Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 July 1993 to 25 September 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

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:
Physical state: Dark brown viscous liquid at 20 °C
Storage conditions: Ambient

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Rat is the preferred species of the test guideline. The study employed a common laboratory strain.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7 weels
- Weight at study initiation: Males 214-252 g; Females 163-193 g
- Fasting period before study: Overnight before dosing
- Housing: Animals were housed individually in suspended stainless steel cages with wire mesh bottom and fron. Bedding was placed in dropping pans under the suspended cages.
- Diet: ad libitum
- Water: ad libitum, tap water untreated with additional chlorine provided by bottles.
- Acclimation period: Minimum of one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-26 °C
- Humidity (%): 41-67 %
- Photoperiod (hrs dark / hrs light): 12 hour light dark cycle (fluorescent lighting)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: 100 g of corn oil was weighed into a jar. According to the dose formulation, different increments of the test material were added and stirred until dissolved. (5 g for high dose, 3 g for mid dose and 1 g for low dose). After the desired weight of the test material was added to the jar, the solutions were stirred magnetically for 30 minutes. Following stirring, the solutions were allowed to stand prior to removing aliquots for analysis.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulations were analysed for concentration, homogeneity and stability in corn oil.
Homogeneity: Six aliquots were removed from each of the test solutions (two samples of each from the top, near the middle and near the bottom). The aliquots were diluted in hexane and analysed.
Stability: The stability of the dosing formulations was analysed of a 13 day period. 8.1 mg/mL and 78.9 mg/mL solutions used for the homogeneity testing. These solutions were analysed on day 7 and 13 after preparation and were compared to the results from the homogeneity testing obtained on Day 0. Solutions were stored at ambient temperatures in the dark. Stability of the dose solutions were also confirmed over the dosing period by analysing the formulations at the beginning and end of the dose interval.
Quantification: Two or three replicate aliquots (approximately 150-300 mg each) of each dose formulation and each solution prepared for homogeneity or stability tests were diluted in volumetric flasks to 10 mL with hexane. Calibration standards were prepared over a range of approximately 0.1 mg to 5 mg/mL in hexane and were analysed with the samples by gas chromatography. Duplicate injections were made for each standard and sample. A linear regression was performed on the calibration standards which was then used to quantify the concentration of the test substance in the dose formulations.
The test material was analysed by gas chromatography under the following conditions:
Column: DB-1 30 M x 0.54 mm, 1.5 µm film
Detector: FID 280 °C
Carrier gas: Helium (ca. 3 mL/min)
Makeup gas: Helium (ca. 34 mL/min)
Split flow: Approximately 23 mL/min
Septum purge flow: 4 mL/min
Hydrogen flow: 33 mL/min
Air flow: 288 mL/min
Run initial temperature: 220 °C
Initial hold time: 1 minute
Ramp: 5 °C/minute
Final temperature: 270 °C, 5 minute hold
Ramp A : 10 °C/minute
Final Temperature A: 300 °C
Final Time A: 1 minutes
Run length: 16 minutes

Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control (corn oil)
Dose / conc.:
83 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
850 mg/kg bw/day (nominal)
Remarks:
Reduced to 425 mg/kg bw/day after 7 days due to mortality and clinical observations.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control, Corn oil (Recovery group)
Dose / conc.:
850 mg/kg bw/day (nominal)
Remarks:
Reduced to 425 mg/kg bw/day after 7 days (Recovery Group).
No. of animals per sex per dose:
5 animals per sex per dose. A further two groups were administered with either the vehicle or the high dose as a satelite recovery group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A dose range finding study was conducted. Animals were dosed with 250, 500 and 1000 mg/kg bw/day for 7 days (3 animals/sex/group). There were no deaths during the range finder, but there was some weight loss observed after one week in all animals dosed with 1000 mg/kg bw /day. Due to the absence of deaths, the doses chosen for the 28 day definitive test were 100, 300 and 1000 mg/kg bw/day.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked:
Mortality
Clinical sign including but not limited to changes to neuromuscular, gastrointestinal, urogenital and respiratory systems, behaviour pattern, integument, secretions and mucous membranes.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed on day 0, day 1 of dosing, weekly thereafter and the day before sacrifice.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was determined weekly for each animal over approximately a 24 hours period.

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: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At scheduled sacrifice
- Anaesthetic used for blood collection: Yes (CO2)
- Animals fasted: Yes (overnight)
- How many animals: All animals
- Parameters checked: Erythrocyte count (#/µL) (RBC), reticulocyte count (1000 cells) (%) (RETIC), haematocrit (%) (HCT), haemoglobin concentration (g/dL) (HGB), total leucocyte count (#/µL) (WBC), differential leucocyte count (lymphocytes, neutrophils, leucocytes, monocytes, eosinophiles, basophils) (#/µL) (LYMPH, SEG, BAND, MONO, EOS, BASO), platelet count (#/µL) (PLATE), mean corpuscular volume (MCV (fL), mean corpuscular haemoglobin (MCH) (pg), mean corpuscular haemoglobin (MCHC) (g/dL).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At scheduled sacrifice
- Animals fasted: Yes (overnight)
- How many animals: All animals
- Parameters checked: Blood urea nitrogen (mg/dL), creatinine (mg/dL), Blood urea nitrogen:Creatinine,

URINALYSIS: Yes
- Time schedule for collection of urine: At scheduled sacrifice.
- Metabolism cages used for collection of urine: No. Urine was collected by holding the rat over a petri dish and exerting gentle pressure of the posterior ventral abdomen.
- Animals fasted: Yes (overnight)
- Parameters checked: pH, protein, glucose, ketones, occult blood.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Body weight and wet weights of liver, kidneys (paired), adrenal glands, brain, testes (paired and without epididymides) and ovaries were measured.

HISTOPATHOLOGY: Yes
The following tissues as well as any gross lesions were preserved in 10 % neutral buffered formalin.
All gross lesions, adrenal glands, bone (femur or sternum), bone marrow, brain, eyes, heart, kidney, liver, lungs and bronchi, ovaries, pituitary, spleen, stomach, testes, thyroid with parathyroids, trachea, urinary bladder.
Representative sections of heart, liver, spleen, kidneys and adrenal glands from animals in the control and high dose groups as well as any gross lesions from all groups were prepared, stained with haematoxylin and eosin, and examined microscopically. Based on liver and kidney lesions in high dose animals, these tissues were examined from all dose groups including recovery groups.

Statistics:
Body weights, food consumption (absolute and relative), organ weights (absolute and relative), clinical chemistry and haematology were analysed among the group means using ANOVA. All groups were analysed for weeks 1-4 and the recovery groups were analysed for weeks 5-6 separately. Where significant difference was observed, the results of the Duncan’s Multiple Rage Test were used to determine which groups were statistically significant compared to the control. All tests were used a significance level of p≤0.05. Males and females were analysed separately. The repeated measure ANOVA was not used as it would not have provided additional information.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Male and females in control group did not show any abnormal clinical signs during the entire study. Minimal, transient abnormal signs were seen in the lowest dose group (83 mg/kg bw/day). Male animals dosed with 250 mg/kg bw/day showed minimal abnormal signs, female animals which survived to terminal necropsy had decreased activity, poor coat and urogenital discolouring. Animals dosed with 425/850 mg/kg bw/day had the most extensive abnormal clinical signs which included decreased activity, nasal and urogenital discharge, poor coat, diarrhoea and in some cases moribundity leading to premature death.
Mortality:
mortality observed, treatment-related
Description (incidence):
Twelve animals either died or were sacrificed during the 28 day dosing period. One high dose female was accidently killed on day 1 due to a gavage error. A replacement animal was added to the group on day 1.
After roughly 1 week of dosing, six high dose animals had either died or were sacrificed due to their moribund condition. Three males and three females from the high dose and recovery groups.
One male and one female were sacrificed moribund on day 8 from the high dose recovery groups to provide a comparison with the high dose animals. A further two females from the high dose and high dose recovery groups were found dead after the dose reduction on days 13 and 16. One 250 mg/kg bw/day female was found dead due to a dosing error.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights for male control animals increased as expected. Body weight declined in animals in the high dose and recovery high dose groups after the first week (when dosed with 850 mg/kg bw/day). After the dose was lowered to 425 mg/kg bw/day, these animals gained weight for the remainder of the study, but remained lower than the control body weights. Animals dosed with 83 or 250 mg/kg bw/day has similar weigh gains to the vehicle control animals.
The body weight gains for the high dose female animals demonstrated a similar trend to the male high dose animals. The initial weight loss was recovered once the high dose was lowered to 425 mg/kg bw/day. The weight gains for the two lower doses (83 and 250 mg/kg bw/day) were slightly lowered compared to the vehicle controls, but only the reduced weight gain for the 250 mg/kg bw/day was statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption values paralleled the results of the body weight gains. For male animals, both absolute and relative food consumption were significantly reduced for animals in the high dose and high dose recovery groups. After the dose was lowered to 425 mg/kg bw/day, the absolute and relative food consumption returned to control values. There were no significant differences in food consumption for either of the two lower dose groups.
The food consumption for female animals were similar to the males. In the high dose and high dose recover groups, both absolute and relative food consumption were reduced, relative to the vehicle controls until the dose was lowered to 425 mg/kg bw/day. Food consumption in the 83 and 250 mg/kg bw/day was not significantly different from the vehicle controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The only significant changes in haematology parameters for male rats was observed on day 29. An increase in the number of white blood cells from 10000/µL in the control to 16100/µLat the high dose group. This increase was primarily due to an increase in lymphocytes. Although this was statistically significant, a dose related trend was noted in the low and mid-dose groups for both white blood cell count and lymphocyte count.
In female rats, this increase in white blood cells was not observed. A statistically significant decrease in mean corpuscular haemoglobin concentration was observed in the high and mid dose groups, but was not considered to be biologically significant. The statistical significance for the high dose groups was attributed to the use of data from only two animals with a standard deviation of 0.00.
Following the recovery period. The increase in white blood cells in high dose males was resolved and a statistically significant decrease was observed from 10000/µL to 8200/µL. The significant increase in the percentages of reticulocytes from 1.4 to 2.4 % was not considered to be biologically significant as these values were observed in the control and high dose groups on day 28. The difference was attributed to the lower standard deviations on day 43.
The significant decrease in the number of monocytes in female rats on was not biologically significant as they were with in the control range established by day 29.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
On day 29, the alkaline phosphatase activity was found to be increased in the high dose group male rats from 578 to 867 U/L, indicating possible cholestasis and toxicity to the bile duct. An increase in phosphorous from 9.0 mg/dL to 11.9 mg/dL was also seen in the high dose group males.
On day 29, female rats in the high dose group had a two-fold increase from 23 U/L to 49 U/L in alanine amino transferase activity, indicating possible liver damage. The alkaline phosphatase activity in high dose animals, while high that the control was not significantly increased. This was attributed to the high standard deviation in the low and mid-dose groups.
In the recovery male animals, alkaline phosphatase activity was similar for both the vehicle and high dose animals. There were no significant changes. In female recovery animals, total bilirubin was significantly decreased in the high dose animals from 0.2 mg/dL to 0.13 mg/dL. This was not considered to be biologically significant as there were no significant differences in total bilirubin in females sacrificed on day 29. Those values ranged from 0.17 to 0.20 for all groups on day 29.
Urinalysis findings:
no effects observed
Description (incidence and severity):
The pH values ranged from 6.0 to 7.0 for males and from 6.0 to 7.5 for females. There were no differences amongst the treatment groups. Glucose levels were negative for all animals.
Protein values for vehicle control animals ranged from tract to 100 mg/dL for males and from trace to 30 mg/dL for females. Although several male animals have values of 300 mg/dL, these appeared in both the control and high dose groups. One female animal from the high dose recovery group had a value of 300 mg/dL. There were no dose-related increases in protein concentration in either sex.
Blood levels ranged from negative, trace moderate and large, but there were no dose-related trends in either sex. Ketones ranged from negative, trace, small and moderate, bit no dose-related trend was observed in this parameter.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute kidney weight was reduced in the high dose recovery females compared to the control recovery group females. When compared to the relative body weight and brain weight, a difference was no longer significant. Female absolute liver weights were not significant but were significantly higher than the control group when compared to the relatively to the body weight. A similar increase in relative liver weight to body weight was also seen in the high dose recovery females compared to the vehicle control recovery group. This relative difference in the high dose females, however did not occur when the liver weights were compared to the relative brain weight.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Gross lesions were not considered to be related to treatment. Some where associated with the method, for example gavage errors.
Gross lesions considered to be related to gavage errors included thoracic cavity fluid or adhesions in on low dose and one high dose male and in one, low mid and high dose group female. Lung perforation was seen in one high dose male and one high dose female. Adhesions of the heart and thymus were observed in one low dose female.
Incidental and spontaneous gross lesions included and adrenal gland nodule in a single group 3 male, an enlarged liver in one high dose male, and enlarged retropharynegeal lymph nodes in one high dose group male. In females, one mid dose female and one high dose recovery female had an enlarged uterus. One low dose female had a liver nodule. One low dose female had an adrenal gland focus. One mid dose group female had hydronephrosis.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver: Two low dose males had minimal biliary hyperplasia. 3 had fatty change of which one was mild and two minimal. All five low dose females had minimal to mild fatty change. One low dose male had minimal portal inflammation.
Three mid dose group males had minimal fatty change. All five mid-dose females had minimal biliary hyperplasia. Four mid dose females had minimal diffuse inflammation, mild fatty change and minimal portal inflammation. One mid dose-female had minimal extramdullary haematopoiesis.
In the high dose males, four had minimal necrosis, all five had minimal to mild biliary hyperplasia, four had minimal fatty change, and all five had minimal to mild portal inflammation. All high dose females had minimal to mild focal to diffuse necrosis, minimal to mild biliary hyperplasia and fatty change, and mild to moderate portal inflammation.
One recovery control group male had minimal necrosis, biliary hyperplasia and portal inflammation, and mild fatty change, this animal was moribund and was sacrificed. One other male from this group had minimal fatty change. Two recovery control group females had mild fatty change.
One high dose recovery male had minimal necrosis. All five recovery control group males had minimal biliary hyperplasia, two had minimal inflammation, two had minimal fatty change, and one had mild portal inflammation. All five high dose recovery females had minimal biliary hyperplasia, minimal to mild fatty change, and minimal to moderate portal inflammation. Two high dose recovery females had mild focal necrosis and three had minimal inflammation.
Kidneys: All control group male kidneys were within normal limits. One control female had minimal focal mineralisation.
Three low dose males had minimal focal tubule dilatation and two had minimal focal interstitial inflammation. One low dose female had minimal focal mineralisation.
One mid dose male had minimal focal tubule dilatation. One mid dose female had minimal focal mineralisation. One mid dose female had minimal diffuse interstitial inflammation.
Three high dose males had minimal tubule dilatation and eosinophilia, two had minimal necrosis. One high dose female had minimal tubule dilatation and one had eosinophilia, one had mild focal mineralisation.
The early sacrifice recovery control male had minimal tubule dilatation, eosinophilia and interstitial inflammation. Two recovery control group females had minimal focal mineralisation.
One high dose recovery male had minimal focal tubule dilatation and interstitial inflammation. Three high dose recovery females had minimal focal to diffuse tubule dilatation, one had mild tubule eosinophilia, one had minimal interstitial inflammation.
Spleen: All control, low- and mid-dose group males had normal spleen sections. Females from the control and low-dose groups were also within normal limits.
One mid-dose female had minimal lymphoid depletion.
Two high-dose males had minimal red pulp depletion. Two high dose females had minimal to mild lymphoid depletion. Three had minimal red pulp depletion.
The spleen sections from the recover control male and female rats were within normal limits.
One male and one female from the high dose recovery groups had minimal lymphoid depletion. Two high dose recovery females had minimal to mild red pulp depletion.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Details on results:
The test substance was determined to be stable at room temperature while stored in the dark for at least 7 days. After 13 days following preparation, the 8.1 mg/mL formulation was approximately 76 % of the initial formulations. After 13 days, residual test material was observed on the glass at the meniscus of the solution. The reduction in concentration with this formulation is attributed to this residue. Based on the results of the stability testing, the dosing solutions were prepared weekly for the duration of the test.
The dose formulation analysis indicated that the average pre- and post-dose analyses indicate that all dosing solutions were within 10 % of the target concentration. No detectable changes in the chromatographic pattern of peaks were observed for any of the analyses.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
83 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Table 1: Mortality

Mortality

Dose

Sex

Result

850 mg/kg

F

Error, replaced

850 mg/kg

F

Dead on day 7

850/mg/kg

M

Moribund sacrifice on day 8

850 mg/kg

F

Moribund sacrifice on day 8

850 mg/kg recovery

M

Moribund sacrifice on day 8

850 mg/kg recovery

F

Dead on day 8

Vehicle recovery

M

Sacrificed to compare to G4/6

Vehicle recovery

F

Sacrificed to compare to G4/6

425/850 mg/kg

M

Dead on day 9

425/850 mg/kg recovery

F

Dead on day 13

425/850 mg/kg

F

Dead on day 16

250 mg/kg

F

Dead on day 18 due to gavage error

 

Table 2: Bodyweights males

Dose

Body weights (g) males

Day 1

Day 8

Day 15

Day 22

Day 28

Day 35

Day 42

Vehicle

Mean

231

264

300

326

347

N/A

N/A

SD

12.8

11.6

13.9

15.1

19.4

n

5

5

5

5

5

83 mg/kg

Mean

231

267

301

324

342

N/A

N/A

SD

14.5

13.7

11.9

21

35.5

n

5

5

5

5

5

250 mg/kg

Mean

229

266

303

332

351

N/A

N/A

SD

10.8

6.2

7

3.9

5.7

n

5

5

5

5

5

425/850 mg/kg

Mean

230

214

257

284

304

N/A

N/A

SD

14

19.4

22

32.9

27.1

n

5

5

3

3

3

Vehicle

Mean

230

268

298

331

361

373

398

SD

13.3

10.9

8.6

13.4

18.6

20.4

25.1

n

5

5

4

4

4

4

4

425/850 mg/kg recovery

Mean

229

209

252

272

284

308

337

SD

9.2

21.9

12.3

19.1

25.4

23.6

23.7

n

5

5

4

4

4

4

4

 

Table 3: Bodyweights females (g)

Dose

Body weights (g)- Females

Day 1

Day 8

Day 15

Day 22

Day 28

Day 35

Day 42

Vehicle

Mean

181

199

219

238

249

N/A

N/A

SD

7.7

6.5

10.9

12.7

15.3

n

5

5

5

5

5

83 mg/kg

Mean

177

193

214

227

231

N/A

N/A

SD

8.4

9.7

13.6

16.8

19.4

n

5

5

5

5

5

250 mg/kg

Mean

179

188

204

208

223

N/A

N/A

SD

8.4

7.6

13.6

16.3

14

n

5

5

5

4

4

425/850 mg/kg

Mean

170

156

195

201

198

N/A

N/A

SD

7.4

8.3

97.5

17

36.1

n

5

4

4

2

2

Vehicle

Mean

176

195

227

241

248

252

263

SD

7.8

9.4

4.9

11

5.6

10.4

11.2

n

5

5

4

4

4

4

4

425/850 mg/kg

Mean

177

160

194

210

221

225

240

SD

6.1

13

3.8

7.6

2.3

8.7

11.7

n

5

5

3

3

3

3

3

 

Table 4: Bodyweight change (females)

Dose

Change in body weights (g)- Females

Day 8

Day 15

Day 22

Day 28

Day 35

Day 42

Vehicle

Mean

17

21

19

11

N/A

N/A

SD

2.7

5.7

6.9

7.6

n

5

5

5

5

83 mg/kg

Mean

17

21

13

4

N/A

N/A

SD

3

6.2

6

15.5

n

5

5

5

5

250 mg/kg

Mean

9

16

0

15

N/A

N/A

SD

10.8

13.5

8.3

8.8

n

5

5

4

4

425/850 mg/kg

Mean

-15

35

7

-4

N/A

N/A

SD

12.9

1.5

17

19.1

n

4

3

2

2

Vehicle

Mean

19

28

14

7

4

11

SD

5.6

8.1

7.7

6.5

8.1

3.4

n

5

4

4

4

4

4

425/850 mg/kg

Mean

-17

28

16

11

4

16

SD

17.4

4.4

11

7.5

9.5

3.2

n

5

3

3

3

3

3

 

Table 5: Absolute food consumption males

Dose

Absolute food consumption (g) - males

Day 4

Day 11

Day 18

Day 25

Day 32

Day 39

Vehicle

Mean

20

20

18

20

N/A

N/A

SD

3

0.4

2.1

3.8

n

5

5

5

5

83 mg/kg

Mean

19

19

18

17

N/A

N/A

SD

2.3

1.5

4.8

4

n

5

5

5

5

250 mg/kg

Mean

21

22

20

19

N/A

N/A

SD

1.5

2.3

2.8

4.2

n

5

5

4

4

425/850 mg/kg

Mean

8

20

16

18

N/A

N/A

SD

3.6

5.1

6.4

7.8

n

5

3

3

3

Vehicle

Mean

23

18

21

17

29

35

SD

0.9

4.7

4.7

4.5

2.4

12

n

5

4

4

4

4

4

425/850 mg/kg

Mean

6

17

18

19

25

30

SD

4.4

4.4

3.4

7.1

9.6

4

n

5

4

4

4

4

4

 

Table 6: Absolute food consumption – females

Dose

Absolute food consumption (g)- Females

Day 4

Day 11

Day 18

Day 25

Day 32

Day 39

Vehicle

Mean

13

15

12

15

N/A

N/A

SD

1.7

0.8

3.3

6.9

n

5

5

5

5

83 mg/kg

Mean

15

16

11

12

N/A

N/A

SD

1.3

1.9

4.1

2.9

n

5

5

5

5

250 mg/kg

Mean

10

15

12

11

N/A

N/A

SD

3.2

3.4

4.6

1.7

n

5

5

4

4

425/850 mg/kg

Mean

6

14

17

13

N/A

N/A

SD

2.6

3.9

0

7.8

n

5

3

1

2

Vehicle

Mean

15

16

14

14

19

23

SD

2.5

1.7

3.8

3.7

5.1

2.4

n

5

4

4

4

4

4

425/850 mg/kg

Mean

6

16

18

19

22

22

SD

4.3

4

5.5

4.7

6

5.7

n

5

4

3

3

3

3

 

Table 7: Haematology males day 29

Dose

WBC 1E+3/µl

RBC 1E+6/µl

HCT %

HGB g/dL

MCV fL

MCH pg

MCHC g/dL

PLATE 1E+3/µl

RETIC %

SEG %

Seg 1E+3/µl

BAND %

BAND 1E+3/µl

LYMPH %

LYMPH 1E+3/µl

MONO %

MONO 1E+3/µl

EOS %

EOS 1E+3/µl

BASO %

BASO 1E+3/µl

NRBC/100 WBC

Vehicle

Mean

10

8.52

50

17.3

61

0.3

34.4

822

1.4

5

0.434

0

0

95

9.543

0

0.021

0

0.041

0

0

0

SD

2.04

0.45

0.8

0.39

2.57

0.76

0.4

99

1.09

3.6

0.2524

0

0

3.4

2.1913

0.4

0.047

0.5

0.0564

0

0

0.4

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

83 mg/kg

Mean

11.9

8.82

52

17.8

60.5

20.2

34.3

747

1.7

6

0.699

0

0

93

11.019

1

0.122

0

0.019

0

0

0

SD

2.1

0.642

2.4

0.77

2.76

0.97

0.4

370.1

1.12

1.4

0.1364

0

0

2.4

2.0713

1.2

0.1383

0.4

0.0434

0

0

0

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

250 mg/kg

Mean

13.7

8.62

50

17.2

59.8

20

34.2

896

1.3

5

0.764

0

0

94

12.916

0

0.019

0

0.045

0

0

0

SD

3.18

0.4

1.3

0.53

3.16

0.86

0.16

93.1

0.3

3.8

0.632

0

0

3.7

2.9535

0.4

0.042

0.5

0.0623

0

0

0.4

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

425/850 mg/kg

Mean

16.1

8.74

50

17

58.7

19.5

34

952

2.4

10

1.675

0

0

88

14.014

2

0.248

1

0.162

0

0

0

SD

2.78

0.254

1

0.26

1.39

0.26

0.26

95.4

0.9

10.7

1.9661

0

0

11.9

2.4929

1.5

0.2156

1

0.1813

0

0

0

n

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

 

Table 8: Haematology females day 29

Dose

WBC 1E+3/µl

RBC 1E+6/µl

HCT %

HGB g/dL

MCV fL

MCH pg

MCHC g/dL

PLATE 1E+3/µl

RETIC %

SEG %

Seg 1E+3/µl

BAND %

BAND 1E+3/µl

LYMPH %

LYMPH 1E+3/µl

MONO %

MONO 1E+3/µl

EOS %

EOS 1E+3/µl

BASO %

BASO 1E+3/µl

NRBC/100 WBC

Vehicle

Mean

11.8

8.49

50

17.5

60.2

20.7

34.9

948

1.3

5

0.639

0

0

93

11.021

0

0.06

1

0.074

0

0

0

SD

4.38

0.554

3

1.06

0.65

0.59

0.22

70.8

0.96

1.5

0.3218

0

0

1.3

4.062

0.5

0.0822

0.8

0.0759

0

0

0.4

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

83 mg/kg

Mean

12.1

8.58

50

17.5

59.9

20.5

35.2

932

1.4

5

0.504

0

0

92

11.28

2

0.171

1

0.124

0

0

0

SD

5.74

0.832

3.6

1.39

1.83

0.37

0.43

75.4

0.42

3.6

0.3945

0

0

4.9

5.4404

2.2

0.0714

0.8

0.1348

0

0

0

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

250 mg/kg

Mean

11.6

8.36

49

16.8

60.1

20.1

34.1

920

1.1

7

0.814

0

0

93

10.967

1

0.064

0

0

0

0

0

SD

1.41

0.331

1.7

0.81

1.21

0.39

0.47

46.8

0.54

3.5

0.483

0

0

3.9

0.9052

1

0.129

0

0

0

0

0

n

4

4

4

4

4

4

4

4

4

4

4

4

4

4

4

4

4

4

4

4

4

4

425/850 mg/kg

Mean

12.6

8.2

49

16.4

62.6

20.4

33.5

920

2

7

0.78

0

0

91

11.518

2

0.202

1

0.1

0

0

0

SD

3.68

0.467

0

0

3.18

1.2

0

4.2

1.13

2.1

0.0283

0

0

2.8

3.7024

0.7

0.1442

1.4

0.1414

0

0

0

n

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

 

Applicant's summary and conclusion

Conclusions:
Tthe NOAEL of the test material was determined to be 250 mg/kg bw/day in male rats and 83 mg/kg bw/day in females, based on the data available in the study.
Executive summary:

The subacute toxicity of the test material was determined in a study conducted in accordance with OECD 407. The test material was administered via oral gavage to male and female rats (5 animals per sex per dose) at 0 (vehicle control, corn oil), 83, 250 and 850 mg/kg bw/day. Due to the toxicity observed in the high dose group, the dose was reduced to 425 mg/kg bw/day after day 7 for the remainder of the study. Recovery groups were included for the high dose and control groups.

Changes in haematological and clinical chemistry parameters were minimal. Alanine aminotransferase was doubled in high dose female rats. Other liver enxymes, aspartate aminotransferase, was not found to be increased. Liver weights relative to body weight were increased in high dose females. This increase remained in the recovery animals. No gross lesions related to treatment were observed. The primary microscopic lesions were found in the liver and were dose-related.These were hepatocellular necrosis, biliary hyperplasia, and portal inflammation. Kidney lesions were also observed and the severity was dose related. These included tubule necrosis, tubule dilatation, tubule eosinophilia and interstitial inflammation.

Under the conditions of the test, the NOAEL of the test material was determined to be 250 mg/kg bw/day in male rats and 83 mg/kg bw/day based on microscopic lesions observed in the liver. Although at the time of the study, these effects were considered adverse and as such were concluded in the study report. However, when reviewing the anatomic physiological observed effects and the individual liver pathological endpoints in line with more current knowledge concerning, these effects are not considered to be adverse and therefore would not be considered for classifcation for specific organ toxicity classification.