Bisisopropyl peroxydicarbonate

Toxicity to reproduction

Administrative data

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:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

corn oil

Duration of treatment / exposure:

In the males:
- 2 weeks before mating,
- during the pairing period,
- until sacrifice (at least 4 weeks in total),

In the females:
- 2 weeks before mating,
- during the mating period,
- during gestation,
- during lactation until Day 13 post-partum inclusive,
- until sacrifice for females with no evidence of mating.

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Control animals:

yes, concurrent no treatment

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Executive summary:

The potential toxic effects of diisopropyl peroxydicarbonate was evaluated following daily oral administration (gavage) to male and female rats from before mating, during mating and, for the females, throughout gestation until Day 13 post partum (p.p.).

This study provides information:

.            on the possible health hazards likely to arise from repeated exposure over a relative limited period of time,

.            on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

Three groups of ten male and ten female Sprague-Dawley rats received the test item,Diisopropyl peroxydicarbonate, daily by the oral route (gavage) at dose levels of 25, 75 or 150 mg/kg/day, under a constant dosage volume of 5 mL/kg/day. Males were treated for 2 weeks before mating, during the mating period and until the day before sacrifice (4 weeks in total). Females were treated 2 weeks before mating, throughout mating and gestation and until Day 13 p.p.inclusive. Another group of ten males and ten females received the vehicle only (corn oil) under the same experimental conditions and acted as a control group. The actual test item concentrations in the dose formulations were determined in Weeks 1 and 6 using a validated HPLC/UV analytical method. Animals were checked daily for clinical signs and mortality. Detailed clinical observations were performed once a week.Body weights and food consumption were recorded weekly until mating (no food consumption during the mating period) and then at designated intervals throughout gestation and lactation. The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until Day 13 p.p. The total litter sizes and numbers of pups of each sex were recorded after birth. The size of each litter was adjusted on Day 4 p.p.by culling extra pups to obtain as nearly as possible four males and four females per litter. The pups were observed daily for clinical signs or abnormal behaviour and were weighed on Days 1, 4, 8 and 13 p.p.The physical development of pups was assessed by measuring the anogenital distance on Day 1 p.p.and by counting the number of nipples and areolae in male pups on Day 12 p.p. Hematology and blood biochemistry investigations as well as urinalysis were performed for five males and females at sacrifice. Thyroid hormones (TSH and T4) were determinedin males at sacrifice and in pups sacrificed on Day 13 p.p. Five males and females were submitted to a functional observation battery (FOB) at the end of the treatment period (Day 13p.p.). Males were sacrificed after 4 weeks of treatment, and dams on Day 14 p.p. A full macroscopic post-mortem examination was performed in adults, with a particular attention to the reproductive organs. Designated organs (adrenals, brain, epididymides, heart, kidneys, liver, pituitary gland, prostate, seminal vesicles, spleen, testes and thymus) were weighed and more tissue specimens were preserved. A microscopic examination was performed on selected tissues from five males and females in the control- and high-dose groups. Pups not selected on Day 4 p.p.were sacrificed and discarded without further examination.Pups selected were sacrificed on Day 13 p.p.and submitted to a detailed external examination with a particular attention to the external genital organs.

The test item concentrations were within an acceptable range of variations (± 10% of the nominal concentrations required). No test item was observed in the control dose formulations.

F0 animals

There were no test item-related unscheduled deaths. Piloerection, round back, emaciated appearance and/or mainly loud/abdominal breathing were transiently observed in 3/20 animals at 150 mg/kg/day. Ptyalism was noted at all dose levels with dose-related incidences. These clinical signs were considered to be test item-related and non-adverse. There were no effects on mean body weight, mean body weight change and mean food consumption. There were no test item-related changes at reactivity to manipulation, to different stimuli and at motor activity evaluation (FOB) at any dose level. At laboratory investigations (hematology, blood biochemistry, urines, thyroid hormones), no differences from controls were attributed to the test item treatment.

At pathology, there were no organ weights, macroscopic or microscopic changes. There were no test item-related effects on estrous cycles during the first 2 weeks of treatment and at termination, and on mean fertility, mating and delivery data.

Pups

There were notest item-related effects in terms ofpup mortality/viability, clinical signs, terminal external examination, development of nipples and areolae in male pups on Day 13 p.p.,percentage of male pups at birthormean T4 and TSH levels on Day 13 p.p.There were no toxicologically significant effects on mean pup body weight.An effect of the test item treatmenton the lower mean anogenital distance at 25 and 150 mg/kg/day compared with controls was considered to be doubtful and non-adverse.

Based on the experimental conditions and results of this study:

.            the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 150 mg/kg/day based on the absence of adverse findings at this high-dose level,

.            the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 150 mg/kg/day based on the absence of test item-related effects on mating and fertility at this dose level,

.            the NOAEL for toxic effects on progeny was considered to be 150 mg/kg/day based on the absence of adverse findings on pups at this high-dose level.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bisisopropyl peroxydicarbonate

EC Number:

203-317-4

EC Name:

Bisisopropyl peroxydicarbonate

Cas Number:

105-64-6

Molecular formula:

C8H14O6

IUPAC Name:

2-[({[(propan-2-yloxy)carbonyl]peroxy}carbonyl)oxy]propane

Constituent 2

Reference substance name:

Ethyl acetate

EC Number:

205-500-4

EC Name:

Ethyl acetate

Cas Number:

141-78-6

Molecular formula:

C4H8O2

IUPAC Name:

ethyl acetate

Test material form:

liquid

Details on test material:

Name: Diisopropyl peroxydicarbonate
Commercial name: LUPEROX IPP50EA Experimental
CAS No.: 105-64-6
Batch No.: CRRA (lot LUG1428)
Description: colorless liquid
Storage condition: in the freezer set at -20°C
Date of analysis: 6 October 2017
Expiry date: 05 April 2018
Peroxide content: 50.2% of peroxide in ethyl acetate

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: the males were approximately 10 weeks old and the females were approximately 11 weeks old
- Mean body weight at study initiation: the males had a mean body weight of 404 g (range: 372 g to 436 g) and the females had a mean body weight of 277 g (range: 251 g to 307 g)
- Housing: F0 animals were individually housed, except during mating and lactation, in polycarbonate cages (Tecniplast 2154, 940 cm²) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing of F0 animals was chosen in order not to jeopardize gestations and lactations and to avoid aggressive behavior between males around mating. Toward the end of gestation and during lactation, autoclaved wood shavings (SICSA, Alfortville, France) were provided to females and their litter as nesting material.
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: the males were acclimated to the study conditions for 7 days before treatment anf the females were acclimated to the study conditions for 7 days before the beginning of estrous cycle monitoring during the pre-treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Concentration in vehicle: 5, 15 and 30 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation (mating period): until mating occurred or 14 days have elapsed
- Proof of pregnancy: vaginal plug or sperm in the morning vaginal lavage referred to as day 0 post-coitum
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: Ultra High Performance Liquid Chromatography with UV detection (UHPLC/UV).
The test item concentrations in the administered dose formulations analyzed in weeks 01 and 04 remained within an acceptable range of variation (-6.4% to -2.6%) when compared to the nominal values (± 10% required).
No test item was observed in the control dose formulation

Duration of treatment / exposure:

In the males:
- 2 weeks before mating,
- during the pairing period,
- until sacrifice (at least 4 weeks in total),

In the females:
- 2 weeks before mating,
- during the mating period,
- during gestation,
- during lactation until Day 13 post-partum inclusive,
- until sacrifice for females with no evidence of mating.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected by the Sponsor, on the basis of the results of 2 week dose-range finding study (Citoxlab France/Study No. 45445 TSR) where Sprague-Dawley male and female rats were treated at 250, 500 and 1000 mg/kg/day:
¿ the dose of 1000 mg/kg/day was reduced to 750 mg/kg/day from Day 5 following poor health condition and premature euthanasia. A total of 1/5 male and 2/5 females were prematurely euthanized in the first week of treatment in this group (breathing difficulties, hunched posture, piloerection, hypoactivity, coldness to the touch and/or increase in size of abdomen). The surviving animals had similar clinical signs, mainly females. Surviving females had a high mean liver weight at euthanasia,
¿ at 500 mg/kg/day, 1/5 female was prematurely euthanized in the second week of treatment for poor health condition too. 2 surviving males had breathing findings,
¿ at 250 mg/kg/day, 1/5 female was prematurely euthanized in the second week of treatment for poor health condition too. Another female given 250 mg/kg/day had hunched posture, piloerection and abdominal breathing but survived,
¿ ptyalism was noted at all doses, and necropsy findings in the GI were seen in prematurely dead and surviving animals of all doses with dose-relationship.

Therefore, 150 mg/kg/day was selected as the high-dose level. The low-dose and mid dose were selected using a ratio representing a 2 or 3-fold interval (i.e. 25 and 75 mg/kg/day).

- Rationale for animal assignment: stratification procedure.

Positive control:

no (not required)

Examinations

Parental animals: Observations and examinations:

MORTALITY/MORBIDITY:
- Time schedule: Each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Detailed clinical examinations were performed on all animals at least once before the beginning of the treatment period and then once a week until the end of the study.

CLINICAL SIGNS:
- Time schedule: From arrival, each animal was observed at least once a day as part of routine examinations. From the start of the treatment period, each animal was observed at least once a day, at approximately the same time of day.

BODY WEIGHT:
- Time schedule: The body weight of each male was recorded on the first day of treatment (Day 1), then once a week until euthanasia including 1 or 2 days before euthanasia.
The body weight of each female was recorded on the first day of treatment (Day 1), then once a week until mated (or until euthanasia for the female with no evidence of mating) and on Days 0, 7, 14 and 20 post coitum (p.c.) and Days 1, 4, 8 and 13 p.p.

FOOD CONSUMPTION:
- Time schedule: The quantity of food consumed by each male was measured once a week from the first day of treatment until the start of the mating period.
The quantity of food consumed by each female was measured once a week from the first day of treatment until the start of the mating period, during gestation for the intervals Days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval Days 1-4, 4-8 and 8-13 p.p.
During the mating period, food consumption was not measured for males or females.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule: The first five males and lactating females from each group euthanized as scheduled were evaluated with a functional observation battery once at the end of the treatment period. For females, this was performed on Day 13 p.p. after euthanasia of the pups.
The following measurements, reflexes and responses were recorded:
¿ touch response,
¿ forelimb grip strength,
¿ pupillary reflex,
¿ visual stimulus response,
¿ auditory startle reflex,
¿ tail pinch response,
¿ righting reflex,
¿ landing foot splay,
¿ at the end of observation: rectal temperature.

HAEMATOLOGY:
The parameters were determined from the first five males and from five to seven females euthanized on Day 15 p.p. from each group on the day of euthanasia, after food fasting.

CLINICAL CHEMISTRY:
The parameters were determined from the first five males and the first five females euthanized on Day 15 p.p. from each group on the day of euthanasia, after food fasting.

URINALYSIS:
The parameters were determined from the first five males and the first five females euthanized on Day 15 p.p. from each group on the day of euthanasia, after food fasting.

THYROID HORMONES:
Blood samples were taken, in the first half of the morning (generally before 10 a.m.),
¿ at termination on Day 14 p.p. from all F0 females (approximately 0.5 mL of blood was collected from the orbital sinus under isoflurane anesthesia),
¿ at termination from all F0 males (approximately 0.5 mL of blood was collected from the orbital sinus under isoflurane anesthesia).
The levels of the thyroid hormone (T4) and thyroid stimulating hormone (TSH) were determined respectively by LC-MS/MS or Luminex MAP® technology for F0 males sampled at termination.

REPRODUCTION (apart from indices):
- Pre-coital time and duration of gestation were recorded.

Oestrous cyclicity (parental animals):

The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning between 8 and 10 a.m.:
- during the 2 weeks of the pre-treatment period (including the two supplementary females per group),
- from the beginning of the treatment period during the pre-mating and mating periods, until the females were mated,
- on the day of sacrifice before euthanasia, to allow correlation with reproductive organs histopathology (i.e. Day 14 p.p.).
- on Day 15 p.p. before euthanasia.

Sperm parameters (parental animals):

Parameters examined in males of parental generation:
- weighing and microscopic examination: see Tissue Procedure Table below
- special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

STANDARDISATION OF LITTERS:
- Performed on Day 4 post-partum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed.

PARAMETERS EXAMINED:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies, weight gain, clinical signs
- anogenital distance (AGD),
- presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS:
- external abnormalities

HORMONES
- blood samples were taken, in the first half of the morning (generally before 10 a.m.),
¿ at termination on Day 4 p.p. from at least two pups/litter culled [chosen by manual randomization; when possible 0.25 mL per pup (to obtain approximately 0.5 mL of blood in total) was collected by decapitation under isoflurane anesthesia and then pooled per litter]. When they were insufficient pups in a litter to have two culled pups, only one pup culled was used for blood collection when available, otherwise there were no blood collection on Day 4 p.p.,
¿ at termination on Day 13 p.p. from at least two pups/litter [chosen by manual randomization; when possible 0.25 mL per pup (to obtain approximately 0.5 mL of blood in total) was collected from the vena cava immediately after sacrifice and then pooled per litter],
The levels of the thyroid hormone (T4) and thyroid stimulating hormone (TSH) were determined respectively by LC-MS/MS or Luminex MAP® technology for pups sampled on Day 13 p.p.

Postmortem examinations (parental animals):

SACRIFICE
On completion of the treatment period, after at least 14 hours fasting, all surviving F0 animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and euthanized by exsanguination:
- males: after the end of the mating period (at least 4 weeks of treatment in total),
- females: on Day 14 p.p.

The following females were euthanized by the same way without overnight fasting:
¿ female M20707 (group 1) which did not deliver: on Day 26 p.c. (after a body weight recording to check for a possible un-noticed delivery),
¿ female M20708 (group 1) with no evidence of mating: 24 days after the end of the mating period since no delivery occurred (after a body weight recording).


ORGAN WEIGHTS:
The body weight of each F0 animal euthanized as scheduled (after the end of the mating period for males or on Day 14 p.p. for females) was recorded before euthanasia. The organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection.

GROSS PATHOLOGY:
A complete macroscopic post-mortem examination was performed on all F0 animals including the female prematurely euthanized.

HISTOPATHOLOGY:
A microscopic examination was performed on:
¿ all tissues listed in the Tissue Procedure Table from five euthanized as scheduled males and lactating females of the control and high-dose groups (groups 1 and 4) (see § Study plan adherence),
¿ all macroscopic lesions of all groups,
¿ all tissues listed in the tissue Procedure Table from the control female that were euthanized prematurely (see § Study plan adherence).
Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Postmortem examinations (offspring):

SACRIFICE: on Day 13 post-partum

GROSS NECROPSY:
- Found dead and prematurely euthanized pups were submitted to a detailed external examination (including orifices and buccal cavity), after euthanasia when applicable. Particular attention was paid to the external genital organs and to whether the pup has been fed (e.g. presence of milk in the stomach) when possible. Then, they were discarded without any further examination..
Pups not selected on Day 4 p.p. were discarded without further examination.
- Pups euthanized on Day 13 p.p. were submitted to a detailed external examination (including orifices and buccal cavity) after euthanasia. Particular attention was paid to the external genital organs. Then, they were discarded without any further examination, or after sampling of thyroids with parathyroids for the selected pups.


HISTOPATHOLOGY: No

ORGAN WEIGTHS: No

Statistics:

Body weight, food consumption and reproductive data
Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).

Hematology, blood biochemistry, urinalysis, hormones, anogenital distance and post-implantation loss data
Citox software was used to perform the statistical analyses.

Organ weight
PathData software was used to perform the statistical analysis of organ weight data.

Reproductive indices:

Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
Post-implantation loss = 100 * (Number of implantation sites - Number of live pups) / Number of implantations
Mating index = 100 * (Number of mated animals / Number of paired animals)
Fertility index = 100 * (Number of pregnant female partners / Number of mated pairs)
Gestation index = 100 * (Number of females with live born pups / Number of pregnant females)

Offspring viability indices:

Live birth index = 100 * (Number of live born pups / Number of delivered pups)
Viability index on day 4 p.p. = 100 * (Number of surviving pups on day 4 p.p. / Number of live born pups)
Lactation index on day 14 p.p. = 100 * (Number of surviving pups on day 14 p.p. / Number of surviving pups on day 4 p.p.)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See Table 1.
Ptyalism was noted at all doses in males and in females. The number of affected animals was dose-related and the duration of affection ranged from 1 day to the whole dosing period. This finding was considered as treatment-related but non-adverse.

At 150 mg/kg/day, loud or abdominal breathing was transiently observed in a limited number of animals of both sexes (male M20233, female M20745 during gestation and lactation, and female M20741 during gestation) for a duration varying from 3 to 12 consecutive days. The most affected female (M20745) also displayed piloerection, round back and emaciated appearance for several days at beginning/mid gestation and then in the second half of the lactation period. These clinical signs were considered to be test item treatment-related and non-adverse (few animals affected, transient clinical signs, no body weight loss associated for at least 2 of these animals, no adverse effects on their food consumption, no impact on maternal behaviour or implantation loss).

Piloerection and round back were also noticed in another female (M20747) at the beginning of lactation (Days 1 to 3 p.p.), concomitantly with pallor of eyes and extremities. Most of its litter was found dead or cannibalized on Day 1 p.p. and the remaining litter had the lowest weight of the study on Day 1 p.p. This was noted just after the delivery, following a long gestation period of 23 days. There was no similarity/no such pattern with another female of the group or the study. Therefore this clinical condition was considered to be likely linked to the delivery and unlikely to the test item treatment.

At 75 mg/kg/day, one female (M20730) presented a growing-up mass on urogenital area from the beginning of the gestation period to the end of the study (correlating with a mammary adenocarcinoma at microscopy). As only one animal was affected in the mid-dose group, this was considered to be not related to the test item treatment.

The other clinical signs observed in test item-treated groups (chromodacryorrhea, chromorhynorrhea, scabs, cutaneous lesions, area of hair loss, reflux at dosing, reddish vaginal discharge during gestation) were considered to be incidental (noted in isolated animals, with no dose-relationship, also observed in control group and/or commonly observed in this species and strain).

Mortality:

no mortality observed

Description (incidence):

In females, one control female (M20712) died during anesthesia before blood sampling on Day 14 p.p.
At 150 mg/kg/day, one female (M20746) was prematurely euthanized on Day 1 p.p. for ethical reasons (vaginal prolapse). This was considered to be related to the parturition and incidental (noted in one isolated animal, no similar findings in other females of the group).
There were no unscheduled deaths in the others groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

See Table 2.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no effects on mean food consumption.
Food consumption was sometimes high for a few females compared with the other weeks or with the other animals from the same group. This was considered to be incidental and related to food spillage which is rather common in rats.

Haematological findings:

no effects observed

Description (incidence and severity):

No differences from control means were attributed to the test item treatment in absence of dose relationship and of statistical significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

In males, at 150 mg/kg/day, a slight increase in circulating potassium level was noted (+7%). Despite a statistical significance (p<0.05), this was considered to be not biologically relevant. No similar effect was observed in females and there were no correlating effects in the other biochemistry parameters. Thus a relationship with test item treatment was considered unlikely.
Any other variations from controls means were considered to be incidental (no dose-relationship, no statistical significance at the high-dose and/or not biologically significant).

Urinalysis findings:

no effects observed

Description (incidence and severity):

At 75 and/or 150 mg/kg/day, there were one to two males with more turbid urines than in controls and one female at 150 mg/kg/day with more urinary ammonium phosphate crystals than in controls. As there were no correlating effects in urinalysis, and in view of the limited number of animals affected and of the slight differences in severity compared with controls, an effect of the test item treatment was considered to be unlikely.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no changes at reactivity to manipulation, to different stimuli and at motor activity which were considered to be test item-related, in view of the comparable scores obtained by the rats given the vehicle or the test item whatever the dose, the absence of dose-relationship, the inter-animal variability and/or the absence of correlating clinical signs.

In particular, at 75 and 150 mg/kg/day, grooming was noted with dose-related incidences (1/5 males and 3/5 males respectively, vs. none in controls), and mean body temperature tended to be lower in females (38.4 and 38.2°C respectively, vs. 38.8°C in controls). Besides, at motor activity, test item-treated male animals had a tendency to a dose-related higher rearing activity, and test item-treated females had a tendency to a lower motor activity (horizontal movements and/or rearing) with no dose-relationship, when compared to the control animals. In absence of correlating findings in other FOB parameters, and in view of the inter-animal variability, the opposite trend between both sexes and/or the absence of observation of similar clinical signs during the study, these results were not considered to be test item-related.
Low values of motor activity obtained in some females, including controls, were considered to be due to the fact that they were submitted to two sessions of motor activity in the same day, following a technical problem during the first session.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

All observations belonged to the spectrum of spontaneous changes in rats of this age and strain and bore no relationship to test item treatment. This includes one case of mammary adenocarcinoma in female M20730 treated at 75 mg/kg/day. There were no test item treatment-related changes in the reproductive organs.
Implantation sites in the uterus and lactation in the mammary gland were not recorded, since they represent expected changes in all females in this type of study.
5/5 control females and 3/5 females treated at 150 mg/kg/day had mucification of the vagina. The remaining two females at the high dose were in diestrus. Vaginal mucification is still mostly observed as expected for lactating females (pups starting to eat solid food from Day 14 p.p.). During this transitory period, diestrus stages are also expected to be observed in a few dams that have started cycling.

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormones
F0 males at termination
Mean thyroid hormone levels (± standard deviation) are presented below:

Dose level (mg/kg/day) 0 25 75 150
T4 (ng/mL) 40.3 ±8.0 36.6 ±6.3 38.7 ±6.0 35.5 ±7.3
(-9) (-4) (-12)
TSH (pg/mL) 2308 ±1242 1805 ±808 1795 ±993 1890 ±939
(-22) (-22) (-18)
In bracket: difference from controls.

There were no differences from control means considered to be test item-related (no dose-relationship and/or slight variations from controls).

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

See Table 3.
There were no test item-related effects on mean estrous cycle data.
At 75 and 150 mg/kg/day, there were one and two females, respectively, with 3 consecutive days of proestrous + estrous (in one female at 150 mg/kg/day one estrous was likely misnamed as proestrous). One female (M20743) at 150 mg/kg/day had this event twice. This was noted in a limited number of females which had no problems to mate, and two consecutive days of estrous can occur spontaneously. Therefore, these findings were considered to be incidental.
On Day 14 p.p.
Diestrous stage was noted in all females observed on Day 14 p.p. [with the exception of two females (M20719 and M20720) at 25 mg/kg/day for which metestrous was noted] correlating with the histopathology of the vagina of the control and high-dose females (see § Microscopic examination).

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating and fertility data
See Table 4.
There were no effects on mean pairing, mating and fertility data.
At 150 mg/kg/day, one pair mated after 12 nights of pairing because the female (M20744) was blocked in diestrous. In view of the isolated incidence and the comparable mean pre-coital time between groups, and as this is of common background in rats, this event was considered to be incidental.

Delivery data
See Table 5.
The mean post-implantation loss tended to be higher in test item-treated groups, especially at 75 mg/kg/day, than in controls. However the number of pups delivered was similar to controls at 25 and 150 mg/kg/day. The variation of mean post-implantation loss was not dose-dependent, and comparable mean post-implantation losses were obtained in control groups of recent studies.
When compared with controls, the mean live birth index tended to be lower at 25 and 150 mg/kg/day. At 150 mg/kg/day, this was mainly due to one litter (M20747) whose dam was in poor clinical condition after delivery and lost 70% of its litter by Day 1 p.p. (not considered to be due to the test item treatment but to condition of the dam); the mean live birth index would be comparable to that of the controls (98.0%) without this litter M20747.
In absence of dose-relationship in mean group data and in the number of females affected, and as at the end the mean number of live pups was not affected, an effect of the test item treatment on both parameters was considered to be unlikely.

Details on results (P0)

Table 1: Body weight

Sex Male Female
Dose-level (mg/kg/day) 0 100 300 1000 0 100 300 1000
Pre-mating or whole study
Day 1 461 457 460 459 276 270 268 282
Day 8 511 509 500 497 280 278 277 293
Day 15 541 548 537 530 291 286 287 299
Day 29 576 584 575 559 / / / /
Days 1-8 +50 +52 +40 +38* +4 +7 +9 +11
Days 1-15 +83 +92 +77 +70 +14 +15 +19 +17
Days 15-29 +35 +35 +38 +29 / / / /
Days 1-29 +118 +127 +115 +99* / / / /
Gestation
Day 0 p.c. / / / / 300 297 289 312
Day 20 p.c. / / / / 465 464 454 473
Days 0-20 p.c. / / / / +165 +167 +165 +161
Lactation
Day 1 p.p. / / / / 373 362 355 369
Day 4 p.p. / / / / 370 367 363 383
Day 13 p.p. / / / / 397 396 384 410
Days 1-4 p.p. / / / / -4 +5 +8 +14**
Days 1-13 p.p. / / / / +24 +34 +30 +40*
/: not applicable, statistical significance *: p<0.05, **: p<0.01.

Table 2: Food consumption

Sex Male Female
Dose-level (mg/kg/day) 0 100 300 1000 0 100 300 1000
Pre-mating or whole study
Days 1-8 32 32 31 33 18 18 18 19
Days 8-15 32 32 34 36* 20 19 20 21
0 +6 +13 -5 0 +5
Gestation
Days 0-7 p.c. / / / / 23 22 23 25
Days 7-14 p.c. / / / / 25 25 26 28
Days 14-20 p.c. / / / / 29 28 29 34**
-3 0 +17
Lactation
Days 1-4 p.p. / / / / 34 38 38 44#
+12 +12 +29
Days 4-8 p.p. / / / / 49 52 52 59**
+6 +6 +20
Days 8-13 p.p. / / / / 63 68 67 69
/: not applicable, statistical significance: *: p<0.05, **: p<0.01, #: p<0.001.
in italic: differences from controls (%).

Table 3: Mating and fertility data

Dose-level (mg/kg/day) 0 100 300 1000
Number of animals paired (M + F) 9 + 10 10 + 10 10 + 10 10 + 10
Number of males mated 9 10 10 9
Number of females mated 10 10 10 9
Mean number of days taken to mate 4.3 3.9 2.8 5.1
Number of pregnant females 10 10 10 9
Fertility index 100% 100% 100% 100%
Number of females with live born pup(s) 10 10 10 9
Gestation index 100% 100% 100% 100%
M: males; F: females.

Table 4: Delivery data

Dose-level (mg/kg/day) 0 100 300 1000
Number of females which delivered 10 10 10 9
Mean duration of gestation (days) 22.0 22.0 22.0 22.0
Mean number of corpora lutea 15.4 15.5 15.5 15.7
Mean number of implantations 14.4 14.8 14.6 15.2
Mean pre-implantation loss (%) 7.9 5.9 5.2 3.0
Mean number of pups delivered 12.3 13.6 12.7 13.1
Mean post-implantation loss (%) 16.7 7.1 12.8 12.2

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Dose descriptor:

NOEL

Remarks:

reproductive performance

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

See Table 8.
There were no test item treatment-related effects on clinical conditions and at external examination of the pups.

Mortality / viability:

no mortality observed

Description (incidence and severity):

See Table 7.
There were no test item treatment-related effects on pup viability.
All pups found dead or cannibalized were found on Day 1 p.p., except one found dead pup at 25 mg/kg/day on Day 3 p.p. A few dead pups had no milk in the stomach at external examination.
As for the mean live birth index, when compared with controls, the mean viability index on Day 4 p.p. tended to be lower at 25 and 150 mg/kg/day. At 150 mg/kg/day, this was mainly due to one litter (M20747) whose dam was in poor clinical condition from Days 1 to 3 p.p. (mean viability index at 99.2% when excluding this litter). In absence of dose-relationship in mean group data and in the number of females affected and as at the end the mean number of live pups was not affected, an effect of the test item treatment was considered to be unlikely.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

See Table 9.
There were no toxicologically significant effects on mean pup body weight; mean body weights of test item groups were at less than ±10% of control mean body weights, there were no statistical significance and mean body weight evolution in test item-treated groups was very close to that of controls.

Other effects:

no effects observed

Description (incidence and severity):

Anogenital distance
See Table 10.
There was no effect of the test item on female AGD but a statistically significant lower AGD in male pups at 25 and 150 mg/kg/day. However, the mean AGD of the control group was higher than in previous similar studies (5.13 to 5.21) and the mean ratios of the treated groups were comparable with control group means of previous studies (2.52 to 2.60). Therefore, the effect was considered not to be test item-related.

Nipples and areolae numbers
At 75 mg/kg/day, pup 1 from M20734 displayed two areolae. No areolae were observed in the other male pups at any dose level. Therefore it was considered that test item treatment had no effect on areola number in male pups.

Thyroid hormones
Mean thyroid hormone levels (± standard deviation) are presented below:

Dose level (mg/kg/day) 0 25 75 150
T4 (ng/mL) 34.5 ±2.3 40.3* ±5.7 37.0 ±6.5 37.0 ±3.6
(+17) (+7) (+7)
TSH (pg/mL) 1732 ±429 1705 ±570 1661 ±461 1842 ±574
(-2) (-4) (+6)
In bracket: difference from controls.
There were no differences from control means considered to be test item-related (no dose-relationship and/or slight variations from controls) .

Sex ratio
There were no test item-related effects on the percentage of male pups at birth which was comparable among groups and close to 50%.

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1.   Clinical signs

The main clinical signs observed in surviving animals during the study are presented in the following table:

 

Sex	Males					Females
Dose level (mg/kg/day)	0	25	75		150	0	25	75	150
Pre-mating (female) or whole study (males)
Ptyalism	1	7	10		10	-	1	4	10
Loud breathing	-	-	-		1	-	-	-	-
Gestation
Ptyalism	/	/	/	/		-	4	5	10
Piloerection, round back, emaciated appearance	/	/	/	/		-	-	-	1
Loud breathing	/	/	/	/		-	-	-	2
Reddish vaginal discharge	/	/	/	/		-	-	1	1
Mass on urogenital area	/	/	/	/		-	-	1	-
Lactation
Ptyalism	/	/	/	/		-	-	1	9
Piloerection, round back	/	/	/	/		-	-	-	2
Emaciated appearance	/	/	/	/		-	-	-	1
Loud or abdominal breathing	/	/	/	/		-	-	-	1
Mass on urogenital area	/	/	/	/		-	-	1	-
Pallor of eye or extremities	/	/	/	/		-	-	-	1

/: not applicable; -: no animal affected.

Table 2. Mean body weights (g) and mean body weight changes (g)

 

Sex	Males				Females
Dose level (mg/kg/day)	0	25	75	150	0	25	75	150
Pre-mating (female) or whole study (males)
Day 1	404	405	404	403	283	272	274	281
Day 15	493	493	490	495	287	282	281	284
Days 1 - 15	+88	+87	+86	+92	+5	+10	+7	+2[RJ1]
Day 36	556	557	546	564	/	/	/	/
Days 1 - 36	+152	+151	+142	+161	/	/	/	/
Gestation
Day 0p.c.	/	/	/	/	292	282	282	287
Day 20p.c.	/	/	/	/	447	453	428	441
Days 0 - 20p.c.	/	/	/	/	+155	+170	+145	+154
Lactation
Day 1p.p.	/	/	/	/	347	342	340	337
Day 13p.p.	/	/	/	/	384	374	369	364
Days 1 - 13p.p.	/	/	/	/	+36	+32	+29	+28

/: not applicable; Inbold: mean body weight change.

Table 3. Estrous cycle

First 2 weeks of the treatment period

Mean data are summarized below:

 

Dose level (mg/kg/day)	0	25	75	150
Number of cycles	3.0	3.1	2.9	3.0
Cycle length (days)	4.0	3.9	4.2	4.1
Number of rats cycling normally^a	10	7	8	8
Number of days of diestrus	4.2	3.8	4.7	4.0
Number of days of proestrus	3.2	3.3	3.3	3.6
Number of days of estrus	3.8	3.9	3.8	3.9
Number of days of metestrus	3.8	4.0	3.1	3.5

a : having a mean cycle of 4-5 days

 

Table 4.    Pairing, mating and fertility data

The summary of mating and fertility datais presented in the following table:

 

Dose level (mg/kg/day)	0	25	75	150
Number of animals paired (M + F)	10 + 10	10 + 10	10 + 10	10 + 10
Number of males mated	10^a	10	10	10
Number of females mated	10^a	10	10	10
Mating index	100%	100%	100%	100%
Mean number of days taken to mate	2.4	1.9	2.4	2.9
Number of pregnant females	9	10	10	10
Fertility index	90%	100%	100%	100%
Number of females with live born pup(s)	9	10	10	10
Gestation index	100%	100%	100%	100%

M: males; F: females.

a:  included male M20207 and female M20708 where the female was pregnant while no evidence of mating was observed.

Table 5.  Delivery and litter data

The summary of delivery and litter datais presented in the following table:

 

Dose level (mg/kg/day)	0	25	75	150
Number of females which delivered	8	10	10	10
Mean duration of gestation (days)	21.9	21.9	21.8	22.0
Mean number ofcorpora lutea	15.6	16.3	16.4	16.2
Mean number of implantations	13.6	16.0	14.9	15.7
Mean pre-implantation loss (%)	11.0	2.0	8.5	2.8
Mean number of pups delivered	13.0	13.9	11.7	13.8
Mean number of live pups on Day 1p.p.	13.0	13.1	11.7	12.6
Mean post-implantation loss (%)	4.7	12.6	20.8	12.4
Live birth index on Day 1p.p.	100	93.8	100	91.2^a

^a:  including data from female M20746, which gave birth to 15 alive and 2 dead pups but was prematurely euthanized on Day 1p.p.due to a vaginal prolapse, therefore its surviving pups were also euthanized.

 

Table 6.  Organ Weights

The following differences between test item-treated and control groups were observed:

 

Selected differences in organ weights (% from controls)

 

Sex	Males			Females
Dose level (mg/kg/day)	25	75	150	25	75	150
Number of animals	10	10	10	10	10	10
Adrenal glands
Absolute (%)	+6	-2	-6	+6	+4	+9
Relative to body weight (%)	+7	+4	-7	+15	+12	+19
Pituitary gland
Absolute (%)	+36	+22	+26	-25	-41##	-31
Relative to body weight (%)	+37	+29	+23	-20	-37	-25
Thymus
Absolute (%)	-14	-15	-12	-4	-1	-6
Relative to body weight (%)	-13	-10	-14	+5	+7	+3

##: p = 0.01 (based on actual values and not on the percentages presented in the table).

Table 7.   Mortality/Viability/Lactation index

The viability and lactation indexes are presented in the table below:

 

Dose level (mg/kg/day)	0	25	75	150^a
Mean number of live pups on Day 4p.p.(pre-culling)	12.9	13.0	11.6	12.3
Viability index on Day 4p.p.(%)	99.1	93.2	99.2	91.6
Mean number of live pups on Day 13p.p.	8.0	8.0	7.8	7.6
Lactation index on Day 13p.p.(%)	100	100	100	100

^a: excluding data from female M20746 (litter euthanized before Day 4p.p.due to dam euthanasia).

 

The distribution of prematurely dead pups is presented in the table below:

 

Dose level (mg/kg/day)	0	25	75	150
Number of cannibalized pups (number of litters affected)	1 (1)	3 (1)	1 (1)	3 (1)
Number of found dead pups (number of litters affected)	0	6 (3)	0	9^b (3)
Number of prematurely euthanized pups^a (number of litters affected)	0	0	0	0

^a: at 150 mg/kg/day, excluding the surviving pups from female M20746 euthanized because of the premature euthanasia of the dam (vaginal prolapse); b: including 2 pups from female M20746

Table 8.    Clinical signsand external observation at euthanasia

Clinical signs and gross external observations observed in surviving pups (culled on Day 4p.p.or euthanized on Day 13p.p.) during the lactation period are summarized in the following table:

 

Dose level (mg/kg/day)	0	25	75	150
Hematoma (head)	1 (1)	1 (1)	1 (1)
Emaciated appearance		1 (1)
Necrose (tail)		1 (1)
Scab (head or neck)			3 (2)
Abnormal color (white) + absence of finger (left hindlimb) + scab (left hindlimb)			1 (1)
Remaining umbilical cord			1 (1)

Pup incidence (litter incidence).

Table 9.   Pup body weight and body weight change

Mean body weight (g) and mean body weight changes (g) in pups are presented in the following table:

 

Sex	Males						Females
Dose level (mg/kg/day)	0	25	75	150		0		25		75		150
Body weight
Day 1p.p.	8.4	7.7 (-8%)	8.2 (-2%)	7.7 (-8%)		7.9		7.3 (-8%)		7.8 (-1%)		7.3 (-8%)
Day 4p.p.(preculling)	12.3	11.2 (-9%)	12.2 (-1%)	11.4 (-7%)		11.7		10.7 (-9%)		11.5 (-2%)		10.8 (-8%)
Day 4p.p.(postculling)	12.3	11.2 (-9%)	12.3 (0%)	11.4 (-7%)		11.8		10.7 (-9%)		11.4 (-3%)		10.7 (-9%)
Day 8p.p.	22.2	20.7 (-7%)	22.0 (-1%)	21.5 (-3%)		21.2		19.9 (-6%)		20.9 (-1%)		20.2 (-5%)
Day 13p.p.	36.3	34.6 (-5%)	35.7 (-2%)	34.9 (-4%)		35.1		33.9 (-3%)		34.1 (-3%)		33.0 (-6%)
Body weight change
Days 1- 4p.p.	3.9	3.5	4.1		3.6	3.7		3.4	3.7		3.5
Days 4 - 13p.p.	24.0	23.4	23.5		23.5	23.3		23.2	22.7		22.3

In brackets: difference from control value.

Table 10.  Anogenital distance

Mean anogenital distance (AGD) data (± standard deviation) are described in the following table:

 

Sex	Males				Females
Dose level (mg/kg/day)	0	25	75	150	0	25	75	150
AGD on Day 1p.p.(mm)	5.37 ±0.42	4.96** ±0.52 (-8%)	5.21 ±0.70 (-3%)	5.00** ±0.53 (-7%)	2.78 ±0.47	2.70 ±0.29 (-3%)	2.74 ±0.34 (-1%)	2.85 ±0.25 (+3%)
Ratio AGD/^3vbody weight (mm/g^1/3)	2.66 ±0.18	2.52** ±0.24	2.59 ±0.31	2.52** ±0.26	1.40 ±0.24	1.42 ±0.17	1.40 ±0.16	1.48* ±0.14

Mean based on individual data and not litter mean. In bracket: difference from controls.

Statistical significance: *: p<0.05, **: p<0.01

Applicant's summary and conclusion

Conclusions:

Based on the experimental conditions and results of this study:
¿ the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 150 mg/kg/day based on the absence of adverse findings at this high-dose level,
¿ the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 150 mg/kg/day based on the absence of test item-related effects on mating and fertility at this dose level,
¿ the NOEL for toxic effects on progeny was considered to be 150 mg/kg/day based on the absence of adverse findings on pups at this high-dose level.

Executive summary:

The potential toxic effects of diisopropyl peroxydicarbonate was evaluated following daily oral administration (gavage) to male and female rats from before mating, during mating and, for the females, throughout gestation until Day 13 post partum (p.p.).

This study provides information:

.            on the possible health hazards likely to arise from repeated exposure over a relative limited period of time,

.            on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

Three groups of ten male and ten female Sprague-Dawley rats received the test item,Diisopropyl peroxydicarbonate, daily by the oral route (gavage) at dose levels of 25, 75 or 150 mg/kg/day, under a constant dosage volume of 5 mL/kg/day. Males were treated for 2 weeks before mating, during the mating period and until the day before sacrifice (4 weeks in total). Females were treated 2 weeks before mating, throughout mating and gestation and until Day 13 p.p.inclusive. Another group of ten males and ten females received the vehicle only (corn oil) under the same experimental conditions and acted as a control group. The actual test item concentrations in the dose formulations were determined in Weeks 1 and 6 using a validated HPLC/UV analytical method. Animals were checked daily for clinical signs and mortality. Detailed clinical observations were performed once a week.Body weights and food consumption were recorded weekly until mating (no food consumption during the mating period) and then at designated intervals throughout gestation and lactation. The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until Day 13 p.p. The total litter sizes and numbers of pups of each sex were recorded after birth. The size of each litter was adjusted on Day 4 p.p.by culling extra pups to obtain as nearly as possible four males and four females per litter. The pups were observed daily for clinical signs or abnormal behaviour and were weighed on Days 1, 4, 8 and 13 p.p.The physical development of pups was assessed by measuring the anogenital distance on Day 1 p.p.and by counting the number of nipples and areolae in male pups on Day 12 p.p. Hematology and blood biochemistry investigations as well as urinalysis were performed for five males and females at sacrifice. Thyroid hormones (TSH and T4) were determinedin males at sacrifice and in pups sacrificed on Day 13 p.p. Five males and females were submitted to a functional observation battery (FOB) at the end of the treatment period (Day 13p.p.). Males were sacrificed after 4 weeks of treatment, and dams on Day 14 p.p. A full macroscopic post-mortem examination was performed in adults, with a particular attention to the reproductive organs. Designated organs (adrenals, brain, epididymides, heart, kidneys, liver, pituitary gland, prostate, seminal vesicles, spleen, testes and thymus) were weighed and more tissue specimens were preserved. A microscopic examination was performed on selected tissues from five males and females in the control- and high-dose groups. Pups not selected on Day 4 p.p.were sacrificed and discarded without further examination.Pups selected were sacrificed on Day 13 p.p.and submitted to a detailed external examination with a particular attention to the external genital organs.

The test item concentrations were within an acceptable range of variations (± 10% of the nominal concentrations required). No test item was observed in the control dose formulations.

F0 animals

There were no test item-related unscheduled deaths. Piloerection, round back, emaciated appearance and/or mainly loud/abdominal breathing were transiently observed in 3/20 animals at 150 mg/kg/day. Ptyalism was noted at all dose levels with dose-related incidences. These clinical signs were considered to be test item-related and non-adverse. There were no effects on mean body weight, mean body weight change and mean food consumption. There were no test item-related changes at reactivity to manipulation, to different stimuli and at motor activity evaluation (FOB) at any dose level. At laboratory investigations (hematology, blood biochemistry, urines, thyroid hormones), no differences from controls were attributed to the test item treatment.

At pathology, there were no organ weights, macroscopic or microscopic changes. There were no test item-related effects on estrous cycles during the first 2 weeks of treatment and at termination, and on mean fertility, mating and delivery data.

Pups

There were notest item-related effects in terms of pup mortality/viability, clinical signs, terminal external examination, development of nipples and areolae in male pups on Day 13 p.p., percentage of male pups at birth, male and female anogenital distances on Day 1p.p.or mean T4 and TSH levels on Day 13p.p.There were no toxicologically significant effects on mean pup body weight.

.            the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 150 mg/kg/day based on the absence of adverse findings at this high-dose level,

.            the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 150 mg/kg/day based on the absence of test item-related effects on mating and fertility at this dose level,

.            the NOEL for toxic effects on progeny was considered to be 150 mg/kg/day based on the absence of adverse findings on pups at this high-dose level.