Dibutyl peroxydicarbonate

Endpoint summary

Description of key information

Dibutyl peroxydicarbonate 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 found on male and female mortality, clinical observations, functional observations, body weight development (except HD males), food consumption, estrous cyclicity, hematology and coagulation, clinical biochemistry, urinalysis and gross macroscopic findings at necropsy in all treatment groups.

There were test item related effects on body weight development in HD males and histopathology in the HD group treated with 1000 mg/kg body weight/day. Based on the results, the NOAEL for repeated dose toxicity is considered to be 300 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

Adopted on 29th July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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 - 251 g
- Housing: Animals were housed in groups of up to 5 animals / sex / cage
- Diet (e.g. ad libitum): Yes, Altromin 1324
- Water (e.g. ad libitum): Yes , tap water
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY: Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich

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 oral 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. The dose formulations were prepared by adding the required volume of corn oil and further vortexing it for 2-3 minutes. Formulates were kept under magnetic stirring during the daily administration. The vehicle corn oil was used as control item (C1). As Peroxan NBC-50 contains the phlegmatizer Isododecane, Isododecane was additionally used as control item (C2). Isododecane was weighed into a tared plastic vial on a precision balance and the vehicle corn oil 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 NBC-50 contains the phlegmatizer isododecane, isododecane was additionally used as control item (C2).

- Lot/batch no. (if required): Corn oil: MKBZ98989V, MKCC0462Z; Isododecane: 1236856

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 (Eurofins Munich Study No. 171438). 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 25 mL, MD 10 mL, HD 6 mL). All A-samples except from the additional control group C2 was analysed at Eurofins Munich (Eurofins Munich Study Phase No. 171439) and until then stored under appropriate conditions based on available stability data (Eurofins Munich study phase no. 171438). The B-samples and all samples of the second control group C2 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, vehicle (group C1) or second control item (group C2, Isododecane) 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:

7 days a week

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

Observations and examinations performed and frequency:

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 at least 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. Any animals prematurely sacrificed were weighed prior to the sacrifice. Inadvertently animal no. 69 (group C2) was weighed on gestation day (GD) 1 instead of GD 0. The value of gestation day 1 was excluded from calculation of mean body weight.

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.

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.

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.

Sacrifice and pathology:

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 (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 (testes, epididymides, prostate with seminal vesicles and coagulating glands, uterus with cervix and ovaries) were weighed from all animals. Thyroid/parathyroid glands from 1 pup/sex/litter/group (if possible) (sacrificed on PND 13) and from all adult males and females were preserved. Weight of thyroid/parathyroid glands was measured after fixation.

HISTOPATHOLOGY: Yes
A full histopathology was carried out on the preserved organs and tissues (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 (Table 7) of all animals which died during the study or which were euthanised due to morbidity.
The organ and tissues specified in Table 8 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 be 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 not extended to animals of all other dosage groups (C2 and MD) for kidney (male C2 and MD group), thymus, stomach (male and female C2, LD and MD group) and mesenteric lymph nodes (C2 and MD females) as treatment-related changes were not observed for these organ/tissues in 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 1).

Other examinations:

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.

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 C1 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).

Clinical signs:

no effects observed

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 in all and or moderately increased salivation in 4 animals of MD group was also observed during mating/post mating day 2-14. In HD group, moving the bedding and moderate to severely increased salivation was observed during premating day 5 to mating and post mating day 14 in almost all animals.
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 1 - PND 12), in all LD animals (between GD 1 and PND 12) and in almost all MD and HD animals (between MD 2 - PND 12 in MD and PMD 8- PND 12 in HD on majority days). A slight to severely increased salivation was observed in almost all MD and HD animals between MD 2 - PND 12 in MD and PMD 8- PND 12 in HD on majority days.
There were also low incidences of the clinical signs like alopecia on various body parts of the 1 each female of C1, C2 and 3 of LD, abnormal breathing, nasal discharge and test item regurgitation in one LD female and swelling under right forelimb in 1 HD 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.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

During the treatment period of this study, one mortality was observed. Macroscopically, fluid filled abdominal cavity, dark abnormal colour of ovaries and mandibular lymph node were observed at necropsy. Histopathologically, the cause of death of the mortal animal could not be established.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In both males and females, there was no statistically significant effect observed on body weight in the treatment groups during the entire study period when compared with the controls (C1). However in males, statistically significantly lower group mean body weight gain was observed during premating day 1 to postmating day 14 and premating day 1 to terminal sacrifice in C2 and HD group when compared with the control (C1). There was also statistically significantly lower group mean body weight during premating day 7-14 in HD and during pre mating day 14 to mating/postmating day 7 in C2 group when compared to the controls. In females, no statistically significant effect on body weight gain was observed in any group when compared to the controls. This effect on body weight gain in C2 and HD males 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. In females also no statistically significant effect was observed throughout the study period on group mean food consumption in treatment groups when compared with the controls (C1).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment related effects were 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. In females sacrificed at the end of treatment period, statistically significantly lower group mean MCHC in C2 and higher group mean monocytes in C2 were observed in 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:

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 respective controls (C1) except statistically significantly lower group mean sodium in MD and HD group females were observed when compared with the controls. As values were within historical control data range (Sodium 74-149 mmol/L), this effect on sodium in female MD and 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, 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 lower supported rearing count and urination count before the initiation of treatment in 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 MD and HD groups before treatment were observed when compared to the controls (C1). As this finding was observed before initiation of the treatment, 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:

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 weights were observed in C2 and HD group (In C2 group , absolute 16.18 g, relative 3.98%, In HD group absolute 15.75 g and relative 3.87%) with statistical significance achieved only for absolute and relative liver in C2 and relative liver weights in HD group when compared with the C1 controls (absolute 13.31 g and relative 3.16%).
In females sacrificed at the end of treatment period, significantly higher group mean absolute and relative liver weights in C2, MD and HD group (In C2 group , absolute 15.88 g, relative 5.47%, In MD group absolute 15.02 g and relative 5.22%, In HD group absolute 16.91 g and relative 5.58 %) when compared to C1 controls (absolute 13.75 g and relative 4.62%). However, statistical significance was achieved only for absolute liver weights in HD and relative liver weights in C2, MD and HD when compared to the controls (C1).
There was a trend toward marginally decreased absolute and relative thymus weights in C2 and HD males and females from C2 and HD groups (range: 10% to 24%). This change was not statistically significant; as it corresponded to low thymus weights in some animals only. Moreover, the change was not dose related.
This effect on liver in males and females in C2 and HD group was considered to be test item related. An increase in absolute and relative liver weights was present of similar magnitude in both groups C2 and HD animals but not LD or MD. However, in absence of histomorphological correlates, this organ weight effect in liver was considered to be related to metabolic adaptation to treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Few specific macroscopic changes as mentioned below 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 finding observed in terminally sacrificed animal was peripelvic dilatation of kidneys both sides (male no. 18 of C2 group). The above mentioned finding was deemed incidental and there were no gross lesions that could be attributed to treatment with the test item. In one decedent (female no. 155 HD), red fluid filled abdominal cavity, dark abnormal coloured ovaries and mesenteric lymph nodes were observed. Histopathologically, the cause of death of female no. 155 could not be established.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In unscheduled (dead) female no.155 of HD group, no treatment-related microscopic findings were noted. Cause of death was undetermined. As this event was sporadic and no sign of general toxicity was noted at this dose level in scheduled animals, this particular death was not attributed to treatment with the compound.
In scheduled (terminally sacrificed) animals, treatment-related microscopic findings were noted in the forestomach (non-glandular stomach), the kidney (males only), the mesenteric lymph nodes (females only) and the thymus of animals sacrificed on schedule.
Among them, all but the forestomach findings were also observed in the C2 group (isododecane at 500 mg/kg bw/day). Minimal to moderate mixed cell (chronic active) inflammation of the forestomach, and parakeratotic hyperkeratosis and/or vacuolation (males only) of the Malpighian epithelium was observed from 300 mg/kg bw/day mid dose (MD) males and females (more pronounced in males). This change was not present in the forestomach of C2 rats.
Isododecane phlegmatizer (C2) and treatment-related alpha2-microglobulin nephropathy associated with hyaline droplet accumulation was observed in the kidney of males (in all C2, MD and HD at similar severity), as well as isododecane-related and treatment-related increased acute or subacute blood drainage was noted in the sinuses of mesenteric lymph nodes of C2, MD and HD females (similar severity in C2 and MD, higher in HD).
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.
No treatment-related findings were noted in reproductive organs. In particular, no treatment-related spermatogenesis maturation abnormalities were noted upon examination of the H-PAS stained sections of testis
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, no major adverse treatment-related tissue findings were noted, correlating with no important changes found in absolute or relative organ weights, and an absence of treatment related gross findings, suggesting the treatment was rather well tolerated. From 300 mg/kg bw/day (MD), adverse inflammation and Malpighian epithelial parakeratotic hyperkeratosis and/or vacuolation, to be related to local irritation, were noted in the forestomach of both sexes. They were not present in controls (C2 or C1).
An adverse, male rat-specific finding was noted in the kidney of males only: there were alpha2-microglobulin nephropathy and/or hyaline droplet accumulation in all C2, MD and HD males, an effect most likely related to the binding of the Isododecane to alpha2-microglobulin, a mechanism which has no relevance to human safety. An Isododecane related and treatment-related increased acute or subacute blood drainage was noted in the sinuses of mesenteric lymph nodes of C2, MD and HD females (similar severity in C2 and MD, higher in HD)
A minor stress related change was noted in thymus in phlegmatizer control and MD males and HD males and females, correlating with lower thymic weight in a few animals.

Histopathological findings: neoplastic:

not examined

Other effects:

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.

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

Remarks on result:

other: No adverse effects

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

histopathology: non-neoplastic

Key result

Critical effects observed:

no

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 98.5 % and 104.8 % of nominal value, for MD group between 99.1 % and 102.2 %, and for HD group between 102.3 % and 104.4 %. Overall recoveries of entire study observed in LD, MD and HD groups were 102.1 %, 100.9 %, and 103.5 % of nominal concentration, respectively.

Conclusions:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening, test item related effects on body weight development and histopathology were observed. Based on the results, the NOAEL 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 Dibutyl peroxydicarbonate (50.6% peroxide) was administered orally 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), food consumption, estrous cyclicity, haematology and coagulation, clinical biochemistry, urinalysis and gross macroscopic findings at necropsy in all treatment groups.

Test item related effect on anogenital distance was observed in all groups. All anogenital values in male and female pups were within historical control data range.No additional finding in the study supporting a possible endocrine disruption modality of the test item. Thus, there is no conclusive evidence of an endocrine disrupting effect of the test item and as a result of that, the findings on AGD cannot be considered as adverse. Furthermore, there was no test item related effect observed on pup thyroid weight, male and PND 13 pup thyroxine hormone (T4) in the treatment groups.

There were test item related effects on body weight development in HD males and histopathology in the HD group treated with 1000 mg/kg body weight/day.

Based on the results, the NOAEL for repeated dose 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 rat. 

Endpoint conclusion

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

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data, the target substance does not warrant classification for specific target organ toxicity in accordance with CLP regulation (EC) No 1272/2008.