded clinical observation categorical data were not analyzed by contingency table methods.

Control versus treated group comparisons were evaluated at the 5.0 %, two tailed probability level. Only data collected on or after the first day of treatment were analyzed statistically. Data for each sex were analyzed separately.

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:

no effects observed

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

no effects observed

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 animals survived to their respective sacrifice. There were no clinical findings considered to be test substance related.

The results of the expanded clinical observations that required hand held or open field behavior assessments were similar across groups. During a given interval, one or two incidences of low locomotor activity in the open field or vocalization during handling were considered to be behaviors representative of normal biological variation. Elicited behaviors were also similar across groups. The significantly higher hind limb grip strength during Week 17 for females that had been given 15 mg/kg was considered incidental. It was not seen in the males or in the other dose groups and was not considered to be test substance related. Minor differences in expanded clinical observations data were attributed to normal biological variation.


BODY WEIGHT AND WEIGHT GAIN- There were no test substance related differences in body weight data across dose groups. There were no remarkable test substance related body weight changes. Females that received 15 mg/kg showed significantly higher body weight gains than the control females during Week 5. Females that received 75 mg/kg had significantly lower body weight gains than control females during Week 11, but had significantly higher body weight gains than control females during Week 13. Although the body weight gains were statistically significant, they were transient and not considered to be test substance related.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)- There were no mean test substance related differences in food consumption values for males or females throughout the study. The overall test substance consumption values (mean across 13 weeks) were 100-105 % of the targeted dose levels. Weekly values ranged from 90-120 % of the target dose level and only during Week 3 did some of the mean test substance values differ by more than 115 % of the targeted dose level.

FOOD EFFICIENCY-N/A


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)-N/A


OPHTHALMOSCOPIC EXAMINATION- Animals selected for the study had no lesions at the prestudy examination. No test substance related ophthalmic observations were noted at the Weeks 14 and 18 examinations. The only finding was inflammation of the left iris in one animal in the control group at Week 14.


HAEMATOLOGY- No significant findings were reported.


CLINICAL CHEMISTRY-Of uncertain relationship to the test substance, at Week 14 females given 75 mg/kg/day exhibited mildly, but statistically significantly (p
At week 19 (recovery animals), females given 75 mg/kg/day continued to have mildly lower mean cholesterol concentration than that of the control females, however the difference was no longer statistically significant. The only significant differences seen at Week 19 (recovery animals) were lower triglycerides for males given 15 or 50 mg/kg/day, lower calcium for males given 50 mg/kg/day, and lower inorganic phosphorus for females given 50 mg/kg/day. All of these were considered incidental and unrelated to administration of the test substance.


URINALYSIS-Of uncertain relationship to the test substance, at Week 14 males given 75 mg/kg/day exhibited a higher incidence of positive urine occult blood results (6 of 18 versus 1 of 18 for control males; reagent strip method) and a higher incidence of the presence of amorphous phosphate crystals in urine sediment (5 of 18 versus 0 of 18 for control males; microscopic examination). Females were not similarly affected, and there were no correlative anatomic or clinical pathology findings. Positive urine occult blood results and the presence of amorphous phosphate crystals are not unusual findings for male rats of this strain and age. Although it was considered likely that the findings were incidental, an association with administration of the test substance could not be completely ruled out. Regardless, these findings were not considered adverse. At Week 19 (recovery animals) there were no differences in the incidences of urine occult blood and amorphous phosphate crystalluria between the control males and males given 75 mg/kg/day.


NEUROBEHAVIOUR-In general, the motor activity results were similar across all groups of each sex at all testing intervals. At the recovery interval (Week 17), males that had received 15 or 75 mg/kg for at least 13 weeks had higher mean motor activity counts during the first 10 minute interval of the 40 minute session than the control males or the males that had been given 50 mg/kg. This was not dose-related, and the motor activity for all other 10 minute intervals and for the entire 40 minute session at Week 17 was similar for males across groups. Moreover, the locomotor activity of the males in the open field, as assessed subjectively, was similar for males across groups at Week 17. Therefore, the differences at Week 17 were considered incidental and not related to the test substance.


ORGAN WEIGHTS- There were no test substance related changes in the terminal and recovery body and organ weights. The relative increases at the terminal sacrifice in the mean pituitary weight for females given 75 mg/kg/day and in the thymus weight for females given 50 and 75 mg/kg/day, with a statistically significant change in the group mean body weight, were not considered biologically significance.


GROSS PATHOLOGY-There were no test substance related macroscopic findings at either the terminal or recovery sacrifice times. There were limited spontaneous tissue changes with no group predominance. Examples were a small testis in only one study animal (1 of 17 animals in the 15 mg/kg/day group, with microscopic correlative finding of atrophy/degeneration) and large pelvis and kidneys of 2 of 18 males given 0 mg/kg/day, 1 of 17 males given 15 mg/kg/day, 1 of 17 males given 50 mg/kg/day and 1 of 18 males given 75 mg/kg/day. The few findings were considered to be incidental.


HISTOPATHOLOGY: NON-NEOPLASTIC- There were no test substance related findings at either the terminal or recovery sacrifice times. The limited findings, such as minimal lymphoplasmacytic infiltrate in the epididymides, lymphohistiocytic infiltrate in the liver and mineralization in the kidney, had no greater incidence or severity in the examined animals receiving test substance than in the control. These tissue changes were considered to be spontaneous or incidental changes.


HISTOPATHOLOGY: NEOPLASTIC (if applicable)-N/A


HISTORICAL CONTROL DATA (if applicable)- N/A


OTHER FINDINGS:
1. Estrous Cycle Evaluation- Vaginal smears done during Weeks 10-18 to evaluate the phase of the estrous cycle revealed no test substance related effects. The mean number of days in each phase of the estrous cycle, the mean number of combined days in proestrus and estrus, and the mean length in days of the estrous cycle (duration of estrus defined as the number of days from the occurrence of estrous stage until the recurrence of the stage) revealed no notable differences among the groups.
2. Male Reproductive Assessment- Mean percent motility, total sperm count and sperm morphology measured at the terminal and recovery sacrifice periods were not affected by treatment with the test substance at any of the dose levels. No biologically meaningful differences were observed between the study groups.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

75 other: mg/kg/day (highest dose tested)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: 50 mg/kg bw/day is considered a NOEL based on the absence of any toxicological relevant effect with statistcally significance. Limited findings are considered to be incidental and / or of spontaneous origin.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Dose Analysis-Overall, the analytical results tended to be lower than theoretical. The diet preparations were stable under the conditions of storage (10 days at room temperature) and the concentrations of the test substance in the low and high diets were maintained within 10% of the initial concentrations. Homogeneity results were good (i.e., consistent for the low, middle, and bottom measurements) for the low dose diet preparation and more variable for the high dose diet preparation, but given the overall lower than theoretical values, these homogeneity results were deemed acceptable.

The concentration verification analyses (routine analyses) of the diets for Weeks 1, 2, 3, 4, 8, and 13 were consistent. The percent of theoretical values averaged 65% for the low dose group diets (range was 56% - 72%), 74% for the mid dose group diet (range 66% - 84%) and 80% for the high dose group diets (range was 72% - 95%).

Applicant's summary and conclusion

Conclusions:

The purpose of the study was to evaluate the toxicity of SS0772.01 when administered in the diet to rats for at least 13 weeks and to evaluate the persistence of, recovery from, or delayed occurrence of any effects of the test substance over a 4-week treatment free period. Based on the findings, the nominal no observed adverse effect level was 75 mg/kg and the nominal no observed effect level was 50 mg/kg.

Executive summary:

The purpose of the study was to evaluate the toxicity of SS0772.01 when administered in the diet to rats for at least 13 weeks and to evaluate the persistence of, recovery from, or delayed occurrence of any effects of the test substance over a 4-week treatment free period.

Male and female Crl:CD(SD)IGS BR rats were assigned to four groups. The control and high dose groups consisted of 18 animals/sex/group and the low and mid dose groups consisted of 17 animals/sex/group. Six animals/sex/group from the control and high dose groups and 5 animals/sex/group from the low and mid dose groups were designated as recovery animals. Animals designated for the recovery sacrifice were observed for 4 weeks after treatment was concluded. Each group received basal diets containing nominal concentrations of 0, 15, 50 or 75 mg of test substance/kg of body weight/day.

Food was provided ad libitum, unless otherwise specified; water was provided ad libitum. The animals were observed twice daily (a.m and p.m.) for mortality and moribundity. Abnormal findings were recorded as they were observed.

Once prior to treatment, on the day of initiation of treatment, and weekly during treatment, each animal was removed from its cage and detailed observations were made; abnormal findings or an indication that the animal appeared normal were recorded.

Expanded clinical observations for hand held and open field observations were conducted on 10 animals/sex/group once prior to treatment and once weekly during Weeks 1 - 13 and on 5 animals/sex/group weekly during Weeks 14 - 17. The same 10 animals/sex/group were tested at each interval. Elicited behavior tests and motor activity testing were included in the behavioral testing of the animals once prior to treatment and once during Weeks 13 and 17. Beginning on Day 64 and continuing daily until the day before the terminal sacrifice (Days 96 and 97), all females had a vaginal smear. This was continued daily for all remaining females until the recovery sacrifice.

Ophthalmic examinations were done prior to initiation of treatment and once during Weeks 14 and 18. Body weights were collected for each animal at arrival, at randomization, on the day of initiation of treatment, and weekly thereafter. Food consumption data were collected weekly.

Animals were fasted overnight and blood and urine samples were collected for hematology, coagulation, clinical chemistry, and urinalysis tests from all animals during Week 14 and for recovery animals during Week 19. Following blood collection, the animals were euthanatized with carbon dioxide, weighed, exsanguinated, and necropsied during Week 14 for the terminal sacrifice and during Week 19 for the recovery sacrifice. At necropsy, macroscopic observations were recorded, selected organs were weighed, and selected tissues were collected and preserved. Microscopic examinations were done on tissues from each animal in the control and high dose groups. In addition, any observed lesions from animals in any group, and the rectum, cecum, colon, duodenum, jejunum, and ileum from each animal in the low and mid dose groups were also examined microscopically. For male reproductive assessment (performed by Pathology Associates, A Charles River Company), the right vas deferens and right epididymis were collected from each male for sperm motility, sperm morphology, and sperm count evaluation.

All animals survived to their respective sacrifices. The clinical observations that were noted throughout the study were not limited to any one dose group and were not considered to be test substance related. There were no remarkable test substance related findings noted during the weekly expanded clinical observations for the hand held and open field testing, during the expanded clinical observation testing that involved elicited behaviors, nor during the motor activity testing.

There were no test substance related differences in body weights, food consumption, or ophthalmic findings. The estrous cycle of the females was similar across all groups and the male reproductive parameters were similar across all groups.

Gonadal tissues were examined for gross changes and histopathology, and no test substance related effects were observed.

Administration of the test substance had no clear effect on clinical pathology test results. Of uncertain relationship to administration of the test substance were mildly lower cholesterol for females given 75 mg/kg and increased incidences of positive urine occult blood results (reagent strip method) and amorphous phosphate crystalluria in urine sediment (microscopic examination) for males given 75 mg/kg. Regardless of their relationship to administration of the test substance, these findings were not considered adverse.

There were no test substance related changes in the body and organ weights in animals from the terminal sacrifice or recovery group. There were no test substance related macroscopic or microscopic findings in any animal at either the terminal or recovery sacrifice times. The few finding were considered to be incidental.

When administered in the diet for at least 13 weeks, the test substance was well tolerated by rats. There were no remarkable test substance related effects on clinical observations, expanded clinical observations, body weights and body weight changes, food consumption, ophthalmic findings, female estrous cycle, male reproductive parameters, terminal or recovery body and organ weights, or macroscopic or microscopic observations. Mildly lower cholesterol values for females given 75 mg/kg were noted after 13 weeks of treatment and at the recovery sacrifice. Increased incidences of positive urine occult blood and amorphous phosphate crystalluria in urine sediment for males given 75 mg/kg were noted after 13 weeks of treatment but were not noted at 19 weeks in recovery rats. These were of uncertain relationship to administration of the test substance, and they were not considered to be adverse. Based on the findings, the nominal no observed adverse effect level was 75 mg/kg and the nominal no observed effect level was 50 mg/kg.