Registration Dossier

Administrative data

Description of key information

In an OECD 408 test (GLP) the No Observed Adverse Effect Level (NOAEL) was established at 300 mg/kg/day and the No Observed Effect Level (NOEL) at 100 mg/kg/day in males and females taking into account that the adverse effects observed in male rat kidneys are not relevant for humans.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 December 2014 - 17 April 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories Italia, Calco, Italy
- Age at study initiation: approximately 6 weeks old on the day of treatment
- Mean body weight at study initiation: the males had a mean body weight of 215 g (range: 196 g to 238 g) and the females had a mean body weight of 180 g (range: 160 g to 205 g)
- Fasting period before study: no
- Housing: the animals were housed in twos 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 study.

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: 11 December 2014 to 17 April 2015.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was put at +2-8°C for thawing and then homogenized before any sampling.
Care was taken to not use glass bottles but HDPE (high-density polyethylene) bottles (for reconditioning if performed) and PP (polypropylene) bottles (for formulations).

The test item was administered as an emulsion in the vehicle. It was mixed with the required quantity of vehicle (considering a test item density of 1). All the formulation preparation process was performed on ice except weighing (test item, vehicle and formulation bottles kept on ice; formulation homogenization and division into daily flasks performed on ice). The test item and formulations were never at room temperature except for test item weighing during formulation preparation.

Dose formulations were prepared for use up to 9 days, stored at +2-8°C and protected from light and delivered to the study room on ice and protected from light.

VEHICLE
- Justification for use and choice of vehicle: emulsion in corn oil stable and homogeneous
- Concentration in vehicle: 0, 2, 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: GC-FID
Test item concentrations: remained within an acceptable range of variation compared to nominal values.
Homogeneity and stability: stable 9 days at +2-8°C protected from light.
Duration of treatment / exposure:
13 weeks followed by a 4-week treatment-free period.
Frequency of treatment:
Daily.
Remarks:
Doses / Concentrations:
10, 100 and 300 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose
6 additional animals per sex in controls and high-dose (investigation of reversibility)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, on the basis of the results of previous toxicology studies performed in the same species.
In a previous study, groups of male and female Sprague-Dawley rats received Luperox 610 daily by gavage at dose-levels of 5, 50 or 750 mg/kg/day (expressed in terms of the active material 3 hydroxy 1,1 dimethylbutyl peroxyneodecanoate) in 0.5% aqueous hydrogel of carboxymethylcellulose for 4 weeks. The test item was clinically well-tolerated inducing only ptyalism at 50 and 750 mg/kg/day. At 50 mg/kg/day, accumulation of intracytoplasmic acidophilic globules was noted in the cortical tubular epithelium of male kidneys. At 750 mg/kg/day, hematology (microcytic anemia, neutrophilia and thrombocytosis) and blood biochemistry (higher urea and creatinine levels and higher alanine aminotransferase and alkaline phosphatase activity) changes were observed, as well as histopathology findings in the liver (higher liver weight, centrolobular hepatocellular hypertrophy).
The No Observed Adverse Effect Level was considered to be 50 mg/kg/day.
In another previous study, Luperox 610 in corn oil was administered daily by gavage to Sprague Dawley rats at dose-levels of 100, 300 or 1000 mg/kg/day for 2 weeks.
At 1000 mg/kg/day, in-life observations were limited to ptyalism in both sexes and lower body weight gain and food consumption at treatment initiation in males. Higher liver weights were recorded in females. At 100 and 300 mg/kg/day, observations were limited to ptyalism in isolated animals.

Thus, based on these available data, the selected dose-levels were 10, 100 and 300 mg/kg/day (expressed in terms of the active material 3 hydroxy 1,1 dimethylbutyl peroxyneodecanoate).

- Rationale for animal assignment: computerized randomization.
Positive control:
no (not required).
Observations and examinations performed and frequency:
MORBIDITY/MORTALITY:
- Time schedule: each animal was checked for mortality and morbidity once a day during the acclimation period and twice a day during the treatment and treatment-free periods, including weekends and public holidays.

CLINICAL OBSERVATIONS:
- Time schedule:once a day.

DETAILED CLINICAL EXAMINATION:
- Time schedule: once before the beginning of the treatment period and then once a week until the end of the study.

FUNCTIONAL OBSERVATION BATTERY:
- Time schedule: all animals (except for the last six group 1 and 4 animals per sex) were evaluated once in Week 12.

BODY WEIGHT:
- Time schedule: once before the beginning of the treatment period, on the first day of treatment and then at least once a week until the end of the study.

FOOD CONSUMPTION:
- Time schedule: once a week, over a 7 day period, during the study.

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule: on all animals, before the beginning of the treatment period and on all control and high-dose animals at the end of the treatment period.

MONITORING OF ESTROUS CYCLE
- Time schedule: the estrous cycle stage was determined for each female planned to be sacrificed at the end of the treatment period.

MOTOR ACITIVITY:
- Time schedule: motor activity was measured by automated infra-red sensor equipment over a 60-minute period.

HAEMATOLOGY, CLINICAL CHEMISTRY, URINALYSIS:
- Time schedule: at the end of the treatment period.

- SEMINOLOGY:
At the end of the treatment period, just before sacrifice, each male was deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and the left epididymis was removed. Animals were then sacrificed.
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.

Epididymal sperm
Sperm from the cauda epididymis was sampled for motility and morphology investigations.
The cauda of the left epididymis was separated from the corpus using a scalpel and subsequently kept at 20°C pending further investigation.

Epididymal sperm motility
The sperm was evaluated on a slide, after appropriate dilution. The number of motile and immotile spermatozoa from a sample of 200 spermatozoa was evaluated under a microscope using a 40-fold magnification. Results were expressed as the proportion of motile and non-motile spermatozoa.

Epididymal sperm morphology
The morphology was determined from a sperm smear, after eosin staining and counting of 100 spermatozoa per slide. Results were expressed as the proportion of spermatozoa in each of the following categories:
- normal,
- normally shaped head separated from flagellum,
- abnormal head separated from flagellum,
- abnormal head with normal flagellum,
- abnormal head with abnormal flagellum,
- normally shaped head with abnormal flagellum.

Epididymal sperm count
After thawing, the left cauda epididymis was weighed, minced and homogenized in a saline-triton solution using a Polytron.
An aliquot of the suspension was sampled and the number of spermatozoa was counted in a microscope slide counting chamber.
Results were expressed as the number of spermatozoa per cauda and per gram of cauda.

Testicular sperm
At necropsy, the left testis was sampled and frozen at -20°C for further sperm count investigation. After thawing, the left testis was weighed (without the albuginea) and ground. The resulting preparation was diluted and sperm heads resistant to homogeneization (i.e. elongated spermatids and mature spermatozoa) were counted in a microscope slide counting chamber.
Results were expressed as a number of sperm heads per gram of testis and the daily sperm production rate was calculated (a time divisor of 6.10 which represents the duration of spermatogenic cycle of homogenization-resistant testicular spermatids).
Sacrifice and pathology:
ORGAN WEIGHTS: see table below
The body weight of each animal was recorded before sacrifice. The organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection.
The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

GROSS PATHOLOGY:
A complete macroscopic post-mortem examination was performed on all animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

HISTOPATHOLOGY:
 all tissues listed in the Tissue Procedure Table for the control and high-dose animals (groups 1 and 4) sacrificed at the end of the treatment period and for animal found dead,
 all macroscopic lesions from all the low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period,
 immunostained kidneys from control and high-dose males (groups 1 and 4) sacrificed at the end of the treatment period.

In addition testicular staging was performed for control and high-dose males (groups 1 and 4). A detailed examination of the testes was performed, using a thorough understanding of tubule development through the different stages of the spermatogenic cycle. Transverse sections of the testes were stained with hematoxylin: PAS in order to detect retained spermatids, missing germ cell layers, multinucleated giant cells or sloughing of spermatogenic cells into the lumen, etc.

According to the microscopic results of the high-dose group, the following tissues were examined:
- kidneys for the low- and intermediate-dose male groups (groups 2 and 3) sacrificed at the end of the treatment period and for the control and high-dose male groups (groups 1 and 4) sacrificed at the end of the treatment-free period,
- liver for the low- and intermediate-dose groups (groups 2 and 3) sacrificed at the end of the treatment period and for the control and high-dose groups (groups 1 and 4) sacrificed at the end of the treatment-free period.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
for food consumption
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
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY:
No test item-related mortality occurred during the study.
One control male was found dead on Day 44 (Week 7). There were no pre-deaths observations.
The cause of the death could not be established at macroscopic post-mortem or microscopic examination.

CLINICAL SIGNS:
Ptyalism was observed in 8/10 males and 3/10 females given 100 mg/kg/day (generally from Week 3) and in 16/16 males and 16/16 females given 300 mg/kg/day (generally from Week 2). This sign, commonly observed when a test item is administered by gavage, was not considered to represent an adverse effect.

The other clinical signs recorded during the study, i.e. scabs, alopecia, chromodacryorrhea, thinning of hair, soiled head or neck, loud breathing, hernia, hunched posture, nodosities on tail and reflux at dosing, were considered to be unrelated to the test item treatment as they were present both in control and test item-treated animals, and/or were reported sporadically in only a few animals and/or without a dose relationship.

BODY WEIGHT (GAIN):
At 300 mg/kg/day in males, slightly lower mean body weight gain was recorded during the first two months of the treatment period (-12% compared with controls), statistically significant only on days 29/36 (+20 g vs. +28 g in controls), leading to a slightly, non significant, lower mean body weight on completion of the treatment period (-8% compared with controls).
This effect was attributed to the test item treatment but considered as non adverse.
A higher mean body weight gain was noted in these animals during the treatment-free period.
At 10 and 30 mg/kg/day in males, no relevant effects were noted on body weight or body weight gain.
At all dose-levels of the test item in females, some instances of statistically significant differences were reported compared with controls. As these were occasional and sometimes showed opposite trends at a same dose-level, they were considered to be unrelated to the test item treatment.

FOOD CONSUMPTION:
At 300 mg/kg/day in males, minimally lower mean food consumption was recorded during the first 6 weeks of the treatment period (-4% compared with controls).
This tendency towards lower food consumption was attributed to the test item treatment and correlated with the lower body weight gain observed in this group.
The mean food consumption was slightly higher in these animals during the treatment-free period.
At 10 and 30 mg/kg/day in males and at all dose-levels of the test item in females, no relevant effects were noted on food consumption.

OPHTHALMOSCOPIC EXAMINATION:
No ophthalmology findings were observed at the end of the treatment period.

NEUROBEHAVIOURAL EXAMINATION:
No behavioral or neurological abnormalities were observed during the tests in any treated animal.
Motor activity (60-minute recording period) was unaffected by the test item treatment.

HAEMATOLOGY:
No test item-related effects on the hematology parameters were observed at the end of the treatment or treatment-free period.

The only statistically significant differences between control and test item-treated animals, namely in red and white blood parameters (both sexes) and prothrombin time (females), were considered of no toxicological importance as they were of low magnitude and/or noted with no dose-relationship.

CLINICAL CHEMISTRY:
At the end of the treatment period, treatment-related blood biochemistry findings (when compared with mean control values) were observed in males given 300 mg/kg/day and consisted of:
- minimally higher mean creatinine level (+21% vs. controls),
- minimally higher mean alanine aminotransferase activity in males (+75% vs. controls).

The changes correlated with microscopic findings observed in the male kidneys.
The other statistically significant differences observed between control and test item-treated animals, namely in glucose (males) and inorganic phosphorus (females) levels and in albumin to globulin ratio (females), were considered to be incidental and not test item-related as they were of low magnitude and/or noted with no dose-relationship.
At the end of the treatment-free period, the changes in creatinine level and alanine aminotransferase activity were no longer observed.

URINALYSIS:
At the end of the treatment period, a lower pH value was noted in males given 300 mg/kg/day (6.3 vs. 7.2 in controls, p<0.05). This change was attributed to the test item treatment and could correlate with the renal microscopic findings.
The other statistically significant differences observed between control and test item-treated animals, i.e. higher mean specific gravity in females given 30 or 100 mg/kg/day, were considered to be incidental and not test item-related as they were of low magnitude.
No relevant changes were noted in the qualitative or semi-quantitative parameters.

At the end of the treatment-free period, the change in pH value was no longer observed.

SEMINOLOGY:
No test item-related effects were noted at seminology investigations.

ORGAN WEIGHTS:
At the end of the treatment period
There were test item-related organ weight increases in kidneys and liver.

The significance concerned the absolute or relative-to body organ weights values and not the percentages.

The final body weight was considered to be minimally decreased with the test item administration.

There were higher absolute and relative-to-body kidney weights in males and females treated at 300 mg/kg/day, and higher relative-to-body kidney weights in males treated at 100 mg/kg/day (p<0.05 or 0.01; up to +29%). These differences correlated with microscopic lesions in males at 100 and 300 mg/kg/day (hyaline droplets, degeneration/necrosis in tubular cells, tubular basophilia, fibrosis and infiltrate of mononuclear cells).

There were higher absolute and/or relative-to-body liver weights in males and females treated at 300 mg/kg/day (p<0.01; up to +23%), and higher absolute liver weights in females treated at 100 mg/kg/day (p<0.05; +12%). These differences correlated with microscopic hepatocellular hypertrophy in males and females.

The other absolute and relative-to-body weight changes were not considered to be test item related because they were of insufficient magnitude, not dose-related and uncorrelated with microscopic findings, including the variations in absolute heart weights in males and females treated at 300 mg/kg/day, of opposite trend, and the decrease in absolute and relative-to-body uterus weights from females treated at 10, 100 or 300 (not dose-related and considered to be related to estrus cycle).

At the end of the treatment-free period
There were test item-related increased kidney weights in males previously treated at 300 mg/kg/day.
The significance concerned the organ weights values and not the percentages.

The final body weight was considered to be unaffected by the test item administration at the end of the treatment-free period.

There were minimally higher relative-to-body kidney weights in males previously treated at 300 mg/kg/day (p<0.01; +10%). These differences were considered to be most probably the contribution of the decreased body weight, although there were some microscopic findings in the kidneys of these males.

The other absolute and relative-to-body weight changes were not considered to be test item related because they were of insufficient magnitude, not dose-related and uncorrelated with microscopic findings, including the minimal variations in relative-to-body left epididymis and right testis.

GROSS PATHOLOGY:
Unscheduled deaths
There were no noteworthy gross findings in one control male but this animal was found cannibalized with absence of the mandibular gland, mandibular lymph node, eye, harderian gland of the right side, and of the tongue.

Terminal sacrifice
At the end of the treatment period
There was test item-related gross tan discoloration of kidneys in 10/10 males treated at 300 mg/kg/day. This correlated with the increased kidney weights and microscopic findings (hyaline droplets, degeneration/necrosis in tubular cells, tubular basophilia, fibrosis and infiltrate of mononuclear cells). This was not seen in females.

The few other gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age because they were non dose related, isolated and/or correlated with common background lesions, including the adrenal gland cyst, the small unilateral testis and epididymis, adhesion of adipose tissue to pancreas, the unilateral focal white discoloration in kidney and the deformed lobe in the liver (in 1/10 males or females treated at 300 mg/kg/day for each of these lesions).

At the end of the treatment-free period
There were no test item-related gross findings.
The few gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age because they were non dose related, isolated and/or correlated with common background lesions, including the unilateral dilated pelvis, the focal small yellow discoloration in the liver and unilateral ovarian cyst (in 1/6 males or females previously treated at 300 mg/kg/day for each of these lesions).

HISTOPATHOLOGY: NON-NEOPLASTIC:
Unscheduled deaths
There were no noteworthy microscopic findings in the cannibalized control male.

Terminal sacrifice
At the end of the treatment period

With a dose-related manner, test item-related minimal to marked hyaline droplets were noted in renal tubular epithelium from all males treated at 100 or 300 mg/kg/day and were positive with α2µ-globulin staining performed at 300 mg/kg/day. This finding was associated with minimal to moderate adverse degeneration/necrosis of tubular cells (containing a high content of hyaline droplets) in 3/10 males treated at 300 mg/kg/day. In addition, test item-related increased tubular basophilia, infiltrated of mononuclear cells, fibrosis and dilation of tubules (deep cortex) were also seen in males treated at 100 or 300 mg/kg/day. These lesions correlated with increased kidney weights and tan discoloration.
The renal lesions including degeneration/necrosis were considered to be adverse in males treated at 300 mg/kg/day.

Minimal test item-related hepatocellular hypertrophy was seen in males and females treated at 300 mg/kg/day and correlated with increased liver weights at this dose-level in both sexes. In the absence of degeneration/necrosis associated with this finding and because of the slight magnitude of the weight increase, it was considered to be non-adverse. This finding was not seen at 10 or 100 mg/kg/day, with the exception of a single female at 100 mg/kg/day with minimal hypertrophy correlated with increased liver weight. Since it was an isolated incidence, this was considered to be of equivocal significance.

There were no test item-related findings in the submitted testes or epididymides at microscopic examination.

There were a few other microscopic observations seen only in males or females treated with test item at 300 mg/kg/day. These findings were seen at low incidence, at minimal severity, were not dose-related and/or correlated with background lesions seen in the rats of these strain and age, including the unilateral severe tubular atrophy/degeneration in testis and associated severe reduced epididymis sperm content seen in one high-dose male, the minimal portal inflammation in the liver from one high-dose female, the focal capsule inflammation in the liver from one high-dose female and the focal chronic inflammation in the renal capsule from two high-dose females. They were considered to be consistent with spontaneous findings and unrelated to test item.

At the end of the treatment-free period
Test item-related increased incidence and severity of tubular basophilia and dilation, together with minimal tubular degeneration/necrosis were noted in the kidneys from the males previously treated at 300 mg/kg/day. These lesions had no gross correlates.
In view of the minimal degeneration/necrosis, although seen in only one male, the renal lesions were considered to be adverse in males previously treated at 300 mg/kg/day. This suggested an ongoing reversibility of these changes.

Conversely, it is noteworthy that there were no hyaline droplets in renal tubular epithelium at the end of the treatment-free period.

There were no hepatocellular hypertrophy in males and females previously treated at 300 mg/kg/day, suggesting complete recovery of this change after the treatment-free period.

Pathology discussion
Presence of hyaline droplets in kidneys of males correlated with increases in kidney weights at necropsy. These hyaline droplets corresponded to α2µ-globulin that were confirmed by immunohistochemistry (positive staining with antibody for this globulin). These were observed with a dose-related trend and were associated at 100 and 300 mg/kg/day with a higher incidence and/or severity of tubular basophilia. Alpha 2µ globulin is known to increase after treatment with a wide range of drugs or chemicals (Greaves, 2012). The prolonged accumulation of hyaline droplets is associated with chronic cell damage and increased cell turnover, which may explain the tubular basophilia seen in groups 3 and 4 and the degeneration/necrosis seen in group 4. These findings were therefore considered to be adverse at 300 mg/kg/day. Although human excretes proteins of a similar nature, they are found in only trace amounts and therefore this finding is considered to be non-relevant for human.

Greaves P. (2012) Histopathology of preclinical toxicity studies, Academic Press, Fourth edition.
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: male rats specific α2µ-globuline hyaline droplets nephropathy
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

Mean body weight (BW) gain /Mean body weight (expressed in g)

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

10

100

300

0

10

100

300

Treatment period

Mean BW gain days 1/29

+209

+210

+208

+194

+68

+76

+85*

+86**

Mean BW gain days 29/57

+92

+87

+80

+71

+30

+29

+32

+26

Mean BW gain days 57/91

+52

+48

+53

+43

+28

+17

+24

+24

Mean BW gain days 1/91

+350

+344

+340

+308

+126

+122

+140

+136

Mean BW gain days 1/91

% from controls

-

-2

-3

-12

-

-3

+11

+8

Mean body weight on day 91

565

560

558

522

304

305

319

315

% from controls

-

-1

-1

-8

-

0

+5

+4

Treatment-free period

Mean BW gain days 91/119

+16

-

-

+26

+3

-

-

-3

Mean BW on day 119

612

-

-

562

306

-

-

307

% from controls

-

-

-

-8

-

-

-

0

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

Mean food consumption (g/animal/day)

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

10

100

300

0

10

100

300

Treatment period

Days 1 to 29

29.1

29.0

29.8

28.0

17.9

18.7

19.0

19.1

Days 29 to 57

26.1

26.3

26.7

25.8

17.5

17.7

18.6

18.1

Days 57 to 91

23.3

24.1

24.3

23.7

16.4

16.6

17.3

17.2

Days 1 to 91

26.2

24.5

26.9

25.8

17.3

17.7

18.3

18.1

% from controls

-

-6

+3

-2

-

+2

+6

+5

Treatment-free period

Days 91 to 119

29.1

-

-

31.1

19.7

-

-

21.0

% from controls

-

-

-

+7

-

-

-

+6

-: not applicable.

Blood biochemistry parameter changes

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

10

100

300

0

10

100

300

End of treatment period

Creatinine (µmol/L)

29.23

29.71

31.58

35.39**

34.38

34.43

33.60

33.91

ALAT (IU/L)

28

30

33

49**

28

27

26

34

End of treatment-free period

Creatinine (µmol/L)

26.18

-

-

26.96

31.95

-

-

32.77

ALAT (IU/L)

35

-

-

32

32

-

-

27

Statistically significant from controls:**: p<0.01, ALAT:alanine aminotransferase,-: not applicable.

Organ weight differences in percentages at the end of the treatment period

 

Sex

Male

Female

Group

2

3

4

2

3

4

Dose-level (mg/kg/day)

10

100

300

10

100

300

Exam. animals / Num. of animals

10/10

10/10

10/10

10/10

10/10

10/10

- Final body weight

+2

+2

-7

-1

+3

+3

- Kidneys

.absolute

+4

+13

+20**

-1

+9

+12**

.relative-to-body

+2

+11*

+29**

0

+6

+9*

- Liver

.absolute

0

+4

+10

+1

+12*

+23**

. relative-to-body

-2

+3

+19**

+2

+9

+20**

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

Organ weight differences in percentages at the end of the treatment-free period

 

Sex

Male

Female

Group

4

4

Dose-level (mg/kg/day)

300

300

Exam. animals / Num. of animals

6/6

6/6

- Final body weight

-9

-1

- Kidneys

. absolute

0

-1

. relative-to-body

+10*

0

Statistically significant from controls: *: p<0.05.

Test item-related gross findings at terminal sacrifice

 

Sex

Male

Group

1

2

3

4

Dose-level (mg/kg/day)

0

10

100

300

Number of animals

9

10

10

10

Tan discoloration of kidneys

0

0

0

10

 

Conclusions:
The toxicity of the test item was evaluated after daily oral administration (gavage) to Sprague Dawley rats at dose levels of 10, 100 or 300 mg/kg/day expressed in terms of the active material (3-hydroxy-1,1-dimethylbutyl peroxyneodecanoate) for 13 weeks followed by a 4-week treatment-free period.
At 300 mg/kg/day, treatment-related changes were restricted to ptyalism in both sexes, a slightly lower body weight, body weight gain and food consumption in males, a higher creatinine level and alanine aminotransferase activity in males associated with microscopic changes in kidneys (male rats specific α2µ-globuline hyaline droplets nephropathy) and a non adverse hypertrophy of liver in both sexes.
At 100 mg/kg/day, the treatment produced only non adverse in-life (ptyalism) and microscopic (kidneys) changes.
At 10 mg/kg/day, no effects were observed.
Consequently, under the experimental conditions of the study, the No Observed Adverse Effect Level (NOAEL) was established at 300 mg/kg/day and the No Observed Effect Level (NOEL) at 100 mg/kg/day in males and females taking into account that the adverse effects observed in male rat kidneys are not relevant for humans.
Executive summary:

The potential toxicity of Luperox 610 was evaluated following daily oral administration (gavage) to rats for 13 weeks. On completion of the treatment period, designated animals were held for a 4-week treatment-free period in order to evaluate the reversibility of any findings. One group of 16 male and 16 female Sprague-Dawley rats was treated daily by the oral route (gavage) with the test item, at the dose-level of 300 mg/kg/day (group 4) for 13 weeks. Two other groups of 10 males and 10 females were treated with the test item at dose-levels of 10 (group 2) or 100 (group 3) mg/kg/day. The dose-levels were expressed in terms of the active material (3-hydroxy-1,1-dimethylbutyl peroxyneodecanoate) using a correction factor of 1.112 to take into account a concentration of 89.9% in Luperox 610. One control group of 16 males and 16 females received the vehicle only (corn oil) under the same experimental conditions, and acted as a control group (group 1). A constant dosage-volume of 5 mL/kg/day was used. At the end of the treatment period, the animals were sacrificed, except for the last six group 1 and 4 animals per sex, which were kept for a 4-week treatment-free period. The actual test item concentrations in the dose formulations prepared for use in Weeks 1, 4, 8 and 13 were determined using a gas chromatography with flame ionization 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 12. Body weight was recorded pre-test, on the first day of treatment and then once a week. Food consumption was recorded weekly. Ophthalmological examinations were performed on all animals before the beginning of the study and on control and high-dose animals at the end of the treatment period (Week 13). Hematology, blood biochemistry and urinalysis investigations were performed at the end of the treatment and treatment-free periods (Weeks 13 and 17). The estrous cycle was determined on all females daily for 21 consecutive days at the end of the treatment period. Seminology investigations (count, motility and morphology) were performed on all males at time of sacrifice at the end of the treatment period. On completion of the treatment or treatment-free 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 (including testicular staging) was performed on designated tissues from control and high-dose animals sacrificed at the end of the treatment period and from prematurely dead animals. Kidneys of control and high-dose males sacrificed at the end of the treatment period were immunostained with an antibody forα2µ-globulin. The kidneys (male) and liver (both sexes) of the low- and intermediate-dose animals (groups 2 and 3) sacrificed at the end of the treatment period and of the control and high-dose animals (groups 1 and 4) sacrificed at the end of the treatment-free period were also microscopically examined as these organs revealed microscopic changes at the end of the treatment period.

Actual concentrations of the active material in the dosage forms administered to animals during the study remained within an acceptable range (-8.2% to +8.1%) compared to the nominal concentrations. No test item-related mortality occurred during the study. Ptyalism was observed with a dose-related incidence at 100 and 300 mg/kg/day. This sign, commonly observed when a test item is administered by gavage, was not considered as an adverse effect. The Functional Observation Battery was unaffected by the test item treatment. Body weight gain was slightly lower in males given 300 mg/kg/day during the first 2 months of the treatment period (-12% vs. controls), leading to a slightly lower body weight on completion of the treatment period (‑8% vs. controls). This change was associated with minimally lower food consumption during the first 6 weeks of the treatment period (-4% vs. controls). No ophthalmology findings were observed at the end of the treatment period. Estrous cycle was not altered by the test item treatment. The epididymal sperm motility and morphology and the spermatozoa count in epididymides or testes were unaffected by the test item treatment. At hematology investigations, no test item-related changes were noted. At blood biochemistry investigations, creatinine level and alanine aminotransferase activity were minimally higher in males given 300 mg/kg/day (+21% vs. controls). At urinary investigations, lower pH value was noted in males given 300 mg/kg/day. Reversibility of these laboratory findings was noted at the end of the treatment-free period. At pathology investigations, increased kidney and liver weights were recorded at 100 and/or 300 mg/kg/day at the end of the treatment period. At macroscopic post-mortem examination, tan discoloration of kidneys was observed in 10/10 males treated at 300 mg/kg/day. This correlated with the increased kidney weights and microscopic findings. It was not seen in females. Minimal to marked test item-related microscopic hyaline droplets in renal tubular epithelium were noted in 10/10 males treated at 100 or 300 mg/kg/day. These hyaline droplets consisted ofα2µ-globulin, confirmed by immunohistochemistry staining performed at 300 mg/kg/day. This finding was associated with adverse minimal to moderate degeneration/necrosis of tubular cells in 3/10 males treated at 300 mg/kg/day. In addition, test item-related increased tubular basophilia, infiltrated of mononuclear cells, fibrosis and dilation of tubules were also seen in males treated at 100 or 300 mg/kg/day. The degeneration/necrosis of tubular cells, tubular basophilia and dilation were seen at the end of the treatment-free period, but without hyaline droplets in renal tubular epithelium. This suggested an ongoing reversibility of these changes. Treatment-related non adverse minimal hepatocellular hypertrophy was seen in males and females treated at 300 mg/kg/day and correlated with increased liver weights at this dose-level in both sexes. Complete recovery of this change was observed at the end of the treatment-free period.

In conclusion:

At 300 mg/kg/day, treatment-related changes were restricted to ptyalism in both sexes, a slightly lower body weight, body weight gain and food consumption in males, a higher (reversible) creatinine level and alanine aminotransferase activity in males associated with microscopic changes in kidneys (male rats specificα2µ-globuline hyaline droplets nephropathy) and a non-adverse hypertrophy of liver in both sexes.  

At 100 mg/kg/day, the treatment produced only non-adverse in-life (ptyalism) and microscopic (kidneys) changes.  

At 10 mg/kg/day, no effects were observed.

Consequently, under the experimental conditions of the study, the No Observed Adverse Effect Level (NOAEL) was established at 300 mg/kg/day and the No Observed Effect Level (NOEL) at 100 mg/kg/day in males and females taking into account that the adverse effects observed in male rat kidneys are not relevant for humans.

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP guideline study

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The potential toxicity of Luperox 610 was evaluated following daily oral administration (gavage) to rats for 13 weeks (Haag, 2015). On completion of the treatment period, designated animals were held for a 4-week treatment-free period in order to evaluate the reversibility of any findings. One group of 16 male and 16 female Sprague-Dawley rats was treated daily by the oral route (gavage) with the test item, at the dose-level of 300 mg/kg/day (group 4) for 13 weeks. Two other groups of 10 males and 10 females were treated with the test item at dose-levels of 10 (group 2) or 100 (group 3) mg/kg/day. The dose-levels were expressed in terms of the active material (3-hydroxy-1,1-dimethylbutyl peroxyneodecanoate) using a correction factor of 1.112 to take into account a concentration of 89.9% in Luperox 610. One control group of 16 males and 16 females received the vehicle only (corn oil) under the same experimental conditions, and acted as a control group (group 1). A constant dosage-volume of 5 mL/kg/day was used. At the end of the treatment period, the animals were sacrificed, except for the last six group 1 and 4 animals per sex, which were kept for a 4-week treatment-free period. The actual test item concentrations in the dose formulations prepared for use in Weeks 1, 4, 8 and 13 were determined using a gas chromatography with flame ionization 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 12. Body weight was recorded pre-test, on the first day of treatment and then once a week. Food consumption was recorded weekly. Ophthalmological examinations were performed on all animals before the beginning of the study and on control and high-dose animals at the end of the treatment period (Week 13). Hematology, blood biochemistry and urinalysis investigations were performed at the end of the treatment and treatment-free periods (Weeks 13 and 17). The estrous cycle was determined on all females daily for 21 consecutive days at the end of the treatment period. Seminology investigations (count, motility and morphology) were performed on all males at time of sacrifice at the end of the treatment period. On completion of the treatment or treatment-free 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 (including testicular staging) was performed on designated tissues from control and high-dose animals sacrificed at the end of the treatment period and from prematurely dead animals. Kidneys of control and high-dose males sacrificed at the end of the treatment period were immunostained with an antibody forα2µ-globulin. The kidneys (male) and liver (both sexes) of the low- and intermediate-dose animals (groups 2 and 3) sacrificed at the end of the treatment period and of the control and high-dose animals (groups 1 and 4) sacrificed at the end of the treatment-free period were also microscopically examined as these organs revealed microscopic changes at the end of the treatment period.

Actual concentrations of the active material in the dosage forms administered to animals during the study remained within an acceptable range (-8.2% to +8.1%) compared to the nominal concentrations. No test item-related mortality occurred during the study. Ptyalism was observed with a dose-related incidence at 100 and 300 mg/kg/day. This sign, commonly observed when a test item is administered by gavage, was not considered as an adverse effect. The Functional Observation Battery was unaffected by the test item treatment. Body weight gain was slightly lower in males given 300 mg/kg/day during the first 2 months of the treatment period (-12% vs. controls), leading to a slightly lower body weight on completion of the treatment period (‑8% vs. controls). This change was associated with minimally lower food consumption during the first 6 weeks of the treatment period (-4% vs. controls). No ophthalmology findings were observed at the end of the treatment period. Estrous cycle was not altered by the test item treatment. The epididymal sperm motility and morphology and the spermatozoa count in epididymides or testes were unaffected by the test item treatment. At hematology investigations, no test item-related changes were noted. At blood biochemistry investigations, creatinine level and alanine aminotransferase activity were minimally higher in males given 300 mg/kg/day (+21% vs. controls). At urinary investigations, lower pH value was noted in males given 300 mg/kg/day. Reversibility of these laboratory findings was noted at the end of the treatment-free period. At pathology investigations, increased kidney and liver weights were recorded at 100 and/or 300 mg/kg/day at the end of the treatment period. At macroscopic post-mortem examination, tan discoloration of kidneys was observed in 10/10 males treated at 300 mg/kg/day. This correlated with the increased kidney weights and microscopic findings. It was not seen in females. Minimal to marked test item-related microscopic hyaline droplets in renal tubular epithelium were noted in 10/10 males treated at 100 or 300 mg/kg/day. These hyaline droplets consisted ofα2µ-globulin, confirmed by immunohistochemistry staining performed at 300 mg/kg/day. This finding was associated with adverse minimal to moderate degeneration/necrosis of tubular cells in 3/10 males treated at 300 mg/kg/day. In addition, test item-related increased tubular basophilia, infiltrated of mononuclear cells, fibrosis and dilation of tubules were also seen in males treated at 100 or 300 mg/kg/day. The degeneration/necrosis of tubular cells, tubular basophilia and dilation were seen at the end of the treatment-free period, but without hyaline droplets in renal tubular epithelium. This suggested an ongoing reversibility of these changes. Treatment-related non adverse minimal hepatocellular hypertrophy was seen in males and females treated at 300 mg/kg/day and correlated with increased liver weights at this dose-level in both sexes. Complete recovery of this change was observed at the end of the treatment-free period.

In conclusion:

At 300 mg/kg/day, treatment-related changes were restricted to ptyalism in both sexes, a slightly lower body weight, body weight gain and food consumption in males, a higher (reversible) creatinine level and alanine aminotransferase activity in males associated with microscopic changes in kidneys (male rats specificα2µ-globuline hyaline droplets nephropathy) and a non-adverse hypertrophy of liver in both sexes.  

At 100 mg/kg/day, the treatment produced only non-adverse in-life (ptyalism) and microscopic (kidneys) changes.  

At 10 mg/kg/day, no effects were observed.

Consequently, under the experimental conditions of the study, the No Observed Adverse Effect Level (NOAEL) was established at 300 mg/kg/day and the No Observed Effect Level (NOEL) at 100 mg/kg/day in males and females taking into account that the adverse effects observed in male rat kidneys are not relevant for humans.

Luperox 610, when administered daily by oral route to Sprague-Dawley rats for 4 weeks (OECD 407 test, GLP study) at 5, 50 or 750 mg/kg/day was clinically well-tolerated at all dose levels, inducing only ptyalism at 50 and 750 mg/kg/day and slight growth retardation in the males given 750 mg/kg/day (Richard, 1993a). The 50 mg/kg/day dose induced the accumulation of intracytoplasmic acidophilic globules in cortical tubular epithelium of kidneys in the males. The 750 mg/kg/day dose induced haematological and blood biochemical changes, and histopathological changes in the liver of the males and the females (centrolobular hepatocellular hypertrophy, bile ductular thrombi, brownish pigment accumulation in hepatic and/or biliary epithelial cells) and in the kidneys (accumulation of acidophilic globules in cortical tubular epithelium) of the males. 


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Key study

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; urogenital: kidneys

Justification for classification or non-classification

According to EU Regulation (EC) N0. 1272/2008 (CLP), the substance is not classified for repeated dose toxicity.