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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The potential toxic effects of n-butyl-4,4-bis(tert-butylperoxy)valerate (Luperox 230) was evaluated following daily oral administration (gavage), to male and female rats from before mating, through mating and, for females, through gestation until Day 14 post-partum (p.p.) inclusive (Bentz, 2017). This study provides initial information on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition. Three groups of 10 male and 10 female (groups 2 to 4) Sprague-Dawley rats received the test item daily by oral (gavage) administration before mating, through mating and, for the females, through gestation until Day 14 p.p. The test item was administered as a solution in the vehicle, corn oil, at dose levels of 100, 300 and 750 mg/kg/day. Another group of 10 males and 10 females received the vehicle alone, under the same experimental conditions and acted as a control group. A constant dose-volume of 5 mL/kg/day was used. The concentrations of the dose formulations were checked in study Weeks 1, 3, 6 and 9. The animals were checked twice daily for mortality and morbidity and once daily for clinical signs during the treatment period. Body weight and food consumption were recorded once a week during pre-mating and mating periods (food consumption not during mating), and during gestation on Days 0, 7, 14 and 20 post-coitum (p.c.) and lactation on Days 1, 4, 8 and 13 p.p. The animals were paired for mating after 4 weeks of treatment and the females were allowed to litter and rear their progeny until Day 14 p.p. The total litter sizes and number of pups of each sex were recorded after birth. Pups were observed daily for clinical signs, abnormal behavior and external abnormalities and weighed on Days 1, 4, 8 and 13 p.p. The physical development of pups was assessed by measuring the anogenital distance on Day 4 p.p. and by counting the number of nipples and areolae in male pups on Day 13 p.p. Litter standardization was performed on Day 4 p.p. in order to reduce the litter size-induced variability in pup growth and development. Thyroid hormone (TSH and T4) plasma levels were determined on the day of sacrifice in parental males and in Day 14 p.p. pups. Males were sacrificed after at least 11 weeks of treatment and females on Day 15 p.p. Final body weights and selected organs weights (kidneys, liver, thyroids with parathyroids and male reproductive organs) were recorded and a macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on ovaries (with oviducts), epididymides and/or testes in the control and high-dose groups, on liver, thyroids and/or kidneys in all dose groups, on a2µ-globulin protein immunostained kidneys of control and high-dose males, and on all macroscopic lesions. A macroscopic post-mortem examination was performed on pups on Day 14 p.p., including those found dead before study termination. 

No test item was observed in the control dose formulations and the test item concentrations in the analyzed dose formulations were within an acceptable range of variations. There were no unscheduled deaths in test item groups of parental animals. Test item-related clinical signs consisted in piloerection and/or round back in a total of four females at 750 mg/kg/day, chromodaccryorrhea in 3/10 males at 300 mg/kg/day and at 750 mg/kg/day, and mainly ptyalism in all males and females at 750 mg/kg/day. There were no toxicologically significant effects on mean body weight in either sex but in males a lower mean body weight gain over the treatment period at 750 mg/kg/day vs. controls (+211 g vs.+255 g, p<0.05) was recorded. At this dose, mean food consumption was lower than in controls in the first week of treatment for both sexes (-18% for males, p<0.01 and -28% for females, p<0.001) but there were no toxicologically significant effects at 100 and 300 mg/kg/day on this parameter. During the 2 weeks of estrous cycle evaluation before mating, 5/10 females had an estrous ending at least after 3 days at 750 mg/kg/day and 2/10 at 300 mg/kg/day, vs. none in controls. There were no effects on estrous cycles at 100 mg/kg/day considered to be of toxicological significance. There were no test item-related effects on mean pairing, mating and fertility data and no toxicologically significant effects on mean delivery data. There were no effects on mean T4 level. At 750 mg/kg/day and when compared with controls, mean TSH plasma concentration in F0 males was 2-fold higher. This result correlated with thyroid microscopic results and was considered to be an indirect effect of the test item through the increase in hepatic metabolism. There were no effects at 100 and 300 mg/kg/day on mean TSH level. At 750 mg/kg/day, there were findings in the liver (mainly adverse hepatocellular degeneration/necrosis; adverse hyperplasia of bile ducts in females only; hepatocellular hypertrophy correlated with increased weights and gross enlargement; periportal inflammatory cells and pigment in females), kidneys in males only (adverse tubular degeneration/necrosis; hyaline droplets in tubular epithelium correlated with increased weights; tubular basophilia and dilation) and thyroid glands (follicular cell hypertrophy correlated with increased weights). At 300 mg/kg/day, there were findings in the liver (non-adverse hepatocellular hypertrophy correlated with increased weights), kidneys in males only (non-adverse hyaline droplets in tubular epithelium correlated with increased weights; non-adverse tubular basophilia and dilation) and thyroid glands (non-adverse follicular cell hypertrophy correlated with increased weights in males). At 100 mg/kg/day, there were findings in the liver (non-adverse hepatocellular hypertrophy correlated with increased weights in males only), kidneys in males only (non-adverse hyaline droplets in tubular epithelium) and thyroid glands (non-adverse follicular cell hypertrophy).

Pups viability index on Day 4 p.p. was low at 750 mg/kg/day (89.6%). This effect could be test item treatment-related and adverse. There were no test item-related pup clinical signs. Mean pup body weight was affected at 750 mg/kg/day on Day 1 p.p. (-14% vs. controls for both sexes, p<0.01) and thereafter (down to -18%vs. controls, p<0.01, or -21%, p<0.001, for males or females on Day 8 p.p., respectively). The same tendency was observed at 300 mg/kg/day, particularly in females on Day 8 p.p. (-11% vs. controls, p<0.05) and on Day 13 p.p. (-10%, p<0.05). Mean body weight gains were lower at 750 mg/kg/day when compared with controls throughout the lactation period. These test item effects were considered to be adverse. There were no toxicologically significant effects at 100 mg/kg/day in mean pup body weight and mean pup body weight change. There were no test item-related effects on pup sex ratio at birth, on male pup development (absence of nipples or areolae) or on mean anogenital distance data. There were no effects on mean T4 level and no toxicologically significant effects on mean TSH level. There were no test item-related necropsy findings in pups.

n-butyl-4,4-bis(tert-butylperoxy)valerate (Luperox 230) was administered daily by gavage to male and female Sprague-Dawley rats for 4 weeks before mating, during mating and, for males until 11 weeks of treatment have elapsed, for females through gestation and lactation until Day 14 p.p., at dose-levels of 100, 300 and 750 mg/kg/day. Under the experimental conditions and results of this study:

.            the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 300 mg/kg/day for males and females based on the absence of adverse effects up to this dose and on the adverse effects on liver (both sexes) and kidneys (males) at microscopy at 750 mg/kg/day,

.            the NOAEL for parental reproductive performance was considered to be 750 mg/kg/day, based on the absence of adverse effects on mean reproductive data up to this dose,

.            the No Observed Effect Level (NOEL) for toxic effects on progeny was considered to be 100 mg/kg/day and the Lowest Observed Adverse Effect Level (LOAEL) was considered to be 300 mg/kg/day, based on the effects on mean pup body weights at 300 and 750 mg/kg/day and on pup viability at 750 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 June 2016 - 15 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France
- Age at the beginning of the treatment period: approximately 8 weeks old (for males) and approximately 11 weeks old (for females)
- Body weight at the beginning of the treatment period: a mean body weight of 383 g (range: 358 g to 404 g) for males and a mean body weight of 266 g (range: 235 g to 293 g) for females
- Housing: F0 animals were individually housed, except during mating and lactation, in polycarbonate cages (Tecniplast 2154, 940 cm2)
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: males were acclimated to the study conditions for 7 days before treatment and females were acclimated to the study conditions for 5 days before the beginning of estrous cycle monitoring during the pre-treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C generally
- 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: 28 June 2016 to 15 September 2016.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING FORMULATIONS:
- Solution in the vehicle
- Concentration in vehicle: 20, 60 and 150 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day

Details on mating procedure:

Females were paired with males from the same dose level group. One female was placed with one male, in the latter's cage, during the night.
Confirmation of mating was made in the morning by checking for the presence of a vaginal plug or for sperm in a vaginal lavage.
The day of confirmed mating was designated Day 0 p.c.
Each female was placed with the same male until mating occurred. The pre-coital time was calculated for each female.

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)
Test item concentrations: remained within an acceptable range of variation (-7.6% to +0.8%) when compared to the nominal values
Homogeneity/Stability: The dose formulations containing the test item and prepared as a solution at 2 mg/mL and 200 mg/mL in corn oil were found to be stable after 11 days of storage at room temperature.

Duration of treatment / exposure:

In the males:
- 4 weeks before mating,
- during the mating period,
- until euthanasia after at least 11 weeks in total.
In the females:
- 4 weeks before mating,
- during the mating period,
- during gestation,
- during lactation until Day 14 p.p. inclusive,
- until euthanasia for females with no delivery.

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for dose level selection:
The dose-levels were selected in agreement with the Sponsor, based on the results of:
- a 2-week dose-range finding study performed in the same species. In this study, three groups of Sprague-Dawley rats 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% vs. Day 1) (followed immediately after by an increase in mean body weight gain), and associated with lower mean food consumption (-57%) were recorded in high-dose females during the first 4 days. Slightly lower mean body weight gain (-32%) and food consumption (-20%) were noted in high-dose males at the same period. 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 mean liver weights recorded in males (all dose levels of the test item; up to 37% at 300 mg/kg/day and 69% at 1000 mg/kg/day, vs. controls) and in females (300 and 1000 mg/kg/day; up to 35% at 300 mg/kg/day and up to 71% at 1000 mg/kg/day),
- a 4-week toxicity study followed by a 2-week treatment-free period, performed in the same species. In this study, three groups of Sprague-Dawley rats were treated with the test item at 30, 100 and 300 mg/kg/day. The most relevant findings at the end of the treatment period were a few variations in blood biochemistry (mainly reduction of glucose and triglycerides in 100 and 300 mg/kg/day males, non-dose-related increase of cholesterol in 100 and 300 mg/kg/day females) and in mean organ weights [mainly increase of liver weight in both sexes at 300 mg/kg/day (up to +35%) and in kidney weight in 100 and 300 mg/kg/day males (up to +28%)],
- mean body weight gain was lower than in controls towards the end of the treatment period in 100 and 300 mg/kg/day males but with no dose relationship. At necropsy, the liver was enlarged and/or with accentuated lobular pattern in 3/5 males at 300 mg/kg/day. Microscopic results are not available yet,
- therefore, 750 mg/kg/day was selected as the high-dose level and is expected to show some toxicity. The low-dose and mid dose were selected as they were used in the 4-week study and as they represent a ratio of a 2.5- and 3-fold interval (i.e. 100 and 300 mg/kg/day).

- Rationale for animal assignment: stratification procedure.

Positive control:

no (not required)

Parental animals: Observations and examinations:

MORTALITY/MORBIDITY:
- Time schedule: each animal were checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

CLINICAL SIGNS:
- Time schedule: from arrival, each animal was observed once a day as part of routine examinations. From the start of the treatment period, each animal was observed once a day, at approximately the same time of day, for the recording of clinical signs.

BODY WEIGHT:
- Time schedule: the body weight of each male was recorded on the first day of treatment (Day 1), then at least once a week until euthanasia.
The body weight of each female was recorded on the first day of treatment (Day 1), then once a week until mated, on Days 0, 7, 14 and 20 p.c. (post-coitum) and on Days 1, 4, 8 and 13 p.p.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by each male was measured once a week from the first day of treatment until the start of the mating period. The quantity of food consumed by each female was measured once a week from the first day of treatment until the start of the mating period, during gestation for the intervals Days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval Days 1-4, 4-8 and 8-13 p.p.
During the mating period, food consumption was not measured for males or females.

HORMONES:
The levels of the thyroid hormone (T4) and Thyroid Stimulating Hormone (TSH) were determined respectively by LC-MS/MS or Luminex MAP® technology for pups sampled on Day 14 p.p. and for F0 males sampled at termination.
Plasma samples obtained on Day 4 p.p. from pups and on Day 15 p.p. from F0 females were kept at -80°C but no analyses were performed as decided by the Sponsor.

REPRODUCTION (apart from indices):
- Pre-coital time and duration of gestation were recorded fo each female.

Oestrous cyclicity (parental animals):

The estrous cycle stage was determined from a fresh vaginal lavage each morning:
- during the 2 weeks before the treatment period,
- during the last 2 weeks of the pre-mating period and during the mating period, until the females were mated,
- on Day 15 p.p. before euthanasia.

Sperm parameters (parental animals):

Parameters examined in males of parental generation:
- testis weight (all groups) + microscopic evaluation (control and high-dose groups)
- epididymis weight (all groups) + microscopic evaluation (control and high-dose groups)
- special emphasis to the stages of the spermatogenic cycle and histopathology of testicular interstitial cells (control and high-dose groups)

Litter observations:

STANDARDISATION OF LITTERS:Yes

PARAMETERS EXAMINED:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies, body weight, clinical signs,
- anogenital distance,
- nipples and areolae numbers in male pups.

GROSS EXAMINATION OF DEAD AND SURVIVING PUPS:
- external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE:
- males: after at least 11 weeks of treatment in total,
- females: on Day 15 p.p.
The following F0 females were euthanized by the same way without overnight fasting:
- females which did not deliver: on Days 25-26 p.c. (after a body weight recording to check for a possible un-noticed delivery),
- females with total litter loss.
Pups were euthanized by an intraperitoneal injection of sodium pentobarbital (or by decapitation, see § Laboratory investigations - thyroid hormones), followed by exsanguination when the thyroids were sampled (see § Preservation of tissues):
- pups not selected on Day 4 p.p.: on Day 4 p.p.,
- surviving pups: on Day 14 p.p.


GROSS NECROPSY:
F0 animals
A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all F0 animals including the female which was euthanized for dead litter. Special attention was paid to the reproductive organs.
The numbers of corpora lutea and implantation sites were recorded for females euthanized as scheduled on Day 15 p.p. and for females euthanized on Days 25-26 p.c. due to no delivery.
For the apparently non-pregnant, the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.

PRESERVATION OF TISSUES:
The tissues of F0 animals specified in the Tissue Procedure Table were preserved in 10% buffered formalin (except for the testes and epididymides which were fixed in Modified Davidson's Fixative).

PREPARATION OF HISTOLOGICAL SLIDES (F0 animals):
All tissues required for microscopic examination were trimmed based on the RITA guidelines, when applicable, embedded in paraffin wax (after 96 hours maximum in formalin for kidneys and liver of all groups and both sexes, even in absence of microscopic examination), sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (except testes and epididymides which were stained with hematoxylin/PAS).
One additional kidney slide of the first five control males and of all group 4 males sacrificed at the end of the treatment period will be immunostained for alpla 2µ-globulin protein.

HISTOPATHOLOGY (F0 animals):
A microscopic examination was performed on:
- all tissues listed in the Tissue Procedure Table from all animals of the control and high-dose groups (groups 1 and 4) euthanized at the end of the treatment period (at the end of the mating period for males or on Day 15 p.p. for females),
- liver (males and females), thyroids (males and females) and kidneys (males) from the low- and mid dose groups (groups 2 and 3) euthanized at the end of the treatment period,
- all immunostained kidney slides from males of the control and high-dose groups (groups 1 and 4) euthanized at the end of the treatment period,
- all macroscopic lesions of all groups.

Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

ORGAN WEIGHTS:
The body weight of each F0 animal euthanized as scheduled (after the end of the mating period for males or on Day 15 p.p. for females) was recorded before euthanasia. The organs of those animals specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection. Thyroids with parathyroids of F0 animals (except groups 1 and 4 females) were weighed after fixation (before processing for preparation of histological slides, when applicable).
The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated.

Postmortem examinations (offspring):

SACRIFICE:
Pups were euthanized by an intraperitoneal injection of sodium pentobarbital (or by decapitation, see § Laboratory investigations - thyroid hormones), followed by exsanguination when the thyroids were sampled (see § Preservation of tissues):
- pups not selected on Day 4 p.p.: on Day 4 p.p.,
- surviving pups: on Day 14 p.p..

GROSS NECROPSY:
On all pups (surviving and found dead)
A macroscopic post-mortem examination of the principal thoracic and abdominal organs, comprising also a detailed external examination (including orifices and buccal cavity) was performed on all found dead pups. Particular attention was paid to the external genital organs and internal reproductive organs and to whether the pup has fed (e.g. presence of milk in the stomach). No tissues were preserved.
Pups not selected on Day 4 p.p. were euthanized and discarded without further examination.

A macroscopic post-mortem examination of the principal thoracic and abdominal organs, comprising also a detailed external examination (including orifices and buccal cavity), was performed on pups sacrificed on Day 14 p.p. Particular attention was paid to the external genital organs and internal reproductive organs. No tissues were preserved except those listed in the Tissue Procedure Table.

PRESERVATION OF TISSUES:
Thyroids with parathyroids of one pup/sex/litter euthanized on Day 14 p.p. were preserved in 10% buffered formalin.

ORGAN WEIGTHS: No

HISTOPATHOLOGY: No

Statistics:

Body weight, food consumption and reproductive data:
Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).

Hormones, anogenital distance and post-implantation loss:
CITOX software was used to perform the statistical analysis of these data.

Organ weight:
PATHDATA software was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01).

Reproductive indices:

Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
Post-implantation loss = 100 * (Number of implantation sites - Number of live pups) / Number of implantations
Mating index = 100 * (Number of mated animals / Number of paired animals)
Fertility index = 100 * (Number of pregnant female partners / Number of mated pairs)
Gestation index = 100 * (Number of females with live born pups / Number of pregnant females)

Offspring viability indices:

Live birth index = 100 * (Number of live born pups / Number of delivered pups)
Viability index on Day 4 p.p. = 100 * (Number of surviving pups on day 4 p.p. / Number of live born pups)
Lactation index: 100 * (Number of surviving pups on day 13 p.p./ number of surviving pups on day 4 p.p.)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The following clinical signs were considered to be test item-related:
- ptyalism was noted in all males and all females at 750 mg/kg/day for the most part of the treatment period (especially males) from Week 3,
- piloerection and/or round back were noted in a total of 4/10 females at 750 mg/kg/day for 1 to 5 days from Day 6, vs. none in controls,
- chromodaccryorrhea was recorded in 3/10 males at 300 mg/kg/day and at 750 mg/kg/day (for 37 to 60 days in two of these three males of each group) vs. one affected control male (during 6 days).
They were considered to be non-adverse and of limited toxicological significance (piloerection/round back, chromodaccryorrhea; low incidences and/or transient) or of minor toxicological significance (ptyalism).

Other clinical signs recorded in test item-treated animals (exophtalmos, opacity of eye, chromorhinorrhea, scab, hair loss, cutaneous lesion, reddish vaginal discharge, nodosities, reflux at dosing, pallor of extremities) were not attributed to the test item treatment (no dose-relationship, of common background and/or at isolated occurrence).

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no unscheduled deaths in test item groups.
One control female was sacrificed on Day 8 p.p following the death of its entire litter (most pups found cannibalized on Day 1 p.p.). A difficult parturition had been observed for this female which delivered on Day 24 p.c. and had abdominal breathing on Day 1 p.p. and brownish vaginal discharge on Days 2 and 3 p.p. Its food consumption during lactation was lower than the other control females.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See Table 1.
In males, there were no toxicologically significant effects on mean body weights but a lower mean body weight gain over the treatment period at 750 mg/kg/day than in controls which was considered to be non adverse and of limited toxicological significance.
In females, there were no test item-related effects on mean body weight and mean body weight changes.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See Table 2.
At 750 mg/kg/day in the first week of treatment, mean food consumption was lower than in controls in both sexes; this test item effect then disappeared (there was a trend toward minimally higher mean food consumption in females) and was considered non-adverse.
There were no toxicologically significant effects at 100 and 300 mg/kg/day, and during gestation and lactation up to 750 mg/kg/day.
The high mean food consumption at 100 mg/kg/day on Days 8-15 was related to food spillage due to two females.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See Tables 14, 14 bis and 14 ter.
There were dose-related findings in the liver (mainly hepatocellular degeneration/necrosis; hyperplasia of bile ducts in females only; hepatocellular hypertrophy; periportal inflammatory cells), kidneys (in males only: tubular degeneration/necrosis; hyaline droplets in tubular epithelium; tubular basophilia and dilation) and thyroid glands (follicular cell hypertrophy) in animals treated at 100, 300 or 750 mg/kg/day.

. Liver
Minimal to moderate dose-related centrilobular hepatocellular degeneration/necrosis, minimal to slight bile duct hyperplasia, minimal to moderate hepatocellular hypertrophy (mainly centrilobular), minimal to slight pigment (mainly located in the bile ducts and Kupffer cells; females only) and minimal infiltrate of mononuclear cells in periportal areas (females only) were noted at 100, 300 and/or 750 mg/kg/day.
In addition, slight subcapsular fibrosis and slight leukocytosis were noted in two males treated at 750 mg/kg/day, and pale vacuoles in a single female treated at 750 mg/kg/day. These findings were considered to be probably related to the test item administration although recorded at very low incidence.
The hepatocellular hypertrophy correlated with the increased mean liver weights.

. Thyroid glands
There was a minimal to slight follicular cell hypertrophy in the thyroid glands from males and females treated at 100, 300 or 750 mg/kg/day.

. Kidneys
In Hematoxylin-Eosin (HE) stained slides, there were minimal tubular degeneration/necrosis in the kidneys of males treated at 750 mg/kg/day, co-localized with minimal to moderate hyaline droplets in the tubular epithelium which were seen at 100, 300 or 750 mg/kg/day, increased severity and/or incidence of tubular basophilia and dilation at the medulla-cortex junction at 300 or 750 mg/kg/day.
The hyaline droplets were characterized by dense eosinophilic droplets in the proximal tubular epithelium.
The a2u-globulin immunostaining showed slight to marked brown globules in the proximal tubules of all high dose males co-localized with the hyaline droplets seen in HE-stained sections, while minimal brown globules were seen in 2/5 control males. This suggested that the hyaline droplets were consistent with a2u globulin deposits.

There were no test item-related findings in the testes, epididymides or ovaries.
The other findings were considered not to be related to test item administration since there were seen with similar incidence and severity in controls and/or were consistent with the spontaneous findings described in the literature.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

See Table 10.
Thyroid hormones
There were no effects on mean T4 level.
At 750 mg/kg/day, mean TSH plasma concentration in F0 males was 2-fold higher than in controls. This result correlated with thyroid cell hypertrophy at microscopy and was considered to be an indirect effect of the test item through the increase in hepatic metabolism.
In absence of statistical significance and dose-relationship, the differences from controls in mean TSH levels at 100 and 300 mg/kg/day were not considered to be test item-related.

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

At 750 mg/kg/day, there were 5/10 females having one estrous cycle with estrous ending after at least 3 days during the pre-mating period (in particular one female was blocked in estrous for about the 2 weeks). At 300 mg/kg/day, one female was blocked in estrous for the 2-week pre-mating period and another female G25911 had estrous ending after 3 days (the second day being noted as proestrous).
None of the females during pre-treatment period and none of the control females during the treatment period had estrous cycles with estrous ending after 3 days or more.
This effect was considered to be test item-related and non-adverse in view of the absence of effect on fertility.

At 100 mg/kg/day, there was one female with an estrous ending after 3 days. An effect of the test item was not excluded but considered of no toxicologically significance in view of the isolated incidence.

At termination on Day 15 p.c., all females were in diestrous (identified as metestrous in a few females) as expected during lactation.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Pairing, mating and fertility data
See Table 3.
There were no effects on mean pairing, mating and fertility data.
The non-pregnant females were not considered to be due to the test item: there were low incidences and no dose-relationship.

Delivery data
See Table 4.
In absence of dose-relationship and/or of statistical significance, there were no toxicologically significant effects on mean delivery data.

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Remarks:

reproductive performance

Effect level:

>= 750 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other:

Organ:

kidney

liver

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

See Table 6.
There were no test item-related pup clinical signs.
The above-mentioned clinical signs of test item groups were of normal background or were recorded in an isolated manner (desquamation).

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

See Tables 5 and 5 bis.
In controls and at 300 mg/kg/day, most prematurely dead pups were from two litters: one from the control group and one from the 300 mg/kg/day group. No such event occurred in the high-dose group.
At 750 mg/kg/day, viability index was lower and the number of found dead/cannibalized pups or of litters affected was higher than controls when excluding one litter. This effect on pup viability until Day 4 p.p. could be test item treatment-related and adverse.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See Table 7.
When compared with controls, mean pup body weight was lower at 750 mg/kg/day on Day 1 p.p. (-14% for both sexes, p<0.01) and thereafter (down to -18%, p<0.01, or -21%, p<0.001, for males or females on Day 8 p.p., respectively). The same tendency was observed at 300 mg/kg/day, particularly in females on Day 8 p.p. (-11%, p<0.05) and on Day 13 p.p. (-10%, p<0.05).
Mean body weight gains were lower than controls at 750 mg/kg/day throughout the lactation period compared with controls; there was no statistically significant effect on mean body weight gains at 300 mg/kg/day.
These test item effects were considered to be adverse.
There were no toxicologically significant effects at 100 mg/kg/day in mean pup body weights and/or mean pup body weight changes.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related necropsy findings in pups.
The few macroscopic findings in scheduled sacrificed pups on Day 14 p.p. (colored area on liver, dilated renal pelvis, dilated ureter) were noted with no dose-relationship and can occur spontaneously in the rat strain.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Pup sex ratio
See Table 8.
There were no effects on sex ratio at birth.

Pup development
Anogenital distance (AGD)
See Table 9.
When compared with controls, in test item groups AGD tended to be higher and ratios of AGD to cube root of body weight were statistically significantly higher. However, taking into account the slight amplitude of differences from controls, the absence of dose-relationship and the intragroup variability, these effects were not considered to be test item treatment-related.

Nipples and areolae numbers
No nipples or areolae were observed in male pups in any groups.

Thyroid hormone levels
See Table 11.
There were no effects on mean T4 level.

At 750 mg/kg/day, there was a tendency towards higher mean TSH plasma levels in litters on Day 14 p.p. This was considered as not toxicologically significant in absence of statistical significance and in view of the slight amplitude of differences from controls and inter-animal variability.

There were no relevant effects at 100 and 300 mg/kg/day on mean TSH level.

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Critical effects observed:

no

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1: Body weight

 

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	750	0	100	300	750
Pre-mating or whole study
Day 1	382	381	385	383	263	272	266	265
		0	+1	0		+3	+1	+1
Day 8	428	427	425	413	269	275	270	274
		0	-1	-4		+2	0	+2
Day 29	518	517	514	498	286	292	285	293
		0	-1	-4		+2	0	+2
Day 78	637	634	622	594	/	/	/	/
		0	-2	-7
Days 1-8	+46	+46	+41	+30**	+6	+3	+4	+10
Days 1-29	+136	+136	+130	+116	+23	+20	+19	+28
Days 29-78	+119	+117	+108	+96*	/	/	/	/
Days 50-57	+20	+18	+12	+5**	/	/	/	/
Days 1-78	+255	+253	+237	+211*	/	/	/	/
Gestation
Day 0p.c.	/	/	/	/	288	298	284	292
Day 7p.c.	/	/	/	/	314	327	315	316
Day 14p.c.	/	/	/	/	348	364	345	346
Day 20p.c.	/	/	/	/	424	452	424	422
Day 0-7p.c.	/	/	/	/	+26	+30	+31	+25
Day 7-14p.c.	/	/	/	/	+34	+37	+30	+29
Day 14-20p.c.	/	/	/	/	+77	+88	+79	+76
Days 0-20p.c.	/	/	/	/	+136	+155	+141	+130
Lactation	/	/	/	/
Day 1p.p.	/	/	/	/	335	346	334	319
Day 4p.p.	/	/	/	/	334	349	328	325
Day 8p.p.	/	/	/	/	346	369	343	344
Day 13p.p.	/	/	/	/	360	384	364	360
Days 1-13p.p.	/	/	/	/	+25	+39	+29	+40

/              : not applicable.

statistically significance: *: p<0.05, **: p<0.01.

in italic  : differences from controls (%).

Table 2: Food consumption

Sex		Male				Female
Dose level (mg/kg/day)		0	100	300	750	0	100	300	750
Pre-mating or whole study
Days 1 - 8		33	31	31	27**	18	18	15	13^#
			-6	-6	-18		0	-17	-28
Days 8 - 15		32	30	32	33	17	21	16	19
			-6	0	+3		+24	-6	+12
Days 15 - 22		30	29	30	31	16	17	17	18*
			-3	0	+3		+6	+6	+13
Days 22 - 29		30	29	32	33	17	18	18	19
			-3	+7	+10		+6	+6	+12
Gestation
Days 0 - 7p.c.		/	/	/	/	20	21	21	21
Days 7 - 14p.c.		/	/	/	/	23	24	22	23
Days 14 - 20p.c.		/	/	/	/	27	28	27	26
Lactation
Days 1 - 4p.p.	/		/	/	/	29	36	27	27
Days 4 - 8p.p.	/		/	/	/	45	52	42	41
Days 8 - 13p.p.	/		/	/	/	65	70	59	56

/              : not applicable.

statistically significance: *: p<0.05, **: p<0.01,^#: p<0.001.

in italic  : differences from controls (%).

Table 3: Pairing, mating and fertility data

 

Dose level (mg/kg/day)	0	100	300	750
Number of animals paired (M + F)	10 + 10	10 + 10	10 + 10	10 + 10
Number of males mated	10	10	10	10
Number of females mated	10	10	10	10
Number of days taken to mate per pair	2.9	2.2	2.4	2.2
Number of pregnant females	10	8	10	9
Number of females with live born pups	10	8	10	9

M: males.

F: females.

Table 4: Delivery data

 

Dose level (mg/kg/day)	0	100	300	750
Number of females which delivered	10	8	10	9
Duration of gestation (days)	22.2	22.3	21.9	22.1
Number ofcorpora lutea	14.0	15.3	14.9	15.1
Number of implantation sites	13.4	14.5	13.4	13.7
Pre-implantation loss (%)	4.0	4.2	9.8	8.6
Number of pups delivered	10.7	12.5	11.0	10.7
Post-implantation loss (%)	19.9	13.5	18.6	22.2

Table 5: Pup mortality

 

Dose level (mg/kg/day)	0	100	300	750
Viability index on Day 4p.p.(%)	90.7 (98.0^a)	99.0*	89.1 (96.0^b)	89.6
Lactation index on Day 13p.p.(%)	98.6	100	100	100

statistically significance: *: p<0.05.

^a: when excluding one litter with a high pup mortality rate

^b: when excluding one litter with a high pup mortality rate

.

Table 5 bis: Distribution of prematurely dead pups

 

Dose level (mg/kg/day)	0	100	300	750
Number of cannibalized pups (litters affected)	8 (1)		1 (1)	4 (2)
Number of found dead pups (litters affected)	3 (3)	1 (1)	11 (4)	6 (4)
Total of prematurely dead pups (litters affected/total of litters)	11 (3/10) [2 (2/9)^a]	1 (1/8)	12 (5/10) [4 (4/9)^b]	10 (5/9)

^a: when excluding one litter with a high pup mortality rate

^b: when excludingone litter with a high pup mortality rate

Table 6: Pup clinical signs

 

Dose level (mg/kg/day)	0	100	300	750
Emaciated appearance	2^a
Dehydration	1^a
Generalized pallor				1
Blackish and/or purplish abdomen		2		1
Blackish dorsal area of abdomen		1
Hematoma (back/head/neck)		2	4
Wound, contusion and/or scab (head/hindlimb)			2	1
Desquamation				1
Total litter affected	1/10	3/8	3/10	2/9

^a: one litter

Table 7: Pup body weight

Sex	Male						Female
Dose level (mg/kg/day)	0	100	300	750		0		100		300		750
Body weight
Day 1 p.p	8.5	8.2	7.8	7.3**		7.8		7.7		7.5		6.7**
		-4	-8	-14				-1		-4		-14
Day 4 p.p (preculling)	12.6	11.9	11.1	10.4*		11.5		11.4		10.7		9.6
		-6	-12	-17				-1		-7		-17
Day 4 p.p (postculling)	12.6	11.8	11.1	10.5		11.5		11.6		10.7		9.7*
		-6	-12	-17				+1		-7		-16
Day 8 p.p	21.6	21.2	19.6	17.8**		20.9		20.9		18.6*		16.6#
		-2	-9	-18				0		-11		-21
Day 13 p.p	35.3	35.6	33.4	29.8**		34.8		34.8		31.3*		28.2#
		+1	-5	-16				0		-10		-19
Body weight change
Days 1 - 4 p.p.	4.1	3.7	3.3		3.1	3.8		3.7	3.3		2.9
Days 4 - 8 p.p.	9.0	9.4	8.4		7.3**	8.9		9.4	7.8		7.0#
Days 4 - 13 p.p.	22.7	23.8	22.3		19.3**	22.8		23.3	20.6		18.5**
Days 8 -13 p.p.	13.7	14.4	13.9		12.0*	13.9		13.9	12.8		11.6*

statistically significance:*: p<0.05, **          : p<0.01, #           : p<0.001.

in italic  : differences from controls (%).

reference data from three contemporaneous studies: study means [min-max] for Day 1 p.p.: males: [8.2-8.4] g, females: [7.8-7.8] g; for Day 4 p.p. preculling: males: [11.7-12.7] g, females: [11.4-12.2] g); for Day 8 p.p.: males: [20.6-23.4] g, females: [19.9-22.8] g; for Day 13 p.p.: males: [33.9-38.5] g, females: [33.0-38.3] g.

Table 8: Pup sex ratio

Dose level (mg/kg/day)	0	100	300	750
Sex ratio on Day 0 p.p.(% of males)	53.5	42.0	54.5	47.9

Table 9: Pup development: AGD  

Sex

Male

Female

Dose level (mg/kg/day)

0

100

300

750

0

100

300

750

AGD (mm)

7.17 ± 0.64

7.43 ± 0.62

7.30 ± 0.53

7.16 ± 0.68

4.53 ± 0.59

4.72 ± 0.57

4.63 ± 0.54

4.31 ± 0.62

Body weight on Day 4 p.p (preculling)

12.6 ± 1.4

11.9 ±1.5

11.1 ±1.8

10.4* ± 1.5

11.5±1.8

11.4 ±1.4

10.7 ±1.6

9.6 ± 1.5

Ratio AGD/^3vBody weight (mm/g^1/3)

3.12 ± 0.27

3.29** ± 0.22

3.35** ± 0.22

3.33** ± 0.28

2.00 ± 0.25

2.11* ± 0.21

2.13* ± 0.21

2.05 ± 0.27

statistically significance: *: p<0.05, **: p<0.01.

mean data ± standard deviation, based on individual pup data.

reference data from three contemporaneous studies (43642 RSR, 43827 RSR, 44042 RSR): study means [min-max] for AGD: males: [5.94-7.22] mm, females: [3.67-4.43] mm; for ratio: males: [2.65-3.12] mm/g^1/3, females: [1.64-1.97] mm/g^1/3.

Table 10: F0 males thyroid hormones

Dose level (mg/kg/day)	0	100	300	750
T4 (ng/mL)	36.7 ± 4.76	40.0 ± 4.44	40.4 ± 5.61	40.2 ± 6.10
		+9%	+10%	+10%
TSH (pg/mL)	1688 ± 965	2904 ± 1516	2485 ± 1823	3512** ± 1680
		+72%	+47%	+108%

statistically significance: **: p<0.01.

in italic: differences from controls.

Table 11: Pups thyroid hormones

Dose level (mg/kg/day)	0	100	300	750
T4 (ng/mL)	36.3 ± 3.69	36.7 ± 5.08	35.2 ± 3.19	34.9 ± 3.39
		+1%	-3%	-4%
TSH (pg/mL)	1397 ± 621	1573 ± 448	1602 ± 739	1912 ± 374
		+13%	+15%	+37%

in italic: differences from controls (%).

Table 12: Organ weights  

Sex	Male			Female
Dose level (mg/kg/day)	100	300	750	100	300	750
Number of examined animals	10	10	10	8	10	9
- Final body weight	-2	-4	-9*	+6	0	-1
- Liver
.absolute	+24**	+48**	+67**	+15	+13	+32**
.relative-to-body	+26**	+54**	+83**	+8	+12**	+32**
- Thyroids
.absolute	+18	+36**	+40**	na	na	na
.relative-to-body	+20	+42**	+54**	na	na	na
- Kidney
.absolute	+7	+18*	+25**	+10	+8	+7*
.relative-to-body	+9	+22**	+37**	+3	+8	+8

statistically significance: *: p<0.05, **: p<0.01.

na: not applicable.The thyroid gland weights of females were not recorded.

The significance concerned the organ weights values and not the percentages.

Table 13: Macroscopic observation

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	750	0	100	300	750
Number of examined animals	10	10	10	10	9	10	10	10
Liver enlargement	0	0	0	5	0	0	0	0

 

Table 14: Microscopic examination - liver 

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	750	0	100	300	750
Number of examined animals	10	10	10	10	9	10	10	10
Degeneration/necrosis; hepatocytes
. grade 1	-	-	-	1	-	-	-	-
. grade 2	-	-	-	1	-	-	-	-
. grade 3	-	-	-	-	-	-	1	-
Hyperplasia; bile duct
. grade 1	-	-	-	-	-	-	-	2
. grade 2	-	-	-	-	-	-	-	1
Hypertrophy; hepatocytes
. grade 1	-	5	2	-	-	-	6	9
. grade 2	-	5	5	2	-	-	2	1
. grade 3	-	-	3	8	-	-	-	-
Inflammatory; mononuclear cell; periportal
. grade 1	-	-	-	-	-	-	-	2
Pigment
. grade 1	-	-	-	-	-	-	-	2
. grade 2	-	-	-	-	-	-	-	3

-: not recorded.

*: out of five grades (minimal; slight; moderate; marked; severe).

 

Table 14 bis - Thyroid glands 

Sex	Male				Female
Dose-level (mg/kg/day)	0	100	300	750	0	100	300	750
Number of examined animals	10	10	10	10	9	10	10	10
Hypertrophy; thyroid glands
. grade 1	-	6	9	6	-	1	5	2
. grade 2	-	2	1	4	-	-	-	8

-: not recorded.

*: out of five grades (minimal; slight; moderate; marked; severe).

Table 14 ter - Kidneys 

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	750	0	100	300	750
Number of examined animals	10	10	10	10	9	0	0	10
Degeneration/necrosis; tubule
. grade 1	-	-	-	9	-	na	na	-
Hyaline droplets; tubular epithelium
. grade 1	-	7	3	-	-	na	na	-
. grade 2	-	2	6	1	-	na	na	-
. grade 3	-	-	1	9	-	na	na	-
Basophilia; tubule
. grade 1	7	7	6	4	-	na	na	-
. grade 2	-	-	4	3	-	na	na	-
. grade 3	-	-	-	2	-	na	na	-
Dilation; tubule
. grade 1	1	-	3	1	3	na	na	-
. grade 2	-	-	1	2	-	na	na	-
. grade 3	-	-	1	3	-	na	na	-

na: not applicable.

-: not recorded.

*: Out of five grades (minimal; slight; moderate; marked; severe)

Conclusions:

n-butyl-4,4-bis(tert-butylperoxy)valerate (Luperox 230) was administered daily by gavage to male and female Sprague-Dawley rats for 4 weeks before mating, during mating and, for males until 11 weeks of treatment have elapsed, for females through gestation and lactation until Day 14 p.p., at dose-levels of 100, 300 and 750 mg/kg/day.
Under the experimental conditions and results of this study:
- the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 300 mg/kg/day for males and females based on the absence of adverse effects up to this dose and on the adverse effects on liver (both sexes) and kidneys (males) at microscopy at 750 mg/kg/day,
- the NOAEL for parental reproductive performance was considered to be 750 mg/kg/day, based on the absence of adverse effects on mean reproductive data up to this dose,
- the No Observed Effect Level (NOEL) for toxic effects on progeny was considered to be 100 mg/kg/day and the Lowest Observed Adverse Effect Level (LOAEL) was considered to be 300 mg/kg/day, based on the effects on mean pup body weights at 300 and 750 mg/kg/day and on pup viability at 750 mg/kg/day.

Executive summary:

The potential toxic effects of n-butyl-4,4-bis(tert-butylperoxy)valerate (Luperox 230) was evaluated following daily oral administration (gavage), to male and female rats from before mating, through mating and, for females, through gestation until Day 14 post-partum (p.p.) inclusive. This study provides initial information on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition. Three groups of 10 male and 10 female (groups 2 to 4) Sprague-Dawley rats received the test item daily by oral (gavage) administration before mating, through mating and, for the females, through gestation until Day 14 p.p. The test item was administered as a solution in the vehicle, corn oil, at dose levels of 100, 300 and 750 mg/kg/day. Another group of 10 males and 10 females received the vehicle alone, under the same experimental conditions and acted as a control group. A constant dose-volume of 5 mL/kg/day was used. The concentrations of the dose formulations were checked in study Weeks 1, 3, 6 and 9. The animals were checked twice daily for mortality and morbidity and once daily for clinical signs during the treatment period. Body weight and food consumption were recorded once a week during pre-mating and mating periods (food consumption not during mating), and during gestation on Days 0, 7, 14 and 20 post-coitum (p.c.) and lactation on Days 1, 4, 8 and 13 p.p. The animals were paired for mating after 4 weeks of treatment and the females were allowed to litter and rear their progeny until Day 14 p.p. The total litter sizes and number of pups of each sex were recorded after birth. Pups were observed daily for clinical signs, abnormal behavior and external abnormalities and weighed on Days 1, 4, 8 and 13 p.p. The physical development of pups was assessed by measuring the anogenital distance on Day 4 p.p. and by counting the number of nipples and areolae in male pups on Day 13 p.p. Litter standardization was performed on Day 4 p.p. in order to reduce the litter size-induced variability in pup growth and development. Thyroid hormone (TSH and T4) plasma levels were determined on the day of sacrifice in parental males and in Day 14 p.p. pups. Males were sacrificed after at least 11 weeks of treatment and females on Day 15 p.p. Final body weights and selected organs weights (kidneys, liver, thyroids with parathyroids and male reproductive organs) were recorded and a macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on ovaries (with oviducts), epididymides and/or testes in the control and high-dose groups, on liver, thyroids and/or kidneys in all dose groups, on a2µ-globulin protein immunostained kidneys of control and high-dose males, and on all macroscopic lesions. A macroscopic post-mortem examination was performed on pups on Day 14 p.p., including those found dead before study termination. 

No test item was observed in the control dose formulations and the test item concentrations in the analyzed dose formulations were within an acceptable range of variations. There were no unscheduled deaths in test item groups of parental animals. Test item-related clinical signs consisted in piloerection and/or round back in a total of four females at 750 mg/kg/day, chromodaccryorrhea in 3/10 males at 300 mg/kg/day and at 750 mg/kg/day, and mainly ptyalism in all males and females at 750 mg/kg/day. There were no toxicologically significant effects on mean body weight in either sex but in males a lower mean body weight gain over the treatment period at 750 mg/kg/day vs. controls (+211 g vs.+255 g, p<0.05) was recorded. At this dose, mean food consumption was lower than in controls in the first week of treatment for both sexes (-18% for males, p<0.01 and -28% for females, p<0.001) but there were no toxicologically significant effects at 100 and 300 mg/kg/day on this parameter. During the 2 weeks of estrous cycle evaluation before mating, 5/10 females had an estrous ending at least after 3 days at 750 mg/kg/day and 2/10 at 300 mg/kg/day, vs. none in controls. There were no effects on estrous cycles at 100 mg/kg/day considered to be of toxicological significance. There were no test item-related effects on mean pairing, mating and fertility data and no toxicologically significant effects on mean delivery data. There were no effects on mean T4 level. At 750 mg/kg/day and when compared with controls, mean TSH plasma concentration in F0 males was 2-fold higher. This result correlated with thyroid microscopic results and was considered to be an indirect effect of the test item through the increase in hepatic metabolism. There were no effects at 100 and 300 mg/kg/day on mean TSH level. At 750 mg/kg/day, there were findings in the liver (mainly adverse hepatocellular degeneration/necrosis; adverse hyperplasia of bile ducts in females only; hepatocellular hypertrophy correlated with increased weights and gross enlargement; periportal inflammatory cells and pigment in females), kidneys in males only (adverse tubular degeneration/necrosis; hyaline droplets in tubular epithelium correlated with increased weights; tubular basophilia and dilation) and thyroid glands (follicular cell hypertrophy correlated with increased weights). At 300 mg/kg/day, there were findings in the liver (non-adverse hepatocellular hypertrophy correlated with increased weights), kidneys in males only (non-adverse hyaline droplets in tubular epithelium correlated with increased weights; non-adverse tubular basophilia and dilation) and thyroid glands (non-adverse follicular cell hypertrophy correlated with increased weights in males). At 100 mg/kg/day, there were findings in the liver (non-adverse hepatocellular hypertrophy correlated with increased weights in males only), kidneys in males only (non-adverse hyaline droplets in tubular epithelium) and thyroid glands (non-adverse follicular cell hypertrophy).

Pups viability index on Day 4 p.p. was low at 750 mg/kg/day (89.6%). This effect could be test item treatment-related and adverse. There were no test item-related pup clinical signs. Mean pup body weight was affected at 750 mg/kg/day on Day 1 p.p. (-14% vs. controls for both sexes, p<0.01) and thereafter (down to -18%vs. controls, p<0.01, or -21%, p<0.001, for males or females on Day 8 p.p., respectively). The same tendency was observed at 300 mg/kg/day, particularly in females on Day 8 p.p. (-11% vs. controls, p<0.05) and on Day 13 p.p. (-10%, p<0.05). Mean body weight gains were lower at 750 mg/kg/day when compared with controls throughout the lactation period. These test item effects were considered to be adverse. There were no toxicologically significant effects at 100 mg/kg/day in mean pup body weight and mean pup body weight change. There were no test item-related effects on pup sex ratio at birth, on male pup development (absence of nipples or areolae) or on mean anogenital distance data. There were no effects on mean T4 level and no toxicologically significant effects on mean TSH level. There were no test item-related necropsy findings in pups.

n-butyl-4,4-bis(tert-butylperoxy)valerate (Luperox 230) was administered daily by gavage to male and female Sprague-Dawley rats for 4 weeks before mating, during mating and, for males until 11 weeks of treatment have elapsed, for females through gestation and lactation until Day 14 p.p., at dose-levels of 100, 300 and 750 mg/kg/day. Under the experimental conditions and results of this study:

.            the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 300 mg/kg/day for males and females based on the absence of adverse effects up to this dose and on the adverse effects on liver (both sexes) and kidneys (males) at microscopy at 750 mg/kg/day,

.            the NOAEL for parental reproductive performance was considered to be 750 mg/kg/day, based on the absence of adverse effects on mean reproductive data up to this dose,

.            the No Observed Effect Level (NOEL) for toxic effects on progeny was considered to be 100 mg/kg/day and the Lowest Observed Adverse Effect Level (LOAEL) was considered to be 300 mg/kg/day, based on the effects on mean pup body weights at 300 and 750 mg/kg/day and on pup viability at 750 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Key study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification is warranted for reproductive toxicity according to CLP/GHS criteria.