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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 April 2001 - 24 May 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted to GLP and the methodology followed the relevant test guidelines of OECD and US EPA

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Principles of method if other than guideline:
The study report does not state the study was conducted in accordance with a particular published guideline, but OECD Test Guideline 407 (1995) and EPA OPPTS 870.3050 and 870.6200 are listed in the references and the reported study methodology does follow these guidelines
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):
- Physical state: Liquid
- Analytical purity: The test substance, as received, was assumed to be the pure substance.
- Lot/batch No.: NDA
- Expiration date of the lot/batch: Dec 2005
- Storage condition of test material: Room temperature
- Other:

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
- Age at study initiation: Males, 7 weeks old; females, 7-8 weeks old.
- Weight at study initiation: Males, 207-255 g; females, 165-208 g.
- Fasting period before study:
- Housing: Individually housed in suspended stainless steel and wire mesh cages.
- Diet (e.g. ad libitum): Ad libitum.
- Water (e.g. ad libitum): Ad libitum.
- Acclimation period: 13-15 days.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 64-72 ºF (17.7-22.2ºC)
- Humidity (%): 30-70%.
- Air changes (per hr): Not stated.
- Photoperiod (hrs dark / hrs light): Approximately 12 hours light / 12 hours dark.


IN-LIFE DATES: From: 2 May 2001 To: 1 June 2001.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Primol 542
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Fresh dosing solutions were prepared weekly.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Not stated.
- Concentration in vehicle: 0, 1, 3 and 10% w/v.
- Amount of vehicle (if gavage): 5 mL/kg.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Representative test substance mixtures that bracketed the concentrations used for the study were analysed for uniformity and stability prior to the experimental start date. Concentration verification was performed during Week 1 and Week 4.
Duration of treatment / exposure:
28 days.
Frequency of treatment:
Daily.
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 150, 500 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A rangefinding study using doses of 50, 125, 250, 500 and 1000 mg/kg/day.
- Section schedule rationale (if not random): Not stated.
Positive control:
None.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
The animals were examined for viability twice daily. All animals also were observed daily for signs of toxicity as well as the nature, onset, severity, and
duration of these effects.


DETAILED CLINICAL OBSERVATIONS: Yes
Once during Weeks 1, 2, and 3, all animals were observed in a standard arena for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture, and response to handling as well as the presence of clonic or tonic movements, stereotypies or unusual behavior also were recorded.

Functional observational batteries (FOBs) were conducted on all animals of each group prior to dose initiation and during Week 4. The FOB consisted of a series of observations and measurements designed to detect behavioral and neurological effects and followed closely the procedures described by Moser et al., (1989, 1991) and the USEPA /AIHC training video and reference manual for FOB (1996). Based on the results of the rangefinding study, the FOBs were performed no earlier than two hours after the end of dosing, which was determined to be the time of maximum effect. Motor activity was assessed for all animals prior to dose initiation and during Week 4. Motor activity was assessed using an automated activity recording apparatus.


BODY WEIGHT: Yes
Body weights were recorded prior to initiation of dosing for group allocation. Body weights also were recorded for all animals on the day of initiation of dosing (Day 0), and on Days 7, 14, 21, and 27, and on the day of death. Body weights were also recorded on Day 28 for the males selected for neuropathology.


FOOD CONSUMPTION: Yes.
Food consumption was measured on Days 7, 14, 21, and 27 (Weeks 1,2, 3, and 4) for all animals. The Week 4 food consumption value was a 6-day value due to the fasting of the animals on Day 27 for blood collection on Day 28.


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
Haematology and clotting potential were performed on all main study animals and certain female neuropathology animals on Day 28. Blood samples were collected from the abdominal aorta of all males and from the retro-orbital sinus of the females while under sodium pentobarbital anesthesia following an overnight fast from food. Haemotology samples were collected in tubes containing EDTA. Clotting potential smaples were collected into tubes containing sodium citrate and placed on ice. The following parameters were examined:
Haematocrit; Haemoglobin; Erythrocyte count; leukocyte count (total and differential); platelet count; reticulocyte count*; Mean corpuscular volume; Mean corpuscular haemoglobin; Mean corpuscular haemoglobin concentration; Prothrombin time; Thromboplastin time.
* Slides were prepared for all animals; slides were not evaluated since other RBC parameters were normal.


CLINICAL CHEMISTRY: Yes
Serum chemistry was performed on all main study animals and certain female neuropathology animals on Day 28. Blood samples were collected from the abdominal aorta of all males and from the retro-orbital sinus of the females while under sodium pentobarbital anesthesia following an overnight fast from food. Serum chermistry samples were collected in tubes without an anticoagulant. The following parameters were measured:
Albumin; Urea nitrogen; Calcium; Creatinine; Electrolytes (Na+, Cl-, K+); Glucose; Phophorus; Gamma glutamyl transpeptidase; Serum alanine aminotransferase; Serum aspartate aminotransferase; Serum alkaline phosphatase; Total protein; total bilirubin; Cholesterol; Triglycerides.


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
Once during Weeks 1, 2, and 3, all animals were observed in a standard arena for changes including autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture, and response to handling as well as the presence of clonic or tonic movements, stereotypies or unusual behavior also were recorded.

Functional observational batteries (FOBs) were conducted on all animals of each group prior to dose initiation and during Week 4. The FOB consisted of a series of observations and measurements designed to detect behavioral and neurological effects and followed closely the procedures described by Moser et al., (1989, 1991) and the USEPA /AIHC training video and reference manual for FOB (1996). Based on the results of the rangefinding study, the FOBs were performed no earlier than two hours after the end of dosing, which was determined to be the time of maximum effect. Motor activity was assessed for all animals prior to dose initiation and during Week 4. Motor activity was assessed using an automated activity recording apparatus.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

NEUROPATHOLOGY:
The animals scheduled for neuropathology were perfused in situ with paraformaldehyde and glutaraldehyde. The following neuropathology were performed on five animals/sex from the control and high dose groups:

- Paraffin embedded tissues
• sections of the four areas of the brain stained with hematoxylin and eosin (H&E)
• transverse and longitudinal sections of the cervical spinal cord stained with H&E
• transverse and longitudinal sections of the lumbar spinal cord stained with H&E
• a section of the biceps femoris stained with H&E
• sections of the four areas of the brain stained with Luxol Fast Blue/Periodic Acid-Schiffs (LFB/PAS)
• transverse and longitudinal sections of the cervical spinal cord stained with LFB/PAS
• transverse and longitudinal sections of the lumbar spinal cord stained with LFB/PAS
• sections of the four areas of the brain stained with glial fibrillary acidic protein (GFAP)
• transverse and longitudinal sections of the cervical spinal cord stained with GFAP
• transverse and longitudinal sections of the lumbar spinal cord stained with GFAP

- Glycol methacrylate embedded tissues
• long and cross sections of the proximal sciatic stained with H&E
• sections of the distal sciatic, tibial branch, sural nerve, cervical dorsal root and ganglion, cervical ventral root, lumbar dorsal root and ganglion, lumbar ventral root, and trigeminal (Gassarian) ganglion stained with H&E
Statistics:
Body weight, organ weight, food consumption and clinical chemistry:
Bartlett’s Test for equal variance. For parametric procedures: one-way ANOVA and standard regression analysis for linear response; Dunnett’s Test. For non-parametric procedures: Kruskal-Wallis Test for equality of means; if significant differences of means, Dunn’s Summed Rank Test was used to determine which groups differed from the control; Jonckheere’s Test for monotonic trend in the dose-response. Bartlett’s Test was conducted at the 1% level of significance; all other tests were conducted at the 5% and 1% levels of significance.

Functional observational data:
One-way ANOVA for continuous variables; residuals tested for normality using the Shapiro-Wilk test; those not exhibiting normally distributed residuals were transformed by Blom’s normalized rank transformation and reanalysed. Nominal and count data were analysed by cumulative logit repeated measures analysis.

Motor activity data:
Analysis used a profile repeated measures analysis with post-hoc tests to test the polynomial response pattern. Blom’s transformation was used to achieve normal distribution of residuals. Residuals tested using Shapiro-Wilk test for normality.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
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:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY
Possible treatment-related effect in females at 150 and 500 mg/kg/day.
Six animals died during the study: 4 high-dose females, 1 high-dose male, and 1 mid-dose female. Four of the 5 high-dose fatalities were attributed to dosing trauma. The high-dose animals became more difficult to dose as the study progressed and this resistance to being dosed was considered to have contributed to these deaths.


CLINICAL SIGNS
No biologically significant treatment-related effects seen.
In-life clinical signs were minimal and not considered related to test material toxicity.


BODY WEIGHT AND WEIGHT GAIN
Effects seen in females at 150 and 500 mg/kg/day.
Statistically significantly increased bodyweight was seen in high-dose females at Days 14, 21 and 27, and mid-dose females on Day 21 only. Statistically significantly increased bodyweight gains were also found for the high-dose females for the intervals Day 0–7 and 7–14. Other bodyweight findings were transient and not considered treatment-related.


FOOD CONSUMPTION
Effects seen in females at 150 and 500 mg/kg/day.
Statistically significantly increased food consumption was seen in high-dose females during Weeks 2, 3 and 4, and mid-dose females during Week 3 only. These increases coincide with the increases in bodyweight.


HAEMATOLOGY
No biologically significant treatment-related effects seen.
There were statistically significant changes in two parameters, but these either did not demonstrate a dose-related trend or were not supported by other related parameters.


CLINICAL CHEMISTRY
No biologically significant treatment-related effects seen.
There were statistically significant changes in several parameters, but these either did not demonstrate a dose-related trend or were not supported by other related parameters or histopathology.


NEUROBEHAVIOUR
Effects seen in females at 150 and 500 mg/kg/day.
Landing foot splay was statistically significantly decreased in mid- and high-dose females, and rectal temperature was also statistically significantly decreased in the high-dose females. There were no statistically significant differences in mean motor activity data.


ORGAN WEIGHTS
Effects seen in females at 500 mg/kg/day.
Statistically significant increases in absolute and relative liver and adrenal weights, and absolute heart weight, were seen in high-dose females. Absolute liver weight was also increased in mid-dose females. The increased liver weight was not considered a biologically significant adverse effect of treatment due to the absence of histopathology findings.


GROSS PATHOLOGY
No biologically significant treatment-related effects seen.
There was no gross pathology that appeared to be treatment-related.


HISTOPATHOLOGY: NON-NEOPLASTIC
Effects seen in females and males at 150 and 500 mg/kg/day.
Adrenal Dose-related increases in cortical vacuolation and hypertrophy were seen in mid- and high-dose animals of both sexes.
Kidneys An increased incidence of increased hyaline droplets was seen in mid- and high-dose males.
Stomach Gastric erosion was seen in one high-dose animal of each sex; forestomach submucosal oedema and inflammation was seen at the high-dose in two males and one female. The study authors considered these effects may have been related to treatment.


HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No biologically significant treatment-related effects seen.
No neoplastic histopathology was reported.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The reported animal housing temperature range for this study (17.8–22.2ºC) is slightly lower than that recommended by OECD (19–25 ºC), but this is not expected to have had any impact on the study.

Bile acids were not measured as part of clinical chemistry and Prostate glands and seminal vesicles with coagulating glands were not weighed at necropsy. These measures were not requirements of the 1995 OECD TG 407, which was current at the time of the study, but are included in the 2008 revised guideline.

Oral administration of the test substance to CD rats produced treatment-related microscopic changes in the adrenal glands of the male and female rats and kidneys of the male rats of the 150 and 500 mg/kg/day groups. Microscopic changes also were noted in the stomach of the male and female rats of the 500-mg/kg/day group and these changes were possibly treatment related. The dosing problems encountered in the 500 mg/kg/day animals probably were related to the changes in the stomach.

The Functional Observational Battery data revealed a statistically significant decrease in landing foot splay in the 150 and 500 mg/kg/day females and a statistically significant decrease in rectal temperature in the 500 mg/kg/day females. However, there were no microscopic changes in the brain, spinal cord, skeletal muscle, peripheral nerves, spinal nerve roots/ganglia and trigeminal ganglion that were considered treatment-related.

Oral administration of the test substance to CD rats did not cause any biologically significant changes in the clinical pathology data or general clinical observations. There was an increase in the adrenal weight parameters in the 500 mg/kg/day females that appears to correlate with the microscopic findings of the adrenals.

Applicant's summary and conclusion

Conclusions:
Oral administration of the test substance to rats by gavage produced treatment related microscopic changes in the adrenal glands of the male and female rats and kidneys of the male rats of the 150 and 500 mg/kg/day groups. Microscopic changes also were noted in the stomach of the male and female rats of the 500 mg/kg/day group and these changes were possibly treatment related. There also was a statistically significant decrease in landing foot splay in the 150 and 500 mg/kg/day females and a statistically significant decrease in rectal temperature in the 500 mg/kg/day females. Therefore the No Observable Effect Level (NOEL) and No Observable Adverse Effect Level (NOAEL) for this study were established at 150 mg/kg/day.
Executive summary:

The test material was investigated for subacute oral toxicity and neurotoxicity/neuropathology in a GLP study conducted in accordance with OECD Test Guideline 407 (1995). Four groups of 10 male and 10 female CD Sprague-Dawley rats were administered the test material (in Primol 542 carrier) by daily gavage at doses of 0, 50, 150 and 500 mg/kg/day for 28 days. The dose volume used was 5 mL/kg body weight. In addition to the observation and measurement of standard parameters, a complete functional observational battery (FOB) was conducted on all animals prior to dosing and during Week 4 of dosing. Additionally, once during Weeks 1, 2 and 3, all animals were observed in a standard arena. Motor activity was assessed using a photobeam activity system at the same intervals as the FOB. Neuropathology was additionally examined in five animals per sex form the control and high-dose groups.

One mid-dose and five high-dose animals died during the study. Four of the high-dose deaths were attributed to dosing trauma. The incidence of clinical in-life observations was minimal and did not appear treatment-related.

Statistically significant increases in body weight, weight gain and/or food consumption were seen in high-dose females and, in Week 3 only, mid-dose females

No treatment-related gross pathology was observed, but microscopic changes were seen variously in the mid- and high-dose groups in the adrenals, kidneys, and stomach/forestomach. The gastric effects in the high-dose were likely to have caused the resistance of these animals to dosing, which led to the dosing trauma. The effects on the adrenals were accompanied by an increased organ weight.

The FOB identified decreased landing foot splay in the mid- and high-dose females, and also decreased rectal temperature in the high-dose females. Microscopic examination of the nervous system revealed no treatment-related changes.

Therefore, the no observable adverse effect level (NOAEL) for this study is 150 mg/kg/day, and the no observable effect level (NOEL) is 50 mg/kg/day. Principal treatment-related effects occurring at higher doses include effects on the adrenals, kidneys, stomach, as well as rectal temperature and landing foot splay.