Butyl 4,4-bis(tert-butyldioxy)valerate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 April 2016 - 27 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Butyl 4,4-bis(tert-butyldioxy)valerate

EC Number:

213-626-6

EC Name:

Butyl 4,4-bis(tert-butyldioxy)valerate

Cas Number:

995-33-5

Molecular formula:

C17H34O6

IUPAC Name:

butyl 4,4-bis(tert-butylperoxy)pentanoate

Test material form:

other: Colorless to yellow liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Italia, Calco, Italy
- Age at study initiation: approximately 5 weeks old
- Mean body weight at study initiation: the males had a mean body weight of 186 g (range: 169 g to 196 g) and the females had a mean body weight of 151 g (range: 129 g to 168 g)
- Fasting period before study: no
- Housing: the animals were housed in fives from the same sex and group in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm2) containing autoclaved sawdust
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 8 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 15 April 2016 to 27 May 2016.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: solution in the vehicle

VEHICLE
- Justification for use and choice of vehicle: suitable formulation in corn oil
- Concentration in vehicle: 6, 20 and 60 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: Gas Chromatography with FID detection (GC-FID)
Once in Weeks 1 and 4.
A sample was taken from control and test item dose formulations and analyzed using the validated method.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 animals per sex per dose (for groups 1 and 4)
5 animals per sex per dose (groups 2 and 3).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, based on the results of a previous 2-week dose-range finding study performed in the same species. In this study, three groups of ten Sprague-Dawley were treated with the test item at 100, 300 and 1000 mg/kg/day.
No clinical signs were observed except for ptyalism and chromodacryorrhea at 1000 mg/kg/day at the end of the treatment period. Body weight loss (-9%) associated with lower food consumption were recorded in high-dose females during the first 4 days. At necropsy, enlargement of the liver was observed in one male at 300 mg/kg/day and in most animals from both sexes at 1000 mg/kg/day, correlating with higher liver weights recorded in males (all dose-levels of the test item) and in females (300 and 1000 mg/kg/day).
- Rationale for animal assignment: computerized randomisation procedure.
- Post-exposure recovery period in satellite groups: 2-week treatment-free period.

Positive control:

no (not required)

Examinations

Observations and examinations performed and frequency:

MORTALITY/MORBIDITY: Yes
- Time schedule: each animal was checked for mortality and morbidity once a day during the acclimation period and at least once a day during the treatment and treatment-free periods, including weekends and public holidays.

CLINICAL OBSERVATIONS: Yes
- Time schedule: each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: detailed clinical examinations were performed on all the animals once before the beginning of the treatment period and then once a week until the end of the study.

BODY WEIGHT: Yes
- Time schedule:
The body weight of each animal was recorded:
- once before the beginning of the treatment period,
- on the first day of treatment,
- at least once a week until the end of the study.

FOOD CONSUMPTION: Yes
- Time schedule: the quantity of food consumed by the animals in each cage was recorded once a week during the study.
Food consumption was calculated per animal and per day.

MONITORING OF ESTROUS CYCLE: Yes
Time schedule: the estrous cycle stage was determined for each female sacrificed at the end of the treatment period daily for 4 consecutive days at the end of the treatment period.
As no treatment-related changes were observed at the end of the treatment period, these examinations were not carried out at the end of the treatment-free period.

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional Observation Battery (FOB)
Each animal was evaluated once in Week 4 before the daily treatment.

Motor activity
For each animal, motor activity was measured once by automated infra-red sensor equipment over a 60 minute period.

HAEMATOLOGY: Yes
Peripheral blood
The parameters were determined for all surviving animals (except recovery animals) before sacrifice.
As no treatment-related changes were observed at the end of the treatment period, these examinations were not carried out at the end of the treatment-free period.

Bone marrow
Two bone marrow smears were prepared from the femoral bone (at necropsy) of each animal sacrificed on completion of the treatment period.
As no relevant abnormalities were observed during the hematological investigations, the bone marrow differential cell count was not determined and smears were archived.

CLINICAL CHEMISTRY: Yes
The parameters were determined for all surviving animals (except recovery animals) before sacrifice.
As changes were observed at the end of the treatment period, these examinations were carried out at the end of the treatment-free period.

URINALYSIS: Yes
The parameters were determined for all surviving animals (except recovery animals) before sacrifice.
As no treatment-related changes were observed at the end of the treatment period, these examinations were not carried out at the end of the treatment-free period.

THYROID HORMONES: Yes
An additional blood sample was taken from each animal at the end of the treatment and treatment-free periods.
The levels of the thyroid hormones and thyroid stimulating hormone was determined from each animal at the end of the treatment and treatment-free periods.

Sacrifice and pathology:

ORGAN WEIGHTS: Yes
The body weight of each animal was recorded before euthanasia at the end of the treatment or treatment free period.

GROSS PATHOLOGY: Yes
Sacrifice
On completion of the treatment or treatment-free period, after at least 14 hours fasting, all surviving animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital.

Macroscopic post-mortem examination
A complete macroscopic post-mortem examination was performed on all study animals.

HISTOPATHOLOGY: Yes
A microscopic examination was performed on:
- all tissues listed in the Tissue Procedure Table for the control- and high-dose animals (groups 1 and 4) euthanized at the end of the treatment period and for all animals that die,
- all macroscopic lesions from all the low- and intermediate-dose animals (groups 2 and 3) euthanized on completion of the treatment period.

Based upon the microscopic results of the high-dose group and after agreement of the Sponsor, liver and thyroids in both sexes and kidneys for males from the low-, intermediate-dose and recovery animals were examined.

In order to characterize the tubular hyaline droplets, microscopic examination of kidneys slides immunostained with an antibody for alpha-2u-globulin protein was performed for all control- and high-dose males at the end of the treatment period.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Chromorhynorrhea were observed both in control and test item-treated animals in male and in one female given 30 mg/kg/day with no dose relationship. This sign can commonly be encountered in rats of this age and strain was unrelated to the test item treatment.
The other clinical signs recorded during the study, i.e. ptyalism, reflux at dosing and thin appearance were observed in one or two females given 300 mg/kg/day. These signs were reported sporadically on a few days but they were considered to be related to the test item treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test item-related deaths occurred during the treatment and treatment-free periods of the study.
One control female died suddenly on Day 21 without ante-mortem clinical signs. Prior to death, body weight, body weight gain and food consumption were unaffected.
The cause of death could not be determined at macroscopic or microscopic examination.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

See Table 1.
In all male treated groups, a statistically significant lower mean body weight changes were observed, when compared to controls, on Days 22/28, without any effect on mean body weight on Day 28 for these groups.
This change was probably due to stress induced by many final investigations and was considered to be unrelated to the test item treatment.
In females, mean body weight and mean body weights gain were unaffected by the test item treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean food consumption was unaffected by the test item treatment in both sexes.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

No test item treatment effects were observed on the hematology parameters at the end of the treatment period in both sexes when compared to control group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See Table 2.
There were no changes in any of the blood biochemistry parameters measured among animals treated at 30 mg/kg/day (the lower glucose levels in males was within the Historical Control Data range).
When compared to controls, test item-related blood biochemistry findings observed at the end of treatment period in animals given 100 and 300 mg/kg/day consisted of:
- lower mean glucose level (males at 100 and 300 mg/kg/day),
- lower mean triglycerides level (males at 100 and 300 mg/kg/day),
- higher mean total protein level (females at 300 mg/kg/day),
- higher mean total cholesterol level (females at 100 and 300 mg/kg/day).

All the above described findings were considered to be of minor toxicological importance as values remained within or close to the range of historical control values.

Test item-related effects observed at the end of the treatment period on the blood biochemistry parameters were no longer observed at the end of the treatment-free period in both sexes, thus indicative of reversibility.
The lower mean inorganic phosphorus and proteins levels observed in males given 300 mg/kg/day at the end of the treatment-free period were slight, of low amplitude and within or close to historical control data.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The only statistically significant differences from controls consisted of slightly lower mean pH value in males given 300 mg/kg/day (6.2 vs. 7.1 in controls; p < 0.05). As this difference was of minimal amplitude and not associated with any other findings, this was considered to be of minor toxicological importance.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

See Table 3.
When compared to control mean values, there were no differences in the motor activity of animals treated with the test item at the end of the dosing period. The apparent higher mean number of rearing movements in males and the lower mean number of horizontal movements in females given 300 mg/kg/day were not considered to be test item treatment-related since these numbers were slight in amplitude and without clear dose-relationship.

Excessive quantity or abnormal appearance of defecation was noted in 1/5 males given 30 mg/kg/day.
In view of the low incidence and in absence of correlating clinical signs during the study, this finding was considered to be unrelated to the test item treatment.
No neurologic, autonomic or behavioral changes that could be related to the treatment were observed during the FOB assessment phase.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See Table 4.
End of the treatment period
Test item-related changes in organ weights were seen in the liver in both sexes and in the kidney and thyroids in males only.

When compared with controls, the mean absolute and relative liver weights were moderately increased in males and females at 300 mg/kg/day. These increased liver weights correlated with hepatocellular hypertrophy at microscopic examination.

There were minimal or slight increases in the mean absolute and relative kidney weights in males at 100 and 300 mg/kg/day. These increased kidney weights correlated with microscopic tubular hyaline droplets at 300 mg/kg/day. In females at 300 mg/kg/day, the minimal, not statistically significant increase in kidney weight was not considered to be toxicologically significant in the absence of microscopic correlates in this sex.

The mean absolute and relative weights of the thyroid glands were minimally increased in males at 300 mg/kg/day. Most individual values were above the range of control data but the variations were not statistically significant. Despite their low magnitude, these increases were considered to be most likely test item-related since they correlated with minimal hypertrophy of the thyroid follicular epithelium but of minor toxicological importance as there were no significant changes in TSH, T3 and T4 levels.

The mean uterus weights were slightly or moderately higher at 100 and 300 mg/kg/day compared to controls. This difference correlated at necropsy with dilatation of the uterus with translucent content, and at microscopic examination with higher incidence of females in proestrus phase of the estrous cycle compared to controls. This was therefore considered to be incidental and unrelated to the test item administration.

The mean weights of the testes and epididymides were minimally or slightly higher in groups treated at all dose-levels compared to controls. This was due to low individual weights of the testes and epididymides in one control male, correlated with spontaneous tubular atrophy/degeneration in the testes and reduced sperm content in the epididymides. Variations in testes and epididymides weights were therefore considered to be incidental and unrelated to the test item administration.

Other organ weight changes were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, and/or were not dose-related in magnitude.

End of the treatment-free period
There was partial recovery of the increases in kidney weights in males at 300 mg/kg/day. Complete recovery was observed for the liver and thyroids.

The mean kidney weights were still minimally increased in males at 300 mg/kg/day compared to controls (absolute weight: +9%, relative weight: +11%; statistically significant for the relative weight only, p < 0.05).

The mean weights of the thyroid glands were minimally higher in males at 300 mg/kg/day compared to controls. This was due to high individual weight in a single male, correlated with macroscopic enlargement.

Other differences in organ weights at the end of the treatment-free period were minor and reflected the usual range of individual variations.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

End of the treatment period
The test item administration induced macroscopic changes in the liver in high-dose males only.

Liver enlargement was noted in 3/5 males at 300 mg/kg/day and was associated with an accentuated lobular pattern. These changes correlated with hepatocellular centrilobular hypertrophy at microscopic examination.
In females, accentuated lobular pattern of the liver was noted in one animal per group in all groups including controls, and correlated with periportal vacuolation of hepatocytes at histology. Given the distribution, this macroscopic change was not considered to be related to the test item administration in females.

The other macroscopic findings had no histologic correlates or correlated with common histologic findings in control rats, and were considered to be incidental.

End of the treatment-free period
There was complete recovery of the liver macroscopic changes.

At 300 mg/kg/day, enlargement of the thyroids was in a single male. In the absence of microscopic correlates, this finding was considered not to be toxicologically significant.

The few macroscopic findings noted at the end of the treatment-free period were of those commonly recorded in the rat and none were considered to be related to the test item administration.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See Table 5.
End of the treatment period
Test item-related microscopic findings were seen in the liver and thyroids in both sexes, and in the kidneys in males only.

In the liver, slight or moderate centrilobular hypertrophy of hepatocytes was found in all males and females at 300 mg/kg/day. Minimal centrilobular hypertrophy was also present in two males and one female at 100 mg/kg/day. There were no significant microscopic findings in the liver at 30 mg/kg/day.

In the thyroids, there was minimal diffuse hypertrophy of the follicular cells in all males and most females at 300 mg/kg/day. There were no test item-related microscopic findings in the thyroids at 30 or 100 mg/kg/day.

In the kidneys, there was a higher incidence and severity of tubular hyaline droplets in males at 300 mg/kg/day compared to controls (5/5 versus 1/5 controls). This was characterized by dense eosinophilic droplets in the proximal tubular epithelium. These droplets were larger and more numerous in high-dose rats compared to the single affected control animal. These droplets were positively stained with an antibody to alpha-2u-globulin. Positive staining of tubular droplets was present in all control and high dose males, but the severity of the staining (i.e. size and number of droplets) was increased in high dose males compared to controls.
There were no significant microscopic changes in the kidneys from males at 30 or 100 mg/kg/day.

Other microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the rat.

End of the treatment-free period
Microscopic changes in the liver, thyroids were totally reversible in both sexes. In males, the increase in renal tubular hyaline droplets was partially reversible.

In the kidneys, the incidence of tubular hyaline droplets was minimally increased in high-dose males compared to controls, but the severity was similar.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Estrous cycle
Mean estrous cycle length and mean numbers of cycles were not affected by the test item treatment.

Thyroid hormones
TSH, T3 and T4 levels were unaffected by the test item at the end of the treatment period.
The only statistically significant difference observed in males given 300 mg/kg/day at the end of the treatment-free period was considered to be incidental and of no toxicological importance as it was not correlated to microscopic findings.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 1: Mean body weight gain / Mean body weight (expressed in g)

 

Sex	Male					Female
Dose-level (mg/kg/day)	0	30	100		300	0	30	100	300
Mean body weight on Day 1	186	182	187	187		148	152	153	152
Mean body weight gain Days 22/28	+29	+17**	+17**	+22*		+15	+15	+10	+10
% from controls	-	-41	-41	-24		-	0	-33	-33
Mean body weight gain Days 1/28	+195	+171	+173	+179		+87	+88	+76	+79
% from controls	-	-12	-11	-8		-	+1	-13	-9
Mean body weight on Day 28	381	353	360	365		235	240	228	230
% from controls	-	-7	-6	-4		-	+2	-3	-2

Statistically significant from controls: *: p<0.05; **: p<0.01; -: not applicable.

In bold: values significantly different from controls.

Table 2: Blood biochemistry

 

Sex	Male				Female
Dose-level (mg/kg/day)	0	30	100	300		0	30	100	300
End of treatment period
Sodium (mmol/L)	144.0	144.7	145.6*	145.6*		-	-	-	-
% from controls	NA	0	+1	+1		-	-	-	-
Glucose (mmol/L)	6.97	6.35	5.49*	5.07**		-	-	-	-
% from controls	NA	-9	-21	-27		-	-	-	-
Total proteins (g/L)	-	-	-	-		56.4	59.1	60.7	62.2*
% from controls	-	-	-	-		NA	+5	+8	+10
Total cholesterol (mmol/L)	-	-	-	-		1.44	1.63	2.01*	1.91*
% from controls	-	-	-	-		NA	+13	+40	+33
Triglycerides(mmol/L)	0.62	0.60	0.32*	0.32*		-	-	-	-
% from controls	NA	-3	-48	-48		-	-	-	-
End of treatment-free period
Sodium (mmol/L)	143.2	NA	NA	144.0		-	NA	NA	-
Inorganic phosphorus (mmol/L)	2.67	NA	NA	2.45**		-	NA	NA	-
Glucose (mmol/L)	6.85	NA	NA	7.38		-	NA	NA	-
Total proteins (g/L)	66.5	NA	NA	63.6*		66.3	NA	NA	66.7
Total cholesterol (mmol/L)	-	NA	NA	-		2.10	NA	NA	2.26
Triglycerides(mmol/L)	0.79	NA	NA	0.81		-	NA	NA	-

Statistically significant from controls: *: p < 0.05; **: p < 0.01; NA: Not Applicable; -: no changes.

In bold: values significantly different from controls.

Table 3: Motor activity

 

Sex	Male				Female
Dose-level (mg/kg/day)	0	30	100	300	0	30	100	300
Horizontal movements	453	451	459	502	698	737	584	519
% from controls	-	0	+1	+11	-	+6	-16	-26
Rearing	143	152	196	184	225	251	310	218
% from controls	-	+6	+37	+29	-	+12	+38	-3

-: not applicable.

 

Table 4: Organ weights

Sex	Male			Female
Group	2	3	4	2	3	4
Dose-level (mg/kg/day)	30	100	300	30	100	300
Exam. animals / Num. of animals	5/5	5/5	5/5	5/5	5/5	5/5
- Final body weight	-7	-6	-6	+2	-2	0
- Liver
.absolute	-5	+2	+27**	+11	+8	+32**
.relative	+2	+8	+36**	+10	+10	+32**
- Kidneys
.absolute	+8	+14	+20**	+7	+7	+13
.relative	+16	+21*	+28**	+5	+9	+13
- Thyroid glands
.absolute	-9	-2	+10	-6	-2	-2
.relative	-2	+4	+18	-8	-1	-2

Statistically significant from controls: *: p < 0.05, **: p < 0.01.

Statistical significance determined for organ weights values and not percent changes

Table 5: Microscopic examination

 

Sex	Male				Female
Group	1	2	3	4	1	2	3	4
Dose-level (mg/kg/day)	0	30	100	300	0	30	100	300
No. animals	5	5	5	5	4	5	5	5
Liver
- Hypertrophy; hepatocyte
Minimal (grade 1)	-	-	2	-	-	-	1	-
Slight (grade 2)	-	-	-	2	-	-	-	2
Moderate (grade 3)	-	-	-	3	-	-	-	3
Thyroids
- Hypertrophy; follicular cell
Minimal (grade 1)	-	-	-	5	-	-	-	4
Kidneys
- Hyaline droplets; tubular  epithelium
Minimal (grade 1)	1	2	2	-	-	NA	NA	-
Slight (grade 2)	-	-	-	5	-	NA	NA	-
- Positive staining;  alpha-2u-globulin
Minimal (grade 1)	5	NA	NA	-	NA	NA	NA	NA
Slight (grade 2)	-	NA	NA	2	NA	NA	NA	NA
Moderate (grade 3)	-	NA	NA	3	NA	NA	NA	NA

-    : no findings.
NA: Not Applicable.

Applicant's summary and conclusion

Conclusions:

The toxicity of the test item was evaluated after daily administration (gavage) to rats at dose-levels of 30, 100 and 300 mg/kg/day for 4 weeks. On completion of the treatment period, designated animals were held for a 2-week treatment-free period in order to evaluate the reversibility of any findings.
At 30 mg/kg/day, the test item was clinically well tolerated. Body weight, food consumption, estrous cycle, hematology, blood biochemistry and urinary parameters were unaffected by the test item. The test item administration did not induce any macroscopic, microscopic or organ weight changes.
At 100 mg/kg/day, the test item was clinically well tolerated. Body weight, food consumption, estrous cycle, hematology and urinary parameters were unaffected by the test item. Minor changes in blood biochemistry consisted of lower glucose, triglycerides and cholesterol levels. Higher absolute and relative kidney weight in males were recorded. Microscopic changes were observed in the liver and consisted of a minimal centrilobular hypertrophy.
At 300 mg/kg/day, test item-related clinical signs such as ptyalism, reflux at dosing and thin appearance were observed in one or two females. Body weight, food consumption, estrous cycle, hematology and urinary parameters were unaffected by the test item. Minor changes in blood biochemistry consisted of lower glucose, triglycerides, total protein and cholesterol levels. Higher absolute and relative liver weight in both sexes, kidneys and thyroid weights in males were recorded. Microscopic changes were observed in the liver (adaptative centrilobular hypertrophy) and thyroids (compensatory minimal follicular hypertrophy) in both sexes and in the kidneys (male rat specific alpha-2u-globulin hyaline droplets in tubular epithelium) in males only.
Consequently, under the experimental conditions of the study, the No Observed Adverse Effect Level (NOAEL) after the 4-week treatment period was established at 300 mg/kg/day.

Executive summary:

The objective of this study was to evaluate the potential toxicity of Luperox 230 following daily oral administration (gavage) to rats for 4 weeks. On completion of the treatment period, designated animals were held for a 2-week treatment-free period in order to evaluate the reversibility of any findings. This study was designed based on OECD No. 407 guideline. Two groups of five male and five female Sprague-Dawley rats and one group of ten male and ten females Sprague-Dawley rats were treated daily by the oral route (gavage) with the test item, at dose-levels of 30, 100 and 300 mg/kg/day, respectively for 4 weeks. Another group of ten animals per sex received the vehicle alone (corn oil) and as acted as a control group. The test item was administered as a solution in the vehicle under a constant dosage-volume of 5 mL/kg/day. The actual test item concentrations in the dose formulations prepared for use in Weeks 1 and 4 were determined using a Gas Chromatography with FID detection analytical method. The animals were checked daily for mortality and clinical signs. Detailed clinical examinations were performed weekly and a Functional Observation Battery (FOB) was conducted in Week 4. Body weight was recorded once pre-test, on the first day of treatment and then weekly. Food consumption was recorded weekly throughout the dosing period. Hematology, blood biochemistry urinalysis and thyroid hormone investigations were performed at the end of the treatment and treatment-free periods (for blood biochemistry and thyroid hormones). The estrous cycle was determined for all females sacrificed at the end of the treatment period, over 4 consecutive days. On completion of the treatment period, the animals were sacrificed and a full macroscopic post-mortem examination was performed. Designated organs were weighed and selected tissues were preserved. A microscopic examination was performed on designated tissues from the control- and high-dose animals and on liver and thyroids in both sexes and kidneys of males from the low- and intermediate-dose animals sacrificed at the end of the treatment period.

Actual concentrations of the test item in the administered dose formulations analyzed in Weeks 1 and 4 remained within an acceptable range (-2% to +1%) when compared to the nominal concentrations. No test item was observed in the control dose formulation. No test item-related deaths occurred during the study. Test item-related clinical signs such as ptyalism, reflux at dosing and thin appearance were observed in females given 300 mg/kg/day.

No neurologic, autonomic or behavioral changes that could be related to the treatment were observed on Day 26 during the FOB assessment phase. Body weight and body weight gain were unaffected in both sexes by the test item treatment. Food consumption was unaffected by the test item treatment in both sexes. Estrous cycle was unaffected by the test item treatment. At hematology investigations, no test item-related changes were noted. Test item-related changes of minor toxicological importance in blood biochemistry parameters consisted of higher sodium, total protein, total cholesterol levels and lower glucose and triglycerides levels. These findings were no longer observed at the end of the treatment-free period in both sexes but lower inorganic phosphorus and proteins levels were observed in males given 300 mg/kg/day. Lower urinary pH value was considered to be of minor toxicological importance due to the minimal amplitude and isolated nature, was noted in males given 300 mg/kg/day. At histopathology, the test item administration at 300 mg/kg/day induced slight or moderate, reversible centrilobular hepatocellular hypertrophy in both sexes. This correlated at 300 mg/kg/day with reversible moderate increases in liver weights in both sexes and macroscopic enlargement and increased lobular pattern in males. There was also minimal, reversible hypertrophy of thyroid follicular cells in both sexes, correlated with increased thyroid weights in males. Both liver and thyroid hypertrophy were not considered as adverse, but rather adaptive changes. In the kidneys from males only, there was an increased incidence and severity of tubular hyaline droplets positively stained with an antibody to alpha-2u-globulin, and correlated with partially reversible increased kidney weights. At the end of the treatment-free period, microscopic tubular hyaline droplets were still increased in incidence compared to controls. This renal finding specific to the male rat is considered to be non-relevant for human. The test item administration at 100 mg/kg/day induced minimal centrilobular hepatocellular hypertrophy in two males and one female in the liver and minimal increases in kidney weights in males only.

The test item administration at 30 mg/kg/day did not induce any macroscopic or organ weight changes.

The toxicity of Luperox 230 was evaluated after daily administration (gavage) to rats at dose-levels of 30,100 and 300 mg/kg/day for 4 weeks. On completion of the treatment period, designated animals were held for a 2-week treatment-free period in order to evaluate the reversibility of any findings. Consequently, under the experimental conditions of the study, the No Observed Adverse Effect Level (NOAEL) after the 4-week treatment period was established at 300 mg/kg/day.