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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant with current guidelines and GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): EC 202-228-8
- Physical state: Solid
- Analytical purity: 98.5%
- Composition of test material, percentage of components: 2-(2-naphthoxy)ethanol >98.5%; Ethylene Carbonate <0.5%; 2-Naphthol <0.5%; Ethylene Glycol <0.5%
- Lot/batch No.: E00173-186
- Expiration date of the lot/batch: Jul 2013
- Storage condition of test material: Room Temperature

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation: 62 days of age (males) and 64 days of age (females)
- Weight at study initiation: The body weight range for the male rats was 228 g to 265 g on the day after arrival at the Testing Facility and was 256 g to 290 g at randomization. The body weight range for the female rats was 172 g to 214 g on the day after arrival at the Testing Facility and was 189 g to 210 g at randomization.
- Housing: The rats were individually housed in stainless steel, wire-bottomed cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days (males) and 8 days (females)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C to 25°C
- Humidity (%): 30% to 70%
- Air changes (per hr): A minimum of 10 changes per hour of 100% fresh air
- Photoperiod (hrs dark / hrs light): 12-hours light: 12-hours dark fluorescent light cycle

IN-LIFE DATES: From: To: 07 Nov 2011 to 19 Dec 2011

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle (if other than water): Commonly used vehicle with test material not soluble in water.
- Concentration in vehicle: 10, 30 and 90 mg/mL
- Amount of vehicle (if gavage): 5 mL/day
- Lot/batch no. (if required): M-631
- Purity: 100%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing suspensions of the test substance prepared in corn oil were analyzed and were found to be acceptable and homogeneous under the conditions of the study. At the initiation of dose administration, all prepared formulations used for dose administration were analyzed and were found to be -0.7%, 0.7% and 1.6% from the target concentrations of 10 mg/mL, 30 mg/mL and 90 mg/mL, respectively. The corresponding percentage differences for the analyses conducted at the end of the study were -0.2%, -3.3% and -5.3%, respectively. The homogeneity values obtained were +0.7%, +1.2% and +2.2% RSD for the 10 mg/mL, 30 mg/mL and 90 mg/mL formulations, respectively.
The stability of the prepared test substance formulations bracketing the range of concentrations used on study was determined in Charles River Laboratories study number 20016972. Stability was determined in the 1 mg/mL and 200 mg/mL dosing solutions at room temperature (20 °C to 25 °C) for 25 hours and refrigerated (2 °C to 8 °C) for 10 days.
Duration of treatment / exposure:
28 days
Frequency of treatment:
The rats received the test substance for 28 consecutive days.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
50 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
450 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 rats/sex/dose group; 5 rats/sex/dose group in the 0 and 450 doses for the satellite group
Details on study design:
- Dose selection rationale: Based on a 14 day range finder (detailed as a supporting study IUCLID 7.5.1).
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: Random
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): Random
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The rats were observed for general appearance once during the acclimation period, daily prior to dose administration and daily during the postdose period.
- There were 29 observations performed on the main study animals and 43 observations performed on the recovery animals.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were recorded once prior to initiation of dose administration and weekly thereafter (with the exception of those weeks in which the Functional Observational Battery (FOB) was conducted since detailed clinical observations were subsumed within the FOB examination).

BODY WEIGHT: Yes
- Time schedule for examinations: Daily prior to dose administration

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Collected on the day of euthanasia from the inferior vena cava.
- Anaesthetic used for blood collection: Yes Isoflurane/oxygen was used as the anaesthetic.
- Animals fasted: Yes They were fasted for no longer than 24 hours.
- How many animals: All animals in the main study and the recovery portion were evaluated.
- Parameters assessed: Red blood cell count, Hemoglobin concentration, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin concentration, Mean corpuscular hemoglobin, Reticulocyte count (absolute), Mean platelet volume, Platelet count, White blood cell count, Neutrophil count, Lymphocyte count, Monocyte count, Eosinophil count, Basophil count, Large unstained cells


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Collected on the day of euthanasia from the inferior vena cava.
- Animals fasted: Yes They were fasted for no longer than 24 hours.
- How many animals: All animals in the main study and the recovery portion as well.
- Parameters assessed: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Gamma-glutamyltransferase, Total bilirubin, Urea nitrogen, Creatinine, Calcium, Phosphorus, Total protein, Albumin, Globulin, Albumin/globulin ratio, Glucose, Cholesterol, Triglycerides, Sodium, Potassium, Chloride


URINALYSIS: Yes
- Time schedule for collection of urine: Day 29 of exposure (main study) and day 43 of study (recovery portion)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters assessed: Colour, Clarity, Specific gravity, Microscopic evaluation of urine sediment, Total Volume, pH, Protein, Glucose, Bilirubin, Ketones, Nitrites, Leukocytes, Blood, Urobilinogen


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional observational battery and motor activity were evaluated on day 28 of the study.
- Dose groups that were examined: All dose groups were examined.
- Battery of functions tested: sensory activity / grip strength / motor activity / other: There were also evaluations of sensorimotor responses to visual, acoustic, tactile and painful stimuli (reactivity and sensitivity) as well as air righting reaction, visual placing response, landing foot splay and body temperature.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Test items in the FOB using interval scales, such as the grip-strength tests and the landing foot splay test, as well as body weight data and feed consumption values were analyzed as described under the Parametric heading of the schematic. Bartlett's Test of Homogeneity of Variances was used to estimate the probability that the groups had different variances. A nonsignificant result (p> 0.001) indicated that an assumption of homogeneity of variance was not inappropriate, and the data was compared using the Analysis of Variance Test. If that test was significant (p≤ 0.05), the groups exposed to the test substance were compared with the control group using Dunnett's Test If Bartlett's Test was significant (p≤ 0.001), the Analysis of Variance Test was not appropriate, and the data were analyzed as described under the Nonparametric heading of the schematic. When 75% or fewer of the scores in all the groups were tied, the Kruskal-Wallis Test was used to analyze the data, and in the event of a significant result (p≤ 0.05), Dunn's Test was used to compare the groups exposed to the test substance with the control group. When more than 75% of the scores in any group were tied, Fisher's Exact Test was used to compare the proportion of ties in the groups.

The remaining information regarding the statistical analyses are provided in the following section.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Male and female rats at 450 mg/kg/day
Mortality:
mortality observed, treatment-related
Description (incidence):
Male and female rats at 450 mg/kg/day
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduction in body weights and body weight gains in the male rats at 450 mg/kg/day
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reduction in absolute and relative food consumption in male rats at 450 mg/kg/day
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Male and female rats at 150 and 450 mg/kg/day
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Increase in movement in female rats at 450 mg/kg/day at the end of the dose period.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY

No test substance-related deaths occurred during the course of the study.
One male rat in the 450 mg/kg/day dose group was found dead on day 10 of study (DS 10). Adverse clinical signs noted for this rat included low carriage (DSs 1 to 3), decreased motor activity (DS 2), mild dehydration (DS 4) and ungroomed coat (DS 8). This rat had comparable body weight and feed consumption values in comparison with other rats in this dose group. Necropsy revealed that all lobes of the lungs were mottled red and dark red; all other tissues evaluated appeared normal. Based on the necropsy observation, this death was considered to be the result of an intubation error and not test substance-related.

There was an increase or statistically significant increase in the number of male and female rats in the 450 mg/kg/day dose groups observed with excess salivation (slight, moderate or extreme) and low carriage in comparison with the control group value. There was also an increase in the number of male and female rats observed with mild or moderate dehydration and the number of male rats observed with urine-stained abdominal fur.

BODY WEIGHT AND WEIGHT GAIN

In the male rats at 450 mg/kg/day, there were statistically significantly reduced (p≤ 0.05 to p≤ 0.01) mean body weights and body weight gains observed at all intervals during the dose period (DSs 1 to 28). In the male rats, body weight gains for the overall dose period (DSs 1 to 28) in the 50, 150 and 450 mg/kg/day dose groups were 86.9%, 102.9% and 61.9%, respectively, as compared with the control group value. The body weight gain for the male rats in the 450 mg/kg/day dose group during the recovery period (DSs 29 to 42) was 89.6% as compared with the control group value.
In the female rats, body weight gains for the overall dose period in the 50, 150 and 450 mg/kg/day dose groups were 112.0%, 81.7% and 98.6%, respectively, as compared with the control group value. The body weight gain for the female rats in the 450 mg/kg/day dose group during the recovery period was 170.7% as compared with the control group value.

HAEMATOLOGY
At the end of the dose period, there was a statistically significant increase (p≤0.05) in the mean platelet volume observed in the female rats at 450 mg/kg/day in comparison with the control group value. In the female rats at 450 mg/kg/day, there was also a statistically significant decrease (p≤0.05) in the mean corpuscular hemoglobin concentration in comparison with the control group value. These differences were not considered to be test substance-related because the values were within the historical control range of the Test Site performing the analysis.

At the end of the recovery period (DS 43), there was a statistically significant decrease (p≤0.05) in hemoglobin observed in the male rats at 450 mg/kg/day in comparison with the control group value. This difference was not considered to be test substance-related because the value was within the 95% spread of the Test Site historical control range.

There were no additional statistically significant or marginally different hematologic changes observed in the male or female rats in this study.

CLINICAL CHEMISTRY

At the end of the dose period (DS 29), changes in serum chemistry that were presumed related to treatment with the test substance included:

• a statistically significant decrease (p≤0.05) in aspartate aminotransferase was observed in the male and female rats at 150 and 450 mg/kg/day;
• an increase or statistically significant increase (p≤0.05) in triglyceride was observed in the male and female rats at 150 and 450 mg/kg/day;
• a statistically significant increase (p≤0.05) in glucose was observed in the male and female rats at 450 mg/kg/day; and
• a statistically significant increase (p≤0.05) in the albumin/globulin ratio and the total bilirubin was observed in the male rats at 450 mg/kg/day.

By the end of the recovery period (DS 43), these changes in serum chemistry had resolved.

All other changes in serum chemistry were not attributed to treatment with the test substance. There was a statistically significant increase (p≤0.05) in cholesterol observed in the female rats at 150 and 450 mg/kg/day in comparison with the control group value. There was also a statistically significant increase (p≤0.05) in creatinine observed in the male rats and calcium observed in female rats at 450 mg/kg/day in comparison with the control group value. These differences were not considered to be test substance-related because the values were within the 95% spread of the Test Site historical control range.

URINALYSIS

There were no biologically important differences between the control and treated groups in the urinalysis parameters evaluated on DSs 29 or 43. In the male rats on DS 29, the specific gravity of the urine in the 50, 150 and 450 mg/kg/day dose groups was significantly lower (p≤ 0.05 to p≤ 0.01) than the control group value (range of 1.0128 to 1.0180 vs. 1.0255 in control group). These differences were not considered to be test substance-related because the control group value appears to be high in comparison with the Testing Facility historical control range (range of 0.8042 to 1.0220).

NEUROBEHAVIOUR

Exposure to the test substance at doses as high as 450 mg/kg/day to male and female rats did not affect any of the parameters evaluated in the functional observational battery (FOB). There was a statistically significant increase (p≤0.05) in the total number of movements observed during the motor activity evaluation performed at the end of the dose period for the female rats at 450 mg/kg/day.

ORGAN WEIGHTS

At the completion of the dose period (DS 29), there was a statistically significant reduction (p≤0.01) in terminal body weights in the male rats at 450 mg/kg/day in comparison to the control group value. Terminal body weights in the male rats at 450 mg/kg/day remained reduced relative to the control group value during the recovery period. Organ weights (absolute and relative) were unaffected by up to 450 mg/kg/day of the test substance. All other statistically significant findings were considered unrelated to the test substance because: 1) the changes were not dosage-dependent; and/or 2) the observation did not persist during the recovery period; and/or 3) the observation was associated with the reduction in body weight.

GROSS PATHOLOGY

There were no test substance-related necropsy observations apparent at the completion of the dose period as well as the recovery period. All necropsy observations were considered unrelated to the test substance because the observations occurred in single rats in the dose groups.

HISTOPATHOLOGY: NON-NEOPLASTIC

No test substance-related microscopic findings were noted on study. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals.

Effect levels

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: At the end of the dose period, changes in serum chemistry that were considered to be test substance-related included a decrease in aspartate aminotransferase and an increase in triglyceride in the male and female rats at 150 and 450 mg/kg/day.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The no-observed-adverse-effect-level (NOAEL) for systemic toxicity produced by oral exposure to the test substance is considered to be at least 50 mg/kg/day for male and female rats. At 150 and 450 mg/kg/day, changes in clinical chemistry parameters were observed in both the male and female rats. In the 450 mg/kg/day dose group, there were also adverse clinical signs observed in both the male and female rats. In the male rats at 450 mg/kg/day, the body weights, body weight changes and food consumption values were reduced throughout the dose period.
Executive summary:

Executive Summary

 

The objectives of this study were to determine the potential toxicity of EC 202-228-8 (test substance), when given orally via gavage for 28 days to rats, to evaluate the potential reversibility of any findings.

The study design was as follows as detailed in Text Table1:

Text Table1
Experimental Design

Group No.

Number of Rats per Sex

Test Material

Dose Level

(mg/kg/day)

Concentration

(mg/mL)

Dose Volume

(mL/day)

Main Study

Recovery Portion

1

10

5

Corn Oil

0

0

5

2

10

-

Test Substance

50

10

5

3

10

-

Test Substance

150

30

5

4

10

5

Test Substance

450

90

5

- = Not applicable

Rats were administered the test substance and/or the vehicle, corn oil, once daily by oral gavage from Days 1 through 28 of study. The dose volume was 5 mL/kg and doses were based on the most recent body weights recorded prior to dose administration and given at approximately the same time each day.

The following parameters and end points were evaluated in this study: viability, clinical signs, body weights, body weight changes, food consumption, functional observational battery, motor activity, clinical pathology, gross necropsy findings, histology and histopathology.

On Day 29 of study (main study) or Day 43 of study (recovery portion), all surviving rats were anesthetized under isoflurane/oxygen and following blood collection from the inferior vena cava, were subsequently euthanized by an injection of sodium pentobarbital into the inferior vena cava. Examination for gross lesions, and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. 

No deaths related to the test substance occurred. One male rat in the 450 mg/kg/day dose group was found dead on Day 10 of study (DS 10). Necropsy revealed that all lobes of the lungs were mottled red and dark red. Based on the necropsy observation, this death was considered to be the result of an intubation error. 

There was an increase or statistically significant increase in the number of male and female rats in the 450 mg/kg/day dose groups observed with excess salivation (slight, moderate or extreme) and low carriage. There was also an increase in the number of male and female rats observed with mild or moderate dehydration and the number of male rats observed with urine-stained abdominal fur. 

In the male rats at 450 mg/kg/day, there were statistically significantly reduced mean body weights and body weight gains observed at all intervals during the dose period. Absolute and relative food consumption values were also reduced or statistically significantly reduced in the male rats at 450 mg/kg/day at all intervals during the dose period. 

At the end of the dose period, changes in serum chemistry that were considered to be test substance-related included a decrease in aspartate aminotransferase and an increase in triglyceride in the male and female rats at 150 and 450 mg/kg/day. There was an increase in glucose observed in the male and female rats at 450 mg/kg/day. In the male rats at 450 mg/kg/day, there was also an increase in the albumin/globulin ratio and the total bilirubin observed at the end of the dose period. These changes in serum chemistry had resolved by the end of the recovery period. 

Doses of the test substance as high as 450 mg/kg/day for 28 consecutive days did not cause any findings during the FOB and motor activity evaluations on DS 28. Likewise, the urinalysis performed at the end of dose period for both male and female rats showed no adverse effects. There were no adverse gross lesions observed at necropsy, effects on the organ weights, hematology or gross or microscopic histopathologic changes that were attributed to exposure to the test substance.   

The no-observed-adverse-effect-level (NOAEL) for systemic toxicity produced by oral exposure to the test substance is considered to be at least 50 mg/kg/day for male and female rats.