Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-03-06 to 2008-10-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Followed acceptable guidelines and GLP requirements.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
according to
Guideline:
other: US OPPTS 870.3050
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): SD10
- Physical state: Clear, colorless liquid
- Analytical purity: 97.17%
- Lot/batch No.: 9147-192-3a
- Expiration date of the lot/batch:
- Stability under test conditions: Formulations in arachis oil were stable for 14 days
- Storage condition of test material: Neat material room temperature in the dark. Formulations were prepared weekly and stored at 4 degrees C in the dark.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan
- Age at study initiation: 6 to 8 weeks at study start
- Weight at study initiation: Females 106 to 143 grams; males 136 to 174 grams
- Housing: solid floor polypropylene cages with stainless steel mesh lids, softwood flake bedding
- Diet (e.g. ad libitum): ad libitum; Rodent 2014C Teklad certified global diet
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 to 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2 degrees C
- Humidity (%): 55 +/- 15%
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

IN-LIFE DATES: From: 06 March 2008 To: 16 October 2008 Dosing 02 May to 29 May 2008

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Remarks:
Arachis Oil BP
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Solutions were prepared in arachis oil. Analytical development work showed that the formulations were stable for 14 days. Thus, dosing formulations were prepared weekly and stored at 4 degrees C until use. Analysis of dosing formulations showed that they were within +/- 10% of nominal concentrations.



VEHICLE
- Justification for use and choice of vehicle (if other than water): Material poorly water soluble. Arachis oil produced acceptable formulations.
- Concentration in vehicle: Nominal: 0, 1.29 mg/l, 2.58 mg/l, 5.15 mg/l
- Amount of vehicle (if gavage): 4 ml/kg/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration of SD10 in test material formulations was determined by gas chromatography using an external standard technique.
Samples: formulations were extracted with acetonitrile to give a final theoretical test material concentration of 0.01 mg/l.
Standards: Standard solutions of test material were prepared in acetonitrile at a nominal concentration of 0.01 mg/l.
Procedure: Standard and ample solutions were analyzed by gas chromatography using the following conditions:
GC System Agilent Technologies 5890, incorporating autosampler and workstation
Column DB-1 (30 m x 0.32 mm id x 0.25 um film
Oven temperature program Initial 150 degrees C for 0 minutes
Rate: 10 degrees C/min
Temp 200 degrees C for 1 minute
Rate 50 degrees C/ minutes
Final 325 degrees C for 10 minutes
Injection temperature 300 degrees C
Flame ionization detector
temperature 300 degrees C
Injection volume 1 ul
Retention time About 4 minutes

Stability of test material formulations: Test formulations were sampled and analyzed initially, and after storage at about 4 degrees C in the dark for fourteen days. Test concentrations analyzed were 0.25, 6.25, and 25 mg test article /l (nominal). Initial analysis concentrations were found to be 0.272 mg/l, 6.44 mg/l, and 26.1 mg/l test article in formulation. After 14 days, the concentrations were found to be 94%, 101% and 99% of initial concentrations.

Verification of concentration of weekly test formulations: test material formulations were sampled and analyzed within 4 days of preparation.

Nominal Wk 1 Wk 2 Wk 3 Wk 4 Wk 5
(mg/l) % nominal % nominal % nominal % nominal % nominal
0 - - - - -
1.29 96 95 96 95 99
2.58 97 98 103 98 103
5.15 103 103 106 103 110

Duration of treatment / exposure:
Single gavage daily for 28 consecutive days
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 (vehicle) and 20 mg/kg/day
Basis:
other: active ingredient gavaged, recovery group
Remarks:
Doses / Concentrations:
5, 10, or 20 mg/kg/day
Basis:
other: active ingredient gavaged, neuropathology group
Remarks:
Doses / Concentrations:
0 (vehicle), 5, 10, 20 mg/kg/day
Basis:
other: active ingredient gavaged, 28 day study
No. of animals per sex per dose:
5 males/5 females per group. 4 groups in the 28 day component, 2 recovery groups, and an additional 3 groups for the neuropathological evaluation
Control animals:
yes
Details on study design:
- Dose selection rationale: based on mortality and clinical signs in preliminary up and down study (all dead at 2000 mgkg, and two animals dead at
550 mg/kg/day.
- Rationale for animal assignment: random assignment
- Rationale for selecting satellite groups: Recovery from clinical signs and pathology; special assessment of neurotoxicity. Animals assigned randomly at beginning of study
- Post-exposure recovery period in satellite groups: 14 days in recovery group.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: immediately before dosing, immediately post dosing, and one to 5 hours after dosing during work week. On weekends and public holidays, animals were observed immediately before and after dosing and within 1 hour after dosing. During the recovery period, animals were observed morning and afternoon (once daily on weekends and holidays).
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: Individual weights recorded day 1 and at weekly intervals. Also performed prior to termination, and in the case of recovery animals, on days 36, and 43 prior to termination.

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


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 / No / No data

WATER CONSUMPTION: Yes
- Time schedule for examinations: observed daily by visual inspection, except for week 3 where it was determined gravimetrically

OPHTHALMOSCOPIC EXAMINATION: Yes / No / No data ?????
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of 28 day treatment in non-recovery group; at end of recovery in recovery group (day 42)
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all non-recovery main test and control animals; all surviving recovery group animals at end of day 42
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of 28 day treatment in non-recovery group; at end of recovery in recovery group
- Animals fasted: No
- How many animals: all non-recovery main animals and control animals, all surviving recovery group animals


URINALYSIS: Yes
- Time schedule for collection of urine: During week 4 for all main non-recovery animals and controls and week 6 for all recovery group animals
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to start of treatment and weekly thereafter, about two hours after dosing
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity: Yes

See Materials and Methods section for examinations performed
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table) in results section
HISTOPATHOLOGY: Yes (see table) in attachments
Other examinations:
Neuropathology group were subjected to whole body perfusion and weighing of brain tissue. Neural tissue from this subgroup were subjected to neuropathological examination. See table in materials and methods for tissues examined
Statistics:
Data processed to give group mean values and standard deviations where appropriate. With the exception of pathology, all data were summarized in tabular form. Where appropriate, quantitative data were analyzed by Provantis Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett's Test. The transformed data were analyzed to find the lowest treatment level that showed a significant effect using Williams Test for parameteric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analyzed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from control group. Finally, if required, pair-wise tets were performed using the Student t-test (parametric) or Mann-Whitney U test (non-parametric). Probability values (p) are presented as follows: p < 0.01 ** p < 0.05 * and p =/> 0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1 high dose female sacrificed in extremis day 27. Other high dose animals had some salivation directly after dosing on days 25 or 27.
Mortality:
mortality observed, treatment-related
Description (incidence):
1 high dose female sacrificed in extremis day 27. Other high dose animals had some salivation directly after dosing on days 25 or 27.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
High dose males and females had reduced gains from week 2 through end. Mid dose males also showed reduced gains between day 15 and 29.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
High dose animals had reduced food consumption from week 2 to end of treatment. This reversed in recovery period.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Decreased food consumption, high dose males and females, weeks 2-4. Increased in recovery period.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Decreased in high dose males and females weeks 3,4. Increased in recovery period.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
High dose males, and to lesser degree, females had < urine volume and > SG.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
See details on results
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
High dose males had decreased liver weights. Females (high and mid dose) had increased kidney weights
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Heart, lungs, liver, kidneys, adrenals, lymph nodes
Details on results:
CLINICAL SIGNS AND MORTALITY: One female treated with 20 mg/kg/day was killed in extremis on Day 27. There were no further unscheduled deaths.
Clinical Observations: The high dose female rat that was killed in extremis displayed pilo-erection from Day 20; from Day 25, tiptoe gait, hunched posture, and dehydration were evident. By Day 27, the animal was emaciated and hypothermic. Incidents of increased salivation were reported for male and female high dose rats on Day 25 and 27. Red stains around the nose were observed in one male and one female on day 22 and one female on day 23. All clinical findings were confined to high dose animals. No clinically observed signs of toxicity were noted in other animals in the study.

BODY WEIGHT AND WEIGHT GAIN: No effects on weight gain were noted in low or middose females. In the high dose group, reduced gains were seen in both sexes from week 2 until the end of treatment. Middose males also showed reduced body weight gains while low dose males only showed a reduced body weight gain between day 16 and 22, but normal weight gain before and after this interval. Body weight gains increased during the recovery period.

FOOD AND WATER CONSUMPTION AND COMPOUND INTAKE (if feeding study) Reduced dietary intake was seen in both sexes in high dose groups during week 3 and 4 of the study. This reversed in recovery animals. No adverse effects on water consumption were seen in mid and low dose animals. Slight reduction in water consumption in the high dose groups were seen in weeks 3 and 4, with recovery after treatment ceased.

HAEMATOLOGY: In high dose males and females statistically significant reductions in hemoglobin, hematocrit, mean cell hemoglobin and mean cell hemoglobin concentration as well as reduced platelet counts were reported. In females also red blood cell counts were statistically significantly lower than those in concurrent controls. In the mid dose animals, reduced mean cell hemoglobin was seen, with females of this group showing increases in clotting times compared to concurrent controls. However, all the numerical values of the findings reported above were within the recent historical control values of the institute and are therfore not considered treatment related.
Increased white blood cell counts, particularly neutrophils, were seen in high dose animals, and females of that group also had increased lymphocytes compared to concurrent controls. The values were also outside the historical control values of the institute and the effect is therefore considered treatment related. These increases persisted after recovery. No treatment related effects were seen in low dose animals.

CLINICAL CHEMISTRY: In all treatment groups, total protein and albumin levels were reduced in males and in high dose females compared to concurrent control levels. Low and mid dose total protein and albumine levels in males were however well within the recent historical control range of the insitute. Cholesterol levels wer decreased significantly compared to concurrent controls in males and females of all dose groups, but the low dose values were within or very close to the recent historical control range of the institute. In the high dose group, glucose levels were reduced in both sexes compared to cuncurrent controls, but within (f) or close to (m) recent historical control levels of the institute. Calcium levels in females of the mid dose group were lower than concurrent control levels. It is however noted, that all Calcium levels in this study, including those of control animals were lower than the recent historical control range of the institute. Recovery high dose males continued to have reduced total proteins, but recovery females had increases in total protein, albumin, and calcium levels. Potassium levels were increased compared to concurrent controls in animals of the high dose group, with the effect still present in recovery females. Liver enzymes (aspartate aminotransferase (m, f) and alanine aminotransferase (f) were elevated in animals of the high dose group, with decreased gamma glutamyltranspeptidase levles in males compared to concurrent controls. These levels were also outside the recent historical control data and are consistent with the histopathological findings in the liver of high dose animals. They are therefore considered treatment related. Alkaline phosphatase levels were higher in high dose females compared to concurrent controls and also outside the recent historical control range of the institute. No significant changes in liver enzymes were reported in the recovery groups indicating a reversibility of the effects.

URINALYSIS: High dose males, and to a lesser extent, females, had reduced urine volume with increased specific gravity. No changes were seen in other groups.
NEUROBEHAVIOUR: Behavioral assessment: In the weekly open-field arena observations, one high dose female showed extremity pallor, hunched posture, and piloerection in last week of study. Another high dose female showed pilo-erection and tonic convulsions in week 4. No such effects were noted in the recovery high dose animals in the recovery period. No toxicologically significant effects were detected in animals of the low and mid-dose groups.
Functional Performance: Slight reductions in motor activity were noted in mid and high dose males during week 2, and in high dose females in week 3 Overall mobile activity was decreased in high dose females during weeks 3 and 4, and in high dose males during week 4. High dose recovery males had a reduction in overall activity during week 5, but there were no differences in comparison to control values during the final week of recovery. Slight reduction in forelimb grip strength was evident for high dose males in Weeks 3 and 4, and mid dose males in week 4. Reduced forelimb grip strength was seen in high dose recovery males in the last week of recovery, although recovery females showed increased forelimb grip strength during this period. No significant effects of treatment were seen in mid dose females, or in low dose males and females.
It is likely that the observed effects can be regarded as secondary to general toxicity, in particular as no treatment related neuropathological changes were observed.

Sensory Reactivity Assessments: The high dose female euthanized in Week 4 had displayed a lower pupil reflex score. No treatment related effects were seen in other animals.

ORGAN WEIGHTS: Absolute and relative adrenal weights were significantly reduced in mid dose males and low and mid dose females compared to concurrent controls. In the absence of similar findings in the high dose group, these are not considered treatment related. High dose group females had statistcally significant increases in both absolute and relative kidney weights. Recovery group females showed decreased absolute and relative liver weihgts comparedo concurrent controls, while recovery males had statistically significant increased spleen weights compared to controls.

GROSS PATHOLOGY: Gross lesions considered to be treatment related consisted of speckled white appearance or patchy pallor of th lungs in single animals of the high dose main and recovery groups.

HISTOPATHOLOGY: NON-NEOPLASTIC: Histopathology: Organs affected by treatment were heart, lungs, liver, kidneys, thyroid glands, adrenals, and mesenteric and cervical lymph nodes. There were no indications of neurotoxicity in perfused tissue from the high dose neuropathology group.
1). Heart: High dose males, and one female in the middose group had myocardial necrosis with inflammatory infiltrate. In the recovery group one male and two females showed myocardial necrosis.
2). Lungs: Foamy macrophages along with increased alveolar wall hyperplasia were seen in the high and mid-dose groups, with increased incidence of alveolitis. Increased macrophages were also seen in the alveoli of some low dose females, but the incidence was close to that of the control group.
3). Liver: Increased hepatocellular eosinophilia was seen in the high dose animals, and in mid-dose females. Hepatocellular apoptosis was seen in high dose animals. Mild hepatocellular hypertrophy was seen in males of all groups with a low incidence in the low dose group (2/5). The recovery group animals did not show liver lesions except for one animal with minimal cellular hypertrophy.
4). Kidneys: High dose animals showed fine-granular eosinophilic cytoplasm in renal cortical tubules, and high dose females also had hyaline inclusions. Recovery group animals did not reveal any histopathological changes in the kindney, indicating full reversibility of the effects.
5). Thyroid: High and mid-dose males showed diffuse follicular thyroid hypertrophy, Recovery group animals except for one male with minimal diffuse hypertrophy did not show histopathological changes in the thyroid. The observed effects in high dose males are consistent with secondary effects of liver enzyme induction resulting from the adaptive liver hypertrophy that are frequently observed with compounds inducing liver hypertrophy.
6). Adrenals: High dose animals showed eosinophilic cytoplasm in the zona fasciculata, with increased apoptosis and minor severity degree fasciculate atrophy. High dose males showed increased fasciculate vacuolation. In the mid-dose animals, diffuse hypertrophy of the fasciculata was seen; this effect was not seen in other dose groups. One female of the mid dose group also had atrophy and apoptosis of the fasciculata. In recovery animals fasciculate atrophy was seen in one male, fasciculata apoptosis in one male and one female and fasciculata hypertrophy in one male.
7). Lymph nodes (mesenteric and cervical): High dose animals showed increased sinusoidal histiocytes, with some incidence seen in mid-dose animals. Two high dose recovery female still showed the effect.
8) Neuropathology: no evidence of neurotoxicity was noted in the tissues from neuropathology animals.



Effect levels

open allclose all
Dose descriptor:
other: No NOEL identified
Effect level:
ca. 5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
ca. 5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Tables for observations, body weights and gains,

KEY

-          =           No change NA     =           Not applicable *         =           Not examined

>         =           Statistically significant increase compared with controls (p < 0.05)

<         =           Statistically significant decrease compared with controls (p < 0.05)

>>       =           Statistically significant increase compared with controls (p < 0.01)

<<       =           Statistically significant decrease compared with controls (p < 0.01)

TI        =           Toxicologically significant increase, but no statistical analysis performed

TR     =           Toxicologically significant decrease, but no statistical analysis performed

+         =           Toxicologically significant change, but no statistical analysis performed

T>       =           Toxicologically significant increase, statistical significance not achieved

T<       =           Toxicologically significant decrease, statistical significance not achieved

( )        =           Statistically significant difference, but of no toxicological importance

[ ]        =           Spontaneous macroscopic abnormality of no toxicological importance

{ }      =           Spontaneous histopathological condition of no toxicological significance,

no difference in incidence/ severity between treated and control animals

Main Groups and Recovery Groups

Control

Low Dose

Mid-dose

High dose

Recovery Groups

Dose Level (mg/kg/day)

0 M

0 F

5 M

5 F

10 M

10 F

20 M

20 F

0 M

0 F

20 M

20 F

Mortality

Sacrificed Day 27 in extremis

-

-

-

-

-

-

-

1

NA

NA

NA

NA

General Appearance (interim death)

Dehydration

Emaciation

Hunched

Hypothermia

Pale extremities

Pilo-erection

Ptosis

Tip-toe gait

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

1

1

1

1

1

1

1

1

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

General appearance (Terminal kill)

Fur loss

> Salivation

-

-

-

-

1

-

-

-

-

-

1

-

-

6

-

4

-

-

-

-

-

-

-

Behavior (interim death)

Week 4

Skin pallor

Pilo-erection

Hunched

Transfer arousal

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

1/1

1/1

1/1

-1.0

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

Behavior (Terminal kill)

Pretest

Transfer arousal

Urination

Defecation

Week 1

Defecation

Week 2

Urination

Defecation

Week 3 Defecation

-

-

-

0.1

-

-

-

0.1

-

-

-

-

-

-

-

0.2

0.2

-

-

0.2

0.4

-

-

-

-

-

-

-

-

-

-

-

0.2

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

Control

Low Dose

Mid-dose

High dose

Recovery Groups

Dose Level (mg/kg/day)

0 M

0 F

5 M

5 F

10 M

10 F

20 M

20 F

0 M

0 F

20 M

20 F

 

Functional Performance

Pretest

Grip strength (hind)

Week 1

Grip strength (fore)

Grip strength (hind)

Activity

Week 2

Activity

Week 3

Grip strength (fore)

Grip strength (hind)

Activity

Week 4

Grip strength (fore)

Activity

Week 5

Activity

Week 6

Grip strength (fore)

-

-

-

-

-

-

-

-

-

(> )

NA

NA

-

-

-

-

-

-

-

-

-

-

NA

NA

(>)

-

-

-

<<

(<<)

-

-

<

-

NA

NA

-

-

-

-

-

-

-

-

-

-

NA

NA

(> )

(> )

(> )

-

<<

-

<

-

<<

-

NA

NA

-

-

-

(> )

-

-

-

<<

-

<<

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

<

<

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

-

> to >>

 

Sensory Reactivity (interim death)

Week 2

Vocalization

Week 4

Pupil reflex

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

1.0

-2.0

NA

NA

NA

NA

NA

NA

NA

NA

 

Control

Low Dose

Mid-dose

High dose

Recovery Groups

Dose Level (mg/kg/day)

0 M

0 F

5 M

5 F

10 M

10 F

20 M

20 F

0 M

0 F

20 M

20 F

 

Sensory Reactivity (Terminal kill)

Week –1

 Vocalization

Week 1

 Vocalization

Week 2

 Vocalization

Week 3

 Vocalization

Week 4

 Vocalization

Week 5

 Vocalization

Week 6

 Vocalization

0.2

-

0.2

0.1

0.1

NA

NA

0.4

0.2

0.4

0.1

-

NA

NA

-

-

-

-

-

NA

NA

0.2

-

0.4

-

0.2

NA

NA

0.2

-

0.2

-

-

NA

NA

0.8

0.4

0.4

-

-

NA

NA

0.2

-

-

-

-

NA

NA

0.7

0.1

0.6

0.1

-

NA

NA

NA

NA

NA

NA

NA

0.4

-

NA

NA

NA

NA

NA

0.2

-

NA

NA

NA

NA

NA

-

0.2

NA

NA

NA

NA

NA

0.8

0.5

 

Body Weight Gains

Week 2

Week 3

Week 4

Week 5

Week 6

Food Consumed

Week 2

Week 3

Week 4

Week 5

Week 6

-

<

-

NA

NA

-

-

-

NA

NA

-

-

(<)

NA

NA

-

-

-

NA

NA

-

<

<<

NA

NA

-

-

-

NA

NA

-

-

(<)

NA

NA

-

-

-

NA

NA

<<

<<

<<

NA

NA

TR

TR

TR

NA

NA

T<

<

<

NA

NA

TR

TR

TR

NA

NA

NA

NA

NA

 >

 >

NA

NA

NA

TI

TI

NA

NA

NA

 >

 -

NA

NA

NA

TI

TI

 

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Applicant's summary and conclusion

Conclusions:
No mortality, except for one animal in the high dose group, was seen in this repeated dose oral gavage 28 day study in rats. Treatment related adverse effects were noted in the high and mid dose groups (20 and 10 mg/kg bw/day). The most prominent effects observed were inflammatory changes in the lungs and changes in the adrenals including fascicular hypertrophy, apoptosis and atrophy. These two organs were already affected at the 10 mg/kg/day dose level. At the high dose level additional changes in the kidney, liver and thyroid became apparent that were also accompanied by corresponding changes in clinical chemistry (e.g. increases in ALAT and ASAT, increased white blood cell counts and neutrophiles). Some adaptive changes that were mostly within the physiological range were already observed at the low dose, but are not considered adverse. A NOAEL of 5 mg/kg bw/d was therefore derived from this study.
Executive summary:

In a guideline conform 28-day study (OECD 407, GLP) the test item was administered by oral gavage in arachis oil to groups of 5 Wistar Han rats per sex at doses of 20, 10 and 5 mg/kg/day. A control group of 5 male and 5 female rats was dosed in the same way with the vehicle. Two recovery groups of 5 animals per sex dosed with either 20 mg/kg/d of the test item or the vehicle were included. In addition to these groups, three additional groups for neuropathological evaluation consisting of 5 animals per sex per group dosed at 5, 10 and 20 mg/kg bw/d and a vehicle control were included. After the 28 -day exposure period these animals were subjected to gross necropsy followed by whole body perfusion and neuropathological examination of the neural tissues.

Results:

Mortality: One female in the high dose group was sacrificed in extremis on day 27. No other unscheduled deaths occurred.

Clinical Observations: The sacrificed high dose female showed piloerection from day 20, tiptoe gait, hunched posture and dehydration from day 25, and emaciation and hypothermia on the day sacrificed. Other animals in the high dose group had scattered incidents of increased salivation on days 25 to 27 that was considered within normal variation for animals of this strain and age. No toxicologically significant clinical signs of toxicity were noted in the low or mid dose animals.

Behavioral assessment: No toxicologically significant adverse effects were noted in high dose recovery groups, or in the low and mid dose treated animals. The sacrificed female in the high dose group was noted to have the aforementioned clinical symptoms in the last week of treatment. One other female in the group showed piloerection, and tonic convulsions in the last week of treatment. Differences in urination, defecation and transfer arousal scores were considered to be within normal variation for the strain and age of animals.

Functional performance: Slightly reduced motor activity were noted in mid dose and high dose males in week 2, and in high dose females in week 3. Overall reduction in mobile activity was noted in high dose females in week 3 and 4. Reduced activity was present in the high dose recovery males in week 5, but not in the final week of recovery. Slight reduction in forelimb grip strength was seen in high dose males in weeks 3 and 4, with the effect seen in mid dose males in week 4. The recovery high dose males still had reduced forelimb grip strength in the last week of recovery, but females showed an increase in grip strength during this time. No significant effects were seen in mid dose females, or in any animals in the low dose group. No histopathology correlates were seen for the changes in functional performance, and in the absence of a convincing dose-response, the differences in functional performance are not likely to be of toxicological concern.

Sensory reactivity: Other than a lower pupil reflex score in the last week in the sacrificed high dose female, there were no treatment related effects. 

 

Body weight: No toxicologically significant effects on weight gain were noted in low or mid dose females. In the high dose group, reduced gains were seen in both sexes from week 2 until the end of treatment.  Mid dose males also showed reduced body weight gains while low dose males only showed a reduced body weight gain at between day 16 and 22, but normal weight gain before and after this interval. Body weight gains increased during the recovery period.

 

Food and water consumption: High dose animals had reduced food consumption from week 2 until end of treatment, although this was not analyzed statistically. The food consumption compared to control animals was increased in the animals during the recovery period. No adverse effects on food consumption were seen in other groups. Slight reduction in water consumption in the high dose groups were seen in weeks 3 and 4, with recovery of water consumption after treatment ceased. No effects on water consumption were seen in the low or mid dose animals.

Hematology:

The only clearly treatment related effects were increased white blood cell counts, particularly neutrophils, seen in high dose animals and increased lymphocyte counts in females of the high dose group compared to concurrent controls. The values were also outside the historical control values of the institute. These increases persisted in the recovery group. No treatment related effects were seen in low dose animals.

 

CLINICAL CHEMISTRY:Total protein and albumin levels were reduced in high dose males and females compared to concurrent control levels and outside historical control values. Cholesterol levels wer decreased significantly compared to concurrent controls in males and females of all dose groups, but the low dose values were within or very close to the recent historical control range of the institute. Recovery high dose males continued to have reduced total proteins, but recovery females had increases in total protein and albumin levels. Potassium levels were increased compared to concurrent controls in animals of the high dose group, with the effect still present in recovery females. Liver enzymes (aspartate aminotransferase (m, f) and alanine aminotransferase (f) were elevated in animals of the high dose group, with decreased gamma glutamyltranspeptidase levels in males compared to concurrent controls. These levels were also outside the recent historical control data and are consistent with the histopathological findings in the liver of high dose animals. They are therefore considered treatment related. Alkaline phosphatase levels were higher in high dose females compared to concurrent controls and also outside the recent historical control range of the institute. No significant changes in liver enzymes were reported in the recovery groups indicating a reversibility of the effects.

Urinalysis: High dose males, and to a lesser extent, females, had reduced urine volume with increased specific gravity. No changes were seen in other groups. No treatment related effects were seen in the low dose group or in recovery animals.

Organ weights: High dose group females had statistically significant increases in both absolute and relative kidney weights. Recovery group females showed decreased absolute and relative liver weights compared to concurrent controls, while recovery males had statistically significant increased spleen weights compared to controls.

Gross Pathology: Gross lesions considered to be treatment related consisted of speckled white appearance or patchy pallor of the lungs in single animals of the high dose main and recovery groups.

Histopathology: Target organs were adrenals, lungs, heart, liver, kidneys, thyroid glands ,and mesenteric and cervical lymph nodes. There were no indications of neurotoxicity in perfused tissue from the high dose neuropathology group.

Adrenals: High dose animals showed eosinophilic cytoplasm in the zona fasciculata, with increased apoptosis and minor severity degree fasciculate atrophy. High dose males showed increased fasciculate vacuolation. In the mid-dose animals, diffuse hypertrophy of the fasciculata was seen; this effect was not seen in other dose groups. One female of the mid dose group also had atrophy and apoptosis of the fasciculata. In recovery animals fasciculate atrophy was seen in one male, fasciculata apoptosis in one male and one female and fasciculata hypertrophy in one male.

Lungs: Foamy macrophages along with increased alveolar wall hyperplasia were seen in the high and mid-dose groups, with increased incidence of alveolitis. Increased macrophages were also seen in the alveoli of some low dose females, but the incidence was close to that of the control group.

Heart: High dose males, and one female in the middose group had myocardial necrosis with inflammatory infiltrate. In the recovery group one male and two females showed myocardial necrosis.

Liver: Increased hepatocellular eosinophilia was seen in the high dose animals, and in mid-dose females. Hepatocellular apoptosis was seen in high dose animals. Mild hepatocellular hypertrophy was seen in males of all groups with a low incidence in the low dose group (2/5). The recovery group animals did not show liver lesions except for one animal with minimal cellular hypertrophy.

Kidneys: High dose animals showed fine-granular eosinophilic cytoplasm in renal cortical tubules, and high dose females also had hyaline inclusions. Recovery group animals did not reveal any histopathological changes in the kindney, indicating full reversibility of the effects.

 Thyroid: High and mid-dose males showed diffuse follicular thyroid hypertrophy, Recovery group animals except for one male with minimal diffuse hypertrophy did not show histopathological changes in the thyroid. The observed effects in high dose males are consistent with secondary effects of liver enzyme induction resulting from the adaptive liver hypertrophy that are frequently observed with compounds inducing liver hypertrophy.

Lymph nodes (mesenteric and cervical): High dose animals showed increased sinusoidal histiocytes, with some incidence seen in mid-dose animals. Two high dose recovery female still showed the effect.