Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The target substance 1-Methyl-1-phenylethyl peroxyneodecanoate was tested in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422). Dose groups chosen were based on the results of a 14-day dose range finding study. No adverse effects were found on male and female mortality, clinical observations, functional observations, body weight development (except HD males and females), food consumption (except HD females), estrous cyclicity, precoital interval and duration of gestation, anogenital distance and nipple retention, pup thyroid weight and thyroid hormone analysis in parental males and pups sacrificed on PND 13, pup external findings, haematology and coagulation, clinical biochemistry, urinalysis and gross macroscopic findings at necropsy in all treatment groups. In the high dose group treated with 1000 mg/kg bw/day treatment related effects on litter data, litter weight data, pre and post-natal data, reproductive indicies and pup survival data were recorded. In addition, effects on organ weights (liver, kidney, uterus with cervix weights, heart, spleen) and histopathology (liver) were observed. Based on the results, the NOAEL for maternal and reproductive/developmental toxicity is considered to be 300 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-06-19 to 2017-09-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline 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:
Adopted, 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
- CAS No.: 26748-47-0
- Batch No.: 1236926-01
- Physical State: fluid
- Colour: colourless - yellowish
- Active Components: 75.1 % peroxide
- Storage Conditions: ≤ -20 °C
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: approx. 12-13 weeks old
- Weight at study initiation: males: 324-392 g, females: 207-253 g
- Housing: housed in groups of up to 5 animals / sex / cage in type IV polysulphone cages during the premating period for both males and females and during post-mating period for males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males were returned to their original cage. In each cage Altromin saw fibre was used as bedding.
- Diet (e.g. ad libitum): ad libitum, Altromin 1324
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Preparation of the Test Item:
The vehicle was selected as suggested by the sponsor based on the test item’s characteristics and testing guideline. The test item was weighed into a tared plastic vial on a precision balance and the vehicle was added to give the appropriate final concentration of the test item and further vortexing and/or stirring it. Formulates were kept under magnetic stirring during the daily administration. The vehicle corn oil was used as control item (C1). Isododecane was weighed into a tared plastic vial on a precision balance and the vehicle was added to give the final concentration of 125 mg/mL (500 mg/kg bw isododecane at an application volume of 4 mL/kg bw) as decided by the sponsor. The control formulation C2 was further vortexed and/or stirred. It was kept under magnetic stirring during the daily administration. The test item and control formulations were prepared freshly on each administration day before the administration procedure.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
The vehicle corn oil was selected as suggested by the sponsor based on the test item’s characteristics and testing guideline. As Peroxan CND contains the phlegmatizer isododecane, isododecane was used additionally as control item (C2).
- Lot/batch no. (if required): Corn oil: MKBZ98989V, MKCC0462Z, Isododecane: 1236856
Details on mating procedure:
Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the mating. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation. The cages were arranged in such a way that possible effects due to cage placement were minimised.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before beginning of the treatment period, formulation samples were prepared and analyzed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle at Eurofins Munich as part of a separate GLP study. Study prestart stability analysis was included on the samples from high dose and low dose group and the investigation was made for 0 h and 6 h (at room temperature). Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups (6 samples).
Since the test item was shown to be homogenous (after 60 min without stirring), during the study samples were not collected for the investigation of homogeneity and only samples were taken for substance concentration in study week 1 (pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) from all groups (20 samples). Each sample taken during the study was retained in duplicate (sample A, sample B; minimum sample volume: LD 30 mL, MD 10 mL, HD 10 mL). All A-samples except from the additional control group C2 were analyzed at Eurofins Munich and until then stored under appropriate conditions based on available stability data. The B-samples and all samples of the second control group C2 will be retained at -15 to -35 °C at BSL Munich (test facility) and discarded after completion of the final study report.
Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle (group C1) or second control item (group C2) on 7 days per week for a maximum period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females. Then in females, treatment was done during the gestation period and up to post-natal day 12. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.
Frequency of treatment:
once daily, 7 days a week
Details on study schedule:
according to guideline
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control 1, corn oil
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Control 2, Isododecane
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Medium dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
10
Control animals:
yes
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a previous dose range finding study
- Rationale for animal assignment: random
Positive control:
No positive control used
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily. Females showing signs of abortion (such as sudden body weight loss combined with bloody discharge from the vagina) or premature delivery prior to gestation day 19 were euthanised and subjected to a thorough macroscopic examination. The reproductive organs, thyroid/parathyroid glands and all organs showing macroscopic lesions were preserved. The number of implantation sites and corpora lutea were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure, and once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed once before the assignment to the experimental groups, on the first day of administration and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 and 13 post-partum along with pups. All animals were weighed directly before termination.

FOOD CONSUMPTION: Yes
Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: Yes, see chapter (neuro)behavioural examination

HAEMATOLOGY: Yes
Haematological parameters from 5 randomly selected males and females (only lactating females were evaluated) from each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in EDTA-coated tubes. Parameters checked in table 2 were examined.

CLINICAL CHEMISTRY: Yes
Parameters of clinical biochemistry from 5 randomly selected males and females (only lactating females were evaluated) from each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in serum separator tubes. Parameters checked in table 3 were examined. From 2 female pups/litter on day 4 after birth, from all dams and 2 pups/litter (1 male and 1 female) at termination on day 13 and from all adult males at termination blood samples were collected from the defined site. All blood samples were stored under appropriate conditions. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4). Further assessment of T4 in blood samples from the dams and day 4 pups was not deemed necessary. Additionally, assessment of TSH in day 4 pups, adult females, day 13 pups, and adult males were not considered necessary based on the fact that no major histopathological findings were observed in thyroid/parathyroid gland of selected male and female adult animals and T4 hormone levels of males and day 13 pups. Pup blood was pooled by litter for thyroid hormone analysis.
Two pups per litter were sacrificed on day 4 after birth and blood samples were taken for possible serum hormone assessments. Two pups per litter should be female pups to reserve male pups for nipple retention evaluations except in the event that removing these pups leaves no remaining females for assessment at termination. No pups were eliminated when litter size dropped below 8 pups. If there was only one pup available above a litter size of 8, only one pup was sacrificed.

URINALYSIS: Yes
A urinalysis was performed with samples from 5 randomly selected males and females (only lactating females were evaluated) prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance were recorded. Parameters checked in table 4 were examined.

(NEURO)BEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and during the lactation period in 5 randomly selected females (only lactating females were evaluated)
- Battery of functions tested: Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye) was measured.

BLOOD COAGULATION:
Coagulation parameters from 5 randomly selected males and females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in citrate tubes. Parameters checked in table 5 were examined.
Oestrous cyclicity (parental animals):
Estrous cycle: Estrous cycles were monitored before treatment starts to select females with regular cyclicity (using vaginal smears) for the study. Further on, vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.
Sperm parameters (parental animals):
not examined
Litter observations:
LITTER OBSERVATIONS:
The duration of the gestation was recorded and was calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted and sexed and litters weighed within 24 hours of littering (PND 0) and on PND 4 and PND 13. Live pups were identified by tattooing. In addition to the observations of the parent animals, any abnormal behaviour of the offspring was recorded.
The anogenital distance (AGD) of each pup was measured on PND 0. Pup body weight measured on PND 0 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD / Cube root of pup weight). The number of nipples/areolae in male pups was counted on PND 12.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
Males were sacrificed any time after the completion of the mating period (after a minimum dosing period of 28 days) and females were sacrificed on the respective PND 13 by using anesthesia (e.g. ketamine/xylazine). All surviving pups were killed by cervical dislocation on PND 13. Vaginal smears were examined on the day of necropsy to determine the stage of estrous cycle. Dead pups and all surviving pups on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice. Non-pregnant females were sacrificed on day 26 from the day of sperm positive vaginal smear or from the last day of mating period. All adult animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved in 4 % neutral-buffered formaldehyde, except for eyes with optic nerves and harderian glands, testes and epididymides which were preserved in modified Davidson’s Solution for 24 hours and then transferred to 70 % ethanol.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. If appropriate and possible, the number of corpora lutea and implantation sites were recorded for any females sacrificed 26 days after the end of the mating period with no evidence of mating and or on day 26 post-coitum due to non-delivery.

ORGAN WEIGHTS: Yes
The wet weight of the organs (see table 6) of 5 randomly selected male and female animals (only lactating females were evaluated) from each group was recorded as soon as possible. Paired organs were weighed together. Organ weights of animals found dead or euthanised for animal welfare reasons were not recorded. Reproductive organs were weighed from all animals. Thyroid/parathyroid glands from 1 pup/sex/litter/group (sacrificed on PND 13) and from all adult males and females were preserved. Weight of thyroid/parathyroid glands were measured after fixation.

HISTOPATHOLOGY: Yes
A full histopathology was carried out on the preserved organs and tissues (see table 7) of the 5 randomly selected animals of the control (C1) and high dose groups which were sacrificed at the end of the treatment period. Histopathological examination of thyroid gland from pups and from the remaining non-selected adult animals was not deemed necessary as no major test item related histopathological findings were observed in thyroid/parathyroid gland of selected animals and there was also no test item related effect observed on T4 hormone level in males and pups sacrificed on PND 13.
A full histopathology was carried out on the preserved organs and tissues of all animals which died during the study or which were euthanised due to morbidity. The organs and tissues specified in table 7 were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and eosin (H&E). In addition, a hematoxylin-Periodic Acid Schiff (H-PAS) was prepared on all processed testes. In addition, ovaries, uterus with cervix and vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were not only examined in all control (C1) and HD animals, but also in non-pregnant female animals of the C2, LD and MD group. Testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland) were also examined in the mating partners (males) of the non-pregnant female C2, LD and MD animals.
These examinations were extended to animals of other dosage groups (C2, and MD) for treatment-related changes that were observed in the high dose group. Only organs and tissues of the other dosage groups showing changes in the high dose group were examined. Organs and tissues examined of the other dosage groups were liver, kidney, stomach, spleen, thymus (male and female C2 and MD group), Thyroid (male C2 and MD group), adrenal (female C2 and MD group), female reproductive organs- ovary, uterus, vagina and cervix (C2 and MD group). Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.
For the testes, a detailed qualitative examination was made; any change noted in this organ was diagnosed according to tubular staging of the spermatogenic cycle (at evaluation of H-PAS stained slides). The histological processing of all preserved tissues to H&E and H-PAS stained microscope slides was performed at the GLP-certified contract laboratory, TPL Path Labs. Histopathological evaluation was performed at the GLP-certified contract laboratory TPL Path Labs, Sasbacher Str. 10, 79111 Freiburg im Breisgau.
Postmortem examinations (offspring):
SACRIFICE
- Dead pups and all surviving pups were carefully examined externally for gross abnormalities before terminal sacrifice on PND 13.

GROSS NECROPSY
- Gross necropsy consisted of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents
Statistics:
A statistical assessment of the results of body weight, food consumption, parameters of haematology, blood coagulation, clinical biochemistry and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights were statistically analyzed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.1.3 software (p< 0.05 was considered as statistically significant).
Reproductive indices:
Copulation, fertility, delivery and viability
Offspring viability indices:
Viability
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
In terminally sacrificed males, predominant clinical signs observed during the treatment period (premating day 1 to mating/post mating day 14) were moving the bedding in all animals and moderate to severely increased salivation in 4 animals of C2 group during mating and post mating day 1-14. Moving the bedding and or moderate to severely increased salivation was also observed during premating day 7 to mating and post mating day 14 in almost all animals of MD and HD group.
In terminally sacrificed females, major clinical signs observed during the treatment period (Premating day 1 to PND 12) were moving the bedding in all C2 animals (on few occasions between MD 2 - PND 12), in two LD animals (on GD 15 and 20), in almost all MD and HD animals (between PMD 10 and PND 12 on majority days). A slight to severely increased salivation was observed in 1 LD (GD 20), in 9 MD (on few occasions between MD 3 and GD 20) and all HD group females (days between PMD 10 and PND 12).
There were also low incidences of the clinical signs like alopecia on various body parts of the 1 each female of C1, C2, LD and HD, abnormal breathing in one each female of MD and HD, moderately reduced spontaneous activity in one HD female, nasal discharge in one LD female, scratch on right shoulder in one LD female, swelling and wound on snout/nose and crust on snout in one MD female observed and considered to be incidental in nature.
The clinical signs salivation and moving the bedding in males and females were observed immediately after the dose administration and therefore were considered to be a sign of a local reaction to the test item rather than a systemic adverse effect and has no toxicological relevance.
None of the females showed signs of abortion or premature delivery. During the weekly detailed clinical observation, no relevant differences between the groups were found.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
During the treatment period of this study, one mortality was observed as follows:
A Female rat was found dead on mating day (MD) 3. Predominant clinical signs observed in this animal before and on day of death were moving the bedding (PMD 11-MD 3), slight increased salivation (MD 1), abnormal breathing, hunched posture, moderate dehydration, moderate piloerection, moderately reduced spontaneous activity (MD 3). Macroscopically, red fluid filled oesophagus, lungs dark abnormal in colour and filled with fluid and organ adhesion with surrounding tissues in thoracic cavity were observed.
Histopathologically, moderate to marked subacute/chronic inflammation was noted on thoracic side of diaphragm, heart epicardium/pericardium, mediastinum and lung pleura, correlating with gross findings. Altogether, lesions in the female rat suggested death secondary to trauma associated with the dosing procedure (misdosing) and esophageal perforation.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In both males and females, there was no test item treatment related effect observed on body weight in the LD and MD dose groups during the entire study period when compared with the controls (C1). However lower group mean body weight in HD males was observed throughout the study from premating day 14 when compared to the controls but statistical significance was achieved only for group mean body weight on day of terminal sacrifice. In females also comparatively lower group mean body weights without achieving statistical significance were observed throughout the study starting from gestation day 7 in HD group when compared to the controls.
In males, statistically significantly lower group mean body weight gain was observed during pre mating day 14 to mating/postmating day 7, premating day 1 to postmating day 14 and premating day 1 to terminal sacrifice in C2 group, during pre mating day 14 to mating/postmating day 7 in MD and during pre mating day 1-7, 7-14, premating day 1 to postmating day 14 and premating day 1 to terminal sacrifice in HD group when compared to the controls. In females, statistically significantly lower group mean body weight gain was observed during gestation day 7-14 in C2, during gestation day 14-20 and 0-20 in HD group when compared with the control. This effect on body weight development in C2 and HD males and HD females was considered as a test item related.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item related effect on food consumption was observed in males during premating period. However, statistically significantly lower food consumption was observed in HD group females during lactation days (post natal days) 0-4, 4-9 and 9-13 when compared with the controls (C1). This effect on food consumption in HD females was considered as test item related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males sacrificed at the end of treatment period, no test item related adverse effects were observed for haematological parameters in treatment groups when compared to the control group. However, statistically significantly higher monocyte count and higher reticulocyte count in HD group was observed when compared with the controls (C1). In the light of all values within historical data range (0.95- 2.90 %) and no effect on RBC values, this effect on reticulocytes was not considered to be adverse. Monocyte values were also within historical control data (0.6- 4.6%).
In females sacrificed at the end of treatment period, statistically significantly lower group mean MCH in HD (within historical control data range- 16.7- 26.3 pg/Ery), MCHC in C2, lymphocytes in MD and higher group mean neutrophils in MD, monocytes in C2 and MD and leucocytes in were observed in C2 group treated with Isododecane phlegmatizer alone when compared with the control group (C1). This effect on few hematology parameters in females was either marginal, within historical control data or with lack of dose dependency and therefore not considered to be test item related.
No statistically significant effect on any other hematology parameter in male and female treatment groups was observed when compared with the respective controls (C1). All other group mean and most of the individual values for haematological parameters in male and females were within the historical control data range.
No test item related effect was observed on coagulation parameters in males and females when compared with the respective controls (C1).
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males and females sacrificed at the end of treatment period, no test item related or statistically significant effect on any clinical biochemistry parameter in treatment groups was observed when compared with the respective controls (C1) except statistically significantly lower group mean alkaline phosphatase and marginally but statistically significantly higher ALB, urea and cholesterol values in HD group males were observed when compared with the controls. In females, statistically significantly lower cholesterol values were observed in HD group when compared with the controls. As values were within historical control data range (AP- 67.27- 483.16 U/L, ALB- 17.26-36.74 g/L, urea- 4.63-45.22 mmol/L, cholesterol- 0.54- 2.62 mmol/L in males and cholesterol - 0.51-3.36 mmol/L in females), this effect on few clinical chemistry parameters in male and female HD group was not considered to be adverse. All group mean and most of the individual values for clinical chemistry parameters in male and females were within the historical control data range.
Urinalysis findings:
no effects observed
Description (incidence and severity):
The urinalysis performed in selected male and female animals sacrificed at the end of treatment period revealed no test item treatment related effect and all urinary parameters were in the normal range of variation. Slightly high protein levels were found in the urine of few male and females of all groups including control group. Therefore, this effect on urine parameters was not considered to be test item related.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
In males and females, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period when compared with the controls. There were no biologically relevant differences observed in body temperature between the groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In males sacrificed at the end of treatment period, significantly higher group mean absolute and relative liver and kidney weights were observed in C2 and HD group with statistical significance achieved only for relative liver and kidney weights in C2 and HD group when compared with the controls (C1).
In females sacrificed at the end of treatment period, statistically significantly higher group mean relative kidney weights in HD, absolute and relative liver weights in C2 and HD, absolute and relative uterus with cervix weights in HD and relative heart weights in HD group were observed when compared with the controls (C1). There was also statistically significantly lower group mean absolute and relative spleen weights were observed in HD group when compared with the controls (C1). This effect on liver, kidney, uterus with cervix weights, heart, spleen in males and females in C2 and HD group was considered to be test item related.
A trend to higher thyroid weights were noted in high dose males (absolute: +14.23 %; relative: +25.86 %). These changes from controls were not statistically significant, but correlated with microscopic findings.
There was a trend toward marginally decreased absolute and relative thymus weights in C2 and HD males and females from C2 group (range: –20 % to –25 %). These changes were not statistically significant; as they corresponded to low thymus weights in some animals only. Moreover, the change was not dose related. Surprisingly, some MD and HD females had higher absolute and relative weights, correlating with microscopic findings. Histopathologically, this effect on thyroid (adoptive) and thymus weights was minor and not considered to be adverse.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Few specific macroscopic changes were recorded in the male and female animals, which based on microscopic examination were not considered to be of test item treatment relevance. The predominant macroscopic changes observed in terminally sacrificed animals were peripelvic dilatation of kidneys- both sides (male no. 18 of C2 group) and 1-2 cm mass on thymus (female no. 85 of MD group). The mentioned findings were deemed incidental and there were no gross lesions that could be attributed to treatment with the test item. In one decedent (female no. 95- HD), red fluid filled oesophagus, lungs dark abnormal in colour and filled with fluid and organ adhesion with surrounding tissues in thoracic cavity were observed. Histopathologically, lesions in female no. 95 suggested death secondary to trauma associated with the dosing procedure (misdosing) and esophageal perforation.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In unscheduled (dead) female rat of HD group, No treatment-related microscopic findings were noted. Moderate to marked subacute/chronic inflammation was noted on thoracic side of diaphragm, heart epicardium/pericardium, mediastinum and lung pleura, correlating with gross findings. Altogether, lesions in female rat suggested death secondary to trauma associated with the dosing procedure (misdosing) and esophageal perforation.
In scheduled (terminally sacrificed) animals, treatment-related microscopic findings were noted in the liver, forestomach (non-glandular stomach), the kidney, the spleen, the thymus, the thyroids (males only), the adrenal (females only) and the female reproductive tract of animals treated in HD group (1000 mg/kg bw/day) sacrificed on schedule. None of these findings were noted in MD dose of 300 mg/kg bw/day, except in the kidney of both genders, where they were also found in vehicle-control (isododecane at 500 mg/kg bw/day) males and females. In the thymus, the lymphoid depletion/atrophy was also noted in vehicle controls.
In the liver, minimal to moderate biliary duct hyperplasia associated with brown pigments in or around the bile duct lumens, that is characterized by red birefringence under polarized light, was noted in both genders at 1000 mg/kg bw/day, slightly more pronounced in females. These main adverse lesions were accompanied by minimal to mild portal tract inflammation, minimal to mild centrilobular hepatocellular hypertrophy and minimal single cell necrosis/apoptosis in 2 males. The main adverse biliary findings were not present at mid dose of 300 mg/kg bw/day, while the adaptive change centrilobular hepatocellular hypertrophy and/or occasional increased mitoses was noted in vehicle control C2 and MD, correlating with significantly or marginally increased liver weights.
Minimal to moderate mixed cell (chronic active) inflammation of the forestomach, and parakeratotic hyperkeratosis of the Malpighian epithelium was observed at 1000 mg/kg bw/day in both males and females (more pronounced in females). Also minimal increased EMH was noted in the red pulp of the spleen of both genders, correlating with marginally increased reticulocytes at high dose. These changes were not present in the forestomach or spleen of vehicle control rats or in treated animals at mid dose.
Vehicle-related and treatment-related alpha2u-globulin nephropathy associated with hyaline droplet accumulation was observed in the kidney of males (in all C2, MD and HD at similar or lower severity than vehicle controls), as well as vehicle-related and treatment-related increased incidence and severity of tubular basophilia – indicative of tubular regerneration – in C2, MD and HD females (similar severity in C2 and MD, higher in HD).
No treatment-related spermatogenesis maturation abnormalities were noted upon examination of the H-PAS stained sections of testis. In the female reproductive tract, no abnormalities were noted, but females with reproductive cycling were frequently encountered in HD females, whereas they were absent in C1, C2 or MD females.
Reproductive cycling was noted in females that were non pregnant at the end of the study. Resumed reproductive cycling was also noted in pregnant animals on sacrifice day, which was not noted in controls (C1 or C2) or in mid-dose treated females.
There were no non-pregnant females in C1, C2 (controls) or MD females, but they were numerous non pregnant females at HD (4/10) and in this situation they were exhibiting reproductive cycling. Cycling could also be seen in lactating post-pregnant females (3/6) when treated at HD (test item at 1000 mg/kg bw/day), whereas this situation was not observed in controls of MD females. As they were non pregnant, some were in proestrus or estrus, with normal uterus dilation with fluid. Also in uterine horns, consequently, metrial glands in non pregnant females were not seen.
Besides non-pregnancy, at least one female without metrial glands was not cycling with moderate vaginal mucification and lack of basophilic corpora lutea . However, this female had a serious procedural accident, and non cycling may be attributed to the associated stress of serious thoracic subacute to chronic inflammation.
Lastly, focal/multifocal minimal/slight lymphoid depletion/atrophy of the thymus was noted in a few C2, MD and HD males and a few C2 and HD females, findings which correlated with low thymus weights in some animals. In HD females, occasional lymphoid hyperplasia correlating with high thymic weights were noted.
Other microscopic findings noted at the end of the treatment period in test-item treated animals were those that are commonly encountered as background in these animals and were not considered to be related to test item effect
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Test item had no biologically significant effect on the estrous cycle analysed during 2 weeks premating period after the first administration in treatment groups when compared to the controls. There were no considerable differences in the length or sequence of cycle stages between the treatment groups and the control group.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
There were no test item related or statistically significant effects observed on the duration of precoital interval and the duration of gestation in the dose groups when compared to the control group. There was test item related lower number of corpora lutea, implantation sites, live pups on PND 0, 4 and 13, percent post implantation loss observed in HD group when compared with the control group (C1). There was no test item related effect observed on the copulation index in the dose groups when compared to the control group (C1). However, a reduced fertility index of 66.67 %, delivery index of 40% and viability index of 22.67 % during PND 0-4 observed in HD group when compared to the control group. This effect on fertility index, delivery index and viability index in HD group was considered as related to treatment with the test item administration.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
other: organ weight
Organ:
liver
kidney
heart
spleen
uterus
cervix
Treatment related:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
No test item related effects were observed.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
A statistically significantly higher pup mortality was observed in HD group during PND 0-4 (77.33 %) when compared to the controls (1.83 %). No test item related effect observed on mean mortality of pups during PND 4-13 in treatment groups when compared to the control group (C1). This effect on pup mortality during PND 0-4 in HD group was considered as related to the treatment with the test item
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was lower group mean pup mean weight, total litter weight, male and female litter weight on PND 0, PND 4 and PND 13 observed in HD group when compared with the controls (C1) although statistical significance was only achieved for pup mean weight on PND 4 and 13, total litter weight on PND 13 in HD and pup mean weight on PND 13 in C2. This effect on litter weight data in HD female was considered to be test item related and toxicologically relevant.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item related gross external abnormalities of toxicological relevance on PND 0-12 were observed in the pups of any of the groups. Few specific findings like pale (pup no. 3 from dam 58 of C1 group), dark snout on PND 0 (pup no. 6 from dam no. 71 of LD), blue snout on PND 0 (pup no. 5 from dam no. 86 of MD and pup no. 1 from dam no. 97) and blue body (pup no. 2 from dam no. 89 of MD).
No external findings were observed in pups of any study group at death/terminal sacrifice on PND 13. However, absent hair coat on body parts was observed in few pups from few dams at terminal sacrifice (pup no. 1-8 from dam no. 55 of C1, pup no. 1 from dam no. 62, pup no. 1-3 from dam no. 65, pup no.1-8, 10, 12 from dam no. 68 of C2, pup no. 1-7, 10-12 from dam no. 73 and pup no. 1-4, 6-9 from dam no. 78 of LD, pup no. 1-4, 7-8 from dam no. 83, pup no. 2-5, 7-10 from dam no. 90 of MD. There were also few pups observed with absent tip tail at the time of terminal sacrifice (pup no. 12 from dam no. 64 of C2, pup no. 9 from dam no. 83 of MD and pup no.11 from 93 of HD). These external findings were considered to be spontaneous in nature and not related to treatment with the test item.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
ANOGENITAL DISTANCE AND NIPPLE RETENTION:
In males, statistically significant lower pup weight and cube root of pup weight on the day of anogenital measurement was observed in male C2, LD and HD group when compared with the controls (C1). There was also statistically significantly lower group mean absolute anogenital distance observed in C2 and HD group when compared with the controls (C1). However, no such effect was observed on relative anogenital distance in any treatment groups when compared with the controls.
In females, statistically significantly lower pup weight and cube root of pup weight on the day of anogenital measurement was observed in female C2, LD and HD group when compared with the controls (C1). However, no such effect was observed on absolute and relative anogenital distance in any treatment groups when compared with the controls.
As effect on pup weight and cube root of pup weight in male and female C2 and LD and absolute anogenital in male C2 was marginal and due to lack of dose dependency in absolute anogenital distance in C2 males, these findings have no toxicological relevance. In the light of absence of effect on absolute and relative anogenital distance, effect on female pup weight and cube root of pup weight in C2, LD and HD has no significance for interpretation of anogenital distance.
Although all anogenital values in male and female pups were within historical control data range and difference is marginal, this effect on anogenital distance, pup weight and cube root of pup weight in HD group males could be considered as test item related. However, toxicological relevance of this finding and possible endocrine effect cannot be decided based on this screening study. No statistically significant effect of toxicological relevance was observed on nipple retention in the pups of any of the groups when compared with the controls (C1).

THYROID HORMONE (T4) ANALYSIS:
No test item related effect of toxicological relevance or statistical significance was observed on male pup thyroid/parathyroid weight, male and PND 13 pup thyroxine hormone (T4) in the treatment groups when compared to the controls (C1). However, statistically significantly lower thyroid/parathyroid weights were observed in C2 and HD female pups when compared with the controls.
LITTER DATA:
There were no test item related or statistically significant effects observed in treatment groups on litter data parameters in C2, LD and MD group when compared with the controls. However, there were low group mean total number of pups born, number of male and females, sex ratio, live pups on PND 0 observed in HD group without achieving statistical significance. There were statistically significantly lower number of males, number of females, live pups (before and after interim sacrifice) on PND 4 and PND 13 observed in HD group when compared to the controls (C1). This effect on litter data parameters in HD group was considered as test item related and toxicologically relevant.

LITTER WEIGHT DATA:
There was lower group mean pup mean weight, total litter weight, male and female litter weight on PND 0, PND 4 and PND 13 observed in HD group when compared with the controls (C1) although statistical significance was only achieved for pup mean weight on PND 4 and 13, total litter weight on PND 13 in HD and pup mean weight on PND 13 in C2. This effect on litter weight data in HD female was considered to be test item related and toxicologically relevant.

PUP SURVlVAL DATA:
A statistically significantly higher pup mortality was observed in HD group during PND 0-4 (77.33 %) when compared to the controls (1.83 %). No test item related effect observed on mean mortality of pups during PND 4-13 in treatment groups when compared to the control group (C1). This effect on pup mortality during PND 0-4 in HD group was considered as related to the treatment with the test item.

PUP EXTERNAL FINDINGS:
No test item related gross external abnormalities of toxicological relevance on PND 0-12 were observed in the pups of any of the groups. Few specific findings like pale (pup no. 3 from dam 58 of C1 group), dark snout on PND 0 (pup no. 6 from dam no. 71 of LD), blue snout on PND 0 (pup no. 5 from dam no. 86 of MD and pup no. 1 from dam no. 97) and blue body (pup no. 2 from dam no. 89 of MD).
No external findings were observed in pups of any study group at death/terminal sacrifice on PND 13. However, absent hair coat on body parts was observed in few pups from few dams at terminal sacrifice (pup no. 1-8 from dam no. 55 of C1, pup no. 1 from dam no. 62, pup no. 1-3 from dam no. 65, pup no.1-8, 10, 12 from dam no. 68 of C2, pup no. 1-7, 10-12 from dam no. 73 and pup no. 1-4, 6-9 from dam no. 78 of LD, pup no. 1-4, 7-8 from dam no. 83, pup no. 2-5, 7-10 from dam no. 90 of MD. There were also few pups observed with absent tip tail at the time of terminal sacrifice (pup no. 12 from dam no. 64 of C2, pup no. 9 from dam no. 83 of MD and pup no.11 from 93 of HD). These external findings were considered to be spontaneous in nature and not related to treatment with the test item.

Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There was test item related effects on litter data, litter weight data, pre and post-natal data, reproductive indices and pup survival data.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes

DOSE FORMULATION ANALYSIS

Concentration analysis of formulation samples was determined at three concentrations, for low dose group (LD, 25 mg/mL), for medium dose group (MD, 75 mg/mL) and for high dose group (HD, 250 mg/mL). Samples were taken in study week 1, week 3, week 5, and in the last week of the study.Mean recoveries observed at the four sampling occasions were for LD group between 93.6 % and 95.0 % of nominal value, for MD group between 97.6 % and 101.0 %, and for HD group between 95.7% and 99.9 %. Overall recoveries of entire study observed in LD, MD and HD groups were 94.2 %, 99.6 %, and 97.6 % of nominal concentration, respectively.

Conclusions:
In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test no adverse effects were found after oral administration of 1-methyl-1-phenylethyl peroxyneodecanoate in male and female Wistar rats and in the male and female pups. Based on the results, the NOAEL for maternal and reproductive/developmental toxicity is considered to be 300 mg/kg bw/day.
Executive summary:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422), the test item 1-methyl-1-phenylethyl peroxyneodecanoate (75.1 % peroxide) was administered to 10 male and female Wistar rats/dose in corn oil by gavage at dose levels of 0, 100, 300, or 1000 mg/kg bw/day. In addition, a second control group was included. Animals of this control group received 500 mg/kg bw/day of the phlegmatizer Isododecane. The animals were treated with the test item formulation on 7 days per week for a period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

No adverse effects were found on male and female mortality, clinical observations, functional observations, body weight development (except HD males and females), food consumption (except HD females), estrous cyclicity, precoital interval and duration of gestation, anogenital distance and nipple retention, pub thyroid weight and thyroid hormone analysis in parental males and pups sacrificed on PND 13, pup external findings, haematology and coagulation, clinical biochemistry, urinalysis and gross macroscopic findings at necropsy in all treatment groups.

In the high dose group treated with 1000 mg/kg bw/day treatment related effects on litter data, litter weight data, pre and post-natal data, reproductive indicies and pup survival data were recorded. In addition, effects on organ weights (liver, kidney, uterus with cervix weights, heart, spleen) and histopathology (liver) were observed. Based on the results, the NOAEL for maternal and reproductive/developmental toxicity is considered to be 300 mg/kg bw/day.

This study is classified as acceptable and satisfies the guideline requirement for an oral repeated dose toxicity study in rats. 

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
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
Additional information

The target substance 1-Methyl-1-phenylethyl peroxyneodecanoate was tested in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422). Dose groups chosen were based on the results of a 14-day dose range finding study. No adverse effects were found on male and female mortality, clinical observations, functional observations, body weight development (except HD males and females), food consumption (except HD females), estrous cyclicity, precoital interval and duration of gestation, anogenital distance and nipple retention, pup thyroid weight and thyroid hormone analysis in parental males and pups sacrificed on PND 13, pup external findings, haematology and coagulation, clinical biochemistry, urinalysis and gross macroscopic findings at necropsy in all treatment groups. In the high dose group treated with 1000 mg/kg bw/day treatment related effects on litter data, litter weight data, pre and post-natal data, reproductive indicies and pup survival data were recorded. In addition, effects on organ weights (liver, kidney, uterus with cervix weights, heart, spleen) and histopathology (liver) were observed. Based on the results, the NOAEL for maternal and reproductive/developmental toxicity is considered to be 300 mg/kg bw/day.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on a NOAEL of 300 mg/kg/day, the target substance does not warrant classification for reproductive toxicity in accordance with CLP Regulation 1272/2008.