Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 421 STUDY conducted in full conformance with GLP.

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test Item
Identification: tert-Butyl peroxybenzoate
Test Item Name for Report: tert-Butyl peroxybenzoate
REACH Name: tert-Butyl perbenzoate
CAS #: 614-45-9
Description: Clear liquid
Batch Number: 0903225181
Purity: 99.2%
Expiry Date (Retest Date): 01-Apr-2011
Stability of the Test Item in the Vehicle: 24 hours based upon Harlan Laboratories Study C57323 (non-GLP)
Storage Conditions: Refrigerator (approx 4 °C)
Safety Precautions: Routine hygienic procedures (gloves, goggles, face mask).

Test animals

Species:
rat
Strain:
other: HanRcc:WIST(SPF)
Sex:
male/female
Details on test animals and environmental conditions:
Test System
Animals: Rat, HanRcc: WIST(SPF)
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
Number of Animals: 40 males: 10 per group
40 females: 10 per group
Age (at Start of Treatment): 11 weeks
Body Weight Range
(at Start of Treatment): Males: 296 to 331 g
Females: 177 to 211 g
Identification: Cage card and individual animal number (ear tattoo).
Randomization: Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.


Allocation
The group identification and animal numbers assigned to treatment are stated in the following table:

Allocation and
Dose Levels
mg/kg bw/day Group 1 Group 2 Group 3 Group 4
control 100 300 750 1000
Males 1 - 10 11 - 20 21 - 30 31 - 40
Females 41 - 50 51 - 60 61 - 70 71 - 80


Husbandry
Room Number, Füllinsdorf: 015/B
Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). There was 12 hour fluorescent light / 12-hour dark cycle with music during the light period.
Accommodation: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J.Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland). During the pre-pairing period, cages with males were inter¬spersed amongst those holding females to promote the development of regular estrus cycles.
Diet: Pelleted standard Kliba Nafag 3433 rodent main¬te¬nance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum (batch no. 83/09). Results of representative analyses for con¬taminants are included in Appendix I on p.{ DA }.
Water: Community tap-water from Füllinsdorf was avail¬able ad libitum in water bottles. Results of bacterio¬logical assay, chemical and contaminant analyses of representative samples are included in Appendix II on p.{ DA }.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Method: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.

Frequency of Administration: Once daily

Target Dose Levels:
Group 1: 0 mg/kg bw/day (control group)
Group 2: 100 mg/kg bw/day
Group 3: 300 mg/kg bw/day
Group 4: 750 mg/kg bw/day (for males)
1000 mg/kg bw/day (for females)

Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rat, Harlan Laboratories Study C57323, using dose levels of 100, 300 and 1000 mg/kg/day, resulting in a NOAEL of 300 mg/kg/day for males and in a NOAEL of 1000 mg/kg/day for females.

Dose Volume: 4 mL/kg body weight

Duration of Acclimatization Period: 7 days

Duration of Treatment Period:
Males: Minimum 4 weeks
Females: Approximately 7 weeks
Details on mating procedure:
During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if:

- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.

The day of mating was designated day 0 post coitum.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of Dose Formulations
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (4 hrs). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Dr. K. Morgenthal (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.

The samples were analyzed by HPLC coupled to an UV detector following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard. Samples were considered accurately prepared and sufficiently stable if the following acceptance criteria were met: ±20% of nominal for sample content, ±15% deviation from mean calculate from top, middle and bottom samples for homogeneity. Analyzed samples were not discarded without written consent from the study director.

The application formulations investigated during the study were found to comprise tert-Butyl peroxybenzoate in the range of 92.2% to 98.2% and, thus, the required content limit of ±20% with reference to the nominal concentration was met. The homogeneous distribution of tert-Butyl peroxybenzoate in the preparations was approved because single results found did not deviate more than 0.9% (<15%) from the corresponding mean. In addition, the test item was found to be stable in application formulations when kept four hours at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.
Details on study schedule:
Study Sequence Females Males
Acclimatization 7 days 7 day
First Test Item Administration
Day 1 of pre-pairing Day 1 of pre-pairing
Pre-Pairing 14 days 14 days
Pairing 14 days maximum 14 days maximum

Gestation Approximately 21 days
Treatment Ends On day 3 post partum On day before sacrifice
Necropsy On day 4 post partum After a minimum of 28 days treatment
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
100 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
300 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
750 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
10m & 10F
Control animals:
yes, concurrent vehicle
Details on study design:
3.9


3.10 Termination of the Study
Males were sacrificed after they had been treated for at least 28 days. Dams and pups were sacrificed on day 4 post partum.

When birth did not occur on the expected date (day 21 post coitum), the dams were sacrificed on day 25 post coitum.


3.10.1 Necropsy
All animals sacrificed or found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

At the scheduled sacrifice, all animals were killed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

Dead pups, except those excessively cannibalized, were examined macroscopically.

All parent animals and pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

For the parent animals, special attention was directed at the organs of the reproductive system.
The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites [see References (1)].

3.10.2 Organ Weights
At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately.


3.10.3 Tissue Preservation
The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution.

The testes and epididymides from all parental males were preserved in Bouin’s fixative. The prostate and seminal vesicles from all males were fixed in neutral phosphate buffered 4% formaldehyde solution.


3.10.4 Histotechnique
All organ and tissue samples to be examined by the study pathologist were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin. Special stains were used at the discretion of the study pathologist.


3.10.5 Histopathology
Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the study pathologist. The same applied to all occurring gross lesions and to all animals, which died spontaneously or had to be terminated in extremis.

Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, if necessary.

A histopathology peer review was performed by Dr. Hans-Jörg Chevalier at the test facility (Harlan Laboratories Ltd., Itingen / Switzerland).

The results were summarized by Dr. T. Razinger and included in Appendix V on p.{ DA }.


3.11 Data Compilation and Processing
The following data were recorded on-line: food consumption, body weights, organ weights, and macroscopic examination data (TOX-control), reproduction and litter data, (RCC-TOX LIMS). Microscopic data were entered into the PathData System. All other data were recorded on data sheets and compiled manually.

From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation losses, mean litter size, pup sex ratios and viability indices.

For reproduction data, group mean values were calculated both on a litter basis and on a percentage per group basis. Mean pup weights were calculated from the individual weights both on a per group and on a per litter basis.

Computer-generated values in the tables represent the rounded-off results of calculations which used the exact raw data values.


3.12 Statistical Analysis
The following statistical methods were used to analyze food consumption, body weights and reproduction data:

• Means and standard deviations of various data were calculated.

• The Dunnett-test [see References (2)] (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.

• The Steel-test [see References (3)] (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.

• Fisher's exact-test [see References (4)] was applied if the variables could be dichotomized without loss of information.

Examinations

Parental animals: Observations and examinations:
Observations
The following observations were recorded:

Viability / Mortality: Twice daily
Clinical Signs: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.
Food Consumption: Males: Weekly during pre-pairing and after pair¬ing periods.
Females: Pre-pairing period days 1 - 8 and 8 - 14, gestation period days 0 - 7, 7 - 14 and 14 - 21 post coitum and lactation period days 1 - 4 post partum.
No food consumption was recorded during the pair¬ing period.
Body Weights: Recorded daily from treatment start to day of ne¬cropsy.
Pup Data: The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups were recorded. Pups were weighed individually (without identification) on days 0 (if possi¬ble), 1 and 4 post partum.
Litter observations:
The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups were recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.
Postmortem examinations (parental animals):
3.10.1 Necropsy
All animals sacrificed or found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

At the scheduled sacrifice, all animals were killed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

Dead pups, except those excessively cannibalized, were examined macroscopically.

All parent animals and pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

For the parent animals, special attention was directed at the organs of the reproductive system.
The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites [see References (1)].
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
• Means and standard deviations of various data were calculated.
• The Dunnett-test [see References (2)] (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test [see References (3)] (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test [see References (4)] was applied if the variables could be dichotomized without loss of information.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Males: At 300 and 750 mg/kg, signs of discomfort such as bedding material in the mouth and salivation before and after administration were noted. Females : At 1000 mg/kg, ruffled fur and soft feces were observed occasionally in few animals. Signs of di
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males at 750 mg/kg, mean body weight gain was statistically significantly lower starting on day 6 of the pre-pairing period. No statistically significant changes were noted in mean body weight.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In males at 750 mg/kg, mean body weight gain was statistically significantly lower starting on day 6 of the pre-pairing period. No statistically significant changes were noted in mean body weight.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

4.2 Parent Animals
4.2.1 Mortality
At 1000 mg/kg, female no. 80 was found dead on day 6 of the pre-pairing period. Beforehand, no clinical signs were observed, and body weight gain was slightly lower on day 5 of the pre-pairing period. At necropsy, lungs discolored reddish, and incompletely collapsed and left lobe margin with greenish foci and thymus discoloured red were observed. Histological evaluation of the organs and tissue examined could not establish the exact cause of death.

4.2.2 Clinical Signs or Observations
Males
At 750 mg/kg, salivation and bedding material in the mouth before and/or after administration were observed in all males starting on day 6 or 7 of the pre-pairing period until the end of the study. These clinical signs were signs of discomfort and not considered to be adverse. During the pre-pairing period, decreased activity and ruffled fur were noted in one male on single occasions, ruffled fur was also noted in another male on one single day. These were considered to be incidental since they occurred occasionally in only two males.

At 300 mg/kg, bedding material in the mouth was observed at the end of the pre-pairing period in three males, in eight males during the pairing period, and in six males during the after pairing period. Salivation after administration was noted in in three males during the pre-pairing period, in seven males during the pairing period and in one male during the after pairing period. These were considered as signs of discomfort rather than adverse clinical signs.

At 100 mg/kg, no clinical signs were noted.

Females
At 1000 mg/kg, soft feces were observed occasionally in three females during the pre-pairing period, in one female during the lactation and in one female on day 2 of the gestation period. Same female was noted to have diarrhea during the pairing and beginning of the gestation period. Ruffled fur was observed occasionally in six females during the pre-pairing and in one female during the lactation period and during the whole second week of the pre-pairing period in two females and at the beginning and during the last week of gestation period in one female. These clinical signs were due to the treatment with the test item although they occurred discontinously. Signs of discomfort such as salivation before and/or after administration and bedding material in the mouth were observed in all females starting on day 6 or 7 of the pre-pairing period until day 2 or 3 of the lactation period. These were considered as signs of discomfort.

At 300 mg/kg, bedding material in the mouth was observed in seven females starting at the end of the pre-pairing period, in nine females during the gestation period and in all females on days 1 and 2 of the lactation period. Salivation after administration was observed occasionally in seven females at the end of the pre-pairing period and in six females during the gestation period.

At 100 mg/kg, no clinical signs were noted.


4.2.3 Food Consumption of Males
Pre-pairing and After Pairing Periods
At 750 mg/kg, mean food consumption was statistically significantly reduced during the first week of the pre-pairing period (-12.9% compared to the control). Afterwards, mean food con¬sumption was similar to the control group.

At 100 and 300 mg/kg, no test item-related effects were noted. The statistically significantly higher mean food consumption at 300 mg/kg was considered to be incidental.

In order of ascending dose levels, the overall differences in food consumption were: +3.5%, +12.5% and -6.7% during pre-pairing period and +5.7%, +11.3% and +5.2% during the after pairing period (percentages refer to the respective values of the control group).


Food Consumption of Females
Pre-pairing, Gestation and Lactation Periods
Mean food consumption was not affected by the treatment with the test item during the entire duration of the treatment.

In order of ascending dose levels, the overall differences in food consumption were: 0.7%, +3.4% and -0.7% during the pre-pairing period and +6.3%, +8.4% and +5.8% during the gestation period and +12.1%, +8.9% and +5.4% during lactation (percentages refer to the respective values of the control group).


Body Weights of Males
Pre-pairing, Pairing and After Pairing Periods

At 750 mg/kg, mean body weight gain was statistically significantly reduced starting on day 6 of the pre-pairing period until the end of the period.

At 100 and 300 mg/kg, there was no indication of any test item-related effect.

In the order of ascending dose levels, the overall mean body weight gains were: +10%, +11%, +12% and +5% during the pre-pairing period, +3%, +4%, +4% and +4% during the pairing period and +5%, +5%, +5% and +5% during the after pairing period (percentages refer to the respective time intervals).


Body Weights of Females
Pre-pairing, Gestation and Lactation Periods
Mean body weight and mean body weight gain were not affected by the treatment with the test item during the entire duration of the study.

In the order of ascending dose levels, the overall mean body weight gain was +7%, +8%, +7% and +8% during the pre-pairing period and +58%, +61%, +60% and +52% during the gestation period, and +4%, +5%, +7% and +5% during the lactation period (percentages refer to the respective time intervals).


Group (mg/kg/day) 1 2 3 4
(0 100 300 1000
Female numbers 41-50 51-60 61-70 71-80
Number of females paired 10 10 10 9
Number of females mated 10 10 10 9
Number of pregnant females 8 10 9 9
Numbers of females
with implantation sites only 1 0 0 0
Number of females which
reared their pups until
day 4 post partum 7 10 9 9



Mating Performance and Fertility
The median and mean precoital times were unaffected by treatment with the test item. Mean precoital times were 2.9, 3.5, 2.1 and 3.4 days in order of ascending dose level. The median precoital time was 3, 4, 3 and 4 days in order of ascending dose level.

Two females (nos. 43 and 50) in the control group and one female (no. 67) in group 3 were not pregnant. Thus, the fertility indices were 80.0%, 100.0%, 90.0% and 100.0% in groups 1, 2, 3 and 4.


Duration of Gestation
The mean duration of gestation was unaffected by treatment with the test item. Mean duration of gestation was 21.6, 21.5, 21.9 and 21.4 days, in order of ascending dose level.


Corpora Lutea Count
The mean number of corpora lutea per dam (determined at necropsy) was not affected by the treatment with the test item. Mean number of corpora lutea was 13.0, 13.8, 12.6 and 14.4 in order of ascending dose level.


Implantation Rate and Post-implantation Loss
The mean number of implantations per dam was not affected by treatment with the test item. The mean numbers of implantations per litter were 11.9, 13.1, 11.7 and 13.7 in order of ascending dose level.

No statistically significant change was observed in mean post-implantation loss and all the values were within the range of the historical control data. The mean incidence of post-implantation loss as a percentage of total implantations was 6.0, 12.2, 11.4 and 12.2% in order of ascending dose level.


Litter Size at First Litter Check
The number of live pups at first litter check was unaffected by treatment with the test item. The mean number of live pups per litter was 11.1, 11.5, 10.3 and 12.0 in order of ascending dose level.


Postnatal Loss Days 0 - 4 Post Partum
The total numbers of pup loss during the first four days were 2, 1, 0 and 0 in order of ascending dose level, corresponding to 3.8, 0.9, 0.0 and 0.0% of living pups.

The resulting viability indices were 96.2%, 99.1%, 100.0% and 100.0% in order of ascending dose levels.


Terminal Findings - Parent Animals
Organ Weights
In males, weights (absolute and relative to body weight) of testes and epididymides were not affected by the treatment with the test item.

Macroscopical Findings
During necropsy of parent animals type and incidence of abnormal findings did not give any indication of a test item-related effect.

Males
No macroscopical findings were observed.

Females
At 1000 mg/kg, lungs discolored reddish, incompletely collapsed and left lobe margin with greenish foci and thymus discoloured red were observed in female no. 80, which was found dead.

At 300 mg/kg, female no. 67 was noted to have ovaries discoloured dark red and both uterine horn dilated containing watery fluid and female no. 68 to have both ovaries discoloured reddish.

At 100 mg/kg, no abnormal finding was noted.

In the control group, female no. 45 was noted to have yellowish firm nodules on adipose tissue of left uterine horn.

Histopathology Findings
No test item-related morphological changes were noted during the histological examination of organ and tissue. All findings recorded are within the range of normal background alterations.

Treatment with the test item did not reveal effects on the completeness of stages or cell populations. There was no indication for maturation arrest, reabsorptions of sperm or any other degenerative type.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the clinical signs observed in females at 1000 mg/kg, on lower food consumption and body weight gain in males at 750 mg/kg, the general NOAEL (No-Observed-Adverse-Effect Level) was considered to be 300 mg/kg.
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOEL
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, mean pup weight was slightly lower on day 1 post partum (5.7 g compared to 6.3 g for combined data of male and female pups) and on day 4 post partum mean pup weight was statistically significantly lower (8.1 g versus 9.7 g). This was consid
Gross pathological findings:
no effects observed

Details on results (F1)

External Examination at First Litter Check and during Lactation
At 1000 mg/kg, one male pup was noted to have a wound on the ear at first litter check. No other findings were noted.


Sex Ratios
Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test item.

The proportion of males on day 4 post partum was 53%, 50%, 55% and 50% in order of ascending dose level.

Pup Weights to Day 4 Post Partum
(See Figures on p.{ DA }, Summary Tables on p.{ DA }, Individual Tables on p.{ DA })

At 1000 mg/kg, mean pup weight was slightly lower on day 1 post partum (5.7 g compared to 6.3 g for combined data of male and female pups) and on day 4 post partum mean pup weight was statistically significantly lower (8.1 g versus 9.7 g). This was considered to be a test item-related effect.

At 100 and 300 mg/kg, no effects were noted in mean pup weight.

Mean pup weights on day 4 post partum were 9.7, 9.6, 9.8 and 8.1 g for combined data of male and female pups in order of ascending dose level.


4.5.4 Macroscopical Findings
See below Summary Table

At 100 mg/kg, one female pup was noted to have no milk in the stomach and autolysis. No abnormal findings were noted at macroscopic examination of the pups.

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the lower mean pup weight at 1000 mg/kg (Female Parent), the NOEL for developmental toxicity was considered to be 300 mg/kg bw/day.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Macroscopical Findings Summary Table:

 Group (mg/kg)  Macroscopical finding(s)  Litter No.  Pup No. / Sex
 1 (0)  No abnormal finding was noted    
 2 (100)  No milk in the stomach and autolysis  51  13/F
 3 (300)  No abnormal finding was noted    
 4 (1000)  No abnormal finding was noted    

Applicant's summary and conclusion

Conclusions:
Based on the clinical signs observed in females at 1000 mg/kg, on lower food consumption and body weight gain in males at 750 mg/kg, the general NOAEL (No-Observed-Adverse-Effect Level) was considered to be 300 mg/kg.

For reproduction toxicity the NOEL (No Observed Effect Level) was at 1000 mg/kg/day for females and at 750 mg/kg for males.

Based on the lower mean pup weight at 1000 mg/kg, the NOEL for developmental toxicity was considered to be 300 mg/kg bw/day.
Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of tert-Butyl peroxybenzoate (CAS # 614-45-9) on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition.

 

Four groups of 10 males and 10 females were treated by gavage with tert-Butyl peroxybenzoate (CAS # 614-45-9) once daily. Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to day 4 post partum.

 

The following dose levels were used:

                       

Group 1:              0 mg/kg body weight/day (control group)

                       Group 2           100 mg/kg body weight/day

                       Group 3:          300 mg/kg body weight/day

                       Group 4           750 mg/kg body weight/day (males only)

                                               1000 mg/kg body weight/day (females only)

 

A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil).

 

The following results were obtained:

 

Mortality and General Tolerability

 At 1000 mg/kg, one female was found dead on day 6 of the pre-pairing period. Slight reduction of body weight was observed on the day before death. At necropsy, thymus was discolored dark red, lungs were incompletely collapsed and discolored reddish and with greenish foci on the left lobe. However, the cause of death could not be established by the organs and tissue examined.

 

Males

 At 300 and 750 mg/kg, signs of discomfort such as bedding material in the mouth and salivation before and after administration were noted. 

 

Females

 At 1000 mg/kg, ruffled fur and soft feces were observed occasionally in few animals. Signs of discomfort such as salivation before or after administration and bedding material in mouth were observed from the second week of the pre-pairing onwards.

 

At 300 mg/kg, bedding material in the mouth was observed in some females at the end of the pre-pairing period until beginning of the lactation. Salivation after administration was observed occasionally at the end of the pre-pairing period and during the gestation period.

 

Food Consumption

 In males at 750 mg/kg, mean food consumption was statistically significantly lower during the first week of the pre-pairing period.

 

In females, mean food consumption was not affected by the treatment with the test item at any dose level.

 

Body Weights

In males at 750 mg/kg, mean body weight gain was statistically significantly lower starting on day 6 of the pre-pairing period. No statistically significant changes were noted in mean body weight.

 

In females, mean body weights and mean body weight gain were not affected by the treatment with the test item at any dose level.

 

Reproductive Data

Mating performance, fertility and duration of gestation were not affected by the treatment with the test item.

 

The mean number of corpora lutea, the mean number of implantations per dam, and the post-implantation losses were also unaffected by treatment with the test item.

 

Organ Weights

Mean absolute and relative weight of testes and epididymides were not affected by the treatment with the test item.

 

Macroscopical Findings and Histopathological Examinations

No abnormal findings were noted in males.Type and incidence of the macroscopical findings observed in females did not give any indication of a test item-related effect. The histopathology examination did not reveal any morphological evidence of toxic effects of the test item.

  

The number of live pups at first litter check and on day 4 post partum was unaffected by treatment with the test item. No test item-related clinical signs were noted at first litter check and during the lactation.

At 1000 mg/kg, mean pup weight was statistically significantly lower on day 4 post partum and this was considered to be due to the treatment of the dams with the test item.

 

        

Based on the clinical signs observed in females at 1000 mg/kg, on lower food consumption and body weight gain in males at 750 mg/kg, the general NOAEL (No-Observed-Adverse-Effect Level) was considered to be 300 mg/kg.

 

For reproduction toxicity the NOEL (No Observed Effect Level) was at 1000 mg/kg/day for females and at 750 mg/kg for males.

 

Based on the lower mean pup weight at 1000 mg/kg, the NOEL for developmental toxicity was considered to be 300 mg/kg bw/day.