Acetic acid, 2-[(5-chloro-8-quinolinyl)oxy]-

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 July 2013 to 28 March 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

September 1998

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

May 2008

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: JMAFF (2000) Subchronic Oral Toxicity Study

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

[(5-chloroquinolin-8-yl)oxy]acetic acid

Cas Number:

88349-88-6

Molecular formula:

C11H8ClNO3

IUPAC Name:

[(5-chloroquinolin-8-yl)oxy]acetic acid

Test material form:

solid: particulate/powder

Remarks:

powder

Specific details on test material used for the study:

- Name of test material (as cited in study report): X204558
- Molecular formula: C11H8ClNO3
- Molecular weight: 237.6
- Analytical purity: 98.3% +/- 0.03% wt/wt by liquid chromatography with identification by liquid chromatography-mass spectrometry, gas chromatography mass spectrometry, and nuclear magnetic resonance
- Lot/batch No.: Lot 2GHB0002, TSN303795

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Details on species / strain selection:

As recommended by the guideline

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Portage, Michigan)
- Age at study initiation: 6-8 weeks
- Weight at study initiation: Males 182.6 - 219.3 g, Females 135.0 - 170.2 g
- Fasting period before study: None
- Housing: two per cage in stainless steel cages. Cages had solid floors with corncob bedding and shredded aspen for enrichment.
- Diet: ad libitum LabDiet Certified Rodent Diet #5002
- Water: ad libitum municipal water
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a tolerance of ± 1°C (and a maximum permissible excursion of ± 3°C)
- Humidity (%): 40-70%
- Air changes (per hr): 10-15 times/hour (average)
- Photoperiod (hrs dark / hrs light): 12-hour light/dark

IN-LIFE DATES: From: 24 September 2013 To: 22 October 2013

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

Oral (dietary) administration was chosen as a possible route of human exposure to the test material could be via accidental ingestion during field application, manufacture or via residues in food.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
Test material was administered as a constant fixed percent in the diet. Diets were prepared by serially diluting a concentrated test material-feed mixture (premix) with ground feed. Premixes and diets were mixed periodically throughout the study based on stability data. The concentration of the test material in diet was not adjusted for purity for either material.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Dose Confirmation and Homogeneity:
Dose confirmation analyses of all dose levels, plus control and premix, were determined pre-exposure, near the middle and end of the study. The homogeneity of the low- and the high-concentration test diets were determined concurrent with dose confirmation. The method used for analyzing the test materials in the diet was a solvent extraction method followed by analysis using liquid chromatography mass spectrometry (LCMS).

- Stability:
X204558 was found to be stable in the feed at concentrations ranging from 0.0005 to 10% for 65 days. Test diets were mixed and used within these stability limits.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily in the diet

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Plain diet controls

Doses / concentrations 2

Dose / conc.:

105 ppm

Doses / concentrations 3

Dose / conc.:

700 ppm

Doses / concentrations 4

Dose / conc.:

2 100 ppm

Remarks:

Administered 700 ppm on days 1-3 to attenutate potential palatability effects

No. of animals per sex per dose:

Ten male and ten female
An additional control group of 10 male and 10 female rats was included and allowed a 28 day recovery period after the 90 day test period

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels selected were based upon the previous toxicity data from a 14-day dietary toxicity and palatability probe study of X204558 conducted in Fischer 344 rats at concentrations ranging from 150 to 10000 ppm, a 14-day dietary palatability probe study of X204558 (2100 ppm) and X204559 (3000 ppm) conducted in Crl:WI(Han) rats and a 90-day dietary toxicity study of X204559 was conducted in Wistar rats at dose concentrations of 30, 150, 1000 or 8000/10000 ppm. The high-concentration of X204558 (2100 ppm) was chosen based on the probe study data wherein 3000 ppm exceeded the MTD as evidenced by the excessive palatability effects resulting in the death (euthanasia) of one female rat. The high concentration of 2100 ppm X204558 represented a molecular weight equivalent concentration of 3000 ppm X204559, a concentration that was not previously evaluated, but represented a concentration higher than the 90-day NOAEL of 1000 ppm for X204559. The mid- and low-concentrations of X204558 represented molecular weight equivalent concentrations of 1000 and 150 ppm X204559 as evaluated in the 90-day study by Kangas (2004). The high-concentration of X204558 and X204559 were both expected to produce decreased feed consumption, some body weight depression and possible renal effects. The mid- and low-concentrations of X204558 were expected to provide dose response data for any treatment-related effects observed in the high-concentration group. The low-concentration was expected to be a no-observed-effect level (NOEL). The high-concentration of X204558 and X204559 were 700 or 1000 ppm, respectively, on test days 1-3 and were increased to 2100 or 3000 ppm on day 4 in order to attenuate any potential palatability effects, as well as maintain consistency in dosing between these two groups.
- Rationale for animal assignment: animals were stratified by body weight and then randomly assigned to treatment groups
- Post-exposure recovery period in satellite groups: The group fed 2100 ppm for 90-days were allowed a 28-day recovery period after exposure, prior to sacrifce. An additional control group (10 rats/sex; fed plain diet only) were also allowed a 28-day recovery period after the exposure period, prior to sacrifce

Positive control:

None, not required

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day.
- Cage side observations included, but were not limited to decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity.
In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on all animals pre-exposure and at least once per week throughout the study.
- Detailed clinical observations included:
Hand-Held Observations: Eye observations (Palpebral closure, Pupil Size, Lacrimation), Salivation, Muscle tone, Extensor-thrust response, Reactivity to stimuli.
Categorical Observations: Abnormal behavior, Abnormalities of the eye, Abnormal urine or feces, Abnormalities of the gastrointestinal (GI) tract, Injury, Missing extremity, Abnormal muscle movements, Palpable mass/swellings, Abnormal posture, Abnormalities of the reproductive system, Abnormal respiration, Abnormal skin or hair-coat/mucous membranes, Excessive soiling, General abnormalities.
Open-Field Observations: Responsiveness to touch, Gait evaluation

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed during the pre-exposure period, daily during the first week of the study, and at least weekly thereafter. Body weight gains were also be calculated.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes, feed consumed was determined pre-exposure, daily during the first week of the study, and at least weekly thereafter for all animals by weighing feed containers at the start and end of a measurement cycle.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the scheduled necropsy
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the scheduled necropsy
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, overnight
- How many animals: All animals except animals that died or were euthanized in a moribund condition
- Parameters examined:
Hematocrit (HCT), Hemoglobin (HGB) concentration, Red blood cell (RBC) count, Total white blood cell (WBC) count, Differential WBC count (Neutrophils (NEUT), Lymphocytes (LYMP), Monocytes (MONO), Eosinophils (EOS), Basophils (BASO), Large Unstained Cells (LUC) which include, atypical lymphocytes, large lymphocytes, plasma cells, and blasts), Platelet (PLT) count, Reticulocyte (RET) count, RBC indices (Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin Concentration (MCHC)) and Prothrombin time (PT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the scheduled necropsy
- Animals fasted: Yes, overnight
- How many animals:All animals except animals that died or were euthanized in a moribund condition
- Parameters examined:
Enzyme Activities of: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (GGT)
Concentrations of: Albumin (ALB), Albumin/Globulin Ratio (A/G) - calculated, Cholesterol (CHOL), Creatinine (CREA), Electrolytes (Calcium (CA), Phosphorus (PHOS), Sodium (NA), Potassium (K), Chloride (CL)), Globulin (GLOB) - calculated, Glucose (GLUC), Total bilirubin (TBIL), Total protein (TP), Triglycerides (TRIG), Urea nitrogen (UN)
Serum Thyroid Hormone: triiodothyronine (T3), thyroxine (T4) and thyroid stimulating hormone (TSH)

URINALYSIS: Yes
- Time schedule for collection of urine: the week prior to the scheduled necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters examined:
Color, appearance, specific gravity (refractometer) and urine volume, pH, Bilirubin, Glucose, Protein, Ketones, Blood, Urobilinogen and Microscopic Exam

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: pre-exposure and during the last week of the treatment period
- Dose groups that were examined: all animals
- Battery of functions tested:
Sensory Evaluation: Responsiveness to sharp noise, Responsiveness to tail pinch,
Measurements: Rectal temperature °C, Hindlimb grip performance in grams, Forelimb grip performance in grams, Motor Activity in square root of counts

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The necropsy included an examination of the external tissues and all orifices. The head were removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin using a handheld syringe and blunt needle.
The brain, liver, kidneys, heart, adrenals, testes, epididymides, uterus, ovaries, thymus and spleen were trimmed and weighed immediately. The thyroid with the
parathyroid gland(s) were trimmed and weighed following fixation and dissection from the trachea. The ratios of organ weight to terminal body weight were calculated.
Liver samples were obtained from all animals at the time of necropsy. After weighing, the upper third of the left lateral liver lobe was stored in RNAlater for possible targeted gene expression analysis. Cross sections through the middle of the left lateral lobe, middle of the right medial lobe, and through the right lateral lobe were preserved in neutral, phosphate-buffered 10% formalin for histological evaluation. All remaining liver tissue were flash frozen and stored at -80ºC for possible biomarker analysis. Transponders were removed and placed in jars with the tissues.
Similar necropsy procedures were followed for animals found dead or moribund, except that body weights, organ weights, and blood samples were not obtained. In addition, liver tissue from animals found dead or euthanized in a moribund condition was not flash frozen or collected in RNAlater.

HISTOPATHOLOGY: Yes
The following tissues collected and preserved at necropsy were examined:
Adrenal, Aorta, Auditory Sebaceous Glands, Bone (Including Joint), Bone Marrow, Brain (Cerebrum, Brainstem, Cerebellum), Cecum, Cervix, Coagulating Glands, Colon, Cranial Nerve - Optic, Duodenum, Epididymides, Esophagus, Eyes, Gross Lesions , Heart, Ileum, Jejunum, Kidneys, Lacrimal/Harderian Glands, Larynx, Liver, Lungs, Mammary Gland - Females Only, Mediastinal Lymph Node, Mediastinal Tissues, Mesenteric Lymph Node, Mesenteric Tissues, Nasal Tissues/Pharynx, Oral, Tissues, Ovaries, Oviducts, Pancreas, Parathyroid Glands, Peripheral Nerve - Tibial, Pituitary, Prostate, Rectum, Salivary Glands, Seminal Vesicles, Skeletal muscle, skin and Subcutis, Spinal Cord (Cervical, Thoracic, Lumbar), Spleen, Stomach, Testes, Thymus, Thyroid Gland, Tongue, Trachea, Urinary Bladder, Uterus, Vagina

Selected histopathologic findings were graded to reflect the severity of specific lesions to evaluate: 1) the contribution of a specific lesion to the health status of an animal, 2) exacerbation of common naturally occurring lesions as a result of the test material, and 3) dose-response relationships for treatment-related effects.

Other examinations:

28-Day Recovery Groups:
Body weights, feed consumption, test material intake, cage-side observations, pre-study ophthalmology, functional tests and detailed clinical observations were conducted on the recovery animals throughout the 90-day dosing periods as previously described for the 90-day group. Weekly body weights, feed consumption, daily cage-side observations were conducted on the animals throughout the 28-day recovery period. A necropsy was also conducted on these animals. Other parameters determined to have treatment-related effects at the end of the dosing period were also examined in the recovery animals. Parameters evaluated included urine volume, urine specific gravity, gross necropsy with spleen weights, urea nitrogen, and platelet counts. Blood smears were collected, and other hematological parameters were measured in conjunction with the platelet counts, but were not subjected to analyses.

Statistics:

Means and standard deviations were calculated for all continuous data. All parameters examined statistically were first tested for equality of variance using Bartlett's test. If the results from Bartlett's test were significant, then the data for the parameter may have been subjected to a transformation to obtain equality of the variances. The transformations that were examined were the common log, the inverse, and the square root, in that order. The data were reviewed and an appropriate form of the data was selected. The selected form of the data was then subjected to the appropriate parametric analysis.
In-life body weights were evaluated using a repeated measures (RM) analysis of variance (ANOVA), the multivariate approach, for time (the repeated factor), sex, and dose. Terminal body weight, organ weight, urine volume, urine specific gravity, hematologic parameters (excluding RBC indices and differential WBC), coagulation, clinical chemistry parameters (excluding globulin and albumin/globulin ratio), and thyroid hormones were evaluated using a two-way ANOVA with the factors of sex and dose. Results for ovaries, uterus, epididymides, and testes (absolute and relative) were analyzed using a one-way ANOVA. Feed consumption data were evaluated by Bartlett's test for equality of variances. DCO and sensory evaluation incidence data (scored observations only) were statistically analyzed by a z-test of proportions comparing each treated group to the control group. Rectal temperature and grip performance data were analyzed by a factorial analysis of covariance (ANCOVA) with the factors of sex and treatment and the covariate of the preexposure time point measure of the variable. Motor activity counts were reported as their square roots to minimize problems of heterogeneity of variance and departure from normality that commonly occur from treatment.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related decreases in body weights and body weight gains noted in both sexes of the 700 and 2100 ppm groups were attributed to decreased palatability. These decreases persisted in males, but not in females given 2100 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Treatment-related decreases in feed consumption of male and female rats given 700 or 2100 ppm were attributed to decreased palatability of the test diets. This was most prominent in males given 2100 ppm and was generally sustained during the study

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Females given 2100 ppm had statistically significant, treatment-related, slightly higher platelet counts as compared to their respective controls that were deemed non-adverse.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Females given 2100 ppm had statistically significant lower BUN levels when compared to controls which was considered treatment-related but non-adverse.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Males and females given 2100 ppm had treatment-related, increases in urine volume and decreases in specific gravity as compared to their respective controls. The higher urine volumes were interpreted to be non-adverse.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males and females given 2100 ppm had statistically significant, treatment-related slightly lower absolute and relative spleen weights when compared to those of the controls which were considered non-adverse.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
All rats survived the 90-day test period and the 28-day recovery period.
There were no treatment-related observations for males or females at any concentration as compared to the controls. Clinical observations (dermatitis, hair loss, thin hair coat, injury) were incidental, unrelated to treatment and typically occurred in 0 to 2 animals/sex/group.

BODY WEIGHT AND WEIGHT GAIN
There was a treatment-related decrease in body weights and body weight gains for males and females given 700 and 2100 ppm on test days 2 through 5, which was attributed to decreased palatability of the test diet. In addition, the treatment-related decreases in body weight and body weight gains persisted in males, but not females given 2100 ppm from test days 29 through 92. At the end of the study, body weight and body weight gains of 2100 ppm males were approximately 3.0 and 6.5%, less than controls, respectively. There were no treatment-related effects on body weight or body weight gain of rats given 105 ppm.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Treatment-related decreases in feed consumption were noted in male and female rats given 700 or 2100 ppm, which occurred initially (test days 1-2) with test diet administration and was attributed to decreased palatability of the test diets. This effect was most prominent in males given 2100 ppm (initially given 700 ppm) and was typically sustained over the course of the study.
Rats were fed diets supplying 0, 105, 700, or 2100 ppm. To reduce initial palatability effects, rats of the 2100 ppm group were given 700 ppm on days 1-3. These values corresponded to time-weighted average doses of 0, 6.05, 38.6, or 116 mg/kg/day for males and 0, 6.42, 42.1, or 127 mg/kg/day for females, respectively.

OPHTHALMOSCOPIC EXAMINATION
There were no treatment-related ophthalmic observations for males or females at any concentration. Pre-exposure ophthalmic examinations indicated all eyes of rats were normal. Pre-terminal ophthalmic examinations also indicated all rats were within normal limits, with the exception of one male from the 2100 ppm group that had a pale fundus of the right eye. This finding was considered unrelated to treatment because it only occurred in one animal and it is a typical finding in control rats of this age.

HAEMATOLOGY
Females given 2100 ppm had statistically significant higher platelet counts as compared to controls. Although these values were only slightly higher than the controls, they were interpreted to be treatment-related because the individual platelet counts of the majority of the animals were slightly higher than that of the concurrent controls. However, this was interpreted to be non-adverse because the platelet counts were only slightly higher, absence of effects in males and there was no effect in any of the other hematological parameters.
There were no statistically significant or treatment-related changes in any of the hematologic parameters for males at all concentrations and females given 105 or 700 ppm as compared to their respective controls.

CLINICAL CHEMISTRY
Males and females given 2100 ppm had statistically significant slightly lower blood urea nitrogen (BUN) levels when compared to those of the controls. However, only the lower blood urea nitrogen level of the high exposure concentration females was interpreted to be treatment-related, because the majority of the individual animal values were consistently lower than that of the concurrent controls. The lower levels of BUN in males given 2100 ppm was considered not to be treatment-related because the values were marginally lower than that of the controls and represents a normal biological variability. In general, slightly lower levels of BUN are typically not indicative of biologically or toxicologically significant effects and, therefore are considered non-adverse.
There were no other statistically significant or treatment-related changes in any of the clinical chemistry parameters for male and female rats of any treated group as compared to their respective controls.
There were no statistically significant or treatment-related effects on any serum thyroid hormones (T3, T4, or TSH) for male and female rats of any treated group as compared to their respective controls.

URINALYSIS
Males and females given 2100 ppm had increased urine volume (not statistically significant) and decreased specific gravity (statistically significant) as compared to their respective controls. These changes were interpreted to be treatment-related because the urine volume and specific gravity values were consistently altered in majority of the animals as compared to their respective controls. The higher urine volumes and lower specific gravities were interpreted to be non-adverse because there were no corresponding adverse clinical chemistry alterations or histopathological evidence of urinary tract toxicity. In addition, such alterations can occur due to increased urine output secondary to possible increased water intake.
There were no other statistically significant or treatment-related alterations in the urinalysis parameters for treated males and females as compared to their respective controls.

NEUROBEHAVIOUR
There were no treatment-related sensory evaluation observations for males or females at any concentration as compared to the controls. There were no treatment-related effects on rectal temperature for males or females at any concentration. There were no treatment-related effects on grip performance for males or females at any concentration. There were no treatment-related effects on motor activity for males or females at any concentration.

ORGAN WEIGHTS
The terminal body weight of males given 2100 ppm was marginally lower (3.3%) than the controls and was interpreted to be treatment-related. There were no treatment related terminal body weight changes in males given 105 or 700 ppm or females at any exposure concentration when compared to those of the controls. Males and females given 2100 ppm had statistically significant slightly lower absolute (12.6 and 10.7 %, respectively) and relative (9.5 and 9.7 %, respectively) spleen weights when compared to those of the controls and were interpreted to be treatment-related because the individual values of the majority of the animals were consistently lower than that of the two control groups used in this study. The lower spleen weights were considered non-adverse because of the absence of a histopathological correlate. A number of statistically significant organ weight differences (increases and/or decreases) were also noted in females given 700 ppm that were interpreted to be spurious and unrelated to treatment because of lack of exposure-concentration response relationship.
There were no other statistically significant or treatment-related alterations in organ weights of treated males and females as compared to their respective controls.

GROSS PATHOLOGY
There were no treatment-related gross pathologic observations. All gross pathologic observations were considered to be spontaneous alterations, unassociated with exposure.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no treatment-related histopathologic observations. All observations were considered to be spontaneous alterations unassociated with exposure.

RESULTS: 28-DAY RECOVERY GROUPS
Detailed Clinical and Cage-side Observations: Similar to the 90-day portion of the study, there were no treatment-related observations in the recovery group animals either during the 90-day treatment period or during the recovery period.
Ophthalmology: Due to the lack of treatment-related effects on ophthalmology during the 90-day portion, recovery group animals were not evaluated at the end of the study.
Functional Tests: Due to the lack of treatment-related effects on functional tests during the 90-day portion, recovery group animals were not evaluated at the end of the study.
BodyWeights/Body Weight Gains: Similar to the main study animals, there was a treatment-related decrease in body weight and body weight gains of males and females of the 2100 ppm X204558 groups, which occurred initially with treatment and was attributed to decreased palatability of the test diet. The treatment-related effects persisted in both males and females given 2100 ppm over the duration of the study. By the end of the 28-day recovery period, the body weights and body weight gains of high-concentration exposure males (5.4 and 7.9%, respectively) and females (4.7 and 7.7%, respectively) were lower than the controls. For the statistical analysis of X204558 recovery group body weights, the treatment by time by sex interaction was not significant on any test day, which indicated that there was no treatment-related sex difference in body weights across days. However, the treatment by time interaction was significant on every measurement day except for day 1. This included all body weight measurement days that spanned the treatment period (days 2-92) and the recovery period (days 99-119). This indicates that the decreased body weights in X204558 rats did not reach control levels by the end of the recovery period, despite increases in feed consumption.
Feed Consumption: Similar to the main study animals, there was a treatment-related decrease in feed consumption of males and females of the 2100 ppm X204558 groups, which occurred initially with treatment and was attributed to decreased palatability of the test diet. While the initial palatability effects became attenuated and feed consumption increased early in the treatment period (by day 3 or 4), a slight decrease in feed consumption was generally sustained throughout the treatment period. After switching to control feed on day 92, the feed consumption of the X204558 rats increased to control levels, which provided further evidence for a slight, but sustained palatability effect in animals given 2100 pm X204558 during the treatment period. Although feed consumption improved during the recovery phase, it was not sufficient enough for complete recovery of body weight effects within 28 day period.
Hematology: Following the 28-day recovery period, there were no statistically significant treatment-related changes in the platelet counts of males and females given 2100 ppm indicating a complete recovery from the treatment-related effects induced following the 90-day phase of the study.
Clinical Chemistry: Following the 28-day recovery period, there were no statistically significant treatment-related changes in the blood urea nitrogen levels of males and females given 2100 ppm indicating a complete recovery from the treatment-related effects induced following the 90-day phase of the study.
Urinalysis: Following the 28-day recovery period, there were no statistically significant treatment-related changes in the urine volume or specific gravity of males and females given 2100 ppm indicating a complete recovery from the treatment-related effects induced following the 90-day phase of the study.
Organ Weights: Following the 28-day recovery period, males and females given 2100 ppm had statistically significant lower terminal body weights (5.2 and 4.5 %, respectively) as compared to those of the controls indicating that the treatment-related decreased body weights did not reach control levels by the end of the recovery period. There were no statistically significant treatment-related differences in the spleen weights. The absolute and relative spleen weights of males and females given 2100 ppm following the 28-day recovery period were similar to those of the controls indicating a complete recovery from the treatment-related effects induced following the 90-day phase of the study.
Gross Pathology: There were no treatment-related gross pathologic observations. All gross pathologic observations were considered to be spontaneous alterations, unassociated with exposure to X204558.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

2 100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Body weight and food consumption effects were considered to be caused by reduced palatability. All other treatment-related effects were considered non-adverse.

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Analyses of all test diets indicated the mean concentration for each dose level ranged from 91.5 to 101% of targeted concentrations and 88.4 to 106.6% of the target concentration for each individual sample, indicating acceptable concentrations of X204558. The relative standard deviations for all diets sampled were between 0.9 and 11.9%, with mean relative standard deviations of 1.3 to 6%, which indicated that the diets were homogeneously mixed.

Selected Body Weight Gain (g) Intervals

Day	0 ppm	105 ppm	700 ppm	2100 ppm
Males
2	5.2	5.2	-3.6	-5.0
3	10.3	10.7	9.4	9.4
29	98.5	104.2	96.6	92.2
57	144.5	152.9	144.3	132.6
92	178.9	186.8	178.6	167.1
Females
2	1.7	3.6	-1.8	-4.9
3	4.5	3.6	2.9	4.1
29	44.1	44.0	51.8	43.4
57	61.5	63.9	71.0	62.4
92	75.8	73.8	83.3	78.2

 

Selected Feed Consumption (g/day) Intervals

Day	0 ppm	105 ppm	700 ppm	2100 ppm
Males
1-2	18.7	17.4	13.1	12.1
2-3	18.0	16.8	19.0	21.2
25-29	18.9	18.7	17.5	17.1
53-57	17.8	18.0	16.7	16.5
88-92	17.0	17.3	15.8	16.2
Females
1-2	13.1	11.8	8.0	6.9
2-3	11.3	11.0	13.6	14.2
25-29	12.4	12.4	12.8	12.4
53-57	12.3	12.6	12.3	12.7
88-92	11.8	11.4	12.0	12.2

 

Hematology – Males and Females

Parameter	0 ppm	105 ppm	700 ppm	2100 ppm
Platelets (E3/UL) – Males	712	763	809	724
Platelets (E3/UL) – Females	782	861	889	963

 

Clinical Chemistry Differences – Males and Females

Parameter	0 ppm	105 ppm	700 ppm	2100 ppm
BUN (mg/dl) – Males	13	15	14	12
BUN (mg/dl) – Females	17	16	15	14

 

Urinalysis – Males and Females

Parameter	0 ppm	105 ppm	700 ppm	2100 ppm
Urine Volume (ml) – Males	4.9	5.7	4.3	7.9
Specific Gravity – Males	1.063	1.063	1.066	1.046
Urine Volume (ml) – Females	3.3	4.1	4.7	5.3
Specific Gravity – Females	1.072	1.059	1.053	1.052

 

Organ Weights – Males and Females

Parameter	0 ppm	105 ppm	700 ppm	2100 ppm
Males
Terminal BodyWeight (g)	358.1	361.0	357.2	346.2
Spleen Weight (g)	0.610	0.636	0.570	0.533
Relative Spleen Weight (g/100)	0.171	0.176	0.160	0.154
Females
Terminal BodyWeight (g)	207.6	203.5	214.9	206.7
Spleen Weight (g)	0.447	0.410	0.427	0.399
Relative Spleen Weight (g/100)	0.215	0.202	0.197	0.194
Adrenal Weight (g)	0.070	0.064	0.059	0.062
Relative Liver Weight (g/100)	2.580	2.432	2.374	2.471
Ovaries Weight (g)	0.122	0.103	0.089	0.102
Relative Ovaries Weight (g/100)	0.059	0.051	0.042	0.050
Relative Thyroid Weight (g/100)	0.0078	0.0080	0.0091	0.0074

Bold text represents effects considered treatment-related

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the NOAEL was the target concentration of 2100 ppm, corresponding to time-weighted average concentrations of 116.0 or 127.0 mg/kg bw/day in males and females, respectively.

Executive summary:

A study was conducted to evaluate the potential toxicity of X204558 (cloquintocet acid), in rats following dietary administration for at least 90 days according to OECD test guideline 408. Recovery groups (additional control group and high concentration of X204558) were given untreated diets for an additional 28-days to evaluate the reversibility or persistence of any potential effects induced after 90 days of dietary exposure. Groups of ten male and ten female Crl:WI(Han) rats were fed diets supplying 0, 105, 700, or 2100 ppm X204558. These values corresponded to time weighted average doses of 0, 6.05, 38.6, or 116 mg/kg/day for males and 0, 6.42, 42.1, or 127 mg/kg/day for females given X204558, respectively. Parameters evaluated were daily cage-side observations, weekly detailed clinical observations, ophthalmic examinations, functional tests, body weights, feed consumption, hematology, prothrombin time, urinalysis, clinical chemistry, thyroid hormone analyses, toxicokinetic analyses of blood and urine (results reported under IUCLID section 7.1.1), organ weights, and gross and histopathologic examinations.

There were no treatment-related effects in clinical signs, functional tests, ophthalmic examinations, prothrombin time, or serum thyroid hormone parameters. There were no treatment related, gross or histopathologic observations. Treatment-related decreases in body weights and body weight gains were noted in both males and females of the 700 and 2100 ppm groups, which occurred initially and were attributed to decreased palatability. The treatment-related decreases in body weights and body weight gains persisted in males, but not in females given 2100 ppm and were 3.0 and 6.5%, less than controls, respectively, at the end of the study. There were treatment-related decreases in feed consumption of male and female rats given 700 or 2100 ppm, which occurred initially with test diet administration and were attributed to decreased palatability of the test diets. The decreases in feed consumption were most prominent in males given 2100 ppm and were generally sustained over the course of the study. Females given 2100 ppm had statistically significant, treatment-related, slightly higher platelet counts as compared to their respective controls that were deemed non-adverse. There were no other statistically significant or treatment-related changes in any of the other hematologic parameters for male and female rats of any treated group as compared to their respective controls. Males and females given 2100 ppm had statistically significant lower blood urea nitrogen (BUN) levels when compared to those of the controls. However, only the lower BUN level of the high-dose females was interpreted to be treatment-related because the individual values were consistently lower than that of the concurrent controls. The lower levels of BUN in males given 2100 ppm was considered not to be treatment-related because the values were similar to that of the controls. In general, lower levels of BUN are typically not indicative of biologically or toxicologically significant effects and, hence are considered non-adverse.

Males and females given 2100 ppm had treatment-related, increases in urine volume and decreases in specific gravity as compared to their respective controls. The higher urine volumes and lower specific gravities were interpreted to be non-adverse because there were no corresponding adverse clinical chemistry alterations or histopathological evidence of urinary tract toxicity.

The terminal body weight of males given 2100 ppm was marginally lower (3.3%) than the controls and was interpreted to be treatment-related. There were no treatment-related terminal body weight changes in males given 105 or 700 ppm or females at any exposure concentration when compared to those of the controls. Males and females given 2100 ppm had statistically significant, treatment-related slightly lower absolute and relative spleen weights when compared to those of the controls. The lower spleen weights were considered non-adverse because of the absence of a histopathological correlate. There were no other statistically significant or treatment-related alterations in organ weights of treated males and females given as compared to their respective controls.

Recovery groups (ten/sex), given either 0 or 2100 ppm X204558 for 90 days, were maintained on control feed for an additional 28-day recovery period to assess the reversibility of treatment-related effects. Parameters evaluated during the recovery period were daily cage-side observations, body weights, feed consumption, limited hematology (platelets), clinical chemistry (BUN), urinalysis (urine volume and specific gravity), selected organ weights (spleen) and gross pathologic observations. Following the 28-day recovery period, the majority of the treatment-related effects noted during the 90-day portion of the study were reversed, except for the body weights and body weight gains, which were slightly lower than the concurrent controls. Although the feed consumption improved during the recovery phase, it was not sufficient for complete recovery of body weight effects within the 28-day period.

Under the conditions of this study, the NOAEL was the target concentration of 2100 ppm that corresponded to time-weighted average concentrations of 116.0 or 127.0 mg/kg bw/day in males and females, respectively.