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Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-01-14 to 2010-03-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
tert-butyl 3,5,5-trimethylperoxyhexanoate
EC Number:
236-050-7
EC Name:
tert-butyl 3,5,5-trimethylperoxyhexanoate
Cas Number:
13122-18-4
Molecular formula:
C13H26O3
IUPAC Name:
tert-butyl 3,5,5-tris(methylperoxy)hexanoate

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (Europe) Laboratories Inc.; Toxi-Coop Zrt. 1103 Budapest; Cserkesz u. 90; Hungary
- Age at study initiation: 10 -11 weeks old (male); 11-12 weeks old (female)
- Weight at study initiation: (P) Males: 277 - 316 g; Females: 182 - 204 g
- Housing: before mating: 2 animals of the same sex/cage; mating: 1 male and 1 female/cage; pregnant females were housed individually; the bedding material was suitable for nesting; Cage Type II polypropylene/polycarbonate
- Diet: ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice (ad libitum)
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3°C
- Humidity: 30 -70 %
- Air changes: 8- 12 air changes/hour by central air-condition system
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Oleum helianthy /Sunflower oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle, sunflower oil, in concentrations of 10, 32 and 100/80 mg/mL. For the treatment of high dose animals, 100 mg/mL was used from days 0-3 of the pre-mating period and 80 mg/mL was used from day 4 of the pre-mating period onwards.

VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water; therefore sunflower oil (oleum helianthy) was used for preparing formulations appropriate for oral administration. Oleum helianthy /sunflower oil was a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 10, 32 and 100/80 mg/mL in sunflower oil
- Lot/batch no.: 2009.11.06
Details on mating procedure:
- M/F ratio per cage: 1:1
Mating was started soon after animals attained full sexual maturity. 2 weeks after the initiation of treatment, one female and one male of the same dose group (1:1 mating) were placed in a single cage. Females remained with the same male until copulation occurred. Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 421). Sperm positive females were caged individually. One female animal was re-mated with a proven male as it failed to mate with male cohabited with at starting. For one animal, mating began on day 19 for completing the 14 days treatment before mating.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
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. Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of gross pathology. Dosing of both sexes began after 6 days acclimatisation and two weeks before mating and was continued up to and including the day before the necropsy.

A constant treatment volume of 5 mL dose preparation/kg body weight was administered in all groups.
Frequency of treatment:
once daily
Details on study schedule:
- Male animals were dosed for 47 days (14 days pre-mating and 16 days mating plus 17 days post mating until the necessary number of pregnant female animals was evident), then they were sacrificed.
- Female animals were dosed for 14 days pre-mating, for up to 16 days mating period, through gestation and up to day 3 post-partum with necropsy on the following day. The day of birth (viz. when parturition was complete) was defined as day 0 post-partum.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
160 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
in the course of the study the dose was reduced to 400 mg/kg bw/d
No. of animals per sex per dose:
12 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
The doses have been chosen by the Sponsor on the basis of a previous 7 day dose range-finding study where TBPIN was given to groups of 5 rats per sex at dose levels of 100, 316 or 1000 mg/kg bw/day. The high dose was chosen with the aim of inducing toxic effects but no death or severe suffering; however 500 mg/kg bw/day proved to be more toxic as it was expected therefore was reduced to 500/400 mg/kg bw/day on day 4 of the pre-mating period. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.

Positive control:
no positive control

Examinations

Parental animals: Observations and examinations:
- Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day)
- Animals showing signs of moribund condition were isolated and sacrificed to prevent suffering, cannibalism or autolysis. These animals were processed in the same way as the animals of the terminal necropsy.

The observations and examinations performed in parental males are detailed in following:
- Clinical observations
- Body weight
- Body weight gain
- Food consumption
- Number of pairings
- Number of fertile pairings
- Number of infertile males
- Male mating index
- Male fertility index
- Necropsy findings
- Organ weights (absolute and relative to the body and brain weights)
- Histopathology findings


The observations and examinations performed in parental females are detailed in following:
- Clinical observations
- Body weight
- Body weight gain
- Food consumption
- Oestrous cycle
- Number of pairings
- Number of pregnant females
- Number of sperm positive, but non-pregnant females
- Number of non mated females
- Female mating index
- Female fertility index
- Gestation index
- Duration of pregnancy (days)
- Number of Corpora lutea / dams
- Number of implantations / dams
- Number of dams with live pups on postpartal days 0 and 4
- Pre-implantation mortality
- Intrauterine mortality
- Total mortality (intra and extra uterine mortality)
- Necropsy findings
- Organ weights (absolute and relative to the body and brain weights)
-Histopathology findings
Oestrous cyclicity (parental animals):
The oestrous stage was determined.
Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymis weight, spermatogenesis, histopathology
Litter observations:
Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. Observations were reported
individually for each animal.
The duration of gestation was recorded and was calculated from day 0 of pregnancy. Dams were observed whether they made a nest from the bedding material and cover their newborns or not. The efficiency of the suckling was observed by the presence of milk in the pups' stomach. All observations were recorded.
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are
significantly smaller than normal pups), and the presence of gross abnormalities.
Live pups were counted, sexed, weighed individually with an accuracy of 0.01 g within 24 hours of parturition (on the first day after parturition was
complete), and day 4 post-partum with an accuracy of 0.1g.
In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.
All the litters were checked and recorded daily for the number of viable and dead pups. The dead pups found were subjected to necropsy with
macroscopic examination.
Postmortem examinations (parental animals):
Gross necropsy was performed on each animal irrespective of the date of its death. Terminally (one day after the last treatment), animals were sacrificed under Isofluran anaesthesia by exsanguination. After examination of the external appearance the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed, and any abnormality was recorded with details of the location, colour, shape and size. Special attention was paid to the organs of the reproductive system.
The number of implantation sites and of corpora lutea was recorded.
The testes, epididymides (total and cauda), seminal vesicles and prostate; female reproductive organs including ovary, uterus, vagina; as well as brain and pituitary of all adult animals were weighed. (Paired organs were weighed individually).
The weighed organs, kidneys of male animals and all organs showing macroscopic lesions of all adult animals were preserved. Testes and epididymides were preserved in Bouin’s solution, all other organs in 8 % buffered formalin solution.
Postmortem examinations (offspring):
Pups were euthanized at postnatal day 4 or shortly thereafter (except some pups euthanized earlier due to cannibalism and moribund condition of pup and moribund condition of dam, and were carefully examined externally for gross abnormalities.
One female pup found dead on day 0 and showing deformed head was processed for skeletal examination after double staining (bone and cartilage).
Statistics:
The statistical evaluation of appropriate data (marked †above) were performed with the statistical 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 Multiple Range test was used to access the significance of intergroup differences. Getting significant result at Bartlett’s test the Kruskal- Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Reproductive indices:
The following reproductive indices were calculated: Male mating index, female mating index, male fertility index, female fertility index, gestation index.
The formulas for calculation can be found below in "Any other information on materials and methods incl. tables"
Offspring viability indices:
The following pup mortality and sex ratio indices were calculated: Survival index, pre - implantation mortality, intrauterine mortality, total mortality, sex ratio. The formulas for calculation can be found below in "Any other information on materials and methods incl. tables"

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Dermal irritation (if dermal study):
not examined
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

MORTALITY (PARENTAL ANIMALS):
Six female animals were found dead or in moribund condition shortly before or after the delivery at 160 mg/kg (2/12) and 500/400 mg/kg (4/12) bw/day dose. There were no preceding clinical signs (salivation only) for two animals. Decreased activity, decreased body tone, piloerection, dyspnoea, paleness, incoordination, lack of reflexes, prone position and colourless discharge in the vagina were noted for other animals from gestational day 20 and 21 to necropsy. One male (500/400 mg/kg bw/day) and two female animals (160 mg/kg bw/day and 500/400 mg/kg bw/day, respectively) animals died shortly after the daily administration on treatment days 27, 3 and gestational day 0, respectively.

CLINICAL SIGNS (PARENTAL ANIMALS):
Tert. butyl 3,5,5-trimethylperoxyhexanoate caused death of pregnant animals at 160 (2/13) and 500/400 mg/kg bw/day (4/12) at the end of pregnancy and at early lactation. Test item related salivation was also observed at 160 and 500/400 mg/kg bw/day in male and female animals in a slight, moderate or marked degree. Male animals were slightly more sensitive than females based on a higher incidence in males at 160 mg/kg bw/day. Alopecia and wounds were noted for some female animals in the control and lower dose groups as species specific findings. These are frequently observed in Hsd.Brl.Han: Wistar rats.

BODY WEIGHT (PARENTAL ANIMALS):
There were no test item related effects at 50, 160 and 500/400 mg/kg bw/day on the body weight development of male groups during the entire treatment period and female groups during the pre-mating and gestation period. In female animals, a test item influence on the body weight gain was noted at 160 and 500/400 mg/kg bw/day during the lactation period. In accordance with clinical observations, the body weight gain of these dams was significantly less than the control value.

FOOD CONSUMPTION (PARENTAL ANIMALS):
There were no test item related effects at 50, 160 and 500/400 mg/kg bw/day on food consumption of male groups during the entire treatment period and female groups during the pre-mating and gestation periods. However, in accordance with clinical observations and the body weight gain, a test item influence on the food consumption was demonstrated at 160 and 500/400 mg/kg bw/day during the lactation period, as the food consumption of these dams was significantly less than the control value.
The lower food consumption in male and female high dose group during the first week was due to the 500 mg/kg bw/day dose and the difference from the control ceased during the second week, after the dose reduction to 400 mg/kg bw/day. The higher food consumption of male groups in the course of post-mating period was not considered to be related to the test item effect. The statistical significances were due to the unusually lower food consumption of the control group.

REPRODUCTIVE FUNCTION: OESTROUS CYCLE (PARENTAL ANIMALS):
No test item effect on the oestrous cycle was found.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
No test item effect was found on the reproductive ability of male and female animals at any dose level tested.

ORGAN WEIGHTS (PARENTAL ANIMALS)
The results of the determination of absolute and relative organ weights did not demonstrate any test item related organ weight alterations.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histological examination of male and female genital organs (ovaries, uterus, cervix vagina, testes, epididymides, prostate, seminal vesicles and pituitary) did not reveal any toxic or other test item related lesions at 50, 160 or 500/400 mg/kg/bw/day doses.
Test item related renal lesions (hyaline droplets, cytoplasmic vacuolization, proteinaceous tubular casts accompanied with multifocal
tubular cell regeneration) were observed in male animals at 160 and 500/400 mg/kg bw/day.

NECROPSY:
Occurrence of pale kidneys was considered to be an indicative of a possible test item related alterations in male animals at 160 and 500/400 mg/kg bw/day. It occurred also in dead female rats with lower incidence. Swollen lungs with foamy liquid content referred to a para-gastric treatment in the three mentioned animals. In dead animals, light red lungs probably were in connection with circulatory disturbance (shock). Sanguineous uterine content, bleeding from vagina refers to disturbances at the delivery, i.e. animals died before the delivery process was fully completed. Smaller than normal seminal vesicles were present in single animals of the control and high dose groups. Pyelectasia and alopecia are species-specific alterations, which commonly occur in untreated animals of this species and strain. Yellow formation liver surface, enlarged adrenal glands and liver, pale liver, and hydrometra were considered as individual alterations.
These are seen occasionally in experimental rats and were not related to the treatment in this study.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING): There was no clear evidence of a direct test item effect on pups’ mortality. Taking into account the lack of dose dependency, the higher mortality was considered to be related to the maternal toxicity at 160 and 500/400 mg/kg bw/day causing indirectly mortality of pups.

CLINICAL SIGNS (OFFSPRING): The number and percent of cold, not suckled and cannibalized pups were higher at 160 and 500/400 mg/kg bw/day when compared to the control value. These findings are the indicative of the maternal nourishing activity therefore were considered to be related to the maternal toxicity at 160 and 500/400 mg/kg bw/day.

BODY WEIGHT (OFFSPRING): The mean body weight and litter weight were less at 160 and 500/400 mg/kg bw/day on postnatal day 0 when compared to the control value. The litter weight remained below the control value on postnatal day 4 at 160 mg/kg bw/day.

GROSS PATHOLOGY (OFFSPRING): The test item related macroscopic alterations were not found in offspring subjected to gross pathological examination. Hydrops fetalis deformed head, dilated and gas filled stomach, right turned Arcus aortae, and urine filled dilated urinary bladder, as well as hydroureter (both sides) occurred in single animals of lower dose groups, these were considered as individual alterations.
No milk in the stomach (not suckled) is frequently observed in newborns. In the lack of any dose relation it has no toxicological importance.
With lung flotation test, air content of the lungs is examined to determine whether newborn died intrauterine (stillborn, airless lungs, opened Ductus Botalli) or extra uterine (liveborn, air in the lungs, closed Ductus Botalli).

OTHER FINDINGS (OFFSPRING):
- Sex distribution: There were no differences between control and test item treated groups in the ratio of genders or in the litter means.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for tert-Butylperoxy-3,5,5-trimethylhexanoat for parental effects was 50 mg/kg bw /day. For reproduction parameters, the NOAELs were 400 mg/kg bw/day for male rats and 50 mg/kg bw/day for female rats. For F1 offspring, the NOAEL was 50 mg/kg bw/day.
Executive summary:

A Reproduction/Developmental toxicity screening test with the test item tert-Butylperoxy-3,5,5-trimethylhexanoat was performed according to OECD 421. The purpose of this study was to obtain initial information on the possible effects of the test item on reproduction and development when administered orally (by gavage) to rats at repeated doses of 50, 160 and 500/400 mg/kg bw/day compared to control animals. As a screening test, it was intended to provide initial information on possible effects on male and female reproductive performance such as gonad function, mating behaviour, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 4 post-partum associated with administration of repeated doses. In this study, 12 animals/sex/dose were involved with exception of the 500/400 mg/kg bw group (13 males) and the 160 mg/kg bw/day group (13 females). All animals of the P generation were treated prior to mating (14 days) and throughout mating (16 days). Male animals were treated 17 days post mating. For females, treatment was continued through the gestation period and up to lactation day 3 or shortly thereafter, i.e. up to the day before the necropsy. Observations included mortality, clinical symptoms, body weight, food consumption, oestrous cycle, mating, and pregnancy and delivery process.

Tert-Butylperoxy-3,5,5-trimethylhexanoat caused death of pregnant Hsd.Brl.HAn: Wistar rats at 160 and 400 mg/kg bw/day at the end of pregnancy and at early lactation due to acute-subacute tubular damage in the kidneys and related pulmonary alterations.

Reproductive performance of males was unaffected by treatment. Reproductive performance of female animals was affected by the treatment at 160 and 400 mg/kg bw/day through the parturition and nourishing and limited to the systemic toxicity at those doses. In the F1 generation, the higher mortality, clinical signs and depressed body weight development was considered to be the consequence of maternal toxicity at 160 and 400 mg/kg bw/day. Effects on reproduction and/or developmental of pups were thus only at doses pronounced in concentration with maternal toxicity and are in conclusion not regarded as relevant or indication for a specific hazard.

The no observed adverse effect level (NOAEL) for tert-Butylperoxy-3,5,5-trimethylhexanoat for parental effects was 50 mg/kg bw /day. For reproduction parameters, the NOAELs were 400 mg/kg bw/day for male rats and 50 mg/kg bw/day for female rats. For F1 offspring, the NOAEL was 50 mg/kg bw/day.