Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The target substance OO-tert-butyl monoperoxymaleate was tested in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422). Dose groups were based on the results of a 14 -day dose range finding study. No adverse effects were observed after oral administration. Based on the results, the NOAEL for maternal and reproductive/developmental toxicity is considered to be 160 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:
2017-06-06 to 2017-08-30
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 29th July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
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. 14-15 weeks old
- Weight at study initiation: males: 322 - 399 g, females: 203 - 246 g
- Housing: Animals were housed in groups of 5 animals/sex/cage in type IV polysulphone cages during the premating period for both males and females and during postmating 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, free access to Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum): ad libitum, free access to 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:
polyethylene glycol
Details on exposure:
Preparation of Test Item Formulations:
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. The dose formulations were prepared by adding the required volume of PEG 400 and further vortexing it for 2-3 minutes. The test item and control formulations were prepared freshly on each administration day before the administration procedure. Formulates were kept under magnetic stirring during the daily administration. The vehicle PEG 400 was used as control item (C1). As Peroxan PM-50 contained the phlegmatizer Triacetin, Triacetin was additionally used as control item (C2). Triacetin was weighed into a tared plastic vial on a precision balance and the vehicle was added to give the final concentration of 20 mg/mL (80 mg/kg bw Triacetin 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.

VEHICLE
- Vehicle used: PEG 400
- Amount of vehicle (if gavage): 4 mL/kg bw
- Lot/batch no. (if required): S7106885

As Peroxan PM-50 contained the phlegmatizer Triacetin (CAS 102-76-1), Triacetin was additionally used as control item (C2).
- Amount of vehicle (if gavage): 4 mL/kg bw
- Lot/batch no. (if required): CH170202A
Details on mating procedure:
- M/F ratio per cage: 1 : 1
- Length of cohabitation: 14 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): After the confirmation of the mating, females were separated and housed individually
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 analysed 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 included 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 included 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 be collected for the investigation of homogeneity and only samples were taken for substance concentration (from C1, C2, LD, MD and HD group) 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 were retained in duplicate (sample A, sample B, minimum sample volume: C1/C2 - 40 mL, LD - 40 mL, MD - 35 mL and HD - 20 mL). All A-samples except from the additional control group C2 were analysed at Eurofins Munich on the same day of sampling within 6 hours and until analysis, samples were stored under appropriate conditions based on available stability data. The B-samples were 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 is completed.
Frequency of treatment:
once daily, 7 days a week
Details on study schedule:
According to OECD test guideline 422
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control 1, PEG 400
Dose / conc.:
80 mg/kg bw/day (nominal)
Remarks:
Control 2, Triacetin
Dose / conc.:
40 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
80 mg/kg bw/day (nominal)
Remarks:
Medium dose
Dose / conc.:
160 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.

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 in serum separator tubes. 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). Total T4 hormone levels were measured by using DRG:HYBRID-XL Analyzer (DRG Instruments GmbH, Division of DRG International, Inc., Frauenbergstrasse 18, D-35039 Marburg, Germany) and DRG:HYBRiD-XL T4 Kit (solid phase enzyme-linked immunosorbent assay (ELISA) based on the principle of competitive binding).
Further assessment of T4 in blood samples from the main study 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 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 were 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 is 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.

OTHER:
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 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 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 die during the study or which were euthanised due to morbidity.
The organ 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, testes, epididymides, ovaries, uterus with cervix, 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. 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 MD dose groups as treatment-related changes were observed in spleen of the high dose 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 (test site).
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:
no effects observed
Description (incidence and severity):
IIn terminally sacrificed males, predominant clinical signs observed during the treatment period (premating day 1 to mating/post mating day 14) were moving the bedding during mating and post mating day 2-14 in all animals of MD and HD group. A moderately increased salivation in one animal of LD (Mating/post mating day 14), two animals of MD (Mating/post mating day 13-14) and one animal of HD during days 7-13 of mating/postmating period.
In terminally sacrificed females, major clinical signs observed during the treatment period (Premating day 1 to PND 12) were moving the bedding in one C1 (GD 0-7), all LD (between GD 1-20), all MD (on few occasions between GD 1 and PND 13) and in all HD females from PMD 6 through majority of gestation and post-natal days. A slight to moderate increased salivation in one MD (GD 20) and 4 HD group females (days between PND 9-12, GD 7- PND 10, PND 9-12 and GD 7-8).
There were also low incidences of the clinical signs like alopecia on various body parts of the 1 each female of C1 and C2 and one female of LD, abnormal breathing and slight reduced spontaneous activity 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, non-treatment-related
Description (incidence):
There was one mortality observed in the study (female no. 87 of HD group) on gestation day 7 and one female (no. 61 from C2 group) was euthanised in moribund condition on mating day (MD) 2 due to animal welfare reasons. Based on histopathological evaluation, cause of the death and moribund condition was considered as technical gavage error.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In both males and females, there was no test item treatment related effect observed on body weight and body weight gain in the dose groups during the entire study period. There were no statistically significant differences observed for body weight and body weight gain between the dose groups and the control group (C1).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
In correlation to the body weight and body weight gain, the food consumption in both males and females tended to increase with the progress of the study in the control, the LD, the MD and the HD group. No test item related or statistically significant effect on food consumption was observed in males and females during the whole study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
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 lower HCT count in C2 group 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.
In females sacrificed at the end of treatment period, no test item related effect or statistically significant effect observed on any of the haematology parameters. However, statistically significantly lower neutrophils and higher lymphocytes group mean values were observed in C2 group treated with Triacetin phlegmatizer alone when compared with the control group (C1) and therefore not considered to be test item related. 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.
Clinical biochemistry findings:
no effects observed
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 control (C1) except statistically significantly lower group mean sodium values in MD group. Due to lack of dose dependency and values within historical control data range (74-149 mmol/L), this effect on sodium was not considered to test item related. 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. 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, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period except statistically significantly higher supported and not supported rearing count before the initiation of treatment in C2, LD, MD and HD group when compared with the controls (C1) which was considered as incidental and toxicologically irrelevant. There were no biologically relevant differences observed in body temperature between the groups. In females, statistically significantly lower supported rearing count in all treatment groups before treatment and in last week of treatment, lower not supported rearing count in all treatment groups before initiation of treatment, lower urination count in C2, LD, MD before imitation of treatment and in MD group in the last week of treatment and lower defecation count in C2, LD, HD group before imitation of the treatment were observed when compared to the controls (C1). LD, MD and HD and statistically significantly lower not supported rearing count in all treatment groups was observed before initiation of the treatment which was not considered to be toxicologically relevant. As this type of difference was either marginal, before imitation of treatment and without dose dependency/consistency, it has no toxicological relevance and could be considered as biological variation. There were no biologically relevant differences in body temperature between the groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
In unscheduled (dead or moribund sacrificed) animals, No treatment-related microscopic findings were noted in one decedent MD female and one moribund sacrificed C2 female.
In scheduled (terminally sacrificed) animals, one treatment-related microscopic finding was noted at the HD group. It consisted of minimal increased extramedullary haematopoiesis in the spleen red pulp of 4/5 HD males and of a single HD female. It correlated with haematological changes (modest individual increases in reticulocytes). These changes revealed increased red blood cell (RBC) turnover secondary to RBC lysis. This change i.e. increased extramedullary haematopoiesis was not noted in the red pulp of the spleen in terminally sacrificed males or females of MD group treated at 80 mg/kg bw/day.
H-PAS stained testicular sections revealed no stage abnormalities in treated males.
In non pregnant females and mating partners, no microscopic findings or abnormalities were noted in reproductive organs of both genders. 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
In conclusion, under the conditions of this study, at 160 mg/kg bw/day (HD), minimal increases in extramedullary haematopoiesis were noted in most males and a single female, correlating with marginal increase in reticulocyte counts, and hence leading to suspect increased red blood cell (erythrocyte) turnover, a compensatory mechanism to an important adverse finding i.e. treatment-related red blood cell destruction, with adequate compensation as no significant RBC decreases were noted.
No other adverse treatment-related tissue findings were noted, correlating with no major changes found in absolute or relative organ weights, and an absence of treatment related gross findings.


Histopathological findings: neoplastic:
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:
no effects observed
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 were no test item treatment related effects observed on the number of corpora lutea, implantation sites, live pups on PND 0, 4 and 13, percent preimplantation loss and post implantation loss in treatment groups when compared with the control group. There were no test item related effects on the reproductive indices (copulation, viability and delivery indices) in the dose groups when compared to the control group. However, a slightly reduced fertility index (number of females pregnant/ No. of females copulated X 100) of 80 % was observed in the HD group as compared to 100 % in control group. Although there was reduction in fertility index in HD group, it was within the standard pregnancy rate of rat i.e. ≥ 80 % and therefore this effect on fertility index was considered as biological variation and not related to treatment with the test item administration.
Key result
Dose descriptor:
NOAEL
Effect level:
160 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at the high dose of 160 mg/kg bw/day
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No test item related effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 in treatment groups when compared to the control group. A marginally higher mean mortality of pups between PND 0 and PND 4 was observed in the C2 (1.25 %), LD (1.25 %) and MD (1.04 %) compared to the control group (0.00 %). This outcome was not dose dependent and did not achieve statistical significance. It was attributed to missing (possible cannibalism) one each pup of C2 dam no. 69 (pup no. 4), LD dam no. 78 (pup no. 2) and MD dam no. 84 (pup no. 1) on PND 1. Due to lack of dose dependency, pup mortality was considered as incidental and not related to the treatment with the test item.
Body weight and weight changes:
no effects observed
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 specified
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
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 HD group when compared with the controls (C1). However, there was no statistically significant effect observed on absolute and relative anogenital distance in treatment groups when compared with the controls (C1).
In females, statistically significantly lower pup weight and cube root of pup weight on the day of anogenital measurement was observed in female LD and HD group when compared with the controls (C1). There was also statistically significantly lower absolute anogenital distance was observed in LD group when compared to the controls (C1).
As effect on pup weight and cube root of pup weight in male HD and female LD and HD was marginal and due to lack of dose dependency in absolute anogenital distance in LD females, these findings have no toxicological relevance.
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 pup thyroid weight, male and PND 13 pup thyroxine hormone (T4) in the treatment groups when compared to the controls (C1).
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
LITTER DATA:
There were no test item treatment related or statistically significant effects observed in treatment groups on litter data parameters like group mean total number of pups born, number of male pups, sex ratio, number of live pups, still birth, runt on PND 0 as well as number of live pups, male pups, number of female pups and sex ratio on PND 4 and PND 13 when compared with the controls (C1) except marginal but statistically significantly higher number of female pups born on PND 0 in HD group. This effect on higher female pups was considered as biological variation as confirmed test article related shift in sex ratio is very rarely observed. Although there is possibility of alterations arising from direct effects on the male germ cell populations that would favor the production or destruction of Y or X chromosome-bearing sperm. However, in the light of absence of histopathological findings and hormonal alterations, this possibility was excluded and hence observed effect has no toxicological relevance.

LITTER WEIGHT DATA:
There was no test item related effect on pup mean weight, total litter weight, female litter weight on PND 0, PND 4 and PND 13 observed in treatment groups when compared with the controls (C1). However, in correlation to low number of male pups and sex ratio, lower group mean male litter weight values in HD group were observed on PND 0, 4 and 13 although statistical significance was achieved only on PND 0 and 13. This decrease in male litter weight in HD group was attributed to low male pups in few females (91, 93 and 98) of the HD group. Therefore this effect on male litter weight was not considered to be test item related and assumed to be biological variation.

PUP SURVIVAL DATA:
No test item related effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 in treatment groups when compared to the control group.
A marginally higher mean mortality of pups between PND 0 and PND 4 was observed in the C2 (1.25 %), LD (1.25 %) and MD (1.04 %) compared to the control group (0.00 %). This outcome was not dose dependent and did not achieve statistical significance. It was attributed to missing (possible cannibalism) one each pup of C2 dam no. 69 (pup no. 4), LD dam no. 78 (pup no. 2) and MD dam no. 84 (pup no. 1) on PND 1. Due to lack of dose dependency, pup mortality was considered as incidental and not 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 dark neck (pup no. 14 from dam 52 of C1 group), pale pups (pup no. 1-7) and stiff abdomen (pup no. 8) from dam no. 78 of LD group, dark snout (pup no. 13 from dam 80 of LD group and pup no. 13 from dam no. 100 of HD group), bitten nose (pup no. 1 from dam no. 84 of MD group), black tag (pup no. 3 from dam no. 89 of MD group), bent tail (pup no. 12 from dam no. 92 of HD group) and dark head (pup no. 12 from dam no. 99 of HD group) on PND 0 were observed.
No external findings were observed in pups of any study group at death/terminal sacrifice on PND 13.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
160 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at the high dose of 160 mg/kg bw/day
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
160 mg/kg bw/day (nominal)
Treatment related:
no

Dose Formulation Analysis:

Concentration analysis of formulation samples was determined at three concentrations, for low dose group (LD, 10 mg/mL), for medium dose group (MD, 20 mg/mL) and for high dose group (HD, 40 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 81.5 % and 103.6 % of nominal value, for MD group between 85.6 % and 105.6 %, and for HD group between 89.9 % and 103.6 %. Overall recoveries of entire study observed in LD, MD and HD groups were 91.7 %, 95.6 %, and 97.1 % of nominal concentration, respectively.

The mean recovery rates for all concentrations are within the acceptance criteria of±10 % deviation from nominal concentration.

Conclusions:
In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test no adverse effects were found after oral administration of OO-tert-butyl monoperoxymaleate 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 160 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 OO-tert-butyl monoperoxymaleate (48.8 % peroxide) was administered orally to 10 male and 10 female Wistar rats/dose in PEG400 by gavage at dose levels of 0, 40, 80 and 160 mg/kg bw/day. In addition, a second control group was included. Animals of this control group received 80 mg/kg bw/day of the phlegmatizer Triacetin. The animals were treated daily 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 of the test item were found on male and female mortality, clinical observations, functional observations, body weight development, food consumption, estrous cyclicity, litter data, litter weight data, precoital interval and duration of gestation, pre and post-natal data, reproductive indices, pup survival data, 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, gross macroscopic findings at necropsy and organ weights in all treatment groups. Histopathologically, at 160 mg/kg bw/day (HD), minimal increases in extramedullary hematopoiesis were noted in most males and a single female, correlating with marginal increase in reticulocyte counts, and hence leading to suspect increased red blood cell (erythrocyte) turnover. No other adverse treatment-related tissue findings were noted, correlating with no major changes found in absolute or relative organ weights, and an absence of treatment related gross findings. Based on the results of this study, the NOAEL for maternal and reproductive/developmental toxicity is considered to be 160 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:
no adverse effect observed
Dose descriptor:
NOAEL
160 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

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422), the target substance OO-tert-butyl monoperoxymaleate (48.8 % peroxide) was administered orally to 10 male and 10 female Wistar rats/dose in PEG400 by gavage at dose levels of 0, 40, 80 and 160 mg/kg bw/day. In addition, a second control group was included. Animals of this control group received 80 mg/kg bw/day of the phlegmatizer Triacetin. The animals were treated daily 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 of the test item were found on male and female mortality, clinical observations, functional observations, body weight development, food consumption, estrous cyclicity, litter data, litter weight data, precoital interval and duration of gestation, pre and post-natal data, reproductive indices, pup survival data, 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, gross macroscopic findings at necropsy and organ weights in all treatment groups. Histopathologically, at 160 mg/kg bw/day (HD), minimal increases in extramedullary hematopoiesis were noted in most males and a single female, correlating with marginal increase in reticulocyte counts, and hence leading to suspect increased red blood cell (erythrocyte) turnover. Based on the results of this study, the NOAEL for maternal and reproductive/developmental toxicity is considered to be 160 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 160 mg/kg/d, the target substance does not warrant classification for reproductive toxicity in accordance with CLP Regulation 1272/2008.