Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

No toxicity for the reproductive organs was observed in a 90-day study on the read-across substance 25155 -25 -3. No histological changes were observed in the reproductive organs of male and female and effects in the sperm parameters of male rats exposed for 90 days to Luperox F (96.7% of structurally analogous [1,3(or 1,4) -phenylenebis(1-methylethylidene) ]bis[tert-butyl] peroxide) up to the dose level of 800 mg/kg bw/day.

In a study with 2212 -81 -9 the NOAEL for reproduction toxicity is at least 1000 mg/kg bw/day, the highest dose tested and no effects on fertility were observed. In an OECD 422 study on the read-across substance 25155 -25 -3: The NOAEL for the systemic toxicity in the parental generation was considered to be 300 mg/kg bw/day, based on decreased body weight gain and food consumption in males and females and microscopic changes in kidneys of females observed at 1000 mg/kg bw/day. TheNOAEL for the fertility was 1000 mg/kg bw/day in males and 300 mg/kg bw/day in females)

Conclusion on fertility

The screening study for 2212 -81 -9 showed no effects on fertility. It has a NOAEL of at least 1000 mg/kg. At the highest dose level for 25155 -25 -3 in the source a reduced fertility index was observed. A smaller number of pregnant dams that were noted with less corpora lutea, less implantation sites, less live embryos at first litter check. The effects on fertility appear only at the highest dose level (1000 mg/kg bw/d). The NOAEL is 300 mg/kg. For both substances the available repeated dose toxicity and screening studies do not indicate adverse effects on reproductive organs or tissues or reveal other concerns in relation with reproductive toxicity.

An OECD 443 has been proposed for the source substance. Based on the available data and tonnage band further testing for the target substance is not required.

For further details on the read across rationale see section 13.2.

Link to relevant study records

Referenceopen allclose all

Endpoint:
reproductive toxicity, other
Remarks:
other: repeated dose toxicity observations
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Justification for type of information:
see read across rationale in Section 13.2
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: RccHan: WIST(SPF)
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
castor oil
Remarks:
Doses / Concentrations:
50, 200 and 800 mg/kg bw/d
Basis:
actual ingested
Control animals:
yes, concurrent vehicle
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
ovaries, uterus (incl. oviducts, cervix and vagina) , testis, epididymides, prostate gland and seminal vesicles incl. coagulating glands
Reproductive function: sperm measures:
no effects observed
Dose descriptor:
NOAEC
Effect level:
> 800 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: repeated dose toxicity observations on sexual organs
Reproductive effects observed:
not specified
Executive summary:

No histological changes were observed in the reproductive organs and effects in the sperm parameters of male and/or female rats exposed for 90 days to Luperox F (96.7% of 2,2-Bis(t-butylperoxy isopropyl)benzene).

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Aug 2017 - Jan 2018
Reliability:
1 (reliable without restriction)
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2000
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Solubility and stability of the test substance in the vehicle: Stability for at least 5 hours at room temperature, 13 days in the refrigerator and 3 weeks in freezer is confirmed over the concentration range 1 to 200 mg/mL
Species:
rat
Strain:
other: Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males were 10-12 weeks old; females were 12-14 weeks old
- Weight at study initiation: males between 275 and 319 g; females between 187 and 230 g
- Fasting period before study: no
- Housing: On arrival and following the pre-test (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages. During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, males were housed in their home cage with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages. During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam. The cages contained appropriate bedding. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cageenrichment, bedding material, food and water.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water: Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours.
- Acclimation period: 6 days prior to start of the pre-test period (females) or 6 days before the commencement of dosing (males)

DETAILS OF FOOD AND WATER QUALITY:
The feed was analysed by the supplier for nutritional components and environmental contaminants.It is considered that there were no known contaminants in the feed that would interfere with
the objectives of the study.
Periodic analysis of the water is performed.It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 21
- Humidity (%): 47 to 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 Sept 2017 To: 15 Nov 2017
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least every 9 days (Groups 1-3 as a clear solution) or every 1-2 days (Group 4 as an opaque suspension), formulated in daily portions. Formulations were heated to a
maximum temperature of 50±5°C for maximally 10 minutes to obtain visual homogeneity. Formulations were stored in the refrigerator. When formulations were used on the day of preparation, formulations were released for dosing when they had reached a temperature of 40°C or lower. When
dosing formulations were prepared and stored in the refrigerator, formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing. The dosing formulations and vehicle were continuously stirred until and during dosing.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 post-coitum
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis in Week 1 for concentration anaysis in all groups and for homogeity in groups 2 and 4 (low and high dose groups).
All samples were stored on dry ice immediately after sampling.
Concentration analysis was performed for duplicate sets of samples (approximately 500 mg). Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions (Group 1-3) and ± 15% for suspensions (Group 4) of target concentration.
Homogeneity analysis was performed on duplicate sets of samples (approximately 500 mg).
Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was # 10%.
Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations used in the present study.
Duration of treatment / exposure:
Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to and including the day before scheduled necropsy.
Females that delivered were treated for 50-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 14 or 16 days after delivery, up to and including the day before scheduled necropsy.
Females which failed to deliver were treated for 41 days.
Frequency of treatment:
daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were selected based on the results of a 17-day dose range finder with oral administration of 1,3-bis(tert-butylperoxyisopropyl)benzene in rats, and in an attempt to produce graded responses to the test item.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical observations were at least conducted immediately after dosing and 3 hours (± 30 minutes) after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. Terminal body weights were recorded on the day of necropsy (fasted for males, non-fasted for females).

FOOD CONSUMPTION:
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: all
- Parameters checked in accordance with Guidelines were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: all
- Parameters checked in accordance with Guidelines were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 6-13).
- Dose groups that were examined: all
- Battery of functions tested: Hearing ability; Pupillary reflex; Static righting reflex; Fore- and hind-limb grip strength; Locomotor activity.

IMMUNOLOGY: No

other: organ weights F0 generation according to Guidelines
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females, except for one female that had died spontaneously.
Sperm parameters (parental animals):
Parameters examined in all P male parental generations:
testis weight, epididymis weight, prostate gland weight, seminal vesicles weight.

For the testes of all selected males of Groups 1 and 4 and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Litter observations:
STANDARDISATION OF LITTERS
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males
and three females) was acceptable.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, plasma T4 level

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Following completion of the mating period (a minimum
of 28 days of administration).
- Maternal animals: - Females which delivered: PND 14 or16; - Females which failed to deliver: With evidence of mating: Post-coitum Day 25-26.

GROSS NECROPSY
- Gross necropsy was performed according to Guidelines

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues according to Guidelines were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
Pups, younger than 7 days were euthanized by decapitation.
All remaining pups (PND 7-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%).
The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.
Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally).
Pups found dead during the weekend were fixed in identified containers containing 70% ethanol as they were not necropsied on the same day.
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

GROSS NECROPSY
- performed according to Guidelines
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Paremetric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. The Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group.
Incidene: An overall Fisher’s exact test was used to compare all groups at the 5% significance level.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
Mating (%): (Number of females mated / Number of females paired) x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): (Number of pregnant females / Number of females mated) x 100
Gestation index (%): (Number of females with living pups on Day 1 / Number of pregnant females) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Post-implantation survival index (%): (Total number of offspring born / Total number of uterine implantation sites) x 100
Offspring viability indices:
Live birth index (%): (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100
Viability index (%): (Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering) x 100
Lactation index (%): (Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling)) x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
about one (males) or two (females) weeks of treatment. In females piloerection occurred mostly in the post-coitum period. Salivation seen among animals treated with the test item, in a dose-related manner, was considered to be a physiological response rather than a sign of systemic toxicity considering its modest severity (slight or moderate) and the time of occurrence (i.e. after dosing).
Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
The premature death of one female of the 1000 mg/kg bw/day group was considered to be related to treatment. All other animals survived until scheduled sacrifice.
The high dose female was found dead, while in labour, on Day 24 of the post-coitum period. On the day prior to death, she showed piloerection. Her body weight development and food consumption were unremarkable. The main macroscopic finding was the presence of dead fetuses in the uterus (10 in total). Main microscopic findings were moderate centrilobular hepatocellular necrosis in the liver and slight bone marrow atrophy. The hepatocellular necrosis was considered to be the main cause of death. As test item-related hepatocellular necrosis was also noted in a surviving male of the 1000 mg/kg bw/day group, the death of the high dose female was attributed to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain of males treated at 1000 mg/kg bw/day was reduced throughout the treatment period (statistically significant), with slight weight loss in several animals in the last week of this period. The resulting reductions in mean body weights were statistically significant at Days 8 and 15 of the mating period (8% difference from controls at the end of treatment). To a lesser extent, reduced weight gain was noted in males treated at 300 mg/kg bw/day (not statistically significant). Mean terminal body weight of 300 mg/kg bw/day males was 5% lower than that of controls.
Body weights and body weight gain of females were considered not to be affected by treatment up to and including 1000 mg/kg bw/day. The statistically significantly higher mean body weight gain noted in 1000 mg/kg bw/day females at Day 7 of the lactation period, mainly due to higher weight gain of one female, was regarded as a chance finding.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males at 1000 mg/kg bw/day consumed less food than controls in treatment weeks 1-2 and, to a lesser extent, weeks 3-4. Their food consumption after correction for body weight was reduced only in weeks 1-2.
Food consumption of females (before and after correction for body weight) was considered not to be affected by treatment. Occasional, slightly higher values noted at 1000 mg/kg bw/day (between Days 11-17 of the post-coitum period and Days 1-4 of the lactation period) were regarded as unrelated to treatment as mean food consumption of 1000 mg/kg bw/day females remained close to that of controls at the other time points.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Red blood cell parameters showed the following statistically significant differences between treated rats and controls (percentage differences from the control group mean are indicated between parentheses):
- Lower haemoglobin concentration in males at 1000 mg/kg bw/day (9%).
- Lower mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) in females at 1000 mg/kg bw/day (5 and 6%, respectively).
There were no treatment-related changes in white blood cell parameters or number of platelets. There were no test item-related differences in coagulation parameters. The lower mean activated partial thromboplastin time (APTT) values noted at 1000 mg/kg bw/day (statistically significant in males only) were considered to have arisen as a result of slightly high concurrent control values and, therefore, regarded as unrelated to treatment. Mean APTT values at 1000 mg/kg bw/day remained in the normal ranges.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes in clinical chemistry parameter, mostly in 1000 mg/kg bw/day males, distinguished treated animals from control animals (percentage differences from the control group mean are indicated between parentheses). Mean values for these parameters in the affected group(s) of males were (slightly) out of the normal ranges.
- Higher alanine aminotransferase (ALAT) in males at 1000 mg/kg bw/day (48%).
- Higher total protein in males at 300 and 1000 mg/kg bw/day (5 and 10%, respectively).
- Higher albumin in males at 1000 mg/kg bw/day (10%).
- Higher creatinine in males at 1000 mg/kg bw/day (23%).
- Higher potassium in males at 1000 mg/kg bw/day (14%). Potassium was also higher (10%) in males at 300 mg/kg bw/day but the difference from controls was not statistically significant.
- Lower cholesterol in males at 1000 mg/kg bw/day (22%).
The other statistically significant variations noted in clinical chemistry values were considered unrelated to treatment due to the lack of a dose-related response, slightly high control value and/or a high value in a single animal.

Thyroid hormone analyses:
Mean serum T4 levels were statistically significantly decreased in F0 males at 300 and 1000 mg/kg bw/day (relative differences from controls: 32 and 51%, respectively), whereas mean serum TSH levels in these males were increased (4.3 and 9.7-fold, respectively; statistically significant at 1000 mg/kg bw/day only). The mean values in these groups of males were out of the historical control ranges.
Serum levels of T4 and TSH in F0 females were considered not to be affected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Females of the 1000 mg/kg bw/day group had lower mean numbers of total movements and ambulations compared to those of the control group (nearly 60% difference, statistically significant for total movements only). Motor activity was particularly low in 3 females which had values below the historical control ranges (2). The other two 1000 mg/kg bw/day females tested had activity values at or just below the concurrent control group ranges.
Activity habituation profiles were similar between the treated and control groups with a decreasing trend in activity over the duration of the test period Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was not affected by treatment.
(2) Historical control data for motor activity in female Wistar Han rats (period 2015-June 2017):
Total movements: mean = 3502, P5 - P95 = 1478 - 5683 (n=195).
Ambulations: mean = 855, P5 - P95 = 301 - 1467 (n=195).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the thymus of males and the thyroid (see also 'any other information').
Kidneys:
- Tubular degeneration was present in males and females at 1000 mg/kg bw/day up to moderate degree.
- Tubular basophilia was present at increased incidence and/or severity in males starting at 300 mg/kg bw/day and in females at 1000 mg/kg bw/day up to slight degree.
- Hyaline droplet accumulation was present at increased severity in males starting at 300 mg/kg bw/ day up to moderate degree.
- Tubular mineralization was present at increased incidence and/or severity in females starting at 300 mg/kg bw/day up to moderate degree.
Liver:
- Hepatocellular hypertrophy was present in males starting at 100 mg/kg bw/day and in females at 1000 mg/kg bw/day up to slight degree. This correlated with the macroscopic enlargement and the increased organ weight.
- Pigment deposition was present in males at 300 and 1000 mg/kg bw/day up to slight degree. This sometimes correlated with the macroscopic black-brown discoloration.
- Hepatocellular necrosis was present in a single male at 1000 mg/kg bw/day at slight degree.
Thyroid gland:
- Follicular cell hypertrophy was present at increased incidence and/or severity in males starting at 100 mg/kg bw/day and in females starting at 300 mg/kg bw/day up to moderate degree. This correlated with the macroscopic enlargement and the increased organ weight.
- Colloid alteration was present in males and females at 1000 mg/kg bw/day up to minimal degree.
Thymus:
- Lymphoid atrophy was present in males at 1000 mg/kg bw/day at minimal degree. This correlated with the decreased organ weight.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were not affected by treatment.
During the treatment period, all females had regular cycles, mostly of four days. The irregular cycle noted in one female of the 1000 mg/kg bw/day group (which was not pregnant) was not test item-related as it occurred prior to initiation of treatment (first cycle in the pretest period).
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
One male of the 300 mg/kg bw/day showed tubular atrophy in the testes and reduced luminal sperm with luminal cell debris in the epididymides which accounted for the observed non-pregnancy. This male had no normal spermatogenic staging profile.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis (except abovementioned 300 mg/kg bw/day male).
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Two females were not pregnant despite evidence of mating (one female at 300 mg/kg bw/day and one female at 1000 mg/kg bw/day). Another female at 1000 mg/kg bw/day was found dead on Day 24 post-coitum while in labour.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis (except one male at 300 mg/kg bw/day).
No effects were observed on mating index, precoital time, number of implantation sites, anf fertility index. The non-pregnancy of one female at 300 mg/kg bw/day was due to infertility of the male she was paired with.The non-pregnancy of one high dose female was not associated with related histopathology changes in reproductive organs. These incidental cases of male infertility/non-pregnancy were regarded as unrelated to treatment as the incidence showed no dose-related trend and was within normal limits.
Key result
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
>= 1 000 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
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment. The clinical signs observed remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices were 100% for the 300 mg/kg bw/day group and 99% for the other groups.
One pup of the control group died spontaneously at PND 1, one pup of the 100 mg/kg bw/day group was euthanized for humane reasons at PND 2 and one pup of the 1000 mg/kg bw/day group went missing (presumably cannibalized) at PND 2. This incidental pup mortality was considered unrelated to treatment as the incidence showed no dose-related trend and remained within the range considered normal for pups of this age.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, treatment related lower body weights of pups were noted on PND 13. This was statistically significant for male pups, female pups and sexes combined, and resulted in a 20% lower mean body weight when compared to concurrent controls.
The remaining statistical significant changes (on PND 7 at 100 and 1000 mg/kg bw/day and on PND 13 at 100 and 300 mg/kg bw/day) were not considered toxicologically relevant as no dose response was apparent, all values were within normal limits7 of which the control group values were at the higher end.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 14-16 pups were judged to be unaffected by treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of findings noted remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Other effects:
no effects observed
Description (incidence and severity):
Sex ratio, anogenital distance (absolute and normalized for body weight) in male and female pups and areola/nipple retention was considered to to be affected by treatment.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects on reproduction observed
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Accuracy

The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with the target concentrations (mean accuracies between 96.2% and 98.7%). No test item was detected in the Group

1 formulation.

Homogeneity

The formulations of Groups 2 and 4 were homogeneous (coefficient of variation of 2.8% and 2.1% in the low and high dose goups, respectively).

Text Table 1 Mean Percent Organ Weight Differences from Control Groups

 

Males

Females

Dose level (mg/kg):

100

300

1000

100

300

1000

 

 

 

 

 

 

 

THYMUS

 

 

 

 

 

 

              Absolute

5

-27

-41

-

-

-

              Relative to body weight

3

-19

-35

-

-

-

 

 

 

 

 

 

 

THYROID GLAND

 

 

 

 

 

 

              Absolute

13

38**

38**

6

13

19*

              Relative to body weight

0

40**

40**

0

17

17**

 

 

 

 

 

 

 

LIVER

 

 

 

 

 

 

              Absolute

28*

26*

63**

-2

15

25**

              Relative to body weight

25**

38**

81**

0

11

39**

 

 

 

 

 

 

 

KIDNEYS

 

 

 

 

 

 

              Absolute

12

16

16

2

4

8

              Relative to body weight

9

27**

28**

3

0

19**

*: P<0.05, **: P<0.01. - no remarkable findings

Text Table 2. Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals

 

Males

Females

Dose level (mg/kg):

0

100

300

1000

0

100

300

1000

 

 

 

 

 

 

 

 

 

KIDNEYSa

5

5

5

5

5

5

5

5

    Tubular degeneration

 

 

 

 

 

 

 

 

        Minimal

-

-

-

-

-

-

-

2/5

        Slight

-

-

-

1/5

-

-

-

-

        Moderate

-

-

-

2/5

-

-

-

-

    Tubular basophilia

 

 

 

 

 

 

 

 

        Minimal

2/5

3/5

2/5

-

-

1/5

-

2/5

        Slight

-

-

2/5

3/5

-

-

-

1/5

a = Number of tissues examined from each group.

 

Males

Females

Dose level (mg/kg):

0

100

300

1000

0

100

300

1000

 

 

 

 

 

 

 

 

 

KIDNEYSa

5

5

5

5

5

5

5

5

    Hyaline droplet accumulation

 

 

 

 

 

 

 

 

        Minimal

3/5

3/5

-

-

-

-

-

-

        Slight

2/5

1/5

1/5

3/5

-

-

-

-

        Moderate

-

1/5

4/5

2/5

-

-

-

-

    Tubular mineralization

 

 

 

 

 

 

 

 

        Minimal

-

-

-

-

2/5

3/5

2/5

-

        Slight

-

-

-

-

-

-

1/5

2/5

        Moderate

-

-

-

-

-

-

1/5

1/5

LIVERa

5

5

7

10

5

5

5

5

    Hepatocellular hypertrophy

 

 

 

 

 

 

 

 

        Minimal

-

3/5

4/7

1/10

-

-

-

2/5

        Slight

-

1/5

1/7

8/10

-

-

-

3/5

    Pigment deposition

 

 

 

 

 

 

 

 

        Minimal

-

-

1/7

2/10

-

-

-

-

        Slight

-

-

1/7

-

-

-

-

-

    Hepatocellular necrosis

 

 

 

 

 

 

 

 

        Slight

-

-

-

1/10

-

-

-

-

THYROID GLANDa

5

5

5

6

5

5

5

5

    Follicular cell hypertrophy

 

 

 

 

 

 

 

 

        Minimal

-

1/5

2/5

1/6

3/5

3/5

1/5

-

        Slight

-

3/5

3/5

5/6

1/5

1/5

4/5

2/5

        Moderate

-

-

-

-

-

-

-

3/5

    Colloid alteration

 

 

 

 

 

 

 

 

        Minimal

-

-

-

1/6

-

-

-

4/5

THYMUSa

5

5

6

5

5

1

-

5

    Lymphoid atrophy

 

 

 

 

 

 

 

 

        Minimal

-

-

-

3/5

-

-

-

-

a = Number of tissues examined from each group.

Conclusions:
The NOAEL for reproduction toxicity is at least 1000 mg/kg bw/day, the highest dose tested.
Executive summary:

In an OECD 422 study, Wistar Han rats were treated with 1,3-bis(tert-butylperoxyisopropyl)benzene by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day (10 rats/sex/dose level). Concurrent

controls (10 rats/sex) received the vehicle, corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and 14 or16 days of lactation (for 50-56 days). Females without offspring were treated for 41 days (two non-pregnant females).

Formulation analysis showed that the formulations were prepared accurately and homogeneously.

Reproductive results

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted the reproductive parameters examined in this study (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Reproduction NOAEL: at least 1000 mg/kg bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
rat
Quality of whole database:
GLP guideline 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

Description of key information

Reduced body weight gain of male and female pups was the only adverse effect in screening studies for both the target and the source substance. No specific developmental toxicity is observed for 2212 -81 -9 or 25155 -25 -3. The NOAEL for the target substance is 300 mg/kg bw/ d and the NOAEL for the source substance 100 mg/kg bw/d in the screening studies. In OECD 414, in rat and rabbit, with the 25155 -25 -3 no adverse developmental effects were observed in the highest dose level tested in the rat, 1000 mg/kg bw/d.

Specific developmental toxicity is not observed for the source and the target substance. Therefore, the read-across of the source substance’s 414 endpoints is deemed appropriate. For further details on the read across rationale see section 13.2.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
see read across rationale in Section 13.2
Reason / purpose:
read-across source
Qualifier:
no guideline available
Principles of method if other than guideline:
Range-finding study
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: RccHanTM: WIST(SPF)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Kreuzelweg 535961 NM Horst / Netherlands
- Age at D0 post coitum: 11 weeks
- Weight at Day 0 Post Coitum: 169 to 203 g
- Fasting period before study: no
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding
- Diet (e.g. ad libitum): Pelleted standard Harlan Teklad 2914C rodent maintenance diet
- Water (e.g. ad libitum): community tap-water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared daily. Vehicle was pre-warmed to a temperature of approximately 40 °C. Luperox F was weighed into a glass beaker on a tared precision balance and approximately 80% of the warm vehicle was added (w/v). The mixture was homogenized using an electrical homogenizer until a clear to colloid-like yellowish solution was obtained. Having obtained a homogeneous mixture, remaining vehicle was added until the required final volume. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle: 25, 75 and 250 mg/ml
- Amount of vehicle (if gavage): 4 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle to confirm the stability (4 hrs and 8 days at room temperature).
The samples were analyzed by HPLC coupled to an UV detector. The test item was used as the analytical standard.
The application formulations investigated during the study were found to comprise Luperox F in the range from 85.0% to 91.5% and, thus, the required content limit of ±20% with reference to the nominal content was met. The homogeneous distribution of the test item in the preparations was approved because maximal coefficient of variation from the corresponding mean was 0.8% (<15%).
In addition, the test item was found to be stable in application formulations when kept for four hours at room temperature due to recoveries which met the variation limit of 10% from the time zero (homogeneity) mean.
Details on mating procedure:
After acclimatization, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if:
- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.
The day of mating was designated day 0 post coitum.
Duration of treatment / exposure:
GD 6 to GD 20
Frequency of treatment:
daily
Duration of test:
up to GD 21
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
8
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on a previous OECD 422 study in Han Wistar rats, using dose levels of 100, 300, and 1000 mg/kg/day, resulting in a NOAEL of 100 mg/kg/day based on effects on kidney in parental generation at 300 mg/kg/day. Moreover treatment at 1000 mg/kg was associated with a smaller number of pregnant dams that were noted with less corpora lutea, less implantation sites, less live embryos at first litter check, and a higher postnatal loss. The living pups at 1000 mg/kg and at 300 mg/kg were also noted with less body weight gain until day 4 post partum.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily,during acclimatization and up to day of necropsy).

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Daily from day 0 until day 21 post coitum.

FOOD CONSUMPTION: Yes
- For the following periods: days 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18 and 18 - 21 post coitum.

WATER CONSUMPTION : No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #21
- Organs examined: gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, corpora lutea count and position of fetuses in the uterus
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Head examinations: No
- Soft tissue examinations:No
- Skeletal examinations: No
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
• Means and standard deviations of various data were calculated and included in the report.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.
Indices:
Pre- and post-implantation losses, embryonic and fetal deaths, live and dead fetuses, abnormal fetuses, fetal sex ratios and fetal body weights.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Viability / Mortality
All females survived scheduled study period.

Clinical Signs
No clinical signs were noted in females at any dose level.

Food Consumption
Mean food consumption from day 6 to 21 of the gestation period was 20.1, 21.0, 19.9 and 18.4 g/animal/day at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.
Treatment with the test item at the dose level 1000 mg/kg bw/day caused a reversible reduction in food consumption. The reduction was statistically significant from day 9 to 15 post coitum. Afterwards it recovered and although remained slightly lower until the completion of the study, the differences to the control values were only minor.
No effects on food consumption were noted in remaining dose groups.

Body Weights
Mean body weight gain from day 6 to 21 post coitum was 50%, 55%, 53% and 48% whereas mean corrected body weight gain was 14.5%, 16.6%, 12.6% and 7.1% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.
Treatment with the test item at the dose level of 1000 mg/kg bw/day caused a reduction in body weight gain and reduction in corrected body weight gain (body weight gain corrected for gravid uterus weight) in the absence of an effect on absolute body weights. Body weight gain was slightly reduced with statistical significance on days 8, 13, 14 and 15 of the gestation period. Also reduction in corrected body weight gain at this dose level was statistically significant if compared to the control value. Despite the reduction in body weight gain, mean absolute body weights at this dose level were similar to the respective control values during the most of the study period.
In one female at the high dose level (no. 28) a body weight loss by 10% was recorded after uterus weight subtraction. Because no body weight loss was recorded in any further female, observation in female no. 28 was considered probably not to be a result of the treatment with the test item but incidental.
In remaining dose groups body weights, body weight gain and corrected body weight gain were not affected by the treatment.
At the dose level of 100 mg/kg bw/day, absolute body weights were statistically significantly higher at the end of the gestation period if compared to the control value. In the absence of similar effect at the higher dose levels, this observation was considered not to be related to the treatment.

Reproduction Data
With exception for one female (no. 9) at the dose level of 100 mg/kg bw/day, all females were pregnant and had living fetuses at termination.
Mean post implantation loss was 0.5, 0.6, 1.0 and 0.3 per dam whereas mean number of living fetuses at termination was 10.4, 11.3, 12.1 and 12.3 per dam, both cited in order of ascending dose levels.
The relevant reproduction data, post-implantation loss and number of fetuses, were not affected by the treatment.

Macroscopic Findings
During macroscopical examination no findings were noted at any dose level.
Remarks on result:
other: dose range finding study for definitive study
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
External Abnormalities and Variations
No findings were noted during external examination of fetuses.

Sex Ratios
Percentage of male fetuses was 45.8%, 48.1%, 53.6% and 60.2% at the dos levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.
In the high-dose group higher portion of male than female fetuses was found; increase in percentage of male fetuses was statistically significant. However, due to the low group size, the significance of the difference may be incidental. Furthermore, at the high dose level a single female (no. 26) had 11 male fetuses while only 2 female fetuses, which contributed considerably to the shift in the fetal sex distribution. For these reasons, increased percentage of male fetuses was considered most probably not to be related to the treatment with the test item.
No significant differences in fetal sex distribution were noted in remaining dose groups.

Body Weights
Mean body weights of fetuses were 5.0 g, 5.1 g, 4.9 g and 4.9 g at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.
Fetal body weights were not affected by the treatment with the test item at any dose level.
Remarks on result:
other: dose range finding study for definitive study
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Treatment with the test item at the dose level of 1000 mg/kg bw/day caused a reversible reduction in food consumption accompanied by slightly reduced body weight gain and a reduction in corrected body weight gain. These effects were not accompanied by any effects on absolute body weight gain and therefore considered not to be adverse. No further signs of general toxicity were observed at any dose level.
The relevant reproduction data, post-implantation loss and number of fetuses, were not affected by the treatment. No indication of an effect on fetal development was noted at any dose level.
Executive summary:

The purpose of this study was to detect effects on the pregnant rat and development of the embryo and fetus consequent to exposure of the female to the Luperox F from day 6 post coitum (implantation) to day 20 post coitum. Luperox F was administered to female rats at the following dose levels: Group 1: 0 mg/kg body weight/day (control group) Group 2: 100 mg/kg body weight/day Group 3: 300 mg/kg body weight/day Group 4: 1000 mg/kg body weight/day. Each group consisted of eight females. A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil).

All females survived scheduled study period. No clinical signs were noted in females at any dose level. At the dose level of 1000 mg/kg bw/day, a reversible reduction in food consumption was observed. At the dose levels of 100 and 300 mg/kg bw/day, food consumption was not affected by the treatment with the test item. At the dose level of 1000 mg/kg bw/day, a reduction in body weight gain and in corrected body weight gain was noted in the absence of an effect on absolute body weights. At the dose levels of 100 and 300 mg/kg bw/day, body weight gain and absolute body weights were not affected by the treatment with the test item. One female at the low dose group was not pregnant. All remaining females were pregnant and had living fetuses at termination. The relevant reproduction data, post-implantation loss and number of fetuses, were not affected by the treatment. During macroscopical examination no findings were noted at any dose level. No findings were noted during external examination of fetuses. Fetal sex ratio was considered not to be affected by the treatment with the test item. No effects on fetal body weights were noted at any dose level.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Justification for type of information:
see read across rationale in Section 13.2
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: RccHanTM: WIST(SPF)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Kreuzelweg 535961 NM Horst / Netherlands
- Age at D0 post coitum: 11 weeks
- Weight at Day 0 Post Coitum: 189 to 236 g
- Fasting period before study: no
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding
- Diet (e.g. ad libitum): Pelleted standard Harlan Teklad 2914C rodent maintenance diet
- Water (e.g. ad libitum): community tap-water
- Acclimation period: >= 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared daily. Vehicle was pre-warmed to a temperature of approximately 40 °C. Luperox F was weighed into a glass beaker on a tared precision balance and approximately 80% of the warm vehicle was added (w/v). The mixture was homogenized using an electrical homogenizer until a clear to colloid-like yellowish solution was obtained. Having obtained a homogeneous mixture, remaining vehicle was added until the required final volume. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle: 25, 75 and 250 mg/ml
- Amount of vehicle (if gavage): 4 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. To confirm the stability (8 days at room temperature) samples of about 2 g of each concentration were taken from the middle of each aliquot used on day 7 of the treatment. At the end of the study, before the first planed necropsies, samples were taken from the middle to confirm concentration. The samples were delivered to the analytical depart¬ment at ambient temperature and analyzed immediately or stored frozen (at -20 ± 5 °C) until analysis.

The samples were analyzed by HPLC coupled to an UV detector following an analytical proce-dure provided by the Sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.
The Luperox F concentrations in the dose formulations ranged from 86.7% to 101.8% with reference to the nominal concentration and were within the accepted range of ±20%.
The homogeneous distribution of Luperox F in the preparations was approved because single results found did not deviate more than 4.7% from the corresponding mean and met the specified acceptance criterion of ≤15%.
In addition, the test item was found to be stable in application formulations when kept eight days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean value.
Details on mating procedure:
After acclimatization, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if:
- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.
The day of mating was designated day 0 post coitum.
Male rats of the same source and strain were used only for mating.
Duration of treatment / exposure:
15 days (during gestation period from day 6 to 20 post coitum)
Frequency of treatment:
daily
Duration of test:
up to GD 21
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous dose range-finding study in Han Wistar rats, Harlan Laboratories study no. D81444, using dose levels of 0, 100, 300 and 1000 mg/kg/day, where no adverse effects were observed up to the highest dose level.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily,during acclimatization and up to day of necropsy).

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Daily from day 0 until day 21 post coitum.

FOOD CONSUMPTION: Yes
- For the following periods: days 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18 and 18 - 21 post coitum.

WATER CONSUMPTION : No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #21
- Organs examined: gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, corpora lutea count and position of fetuses in the uterus. The uteri (and contents) of all females with live fetuses were weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain. If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide to accentuate possible hemorrhagic areas of implantation sites.
At the scheduled sacrifice, placentas were trimmed from any adherent tissue, and their wet weight taken.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
Fetuses were removed from the uterus by Caesarean section, sexed, weighed individually, exam-ined for gross external abnormalities, sacrificed and allocated to one of the following procedures:

1. Microdissection technique (sectioning/dissection technique). At least one half of the fetuses from each litter was fixed in Bouin's fixative (one fetus per container). They were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys. After examination, the tissue was preserved in a solution of glycerin/ethanol (one fetus per container). Descrip-tions of any abnormalities and variations were recorded.

2. The remaining fetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. Carcasses were processed through solutions of ethanol, glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for pres-ervation and storage. The skeletons were examined and all abnormal findings and variations were recorded. The specimens were preserved individually in small containers.
Statistics:
The following statistical methods were used to analyze food consumption, body weights, reproduction and skeletal examination data:

• Means and standard deviations of various data were calculated and included in the report.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.
Indices:
Pre- and post-implantation losses, embryonic and fetal deaths, live and dead fetuses, abnormal fetuses, fetal sex ratios and fetal body weights.
Historical control data:
See Attached backgroung material
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Summary of Performance of Mated FemalesViability / Mortality
See Table 1.

All females survived until the scheduled necropsy.

Clinical Signs
No clinical signs were observed at any dose level during the study.

Food Consumption
Mean food consumption from day 6 to 21 post coitum was 22.3, 22.2, 21.9 and 20.9 g/animal/day in order of ascending dose levels.
Treatment with the test item caused a reversible reduction in food consumption at the dose level of 1000 bw/day. The reduction was statistically significant from day 9 to 15 post coitum. Afterwards food consumption recovered and was similar to the control values towards the end of the study.
At the dose levels of 100 and 300 mg/kg bw/day, food consumption was similar to the control values during the entire treatment period.

Body Weights
(See attached figures)
Mean body weight gain from day 6 to 21 post coitum was 48%, 49%, 46% and 41% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively. Mean corrected body weight gain (body weight gain at termination corrected for the gravid uterus weight) was 10.8%, 10.1%, 8.1% and 4.8% in order of ascending dose levels.
Treatment with the test item caused a statistically significant reduction in body weights, body weight gain and corrected body weight gain at the dose level of 1000 mg/kg bw/day. The statistical significance of the effect was observed from day 11 of the gestation period onwards for the body weights and from day 8 of the gestation period onwards for the body weight gain.
A reversible reduction in body weight gain was observed at the dose level of 300 mg/kg bw/day. This effect was statistically significant day 11 to 16 of the gestation period. No statistically significant changes were observed in absolute body weights or in corrected body weight gain.
At the dose level of 100 mg/kg bw/day, body weights, body weight gain and corrected body weight gain were similar to the respective values in the control group.

Reproduction Data
(See Summary Table 2)

The corresponding historical control data are included in the Attached background material.

The relevant reproduction data (post implantation loss and number of fetuses per dam) were not affected by treatment with the test item. Mean number of implantations lost was 4.5, 3.3, 5.5 and 4.1 per dam whereas mean number of live fetuses was 12.6, 12.6, 12.0 and 11.8 per dam at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day.

The slightly lower mean number of fetuses in the high-dose group was due to the lower number of implantation sites. Because implantation process is considered to be completed before the treatment start, this observation was not related to the treatment.

Pathology (Maternal Data)
Macroscopic Findings
No treatment-related findings were noted during macroscopical examination of females at any dose level.

In group 4, a watery fluid was observed in one non-pregnant female (no. 75). Due to isolated occurrence, this finding was considered not to be related to the treatment.

No further findings were noted during necropsy in any group.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEC
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Placenta Weights
Placenta weights (TABLE 3) were not affected by the treatment with the test item at any dose level. Mean placenta weights calculated on a litter basis were: 0.515 g, 0.530 g, 0.549 g and 0.538 g whereas calculated on an individual basis, they were 0.511 g, 0.527 g, 0.542 g and 0.539 g, both cited in order of ascending dose level.
Mean values calculated on an individual basis were statistically significantly higher in all dose groups if compared to the control. The differences were only minor and not dose-dependent. Therefore this observation was considered not to be related to the treatment.

External Abnormalities and Variations
No findings were noted during external examination of the fetuses in any group.

Sex Ratios
No test item-related differences in the sex ratio (TABLE 2) of the fetuses were observed in any group.
The proportion of male fetuses was 47.5%, 53.3%, 46.4% and 49.8% in order of ascending dose level.

Body Weights
Fetal body weights (TABLE 4) were not affected by the treatment with the test item at any dose level. Mean fetal body weights for male and female fetuses calculated on a litter basis were: 5.0 g, 5.1 g, 5.2 g and 5.1 g whereas calculated on an individual basis, they were 5.0 g, 5.1 g, 5.2 g and 5.1 g, both cited in order of ascending dose level.
Mean body weights of live fetuses calculated on individual basis were statistically significantly higher in mid-dose group and calculated on an individual basis were statistically significantly higher in all dose groups compared to the respective control values. The differences were however only minor and did not follow a dose-dependency. Therefore this observation was considered not to be related to the treatment.

Visceral Abnormalities and Variations
The corresponding historical control data are in an attached background material.
During visceral examination of the fetuses, findings were noted in:
36% examined fetuses (in 96% litters) in the control group
34% examined fetuses (in 96% litters) at the dose level of 100 mg/kg bw/day
35% examined fetuses (in 100% litters) at the dose level of 300 mg/kg bw/day
41% examined fetuses (in 100% litters) at the dose level of 1000 mg/kg bw/day
Fetuses with anophthalmia (unilateral) were found in all dose groups; one fetus (in litter no. 42), one fetus (in litter no. 72) and two fetuses (in litter nos. 76 and 95) with this finding were observed at the dose levels of 100, 300 and 1000 mg/kg bw/day, respectively. No fetuses with this observation were found in the control group. In addition to the unilateral anophthalmia found in fetuses in groups 2 and 3 and one of the fetuses in group 4 (from litter nos. 42, 72 and 76, respectively), reduced body weights but no other visceral findings were recorded. The second affected fetus in group 4 (from litter no. 95) had one eye missing, one eye small, reduced body weight as well as multiple abnormalities of other organs (absent kidneys and ureter and malpositioned ovaries). Based on this observation, a different mechanism causing the developmental disturbance may be concluded for the two fetuses in group 4. Considering the type and distribution of fetuses with the eye finding among groups, there were no apparent dose dependency. Although anophthalmia was not recorded in the most recent historical controls, this abnormality is known to occur spontaneously in the rat strain used in the study and therefore the observation in this study was considered to be incidental and not related to the treatment.

Further abnormalities occurred in single fetuses and therefore were considered not to be related to the treatment; a situs inversus found in one fetus in litter no. 27 at the dose level of 100 mg/kg bw/day as well as severely thin diaphragm tendinous region in one fetus in litter no. 93, severely dilated renal pelvis in one fetus in litter no. 87, and absent kidney and ureter together with severely malpositioned ovary adjacent to adrenal in one fetus in litter no. 95 at the dose level of 1000 mg/kg bw/day.
Type and distribution of variations recorded during the visceral examination did not give any indication of a test item-related effect. The variations were distributed among groups without dose dependency and their frequency remained within the historical control range.

Bone and Cartilage Abnormalities and Variations
The corresponding historical control data are in an attached background material.
During skeletal examination of the fetuses, findings were noted in:
10% examined fetuses (in 42% litters) in the control group
20% examined fetuses (in 52% litters) at the dose level of 100 mg/kg bw/day
16% examined fetuses (in 61% litters) at the dose level of 300 mg/kg bw/day
35% examined fetuses (in 80% litters) at the dose level of 1000 mg/kg bw/day
Abnormalities were found in individual fetuses distributed without dose dependency among groups and therefore they were considered not to be test item-related. In particular, multiple findings (misshapen thoracic vertebral body 1, split cervical vertebral bodies 5 and 6 split and fused cervical vertebral bodies 5 to 7 and thoracic vertebral body 1) were found in one fetus in litter no. 24 in the control group, severe fused sternebrae 1 and 2 and short left costal cartilage 1 were found in one fetus in litter no. 31 at the dose level of 100 mg/kg bw/day as well as multiple findings (disorganized, misshapen, absent or fused cervical or upper thoracic vertebral structures as well as long cervical rib, misshapen clavicle and scapula, split sternebra and xyphoid cartilage and fused ribs) were found in one fetus in litter no. 53 and short costal cartilage 2 in another fetus in litter no. 55 at the dose level of 300 mg/kg bw/day. No skeletal abnormalities were found at the dose level of 1000 mg/kg bw/day.
At the dose level of 1000 mg/kg bw/day, slightly higher number of fetuses with fused zygomatic arch was recorded; 23%/60% fetuses/litters compared to 5% /29% fetuses/litters in the control group. Further, higher number of fetuses had malpositioned pelvic girdle: 14%/40% fetuses/litters compared to 3%/13% fetuses/litters in the control group. Values in the high-dose group were slightly above the historical control background and therefore this observation was considered to be related to the treatment.

Ossification and Supernumerary Ribs
The corresponding historical control data are in an attached background material.
No test item-related effects on ossification stage or incidence of supernumerary ribs were observed at any dose levels.
Incidentally, slightly but statistically significantly lower incidence of non-ossified limb bones was observed in the mid-dose group. Due to the lack of a dose dependency, this finding was considered not to be related to the treatment.

Additional Cartilage Variations
The corresponding historical control data are in an attached background material.
No test item-related additional cartilage variations were observed at any dose levels.
Incidentally, slightly but statistically significantly higher incidence of interrupted costal cartilage was observed in the mid-dose group. Due to the lack of a dose dependency, this finding was considered not to be related to the treatment.
Further, statistically significantly higher number of fetuses with branched xiphoid cartilage was observed in all dose groups when calculated on a fetus basis. The differences were only minor and they were not statistically significant when calculated on a litter basis. Further, values were distributed without dose-dependency. For these reasons this observation was considered not be related to the treatment with the test item.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity sign of minor delay in development, probably secondary to the maternal toxicity
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1: Summary of Performance of Mated Females

Group
Dose (mg/kg)

1
(0)

2
(100)

3
(300)

4
(1000)

Female numbers

1 - 24

25 - 48

49 - 72

73 - 96

Number of mated females

24

24

24

24

Not pregnant (A)

0

1

1

4

Number of females with live fetuses at termination (B)

24

23

23

20

 (A)  Female nos. 36, 56, 75, 83, 85 and 88.

(B)   Only dams with at least one live fetus at Caesarean section were used for the calculations of food consumption, body weight gain and corrected body weight gain data

TABLE 2: REPRODUCTION DATA SUMMARY
PARENTAL GENERATION - POST COITUM

 

 

 

GROUP 1

0 MG/KG

 

GROUP 2

100 MG/KG

 

GROUP 3

300 MG/KG

 

GROUP 4

1000 MG/KG

 

 

 

NUMBER OF DAMS

 

24

 

23

 

23

 

20

 

 

 

CORPORA LUTEA

 

320

 

308

 

297

 

254

 

 

MEAN (+)

13.3

13.4

12.9

12.7

 

 

ST.DEV.

1.6

2.0

1.9

1.9

 

 

 

PRE-IMPLANTATION LOSS

 

3

 

9

 

5

 

9

 

 

% OF CORP. LUTEA (#)

0.9

2.9

1.7

3.5 #

 

 

MEAN (+)

0.1

0.4

0.2

0.5

 

 

ST.DEV.

0.4

0.7

0.6

1.4

 

 

NUMBER OF DAMS AFFECTED

2

6

3

3

 

 

 

IMPLANTATION SITES

 

317

 

299

 

292

 

245

 

 

% OF CORP. LUTEA (#)

99.1

97.1

98.3

96.5 #

 

 

MEAN (+)

13.2

13.0

12.7

12.3

 

 

ST.DEV.

1.6

2.0

1.8

1.8

 

 

 

POST-IMPLANTATION LOSS

 

14

 

10

 

16

 

10

 

 

% OF IMPL. SITES (#)

4.4

3.3

5.5

4.1

 

 

MEAN (+)

0.6

0.4

0.7

0.5

 

 

ST.DEV.

1.1

0.7

0.8

0.8

 

 

NUMBER OF DAMS AFFECTED

7

7

12

7

 

 

IMPLANTATION SITE SCARS

0

0

0

0

 

 

EMBRYONIC/FETAL DEATHS TOTAL

14

10

16

10

 

 

EMBRYONIC RESORPTIONS

14

10

16

10

 

 

FETAL RESORPTIONS

0

0

0

0

 

 

 

 

 

 

 

 

 

TOTAL FETUSES

303

289

276

235

 

% OF IMPL. SITES (#)

95.6

96.7

94.5

95.9

 

MEAN (+)

12.6

12.6

12.0

11.8

 

ST.DEV.

2.2

2.1

2.2

1.7

 

LIVE FETUSES

303

289

276

235

 

DEAD FETUSES

0

0

0

0

 

ABNORMAL FETUSES

0

0

0

0

SEX OF FETUSES

TOTAL MALES

 

 

144

 

 

154

 

 

128

 

 

117

% OF FETUSES (#)

47.5

53.3

46.4

49.8

MEAN (+)

6.0

6.7

5.6

5.9

ST.DEV.

2.4

2.6

2.1

2.0

TOTAL FEMALES

159

135

148

118

% OF FETUSES (#)

52.5

46.7

53.6

50.2

MEAN (+)

6.6

5.9

6.4

5.9

ST.DEV.

2.6

2.1

2.3

2.1

*/** : Dunnett-Test based on pooled variance significant at level 5% (*) or 1% (**)

#/## : Fisher's Exact Test significant at level 5% (#) or 1% (##)

+ : Steel Test significant at level 5%

 

TABLE 3. PLACENTA WEIGHTS SUMMARY

PARENTAL GENERATION - POST COITUM

 

 

 

GROUP 1

0 MG/KG

 

GROUP 2

100 MG/KG

 

GROUP 3

300 MG/KG

 

GROUP 4

1000 MG/KG

PLACENTA WEIGHTS (G) (LITTER BASIS)

TOTAL FETUSES N (LITTERS)

 

24

 

23

 

23

 

20

MEAN (*)

0.515

0.530

0.549

0.538

ST.DEV.

0.043

0.052

0.061

0.052

PLACENTA WEIGHTS (G) (INDIVIDUAL BASIS)

TOTAL FETUSES N (FETUSES)

 

303

 

289

 

276

 

235

MEAN (*)

0.511

0.527 *

0.542 **

0.539 **

ST.DEV.

0.074

0.080

0.085

0.081

 

TABLE 4. REPRODUCTION DATA SUMMARY

PARENTAL GENERATION - POST COITUM

 

 

GROUP 1

0 MG/KG

 

GROUP 2

100 MG/KG

 

GROUP 3

300 MG/KG

 

GROUP 4

1000 MG/KG

 

NUMBER OF DAMS

 

24

 

23

 

23

 

20

SEX OF FETUSES (CONT.)

LIVE MALES

144

154

128

117

LIVE FEMALES

159

135

148

118

 

 

 

 

 

WEIGHTS OF LIVE FETUSES (G) (LITTER BASIS)

 

TOTAL FETUSES

 

N (LITTERS)

24

23

23

20

 

MEAN (*)

5.0

5.1

5.2+

5.1

 

ST.DEV.

0.2

0.3

0.3

0.4

 

MALES

 

N (LITTERS)

24

23

23

20

 

MEAN (*)

5.1

5.2

5.3*

5.2

 

ST.DEV.

0.2

0.3

0.3

0.3

 

FEMALES

 

N (LITTERS)

24

23

23

20

 

MEAN (*)

4.9

4.9

5.1

4.9

 

ST.DEV.

0.2

0.3

0.3

0.3

 

WEIGHTS OF LIVE FETUSES (G) (INDIVIDUAL BASIS)

 

TOTAL FETUSES

 

N (FETUSES)

303

289

 

276

 

MEAN (*)

5.0

5.1**

5.2**

5.1*

 

ST.DEV.

0.4

0.4

0.4

0.4

 

MALES

 

N (FETUSES)

144

154

128

117

 

MEAN (*)

5.1

5.2**

5.3**

5.2**

 

ST.DEV.

0.3

0.4

0.4

0.5

 

FEMALES

 

N (FETUSES)

159

135

148

118

 

MEAN (*)

4.8

4.9

5.0**

4.9

 

ST.DEV.

0.3

0.4

0.4

0.4

 

*/** : Dunnett-Test based on pooled variance significant at level 5% (*) or 1% (**)

#/## : Fisher's Exact Test significant

+ : Steel Test significant at level 5%

Fetal Examination: External Abnormalities and Variations: see Attached background material

Fetal Examination: Visceral Abnormalities and Variations

see Attached background material

Fetal Examination: Bone and Cartilage Abnormalities and Variations:

see Attached background material
Conclusions:
The purpose of this study was to detect effects on the pregnant rat and development of the embryo and fetus consequent to exposure of the female to Luperox F from day 6 post coitum (implantation) to day 20 post coitum (the day prior to Caesarean section). Luperox F was administered orally by gavage once daily at dose levels of 0, 100, 300 and 1000 mg/kg bw/day.
All females survived until the scheduled necropsy. No clinical signs were recorded in any group.
Sins of general toxicity were observed in females at the dose levels of 1000 mg/kg bw/day. A reduction in food consumption was observed in this group together with reduced body weights, reduced body weigh gain and reduced corrected body weight gain. Effect on food consumption was reversible and at the end of the gestation it was similar to the control values despite continued treatment and was therefore considered not to be adverse. Different from the changes in food consumption, effects on body weight and body weight development remained statistically significant until the completion of the study and therefore they were considered to be adverse.
Reduction in body weight gain was observed also at the dose level of 300 mg/kg bw/day. This effect was reversible and did not result in any significant changes in absolute body weights. For these reasons it was considered not to be adverse.
Relevant reproduction data (post-implantation loss and number of fetuses per dam) were not affected by the treatment with the test item at any dose level.
No test item-related effects on placenta weights were observed at any dose level.
During visceral examination, anophthalmia was found in one fetus in each group 2 and 3 and two fetuses in group 4. In one of the fetuses at the high dose level, the eye finding was accompanied by small other eye and abnormalities in other organs indicating possible another mechanism leading to the developmental disturbance than that observed in the remaining three fetuses, in which anophthalmia was the only identified visceral change. Due to the type and distribution of this finding, which did not clearly follow dose dependency, it was considered to be incidental and not related to the treatment. Also the type and distribution of the remaining visceral findings gave no indication of a test item-related effect.
No adverse effects on fetal skeleton development occurred in the study at any dose level. A slightly higher number of fetuses with fused zygomatic arch and slightly higher number of fetuses with malpositioned pelvic girdle were found at the dose level of 1000 mg/kg bw/day. The differences to the control values were only minor and frequency of these findings was close to the historical control range. Therefore these findings were considered to be a sign of minor delay in development, probably secondary to the maternal toxicity.
Based on these results, the NOAEL (No Observed Adverse Effect Level) for maternal toxicity was considered to be 300 mg/kg bw/day whereas NOEL (No Observed Effect Level) was 100 mg/kg bw/day. For prenatal developmental toxicity, the NOEL was considered to be 300 mg/kg bw/day and the NOAEL was considered to be 1000 mg/kg bw/day.
Executive summary:

The possible toxic effects of Luperox F on the pregnant rat and development of the embryo and fetus was evaluated by continuous oral administration to female rat from day 6post coitum(implantation) to day 20post coitum(the day prior to Caesarean section). This study was performed following the OECD test guideline no. 414.

Four groups of 24 mated females per group were treated by gavage with Luperox F once daily at dose levels of 0, 100, 300 and 1000 mg/kg body weight/day (groups 1, 2, 3 and 4, respectively). A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil). All females were sacrificed on day 21post coitumand the fetuses were removed by Caesarean section.

All females survived until the scheduled necropsy. No clinical symptoms or signs were observed at any dose level during the study. Treatment with the Luperox F caused a reversible reduction in food consump­tion at the dose level of 1000 bw/day. This reduction was considered not to be adverse. Food consumption was not affected by the treatment at the dose levels of 100 and 300 mg/kg bw/day. Treatment with the Luperox F caused a statistically significant reduction in body weights, body weight gain and corrected body weight gain (body weight gain corrected for the gravid uterus weight) at the dose level of 1000 mg/kg bw/day. This effect was considered to be adverse. A reversible reduction in body weight gain in the absence of significant effects on absolute body weights or corrected body weight gain was recorded at the dose level of 300 mg/kg bw/day. This effect was considered not to be adverse. No effects on body weight gain or body weights were observed at the dose level of 100 mg/kg bw/day.The relevant reproduction data (post implantation loss and number of fetuses per dam) were not affected by treatment with the Luperox F. No Luperox F-related findings were observed during the macroscopical examination at any dose level.

No effects on placenta weights were observed at any dose level. No findings were observed during the external examination of the fetuses. No Luperox F-related effects on the sex ratio of the fetuses were noted in any group. No Luperox F-related effects on fetal body weights were noted.

Type and distribution of findings recorded during visceral examination of fetuses gave no indication of a test item-related effect.

A slight increase in the incidence of fused zygomatic arch and malpositioned pelvic girdle was observed at the dose level of 1000 mg/kg bw/day. This finding was considered not to be adverse. No effects on ossification stage or incidence of supernumerary ribs were observed at any dose level. No Luperox F-related additional cartilage variations were observed at any dose level.

Based on these results, the NOAEL (No Observed Adverse Effect Level) for maternal toxicity was considered to be 300 mg/kg bw/day whereas NOEL (No Observed Effect Level) was 100 mg/kg bw/day. For prenatal developmental toxicity, the NOEL was considered to be 300 mg/kg bw/day and the NOAEL was considered to be 1000 mg/kg bw/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Aug 2017 - Jan 2018
Reliability:
1 (reliable without restriction)
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developme ntal Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD 421, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2016
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral)
Version / remarks:
2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents.
Version / remarks:
2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Solubility and stability of the test substance in the vehicle: Stability for at least 5 hours at room temperature, 13 days in the refrigerator and 3 weeks in freezer is confirmed over the concentration range 1 to 200 mg/mL
Species:
rat
Strain:
other: Crl: WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males were 10-12 weeks old; females were 12-14 weeks old
- Weight at study initiation: males between 275 and 319 g; females between 187 and 230 g
- Fasting period before study: no
- Housing: On arrival and following the pre-test (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages. During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon
plastic cages. During the post-mating phase, males were housed in their home cage with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages. During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam. The cages contained appropriate bedding. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cageenrichment, bedding material, food and water.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water: Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours.
- Acclimation period: 6 days prior to start of the pre-test period (females) or 6 days before the commencement of dosing (males)

DETAILS OF FOOD AND WATER QUALITY:
The feed was analysed by the supplier for nutritional components and environmental contaminants. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
Periodic analysis of the water is performed. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 21
- Humidity (%): 47 to 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 Sept 2017 To: 15 Nov 2017
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least every 9 days (Groups 1-3 as a clear solution) or every 1-2 days (Group 4 as an opaque suspension), formulated in daily portions. Formulations were heated to a
maximum temperature of 50±5°C for maximally 10 minutes to obtain visual homogeneity. Formulations were stored in the refrigerator. When formulations were used on the day of preparation, formulations were released for dosing when they had reached a temperature of 40°C or lower. When
dosing formulations were prepared and stored in the refrigerator, formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing. The dosing formulations and vehicle were continuously stirred until and during dosing.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis in Week 1 for concentration anaysis in all groups and for homogeity in groups 2 and 4 (low and high dose groups).
All samples were stored on dry ice immediately after sampling.
Concentration analysis was performed for duplicate sets of samples (approximately 500 mg). Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions (Group 1-3) and ± 15% for suspensions (Group 4) of target concentration.
Homogeneity analysis was performed on duplicate sets of samples (approximately 500 mg).
Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was # 10%.
Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations used in the present study.
Details on mating procedure:
- M/F ratio per cage: 1/1
Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were
separated.
Duration of treatment / exposure:
Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to and including the day before scheduled necropsy.
Females that delivered were treated for 50-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 14 or 16 days after delivery, up to and including the day before scheduled necropsy.
Females which failed to deliver were treated for 41 days.
Frequency of treatment:
daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on the results of a 17-day dose range finder with oral administration of 1,3-bis(tert-butylperoxyisopropyl)benzene in rats, and in an attempt to produce graded responses to the test item.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical observations were at least conducted immediately after dosing and 3 hours (± 30 minutes) after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. Terminal body weights were recorded on the day of necropsy (fasted for males, non-fasted for females).

FOOD CONSUMPTION:
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND
1, 4, 7, and 13.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: all
- Parameters checked in accordance with Guidelines were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: all
- Parameters checked in accordance with Guidelines were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 6-13).
- Dose groups that were examined: all
- Battery of functions tested: Hearing ability; Pupillary reflex; Static righting reflex; Fore- and hind-limb grip strength; Locomotor activity.

IMMUNOLOGY: No

other: organ weights F0 generation according to Guidelines

Sacrifice and pathology
GROSS PATHOLOGY: Yes, according to Guidelines
HISTOPATHOLOGY: Yes, according to Guidelines

Other examinations
F0 generation: Estrous cycle determination, cohabitating/mating procedure, general reproduction data, TSH and total T4 measurement
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: No
Fetal examinations:
Post implantation survival index, litter size, live birth index, viability index, lactation index, clinical signs, body weights, sex ratio, AGD, areola/nipple retention, serum T4 levels, macroscopy
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Paremetric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. The Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group.
Incidene: An overall Fisher’s exact test was used to compare all groups at the 5% significance level.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Indices:
Mating (%): (Number of females mated / Number of females paired) x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): (Number of pregnant females / Number of females mated) x 100
Gestation index (%): (Number of females with living pups on Day 1 / Number of pregnant females) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Post-implantation survival index (%): (Total number of offspring born / Total number of uterine implantation sites) x 100

Live birth index (%): (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100
Viability index (%): (Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering) x 100
Lactation index (%): (Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling)) x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Piloerection was observed in about half of the animals treated at 1000 mg/kg bw/day, starting after about one (males) or two (females) weeks of treatment. In females piloerection occurred mostly in the post-coitum period. Salivation seen among animals treated with the test item, in a dose-related manner, was considered to be a physiological response rather than a sign of systemic toxicity considering its modest severity (slight or moderate) and the time of occurrence (i.e. after dosing).
Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated
to treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
The premature death of one female of the 1000 mg/kg bw/day group was considered to be related to treatment. All other animals survived until scheduled sacrifice.
The high dose female was found dead, while in labour, on Day 24 of the post-coitum period. On the day prior to death, she showed piloerection. Her body weight development and food consumption were unremarkable. The main macroscopic finding was the presence of dead fetuses in the uterus (10 in total). Main microscopic findings were moderate centrilobular hepatocellular necrosis in the liver and slight bone marrow atrophy. The hepatocellular necrosis was considered to be the main cause of death. As test item-related hepatocellular necrosis was also noted in a surviving male of the 1000 mg/kg bw/day group, the death of the high dose female was attributed to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain of males treated at 1000 mg/kg bw/day was reduced throughout the treatment period (statistically significant), with slight weight loss in several animals in the last week of this per iod. The resulting reductions in mean body weights were statistically significant at Days 8 and 15 of the mating period (8% difference from controls at the end of treatment). To a lesser extent, reduced weight gain was noted in males treated at 300 mg/kg bw/day (not statistically significant). Mean terminal body weight of 300 mg/kg bw/day males was 5% lower than that of controls.
Body weights and body weight gain of females were considered not to be affected by treatment up to and including 1000 mg/kg bw/day. The statistically significantly higher mean body weight gain noted in 1000 mg/kg bw/day females at Day 7 of the lactation period, mainly due to higher weight gain of one female, was regarded as a chance finding.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males at 1000 mg/kg bw/day consumed less food than controls in treatment weeks 1-2 and, to a lesser extent, weeks 3-4. Their food consumption after correction for body weight was reduced only in weeks 1-2.
Food consumption of females (before and after correction for body weight) was considered not to be affected by treatment. Occasional, slightly higher values noted at 1000 mg/kg bw/day (between Days 11-17 of the post-coitum period and Days 1-4 of the lactation period) were regarded as unrelated to treatment as mean food consumption of 1000 mg/kg bw/day females remained close to that of controls at the other time points.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Red blood cell parameters showed the following statistically significant differences between treated rats and controls (percentage differences from the control group mean are indicated between parentheses):
- Lower haemoglobin concentration in males at 1000 mg/kg bw/day (9%).
- Lower mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) in females at 1000 mg/kg bw/day (5 and 6%, respectively).
There were no treatment-related changes in white blood cell parameters or number of platelets. There were no test item-related differences in coagulation parameters. The lower mean activated partial
thromboplastin time (APTT) values noted at 1000 mg/kg bw/day (statistically significant in males only) were considered to have arisen as a result of slightly high concurrent control values and, therefore, regarded as unrelated to treatment. Mean APTT values at 1000 mg/kg bw/day remained in the normal ranges.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes in clinical chemistry parameter, mostly in 1000 mg/kg bw/day males, distinguished treated animals from control animals (percentage differences from the control group mean are indicated between parentheses). Mean values for these parameters in the affected group(s) of males were (slightly) out of the normal ranges.
- Higher alanine aminotransferase (ALAT) in males at 1000 mg/kg bw/day (48%).
- Higher total protein in males at 300 and 1000 mg/kg bw/day (5 and 10%, respectively).
- Higher albumin in males at 1000 mg/kg bw/day (10%).
- Higher creatinine in males at 1000 mg/kg bw/day (23%).
- Higher potassium in males at 1000 mg/kg bw/day (14%). Potassium was also higher (10%) in males at 300 mg/kg bw/day but the difference from controls was not statistically significant.
- Lower cholesterol in males at 1000 mg/kg bw/day (22%).
The other statistically significant variations noted in clinical chemistry values were considered unrelated to treatment due to the lack of a dose-related response, slightly high control value and/or a high value in a single animal.
Thyroid hormone analyses:
Mean serum T4 levels were statistically significantly decreased in F0 males at 300 and 1000 mg/kg bw/day (relative differences from controls: 32 and 51%, respectively), whereas mean serum TSH levels in these males were increased (4.3 and 9.7-fold, respectively; statistically significant at 1000 mg/kg bw/day only). The mean values in these groups of males were out of the historical control ranges.
Serum levels of T4 and TSH in F0 females were considered not to be affected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Females of the 1000 mg/kg bw/day group had lower mean numbers of total movements and ambulations compared to those of the control group (nearly 60% difference, statistically significant for total movements only). Motor activity was particularly low in 3 females which had values below the historical control ranges (2). The other two 1000 mg/kg bw/day females tested had activity values at or just below the concurrent control group ranges.
Activity habituation profiles were similar between the treated and control groups with a decreasing trend in activity over the duration of the test period Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was not affected by treatment.
(2) Historical control data for motor activity in female Wistar Han rats (period 2015-June 2017):
Total movements: mean = 3502, P5 - P95 = 1478 - 5683 (n=195).
Ambulations: mean = 855, P5 - P95 = 301 - 1467 (n=195).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related changes were noted in the weights of the thymus in males and of the thyroid gland, liver and kidneys in both sexes, as described below. Relative differences from control values and statistical significances are shown in the Text Table 1 (see any other information).
- Thymus (absolute and relative to body weight): lower (not statistically significant) in males at 300 and 1000 mg/kg bw/day.
- Thyroid gland (absolute and relative to body weight): higher (statistically significant) in males at 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day.
- Liver (absolute and relative to body weight): higher (statistically significant) in males at 100, 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day.
- Kidneys (relative to body weight): higher (statistically significant) in males at 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day.
There were no other test item-related organ weight changes. A few statistically significant organ weight differences noted at 1000 mg/kg bw/day were regarded as secondary to differences in terminal body weight rather than as primary effects of the test item (lower absolute and relative prostate gland weights, higher relative brain weight in females).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related macroscopic findings consisted of:
- Liver: Enlarged at 300 mg/kg bw/day (1/10 females) and 1000 mg/kg bw/day (9/10 males, 2/10 females).
- Liver: Black-brown discoloration at 300 mg/kg bw/day (2/10 males) and 1000 mg/kg bw/day (10/10 males, 3/10 females).
- Thyroid gland: Enlarged at 1000 mg/kg bw/day (3/10 males).
- General: Emaciated appearance at 300 mg/kg bw/day (1/10 females) and 1000 mg/kg bw/day (5/10 females). However, as terminal body weights were within normal limits, this finding was not considered toxicologically significant.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These necropsy findings were herefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the thymus of males and the thyroid (see also 'any other information').
Kidneys:
- Tubular degeneration was present in males and females at 1000 mg/kg bw/day up to moderate degree.
- Tubular basophilia was present at increased incidence and/or severity in males starting at 300 mg/kg bw/day and in females at 1000 mg/kg bw/day up to slight degree.
- Hyaline droplet accumulation was present at increased severity in males starting at 300 mg/kg bw/ day up to moderate degree.
- Tubular mineralization was present at increased incidence and/or severity in females starting at 300 mg/kg bw/day up to moderate degree.
Liver:
- Hepatocellular hypertrophy was present in males starting at 100 mg/kg bw/day and in females at 1000 mg/kg bw/day up to slight degree. This correlated with the macroscopic enlargement and the increased organ weight.
- Pigment deposition was present in males at 300 and 1000 mg/kg bw/day up to slight degree. This sometimes correlated with the macroscopic black-brown discoloration.
- Hepatocellular necrosis was present in a single male at 1000 mg/kg bw/day at slight degree.
Thyroid gland:
- Follicular cell hypertrophy was present at increased incidence and/or severity in males starting at 100 mg/kg bw/day and in females starting at 300 mg/kg bw/day up to moderate degree. This correlated with the macroscopic enlargement and the increased organ weight.
- Colloid alteration was present in males and females at 1000 mg/kg bw/day up to minimal degree.
Thymus:
- Lymphoid atrophy was present in males at 1000 mg/kg bw/day at minimal degree. This correlated with the decreased organ weight.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Number of abortions:
no effects observed
Description (incidence and severity):
No signs of difficult or prolonged parturition were noted among the other pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was considered not to be affected by treatment. The survival indices were 81, 94, 88 and 81% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
It was noted that the post-implantation survival indices for the control and 1000 mg/kg bw/day groups were lower than normal. Two control females had more unaccounted for sites (i.e. difference between number of implantation sites and total number of pups born) than observed normally (2 females with 6 and 7 unaccounted for sites, respectively). The low post-implantation survival index for the 1000 mg/kg bw/day group was due to one female which died while in labour. In her uterus 13 implantation sites, 10 dead fetuses and 3 early resorptions were noted. As the death of that females was related to liver toxicity and the other 1000 mg/kg bw/day females had zero (4 females) or only 1-2 (4 females) unaccounted for sites, postimplantation survival at 1000 mg/kg bw/day was judged to be unaffected by treatment.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
In the uterus of one high dose female 3 early resorptions were noted, which is considered not relatd to treatment, and is within normal limits.
Dead fetuses:
no effects observed
Description (incidence and severity):
The mean number of living pups at first litter check (live litter size) was not affected by treatment.
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment. The live birth indices were 99-100%. The numbers of dead pups at first litter check in the control and 100 mg/kg bw/day groups were within normal limits (one dead pup in two liters) and the incidental pup mortality in the lowest dose group (100 mg/kg bw/day) was regarded as unrelated to treatment.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Fertility index was considered not to be affected by treatment.
Except for one female at 300 mg/kg bw/day and one female at 1000 mg/kg bw/day, all mated females were pregnant. The fertility indices were 100% for the control and 100 mg/kg bw/day groups and 90% for the 300 and 1000 mg/kg bw/day groups.
The non-pregnancy of one female was due to infertility of the male she was paired with (see histopathology changes in the testes and epididymides of this male). The non-pregnancy of another female was not associated with related histopathology changes in reproductive organs. These incidental cases of male infertility/non-pregnancy were regarded as unrelated to treatment as the incidence showed no dose-related trend and was within normal limits.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
histopathology: non-neoplastic
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, treatment related lower body weights of pups were noted on PND 13. This was statistically significant for male pups, female pups and sexes combined, and resulted in a 20% lower mean body weight when compared to concurrent controls.
The remaining statistical significant changes (on PND 7 at 100 and 1000 mg/kg bw/day and on PND 13 at 100 and 300 mg/kg bw/day) were not considered toxicologically relevant as no dose response was apparent, all values were within normal limits7 of which the control group values were at the higher end.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment. The live birth indices were 99-100%. The numbers of dead pups at first litter check in the control and 100 mg/kg bw/day groups were within normal limits (one dead pup in two litters nos. 47 and 51) and the incidental pup mortality in the lowest dose group (100 mg/kg bw/day) was regarded as unrelated to treatment.
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices were 100% for the 300 mg/kg bw/day group and 99% for the other groups. One pup of the control group died spontaneously at PND 1, one pup of the 100 mg/kg bw/day group was euthanized for humane reasons at PND 2 and one pup of the 1000 mg/kg bw/day group went missing (presumably cannibalized) at PND 2. This incidental pup mortality was considered unrelated to treatment as the incidence showed no dose-related trend and remained within the range considered normal for pups of this age.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was considered not to be affected by treatment.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean number of living pups at first litter check (live litter size) was not affected by treatment.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
see reduction in number of live offspring
External malformations:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of findings noted remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.
Treatment up to and including 1000 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Serum T4 levels in male and female PND 14-16 pups were judged to be unaffected by treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes

Accuracy

The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with the target concentrations (mean accuracies between 96.2% and 98.7%). No test item was detected in the Group

1 formulation.

Homogeneity

The formulations of Groups 2 and 4 were homogeneous (coefficient of variation of 2.8% and 2.1% in the low and high dose goups, respectively).

Text Table 1 Mean Percent Organ Weight Differences from Control Groups

 

Males

Females

Dose level (mg/kg):

100

300

1000

100

300

1000

 

 

 

 

 

 

 

THYMUS

 

 

 

 

 

 

              Absolute

5

-27

-41

-

-

-

              Relative to body weight

3

-19

-35

-

-

-

 

 

 

 

 

 

 

THYROID GLAND

 

 

 

 

 

 

              Absolute

13

38**

38**

6

13

19*

              Relative to body weight

0

40**

40**

0

17

17**

 

 

 

 

 

 

 

LIVER

 

 

 

 

 

 

              Absolute

28*

26*

63**

-2

15

25**

              Relative to body weight

25**

38**

81**

0

11

39**

 

 

 

 

 

 

 

KIDNEYS

 

 

 

 

 

 

              Absolute

12

16

16

2

4

8

              Relative to body weight

9

27**

28**

3

0

19**

*: P<0.05, **: P<0.01. - no remarkable findings

Text Table 2. Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals

 

Males

Females

Dose level (mg/kg):

0

100

300

1000

0

100

300

1000

 

 

 

 

 

 

 

 

 

KIDNEYSa

5

5

5

5

5

5

5

5

    Tubular degeneration

 

 

 

 

 

 

 

 

        Minimal

-

-

-

-

-

-

-

2/5

        Slight

-

-

-

1/5

-

-

-

-

        Moderate

-

-

-

2/5

-

-

-

-

    Tubular basophilia

 

 

 

 

 

 

 

 

        Minimal

2/5

3/5

2/5

-

-

1/5

-

2/5

        Slight

-

-

2/5

3/5

-

-

-

1/5

a = Number of tissues examined from each group.

 

Males

Females

Dose level (mg/kg):

0

100

300

1000

0

100

300

1000

 

 

 

 

 

 

 

 

 

KIDNEYSa

5

5

5

5

5

5

5

5

    Hyaline droplet accumulation

 

 

 

 

 

 

 

 

        Minimal

3/5

3/5

-

-

-

-

-

-

        Slight

2/5

1/5

1/5

3/5

-

-

-

-

        Moderate

-

1/5

4/5

2/5

-

-

-

-

    Tubular mineralization

 

 

 

 

 

 

 

 

        Minimal

-

-

-

-

2/5

3/5

2/5

-

        Slight

-

-

-

-

-

-

1/5

2/5

        Moderate

-

-

-

-

-

-

1/5

1/5

LIVERa

5

5

7

10

5

5

5

5

    Hepatocellular hypertrophy

 

 

 

 

 

 

 

 

        Minimal

-

3/5

4/7

1/10

-

-

-

2/5

        Slight

-

1/5

1/7

8/10

-

-

-

3/5

    Pigment deposition

 

 

 

 

 

 

 

 

        Minimal

-

-

1/7

2/10

-

-

-

-

        Slight

-

-

1/7

-

-

-

-

-

    Hepatocellular necrosis

 

 

 

 

 

 

 

 

        Slight

-

-

-

1/10

-

-

-

-

THYROID GLANDa

5

5

5

6

5

5

5

5

    Follicular cell hypertrophy

 

 

 

 

 

 

 

 

        Minimal

-

1/5

2/5

1/6

3/5

3/5

1/5

-

        Slight

-

3/5

3/5

5/6

1/5

1/5

4/5

2/5

        Moderate

-

-

-

-

-

-

-

3/5

    Colloid alteration

 

 

 

 

 

 

 

 

        Minimal

-

-

-

1/6

-

-

-

4/5

THYMUSa

5

5

6

5

5

1

-

5

    Lymphoid atrophy

 

 

 

 

 

 

 

 

        Minimal

-

-

-

3/5

-

-

-

-

a = Number of tissues examined from each group.

Conclusions:
The NOAEL for developmental toxicity is 300 mg/kg bw/day, based on reduced body weight gain of male and female pups at 1000 mg/kg bw/day.
Executive summary:

In an OECD 422 study, Wistar Han rats were treated with 1,3-bis(tert-butylperoxyisopropyl)benzene by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the vehicle, corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and 14 or16 days of lactation (for 50-56 days). Females without offspring were treated for 41 days (two non-pregnant females). Formulation analysis showed that the formulations were prepared accurately and homogeneously.

Developmental results

Developmental toxicity occurred at 1000 mg/kg bw/day and was characterized by reduced body weights gain of male and female pups. This resulted in about 20% lower mean body weights at PND 13. Birth weight of 1000 mg/kg bw/day pups was not affected by treatment. No treatment-related changes were noted in the other developmental parameters examined (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, clinical signs, anogenital distance, areola/nipple retention, serum T4 thyroid hormone levels and macroscopic examination).

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Level (NOAELs) of 1,3-bis(tert-butylperoxyisopropyl)benzene was established: Developmental NOAEL: 300 mg/kg bw/day, based on reduced body weight gain of male and female pups at 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day

Justification for classification or non-classification

According to regulation (CE) n°1272/2008, [1,3 -phenylenebis(1-methylethylidene) ]bis[tert-butyl] peroxide) is not classified as toxic for the reproduction based on the test results of structurally analogous 1,3(or1,4)-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide.