tert-pentyl 2-ethylperoxyhexanoate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The possible effects of CAS# 686 -31 -7on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus, parturition and early lactation of the offspring (Day 6 post partum) was evaluated in an OECD 421 study (Sisti, 2015). The test item was given to Sprague Dawley rats by oral administration (gavage) before and during mating and throughout the gestation period until Day 6 post partum at dosages of 50, 250 and 1000 mg/kg/day. One female with litter, receiving 50 mg/kg/day was sacrificed for humane reasons on Day 1 post partum. Histopathological evaluation revealed a severe atrophy of thymus, lymphoid depletion of spleen, mucosal ulceration of forestomach (non glandular region), villous atrophy of jejunum and ileum and cortical vacuolation and nephropathy of kidneys. These changes were considered as factors contributory to the illness status of the animal. The major clinical signs noted in treated males receiving 1000 mg/kg/day were matted fur and salivation, while only salivation was recorded in females receiving the same dose level. Soft faeces were observed in the cage tray of animals of both sexes receiving 1000 mg/kg/day and sometimes in males receiving 250 mg/kg/day. Body weight of males receiving 1000 mg/kg/day was slightly lower (<10%) than the control group, as well as, body weight gain on Days 8 (before pairing) and 15 (mating phase) of the study. Food consumption was decreased in females receiving 1000 mg/kg/day before pairing (Day 8 of study) and on Day 6 post partum. Concerning the reproductive parameters, no relevant differences were found in terms of mating performance including the pre-coital interval (number of days paired to sperm positive day) and the copulatory evidence (the positive identification of mating i.e. the presence of sperm and/or copulation plug in situ or in the cage). The resulting copulatory index and fertility index did not show intergroup differences. Post-implantation loss was slightly increased in females receiving 1000 mg/kg/day when compared to the control group. Stillbirths or total litter loss were noted in 5 females receiving 1000 mg/kg/day the day of parturition or the day after parturition. Increased incidences of pup loss at birth and cumulative loss on Day 6 post partum were also noted. Decreases in the number of males and consequently in the total number of pups were noted on Day 6 post partum in females receiving 1000 mg/kg/day when compared to controls. Sex ratios on Day 6 post partum was also decreased when calculated as the percentage of males. Marked mortality of pups or missing pups were noted at 1000 mg/kg/day. Cold to touch, apparently no food intake (milk) and small appearance were noted in the surviving pups of dams receiving 1000 mg/kg/day, in control pups and in those receiving the dose levels ≥ 50 mg/kg/day. Necropsy findings observed in decedent control and treated pups, were similar. No necropsy findings were observed in all pup of control and treated groups, sacrificed on Day 6 post partum. Slight decrease in terminal body weight was observed in high dose animals of both sexes receiving 1000 mg/kg/day. Some changes in absolute and relative organ weights (adrenals, liver, kidneys, uterus, testes and thymus) were noted in treated animals mainly in those receiving 1000 mg/kg/day. However, the differences were not accompanied by histological findings, with the exception of those observed in thymus of high dose females. At macroscopic observations the most remarkable change was an increased incidence of reduced size of the thymus in high dose females. At microscopic observations treatment-related atrophic changes were noted in the thymus of female treated at 1000 mg/kg/day. On the basis of the results obtained, the NOAEL (No Observed Adverse Effect Level) for parental toxic ity and fertility could be considered to be 1000 mg/kg/day for males and 250 mg/kg/day for females. The NOAEL for the toxicity on development could be considered to be 250 mg/kg/day.

A Reproduction/Developmental Toxicity Screening Test with the read-across test item, CAS# 3006-82-4, tert.-Butylperoxy- 2-ethylhexanoat was performed according to OECD 421.

The CAS# 3006 -82 -4 was administered once daily orally (by gavage) to male and female rats throughout the pre-pairing, pairing and lactation periods until necropsy (day 4 of lactation). The dose levels were applied 0, 100, 300 and 1000 mg/kg body weight/day to male rats for 41 days in total and female rats throughout the pre-pairing, the pairing, the gestation and the lactation periods until day 3 post partum (last dosing). Treatment at 1000 mg/kg was associated with decreased food consumption in male and female animals during the first week of the pre-pairing period. Some dams were noted with ruffled fur after parturition and generally bad conditions. No test item-related effects were noted during necropsy and for macroscopic findings. Treatment at 1000 mg/kg was associated with an increase of pre-implantation-, post-implantation-, and post-natal loss, and a reduction of live pups limited to general systemic toxicity in parental animals. The mean body weight of pups also was reduced at this systemic maternal toxic dose. Based on these data, it can be concluded that the NOEL was at 300 mg/kg body weight/day for parental generation as well as for the offspring.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

A total of 90 Sprague Dawley [Crl:CD(SD)BR] rats (45 males and 45 virgin females), 7 to 8 weeks old and weighing 220 to 235 g for males and 189 to 195 g for females, were received from Charles River Italia S.p.A., Calco (Lecco), Italy. After arrival the weight range for each sex was determined and the animals were temporarily identified within the cage by means of a coloured mark on the tail. A health check was then performed by a veterinarian. An acclimatisation period of 20 days was allowed before the start of treatment, during which time the health status of the animals were assessed by thorough observations.
he animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22 °C+/-2°C
and 55 % +/- 15 % respectively; actual conditions were monitored, recorded and the records retained. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day. From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5x38x20 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week.

During mating animals were housed one male to one female in clear polysulfone cages measuring 42.5x26.6x18.5 cm with a stainless steel mesh lid and floor (Techniplast – Gazzada S.a.r.l.). Each cage tray held absorbent material which was inspected and changed daily. After mating the males were re-caged as they were before mating. The females were transferred to individual polysulfone solid bottomed cages measuring 42.5x26.6x18.5 cm (Techniplast Gazzada S.a.r.l.) for the gestation period, birth and lactation. Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary. Nesting material was changed at least 2 times a week.

Drinking water was supplied ad libitum to each cage via water bottles. A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) was offered ad libitum throughout the study.

The animals arrived on 08 January 2015 and were allocated to groups on 21 January 2015. Dosing commenced on 28 January 2015 and the last necropsy was performed on 20 MArch 2015.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on mating procedure:

Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray). The female was paired with the same male until positive identification occurred.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before treatment, analyses were performed to confirm that the proposed formulation procedure was acceptable. Samples of the formulations prepared during the study were analysed to check the concentration and homogeneity (the first and the last week of treatment). Chemical analyses were carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study number 88800) in the range from 1 to 200 mg/mL in corn oil. In addition, the stability of formulated samples at 1 and 200 mg/mL was verified after 24 hours and 8 days at +4°C in the same study. The software used for this activity was Empower®2 Build No. 2154.

Duration of treatment / exposure:

Males for 2 consecutive weeks prior to pairing and thereafter through the day before necropsy (Days 29 and 30 of study). Females for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 5 post partum.

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
50, 250 and 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

Mortality
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holiday a similar procedure was followed except that the final check was carried out at approximately mid-day. This allowed post mortem examinations to be carried out during the working period of that day. A complete necropsy was performed as detailed in section [sub:Necropsy] below.

Clinical signs
All clinical signs were recorded for individual animals. Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions (15-30 minutes and 1 - 1.5 hours after dosing).

Observations of the cage tray
Observations of the cage tray, during the pre-mating (males and females) and after mating periods (only males), were performed and recorded three times weekly. During mating and gestation periods (only females until Day 11 post coitum), these observations were performed and recorded daily.

Body weight
Males were weighed on the day of allocation to treatment groups, on the day of treatment commencement, weekly thereafter and at termination.
Females were weighed on the day of allocation to treatment groups, on the day of treatment commencement, weekly thereafter until positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 6 post partum.

Food consumption
The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period following allocation. Individual food consumption for the females were measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 6 post partum starting from Day 1 post partum.

Parturition check and duration of gestation
A parturition check was performed from Day 20 to Day 25 post coitum. Female nos. X0080059 (Group 3) and X0080071 (Group 4) which did not give birth after 25 days of post coitum period were sacrificed shortly after (Days 26 and 27 post coitum, respectively).
Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring were first detected in the cage was considered Day 0 post partum.

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily in the morning starting from two weeks before pairing throughout the mating period until a positive identification of copulation was made. The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)

Sperm parameters (parental animals):

Not done

Litter observations:

Pups identification, weight and observation
As soon as possible, after parturition was considered complete (Days 0 or 1 post partum), all pups (live and dead) were counted, sexed and live pups were identified. Live pups were individually weighed on Days 1 and 6 post partum. Pups killed or dying during the lactation period were weighed before the despatch to necropsy. Observation was performed once daily for all litters.

Postmortem examinations (parental animals):

Parental animal were euthanised with carbon dioxide. The males were killed after the mating of all females after 29 and 30 days of treatment. The females with live pups were killed on Day 6 post partum. The females with total litter loss were killed on the day of the occurrence of total litter loss or shortly after. The females showing no evidence of copulation were killed after 25 days of the last day of the mating session (Days 26 and 27 post coitum).

Gross observation
The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed (excluding animals sacrificed for humane reasons) and the required tissue samples preserved in fixative and processed for histopathological examination.
All females were examined also for the following:
• external and internal abnormalities;
• number of visible implantation sites (for pregnant animals);
• number of corpora lutea (if detectable).
Uteri of apparently non-pregnant females or uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

Organ weights
From all parental animals completing the scheduled test period the organs indicated in Annex 1 of the study ptotocol were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal. U

Tissues fixed and preserved
Samples of all the tissues indicated in annex 1 of the study protoocol were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in Modified Davidson's fluid and preserved in 70% ethyl alcohol).

Histopathological examination
The tissues required for histopathological examination are listed in Annex 1 of Study Protocol . After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides of all males in the control and high dose groups were cut at 2-3 micrometre thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
The examination was restricted as detailed below:
Tissues specified in Annex 1 of Study Protocol from all animals in the control and high dose group killed at term
Tissues specified in Annex 1 of Study Protocol from the animal killed during the treatment period
All abnormalities in all groups
On the basis of the treatment-related changes detected in the thymus of high dose treated females, the histopathological evaluation of the thymus was extended to the remaining low and mid-dose females.

Postmortem examinations (offspring):

Pups were euthanised by intraperitoneal injection of sodium thiopenthal.
All pups found dead in the cage or sacrificed for humane reasons were examined for external and internal abnormalities. All live pups sacrificed at termination (Day 6 post partum) were killed and examined for external abnormalities and sex confirmation by gonadal inspection.

Statistics:

Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test. The non-parametric Kruskal- Wallis analysis of variance (non-continuous variables) was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p < 0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off. Further tests were used as considered appropriate.

Reproductive indices:

Males
Copulatory Index (%) = no. of animals mated / no. of animals paired * 100
Fertility Index (%) = no. of males which induced pregnancy / no. of animals paired * 100

Females
Copulatory Index (%) = no. of animals mated / no. of animals paired * 100
Fertility Index (%) = no. of pregnant females / no. of females paired * 100
Pre-birth loss was calculated as a percentage from the formula: no. of visible implantations - total litter size at birth / no. of visible implantations * 100
Pre-implantation loss was calculated as a percentage from the formula: no. of corpora lutea - no. of visible implantations / no. of corpora lutea * 100

Males and females
Pre coital Interval = Mean number of days between pairing and mating

Offspring viability indices:

Pup loss at birth was calculated as a percentage from the formula: Total litter size - live litter size / Total litter size * 100
Cumulative pup loss on Day 6 post partum was calculated as a percentage from the formula: Total litter size at birth - live litter size at Day 6 / Total litter size at birth * 100
Sex ratios were calculated at birth and on Day 6 post partum and were presented as the percentage of males per litter.

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Mortality and fate of females
One female with litter, receiving 50 mg/kg/day was sacrificed for humane reasons on Day 1 post partum.
The day before the unscheduled death the following clinical signs were noted:
- hunched posture, emaciated aspect, piloerection and teeth missing. On the day of sacrifice, decreased activity, cold to touch, staining on perigenital region and pale aspect were recorded. The most relevant changes observed at post mortem examination were distention with gas content in stomach, duodenum, ileum and jejunum; small size of thymus, spleen and pancreas. Histopathological evaluation revealed a severe atrophy of thymus, lymphoid depletion of spleen, mucosal ulceration of forestomach (non glandular region), villous atrophy of jejunum and ileum and cortical vacuolation and nephropathy of kidneys. The above mentioned pathological changes were considered as factors contributory to the illness status of the animal.
One female (X0080071) receiving 1000 mg/kg/day was found not pregnant at necropsy. One female (X0080059) receiving 250 mg/kg/day showed unilateral implantation. This female with unilateral implantation in the right horn had also total resorption in that horn and was not pregnant in the left one. The number of females with live pups on Day 6 post partum was: 10 in the control, 9 in the low dose (50 mg/kg/day), 9 in the mid- dose group (250 mg/kg/day) and 4 in the high dose group (1000 mg/kg/day).

Clinical signs
Matted fur and salivation were the principal clinical signs observed in treated males receiving 1000 mg/kg/day. Occasionally salivation was also noted in one male receiving 250 mg/kg/day. One control male (animal no. X0080010) showed scabs on body surface (thoracic region, lumbar region and upper hindlimb starting from Day 10 of the mating phase) and was isolated an individual cage.
Salivation was mainly noted in females receiving 1000 mg/kg/day before pairing, during gestation and post partum period. Hunched posture was occasionally observed in this group. \vphantom{} Observations of the cage tray Soft faeces, slight, were observed throughout the study in males and females receiving 1000 mg/kg/day. Occasionally this sign was recorded in males receiving 250 mg/kg/day.

Observations of the cage tray
Soft faeces, slight, were observed throughout the study in males and females receiving 1000 mg/kg/day. Occasionally this sign was recorded in males receiving 250 mg/kg/day.

Body weight
Slight decrease in body weight was noted in treated males receiving 1000 mg/kg/day throughout the study. However this change was always below 10%. Body weight of treated females was, in general, comparable to the control group during the study. Body weight gain of males receiving 1000 mg/kg/day was decreased compared to the control group, both on Day 8 of the study (before pairing) and on Day 15 (mating phase). Changes were of -37% and -79%, respectively.

Food consumption
Food consumption was decreased in females receiving 1000 mg/kg/day before pairing (Day 8 of study) and on Day 6 post partum (-20% and -19%, respectively). Food consumption of treated males were comparable to the control group throughout the study, as well as, for females during the gestation period.

Oestrous cycle, reproductive parameters, pairing combination and mating performance
Oestrus cycle and reproductive parameters (pre-coital intervals, copulatory and fertility indices) were similar in treated and control groups.
Implantation, pre-implantation loss data, pre-birth loss data (or post- implantation loss) and gestation length of females
Gestation periods of control and treated groups were similar and dams gave birth on Day 22 post coitum (mean value). Implantation sites, pre-implantation loss data and total litter size at birth were, in general, comparable between groups. Post- implantation loss was slightly increased in females receiving 1000 mg/kg/day.

Litter data at birth, on Day 1 and on Day 6 post partum and sex ratio of pups
Stillbirths (X0080069) or total litter loss (X0080061, X0080063, X0080077, X0080079) were noted the day of parturition or the day after parturition in females receiving 1000 mg/kg/day. In addition, an increased incidence of pup loss at birth and cumulative loss on Day 6 post partum were noted. Live litter size was reduced at birth, on Days 1 and 6 post partum and consequently litter weight was also decreased. Decreases in the number of male pups and consequently in total number of pups were noted on Day 6 post partum in females receiving 1000 mg/kg/day when compared to controls. Sex ratio on Day 6 post partum was also decreased when calculated as the percentage of males.

Terminal body weight and organ weights
Slight decrease in terminal body weight was observed in high dose animals of both sexes (-6% to -8 %) receiving 1000 mg/kg/day. Some differences, sometimes statistically significantly, were noted in the absolute and/or relative organ weight, such as: - Increased absolute kidneys (+18%) and liver (+21%) weights in males receiving 1000 mg/kg/day - Increased absolute adrenals (+16%), liver (+12%) and uterus weights (+107%) in females receiving 1000 mg/kg/day. Absolute thymus (+23%) weight was increased in females receiving 250 mg/kg/day and decreased in females receiving 1000 mg/kg/day (-18%). - Increase in relative adrenals, kidneys, liver and testes weights in males receiving 1000 mg/kg/day (+24%, +27%, +30%, +9%, respectively). Relative kidneys weight was also increased in males receiving 250 mg/kg/day (+13%). - Increase in relative adrenals, kidneys, liver, uterus weights and decrease relative thymus weight in females receiving 1000 mg/kg/day (+28%, +13%, +21%, +126%, -11%, respectively). No concurrent histological findings were noted in the above mentioned organs with the exception of those observed in the thymus of high dose females.

Macroscopic observations
The most remarkable change noted at post mortem examination was an increased incidence of reduced size of the thymus in high dose females, when compared with the control and low dose females. In addition, enlarged adrenals or kidneys were observed in few treated animals.

Microscopic observations
Treatment-related changes were noted in the thymus of female rats treated at 1000 mg/kg/day. The histopathological change detected in the thymus was atrophy, represented by: a minimal to marked reduction in cortical lymphocytes, shrinkage of the thymic lobules, increased prominence of interlobular septae and an inverse cellular density ratio cortex/medulla. The females treated at 250 and 50 mg/kg/day did not show any remarkable changes in the thymus.

Spermatogenic cycle
A detailed qualitative examination of the testes was performed in all control and high dose group males. The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted in order to identify treatment related effects, such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Identification of the stages of the spermatogenic cycle was carried out as described by Leblond and Clermont, 1952 and referred to the comprehensive reviews on the subject Russell, 1990; Creasy, 1997; Creasy, 2002. The PAS-H stained sections were used to identify the spermatogenic stages.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted. The remaining sporadic lesions, reported in control and treated animals, were considered to be an expression of spontaneous and/or incidental pathology, seen in this species and age of untreated animals.

Key result

Dose descriptor:

NOAEL

Remarks:

toxicity

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No treatment-related adverse effects noted

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No treatment-related adverse effects noted

Key result

Dose descriptor:

NOAEL

Remarks:

toxicity

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Treatment-related changes in the thymus at 1000 mg/kg/day

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

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

Based on:

act. ingr.

Sex:

female

Basis for effect level:

other: Post-implantation loss, stillbirths or total litter loss at 1000 mg/kg/d

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings:

no effects observed

Clinical signs of pups
Marked mortality of pups or missing pups were noted at 1000 mg/kg/day. Cold to touch, apparently no food intake (milk) and small appearance were noted in the remaining pups of the dams treated at 1000 mg/kg/day and reaching Day 6 post coitum. These clinical signs were also observed in control and treated pups receiving dose levels ≥ 50 mg/kg/day. Found dead and missing pups were observed on control, low and mid-dose groups, with similar incidence.

Necropsy findings in decedent pups and in pups sacrificed on Day 6 post partum.
Autolysed abdominal/thoracic organs were generally observed in control and treated pups which died during the lactation period. No milk in stomach and dark staining (abnormal colour) on the abdominal region were also noted in pups at 1000 mg/kg/day. No necropsy findings were observed in all pups of control and treated groups, sacrificed on Day 6 post partum.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Pups mortality at 1000 mg/kg/d

Reproductive effects observed:

not specified

Conclusions:

On the basis of the results obtained, the NOAEL (No Observed Adverse Effect Level) for parental toxicity and fertility could be considered to be 1000 mg/kg/day for males and 250 mg/kg/day for females. The NOAEL for the toxicity on development could be considered to be 250 mg/kg/day.

Executive summary:

The possible effects of CAS# 686 -31 -7 on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus, parturition and early lactation of the offspring (Day 6 post partum) was evaluated in an OECD 421 study (Sisti, 2015). The test item was given to Sprague Dawley rats by oral administration (gavage) before and during mating and throughout the gestation period until Day 6 post partum at dosages of 50, 250 and 1000 mg/kg/day. One female with litter, receiving 50 mg/kg/day was sacrificed for humane reasons on Day 1 post partum. Histopathological evaluation revealed a severe atrophy of thymus, lymphoid depletion of spleen, mucosal ulceration of forestomach (non glandular region), villous atrophy of jejunum and ileum and cortical vacuolation and nephropathy of kidneys. These changes were considered as factors contributory to the illness status of the animal. The major clinical signs noted in treated males receiving 1000 mg/kg/day were matted fur and salivation, while only salivation was recorded in females receiving the same dose level. Soft faeces were observed in the cage tray of animals of both sexes receiving 1000 mg/kg/day and sometimes in males receiving 250 mg/kg/day. Body weight of males receiving 1000 mg/kg/day was slightly lower (<10%) than the control group, as well as, body weight gain on Days 8 (before pairing) and 15 (mating phase) of the study. Food consumption was decreased in females receiving 1000 mg/kg/day before pairing (Day 8 of study) and on Day 6 post partum. Concerning the reproductive parameters, no relevant differences were found in terms of mating performance including the pre-coital interval (number of days paired to sperm positive day) and the copulatory evidence (the positive identification of mating i.e. the presence of sperm and/or copulation plug in situ or in the cage). The resulting copulatory index and fertility index did not show intergroup differences. Post-implantation loss was slightly increased in females receiving 1000 mg/kg/day when compared to the control group. Stillbirths or total litter loss were noted in 5 females receiving 1000 mg/kg/day the day of parturition or the day after parturition. Increased incidences of pup loss at birth and cumulative loss on Day 6 post partum were also noted. Decreases in the number of males and consequently in the total number of pups were noted on Day 6 post partum in females receiving 1000 mg/kg/day when compared to controls. Sex ratios on Day 6 post partum was also decreased when calculated as the percentage of males. Marked mortality of pups or missing pups were noted at 1000 mg/kg/day. Cold to touch, apparently no food intake (milk) and small appearance were noted in the surviving pups of dams receiving 1000 mg/kg/day, in control pups and in those receiving the dose levels ≥ 50 mg/kg/day. Necropsy findings observed in decedent control and treated pups, were similar. No necropsy findings were observed in all pup of control and treated groups, sacrificed on Day 6 post partum. Slight decrease in terminal body weight was observed in high dose animals of both sexes receiving 1000 mg/kg/day. Some changes in absolute and relative organ weights (adrenals, liver, kidneys, uterus, testes and thymus) were noted in treated animals mainly in those receiving 1000 mg/kg/day. However, the differences were not accompanied by histological findings, with the exception of those observed in thymus of high dose females. At macroscopic observations the most remarkable change was an increased incidence of reduced size of the thymus in high dose females. At microscopic observations treatment-related atrophic changes were noted in the thymus of female treated at 1000 mg/kg/day. On the basis of the results obtained, the NOAEL (No Observed Adverse Effect Level) for parental toxic ity and fertility could be considered to be 1000 mg/kg/day for males and 250 mg/kg/day for females. The NOAEL for the toxicity on development could be considered to be 250 mg/kg/day.

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1 study, GLP compliant.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Short description of key information:
In an OECD 421 study, the NOAEL (No Observed Adverse Effect Level) for parental toxic ity and fertility could be considered to be 1000 mg/kg/day for males and 250 mg/kg/day for females. The NOAEL for the toxicity on development could be considered to be 250 mg/kg/day.

Justification for selection of Effect on fertility via oral route:
Key study

Effects on developmental toxicity

Description of key information

Oral treatment of pregnant Hsd. Brl. Han: WISTAR rats from gestation day 5 up to day 19 with the read-across substance, CAS# 3006-82-4, Tert.-Butylperoxy- 2-ethylhexanoat at the dose levels of 200, 400 and 1000 mg/kg bw/day did not cause death and necropsy findings. The test item did not reveal any adverse effect on the pregnancy and the intrauterine mortality of the conceptuses, the number of viable fetuses and their sex distribution. Further the test substance did not increase significantly the incidence of external and visceral variations, and caused no skeletal malformations. The slight delay in ossification in fetuses of the 1000 mg/kg bw/day dose group is considered to be non-adverse.
Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:
NOAEL maternal toxicity: 1000 mg/kg bw/day
NOAEL developmental toxicity: 1000 mg/kg bw/day

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2012-10-17 to 2013-02-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in accordance with GLP regulation and the respective OECD/EU guideline.

Justification for type of information:

see cross-referenced read-across:supporting information in Section 13.2 attachment

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

22 January 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Toxi-Coop Zrt. 1103 Budapest Cserkesz u. 90. Hungary
- Age at study initiation: Females: Young adult and nulliparous females, 10-11 weeks of age at start of the mating period. Males: experienced males 35-37 weeks of age at start of the mating period.
- Fasting period before study: no
- Housing: Before mating: 1-3 females per cage, 1-2 males per cage. Mating: 1 male and 1-3 females / cage. During gestation: 2-3 sperm positive females per cage, if not possible 1 sperm positive female per cage.
- Diet: ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" ad libitum
- Water: tap water ad libitum
- Acclimation period: 20 days for females and 181 days for males

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 36-46
- Air changes (per hr): 8-12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2012-10-17 To: 2012-11-13

Route of administration:

oral: gavage

Vehicle:

other: Helianthy Annui Oleum Raffinatum / Sunflower oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations were prepared in the laboratory of Toxi-Coop Zrt. daily or according to the stability data of the formulations (based on previous analytical measurements performed in the Laboratory of Toxi-Coop Zrt).

VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble and not stable in water therefore oleum helianthy was used for preparing formulations appropriate for oral administration. Oleum helianthy /sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 100, 200, 500 mg/mL
- Amount of vehicle: 2 mL/kg bw
- Lot/batch no.: 19T3

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control (concentration, homogeneity) of dosing solutions was performed in the Laboratory of Toxi-Coop Zrt. on the first and last treatment weeks.
The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. A sufficient stability and homogeneity in the chosen vehicle was verified over the range of relevant concentrations at the appropriate frequency of preparation. The test item concentrations in the samples were found to be 94 – 106 % in comparison to the nominal values.

Analytical method:
- HPLC-UV
- Detector: 210 nm
- Column: HyperPrep HS C18, 250 x 4.6 mm, 8 μm,
- Mobil Phase: Acetonitrile: Water = 9 : 1 (v/v)
- Flow: 1.2 mL/min
- Injection volume: 50 μL
- Temperature: 25 °C
- Retention time: 5.3 min ± 10 %

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/3
- Length of cohabitation: in the mornings for two to four hours
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

GD 5 to GD 19

Frequency of treatment:

The test item was administered in a single dose by oral gavage (stomach tube) on a 7 days/week basis every day at similar time.

Duration of test:

GD 5 to GD 19

No. of animals per sex per dose:

24 Females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The doses have been chosen by the Sponsor on the basis of a previous study (GLP OECD 421 Reproduction/Developmental Toxicity Screening Study of Tert.-Butylperoxy- 2-ethylhexanoat in the Wistar Rat).
- Rationale for animal assignment: The sperm positive females were allocated to each experimental group on each mating day in such a way that the group averages of the body weight were as similar as possible on the first day of gestation. If possible, females paired with the same male were allocated to different groups on the same mating day.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for signs of morbidity and mortality were made twice daily, at the beginning of the working period and in the afternoon.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a day, after treatment at approximately the same time. Individual observation included the check of behavior and general condition.
Duration and severity of the clinical signs were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Gestation days 0, 3, 5, 8, 11, 14, 17 and 20

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined:
the uterus with cervix and the left ovary were removed and weighed. The right ovary was placed into a Petri dish after removal. After removing the uterus gross pathology of dams' viscera was performed. The number of corpora lutea in each ovary and implantation sites in each uterine horn, live fetuses, early and late embryonic death and fetal death were counted. Animals, in which unambiguous implantation sites, but not fetuses have been found, were considered as pregnant.

EXAMINATION OF PLACENTAL SIGNS:
All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the 13th gestational day. If negative on day 13, the examination was repeated on day 14 of gestation.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

The statistical evaluation of data was performed with the program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan’s Multiple Range test was used to access the significance of inter-group differences. If significance was the result of the Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.
Dams or litters were excluded from the data evaluation in cases of:
- Any disease or death of the dam unrelated to the treatment (total exclusion)
- Non pregnant females or dams with 3 or less implantations independent of their viability (total exclusion)
Although these animals were excluded from the data evaluation the final report contains all data of these animals, too.
A male/female fetus was considered as retarded in body weight, when its weight is below the average minus twofold standard deviation of the control male/female fetuses.

Indices:

- Number of corpora lutea
- Number of implantations
- Number and percent of live fetuses
- Pre-implantation loss (%):
(Number of corpora lutea - Number of implantation) / Number of corpora lutea x 100
- Post-implantation loss (%):
(Number of implantations - Number of live foetuses) / Number of implantations x 100
- Sex distribution (%):
Number of Male (Female) foetuses / Number of foetuses x 100
- External abnormalities per litter (%)
Number of fetuses with abnormality / Number of fetuses x 100
- Visceral abnormalities per litter (%)
Number of fetuses with abnormality / Number of foetuses examined x 100
- Skeletal abnormalities per litter (%)
Number of fetuses with abnormality / Number of foetuses examined x 100

Historical control data:

Historical contral data are available and were used for evaluation of study results.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Clinical symptoms, mortality:
One pregnant female in the control group died in the course of the study on gestational day 20. The death was considered to be due to the intrauterine autolyzing of dead embryos and fetuses. This dam had no clinical signs before death but lost weight.
Alopecia was found sporadically without a dose response in the females. Salivation was recorded in association the treatment in nine of 23 females in the 400 mg/kg bw/day group and in all of the dams of the 1000 mg/kg bw/day dose group, directly after treatment. This was judged to be in relationship with the taste of the test item.

Body weight, body weight gain and corrected body weight:
There was no indication of an effect of the test item on body weight development of the dams in the 200 and 400 mg/kg bw/day dose groups. The body weight gain was statistically significant (p<0.01) reduced on the first three days of treatment in the 1000 mg/kg bw/day group. This finding was considered as test item related but not adverse. Between gestational days 8 and 11 it turned to an increased body weight gain with a statistical significance (p<0.05).
There were no dose related differences in the corrected body weight and corrected body weight gain of the dams in the experimental groups.

Food Consumption:
There was no indication of an effect of the test item on the food consumption of the dams in the 200 and 400 mg/kg bw/day dose groups. There was a statistically significantly (p<0.01) reduced food consumption on the first six days of treatment in the 1000 mg/kg bw/day dose group related to the treatment with the test item. Statistical significant increases (p<0.05) were indicated in two occasions (once before the treatment period and once at the beginning of it) in the food consumption of the animals in the 200 mg/kg bw/day dose group which are not associated with the test item.

Necropsy
There were no macroscopic alterations recorded for the dams during necropsy.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Intrauterine mortality, viable fetuses and their sex distribution:
There was no dose related significant difference in the intrauterine mortality of the conceptuses, the number of implantations, viable fetuses and their sex distribution. The number of late embryonic death increased slightly but statistically significantly (p<0.05) in the 400 mg/kg bw/day dose group and without a statistical significance in the 1000 mg/kg bw/day group. No dose response was indicated and the values are in the range of the historical control data. There was no statistical significance indicated in the mean percentage value of the late embryonic death in the experimental groups.

Body weight of fetuses, placental weight
The mean fetal weight was similar in the control, 200 and 400 mg/kg bw/day groups. The slight but statistically significant (p<0.01) reduction in the mean body weight of the male and female fetuses in the 1000 mg/kg bw/day group might be attributed to the statistically significant reduced body weight gain and statistically significant lower food consumption of the dams between gestational day 5 and 8 and 5 and 11 respectively in this dose group.
Although a statistical significance in the fetal weight in the 1000 mg/kg bw/day group was noted, the value was in the range of the historical control data and therefore considered to be non-adverse.
Placental weight was similar in all experimental groups. There was a statistically significant increase indicated in the relative placental weight in the 1000 mg/kg bw/day dose group (p<0.05), however it was below the historical control level.

External and visceral examination
The number of evaluated fetuses was 228, 190, 239 and 192 at external and 114, 96, 120 and 97 at visceral examination in the control, 200, 400 and 1000 mg/kg bw/day groups, respectively.
The incidence of visceral abnormalities (malformations and variations) was statistically significant (p<0.05) higher in the 1000 mg/kg bw/day dose group. However, the number of affected fetuses is well within the historical control range and therefore considered to be of no biological relevance.

- Malformations
Umbilical hernia was found in one fetus as a malformation at external and visceral examination in the 1000 mg/kg bw/day dose group which was neither proven nor closed out to be in relationship with an effect of the test item. According to the experience of this laboratory and the scientific literature umbilical hernia is a rather common finding in the tester strain used. Therefore, this single event is not considered to be an indication for a test substance related effect.

- Variation
There was no increased incidence of external and visceral variations in the test item treated groups. Visceral variations such as bilateral hydroureter or hydroureter with dilated renal pelvis occurred with a very low incidence without significant difference among the experimental groups, including control.

Skeletal examination
The number of examined fetuses was 114, 94, 119 and 95 in the control, 200, 400 and 1000 mg/kg bw/day respectively.
The incidence of skeletal abnormalities (malformations and variations) increased with a statistical significance (p<0.01) due to the increase in the variations (p<0.01) in the 1000 mg/kg bw/day dose group.

-Malformation
Malformations were recorded such as a bipartite thoracic centrum and dumb-bell shaped cartilage of thoracic centrum in two fetuses in the control and in one in the 1000 mg/kg bw/day dose group without a relationship with the test item.

- Variation
Incomplete ossification of the skull, bipartite supraoccipital, incompletely ossified or misaligned sternebrae, wavy ribs, dumb-bell shaped or bipartite vertebral centra, incomplete or asymmetric ossification of sacral arches and asymmetric or incomplete ossification of metacarpal or metatarsal, were evaluated as variations during the skeletal examination.
There was a slightly but statistically significant (p<0.01) increase in the incidence of fetuses with incomplete ossification of the skull-bones and metacarpal/metatarsal in the 1000 mg/kg bw/day dose group. At this dose level, reduced body weight and food consumption of the dams might explain this slight delay in ossification. Therefore, this variation is not considered to be an indication for developmental toxicity. This assumption is supported by Mylchreest et al. (2005), who stated that retarded skull bone ossification is a relatively common observation and may not be a reliable indicator for developmental toxicity.

References
- Mylchreest, E., Munley, S.M., and Kennedy Jr., G.L. (2005) Evaluation of the Developmental Toxicity of 8-2 Telomer B Alcohol. Drug and Chemical Toxicology, 28:315-328.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Key result

Dose descriptor:

NOEC

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Oral treatment of pregnant Hsd. Brl. Han: WISTAR rats from gestation day 5 up to day 19 with Tert.-Butylperoxy- 2-ethylhexanoat at the dose levels of 200, 400 and 1000 mg/kg bw/day did not cause death and necropsy findings. The test item did not reveal any adverse effect on the pregnancy and the intrauterine mortality of the conceptuses, the number of viable fetuses and their sex distribution. Further the test substance did not increase significantly the incidence of external and visceral variations, and caused no skeletal malformations. The slight delay in ossification in fetuses of the 1000 mg/kg bw/day dose group is considered to be non-adverse.
Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:
NOAEL maternal toxicity: 1000 mg/kg bw/day
NOAEL developmental toxicity: 1000 mg/kg bw/day
Based on these observations the No Observed Effect Level (NOEL) was determined as follows:
NOEL maternal toxicity: 400 mg/kg bw/day
NOEL developmental toxicity: 400 mg/kg bw/day

Executive summary:

Groups of 24 sperm-positive female Hsd. Brl. Han: WISTAR rats were treated with Tert.-Butylperoxy- 2-ethylhexanoat by oral administration daily at three dose levels of 200, 400 and 1000 mg/kg bw/day from day 5 up to and including day 19 post coitum. A control group of 24 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 ml/kg bw.

During the study, mortality was checked for and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day when sperm was detected in the vaginal smear was regarded as day 0 of gestation. A Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.

After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

In total, there were 21, 19, 23 and 19 evaluated litters in the control, 200, 400 and 1000 mg/kg groups, respectively.

One pregnant female died in the course of the study in the control group which was found dead on gestational day 20 due to total intrauterine death.

No other clinical signs than alopecia in a few females unrelated to the treatment and salivation in the 400 and 1000 mg/kg bw/day dams immediately after treatment were observed. This was attributed to be an effect of the treatment, however as non-adverse.

There were no findings observed at necropsy.

There was no indication of an effect of the test item on body weight development and food consumption of the dams in the 200 and 400 mg/kg bw/day dose groups. The statistically significantly (p<0.01) reduced body weight gain on the first three days of treatment and the statistically significantly (p<0.01) reduced food consumption in the first week of treatment in the 1000 mg/kg bw/day dose were considered as test item related but not adverse.

There was no dose related significant difference in the intrauterine mortality of the conceptuses, the number of implantations, viable fetuses and their sex distribution.

The number of late embryonic death increased slightly but statistically significant (p<0.05) in the 400 mg/kg bw/day dose group (and without a statistical significance in the 1000 mg/kg bw/day group) and was around the historical control level. There was no statistical significance indicated in the mean percentage value of the late embryonic death in the experimental groups.

The statistically significant (p<0.01) reduction in the body weight of the male and female fetuses in the 1000 mg/kg bw/day group, which was in the range of historical control data, might be a consequence of the statistically significant reduction of the food consumption between gestation day 5 and 11 and lower mean body weight gain of the dams between gestation day 5 and 8. Placental weight was similar in all experimental groups. There was a statistically significant increase indicated in the relative placental weight in the 1000 mg/kg bw/day dose group, however it was below the historical control level.

The distribution of external and visceral variations was homogenous in the test item treated groups, however the incidence of visceral abnormalities (variations and malformation) increased statistically significant. The occurrence of single type of variations was in the historical control range. Umbilical hernia as a malformation occurred in one fetus in the 1000 mg/kg bw/day dose group which was neither proven nor closed out to be in relationship with an effect of the test item. According to the experience of the formar laboratory of this facility and the scientific literature umbilical hernia is a rather common finding in the rat strain used.

There was no test item related effect indicated at skeletal examination of the fetuses in the 200 and 400 mg/kg bw/day dose group. The incidence of the fetuses with skeletal variations increased significantly (p<0.01) in the 1000 mg/kg bw/day dose group due to the higher incidence of the delayed ossification of skull and metacarpal/metatarsal. These findings might be attributed to the effects on body weight gain and food consumption of the dams observed in the 1000 mg/kg bw/day dose group.

Based on these observations, and the assumptions in the international literature that retarded skull bone ossification is a relatively common observation and may not be a reliable indicator for developmental toxicity (Mylchreest et al. (2005)), the slight delay in ossification is considered to be non-adverse.

Skeletal malformations were found only in the control and 1000 mg/kg bw/day dose group with an incidence of 2 and 1 respectively, thus the test item did not induced skeletal malformations.

 

Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAEL maternal toxicity: 1000 mg/kg bw/day

NOAEL developmental toxicity: 1000 mg/kg bw/day

 

Based on these observations the No Observed Effect Level (NOEL) was determined as follows:

NOEL maternal toxicity: 400 mg/kg bw/day

NOEL developmental toxicity: 400 mg/kg bw/day

 

Reference 2

Endpoint:

developmental toxicity

Remarks:

screening study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 421

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

A total of 90 Sprague Dawley [Crl:CD(SD)BR] rats (45 males and 45 virgin females), 7 to 8 weeks old and weighing 220 to 235 g for
males and 189 to 195 g for females, were received from Charles River Italia S.p.A., Calco (Lecco), Italy. After arrival the weight range for
each sex was determined and the animals were temporarily identified within the cage by means of a coloured mark on the tail. A health
check was then performed by a veterinarian. An acclimatisation period of 20 days was allowed before the start of treatment, during which
time the health status of the animals were assessed by thorough observations.
he animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at
22 °C+/-2°C
and 55 % +/- 15 % respectively; actual conditions were monitored, recorded and the records retained. There were approximately 15 to 20
air changes per hour and the rooms were lit by artificial light for 12 hours each day. From arrival to pairing, animals were housed up to 5
of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5x38x20 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese).
Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During mating animals were housed one male to one female in clear polysulfone cages measuring 42.5x26.6x18.5 cm with a stainless
steel mesh lid and floor (Techniplast – Gazzada S.a.r.l.). Each cage tray held absorbent material which was inspected and changed daily.
After mating the males were re-caged as they were before mating. The females were transferred to individual polysulfone solid bottomed
cages measuring 42.5x26.6x18.5 cm (Techniplast Gazzada S.a.r.l.) for the gestation period, birth and lactation. Nesting material was
provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary. Nesting
material was changed at least 2 times a week.
Drinking water was supplied ad libitum to each cage via water bottles. A commercially available laboratory rodent diet (4 RF 21, Mucedola
S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) was offered ad libitum throughout the study.
The animals arrived on 08 January 2015 and were allocated to groups on 21 January 2015. Dosing commenced on 28 January 2015 and
the last necropsy was performed on 20 MArch 2015.

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before treatment, analyses were performed to confirm that the proposed formulation procedure was acceptable. Samples of the
formulations prepared during the study were analysed to check the concentration and homogeneity (the first and the last week of
treatment). Chemical analyses were carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC
Study number 88800) in the range from 1 to 200 mg/mL in corn oil. In addition, the stability of formulated samples at 1 and 200 mg/mL
was verified after 24 hours and 8 days at +4°C in the same study. The software used for this activity was Empower®2 Build No. 2154.

Details on mating procedure:

Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive
identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray). The female was paired
with the same male until positive identification occurred.

Duration of treatment / exposure:

Males for 2 consecutive weeks prior to pairing and thereafter through the day before necropsy (Days 29 and 30 of study). Females for 2
consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 5 post partum.

Frequency of treatment:

Daily

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Parental animals: Observations and examinations
Mortality
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holiday a
similar procedure was followed except that the final check was carried out at approximately mid-day. This allowed post mortem
examinations to be carried out during the working period of that day. A complete necropsy was performed as detailed in section
[sub:Necropsy] below.
Clinical signs
All clinical signs were recorded for individual animals. Once before commencement of treatment and at least once daily during the study,
each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the
interval was selected taking into consideration the presence of post-dose reactions (15-30 minutes and 1 - 1.5 hours after dosing).
Observations of the cage tray
Observations of the cage tray, during the pre-mating (males and females) and after mating periods (only males), were performed and
recorded three times weekly. During mating and gestation periods (only females until Day 11 post coitum), these observations were
performed and recorded daily.
Body weight
Males were weighed on the day of allocation to treatment groups, on the day of treatment commencement, weekly thereafter and at
termination.
Females were weighed on the day of allocation to treatment groups, on the day of treatment commencement, weekly thereafter until
positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 6 post partum.
Food consumption
The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period following allocation.
Individual food consumption for the females were measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on
Day 6 post partum starting from Day 1 post partum.
Parturition check and duration of gestation
A parturition check was performed from Day 20 to Day 25 post coitum. Female nos. X0080059 (Group 3) and X0080071 (Group 4) which
did not give birth after 25 days of post coitum period were sacrificed shortly after (Days 26 and 27 post coitum, respectively).
Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of
birth (i.e. first detected presence of offspring in the cage). The day that offspring were first detected in the cage was considered Day 0
post partum.
Estrous cyclicity (Parental animals)
Vaginal smears were taken daily in the morning starting from two weeks before pairing throughout the mating period until a positive
identification of copulation was made. The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
Sperm parameters (Parental animals)
Not done
Litter observations
Pups identification, weight and observation
As soon as possible, after parturition was considered complete (Days 0 or 1 post partum), all pups (live and dead) were counted, sexed
and live pups were identified. Live pups were individually weighed on Days 1 and 6 post partum. Pups killed or dying during the lactation
period were weighed before the despatch to necropsy. Observation was performed once daily for all litters.
Postmortem examinations (Parental animals)
Parental animal were euthanised with carbon dioxide. The males were killed after the mating of all females after 29 and 30 days of
treatment. The females with live pups were killed on Day 6 post partum. The females with total litter loss were killed on the day of the
occurrence of total litter loss or shortly after. The females showing no evidence of copulation were killed after 25 days of the last day of
the mating session (Days 26 and 27 post coitum).
Gross observation
The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was
conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed (excluding
animals sacrificed for humane reasons) and the required tissue samples preserved in fixative and processed for histopathological
examination.
All females were examined also for the following:
• external and internal abnormalities;
• number of visible implantation sites (for pregnant animals);
• number of corpora lutea (if detectable).
Uteri of apparently non-pregnant females or uteri of females with no visible implantations were immersed in a 20% solution of ammonium
sulphide to reveal evidence of implantation.
Organ weights
From all parental animals completing the scheduled test period the organs indicated in Annex 1 of the study ptotocol were dissected free
of fat and weighed. The ratios of organ weight to body weight were calculated for each animal. U
Tissues fixed and preserved
Samples of all the tissues indicated in annex 1 of the study protoocol were fixed and preserved in 10% neutral buffered formalin (except
testes and epididymides which were fixed in Modified Davidson's fluid and preserved in 70% ethyl alcohol).
Histopathological examination
The tissues required for histopathological examination are listed in Annex 1 of Study Protocol . After dehydration and embedding in
paraffin wax, sections of the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin. In addition, the testes
and epididymides of all males in the control and high dose groups were cut at 2-3 micrometre thickness and stained with Periodic Acid
Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
The examination was restricted as detailed below:
Tissues specified in Annex 1 of Study Protocol from all animals in the control and high dose group killed at term
Tissues specified in Annex 1 of Study Protocol from the animal killed during the treatment period
All abnormalities in all groups
On the basis of the treatment-related changes detected in the thymus of high dose treated females, the histopathological evaluation of the
thymus was extended to the remaining low and mid-dose females.
Postmortem examinations (Offspring)
Pups were euthanised by intraperitoneal injection of sodium thiopenthal.
All pups found dead in the cage or sacrificed for humane reasons were examined for external and internal abnormalities. All live pups
sacrificed at termination (Day 6 post partum) were killed and examined for external abnormalities and sex confirmation by gonadal
inspection.

Statistics:

Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst
groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s
test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found
to be inhomogeneous a Modified t test (Cochran and Cox) was applied.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test. The nonparametric
Kruskal- Wallis analysis of variance (non-continuous variables) was used for the other parameters. Intergroup differences
between the control and treated groups were assessed by the non-parametric version of the Williams test. The criterion for statistical
significance was p < 0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the
computer without rounding off. Further tests were used as considered appropriate.

Indices:

Males
Copulatory Index (%) = no. of animals mated / no. of animals paired * 100
Fertility Index (%) = no. of males which induced pregnancy / no. of animals paired * 100
Females
Copulatory Index (%) = no. of animals mated / no. of animals paired * 100
Fertility Index (%) = no. of pregnant females / no. of females paired * 100
Pre-birth loss was calculated as a percentage from the formula: no. of visible implantations - total litter size at birth / no. of visible
implantations * 100
Pre-implantation loss was calculated as a percentage from the formula: no. of corpora lutea - no. of visible implantations / no. of corpora
lutea * 100
Males and females
Pre coital Interval = Mean number of days between pairing and mating
Offspring viability indices
Pup loss at birth was calculated as a percentage from the formula: Total litter size - live litter size / Total litter size * 100
Cumulative pup loss on Day 6 post partum was calculated as a percentage from the formula: Total litter size at birth - live litter size at
Day 6 / Total litter size at birth * 100
Sex ratios were calculated at birth and on Day 6 post partum and were presented as the percentage of males per litter.

Abnormalities:

not specified

Developmental effects observed:

not specified

See endpoint study record 7.8.1

Conclusions:

On the basis of the results obtained, the NOAEL (No Observed Adverse Effect Level) for parental toxicity and fertility could be considered
to be 1000 mg/kg/day for males and 250 mg/kg/day for females. The NOAEL for the toxicity on development could be considered to be
250 mg/kg/day.

Executive summary:

The possible effects of LUPEROX® 575 on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus, parturition and early lactation of the offspring (Day 6 post partum) was evaluated in an OECD 421 study (Sisti, 2015). The test item was given to Sprague Dawley rats by oral administration (gavage) before and during mating and throughout the gestation period until Day 6 post partum at dosages of 50, 250 and 1000 mg/kg/day. One female with litter, receiving 50 mg/kg/day was sacrificed for humane reasons on Day 1 post partum. Histopathological evaluation revealed a severe atrophy of thymus, lymphoid depletion of spleen, mucosal ulceration of forestomach (non glandular region), villous atrophy of jejunum and ileum and cortical vacuolation and nephropathy of kidneys. These changes were considered as factors contributory to the illness status of the animal. The major clinical signs noted in treated males receiving 1000 mg/kg/day were matted fur and salivation, while only salivation was recorded in females receiving the same dose level. Soft faeces were observed in the cage tray of animals of both sexes receiving 1000 mg/kg/day and sometimes in males receiving 250 mg/kg/day. Body weight of males receiving 1000 mg/kg/day was slightly lower ( than the control group, as well as, body weight gain on Days 8 (before pairing) and 15 (mating phase) of the study. Food consumption was decreased in females receiving 1000 mg/kg/day before pairing (Day 8 of study) and on Day 6 post partum. Concerning the reproductive parameters, no relevant differences were found in terms of mating performance including the pre-coital interval (number of days paired to sperm positive day) and the copulatory evidence (the positive identification of mating i.e. the presence of sperm and/or copulation plug in situ or in the cage). The resulting copulatory index and fertility index did not show intergroup differences. Post-implantation loss was slightly increased in females receiving 1000 mg/kg/day when compared to the control group. Stillbirths or total litter loss were noted in 5 females receiving 1000 mg/kg/day the day of parturition or the day after parturition. Increased incidences of pup loss at birth and cumulative loss on Day 6 post partum were also noted. Decreases in the number of males and consequently in the total number of pups were noted on Day 6 post partum in females receiving 1000 mg/kg/day when compared to controls. Sex ratios on Day 6 post partum was also decreased when calculated as the percentage of males. Marked mortality of pups or missing pups were noted at 1000 mg/kg/day. Cold to touch, apparently no food intake (milk) and small appearance were noted in the surviving pups of dams receiving 1000 mg/kg/day, in control pups and in those receiving the dose levels = 50 mg/kg/day. Necropsy findings observed in decedent control and treated pups, were similar. No necropsy findings were observed in all pup of control and treated groups, sacrificed on Day 6 post partum. Slight decrease in terminal body weight was observed in high dose animals of both sexes receiving 1000 mg/kg/day. Some changes in absolute and relative organ weights (adrenals, liver, kidneys, uterus, testes and thymus) were noted in treated animals mainly in those receiving 1000 mg/kg/day. However, the differences were not accompanied by histological findings, with the exception of those observed in thymus of high dose females. At macroscopic observations the most remarkable change was an increased incidence of reduced size of the thymus in high dose females. At microscopic observations treatment-related atrophic changes were noted in the thymus of female treated at 1000 mg/kg/day. On the basis of the results obtained, the NOAEL (No Observed Adverse Effect Level) for parental toxic ity and fertility could be considered to be 1000 mg/kg/day for males and 250 mg/kg/day for females. The NOAEL for the toxicity on development could be considered to be 250 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Quality of whole database:

Key study. Apparently well conducted GLP study on the read-across substance CAS# 3006-82-4.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Oral treatment of pregnant Hsd. Brl. Han: WISTAR rats from gestation day 5 up to day 19 with the read-across substance, CAS# 3006-82-4, Tert.-Butylperoxy- 2-ethylhexanoat at the dose levels of 200, 400 and 1000 mg/kg bw/day did not cause death and necropsy findings. The test item did not reveal any adverse effect on the pregnancy and the intrauterine mortality of the conceptuses, the number of viable fetuses and their sex distribution. Further the test substance did not increase significantly the incidence of external and visceral variations, and caused no skeletal malformations. The slight delay in ossification in fetuses of the 1000 mg/kg bw/day dose group is considered to be non-adverse.

Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAEL maternal toxicity: 1000 mg/kg bw/day

NOAEL developmental toxicity: 1000 mg/kg bw/day

The possible effects of CAS# 686 -31 -7on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus, parturition and early lactation of the offspring (Day 6 post partum) was evaluated in an OECD 421 study (Sisti, 2015). The test item was given to Sprague Dawley rats by oral administration (gavage) before and during mating and throughout the gestation period until Day 6 post partum at dosages of 50, 250 and 1000 mg/kg/day. One female with litter, receiving 50 mg/kg/day was sacrificed for humane reasons on Day 1 post partum. Histopathological evaluation revealed a severe atrophy of thymus, lymphoid depletion of spleen, mucosal ulceration of forestomach (non glandular region), villous atrophy of jejunum and ileum and cortical vacuolation and nephropathy of kidneys. These changes were considered as factors contributory to the illness status of the animal. The major clinical signs noted in treated males receiving 1000 mg/kg/day were matted fur and salivation, while only salivation was recorded in females receiving the same dose level. Soft faeces were observed in the cage tray of animals of both sexes receiving 1000 mg/kg/day and sometimes in males receiving 250 mg/kg/day. Body weight of males receiving 1000 mg/kg/day was slightly lower (<10%) than the control group, as well as, body weight gain on Days 8 (before pairing) and 15 (mating phase) of the study. Food consumption was decreased in females receiving 1000 mg/kg/day before pairing (Day 8 of study) and on Day 6 post partum. Concerning the reproductive parameters, no relevant differences were found in terms of mating performance including the pre-coital interval (number of days paired to sperm positive day) and the copulatory evidence (the positive identification of mating i.e. the presence of sperm and/or copulation plug in situ or in the cage). The resulting copulatory index and fertility index did not show intergroup differences. Post-implantation loss was slightly increased in females receiving 1000 mg/kg/day when compared to the control group. Stillbirths or total litter loss were noted in 5 females receiving 1000 mg/kg/day the day of parturition or the day after parturition. Increased incidences of pup loss at birth and cumulative loss on Day 6 post partum were also noted. Decreases in the number of males and consequently in the total number of pups were noted on Day 6 post partum in females receiving 1000 mg/kg/day when compared to controls. Sex ratios on Day 6 post partum was also decreased when calculated as the percentage of males. Marked mortality of pups or missing pups were noted at 1000 mg/kg/day. Cold to touch, apparently no food intake (milk) and small appearance were noted in the surviving pups of dams receiving 1000 mg/kg/day, in control pups and in those receiving the dose levels ≥ 50 mg/kg/day. Necropsy findings observed in decedent control and treated pups, were similar. No necropsy findings were observed in all pup of control and treated groups, sacrificed on Day 6 post partum. Slight decrease in terminal body weight was observed in high dose animals of both sexes receiving 1000 mg/kg/day. Some changes in absolute and relative organ weights (adrenals, liver, kidneys, uterus, testes and thymus) were noted in treated animals mainly in those receiving 1000 mg/kg/day. However, the differences were not accompanied by histological findings, with the exception of those observed in thymus of high dose females. At macroscopic observations the most remarkable change was an increased incidence of reduced size of the thymus in high dose females. At microscopic observations treatment-related atrophic changes were noted in the thymus of female treated at 1000 mg/kg/day. On the basis of the results obtained, the NOAEL (No Observed Adverse Effect Level) for parental toxic ity and fertility could be considered to be 1000 mg/kg/day for males and 250 mg/kg/day for females. The NOAEL for the toxicity on development could be considered to be 250 mg/kg/day.

A Reproduction/Developmental Toxicity Screening Test with the read-across test item, CAS# 3006-82-4, tert.-Butylperoxy- 2-ethylhexanoat was performed according to OECD 421.

The CAS# 3006 -82 -4 was administered once daily orally (by gavage) to male and female rats throughout the pre-pairing, pairing and lactation periods until necropsy (day 4 of lactation). The dose levels were applied 0, 100, 300 and 1000 mg/kg body weight/day to male rats for 41 days in total and female rats throughout the pre-pairing, the pairing, the gestation and the lactation periods until day 3 post partum (last dosing). Treatment at 1000 mg/kg was associated with decreased food consumption in male and female animals during the first week of the pre-pairing period. Some dams were noted with ruffled fur after parturition and generally bad conditions. No test item-related effects were noted during necropsy and for macroscopic findings. Treatment at 1000 mg/kg was associated with an increase of pre-implantation-, post-implantation-, and post-natal loss, and a reduction of live pups limited to general systemic toxicity in parental animals. The mean body weight of pups also was reduced at this systemic maternal toxic dose. Based on these data, it can be concluded that the NOEL was at 300 mg/kg body weight/day for parental generation as well as for the offspring.

Justification for selection of Effect on developmental toxicity: via oral route:
Apparently well conducted GLP study on the read-across substance CAS# 3006-82-4.

Justification for classification or non-classification

According to EU Regulation (EC) N0. 1272/2008 (CLP), no classification for reproductive toxicity is warranted. Effects noted in the OECD 421 studies, occurred in the presence of maternal toxicity and effects on the pups were not duplicated in the OECD 414 study conducted on the read-across substance CAS# 3006 -82 -4. In addition, no effects on reproductive organs were reported in the 90-day study conducted on the read-across substance as well as no adverse effects reported in the OECD 414 study conducted on the read-across substance CAS# 3006-82-4.