5,7-dichloro-4-(4-fluorophenoxy)quinoline;quinoxyfen (ISO)

Endpoint summary

Description of key information

90-Day mouse feeding study LOAEL: 500 mg/kg bw/day for males and females; OECD 408; Reliability = 1

90-Day rat feeding study LOAEL: 100 mg/kg/day; OECD 408; Reliability = 1

90-Day dog feeding study LOAEL: >100 mg/kg/day (highest concentration tested); OECD 409; Reliability = 1

12-Month Neurotox Study Rat Diet Systemic LOAEL: 80 mg/kg, Neurotox LOEL: >80 mg/kg (highest dose tested); not neurotoxic. EPA 83-1; Reliability = 1

28-Day rat feeding study LOAEL: 250 mg/kg (lowest dose tested); FIFRA 82-1; Reliability = 2

28-day dog feeding study LOAEL = 250 mg/kg/day; FIFRA 82-1; Reliability = 2

28-Day Immunotox Study Rat Diet LOAEL: 100 mg/kg/day; no humoral immune response. EPA OPPTS 870.7800; Reliability = 1

28-day rat dermal study LOAEL > 1000 mg/kg/day; OECD 410; Reliability = 1

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC Directive 87/302 EEC: Subchronic Oral Toxicity

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MAFF Subchronic Study Guidelines

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Test substance: XR-795
Test substance ID: TSN100010
Purity: 98.7%

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

This strain of mouse was selected because of its general acceptance and suitability for toxicity testing, the availability of historical background data, and the reliability of the commercial supplier.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory, Kingston, New York
- Age at study initiation: 8 weeks
- Weight at study initiation: Male: 25.8 to 28.2 grams; Female: 21.7 to 22.1 grams
- Housing: Animals were housed 2- 3/cage prior to randomization and allocation to test groups and 1/cage thereafter. Cages were of stainless steel construction with wire mesh floors and each contained a feed crock and pressure-activated water nipple
- Diet: Purina Certified Rodent Chow #5002, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: Atleast 7 days

DETAILS OF FOOD AND WATER QUALITY: The feed was certified by the manufacturer to contain no more than specified maximum concentrations of certain heavy metals, chlorinated hydrocarbons, PCBs, aflatoxin, and organophosphates, and to have been manufactured in a plant in which the use of antibiotics and synthetic estrogens is prohibited. in which the use of antibiotics and synthetic estrogens is prohibited. Analyses of Purina Certified Rodent Chow #5002 feed revealed no contaminants which may have interfered with the results of this study.

ENVIRONMENTAL CONDITIONS
- Temperature: Approximately 72°F
- Air changes (per hr): 13
- Photoperiod (hrs dark / hrs light): 12

Route of administration:

oral: feed

Details on route of administration:

The probable routes of human exposure to test substance are via ingestion of foodstuffs that might contain low residues of the test material or from accidental ingestion or dermal contact during manufacture or use. Thus, formulation with feed was the desired method of delivery to assess the potential systemic toxicity of the test material following oral administration

Vehicle:

other: Feed

Details on oral exposure:

DIET PREPARATION
Diets and premixes were prepared weekly by serially diluting a concentrated test substance-feed mixture (premix). These dilutions were made by following computer-generated diet mixing instructions, with the concentrations of test substance in the diets based on group mean body weights and feed consumption. Initial concentrations of test material in the feed were calculated from pretest body weights and feed consumption data targeted on the desired dose levels on a mg/kg bw/day basis. Thereafter, the most recent body weight and feed consumption data were used to adjust the concentration of the test substance in the feed to maintain those targeted dose levels. Test substance concentrations in the feed were adjusted weekly to maintain the targeted exposure levels.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Data generated from the analyses indicated that appropriate concentrations of test substance were maintained such as to closely approximate the targeted dose levels

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The high dose of 500 mg/kg bw/day was selected based upon the results of the previous 2-week study. Body weight and body weight gain reductions as well as varying degrees of hepatotoxicity were expected at this dose level. The intermediate dosages of 100 and 50 mg/kg bw/day were selected to demonstrate a pattern of dose response, while the low dosage of 10 mg/kg bw/day was expected to establish a no-observed-effect level for test substance.
- Rationale for animal assignment: Prior to the start of the study, the animals were weighed and randomly allocated to study groups using a computerized, weight-stratification and random-number based procedure.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: All animals were observed cageside at least once daily during the workweek. These observations included evaluation of the skin, fur, mucous membranes, respiration, nervous system, and behavior patterns. Observations on weekends and holidays were limited to a check of all cages for dead animals and the availability of feed and water.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Prior to the start of the study and weekly thereafter through the duration of the study

BODY WEIGHT:
- Time schedule for examinations: Prestudy period and weekly during the dosing period

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Ophthalmological examinations were conducted on all animals during their scheduled necropsy

HAEMATOLOGY:
- Time schedule for collection of blood: At the time of necropsy, blood specimens were taken by orbital eye puncture from anesthetized animals for hematology determinations.
- Anaesthetic used for blood collection: Methoxyflurane
- Hematologic values which were measured were packed cell volume, hemoglobin concentration, red and white blood cell counts, and platelet counts using an ELT-IS Hematology Analyzer. Complete blood smear examinations were conducted which included differential leukocyte counts and an assessment of erythrocyte, leukocyte, and platelet morphology. Any morphologic abnormalities observed were reported.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: At the time of necropsy, blood specimens were taken by orbital eye puncture from anesthetized animals for clinical chemistry determinations.
- Clinical chemistry/electrolyte determinations which were measured are alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, blood urea nitrogen, creatinine, total bilirubin, glucose, total protein, calcium, phosphorus, chloride, sodium, potassium, and albumin using a Spectrum Analyzer. The globulin concentrations were calculated from the total protein and albumin values.

Sacrifice and pathology:

GROSS PATHOLOGY: A complete gross necropsy was performed by a veterinary pathologist. Weights of the brain, heart, liver, kidneys, adrenal glands, and testes were recorded and the organ weight to final body weight ratios calculated for all animals. Lungs were distended to an approximately normal inspiratory volume with neutral, phosphatebuffered 10% formalin solution by tracheal instillation using a handoperated syringe.

HISTOPATHOLOGY: The tissues examined for histopathology are adrenal glands, aorta, bone, bone marrow, brain, cecum, cervix, coagulating gland, colon, duodenum, epididymides, esophagus, eyes & optic nerve, gall bladder, heart, ileum, jejunum, kidneys, lacrimal glands, larynx, liver, lungs, mammary glands, mediastinal lymph node, mediastinal tissue, nasal tissue, oral tissue, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve, pituitary gland, prostate, rectum, salivary glands, seminal vesicles, skeletal muscle, skin and subcutis, spinal cord-cervical, spinal cord-thoracic, spinal cord-lumbar, spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus and vagina.

Statistics:

Hematology (excluding differential counts), electrolytes, clinical chemistry data, body weights, and absolute (grams) and relative (g to 100 g terminal body weight) organ weights were evaluated by Bartlett's test for equality of variances. Based on the outcome of Bartlett's test, exploratory data analysis was performed by a parametric or non-parametric analysis of variance (ANOVA), followed, if appropriate, by Dunnett's test or Wilcoxon rank-sum test with Bonferroni's correction for multiple comparisons. Statistical outliers were identified by a sequential test described by Grubbs (1969). Feed consumption data, which was used in the computation of desired test material concentrations and shown in this final report, was not analyzed for differences of statistical significance. Descriptive statistics were performed on feed efficiency and white blood cell differential data.
Differences found to be statistically significant were not necessarily accepted as toxicologically significant, i.e., final interpretation of the numerical data did consider the statistical outcomes together with other factors, such as dose-response relationships, biologic plausibility, and pathological observations.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical observations related to test substance administration were present for any animal. Several random instances of skin ulceration were observed during the course of the study but posed no serious health problems.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weight values for male and female mice revealed no changes that were interpreted as a result of test substance toxicity. The mean body weights in male mice administered 500 mg/kg bw/day were slightly lower than controls throughout the study. Statistically lower mean body weights were evident in male mice administered 10 mg/kg bw/day. However, due to the lack of a dose response relationship, these differences were considered to be incidental and unrelated to compound administration. The mean body weights for female mice were unaffected at all dose levels.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

There were no discernible differences in feed efficiency values for any of the male or female treatment groups, when compared to corresponding control groups. There was a high degree of variability associated with the feed efficiency data generated during this study and no dose response relationships were evident. Intervals of negative weight gain with resulting negative feed efficiency values, were excluded from analysis.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In both male and female mice, after 13 weeks of dietary exposure to test substance, no statistically significant differences were observed for any hematology or leukocyte parameters measured at any dose level. Slightly lower erythrocyte (RBC), hemoglobin (HGB), and hematocrit (PVC) values were seen in both male and female treatment groups and slightly higher white blood cell counts (WBC) were seen in male treatment groups. However, all of these differences were judged to be within normal limits for CD-1 mice of this age and of no biological significance. All other parameters were judged unchanged and normal.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Several parameters demonstrated statistical significance even though no toxicological significance was indicated. Parameters shown to be statistically different from control in male mice were lower albumin (ALB), 10, 50, and 100 mg/kg bw/day groups; lower total protein (TPRO), 50 mg/kg bw/day group; and lower calcium (CA), 50 mg/kg bw/day group. None of these differences reflect any definite response to test susbtance administration as affected values were random with no dose response relationships evident.
In female animals, the only parameter which was statistically different from controls was lower albumin (ALB) in the 100 and 500 mg/kg bw/day groups, however these differences were minor. Examination of individual animal data showed albumin concentrations for animals in the 100 and 500 mg/kgbw/day dose levels identical with those reported for control animals. Therefore, the lower albumin mean values reported for these dose groups were interpreted as incidental and random, rather than treatment related.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In male mice, organ weight values shown to be statistically different from controls were lower absolute heart weight in 10 mg/kg bw/day group, lower absolute kidney weights in 10 and 50 mg/kg bw/day groups, lower relative kidney weight in 50 mg/kg bw/day group, lower absolute liver weights in 10 and 50 mg/kg bw/day groups and higher relative liver weight in 500 mg/kg bw/day group. All of these differences except for the higher relative liver weight in the 500 mg/kg bw/day dose group, were judged to be reflections of lower terminal body weights in these groups, rather than toxic effects. The statistically higher relative liver weight in the male 500 mg/kg bw/day dose group coupled with a lower terminal body weight compared to controls, was judged to be a definite toxic effect of test substance administration in these animals. This conclusion is further supported by hepatic alterations seen histologically in this dose group.
In female mice, the only organ weight data demonstrating statistical significance are absolute and relative liver weights in the 500 mg/kg bw/day dose group. Although the terminal body weight for this group was slightly higher than controls, the differences between the absolute and relative mean liver weight values of the high dose females and controls is substantial and, as in male mice, was judged to represent a toxic response to test material ingestion. Histologic alterations seen in the livers of high dose female mice further supported this conclusion.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No gross lesions attributable to dietary administration of test susbtance were observed at any dose level. The splenomegaly seen in several mice was associated with dermal inflammatory lesions in these animals. These dermal lesions were considered to be secondary to localized skin reactions caused by ear tags in some of these animals.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-associated tissue alterations were confined to the liver of mice administered 500 mg/kg bw/day. These consisted of slight or moderate hypertrophy of centrilobular and midzonal hepatocytes. Additionally, 3 of 10 males and 4 of 10 females demonstrated minimal individual cell necrosis of hepatocytes. Dermal inflammatory lesions noted grossly were characterized by chronic inflammatory changes and ulcerative necrosis of the affected tissues. In one high dose level female mouse this was also accompanied by reactive lymphoid hyperplasia of the cervical lymph node. Splenomegaly was characterized by extramedullary hematopoiesis secondary to the dermal inflammatory changes.

Key result

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Conclusions:

NOEL (Mice) (Male/Female): 100 mg/kg bw/day

Executive summary:

The test was conducted according to guideline OECD 408 to evaluate 13-week dietary toxicity of test substance in mice. Ten male and ten female CD-1 mice were administered test substance, in their feed at concentrations of 0 (control), 10, 50, 100, or 500 mg/kg bw/day for 13 weeks. Parameters measured included: clinical observations, feed consumption, feed efficiency, body weight, hematology, clinical chemistries and electrolytes, organ weights, and gross and histopathologic evaluation of tissues.

The only toxic effects following administration of test substance for 13 weeks at the prescribed dosages were seen in male and female mice in the 500 mg/kg bw/day dose group. These effects were limited to the liver and consisted of organ weight changes and histologic alterations. Both absolute and relative liver weights in male and female 500 mg/kg bw/day mice were greater than controls. Histologic alterations observed in animals administered 500 mg/kg bw/day consisted of slight to moderate hypertrophy of centrilobular and midzonal hepatocytes and minimal individual cell necrosis. All other parameters measured: feed consumption, feed efficiency, body weight, hematology, clinical chemistries and electrolytes revealed no changes suggestive of test substance toxicity.

Animals in the other dose groups, 10, 50 and 100 mg/kg bw/day, were judged to have been unaffected by 13 weeks of test substance administration. Based on the data generated in this study, the no-observed-effect-level (NOEL) was 100 mg/kg bw/day in male and female CD-1 mice.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: European Economic Community, Subchronic Oral Toxicity Test 87/302

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japan MAFF, Subchronic Toxicity Study

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Test substance ID: TSN 100010
Lot number: DECO-104-116
Purity: 99.0 ± 0.2%

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

This strain of rat was selected because of its general acceptance and suitability for toxicity testing and the reliability of its commercial supplier.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, New York
- Weight at study initiation: Males: 170.6 to 171.4 grams; Females: 131.8 to 134.3 grams
- Housing: The rats were housed 1/cage during the pre-randomization period and 1/cage during the study
- Diet: Purina Certified Rodent Chow #5002, ad libitum
- Water: Municipal water, ad libitum
- Acclimation period: 15 days

DETAILS OF FOOD AND WATER QUALITY: The feed was certified by Purina Mills, Inc. to contain specified minimum levels of protein, fats, and fiber and to contain no more than specified maximum concentrations of certain heavy metals, chlorinated hydrocarbons, PCBs, aflatoxin, organophosphates, and to have been manufactured in a plant in which the use of antibiotics and synthetic estrogens is prohibited. Routine periodic analyses of the water have revealed no contaminants at levels likely to interfere with the interpretation of the results of this study.

ENVIRONMENTAL CONDITIONS
- Temperature: 72°F
- Air changes (per hr): 13
- Photoperiod (hrs dark / hrs light): 12

Route of administration:

oral: feed

Details on route of administration:

The probable route of human exposure to the test substance is via ingestion of foodstuffs that might contain low residues of the test material or from accidental ingestion or dermal contact during manufacture or use. Thus, formulation with feed was the desired method of delivery to assess potential systemic toxicity following oral administration.

Vehicle:

other: Feed

Details on oral exposure:

DIET PREPARATION
Diets mixed on study day 1 employed a 4 hour ball-milled premix and a 15 minute paddle mixing of the actual diet. Homogeneity analysis of test substance in Purina Certified Rodent Chow #5002 assessed on the 10 mg/kg bw/day female diet suggested incomplete dispersion of the test substance in the rodent chow. Diets mixed on study day 8, as per protocol, employed a 15 minute premix and a 15 minute paddle mixing of the actual diet. Homogeneity analysis assessed on the 10 mg/kg bw/day female diet demonstrated homogeneous dispersion of the diet within the parameters for Subchronic/Chronic Dietary area of this laboratory; however the decision of the study director with the concurrence of the analytical chemist was to increase the premix time to 4 hours and paddle mix the diets for 20 minutes for study week 3. Homogeneity analysis of diets mixed by the latter method. was performed on study day 43. Results of these analyses showed homogeneous dispersion of the test material in Purina Certified Rodent Chow #5002.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis showed that the test substance stayed within the targeted 90 percent range through 26 days.

Duration of treatment / exposure:

13 Weeks

Frequency of treatment:

Daily

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Rationale for animal assignment (if not random): Rats were randomly allocated to study groups using a computerized. system based on random numbers.
- Rationale for selecting satellite groups: Satellite groups of 10 male and female rats were fed 0 or 250 mg/kg bw/day for 13 weeks and then maintained for an additional four weeks on plain rodent chow.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: At least once daily

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: At least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on study Days 8 and 1 (for prestudy values) and at seven day intervals beginning on study Day 7.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

HAEMATOLOGY:
- Time schedule for collection of blood: At the time of necropsy
- Anaesthetic used for blood collection: Methoxyflurane.
- Animals fasted: Rats were fasted overnight prior to necropsy
- Parameters like Packed cell volume (PCV) (hematocrit), hemoglobin concentration (HGB), erythrocyte (RBC), leukocyte (WBC), and platelet counts (PLAT) were examined. Complete blood smear examinations were conducted, including differential leukocyte counts on 100 cells and an assessment of erythrocyte, leukocyte, and platelet morphology. Any morphologic abnormalities were reported.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: At the time of necropsy
- Animals fasted: Rats were fasted overnight prior to necropsy
- Parameters like albumin (ALB),alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), creatine phosphokinase (CK), creatinine (CREA), glucose (GLU), total protein (TPRO), (blood) urea nitrogen (UREA), sodium (NA), potassium (K), chloride (CL), calcium (CA), and phosphorus (P) were examined. Globulin values were calculated from the total protein and albumin values. Clinical chemistry tests performed on rats

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalysis parameters were determined for all rats randomized to the 13-week portion of this study on study Day 87
- Urine specific gravity was determined using a Goldberg refractometer. Commercially available reagent strips were used to semiquantitatively measure pH, protein, glucose, ketones, bilirubin, occult blood, and urobilinogen. Urinary sediment was examined microscopically on pooled samples by exposure group.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: A functional observational battery was conducted on study day 83 on all rats randomized to the 13-week portion of this study.
- The rats were carefully examined in a random order and in such a way that the observer was not aware of the exposure level or identification number of the rat. Observations noted
were any unusual responses with respect to body position, activity level, coordination of movement, and gait. Any unusual behavior induding but not limited to head flicking, headsearching, compulsive biting or licking, selfmutilation, circling, and walking backwards were observed. The presence of convulsions, tremors, increased levels of lacrimation and/ or red-colored tears, piloerection, pupillary dilation or constriction, unusual respiration (shallow, labored, gasping, and retching) and/or mouth breathing, diarrhea, excessive or diminished urination, vocalization and sensory function (audition, pain perception, and visual placing). Most observations were recorded after placing the rat in a clear plastic observation box measuring approximately 50x50x25 cm. If movement was not judged adequate within the allotted 20 second interval, the rat was gently prodded with the blunt end of a probe.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: The tissues examined for histopathology are adrenal glands, aorta, bone (including joint), bone marrow, brain, cecum, cervix, coagulating gland, colon, duodenum, epididymides, esophagus, eyes & optic nerve, gross lesions, heart, ileum, jejunum, kidneys, lacrimal glands, larynx, liver, lungs, mammary glands, mediastinal lymph node, mediastinal tissue, mesentric lymph node, mesentric tissue, nasal tissue, oral tissue, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve, pituitary gland, prostate, rectum, salivary glands, seminal vesicles, skeletal muscle, skin and subcutis, spinal cord-cervical, spinal cord-thoracic, spinal cord-lumbar, spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus and vagina.

Statistics:

Descriptive statistics only (means and standard deviations) were reported for feed consumption, feed efficiency, and white blood cell differential counts. Body weights, organ weights, clinical chemistry data, appropriate hematologic data and urinary specific gravity were evaluated by Bartlett's test for equality of variances. Based on the outcome of Bartlett's test, exploratory data analysis was performed by a parametric or nonparametric analysis of variance (ANOVA), followed respectively by Dunnett's test or the Wilcoxon Rank-Sum test with a Bonferroni correction for multiple comparisons. Statistical outliers were identified by a sequential test, but routinely excluded only from feed consumption. Negative values and those values with a divisor of zero were excluded from feed efficiency calculations.
Because numerous measurements were statistically compared on the same group of animals, the overall false positive rate (Type 1 errors) may have been much greater than the above-cited alpha levels would suggest. As a consequence, the final interpretation of numerical data considered statistical analyses along with other factors, such as dose response relationships and whether the results were plausible in light of other biological and pathological findings.

Clinical signs:

no effects observed

Description (incidence and severity):

No systemic treatment-related effects were noted during the daily or weekly clinical observations.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the 13-week portion of this study, mean body weights in the high dose males were slightly lower, although not statistically significant, than the controls (<3%). In female rats administered 100 mg/kg bw/day, body weights were statistically lower than the controls beginning on study day 63 (~96% of controls at study day 91). In female rats given 250 mg/kg bw/day, body weights were adversely affected early in the study and were approximately 95% of controls at study Day 91. Body weights in males administered 10 or 100 mg/kg bw/day and females administered 10 mg/kg bw/day were unaffected by test substance administration.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Mean feed efficiency values in males and females were highly variable throughout the study; however, there was no apparent effect of test substance administration in male rats. Mean feed efficiency values in the high dose males and females in the recovery portion of this study were lower than controls reflecting the greater body weight gain in these animals.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In male rats administered 250 mg/kg bw/day, hemoglobin concentration was shown to be statistically lower than controls. This value was not considered to reflect test material toxicity but rather random variation due to the lack of correlative changes in the erythrocyte count and hematocrit. In female rats administered 250 mg/kg bw/day, the platelet count was shown to be statistically lower. Again this was considered to be incidental and to have no biological or toxicological significance. No hematologic effects were observed in either males or females administered 10 or 100 mg/kg bw/day.
In the recovery portion of this study, the hemoglobin concentration and hematocrit for male high dose level rats were statistically lower and the erythrocyte count was lower than controls. This was considered to reflect the rapid growth of these animals during the 4 week recovery period and to be unrelated to test material toxicity. In male and female high dose level rats, the platelet count was shown to be statistically elevated compared to the controls. As in the 13-week portion of this study, this was considered to be an incidental finding and to have no biological or toxicological significance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males, after 13-week dietary administration of test substance caused elevation of albumin, total protein, potassium at 100 and 250 mg/kg bw/d. Elevation of levels of alkaline phosphatase, aminotransferase and aspartate aminotransferase were observed at 10 and 250 mg/kg bw/day. In females, after 13-week dietary administration of test substance caused elevation of albumin, total protein, potassium at 250 mg/kg bw/day. Elevation of levels of alkaline phosphatase, aminotransferase and aspartate aminotransferase were observed at 10 and 250 mg/kg bw/day. Although associated with test substance administration, the changes in the above parameters were minor and are considered unlikely to represent significant biologic or toxicologic effects.
Although shown to be statistically different from controls, the lower mean creatine phosphokinase values in the low dose males and females and the elevated mean glucose concentration in the low dose males were considered incidental and due to random variation. No clinical chemistry effects were observed in either males or females administered 10 or 100 mg/kg bw/day.
Examination of the above parameters after a 4-week recovery period showed that in males alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase activities were still slightly lower than controls and total protein and potassium concentrations remained Slightly elevated. In females, alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase activities were still slightly lower than controls whereas total protein and potassium concentrations were slightly lower than controls. These slight variations were considered to be of no toxicologic or biologic significance and would have disappeared over a longer recovery period.

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

After 13 weeks of dietary administration, the terminal body weights in the high dose male and the middle and high dose female rats were lower than controls. Absolute and relative liver weights in the middle and high dose males and females were shown to be significantly higher than those of controls. In high dose males, the absolute and relative kidney weights, were elevated; in high dose females, the relative kidney weights were elevated. Although associated with test substance administration, the kidney weight differences (reversible, i.e., not seen in the recovery animals) were unassociated with changes in urinalysis, clinical chemistry parameters, gross observations, or histopathologic alterations in the kidneys. The higher relative brain weight in the high dose female rats was considered to be the result of the lower terminal body weight in this group of rats rather than a toxicologic effect of test substance administration.
Parameters still affected after a 4-week recovery period were terminal body weight in high dose female rats; and absolute and relative liver weights in high dose male rats. As with clinical chemistry parameters, these slight differences from control most likely would have disappeared over a longer recovery period.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormalities attributable to test substance administration were found at any dose level.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Alterations attributed to test substance administration were found in the livers of male and female rats administered 100 and 250 mg/kg bw/day. In 10 of 10 males and 3 of 10 females administered 100 mg/kg bw/day and 3 of 10 males administered 250 mg/kg bw/day, these alterations consisted of a slight hypertrophy with increased basophilia of centrilobular and mid zonal hepatocytes. In 7 of 10 males and 10 of 10 females administered 250 mg/kg bw/day, the alteration consisted of a slight panlobular hypertrophy with increased basophilia of hepatocytes. In 2 of 10 males and 9 of 10 female rats administered 250 mg/kg bw/day, a few randomly scattered foci of individual cell hepatocellular necrosis were present.
Histopathologic alterations in rats randomized to the 4-week recovery period consisted of a very slight hypertrophy of centrilobular hepatocytes in male rats. Hepatocellular alterations seen earlier in the females in the 13-week portion of this study were not apparent after a 4 week recovery period. The lower grade of hypertrophy seen after 4 weeks in these animals suggests that hypertrophy would have been inapparent over a longer recovery period.

Other effects:

no effects observed

Description (incidence and severity):

Inaddition to the daily and weekly recording of animal observations, a functional observational battery was performed on study Day 83. All observations in both male and female rats were considered to be normal for Fischer 344 rats.

Key result

Dose descriptor:

NOEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Conclusions:

NOEL (Rat) (Male/Female): 10 mg/kg bw/day

Executive summary:

The test was conducted according to OECD guideline 408 to evaluate 13-week dietary toxicity of test substance in rats. Test substance was fed to groups of male and female Fischer 344 rats at targeted dosages of 0 (control), 10, 100, or 250 mg/kg bw/day for 13 weeks. Satellite groups of 10 male and female rats were fed 0 or 250 mg/kg bw/day for 13 weeks and then maintained for an additional 4 weeks on plain rodent chow. Parameters evaluated included in-life observations, functional observational battery, body weights, feed consumption and feed efficiency, hematology, clinical chemistries, urinalysis, organ weights, and gross and histopathologic alterations.

In-life and terminal fasted body weights for males administered 250 mg/kg bw/day and females administered 100 or 250 mg/kg bw/day were slightly lower than controls. Organ-specific effects of test substance administration were in the liver as evidenced by histopathologic alterations and elevated absolute and relative liver weights in male and female rats given 100 or 250 mg/kg bw/day. Minor changes in hepatic-associated clinical chemistries were evident in male and female high dose level rats. The histopathologic lesion was characterized by a dose-related distribution of hepatocellular hypertrophy. In 10 of 10 males and 3 of 10 females administered 100 mg/kg bw/day and 3 of 10 males administered 250 mg/ kg bw/day, these alterations consisted of a slight hypertrophy with increased basophilia of centrilobular and midzonal hepatocytes. In 7 of 10 males and 10 of 10 females administered 250 mg/kg bw/day, the alteration consisted of a slight panlobular hypertrophy with increased basophilia of hepatocytes. In 2 of 10 male and 9 of 10 female rats administered 250 mg/kg bw/day, a few randomly scattered foci of individual cell hepatocellular necrosis were present.

Rats in the satellite group had amelioration of the effects of test substance treatment; however, recovery was not complete over the 4-week period suggesting that complete recovery could occur following a longer recovery period.

Based on the data generated in this study, the no-observed-effect-level (NOEL) for sub chronic dietary administration of test substance in Fischer 344 rats was 10 mg/kg bw/day.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: European Economic Community, Subchronic Oral Toxicity Test 87/302

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japan MAFF, Subchronic Toxicity Study

Deviations:

no

GLP compliance:

yes

Limit test:

yes

Specific details on test material used for the study:

Test substance ID: TSN 100010
Lot number: DECO 104-116
Purity: 99.0%

Species:

dog

Strain:

Beagle

Details on species / strain selection:

The dog (Canis familiaris) was selected for this study because of its general acceptance and suitability as a non-rodent species for toxicological testing. The Beagle dog was used because of its availability, its historical background data, its familiarity with human handling, and it can be obtained in reasonably uniform size, weight, and age from a reliable commercial supplier.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazleton Research Products, Kalamazoo
- Age at study initiation: Approximately 6 months
- Weight at study initiation: Females: 7478 to 9300 grams; Males: 8993 to 12689 grams
- Housing: The dogs were placed in the kennel and given water and feed ad libitum
- Diet: Purina Certified Canine Diet #5007, ad libitum
- Water: Municipal water, ad libitum
- Acclimation period: 5 weeks

DETAILS OF FOOD AND WATER QUALITY: The feed was certified by the manufacturer to contain specified minimum levels of protein, fats, and fiber and to contain no more than specified maximum concentrations of certain heavy metals, chlorinated hydrocarbons, PCBs, aflatoxin, organophosphates, and to have been manufactured in a plant in which the use of antibiotics and synthetic estrogens are prohibited. The levels of these contaminants in the canine diet were judged not to be an interfering factor in the interpretation of the results of this study. Routine periodic analyses of the water have revealed no contaminants at levels likely to interfere with the interpretation of the results of this study

ENVIRONMENTAL CONDITIONS
- Temperature: 75 ± 5°F
- Photoperiod (hrs dark / hrs light): 12

Route of administration:

oral: feed

Details on route of administration:

The probable routes of human exposure to the test material are via ingestion of foodstuffs that might contain low residues of the test substance or from accidental ingestion or dermal contact during manufacture or use. Thus, formulation with feed was the desired method of delivery to assess the potential systemic toxicity of the test material following oral administration.

Vehicle:

other: Feed

Details on oral exposure:

DIET PREPARATION
Separate male and female dietary formulations of test substance were prepared weekly. A test substance/feed concentrate (premix) was prepared by using the ball-mill method. This method involves putting a determined amount of test substance together with Purina Certified Canine Diet #5007 into a grinding jar containing a preset amount of carborundum grinding pellets. The jar is sealed and rolled for at least 4 hours. Dietary formulations were prepared by serial dilution with canine chow beginning with the premix. Mixing was done in stainless-steel bowls using paddle mixers. The dietary concentrations and amounts were calculated using a computer program, based on the previous weeks mean body weights, calculated daily feed consumptions, premix concentrations, and targeted dose levels.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneous dispersion of test substance was determined on a diet mixed on Study Day 1; results were judged to show that the mixing method employed resulted in homogeneous dispersion of the test substance. Stability of test substance in Purina certified canine dietwas established over a 28 day period.
Concentration analyses were performed on feed mixed on study Days 1, 50, and 85. The results of these analyses indicated that appropriate concentrations of test substance were maintained such as to closely approximate the targeted dose levels.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

4

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Four dose levels, 0 (control), 10, 50, and 100 mg/kg bw/day were selected based on previous studies performed with test substance. The high dose of 100 mg/kg bw/day was expected to induce hepatic alterations. Although the diet was anticipated to be palatable, based on results of the previous study, a decrease in body weight gain and feed consumption at the high dose was expected. The middle dose was selected to show a dose-response in treatment-related effects. The low dose was chosen to demonstrate a no observed-effect-level (NOEL).
- Rationale for animal assignment: Randomization of the dogs into dose level groups was done using a computerized procedure, the dogs were stratified by weight and randomly assigned to experimental groups.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: The animals were observed at least once daily, including weekends and holidays, by laboratory personnel for evidence of treatment-related effects or changes in demeanor. Only abnormal observations were noted in the studyroom notebook. The observations were directed towards the animal's general appearance, pattern of movement and demeanor, to the presence of feces, and to the amount, if any, of excessive feed wastage. In addition, the condition of the mucous membranes; the respiration and circulation; the autonomic and central nervous\systems; somatomotor activity; and behavior/demeanor were assessed

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: All dogs were also given a weekly clinical observation by the study director or her designate. These observations included detailed evaluation of skin, ocular and nasal discharges, vaginal and penile discharges, mucous membrane color and condition, feces, central nervous system function, swelling, masses, and behavior for each animal.

BODY WEIGHT:
- Time schedule for examinations: All animals were weighed prior to the first exposure and weekly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

HAEMATOLOGY:
- Time schedule for collection of blood: Blood was collected from dogs prior to the administration of the test substance (Study Day -6), after approximately 6 weeks on study (Study Day 44), and immediately prior to the study termination (Study Day 86).
- Anaesthetic used for blood collection: No
- Animals fasted: Yes, overnight
- How many animals: All animals
- The hematologic parameters measured were hematocrit (PCV), hemoglobin (HGB) , erythrocyte count (RBC), leukocyte count (WBC) , leukocyte differential count, and platelet count (PLAT). PCV, HGB, RBC, WBC, and PLAT values were determined using an Ortho ELT-IS. Complete blood smear examinations which included a differential count of 100 white blood cells and an assessment of erythrocyte, leukocyte, and platelet morphology, were conducted on all dogs. Any morphological abnormalities were reported.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected from dogs prior to the administration of the test substance (Study Day -6), after approximately 6 weeks on study (Study Day 44), and immediately prior to the study termination (Study Day 86).
- Animals fasted: Yes
- How many animals: All animals
- The clinical chemistry parameters measured were albumin (ALB), alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST) , blood urea nitrogen (UREA), calcium (CALC), chloride (CL), creatinine (CREA), glucose (GLUC), inorganic phosphorus (PHOS), potassium (K), sodium (NA), total bilirubin (TBIL), and total protein (TPRO). Globulin (GLOB) values were calculated, by the Spectrum Analyzer, from the total protein and albumin values.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was aspirated directly from the urinary bladder at necropsy.
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Commercially available reagent strips were used to semiquantitatively measure pH, protein, ketones, bilirubin, occult blood, glucose, and urobilinogen. Urine specific gravity was determined using a Goldberg refractometer. Urinary sediment was examined microscopically.

Sacrifice and pathology:

GROSS PATHOLOGY: All test animals were examined externally and internally by a veterinary pathologist. The adrenal glands, brain, heart, kidneys, liver, pituitary gland, testes (males), ovaries (females), and thyroid
land were weighed immediately after dissection and the organ to body weight ratios were subsequently calculated. After gross examination, the lungs were distended to their approximate normal inspiratory volume by tracheal infusion of neutral, phosphate-buffered 10% formalin. The intestinal tract and hollow organs were incised for internal examination.

HISTOPATHOLOGY: The tissues examined for histopathology are adrenal glands, aorta, bone (including joint), bone marrow, brain, cervical lymph node, cecum, cervix, colon, duodenum, epididymides, esophagus, eyes & optic nerve, gall bladder, gross lesions, heart, ileum, jejunum, kidneys, larynx, liver, lungs, mammary glands, mediastinal lymph node, mediastinal tissue, mesentric lymph node, mesentric tissue, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve, pituitary gland, prostate, rectum, salivary glands, skeletal muscle, skin and subcutis, spinal cord-cervical, spinal cord-thoracic, spinal cord-lumbar, spleen, stomach, testes, thymus, thyroid gland, tongue, tonsils, trachea, urinary bladder, uterus and vagina.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Thirteen week dietary administration of test susbtance at dose levels less than or equal to 100 mg/kg bw/day had no effect on body weights. Although body weights for the male 50 mg/kg bw/day dose group were shown to be statistically different from controls, this was not judged to be the result of test substance administration due to the lack of a dose-response effect.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males (100 mg/kg bw/day), feed consumption values during the middle third of this study approximated those recorded for control males. During the last weeks on study, feed consumption values for males (100 mg/kg bw/day) were lower than controls.
In females (100 mg/kg bw/day), feed consumption values approximated or exceeded those of controls, suggesting adaptation to an initially unpalatable diet. Feed consumption was unaffected by test substance administration at the low and middle dose levels (10 and 50 mg/kg bw/day respectively).

Food efficiency:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Parameters shown to have statistical difference were: increased relative kidney weight (male 10 mg/kg bw/day) and increased relative liver weight (male and female 50 mg/kg bw/day). Due to the lack of a dose-response relationship, the lack of positive correlative changes in related clinical chemistry parameters, and the absence of histopathologic alterations, the slight increase in relative kidney weight in males given 10 mg/kg bw/day and the minimal increase in relative liver weights in males and females administered 50 mg/kg bw/day were considered incidental and due to random- variation.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No morphological alterations, attributed to test substance, were present in any dog. Hematocysts noted on the mitral valves of two male dogs, a control and a high dose animal, were considered incidental and embryonic in origin. The skin mass/nodule noted in a control female dog was judged to be a localized inflammatory reaction. Also, the congested kidneys on a female middle dose dog had no histopathologic alterations which correlated positively to the gross observation.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopatholigic alterations were judged to be incidental and consistent with Beagle dogs of this age. These included epididymal sperm granulomas, adrenal cortical cysts, cardiac valve hematocysts, vacuolation of renal proximal tubule epithelial cells, parathyroid embryonic cysts, pituitary cysts, salivary gland inflammation, dermal inflammation, minimal testicular degeneration, hepatic mononuclear and hematopoietic cell aggregations, hepatocellular vacuolation, and pulmonary fibrosis. Pyogranulomatous inflammatory foci in the lungs of dogs in all treatment groups were consistent with inhalation of meal form dog chow.
Most tissue alterations recorded in the livers of control and treated dogs in this study were considered to be incidental and consistent with dogs of this age.

Key result

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: Highest dose tested

Key result

Critical effects observed:

no

Conclusions:

NOEL (Dog): 100 mg/kg bw/day (Highest dose tested)

Executive summary:

The test was conducted according to OECD guideline 409 to evaluate 13 -week toxicity in dogs. Test substance was administered to male and female Beagle dogs approximately 6 months of age at targeted dose levels of 0 (control), 10, 50, and 100 mg/kg bw/day for 13 weeks. Parameters evaluated were: body weight, feed consumption and feed efficiency, in-life observations, hematology, clinical chemistries, urinalysis, organ weights, and gross and histopathologic observations.

All parameters were unaffected following 13 weeks dietary administration of test substance at all dose levels. In this study, the no observed- effect-level (NOEL) for test substance administration to male or female Beagle dogs was 100 mg/kg bw/day.

Reference 4

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EPA (FIFRA), Series 82-1, Subchronic Testing - Supplemental

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Substance ID: TSN 100003
- Lot number: DECO-36-106
- Purity: 97.6%

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Raleigh, North Carolina
- Weight at study initiation: Male: 120.9 to 123.2 g; Female: 110.9 to 113.9 g
- Housing: One per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: The feed was certified by Purina Mills, Inc. to contain specified minimum levels of protein, fats, and fiber and to contain no more than specified maximum concentrations of certain heavy metals, chlorinated hydrocarbons, PCBs, aflatoxin, organophosphates, and to have been manufactured in a plant in which the use of antibiotics and synthetic estrogens is prohibited. The levels of these contaminants in the rodent diet were judged not to be an interfering factor in the interpretation of the results of this study. Routine periodic analyses of the water have revealed no contaminants at levels likely to interfere with the interpretation of the results of this study.

ENVIRONMENTAL CONDITIONS
- Temperature: Approximately 72°F
- Air changes: 13 per hour
- Photoperiod: 12 hour photocycle

Route of administration:

oral: feed

Vehicle:

other: diet

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Four weeks

Frequency of treatment:

daily

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS
- Time schedule: At least once daily

DETAILED CLINICAL OBSERVATIONS
- Time schedule: At least once daily

BODY WEIGHT
- Time schedule for examinations: All animals were weighed prior to the first exposure and approximately 2 times/week thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE
- Time schedule: Feed consumption was determined prior to the first exposure and approximately 2 times/week thereafter

HAEMATOLOGY
- Time schedule for collection of blood: At the time of necropsy
- Anaesthetic used for blood collection: Yes (with methoxyflurane)

CLINICAL CHEMISTRY
- Time schedule for collection of blood: At the time of necropsy
- Anaesthetic used for blood collection: Yes (with methoxyflurane)

URINALYSIS
- Time schedule for collection of urine: Study day 28
- Metabolism cages used for collection of urine: No

Sacrifice and pathology:

GROSS PATHOLOGY: Rats were fasted overnight prior to necropsy. At the time of necropsy, the rats were weighed and anesthetized with methoxyflurane. All rats were examined externally and internally by a veterinary pathologist for gross pathological alterations. The tracheas were clamped to prevent aspiration of blood; the animals were decapitated. The organs and tissues were collected and preserved in neutral, phosphate-buffered 10% formalin. After gross examination, the lungs were distended to their approximate normal inspiratory volume by tracheal infusion of neutral, phosphate-buffered 10% formalin. The intestinal tract and hollow organs were incised for internal examination. The nasal cavities were flushed with formalin via the pharyngeal duct. Weights of brain, heart, kidneys, liver, and testes were recorded and the organ-to-fasted-body-weight ratios were subsequently calculated.

HISTOPATHOLOGY: Histopathologic examination was limited to kidneys, liver, and testes. Representative sections of these tissues were processed conventionally, sectioned (4-6 microns), mounted on glass slides, stained with hematoxylin and eosin, and examined microscopically.

Statistics:

All parameters examined statistically were first tested for equality of variance using Bartlett's test. If the results of Bartlett's test were significant, then the data for the parameter were subjected to a transformation to obtain equality of the variances. When Bartlett's test was satisfied no further transformations were applied, or, if none of the transformations resulted in homogeneous variances the transformed data or raw data with the lowest Bartlett's statistic were used. The selected form of the data was then subjected to the appropriate parametric analysis as described below.
In-life body weights were evaluated using a three-way repeated measures (RM) analysis of variance (ANOVA).
Parameters analyzed by a three-way RM-ANOVA involved several preliminary examinations.
Terminal body weight, organ weight (absolute and relative, excluding testes), hematologic parameters (excluding differential WBC), clinical chemistry parameters, and urine specific gravity were evaluated using a two-way ANOVA with the factors of sex and dose; differences between the groups were primarily detected by the dose factor. For these parameters, the first examination was whether the sex-dose interaction was significant; if it was, a one-way ANOVA was done separately for each sex. Comparisons of individual dose groups to the control group were made with Dunnett's test only when a statistically significant dose effect existed; this was subsequent to the evaluation of the sex-dose interaction. The form of the ANOVA, one-way or two-way, was determined by whether or not the analysis had been separated by sex or not.
Results for testes weights (absolute and relative) were analyzed using a one-way ANOVA. If significant dose effects were determined in the one-way ANOVA, then separate doses were compared to controls using Dunnett's test.
Descriptive statistics only (means and standard deviations) were reported for feed consumption and WBC differential counts.

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights for male and female rats administered 250, 500, or 1000 mg/kg bw/day were shown to be statistically lower than controls. The body weights for the high dose male rats were approximately 67% of the controls; for the low and middle dose level males, the body weights were approximately 89% and 83% those of controls, respectively. The body weights of female rats were not as markedly affected; the body weights for the high dose female rats were approximately 84% of controls. The low and middle dose level female rats were 93% and 89% of controls, respectively. In both male and female rats this effect corresponded to lower feed consumption in these dose levels.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Feed consumption in male and female rats administered 250, 500, or 1000 mg/kg/day were generally lower throughout the four-week test period, suggesting a degree of unpalatability of the test compound diet mixture at all dose levels. With the exception of study days 10 and 14, feed consumption for the high dose male and female rats was markedly lower than the control rats. Body weight data for this same period of the study would suggest that the high feed consumption values for study day 10 and 14 in the high dose animals were due to feed wastage rather than consumption.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Although some parameters were shown to be statistically different from control, due primarily to the lack of a dose-response effect these parameters were considered incidental and due to random variation. Packed cell volume (hematocrit) shown to be statistically different in both sexes (middle dose only) was the same as controls in males and greater than controls in females. The lower platelet count in male and female rats at the 1000 mg/kg bw/day dose level was also statistically significant. Hemoglobin concentration in male rats given 500 or 1000 mg/kg bw/day was lower than controls but not different in females at these dose levels. The erythrocyte count in the middle dose females was statistically different.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Parameters shown to be statistically different from controls were: glucose - both sexes, low and high dose; blood urea nitrogen - male, high dose; and creatinine - male, low dose. Glucose values in the low dose rats were not judged to reflect test substance toxicity due to the lack of a dose-response effect. Lower glucose and blood urea nitrogen values in the high dose rats were considered to be a reflection of the lower feed consumption and markedly lower body weights in these rats. Creatinine values did not show a dose-response relationship.

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The terminal body weights in male and female rats administered 250, 500, or 1000 mg/kg bw/day were lower than controls, reflecting the lower in-life body weights. Absolute and relative testicular weights in the high dose males were shown to be significantly lower than those of controls. These differences were judged to be the result of the markedly lower body weights in this group of rats (67% of control).

Differences in the following parameters were judged to most likely represent the lower terminal body weights in these groups of animals; the parameters were shown to be statistically different in both sexes: absolute brain weight, high dose; relative brain weight, all doses; absolute kidney weight, high dose; and relative liver weight, all doses. Parameters shown to be statistically different with sex differences were: absolute heart weight, male - middle and high doses; absolute heart weight, female - all doses; relative kidney weight, male - all doses; relative kidney weight, female - middle and high doses; and, inverse absolute liver weight, female - all doses.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Morphological alterations attributable to test substance administration were only noted in male rats administered 1000 mg/kg bw/day. These consisted of bilateral, atrophic/ small testes in 3 of 5 animals at this dose level.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Lesions attributed to test substance administration were found in the testes of animals administered 1000 mg/kg bw/ day. These alterations consisted of a moderate to severe decrease in spermatogenesis in 4 out of 5 animals. As with absolute and relative testicular weights, the histopathologic alteration was judged to be consistent with the markedly lower body weights in this group of rats (67% of control).

Key result

Dose descriptor:

LOEL

Effect level:

250 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Table-1: Gross observations in male rats

Dose (mg/kg/d)	0	250	500	1000
No. of animals examined	5	5	5	5
Testes (Atrophic/small, bilaterally)	0	0	0	3

Table-2: Histopathologic observations in male rats

Dose (mg/kg/d)	0	250	500	1000
No. of animals examined	5	5	5	5
Testes (Decreased spermatogenesis, seminiferous epithelium, diffuse, moderate)	0	0	0	3
Testes (Decreased spermatogenesis, seminiferous epithelium, diffuse, severe)	0	0	0	1

Conclusions:

No NOEL established

Executive summary:

Groups of 5 male and 5 female Fischer 344 rats were administered test substance in their diet for 4 weeks at doses targeted to deliver 0 (control), 250, 500, or 1000 mg/kg bw/day following EPA (FIFRA) Series 82-1, Subchronic Testing-Supplemental. Parameters measured were: in-life observations, body weights, feed consumption, hematology, selected clinical chemistries, urinalysis, organ weights, gross observations, and histopathologic evaluations of kidneys, liver, and testes.

Administration of test substance at dose levels of 250, 500, or 1000 mg/kg bw/day to male and female rats resulted in markedly lower in-life and terminal fasted body weights with lower feed consumption values consistent with their lower body weight. In male rats, their body weights were 83%, 89%, and 67% of control in the 250, 500, and 1000 mg/kg bw/ day dose levels, respectively. In female rats, the body weights for animals administered 250, 500, and 1000 mg/kg bw/day were 93%, 89%, and 84% of control values, respectively.

Hematology, clinical chemistry, urinalysis, and organ weight parameters were either unaffected by treatment or differences were attributed to be secondary to the markedly lower body weights.

In this study, a no-observed-effect-level was not established for test substance administration to male or female Fischer 344 rats.

Reference 5

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: EPA (FIFRA), Series 82-1, Subchronic Testing - Supplemental

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Substance ID: TSN 100010
- Name of substance: XR-795
- Lot number: DECO-104-116
- Purity: 98.7%

Species:

dog

Strain:

Beagle

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

other: diet

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Four weeks

Frequency of treatment:

Daily

Dose / conc.:

250 mg/kg bw/day (nominal)

No. of animals per sex per dose:

2

Control animals:

yes, plain diet

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Both male and female dogs administered 250 mg/kg bw/day lost body weight during the 4-week study period. In males, body weight loss for one dog was 4.2% and for another dog was 8.2%. In female dogs, body weight loss for a dog was 16.3% and for another dog was 21.9%. Mean body weight gain for control males was 6.6%; mean body weight gain for control females was 10.6%.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Excessive feed wastage was not observed with either male or female dogs. In males, mean feed consumption data for dogs administered 250 mg/kg bw/day were slightly lower at the 8 and 22 day study intervals. Individual feed consumption values for males given 250 mg/kg bw/day were more variable on a week to week basis than individual feed consumption values for control males.

In females, mean feed consumption values for dogs administered 250 mg/kg bw/day were lower at all data intervals. Individual feed consumption values in females given 250 mg/kg bw/day were markedly lower than feed consumption values recorded for female control dogs.

Lower feed consumption values in males and females resulted in a lower "calculated" dose administered to these dogs than the targeted dose. To compensate for the lower feed consumption, the percent concentration in diet was increased which may have increased the unpalatability of the diets.

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathologic alterations attributed to test substance administration were present in the liver of animals (both sexes) administered 250 mg/kg bw/day. These consisted of a slight vacuolation of centrilobular and midzonal hepatocytes. Vacuoles were of variable size and had a foamy appearance suggestive of dilated endoplasmic reticulum rather than lipid accumulation.

Key result

Dose descriptor:

LOEL

Effect level:

250 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

histopathology: non-neoplastic

Critical effects observed:

no

Lowest effective dose / conc.:

250 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Conclusions:

No NOEL estabilished. Dietary administration at 250 mg/kg/d to dogs for 4-weeks resulted in lower body weight, body weight gain and feed consumption. Primary organ toxicity was confined to the liver and was characterized by slight vacuolation of centrilobular and midzonal hepatocytes.

Executive summary:

The study was conducted following EPA (FIFRA), Series 82-1, Subchronic Testing-Supplemental. The test substance was administered in the diet to Beagle dogs, 2 animals/ sex/ dose level, for 4-weeks at doses targeted to deliver 0 (control) or 250 mg/kg bw/day. Parameters measured were in-life observations, body weights and body weight gains, feed consumption, hematology, clinical chemistries, urinalysis, organ weights, gross observations, and histopathologic evaluations.

Body weight, body weight gain, and feed consumption in male and female dogs given 250 mg/kg bw/day were decreased. In addition to test substance toxicity, feed consumption data from these animals suggested some degree of unpalatability of the test diet.

Slight vacuolation of centrilobular and midzonal hepatocytes was present in the liver of male and female dogs given 250 mg/kg bw/day. The variably sized vacuoles had a foamy appearance suggestive of dilated endoplasmic reticulum rather than lipid accumulation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Data for 28-day exposure are available for rats and dogs. 90-day data are available for rats, mice, and dogs. In addition, studies for immunotoxicity and neurotoxicity are also available in their respective IUCLID sections.

System:

hepatobiliary

Organ:

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Requirements for Safety Evaluation of Agricultural Chemicals, JMAFF

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Substance ID: TSN 100097
- Name of substance: XDE-795
- Lot number: DECO-97-152-1
- Purity: 97.4%

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Inc., Raleigh, North Carolina
- Age at study initiation: 8 weeks old
- Weight at study initiation: Males: 171.0-179.3 g; Females: 115.6-118.1 g
- Housing: One animal per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 15 days

DETAILS OF FOOD AND WATER QUALITY: Analysis of the feed was performed to confirm the diet provided adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility.

ENVIRONMENTAL CONDITIONS
- Temperature: 21.9-22.2°C
- Humidity: 48.7-60%
- Air changes: Approximately 12-15 times per hr
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Vehicle:

methylcellulose

Details on exposure:

TEST SITE
- Area of exposure: Back of each animal starting at the scapulae (shoulders) to the wing of the ileum (hip bone) and half way down the flank on each side
- % coverage: 10
- Type of wrap if used: Absorbent gauze pad and non-absorbent cotton and wrapped with elastic bandage
- Time intervals for shavings or clipplings: 24 hours prior to initiation of dosing

REMOVAL OF TEST SUBSTANCE
- Washing: Application site was wiped with a water-dampened towel
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 4 mL/kg
- Constant volume or concentration used: Yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration verification: Concentration analyses of all dose levels were conducted at the study start and during week 2 of the study. The test substance in 0.5% methylcellulose was analyzed using HPLC with ultraviolet detection and external standards.
Homogeneity: Homogeneity of the low- and high-dose suspensions was determined prior to the start of the study.
Stability: The stability of test substance in 0.5% methylcellulose was established prior to the study start by reanalyzing the low- and high-dose concentrations 12 days after the initial concentration verification.

Duration of treatment / exposure:

6 hrs per day for 4 weeks

Frequency of treatment:

5 days per week

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The high-dose level of 1000 mg/kg/day represents the limit test as specified by several governmental guidelines. The mid- and low-dose levels were expected to provide dose response data for any treatment-related effect(s) observed in the high-dose group. The low-dose was also expected to ensure definition of a no-observed-effect level (NOEL).
- Rationale for animal assignment: Animals were stratified by body weight and then randomly assigned to treatment groups using a computer program

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS
- Time schedule: Twice each day

DETAILED CLINICAL OBSERVATIONS
- Time schedule: Pre-exposure and weekly throughout the study

DERMAL IRRITATION
- Time schedule for examinations: The dermal test site was subjectively evaluated when wraps were removed on the last day of a dosing week and on the afternoon prior to necropsy

BODY WEIGHT
- Time schedule for examinations: Pre-exposure and weekly throughout the study

FOOD CONSUMPTION
- Time schedule for examinations: Pre-exposure and weekly throughout the study
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION
- Time schedule for examinations: Prior to the study start and prior to termination
- Dose groups that were examined: All treatment groups and control group

HAEMATOLOGY
- Time schedule for collection of blood: At the scheduled necropsy
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: Yes

CLINICAL CHEMISTRY
- Time schedule for collection of blood: At the scheduled necropsy
- Animals fasted: Yes

URINALYSIS
- Time schedule for collection of urine: During the week prior to necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No

Sacrifice and pathology:

GROSS PATHOLOGY: Fasted rodents submitted alive for necropsy were anesthetized by the inhalation of carbon dioxide (CO2) vapors, weighed, and blood samples were obtained from the orbital sinus. Their tracheas were exposed and clamped, and the animals were euthanized by decapitation. The necropsy included an examination of the external tissues and all orifices. The head was 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 glass slide to each cornea. 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 hand-held syringe and blunt needle. 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 brain, liver, kidneys, heart, adrenals, testes, epididymides, uterus, ovaries, thymus and spleen were trimmed and weighed immediately. The ratios of organ weight to terminal body weight were calculated.

HISTOPATHOLOGY: Representative samples of tissues were collected and preserved in neutral, phosphate-buffered 10% formalin. Transponders were removed and placed in jars with the tissues. The number of sections from all preserved tissues were processed by standard histologic procedures from control and high-dose group animals. The following tissues from the remaining groups were processed and histopathologically examined: liver, kidneys, lungs, dermal test site, and skin adjacent to the dermal test site.

Statistics:

Means and standard deviations were calculated for all continuous data. Body weights, feed consumption, organ weights, urine volume, urine specific gravity, clinical chemistry data, and appropriate hematologic data were evaluated by Bartlett's test (alpha=0.01) for equality of variances. Based on the outcome of Bartlett's test, exploratory data analysis was performed by a parametric (alpha=0.05) or nonparametric (alpha=0.05) analysis of variance (ANOVA) and if significant, followed respectively by Dunnett's test (alpha=0.05) or the Wilcoxon Rank-Sum test (alpha=0.05) with a Bonferroni correction for multiple comparisons to the control. The experiment-wise alpha level was reported for these two tests. Detailed clinical observations incidence scores were statistically analyzed by a z-test of proportions comparing each treated group to the control group (alpha=0.05). The data were analyzed separately for each time point. Descriptive statistics only (means and standard deviations) were reported for body weight gains, RBC indices, and white blood cell differential counts. Statistical outliers were identified by a sequential test (alpha=0.02) but routinely excluded only from feed consumption statistics. Outliers may have been excluded from other analyses only for documented, scientifically sound reasons.
Because numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) was greater than the nominal alpha levels. Therefore, the final interpretation of the data considered statistical analyses along with other factors, such as dose-response relationships and whether the results were consistent with other biological and pathological findings and historical control values.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Weekly examinations revealed infrequent occurrences of lacrimation, and soiling (periocular, perineal, and perinasal). Since these observations did not occur in a dose-responsive manner, and are typical reactions to the semi-occlusive bandaging process, they were interpreted to not be treatment related.

Dermal irritation:

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

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Examinations performed on all animals pre-exposure revealed a few animals with cloudy corneas, while examinations performed during Week 3 of the study revealed an increased incidence of cloudy corneas. The distribution of cloudy cornea observations occurred across all dose levels, included both sexes, and did not occur in a dose-responsive manner, and therefore was interpreted not to be treatment related. Additionally, during the Week 3 examination, pale fundus was observed in a few animals from both sexes. However, this observation did not occur in a dose-responsive manner and therefore, was also interpreted to not be treatment-related.

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related changes in absolute or relative organ weights in any dose group. Relative testes weight means for males given 100 mg/kg/day were slightly higher than the control values and statistically identified. Although the group mean relative testes weight of 1.435 g/100 was heavier than the control mean of 1.330 g/100, it was also heavier than the high-dose group mean of 1.391 g/100. Additionally, both the absolute and relative testes weights for males given 100 mg/kg/day were within test facility’s historical control range. Therefore, due to the lack of a dose-responsive manner the increase in relative testes weight for males given 100 mg/kg/day was interpreted not to be treatment-related.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related gross pathologic observations. All gross pathologic observations were considered unassociated with exposure to treatment.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related microscopic changes due to exposure to the test substance in any of the tissues examined. The observations present were considered to be spontaneous alterations, unassociated with exposure to treatment.

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no treatment-related effects on any of the measured parameters

Remarks on result:

other: highest dose tested

Critical effects observed:

no

Conclusions:

NOEL (Rat): 1000 mg/kg bw/day (highest dose tested)

Executive summary:

The study was conducted following OECD guideline 410 and US EPA guideline 870.3200. Four groups of 10 Fischer 344 rats/sex/dose were dermally exposed at a semi-occluded test site to 0, 10, 100, or 1000 mg/kg bw/day 6hr/day, 5 days/week for 4 weeks to evaluate the potential for systemic toxicity. Daily cage-side, body weight, feed consumption, detailed clinical observation, dermal observation, ophthalmologic examination, hematology, clinical chemistry, urinalysis and organ weight data were evaluated. In addition, complete gross and histopathologic examinations were conducted.

There were no treatment-related effects on any of the measured parameters. The no-observed-effect level (NOEL) for Fischer 344 rats following dermal exposure to test substance for 4 weeks was 1000 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Only one study was available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Requirements for Safety Evaluation of Agricultural Chemicals, JMAFF

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Substance ID: TSN 100097
- Name of substance: XDE-795
- Lot number: DECO-97-152-1
- Purity: 97.4%

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Inc., Raleigh, North Carolina
- Age at study initiation: 8 weeks old
- Weight at study initiation: Males: 171.0-179.3 g; Females: 115.6-118.1 g
- Housing: One animal per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 15 days

DETAILS OF FOOD AND WATER QUALITY: Analysis of the feed was performed to confirm the diet provided adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility.

ENVIRONMENTAL CONDITIONS
- Temperature: 21.9-22.2°C
- Humidity: 48.7-60%
- Air changes: Approximately 12-15 times per hr
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Vehicle:

methylcellulose

Details on exposure:

TEST SITE
- Area of exposure: Back of each animal starting at the scapulae (shoulders) to the wing of the ileum (hip bone) and half way down the flank on each side
- % coverage: 10
- Type of wrap if used: Absorbent gauze pad and non-absorbent cotton and wrapped with elastic bandage
- Time intervals for shavings or clipplings: 24 hours prior to initiation of dosing

REMOVAL OF TEST SUBSTANCE
- Washing: Application site was wiped with a water-dampened towel
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 4 mL/kg
- Constant volume or concentration used: Yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration verification: Concentration analyses of all dose levels were conducted at the study start and during week 2 of the study. The test substance in 0.5% methylcellulose was analyzed using HPLC with ultraviolet detection and external standards.
Homogeneity: Homogeneity of the low- and high-dose suspensions was determined prior to the start of the study.
Stability: The stability of test substance in 0.5% methylcellulose was established prior to the study start by reanalyzing the low- and high-dose concentrations 12 days after the initial concentration verification.

Duration of treatment / exposure:

6 hrs per day for 4 weeks

Frequency of treatment:

5 days per week

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The high-dose level of 1000 mg/kg/day represents the limit test as specified by several governmental guidelines. The mid- and low-dose levels were expected to provide dose response data for any treatment-related effect(s) observed in the high-dose group. The low-dose was also expected to ensure definition of a no-observed-effect level (NOEL).
- Rationale for animal assignment: Animals were stratified by body weight and then randomly assigned to treatment groups using a computer program

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS
- Time schedule: Twice each day

DETAILED CLINICAL OBSERVATIONS
- Time schedule: Pre-exposure and weekly throughout the study

DERMAL IRRITATION
- Time schedule for examinations: The dermal test site was subjectively evaluated when wraps were removed on the last day of a dosing week and on the afternoon prior to necropsy

BODY WEIGHT
- Time schedule for examinations: Pre-exposure and weekly throughout the study

FOOD CONSUMPTION
- Time schedule for examinations: Pre-exposure and weekly throughout the study
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION
- Time schedule for examinations: Prior to the study start and prior to termination
- Dose groups that were examined: All treatment groups and control group

HAEMATOLOGY
- Time schedule for collection of blood: At the scheduled necropsy
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: Yes

CLINICAL CHEMISTRY
- Time schedule for collection of blood: At the scheduled necropsy
- Animals fasted: Yes

URINALYSIS
- Time schedule for collection of urine: During the week prior to necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No

Sacrifice and pathology:

GROSS PATHOLOGY: Fasted rodents submitted alive for necropsy were anesthetized by the inhalation of carbon dioxide (CO2) vapors, weighed, and blood samples were obtained from the orbital sinus. Their tracheas were exposed and clamped, and the animals were euthanized by decapitation. The necropsy included an examination of the external tissues and all orifices. The head was 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 glass slide to each cornea. 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 hand-held syringe and blunt needle. 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 brain, liver, kidneys, heart, adrenals, testes, epididymides, uterus, ovaries, thymus and spleen were trimmed and weighed immediately. The ratios of organ weight to terminal body weight were calculated.

HISTOPATHOLOGY: Representative samples of tissues were collected and preserved in neutral, phosphate-buffered 10% formalin. Transponders were removed and placed in jars with the tissues. The number of sections from all preserved tissues were processed by standard histologic procedures from control and high-dose group animals. The following tissues from the remaining groups were processed and histopathologically examined: liver, kidneys, lungs, dermal test site, and skin adjacent to the dermal test site.

Statistics:

Means and standard deviations were calculated for all continuous data. Body weights, feed consumption, organ weights, urine volume, urine specific gravity, clinical chemistry data, and appropriate hematologic data were evaluated by Bartlett's test (alpha=0.01) for equality of variances. Based on the outcome of Bartlett's test, exploratory data analysis was performed by a parametric (alpha=0.05) or nonparametric (alpha=0.05) analysis of variance (ANOVA) and if significant, followed respectively by Dunnett's test (alpha=0.05) or the Wilcoxon Rank-Sum test (alpha=0.05) with a Bonferroni correction for multiple comparisons to the control. The experiment-wise alpha level was reported for these two tests. Detailed clinical observations incidence scores were statistically analyzed by a z-test of proportions comparing each treated group to the control group (alpha=0.05). The data were analyzed separately for each time point. Descriptive statistics only (means and standard deviations) were reported for body weight gains, RBC indices, and white blood cell differential counts. Statistical outliers were identified by a sequential test (alpha=0.02) but routinely excluded only from feed consumption statistics. Outliers may have been excluded from other analyses only for documented, scientifically sound reasons.
Because numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) was greater than the nominal alpha levels. Therefore, the final interpretation of the data considered statistical analyses along with other factors, such as dose-response relationships and whether the results were consistent with other biological and pathological findings and historical control values.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Weekly examinations revealed infrequent occurrences of lacrimation, and soiling (periocular, perineal, and perinasal). Since these observations did not occur in a dose-responsive manner, and are typical reactions to the semi-occlusive bandaging process, they were interpreted to not be treatment related.

Dermal irritation:

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

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Examinations performed on all animals pre-exposure revealed a few animals with cloudy corneas, while examinations performed during Week 3 of the study revealed an increased incidence of cloudy corneas. The distribution of cloudy cornea observations occurred across all dose levels, included both sexes, and did not occur in a dose-responsive manner, and therefore was interpreted not to be treatment related. Additionally, during the Week 3 examination, pale fundus was observed in a few animals from both sexes. However, this observation did not occur in a dose-responsive manner and therefore, was also interpreted to not be treatment-related.

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related changes in absolute or relative organ weights in any dose group. Relative testes weight means for males given 100 mg/kg/day were slightly higher than the control values and statistically identified. Although the group mean relative testes weight of 1.435 g/100 was heavier than the control mean of 1.330 g/100, it was also heavier than the high-dose group mean of 1.391 g/100. Additionally, both the absolute and relative testes weights for males given 100 mg/kg/day were within test facility’s historical control range. Therefore, due to the lack of a dose-responsive manner the increase in relative testes weight for males given 100 mg/kg/day was interpreted not to be treatment-related.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related gross pathologic observations. All gross pathologic observations were considered unassociated with exposure to treatment.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related microscopic changes due to exposure to the test substance in any of the tissues examined. The observations present were considered to be spontaneous alterations, unassociated with exposure to treatment.

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no treatment-related effects on any of the measured parameters

Remarks on result:

other: highest dose tested

Critical effects observed:

no

Conclusions:

NOEL (Rat): 1000 mg/kg bw/day (highest dose tested)

Executive summary:

The study was conducted following OECD guideline 410 and US EPA guideline 870.3200. Four groups of 10 Fischer 344 rats/sex/dose were dermally exposed at a semi-occluded test site to 0, 10, 100, or 1000 mg/kg bw/day 6hr/day, 5 days/week for 4 weeks to evaluate the potential for systemic toxicity. Daily cage-side, body weight, feed consumption, detailed clinical observation, dermal observation, ophthalmologic examination, hematology, clinical chemistry, urinalysis and organ weight data were evaluated. In addition, complete gross and histopathologic examinations were conducted.

There were no treatment-related effects on any of the measured parameters. The no-observed-effect level (NOEL) for Fischer 344 rats following dermal exposure to test substance for 4 weeks was 1000 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/cm²

Study duration:

subchronic

Species:

rat

Additional information

Groups of 10 male and 10 female CD-1 mice were fed diets containing 0 (control), 10, 50, 100, or 500 mg/kg/day test substance for 90 consecutive days following OECD guideline 408. Parameters measured included clinical observations, feed consumption, feed efficiency, body weight, hematology, clinical chemistries and electrolytes, organ weights, and gross and pathological evaluation of tissues. All animals survived to scheduled termination. The only toxic effects following administration of test substance for 13 weeks at the prescribed dosages were seen in male and female mice in the 500 mg/kg bw/day dose group. These effects were limited to the liver and consisted of organ weight changes and histologic alterations. Both absolute and relative liver weights in male and female 500 mg/kg bw/day mice were greater than controls. Histologic alterations observed in animals administered 500 mg/kg bw/day consisted of slight to moderate hypertrophy of centrilobular and midzonal hepatocytes and minimal individual cell necrosis. All other parameters measured: feed consumption, feed efficiency, body weight, hematology, clinical chemistries and electrolytes revealed no changes suggestive of test substance toxicity.

 

Groups of 10 male and 10 female Fischer 344 rats were fed targeted dosages of 0 (control), 10, 100, or 250 mg/kg bw/day for 13 weeks. following OECD guideline 408. Satellite groups of 10 male and female rats were fed 0 or 250 mg/kg bw/day for 13 weeks and then maintained for an additional 4 weeks on plain rodent chow. Parameters evaluated included in-life observations, functional observational battery, body weights, feed consumption and feed efficiency, hematology, clinical chemistries, urinalysis, organ weights, and gross and histopathologic alterations. In-life and terminal fasted body weights for males administered 250 mg/kg bw/day and females administered 100 or 250 mg/kg bw/day were slightly lower than controls. Organ-specific effects of test substance administration were in the liver as evidenced by histopathologic alterations and elevated absolute and relative liver weights in male and female rats given 100 or 250 mg/kg bw/day. Minor changes in hepatic-associated clinical chemistries were evident in male and female high dose level rats. The histopathologic lesion was characterized by a dose-related distribution of hepatocellular hypertrophy. In 10 of 10 males and 3 of 10 females administered 100 mg/kg bw/day and 3 of 10 males administered 250 mg/ kg bw/day, these alterations consisted of a slight hypertrophy with increased basophilia of centrilobular and midzonal hepatocytes. In 7 of 10 males and 10 of 10 females administered 250 mg/kg bw/day, the alteration consisted of a slight panlobular hypertrophy with increased basophilia of hepatocytes. In 2 of 10 male and 9 of 10 female rats administered 250 mg/kg bw/day, a few randomly scattered foci of individual cell hepatocellular necrosis were present. Rats in the satellite group had amelioration of the effects of test substance treatment; however, recovery was not complete over the 4-week period suggesting that complete recovery could occur following a longer recovery period. Based on the data generated in this study, the no-observed-effect-level (NOEL) for sub chronic dietary administration of test substance in Fischer 344 rats was 10 mg/kg bw/day.

 

Groups of 4 male and 4 female Beagle dogs were fed dose levels of 0 (control), 10, 50, and 100 mg/kg bw/day for 13 weeks following OECD guideline 409. Parameters evaluated were: body weight, feed consumption and feed efficiency, in-life observations, hematology, clinical chemistries, urinalysis, organ weights, and gross and histopathologic observations. All parameters were unaffected following 13 weeks dietary administration of test substance at all dose levels. Therefore, the no observed- effect-level (NOEL) for test substance administration to male or female Beagle dogs was 100 mg/kg bw/day.

Groups of 5 male and 5 female Fischer 344 rats were administered test substance in their diet for 4 weeks at doses targeted to deliver 0 (control), 250, 500, or 1000 mg/kg bw/day following EPA (FIFRA) Series 82-1, Subchronic Testing-Supplemental. Parameters measured were: in-life observations, body weights, feed consumption, hematology, selected clinical chemistries, urinalysis, organ weights, gross observations, and histopathologic evaluations of kidneys, liver, and testes. Administration of test substance at dose levels of 250, 500, or 1000 mg/kg bw/day to male and female rats resulted in markedly lower in-life and terminal fasted body weights with lower feed consumption values consistent with their lower body weight. In male rats, their body weights were 83%, 89%, and 67% of control in the 250, 500, and 1000 mg/kg bw/ day dose levels, respectively. In female rats, the body weights for animals administered 250, 500, and 1000 mg/kg bw/day were 93%, 89%, and 84% of control values, respectively. Hematology, clinical chemistry, urinalysis, and organ weight parameters were either unaffected by treatment or differences were attributed to be secondary to the markedly lower body weights. In this study, a no-observed-effect-level was not established for test substance administration to male or female Fischer 344 rats.

Groups of 2 male and 2 female Beagle dogs were fed dose levels of 0 (control) and 1250 mg/kg bw/day for 4 weeks following FIFRA 82-1. Parameters measured were in-life observations, body weights and body weight gains, feed consumption, hematology, clinical chemistries, urinalysis, organ weights, gross observations, and histopathologic evaluations. Body weight, body weight gain, and feed consumption in male and female dogs given 250 mg/kg bw/day were decreased. In addition to test substance toxicity, feed consumption data from these animals suggested some degree of unpalatability of the test diet. Slight vacuolation of centrilobular and midzonal hepatocytes was present in the liver of male and female dogs given 250 mg/kg bw/day. The variably sized vacuoles had a foamy appearance suggestive of dilated endoplasmic reticulum rather than lipid accumulation. A NOAEL was not determined in this study.

Groups of 10 male and 10 female Fischer 344 rats were dermally exposed to targeted dosages of 0 (control), 10, 100, or 1000 mg/kg bw/day for 4 weeks. following OECD guideline 410. Daily cage-side, body weight, feed consumption, detailed clinical observation, dermal observation, ophthalmologic examination, hematology, clinical chemistry, urinalysis and organ weight data were evaluated. In addition, complete gross and histopathologic examinations were conducted. There were no treatment-related effects on any of the measured parameters. The no-observed-effect level (NOEL) for Fischer 344 rats following dermal exposure to test substance for 4 weeks was 1000 mg/kg/day.

Justification for classification or non-classification

Under the criteria of CLP Regulation [EC] No. 1272/2008, STOT RE may be assigned on the basis of a substance demonstrating evidence of significant or severe specific organ toxicity in a 90-day oral study at or below a guidance value of 100 mg/kg bw/day (basis of Category 2). This guidance value is adjusted in accordance with the Haber’s rule for studies of different durations. ‘Significant’ toxicity is taken to mean changes that clearly indicate functional disturbance or morphological changes that are toxicologically relevant. ‘Severe’ toxicity is considered to be more profound or serious and indicates changes that are of a considerably adverse nature with a significant impact on health.

There is no evidence for any adverse findings or serious target organ toxicity in 28-day and/or 90-day repeat dosing studies in rats, mice or dogs that meet the criteria of CLP Regulation [EC] No. 1272/2008 for STOT RE. The primary effects in these feeding studies were reductions in body weight and food consumption. . Organ-specific effects of test substance administration were in the liver as evidenced by histopathologic alterations and elevated absolute and relative liver weights in male and female rats given 100 or 250 mg/kg bw/day. Minor changes in hepatic-associated clinical chemistries were evident in male and female high dose level rats. The histopathologic lesion was characterized by a dose-related distribution of hepatocellular hypertrophy. In the absence of liver cell toxicity, these changes are attributed to no adverse induction of hepatic metabolising enzymes, an adaptive response to xenobiotic exposure. The test substance did not induce any adverse effects on the nervous system or immune system, nor when tested by the dermal route in short-term repeat exposure studies.  Therefore, the test substance is not classified for STOT RE according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.