Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Further information on fertility

An OECD443 study with tert-butyl peroxybenzoate (t-BP) is not available. There is other data available that enables the registrant to conclude on this endpoint. A weight of evidence approach as specified in REACH Annex XI, Section 1.2. is taken.

 

In this approach data from the substance itself, the breakdown products and QSARs are used together to conclude on the hazard of tert-butyl peroxybenzoate to cause effects on fertility.

 

Tert-butyl peroxybenzoate

Available animal studies with t-BP give no indication of an effect on fertility. A OECD 421 reproductive and developmental toxicity screening study is negative for reproductive toxicity. 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.

Four groups of 10 males and 10 females were treated by gavage once daily at 0, 100, 300, 750 (males) and 1000 mg/kg (females). 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. For reproduction toxicity the NOEL (No Observed Effect Level) was at 1000 mg/kg/day for females (the highest dose administered) and at 750 mg/kg for males (the highest dose administered). Moreover, results from the 90-studies do not indicate effects on reproductive organs. 

Developmental delays, in an OECD 414 study, in the presence of reduced maternal weight gain and food consumption were the only effects reported. In an OECD 414 in the rabbit no adverse effects were observed up to the highest dose tested; 250 mg/kg bw/day.

OECD ToolBox (4.2) does not give any alerts for reproductive toxicity. DART: “Not known precedent reproductive and developmental toxic potential”

 

Toxicokinetics

There is reliable data from the National Toxicology Program for t-BP. In vitro studies conducted by the National Toxicology Program showed that concentrations of 1.1, 0.11, and 0.011 mg/ml degraded by 0, 31, and 74%, respectively, in 1 hour at 37°C in a suspension of stomach contents. However, the substance is extremely rapidly degraded in rat and human blood. These experiments resulted in half lives of 4mg/L to be 10.4 and 4.0 minutes, respectively. When in incubated with microsomal or soluble enzyme preparations from rat liver less than 1% of the parent substance remained after 15 minutes. This shows that degradation is extremely rapid also in contact with liver enzymes. Also incubation with glutathione showed that t-BP solutions of 0.011, 0.11, and 1.1 mg/ml in HEPES buffer, pH 7.4, containing 5 mM glutathione, were degraded by 22, 18, and 10% in 15 minutes, respectively. Overall these results indicating that any substance absorbed in the body will have a very short half-life.

Dermal studies determined that approximately 16% of dermal doses administered to rats was absorbed and rapidly eliminated without tissue accumulation.

Similarly, t-BP given intravenously was rapidly degraded and eliminated, primarily in urine, with no apparent accumulation in any tissue.

The toxicity of parent substance, t-BP, is limited to the site of contact. This can be seen in the results from oral repeated dose studies where effects on the stomach were observed. t-BP would not be expected to gain access to the systemic circulation of humans.

Benzoic acid and t-butanol made up 93% of the major degradation and/or metabolic products. As supporting data the OECD ToolBox (4.2) was also run and the results are in line with the in vivo findings. Next to Benzoic acid and t-butanol 7 additional metabolites are predicted. Just as the two major metabolites none of these metabolites are classified for long term systemic effects including reproductive toxicity (see appendix 1).

The degradation products of t-BP, which are t-butanol and benzoic acid, are likely to be absorbed into the systemic circulation, but they are relatively nontoxic. Both t-butanol (CAS#75-65-0) and benzoic acid (CAS#65-85-0) have been the subjects of other studies and they do not appear to produce other (chronic) toxicity (NTP, Mathews, 1992).

Based on this information the data available for -butanol and benzoic acid about effects on fertility are described below and are relevant for assessment of effects on fertility of t-BP.

 

t-butanol

“There were no adverse effects on mating performance, fertility, or reproductive organs in a reproduction and developmental toxicity screening test in which male and female Sprague-Dawley rats received tertiary butyl alcohol by gavage at dose levels up to 1000 mg/kg bw/day. Developmental toxicity was evidenced by reduced body weights and increased mortality in the offspring of dams receiving 1000 mg/kg bw/day. Mild to moderate toxicity, including CNS effects, decreased body weight gain, and decreased food consumption, was observed in adult males and females at that dose level. The NOAEL for developmental/ reproductive effects was 400 mg/kg bw/day while the overall NOAEL for the study was 160 mg/kg bw/day.

In a well-conducted reproductive and developmental screening study in rats, there was no clear evidence of an adverse effect on sexual function, fertility or post-natal development in the absence of toxicity in the parental generation. Fetotoxicity was evidenced by reductions in mean litter size, a decrease in number of live born pups, an increase in the number of stillborn pups, reduced pup body weights, and increased mortality in the offspring of dams receiving approximately 1000 mg/kg bw/day by oral gavage. This dose level had been selected to cause some toxicity in the F0 animals. At this dose level, systemic toxicity, including reversible CNS effects, reduced feed consumption, and reduced weight gain was observed in both sexes of the parental generation in the present study. Based on this information, tertiary butyl alcohol is not a reproductive toxicant and is not selectively toxic to the developing fetus.”

 

“Based on metabolism studies which demonstrate that MTBE is metabolized to tertiary butyl alcohol in vivo, data from the two-generation study with MTBE is relevant for evaluation of reproductive and developmental toxicity of tertiary butyl alcohol.

There were no adverse effects on mating performance, fertility or reproductive organs in a two-generation whole-body inhalation study in which Sprague-Dawley rats were exposed to MTBE at target exposure concentrations of 400, 3000 and 8000 ppm. General signs of toxicity were observed at 3000 and 8000 ppm in both generations of parental animals. Some developmental toxicity (lowered body and body weight gains in both the F1 and F2 litters) was observed in the offspring of both generations (at 3000 and 8000 ppm), but only in the presence of maternal toxicity. The NOAEC for parental toxicity and developmental toxicity was 400 ppm while the NOAEC for reproductive toxicity was 8000 ppm. Because these data demonstrate that MTBE is not a reproductive or developmental toxicant, it is reasonable to conclude that the metabolite tertiary butyl alcohol is also not a reproductive toxicant and is also not selectively toxic to the developing fetus.”

 

Furthermore, as part of two 13-week studies performed by the NTP evaluation of estrous cycle and sperm were made. In ratsno effect were seen on estrous cycle length or percentage of time spent in the various estrous stages and no effect on sperm motility or sperm morphology. In ice there where no effect on percentage of time spent in various stages of estrous, but increased estrous cycle length at the highest dose level tested, 40 mg/ml. At this dose also 40% of the animals died and reduced body weight was seen.

REACH:https://echa.europa.eu/nl/registration-dossier/-/registered-dossier/14112

 

Benzoic acid

“While the design of this four-generation study for reproductive toxicity does not exactly match the endpoints included within the EOGRTS, it does include almost all of the endpoints associated with the OECD Guideline 416 – two generation reproductive toxicity that was previously considered adequate to satisfy the reproductive toxicity endpoint under the REACh regulation. The only missing reproductive endpoints within the Kiekebusch and Lang publication are collection of sperm parameters or estrous cyclicity data. However, the endpoints that are missing from the four-generation study with benzoic acid are available for benzyl acetate, a chemical that is metabolized completely to benzoic acid. In the National Toxicology Program (Morrissey et al., 1988) 13-week feeding benzyl acetate studies in rats and mice, both sperm parameters (epididymis, cauda epididymis, testis weights, and sperm motility, density and percent abnormal sperm) and estrus cyclicity measurements were collected. Dose levels of 2000 to 3000 mg/kg/day did not affect any endpoints of male reproduction. Estrus cycles were lengthened in female mice receiving >3000 mg/kg/day but this only occurred at a dose levels also causing significant decreases in growth (body weights and body weight gain). Therefore, it appears that all of the reproductive toxicity endpoints have been collected, albeit in two separate studies on benzoic acid and benzyl acetate.”

“Combining the Weight of Evidence across all studies, benzoic acid is not considered to be reproductive or developmental toxicant, and is therefore not classified.”

REACH:https://echa.europa.eu/nl/registration-dossier/-/registered-dossier/13124

 

Conclusion

In summary there are no indications of any effect on fertility based on:

·        Results from the 90-studies do not indicate effects on reproductive organs.

·        Results from a reproductive/developmental screening study do not indicate effects on reproduction.

·        OECD ToolBox (4.2) (DART) does not give any alerts for reproductive toxicity for t-BP.

·        t-BP does not reach the systemic circulation. The degradation products of t-BP, t-butanol (CAS#75-65-0) and benzoic acid (CAS#65-85-0), are likely to be absorbed into the systemic circulation, but they are relatively nontoxic.

·        t-butanol and benzoic acid have no effects on fertility.

·        The OECD Toolbox (4.2) predicts several metabolites, next tot-butanol (CAS#75-65-0) and benzoic acid (CAS#65-85-0). None of these metabolites are classified for reproductive toxicity.

Based on this information it can be concluded that the weight of the evidence shows that t-BP has no effect on fertility and no further testing with t-BP to investigate the effect of this substance on fertility is not warranted.

Link to relevant study records

Referenceopen allclose all

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.
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no
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 }.
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
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.
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.
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: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
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.

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
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
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.
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.
Reproductive effects observed:
not specified

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    
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.

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
no guideline followed
Principles of method if other than guideline:
No specific guideline was reported. However, the study was generally well conducted.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Methyl tertiary-butyl ether (MTBE, CAS no. 1634-04-4) was supplied by Texas Petrochemicals Corporation (Houston, TX). Prior to and following the study, the purity of the test material was determined by gas chromatography to be at least 99% pure.
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
-Source: Charles River Breeding Laboratories
-Age at study initiation: 6 weeks
-Housing: individually
-Diet: ad libitum
-Water: ad libitum

ENVIRONMENTAL CONDITIONS
-Temperature (°F): 66-77
-Humidity (%): 40-70
-Air changes (per hr): 14
-Photoperiod: (hrs dark/ hrs light): 12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Details on exposure:
All exposures were conducted in 4.3 m3 stainless-steel and glass inhalation chambers. Airflow in each chamber was approximately 1000 L/min (14 air changes per hour). For the generation of atmospheres, liquid MTBE was metered from a piston pump into a heated glass evaporator. The temperature was maintained at the lowest level sufficient to vaporize liquid. The resulting vapor was carried into the chamber by a countercurrent air stream that entered the bottom of the evaporator.
Details on mating procedure:
During the mating period, one male was co-housed with one female selected randomly within the same treatment group for a maximum of 7 days. After 7 days, the females of those pairs that were not confirmed as having successfully mated were placed with males of other unmated pairs within the same treatment group for the remainder of the 21-day mating period. Mating was confirmed by observation of a copulatory plug or by the presence of sperm in a vaginal rinse. The day on which mating was confirmed was designated as the female’s Day 0 of gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Atmosphere was analyzed once every 25 minutes during each 6 hours of exposure using a Perkin-Elmer gas chromatograph equipped with a flame ionization detector.
Duration of treatment / exposure:
Males: 10 weeks before mating until delivery of the F1-litters
Females: 10 weeks before mating, during 21-day mating period, gestation and lactation starting Day 5 and until the day of sacrifice, which followed the weaning of the offspring.
Frequency of treatment:
6 hours/day, 5 days/week.
Details on study schedule:
The exposure started when the rats were about 6 weeks old and lasted for 10 weeks before mating. Exposure of the females continued through the 21-day mating period, gestation and lactation starting on Day 5 and continued until the day of sacrifice, which followed the weaning of the offspring. Male exposure continued until the delivery of the F1 litters. The new parents were randomly selected from the F1-litters at weaning on postnatal day 28 (PND 28), which was also the day they started receiving exposure. The exposure procedure was the same as it was for the P-animals.
Dose / conc.:
400 ppm
Remarks:
target concentration, actual: 402 ± 8
Dose / conc.:
3 000 ppm
Remarks:
target concentration, actual: 3019 ± 56
Dose / conc.:
8 000 ppm
Remarks:
target concentration, actual: 8007 ± 137
No. of animals per sex per dose:
25
Control animals:
yes
Parental animals: Observations and examinations:
Viability was observed twice a day, detailed clinical examinations were performed once daily and body weights were recorded weekly. Female body weights were recorded before mating, during gestation and in the post-natal days until weaning. Food consumption was recorded weekly and for females at every 3-4 days of gestation.
Litter observations:
Litters were examined twice daily. On PND 0, 1, 4, 7, 14, 21 and 28, litters were counted, weighed and sexed and checked for abnormalities. On PND 4, the size of each litter was reduced by random selection to yield as nearly as possible four male and four female pups per litter. Culled pups were euthanized and given an external examination (including an examination for cleft palate). Abnormal pups were given a gross post-mortem examination.
Postmortem examinations (parental animals):
All adult F0 and F1 animals used for mating went through a post-mortem examination (gross necropsy). Pituitary, testes, epididymis, prostate and seminal vesicles, vagina, uterus, ovaries and respiratory tract were examined microscopically from all parent animals of the control and high-dose group as well as tissues with gross lesions.

Livers from F1- animals were weighed and those of the control and high-dose group were examined microscopically.
Postmortem examinations (offspring):
On PND 4, culled pups were euthanized and given an external examination (including an examination for cleft palate). Abnormal pups were given a gross post-mortem examination.
Statistics:
The unit of comparison was the male, the female (during the pre-mating exposure period), the pregnant female or the litter. Results of quantitative continuous variables were intercompared for the three treatment groups and one control group by use of Levene’s test for equal variances, analysis of variance (ANOVA), and t-tests. Non-parametric data were statistically evaluated using the Kruskal-Wallis test followed by the Mann-Whitney U test for pairwise comparisons when appropriate. Frequency data were compared using the Fisher’s exact test.
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Toxic effects observed in parent F0 animals: Hypoactivity and lack of startle reflex were observed in the parental rats at 3000 and 8000 ppm. Parental animals at 8000 ppm were also ataxic. The 8000 ppm dose group males had a significantly reduced body weight throughout the 10-week pre-mating period. In the beginning of the mating period, the reduction in male bodyweight was almost 14%. There was also a significant reduction in the bodyweight gain in that group during the first 7 weeks. In the first three pre-mating weeks, male food consumption was reduced on average by 11%. Female body weights did not differ from controls in the pre-mating period but the body weight gain was significantly increased in the 8000 ppm group during post-natal days 21-28. The overall difference in lactation-period weight gain was more than two-fold (p<0.05).

The reproduction data, such as pregnancy rates, gestation days and mating and fertility indices showed no meaningful differences from the control.

Toxic effects observed in parent F1 animals: Hypoactivity and lack of startle reflex were observed in the parental rats at 3000 and 8000 ppm. Parental animals at 8000 ppm were also ataxic. In the adult animals, the highest dose group of F1-males had about 15% less weight gain (p<0.01) during the first two weeks, accompanied by a reduced absolute body weight throughout the pre-mating period. Although no decrease in weight gain was noted with either male or female rats in the 3000 ppm exposure group, they still had significantly reduced body weight in the first 3-4 pre-mating weeks. Similarly to the F0 parent animals, no differences were noted in female body weights during the gestation period. However, the high-dose group had a significantly increased (4-fold) body weight during PND 14-21 and the lactation period. Nevertheless, the food consumption was significantly (p<0.01) reduced by approximately 10% during PND 7-14 in these females. Gross examination at necropsy revealed statistically significantly increased relative liver weights in both sexes of the F1-animals at 8000 ppm and in males at 3000 ppm. However, no treatment-related histopathological changes were noted.

Pregnancy incidence, mating and fertility and gestation indices were within the control values.
Key result
Dose descriptor:
NOAEC
Remarks:
parental toxicity
Effect level:
400 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on general toxicity signs (reduced bodyweight, hypoactivity, blepharospasms) at 3000 and 8000 ppm in parental animals.
Key result
Dose descriptor:
NOAEC
Remarks:
fertility
Effect level:
8 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No significant changes in the reproduction parameters.
Key result
Dose descriptor:
NOAEC
Remarks:
developmental toxicity
Effect level:
400 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
no effects observed
Developmental toxicity observed in F1 litters: There was a statistically significant (p<0.01) increase of dead pups in the 8000 ppm group. This followed from the death of an entire litter of 16 pups. However, there was no significant change in the survival index of the F1 generation. The high-dose group male and female offspring body weights were significantly lower than the controls during PND 14-28. The female offspring of the 3000 ppm exposure group had significantly lower body weights also on Day 14. In general, when compared to controls, no significant differences were evident in the number of pups stillborn relative to live ones, judging by the survival indices on Days 4, 7, 14, 21, 20 or the lactation index.

Developmental toxicity observed in F2 litters: As in the F1-offspring, the F2-pups also had a significantly increased incidence of dead pups on PND 4 in the 8000 ppm exposure group. Again, in a manner similar to F1-pups, the F2-offsping had no significant difference in the survival indices when compared to controls. Male body weights of the 3000 and 8000 ppm dose groups were statistically significantly (p<0.05) reduced when compared to the control group. Weight loss was seen in males of the 8000 ppm group during PND 7-28 and in the males of the 3000 ppm group during PND 14-28.
Remarks on result:
other: See P0
Key result
Reproductive effects observed:
no
Conclusions:
Based on metabolism studies which demonstrate that MTBE is metabolized to tertiary butyl alcohol in vivo, data from the two-generation study with MTBE is relevant for evaluation of reproductive and developmental toxicity of tertiary butyl alcohol.

There were no adverse effects on mating performance, fertility or reproductive organs in a two-generation whole-body inhalation study in which Sprague-Dawley rats were exposed to MTBE at target exposure concentrations of 400, 3000 and 8000 ppm. General signs of toxicity were observed at 3000 and 8000 ppm in both generations of parental animals. Some developmental toxicity (lowered body and body weight gains in both the F1 and F2 litters) was observed in the offspring of both generations (at 3000 and 8000 ppm), but only in the presence of maternal toxicity. The NOAEC for parental toxicity and developmental toxicity was 400 ppm while the NOAEC for reproductive toxicity was 8000 ppm. Because these data demonstrate that MTBE is not a reproductive or developmental toxicant, it is reasonable to conclude that the metabolite tertiary butyl alcohol is also not a reproductive toxicant and is also not selectively toxic to the developing fetus.
Executive summary:

In a two-generation reproductive toxicity study, MTBE was administered to 25 Sprague-Dawley rats/sex/group by whole body inhalation at target concentrations of 0, 400, 3000 and 8000 ppm for 10 weeks prior to mating. Males continued exposure until delivery of the F1-litters. Females continued exposure during mating, gestation and lactation until weaning of the offspring. There were no adverse effects on any reproductive parameters including pregnancy rates, gestation days, and mating and fertility indices. Toxic effects including clinical signs of CNS depression, and reduced body weight and/or body weight gain were observed in the F0 and F1 parental generations at 3000 and 8000 ppm. Signs of developmental toxicity such as lowered body weight and body weight gains were observed in the F1 and F2 litters when parents were exposed to 3000 or 8000 ppm but adverse effects occurred only in the presence of maternal toxicity. MTBE and by analogy its metabolite tertiary butyl alcohol are not considered to be reproductive or developmental toxicants.      

Endpoint:
multi-generation reproductive toxicity
Remarks:
The effect of benzoic acid on reproduction was studied through four generations.
Type of information:
experimental study
Remarks:
In a chronic feeding study, male and female rats were exposed to up to 1% benzoic acid in the diet through four generations
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: See remarks
Remarks:
While the design of this four-generation study for reproductive toxicity does not exactly match the endpoints included within the EOGRTS, it does include almost all of the endpoints associated with the OECD Guideline 416 – two generation reproductive toxicity that was previously considered adequate to satisfy the reproductive toxicity endpoint under the REACh regulation. The only missing reproductive endpoints within the Kiekebusch and Lang publication are collection of sperm parameters or estrous cyclicity data. However, the endpoints that are missing from the four-generation study with benzoic acid are available for benzyl acetate, a chemical that is metabolized completely to benzoic acid. In the National Toxicology Program (Morrissey et al., 1988) 13-week feeding benzyl acetate studies in rats and mice, both sperm parameters (epididymis, cauda epididymis, testis weights, and sperm motility, density and percent abnormal sperm) and estrus cyclicity measurements were collected. Dose levels of 2000 to 3000 mg/kg/day did not affect any endpoints of male reproduction. Estrus cycles were lengthened in female mice receiving >3000 mg/kg/day but this only occurred at a dose levels also causing significant decreases in growth (body weights and body weight gain). Therefore, it appears that all of the reproductive toxicity endpoints have been collected, albeit in two separate studies on benzoic acid and benzyl acetate
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
Study predates current published regulatory guidelines for GLP compliance and reproductive toxicity studies; study contains most of the same elements found in a present-day two generation reproductive toxicity test and continued exposure through four generations. The premating exposure intervals were at least 10 weeks and the dose levels used were comparable to the limit dose level of 1000 mg/kg/day required under the current regulatory guidelines. Endpoints examined included collection of body weight and feed consumption data, determination of effects on sexual maturity and fertility, collection of litter and pup data, necropsy data including organ weights and histopathology, and effect on onset of sexual senescence, and lifespan. The study did not collect sperm parameters and estrous cyclicity data but these endpoints are addressed in a separate 13-week NTP feeding study in rats with the analogue benzyl acetate.
Deviations:
yes
Remarks:
See field: Version / remarks
Principles of method if other than guideline:
A feeding study was performed, in which 4 generations of rats received the test substance (0.5% or 1%). The first generation was exposed for 8 weeks an then allowed to mate (1/1 for a period of 14 days). Mating was repeated in week 48 to raise a second litter. Survival of the first and second generation was measured. The third generation was terminated after 16 weeks and examined histopathologically. In this generation weights of brain, heart, liver, spleen, kidneys and testes were determined. The fourth generation was terminated after weaning of the pups. Body weights were determined in week 4, 8 and 12 weeks of each generation (week 12 males only). Feeding efficiency was measured in all generations after 2,4, 6 and 8 weeks. Some reproduction parameters were assessed: percentage of infertility, delayed sexual maturation, litter size, total pups, surviving pups. These parameters were assessed for all generations (summed) and for thefirst two generations separately.
GLP compliance:
no
Remarks:
Study pre-dated GLPs.
Limit test:
no
Specific details on test material used for the study:
No specified
Species:
rat
Strain:
not specified
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Farbwerken Bayer (Elherfeld)- Females (if applicable) nulliparous and non-pregnant: yes- Age at study initiation: Not provided in publication.- Weight at study initiation: 40-50g- Fasting period before study: Not provided in publication.- Housing: Rats were kept in double cages in a simplified Columbus-type feed apparatus.- Diet (e.g. ad libitum): For the first 8 weeks, they were fed according to the “paired feed” method, and then ad libitum.- Water (e.g. ad libitum): ad libitum.- Acclimation period: Not provided in publication.ENVIRONMENTAL CONDITIONS- Temperature (°C): Not provided in publication.- Humidity (%): Not provided in publication.- Air changes (per hr): Not provided in publication.- Photoperiod (hrs dark / hrs light): Not provided in publication.IN-LIFE DATES: From: To: Not provided in publication.
Route of administration:
oral: feed
Details on exposure:
DIET PREPARATION- Rate of preparation of diet (frequency): Not provided in publication.- Mixing appropriate amounts with (Type of food): Feed mixtures were produced in a feed mixing machine made of stainless steel. Diet consisted of commercial standard rat feed made by Lutz (Euskirchen) with sufficient benzoic acid added to achieve feed concentrations of 0.5% and 1.0%- Storage temperature of food: Not provided in publication.
Details on mating procedure:
- M/F ratio per cage: 1M/1F- Length of cohabitation: 14 days- Proof of pregnancy: Not provided in publication.- After 14 days of unsuccessful pairing ,the pairing was repeated 8 weeks later to investigate delays in sexual maturity or full sterility.- Further matings after two unsuccessful attempts: no - After successful mating each pregnant female was caged (how): Not provided in publication
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Not provided in publication
Duration of treatment / exposure:
11-12 weeks prior to mating and through 4 generations.
Frequency of treatment:
Feed containing test material provided ad libitum.
Details on study schedule:
Details on study schedule were not provided in the publication.
Dose / conc.:
0 other: % in diet
Dose / conc.:
0.5 other: % in diet
Remarks:
Overall, the dose level from the 0.5% diet for the entire premating period was approximately 450 and 600 mg/kg/day for the male and female rats, respectively.
Dose / conc.:
1 other: % in diet
Remarks:
Overall, the dose level from the 1% diet for the entire premating period was approximately 900 and 1176 mg/kg/day for the male and female rats, respectively
No. of animals per sex per dose:
20
Control animals:
yes, plain diet
Details on study design:
Dose selection rationale: The dietary concentrations were selected based upon a previous probe study where rats consuming 5% benzoic acid in the diet died within 3 weeks due to lack of palatability of the diet and corrosive effects of the free acid on the digestive tract.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data DETAILED CLINICAL OBSERVATIONS: No data BODY WEIGHT: Yes - Time schedule for examinations: During the first 8 weeks of the experiment, weight checks were done weekly; after that, weight was checked in 4-week intervals. FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes - Food consumption for each animal determined: Yes; feed consumption data were collected throughout the exposure period over the four generations; for reporting purposes in the publication, feed consumption data are presented as “Protein efficiency (weight increase per gram of dietary protein)” - Compound intake calculated as time-weighted averages from the consumption and body weight gain data: calculated from body weight and feed consumption data include within the publication.
Oestrous cyclicity (parental animals):
Not examined.
Sperm parameters (parental animals):
Testis weight was examined in the 3rd generation.
Litter observations:
STANDARDISATION OF LITTERS- Performed on day 4 postpartum: No; Litter size was recorded on the 1st and 2nd day as well as the number of surviving young on the 21st day. PARAMETERS EXAMINEDThe following parameters were examined in [F1 / F2 / F3] offspring: Since there was no demonstratable effect of benzoic acid on reproduction and since the data from all four generations were similar, the data were combined. Parameters examined included total number of pups born, pup survival, and litter size. GROSS EXAMINATION OF DEAD PUPS: No information provided.ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Not examined.ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Not examined.
Postmortem examinations (parental animals):
SACRIFICE- Male animals: The third generation of animals was necropsied after 16 weeks of exposure. Organ weights of the brain, heart, spleen, liver, kidneys and testis were collected. Organs were examined histopathologically.- Maternal animals: The third generation of animals was necropsied after 16 weeks of exposure. Organ weights of the brain, heart, spleen, liver, and kidneys were collected. Organs were examined histopathologically.HISTOPATHOLOGY / ORGAN WEIGHTS: The following organs from the third generation animals were prepared for microscopic examination and weighed, respectively.: brain, heart, spleen, liver, kidneys, testis.
Postmortem examinations (offspring):
None stated.
Statistics:
Not provided in publication.
Reproductive indices:
Not provided in publication.
Offspring viability indices:
No differences were observed between generations so all data were combined.
Clinical signs:
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
There was no difference between the 1% group and the control group in terms of lifespan (similar number of short vs long-lived animals) but there was a statistically significant increase in the number of long-lived animals in the 0.5% group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on body weight gain or body weights over the four generations of rats tested in this study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on feed consumption (efficiency) over the four generations of rats tested in this study.
Food efficiency:
no effects observed
Description (incidence and severity):
Feed consumption data in this study are presented as “Protein efficiency (weight increase per gram of dietary protein)”. There was no effect on feed consumption (efficiency) in either test group.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Organ weights were only reported for the 3rd generation animals.
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathology was only reported for 3rd generation animals.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
For all four generations, there were no adverse effects on food consumption or efficiency, body weights, life span, organ weights, or organ pathology in male and female rats receiving 1% benzoic acid in the diet when compared to the control group. There were no differences in reproduction or development of the young in the 4 observed generations exposed to 1% benzoic acid in the diet. See Table 1 in "Any other information on results incl. tables".
Key result
Dose descriptor:
NOEC
Effect level:
> 1 other: %
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Generational data were not reported separately. At concentrations up to 1% benzoic acid in the diet, there were no adverse effects on parents or offspring through four generations of test substance administration. Overall, the dose level from the 1% diet for the entire premating period was approximately 900 and 1176 mg/kg/day for the male and female rats, respectively.
Key result
Critical effects observed:
no
Clinical signs:
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
There was no difference between the 1% group and the control group in terms of lifespan (similar number of short vs long-lived animals) but there was a statistically significant increase in the number of long-lived animals in the 0.5% group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on body weight gain or body weights over the four generations of rats tested in this study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on feed consumption (efficiency) over the four generations of rats tested in this study.
Food efficiency:
no effects observed
Description (incidence and severity):
Feed consumption data in this study are presented as “Protein efficiency (weight increase per gram of dietary protein)”. There was no effect on feed consumption (efficiency) in either test group.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathology was only reported for 3rd generation animals.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
For all four generations, there were no adverse effects on food consumption or efficiency, body weights, life span, organ weights, or organ pathology in male and female rats receiving 1% benzoic acid in the diet when compared to the control group.There were no differences in reproduction or development of the young in the 4 observed generations exposed to 1% benzoic acid in the diet. See Table 1 in "Any other information on results incl. tables".
Dose descriptor:
NOEC
Effect level:
> 1 other: %
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Generational data were not reported separately. At concentrations up to 1% benzoic acid in the diet, there were no adverse effects on parents or offspring through four generations of test substance administration. Overall, the dose level from the 1% diet for the entire premating period was approximately 900 and 1176 mg/kg/day for the male and female rats, respectively.
Critical effects observed:
no
Clinical signs:
not specified
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
There was no difference between the 1% group and the control group in terms of lifespan (similar number of short vs long-lived animals) but there was a statistically significant increase in the number of long-lived animals in the 0.5% group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on body weight gain or body weights over the four generations of rats tested in this study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on feed consumption (efficiency) over the four generations of rats tested in this study.
Food efficiency:
no effects observed
Description (incidence and severity):
Feed consumption data in this study are presented as “Protein efficiency (weight increase per gram of dietary protein)”. There was no effect on feed consumption (efficiency) in either test group.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Description (incidence and severity):
Histopathology was only reported for the 3rd generation animals.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
For all four generations, there were no adverse effects on food consumption or efficiency, body weights, life span, organ weights, or organ pathology in male and female rats receiving 1% benzoic acid in the diet when compared to the control group.
Dose descriptor:
NOEC
Generation:
F1
Effect level:
> 1 other: %
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
There were no adverse effects on viability, sexual maturation, mortality, body weight and weight gain, food consumption and efficiency, body weights, select organ weight and pathology for any of the four generations in this study receiving up to 1% benzoic acid in the diet. Overall, the dose level from the 1% diet for the entire premating period was approximately 900 and 1176 mg/kg/day for the male and female rats, respectively.
Critical effects observed:
no
Clinical signs:
not specified
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
There was no difference between the 1% group and the control group in terms of lifespan (similar number of short vs long-lived animals) but there was a statistically significant increase in the number of long-lived animals in the 0.5% group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on body weight gain or body weights over the four generations of rats tested in this study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of feeding 0.5 or 1.0% benzoic acid in the diet on feed consumption (efficiency) over the four generations of rats tested in this study.
Food efficiency:
no effects observed
Description (incidence and severity):
Feed consumption data in this study are presented as “Protein efficiency (weight increase per gram of dietary protein)”. There was no effect on feed consumption (efficiency) in either test group.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
Organ weights were only reported for the 3rd generation animals.
Gross pathological findings:
not specified
Histopathological findings:
no effects observed
Description (incidence and severity):
Histopathology was only reported for 3rd generation animals.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
For all four generations, there were no adverse effects on food consumption or efficiency, body weights, life span, organ weights, or organ pathology in male and female rats receiving 1% benzoic acid in the diet when compared to the control group.
Dose descriptor:
NOEC
Generation:
F2
Effect level:
> 1 other: %
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
There were no adverse effects on viability, sexual maturation, mortality, body weight and weight gain, food consumption and efficiency, body weights, select organ weight and pathology for any of the four generations in this study receiving up to 1% benzoic acid in the diet. Overall, the dose level from the 1% diet for the entire premating period was approximately 900 and 1176 mg/kg/day for the male and female rats, respectively.
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Table 1: Reproduction of rats given chronic feeding of benzoic acid over 4 generations

      % Benzoic Acid in Feed   
   0 0.5 1.0 
 Total number of females used 80  78  80 
 Sterile females in % 14% 10% 4%
       
 Females with delayed sexual maturity

 7.5

17 

9

 Litter Size

 9.0+0.33

9.5+0.26 

9.6+0.29 

 

 

 

 

 Total number of pups born

625 

688 

741 

 Surviving young in % of number of pups born

74%

66%

65%

       
 Age Pairing (2nd Generation)      
 Number of females used 37 38 38
 Litter size

6.9

7.7

7.5

 

 

 

 

 Total number of pups born  173 139  171 
 Surviving young in % of number of pups born  72% 61%  73% 
Conclusions:
In a chronic feeding study, there were no adverse effects on reproductive parameters including fertility measures, delayed sexual maturity, total number of pups born, pup survival, onset of reproductive senescence or litter size when male and female rats were fed up to 1% benzoic acid in the diet over four generations. Under conditions of this study, benzoic acid is not a reproductive toxicant.
Executive summary:

In a long-term feeding study, benzoic acid was added to standard feed to achieve concentration of 0, 0.5% or 1.0% in the diet. Diets were provided ad libitum to groups of 20 rats/sex through four generations. Body weights and feed consumption data were collected throughout the exposure period. Other endpoints examined included: onset of sexual maturity, evidence of permanent sterility, onset of menopause, litter sizes, number of pups born, surviving young, organ weights and histology, and effect on lifespan. Feed consumption, body weights, and weight gain were unaffected by exposure to benzoic acid at concentrations up to 1% in the diet. There was actually an unexplained statistically significant increase in the lifespan of rats in the 0.5% exposure group, i.e., a higher percentage of rats lived longer. There were no differences between the groups exposed to benzoic acid and the control group for fertility measures, delayed sexual maturity, total number of pups born, pup survival, onset of reproductive senescence or litter size. In addition, organ weights and histopathologic findings were similar for all groups. Under conditions of this study, there were no dose-related adverse effects on either reproductive or developmental parameters in both sexes of rats fed 1% benzoic acid in the diet over four generations.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
June - September 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
-Test substance name (as cited in study report): Tertiary butanol (t-butyl alcohol, TBA)
-Test substance category: Chemical intermediate
-Supplier: Haltermann Ltd. (Houston, TX)
-Lot number: HL30408005
-Purity: 99.6%
-Physical description: Waxy solid at room temperature. Forms a clear, colorless solution in water
-Date received: April 8, 2003
-Expiration date: No certification of stability was available. A nominal expiry date of 1 year following packaging was allocated by the Sponsor.
-Storage: Room temperature
-Analysis: Documentation of the identity, purity, composition, and other characteristics that define the test substance and the maintenance of these records was the responsibility of the Sponsor. A certificate of analysis was provided to the testing laboratory.
Species:
rat
Strain:
other: Albino rats, Sprague-Dawley derived strain (outbred), Crl:CD(SD) IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Kingston, New York
- Age at study initiation: approx. 8 weeks
- Acclimation period: 14 days
- Identification: Ear-Tag
- Housing: F0 animals were housed individually in clean, suspended, stainless steel wire-mesh cages in an environmentally controlled room (except during mating when one male and one female were housed together). Starting on gestation day 18 and during lactation, each dam was housed with its litter in a plastic shoebox cage with ground corncob bedding (Bed-O’-Cobs). Fresh bedding was provided every 10-11 days or as needed throughout lactation. Each cage was supplied with a glass feeder jar with a stainless steel lid. F1 weanlings retained after PND 21 were housed initially in pairs (males and females separate), then individually after PND 22 (as F0 animals).
- Diet: PMI Nutrition International, LLC Certified Rodent Lab Diet® 5002 (meal) ad libitum. Clean jars of fresh food were provided at least weekly and at each interval when feed consumption was recorded.
- Water: Tap water (Elizabethtown Water Company, Westfield NJ) was available ad libitum through an automated watering system
-Weight at study initiation: 248-300 g (males); 165-235 g (females)
-Randomization: Animals judged suitable based on body weight and other pretest evaluations were randomly assigned to control or test groups to provide similar group and individual body weight means.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24°C
- Humidity (%): 44-81%.
- Photoperiod (hrs dark / hrs light): 12 hours light/dark

IN-LIFE DATES
-Study initiation: May 23, 2003
-Date of animal receipt: June 3, 2003
-Dosing initiation: (F0 animals): June 17, 2003
(F1 treated animals): August 27, 2003
-Dosing termination: (F0 males): August 19, 2003
(F0 females): September 4, 2003
(F1 treated pups): September 11, 2003
-Terminal sacrifice: (F0 males): August 20, 2003
(F0 dams and excess pups): August 11-September 07, 2003
(F1 treated pups): September 3-12, 2003
Route of administration:
oral: gavage
Vehicle:
other: deionized water
Details on exposure:
DOSE PREPARATION:
Formulations were prepared weekly. The bulk container of tertiary butyl alcohol was heated to 30 °C to liquefy the test material. The requisite quantity of test material was weighed into a pre-warmed calibrated beaker and the necessary amount of warmed vehicle (deionized water) added with mixing to prepare the specific dose. Formulations were stored in labeled containers with nitrogen caps at room temperature. Appropriate aliquots were removed each day. Dosing formulations were re-mixed by inversion prior to opening for dosing.

DOSE ADMINISTRATION:
Dose levels were 0, 64, 160, 400, or 1000 mg/kg bw/day of the test material. Animals were dosed by oral gavage seven days per week including holidays. The dose volume was 5 mL/kg body weight.

Dosing volumes were calculated for each animal using the most recent body weight recorded, except for F0 females during the period of gestation after gestation day (GD) 17, when the dose volume remained fixed according to the GD 17 body weight until the animal had completed parturition. If at the time of dosing an animal had started parturition, but had not completed the process by approximately 1400 hours, it was not dosed that day. For females that had completed parturition, dosage volume reverted to being determined by the most recent body weight recorded. Controls received a 5 mL/kg body weight dose of distilled water.

F0 males were dosed once daily, seven days/wk for 4 weeks prior to first pairing for mating. Dosing continued through the mating and post-mating periods until sacrifice for necropsy after an overall 9 weeks (63 doses) of treatment; males were euthanized the following day. F0 females were dosed once daily, seven days/wk for 4 weeks prior to first pairing for mating. Dosing continued through mating, gestation and lactation until PND 20. Females that did not produce a litter continued treatment for up to 24 days following completion of the mating period and were terminated on the 25th or 26th day after the last exposure to a male. F1 offspring were not treated directly prior to weaning. Selected F1 weanlings, 1 male and 1 female from each available litter, were dosed once daily on PND 21-27, at the same dosages used for the F0 parents, with termination on PND 28.
Details on mating procedure:
Within each treatment group the animals were paired, 1 male to 1 female in the male’s cage, until evidence of mating was seen or for a maximum of 2 consecutive weeks. The females were observed early each morning for the presence of a vaginal plug or of sperm in a vaginal smear. The day on which positive evidence of mating was observed was defined as GD 0. Once mated, the female was removed from the mating cage and was housed individually throughout the remainder of the study. After the mating period was over, females without evidence of copulation were also returned to individual housing and monitored for visible signs of pregnancy.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation concentrations and stability analyses were performed by the Huntingdon Life Sciences Analytical Chemistry Laboratory. As the formulations comprised true aqueous solutions, homogeneity analysis was not necessary.
Trial formulations were prepared 8 days prior to the start of dose administration, allowed to stand in sealed containers for 4 hours at room temperature, remixed and then sampled in duplicate. One set of samples was analyzed for concentration using a method previously validated by the Huntingdon Life Sciences Analytical Chemistry Laboratory. This analysis was intended to demonstrate 4-hr stability in support of daily formulation preparation. Briefly, the analytical method involved the dilution of tertiary butyl alcohol with methanol, followed by quantification of the compound using gas chromatography-FID detection. The second set of samples was frozen at -70 °C or below pending a satisfactory outcome of the first analyses. The trial formulations were then stored at room temperature for 8 days, remixed, again sampled in duplicate and analyzed for concentration in the same manner as samples taken from the first day. These data were intended to support weekly formulation preparation. Dosing formulation samples were taken at the beginning, around week 5, and near the end of the study. These samples were analyzed for concentration in the same manner as previous samples.
Duration of treatment / exposure:
F0 males: 9 weeks
F0 females: 4 weeks prior to mating through lactation day 21
F1 directly-treated pups: PND 21-27
Frequency of treatment:
daily
Details on study schedule:
Randomly assigned groups of 12 male and 12 female rats received tertiary butyl alcohol by oral gavage at dose levels of 0, 64, 160, 400, or 1000 mg/kg bw/day; animals were dosed 7 days a week. Males were dosed from 4 weeks prior to mating until termination (approximately 63 doses). Females were treated from the beginning of the premating period through gestation until termination on PND 21. Selected weanlings (1/sex/litter) were dosed once daily on PND 21-27 at the same dose levels received by their parents, then terminated on PND 28. Pups not selected for test substance administration were euthanized on PND day 21 or 28.

Viability and post-dose observations were checked daily. Physical observations, body weights, and feed consumption were performed on a weekly basis for males and non-gravid females throughout the study; similar examinations were made for females during the pre-mating period and at select intervals for gravid females during gestation and lactation. Feed consumption measurements were suspended during the mating trial.

From GD 18 onward, gravid dams were examined twice daily for signs of parturition. After parturition, litters were observed twice daily during lactation. Pups were given a gross physical exam and sexed on PND 0, then examined at select times throughout lactation. On PND 4, litters were culled to 10 pups/sex. Pre-weaning pups were weighed on PND 1, 4, 7, 14 and 21. Directly-dosed pups were weighed on PND 21, 24 and 28; food consumption for this group was recorded for PND 21-24 and 24-28.

At necropsy, macroscopic and microscopic examinations were performed on all parental animals. The liver, kidneys, testes and epididymides were weighed. The ovaries with oviducts, testis, epididymis, seminal vesicle with coagulating gland, prostate, thyroids with parathyroids, kidneys, and gross lesions were fixed in 10% buffered formalin. The testis, epididymis, ovaries and thyroids with parathyroids were examined microscopically from the control and high-dose animals. Sperm parameters were evaluated.

All pups were subjected to a macroscopic examination on their scheduled day of euthanasia or for pups found dead or euthanized in extremis.
Dose / conc.:
64 mg/kg bw/day (nominal)
Remarks:
in water
Dose / conc.:
160 mg/kg bw/day (nominal)
Remarks:
in water
Dose / conc.:
400 mg/kg bw/day (nominal)
Remarks:
in water
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
in water
No. of animals per sex per dose:
F0: 12 animals/sex/group
F1: 1 animal/sex/litter (10/sex/group)
Control animals:
yes, concurrent vehicle
Details on study design:
DOSE LEVEL SELECTION:
Previous studies performed under the National Toxicology Program included a 13-week and a 2-year toxicity study in rats, both by administration in the drinking water at levels of 2.5 to 40 mg/mL (13 week study) and 1.25 to 10 mg/mL (2 year study). The 40 mg/mL dosage was lethal for most animals in the 13-week study and final body weight of males at 10 and 20 mg/mL was 12% and 17% less, respectively, compared to controls. Liver and kidney weights, and the incidence of mineralization in the kidney, were increased at all dosages and there was increased incidence/severity of nephropathy. There was also increased activity of sorbitol dehydrogenase and alanine aminotransferase, with decreased urine volume and increased urine specific gravity. Hyperplasia and inflammation of the urinary bladder epithelium was observed only at dosages in excess of 10 mg/mL.

The 10 mg/mL dosage in drinking water from the 13-week study equated to approximately 750 mg/kg bw/day received dose. Based on the results of that study, the upper dosage in the present study, 1000 mg/kg bw/day, was expected to cause some toxicity in the F0 animals, in terms of effects on body weight, liver and kidney weight and more particularly in males, possibly the exacerbation of chronic progressive nephropathy. The 400 mg/kg bw/day dosage was expected to cause some lesser/marginal toxicity, while the lower dosages were expected to be without discernible toxicity.
Parental animals: Observations and examinations:
VIABILITY:
Observations were made at least twice daily, prior to dosing during the treatment period, and again late in the day.

POST-DOSE OBSERVATIONS:
Each animal was checked for overt signs of toxicity prior to dosing and any changes from the preceding observations were recorded. Each dosed animal was observed approximately one to two hours after dosing. An additional observation was made at approximately 4 hours after dosing for the first 4 days of test substance administration.

PHYSICAL OBSERVATIONS:
Animals were examined prior to randomization into treatment groups, and then once weekly until euthanasia, for any abnormality or sign of toxicity. During the treatment period, this evaluation was performed prior to dosing. For mated females, this corresponded to GD 0, 7, 14, 20 and PND 1, 7, 14 and 21.

BODY WEIGHTS:
Body weights were recorded at the time of randomization into test groups, on the day treatment was initiated, and weekly thereafter until pairing for mating. Males continued to be weighed weekly until termination. Mated females were weighed on gestation day 0, 7, 14, 17, and 20 and for those animals that delivered litters on PND 1, 4, 7, 14, and 21. If parturition was complete, females were also weighed on PND 0. Females without evidence of mating were weighed weekly until termination unless they delivered a litter. A terminal body weight (non-fasted) was also recorded for each animal.

FEED CONSUMPTION:
Food consumption was recorded weekly during the pre-mating period but not during the mating period. Weekly measurements continued for males after the mating period and for females without signs of mating until termination. Food consumption for mated females was recorded for GD 0-7, 7-14, and 14-20 and for those animals that delivered litters for PND 1-4, 4-7, and 7-14.

PARTURITION AND LACTATION:
After GD 18, examination for signs of parturition was made twice daily (morning and afternoon). Litters were observed as soon as possible after parturition for number of live and dead pups, any pup abnormalities, and sex of each pup. After completion of parturition, litters were observed twice daily. Number of dead pups, unusual observations, signs of deficient maternal care, and pups without milk in the stomach were recorded. On PND 4, litters of more than 10 pups were randomly culled to that number with equal sex distribution where possible.
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
For F0 males at scheduled necropsy, the following sperm evaluations were performed by Pathology Associates International, Frederick, Maryland.

The right vas deferens was excised and placed in a pre-warmed solution of phosphate buffered saline and 1% Bovine Serum Albumin. After a minimum 3 minutes, a sample was placed in a Hamilton Thorne IVOS sperm analyzer and five microscope field images were stored electronically. For the control and high dose animals, these fields were analyzed for percent motility.

The right testis and epididymis were frozen on dry ice and stored at -70 °C until evaluation for sperm count. For the control and high dose animals, the epididymis was thawed and the caudal portion removed and weighed. Homogenized samples of the caudal epididymis and the testis were stained and examined using a Hamilton Thorne IVOS sperm analyzer. For each stained preparation, 20 fields were counted. The total number of sperm in the caudal epididymis, or spermatids in the testis, were calculated and reported adjusted for organ weights.

Two Sperm morphology slides were prepared for each male, stained with Eosin and then evaluated for morphological development (at least 200 cells evaluated for each animal.)
Litter observations:
VIABILITY/CLINICAL OBSERVATIONS:
Litters were observed as soon as possible after parturition for number of live and dead pups and any pup abnormalities; thereafter, litters were observed twice daily during the lactation period. F1 animals were also observed for clinical signs and mortality after direct treatment with tertiary butyl alcohol.

PHYSICAL EXAMINATION:
Each pup was given a gross physical examination and sexed on PND 0 (if parturition was complete), 1, 4 (pre-cull), 7, 14 and 21. The pups were also observed for abnormal behavior.

BODY WEIGHTS:
Pre-weaning F1 pups were weighed individually on PND 1, 4 (pre-cull), 7, 14 and 21. A terminal body weight (non-fasted) was recorded for each F1 (post-weaning) animal. Directly-dosed F1 offspring were weighed on PND 21, 24, and 28.

FEED CONSUMPTION:
Food consumption was recorded for cages of directly dosed offspring on PND 21-24 and 24-28.
Postmortem examinations (parental animals):
animals):EUTHANASIA:
Males were terminated after at least 9 weeks of treatment. Females with litters were terminated on lactation day 21. Females that mated but failed to deliver a litter were terminated at 25 to 26 days after evidence of mating. The single female that showed total litter loss before weaning was retained on treatment until it was clear that further mating in the study was not necessary, and then terminated on lactation day 9.

MACROSCOPIC EVALUATION:
A macroscopic examination was performed on all animals including examination of: external surfaces; all orifices; cranial cavity; neck and its associated tissues and organs; thoracic, abdominal and pelvic cavities and their associated tissues and organs; and external surfaces of the brain. The number of implantation sites was recorded for each female. Apparently non-pregnant status was confirmed by ammonium sulphide staining. Any gross lesions or tissues with significant findings were preserved in 10% neutral buffered formalin. The testis, epididymis, seminal vesicles with coagulating gland, prostate, ovaries with oviduct, thyroids with parathyroids, and kidneys were preserved in formalin.

ORGAN WEIGHTS:
The kidneys (for both sexes), liver (for both sexes), testes and epididymides were removed at necropsy, trimmed to remove fat and other contiguous tissue and were weighed as soon as possible after dissection. Paired organs were weighed separately.

MICROSCOPIC EVALUATION:
The ovaries (2), testis (1), epididymis (1) and thyroids with parathyroids (for both sexes) were examined microscopically from the control and high-dose groups.
Postmortem examinations (offspring):
EUTHANASIA:
Offspring not selected for direct treatment were sacrificed on PND 21. Offspring selected as reserves and not directly treated were sacrificed on PND 23 or 28. Offspring treated directly were sacrificed on PND 28.

NON-SELECTED AND DIRECTLY TREATED:
A necropsy was performed on all animals. This included a macroscopic examination of: external surfaces; all orifices; cranial cavity; neck and its associated tissues and organs; thoracic, abdominal and pelvic cavities and their associated tissues and organs; and external surfaces of the brain. Any gross lesions or tissues with significant findings were preserved in 10% neutral buffered formalin.

PUPS CULLED ON PND 4:
A macroscopic external examination was performed for all pups culled on postnatal day 4. All pups were externally normal and were discarded without necropsy. Absence of milk in the stomach (visible through the skin) was noted.

PUPS FOUND DEAD OR EUTHANIZED IN EXTREMIS:
A macroscopic external examination was performed for all pups found dead or sacrificed prior to weaning. A necropsy was performed where practical, including examination of the thoracic, abdominal and cranial contents for any grossly apparent abnormalities, but no tissues were preserved. Where possible, the presence or absence of milk in the stomach was determined.
Statistics:
CONTINUOUS DATA:
Evaluation of equality of group means was made by an appropriate statistical method, followed by a multiple comparison test if needed. Bartlett’s test was performed to determine if group variances were equivalent. For all parameters, the variances were equivalent and parametric analysis procedures were used. The parametric method used was the standard one-way analysis of variance (ANOVA) using the F ratio to assess significance. If significant differences among the means were indicated, additional tests were used to determine which means were significantly different from the control: Dunnett’s. Bartlett’s test for equality of variance was conducted at the 1% significance level; all other statistical tests were conducted at the 5% and 1% significance levels. The following parameters were analyzed statistically: body weights, body weight changes, feed consumption, organ weights, organ weight ratios, mean number of uterine implantations, pup weights [by sex and composite (each weighing interval during lactation)], and litter size.

INCIDENCE DATA:
A Fischer Exact Test with Bonferroni correction was performed to identify differences between control and treatment groups. All such tests were conducted at the 5% and 1%, two-sided risk levels. The following parameters were analyzed: mortality incidence, mating indices, pregnancy rates, and fertility indices.

Statistical analysis of pathological abnormalities was not necessary as there were no abnormalities present in the treated animals.
Reproductive indices:
MATING INDEX (MI):
Female MI = No. of females mated/No. of females exposed to males
Male MI = No. of males with confirmed mating/No. of males placed with females

FERTILITY INDEX (FI):
Female FI = No. of females pregnant/No. of females exposed to males
Male FI = No. of males with females pregnant/No. of males placed with females

PREGNANCY INDEX (PI):
PI = No. of females pregnant/No. of females mated

GESTATION INDEX (GI):
GI = No. of females with liveborn/No. of females with confirmed pregnancy
Offspring viability indices:
VIABILITY INDEX (VI):
VI = No. of pups alive on lactation day 4[precull]/No. of liveborn pups

LACTATION INDEX (LI):
LI = No. of pups alive on PND day 21/No. of pups alive on PND 4 [postcull]

LIVE BIRTH INDEX (LBI):
LBI = Total No. of liveborn pups/total No. of pups born
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
no effects observed
MORTALITY:
There were no incidences of mortality during the study.

CLINICAL OBSERVATIONS:
1000 mg/kg bw/day: apparent central nervous system (CNS) toxicity from the first day of dosing, characterized by unresponsiveness/lethargy and some ataxia; some animals also exhibited increased vocalization and rapid breathing. Effects were generally of moderate intensity in males and mild intensity among females with the effects generally appearing 1-2 hours after dosing and resolving within the 24-hour period between doses. Intensity of symptoms was similar during the study.
400 mg/kg bw/day: similar CNS findings of mild intensity in about half of the females, occurring after about 2 weeks of dosing. Effects became indiscernible after about the fourth week of dosing.
No discernible clinical signs of toxicity at lower dosages of the test substance.

BODY WEIGHT:
Males: High-dose males showed reduced weight gain during week 1 of treatment, leading to an approximate 5% deficit in body weight. This reduction was not recovered during the 9 weeks of test substance administration and resulted in a 7% deficit as compared to control at the end of the study. Deficit in initial body weight gain was statistically significant but the differences in absolute weight were not. No effect occurred at lower doses.

Females: No effect on female body weight development at any of the dosages until pregnancy was established. At 1000 mg/kg bw/day, there was a decrease in body weight gain in the last few days of gestation, leading to an approximate 6% deficit in body weight at GD 20. The difference in weight gain but not the absolute difference was statistically significant. This slight weight difference remained until the third week of lactation when the high dose animals showed a net gain in weight, while the other groups lost some body weight.

FEED CONSUMPTION:
No effect in males. No effect until the lactation period for females in the highest dose group when consumption over the first 2 weeks of lactation was reduced approximately 15%.

SEMINOLOGY:
No effect on numbers of epididymal sperm, testicular spermatids, or incidences of sperm abnormalities. A very marginal decrease in mean sperm motility in the 1000 mg/kg bw/day dose-group reached statistical significance. This difference was considered not to be of importance since the value of 91% was only -3% different from the control and the value of 91% was within range of the laboratory’s historical control values of 89-96% mean motility.

REPRODUCTIVE PERFORMANCE:
There was no statisitically significant effect on mating index, fertility index, pregnancy index, gestation index or length of gestation.

MACROSCOPIC EXAMINATION:
No test substance-related effects were observed.

ORGAN WEIGHTS:
There was no test substance-related effect on female organ weights that were collected (kidneys and liver). For males in the 1000 mg/kg bw/day dose group, there was a statistically significant increase in absolute (18-20%) and relative (27-30%) kidney weights, relative liver weight (16%), and relative testis weight (13-16%). In the 400 and 160 mg/kg bw/day male groups, relative kidney weights were statistically significantly increased 14-17% and 11-14%, respectively.

MICROSCOPIC EXAMINATION:
There were no microscopic findings in the reproductive tissues or thyroids associated with the administration of the test substance at 1000 mg/kg bw/day. The left testis of one animal in the high-dose group contained a small number of tubules totally depleted of germ cells. This is a relatively common background finding in rats of this strain and the single occurrence was considered to be unrelated to tertiary butyl alcohol administration.
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on no effects on reproduction at all dosage levels.
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
160 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: kidney weights, general transient toxicity
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
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:
no effects observed
VIABILITY:
There was no effect on the number of implantations per pregnancy. There was a significant reduction in the number of live born per pregnancy at 1000 mg/kg bw/day and an increase in the number of stillborn pups. The mean litter size for the high-dose group was only 10 per litter on PND 1 as compared with 14 or 15 in other groups. Subsequently, there was significantly reduced pup survival at the high dose with only 80% survival to PND 4 as compared with close to 100% in the other groups. Most of this was attributed to one early total litter loss in the group. After PND 4, pup survival was optimal or near optimal in all the groups. At the normal weaning date of PND 21, litter size in the high-dose group was substantially reduced (by 50%) for 3/10 surviving litters in the groups as a result of early pup losses. There was no meaningful inter-group differences in live pup sex ratio for the litters.

CLINICAL OBSERVATIONS:
No effects on pups were observed during the lactation period. There were no clinical signs of toxicity or mortality among the offspring after direct tertiary butyl alcohol treatment at any of the dose levels.

BODY WEIGHTS:
Offspring born to dams treated with tertiary butyl alcohol at 1000 mg/kg bw/day exhibited lower mean body weight than the control offspring. For both sexes, there was an approximate 10% deficit in weight at PND day 1, increasing to approximately 15% by PND 7 at which point it achieved statistical significance. This deficit was still present at PND day 14 but then decreased in late lactation such that by PND 21, the mean weight for males and females was about 11% and 6% less than controls, respectively. No effects were observed at lower doses.

Pups born to dams treated with tertiary butyl alcohol at 1000 mg/kg bw/day during pregnancy and lactation, then directly treated with tertiary butyl alcohol daily at the same dose level from PND 21-27, exhibited lower mean body weight than the control group. The initial differences from control were approximately -13% and -6% for males and females, respectively, arising from the deficits acquired peri-natally. Direct tertiary butyl alcohol had no further effect on body weight gain of either male or female offspring over the one-week direct dosing period after weaning.

FEED CONSUMPTION FOR DIRECTLY-TREATED PUPS:
There appeared to have been no effect of direct tertiary butyl alcohol treatment on food consumption, although substantial contamination of the feed jars by the pups made unequivocal judgments impossible.

MACROSCOPIC EVALUATION:
For all pups, both non-selected and treated, there was only a low, sporadic incidence of minor findings.
Remarks on result:
other: See P0
Reproductive effects observed:
no

STABILITY AND DOSE CONFIRMATION TESTS:

Stability: Stability was determined for the low and high dose levels.  The preliminary mixes of tertiary butyl alcohol in water were stable for 8 days when stored at room temperature and the mean % nominal values of all samples were within ±10% of initial concentrations.

  

Dose Confirmation: All groups were assayed in duplicate on 3 occasions.  The % nominal values for all dose groups were within the test laboratories SOP specified limits.  The concentrations of duplicates of the samples were within ±10% of each other, and the means of concentration were within ±10% of nominal.

Conclusions:
There were no adverse effects on mating performance, fertility, or reproductive organs in a reproduction and developmental toxicity screening test in which male and female Sprague-Dawley rats received tertiary butyl alcohol by gavage at dose levels up to 1000 mg/kg bw/day. Developmental toxicity was evidenced by reduced body weights and increased mortality in the offspring of dams receiving 1000 mg/kg bw/day. Mild to moderate toxicity, including CNS effects, decreased body weight gain, and decreased food consumption, was observed in adult males and females at that dose level. The NOAEL for developmental/ reproductive effects was 400 mg/kg bw/day while the overall NOAEL for the study was 160 mg/kg bw/day.

In a well-conducted reproductive and developmental screening study in rats, there was no clear evidence of an adverse effect on sexual function, fertility or post-natal development in the absence of toxicity in the parental generation. Fetotoxicity was evidenced by reductions in mean litter size, a decrease in number of live born pups, an increase in the number of stillborn pups, reduced pup body weights, and increased mortality in the offspring of dams receiving approximately 1000 mg/kg bw/day by oral gavage. This dose level had been selected to cause some toxicity in the F0 animals. At this dose level, systemic toxicity, including reversible CNS effects, reduced feed consumption, and reduced weight gain was observed in both sexes of the parental generation in the present study. Based on this information, tertiary butyl alcohol is not a reproductive toxicant and is not selectively toxic to the developing fetus.
Executive summary:

In a reproduction/developmental toxicity screening study, tertiary butyl alcohol was administered to 12 Sprague-Dawley rats/sex/group by oral gavage at dose levels of 0, 64, 160, 400, and 1000 mg/kg bw/day for up to 63 days in males and from 4 weeks prior to mating through PND 21 in females.  There were no adverse effects on any reproductive parameters including mating index, fertility index, pregnancy index, or gestation index. 

For dams receiving 1000 mg/kg bw/day tertiary butyl alcohol through gestation and lactation, there was a significant reduction in mean litter size, a decrease in the number of live born per pregnancy, an increase in the number of stillborn pups, increased pup mortality up to PND 4, and a decrease in mean pup body weight at birth which continued to weaning. At this dose level, mild to moderate transient systemic toxicity was observed in both sexes in the parental generation including reversible CNS effects such as lethargy and ataxia, and reduced feed consumption and weight gain.

  

No significant toxicity was observed at any other dose level.  The NOAEL for developmental/reproductive effects was 400 mg/kg bw/day.  The NOAEL for overall toxicity was 160 mg/kg bw/day.   

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
Fertility
An OECD443 study with tert-butyl peroxybenzoate (t-BP) is not available. There is other data available that enables the registrant to conclude on this endpoint. A weight of evidence approach as specified in REACH Annex XI, Section 1.2. is taken.
 
In this approach data from the substance itself, the breakdown products and QSARs are used together to conclude on the hazard of tert-butyl peroxybenzoate to cause effects on fertility.
 
Tert-butyl peroxybenzoate
Available animal studies with t-BP give no indication of an effect on fertility. A OECD 421 reproductive and developmental toxicity screening study is negative for reproductive toxicity. 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. Moreover, results from the 90-studies do not indicate effects on reproductive organs.
Developmental delays, in an OECD 414 study, in the presence of reduced maternal weight gain and food consumption were the only effects reported. An OECD 414 in the rabbit is ongoing (2018).
The lack of reproduction and developmental toxicity is further supported by QSAR evaluations. QSARs do not show alerts for reproduction/developmental toxicity. (QSAR Toolbox DART Scheme v.1.2;Derek Nexus v.6.0.1; VEGA - Developmental Toxicity model (CAESAR) v.2.1.7; VEGA - Developmental/Reproductive Toxicity library (PG), v.1.0.0; TOPKAT Developmental Toxicity Potential)

Toxicokinetics
There is reliable data from the National Toxicology Program for t-BP. In vitro studies conducted by the National Toxicology Program showed that concentrations of 1.1, 0.11, and 0.011 mg/ml degraded by 0, 31, and 74%, respectively, in 1 hour at 37°C in a suspension of stomach contents. However, the substance is extremely rapidly degraded in rat and human blood. These experiments resulted in half lives of 4mg/L to be 10.4 and 4.0 minutes, respectively. When in incubated with microsomal or soluble enzyme preparations from rat liver less than 1% of the parent substance remained after 15 minutes. This shows that degradation is extremely rapid also in contact with liver enzymes. Also incubation with glutathione showed that t-BP solutions of 0.011, 0.11, and 1.1 mg/ml in HEPES buffer, pH 7.4, containing 5 mM glutathione, were degraded by 22, 18, and 10% in 15 minutes, respectively. Overall these results indicating that any substance absorbed in the body will have a very short half-life.
Dermal studies determined that approximately 16% of dermal doses administered to rats was absorbed and rapidly eliminated without tissue accumulation.
Similarly, t-BP given intravenously was rapidly degraded and eliminated, primarily in urine, with no apparent accumulation in any tissue.
The toxicity of parent substance, t-BP, is limited to the site of contact. This can be seen in the results from oral repeated dose studies where effects on the stomach were observed. t-BP would not be expected to gain access to the systemic circulation of humans.
Benzoic acid and t-butanol made up 93% of the major degradation and/or metabolic products. As supporting data the OECD ToolBox (4.2) was also run and the results are in line with the in vivo findings. Next to Benzoic acid and t-butanol 7 additional metabolites are predicted. Just as the two major metabolites none of these metabolites are classified for long term systemic effects including reproductive toxicity (see appendix 1 of attached justification).
The degradation products of t-BP, which are t-butanol and benzoic acid, are likely to be absorbed into the systemic circulation, but they are relatively nontoxic. Both t-butanol (CAS#75-65-0) and benzoic acid (CAS#65-85-0) have been the subjects of other studies and they do not appear to produce other (chronic) toxicity (NTP, Mathews, 1992).
Based on this information the data available for -butanol and benzoic acid about effects on fertility are described below and are relevant for assessment of effects on fertility of t-BP.

t-butanol
“There were no adverse effects on mating performance, fertility, or reproductive organs in a reproduction and developmental toxicity screening test in which male and female Sprague-Dawley rats received tertiary butyl alcohol by gavage at dose levels up to 1000 mg/kg bw/day. Developmental toxicity was evidenced by reduced body weights and increased mortality in the offspring of dams receiving 1000 mg/kg bw/day. Mild to moderate toxicity, including CNS effects, decreased body weight gain, and decreased food consumption, was observed in adult males and females at that dose level. The NOAEL for developmental/ reproductive effects was 400 mg/kg bw/day while the overall NOAEL for the study was 160 mg/kg bw/day.
In a well-conducted reproductive and developmental screening study in rats, there was no clear evidence of an adverse effect on sexual function, fertility or post-natal development in the absence of toxicity in the parental generation. Fetotoxicity was evidenced by reductions in mean litter size, a decrease in number of live born pups, an increase in the number of stillborn pups, reduced pup body weights, and increased mortality in the offspring of dams receiving approximately 1000 mg/kg bw/day by oral gavage. This dose level had been selected to cause some toxicity in the F0 animals. At this dose level, systemic toxicity, including reversible CNS effects, reduced feed consumption, and reduced weight gain was observed in both sexes of the parental generation in the present study. Based on this information, tertiary butyl alcohol is not a reproductive toxicant and is not selectively toxic to the developing fetus.”

“Based on metabolism studies which demonstrate that MTBE is metabolized to tertiary butyl alcohol in vivo, data from the two-generation study with MTBE is relevant for evaluation of reproductive and developmental toxicity of tertiary butyl alcohol.
There were no adverse effects on mating performance, fertility or reproductive organs in a two-generation whole-body inhalation study in which Sprague-Dawley rats were exposed to MTBE at target exposure concentrations of 400, 3000 and 8000 ppm. General signs of toxicity were observed at 3000 and 8000 ppm in both generations of parental animals. Some developmental toxicity (lowered body and body weight gains in both the F1 and F2 litters) was observed in the offspring of both generations (at 3000 and 8000 ppm), but only in the presence of maternal toxicity. The NOAEC for parental toxicity and developmental toxicity was 400 ppm while the NOAEC for reproductive toxicity was 8000 ppm. Because these data demonstrate that MTBE is not a reproductive or developmental toxicant, it is reasonable to conclude that the metabolite tertiary butyl alcohol is also not a reproductive toxicant and is also not selectively toxic to the developing fetus.”

Furthermore, as part of two 13-week studies performed by the NTP evaluation of estrous cycle and sperm were made. In rats no effect were seen on estrous cycle length or percentage of time spent in the various estrous stages and no effect on sperm motility or sperm morphology. In ice there where no effect on percentage of time spent in various stages of estrous, but increased estrous cycle length at the highest dose level tested, 40 mg/ml. At this dose also 40% of the animals died and reduced body weight was seen.
REACH: https://echa.europa.eu/nl/registration-dossier/-/registered-dossier/14112

Benzoic acid
“While the design of this four-generation study for reproductive toxicity does not exactly match the endpoints included within the EOGRTS, it does include almost all of the endpoints associated with the OECD Guideline 416 – two generation reproductive toxicity that was previously considered adequate to satisfy the reproductive toxicity endpoint under the REACh regulation. The only missing reproductive endpoints within the Kiekebusch and Lang publication are collection of sperm parameters or estrous cyclicity data. However, the endpoints that are missing from the four-generation study with benzoic acid are available for benzyl acetate, a chemical that is metabolized completely to benzoic acid. In the National Toxicology Program (Morrissey et al., 1988) 13-week feeding benzyl acetate studies in rats and mice, both sperm parameters (epididymis, cauda epididymis, testis weights, and sperm motility, density and percent abnormal sperm) and estrus cyclicity measurements were collected. Dose levels of 2000 to 3000 mg/kg/day did not affect any endpoints of male reproduction. Estrus cycles were lengthened in female mice receiving >3000 mg/kg/day but this only occurred at a dose levels also causing significant decreases in growth (body weights and body weight gain). Therefore, it appears that all of the reproductive toxicity endpoints have been collected, albeit in two separate studies on benzoic acid and benzyl acetate.”
“Combining the Weight of Evidence across all studies, benzoic acid is not considered to be reproductive or developmental toxicant, and is therefore not classified.”
REACH: https://echa.europa.eu/nl/registration-dossier/-/registered-dossier/13124

Exposure
The substance does not have consumer or professional users. The substance only has industrial uses.

Conclusion
In summary there are no indications of any effect on fertility based on:
• Results from the 90-studies do not indicate effects on reproductive organs.
• Results from a reproductive/developmental screening study do not indicate effects on reproduction.
• QSARs do not give any alerts for reproductive toxicity for t-BP.
• t-BP does not reach the systemic circulation. The degradation products of t-BP, t-butanol (CAS#75-65-0) and benzoic acid (CAS#65-85-0), are likely to be absorbed into the systemic circulation, but they are relatively nontoxic.
• t-butanol and benzoic acid have no effects on fertility.
• The OECD Toolbox (4.2) predicts several metabolites, next to t-butanol (CAS#75-65-0) and benzoic acid (CAS#65-85-0). None of these metabolites are classified for reproductive toxicity.
• The substance has industrial use only.
Based on this information it can be concluded that the weight of the evidence shows that t-BP has no effect on fertility and further testing with t-BP to investigate the effect of this substance on fertility is not warranted.
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Principles of method if other than guideline:
See summary below
Remarks on result:
other: see summary below
Remarks on result:
other: see summary below
Conclusions:
In summary there are no indications of any effect on fertility based on:
• Results from the 90-studies do not indicate effects on reproductive organs for t-BP.
• Results from a reproductive/developmental screening study do not indicate effects on reproduction for t-BP.
• QSARs do not give any alerts for reproductive toxicity for t-BP.
• t-BP does not reach the systemic circulation. The degradation products of t-BP, t-butanol (CAS#75-65-0) and benzoic acid (CAS#65-85-0), are likely to be absorbed into the systemic circulation, but they are relatively nontoxic.
• t-butanol and benzoic acid have no effects on fertility and studies do not indicate effects on reproductive organs.
• The OECD Toolbox (4.2) predicts several metabolites, next to t-butanol (CAS#75-65-0) and benzoic acid (CAS#65-85-0). None of these metabolites are classified for reproductive toxicity.
• t-BP has industrial use only.
Based on this information it can be concluded that the weight of the evidence shows that t-BP and its degradation products have no effect on fertility and no further testing with t-BP to investigate the effect of this substance on fertility is not warranted.
Endpoint:
fertility, other
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
accepted calculation method
Justification for type of information:
See summary below
Principles of method if other than guideline:
See summary below
Remarks on result:
other: see summary below
Remarks on result:
other: see summary below
Key result
Reproductive effects observed:
no
Conclusions:
The lack of reproductive toxicity is supported by QSAR evaluations. QSARs do not show alerts for reproductive toxicity. (QSAR Toolbox DART Scheme v.1.2;Derek Nexus v.6.0.1; v.2.1.7; VEGA - Developmental/Reproductive Toxicity library (PG), v.1.0.0)
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
rat
Quality of whole database:
The study is reliable without restrictions.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Quality of whole database:
Justification for selection of Effect on fertility via oral route: Apparently well conducted OECD 421 GLP study.

Effects on developmental toxicity

Description of key information

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 3 and 19 of gestation inclusive at dose levels 100, 300, and 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Corn Oil) to serve as a control.  

Treatment at 1000 mg/kg bw/day was associated with lower maternal body weight gain during gestation and an initial effect on food consumption. No similar effects were apparent at 300 mg/kg bw/day and this dosage is considered to represent the No Observed Effect Level (NOEL) for the pregnant female. In-utero survival of the developing conceptus was unaffected by maternal treatment at 1000 mg/g bw/day although reduced fetal weight and external, visceral and skeletal findings indicated an adverse effect on fetal growth. The absence any structural defects indicated that development per se was unaffected at this dosage.

Kinked/dilated uretus are considered considered reversible variations (Solecki, R. et al. Reproductive Toxicology 17 (2003) 635 -637) and therefore not considered adverse.  The NOAEL was therefore 300 mg/kg bw/day.

Only an equivocal increase in the incidence of fetuses/litter showing kinked/dilated ureter(s) prevented 300 mg/kg bw/day being classified as a fetal No Observed Effect Level and a dosage of 100 mg/kg bw/day is therefore considered to be a clear No Observed Effect Level (NOEL) for the developing conceptus.

In the preliminary embryo-fetal development study New Zealand White rabbits received doses of 100, 200 or 300 mg/kg/day during Gestation Days (GD) 6-28. The primary effect was restricted to low food consumption at 200 and 300 mg/kg/day. The effect on food consumption at 200 mg/kg/day was minimal, however at 300 mg/kg/day one female was killed for welfare reasons after a period of marked inappetence with associated clinical signs and body weight loss. For the surviving females there was no adverse effect on maternal clinical condition or bodyweight and the embryo-fetal survival and development was unaffected by treatment. In view of the mortality a dose of 250 mg/kg/day was considered suitable as the high dose for the main study with low and intermediate dose levels of 25 and 80 mg/kg/day to provide approximate 3-fold dose intervals.

In the main study oral administration of tert-butyl perbenzoate to pregnant New Zealand White rabbits during organogenesis and the fetal growth phase at 25, 80 and 250 mg/kg/day was well tolerated with no adverse effects on maternal condition, body weight, food consumption and macropathology or on embryo-fetal survival and development. Effects were limited to slight but transient effects on maternal food consumption and body weight. It is therefore concluded that the no observed adverse effect level (NOAEL) in this study for both maternal toxicity and embryo-fetal survival/development is 250 mg/kg/day.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 13 June 2013; Experimental Completion Date: 06 January 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidenlines and/or minor methodlogical deficiences, which do not affect the quality of relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147 (24 November 2000)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited. Animals were received in two deliveries each containing two separate batches of females on either Day 0 or Day 1 of presumed gestation. The day that positive evidence of mating was observed at the supplier was designated Day 0 of gestation. On arrival the females weighed 194 to 262g.

The animals were housed individually in solid-floor polypropylene cages with stainless steel lids furnished with softwood flakes. The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly mean temperatures and humidity were included in the study records. The target ranges for temperature and relative humidity were 22 ± 3 ºC and 50 ± 20% respectively; there were no deviations from the target range for temperature. Deviations from the target range for relative humidity were observed but were considered not to have affected the purpose or integrity of the study.

The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.






Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Test Item Formulation:
For the purpose of the study the test item was prepared at the appropriate concentrations as a solution in Corn Oil. Corn oil was used as the vehicle for this study, as it had been successfully used in previously toxicity work. The stability and homogeneity of the test item formulations were determined as part of this study. Results showed the formulations to be stable for at least twenty one days. Bulk formulations were therefore prepared twice and divided into daily aliquots which were stored at approximately +4 °C in the dark.

Representative samples were taken of each bulk test item formulation and were analyzed for concentration of Tert-butyl perbenzoate CAS# 614-45-9.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Representative samples were taken of each bulk test item formulation and were analyzed for concentration of Tert-butyl perbenzoate CAS# 614-45-9. The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLC/UV) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.

The results indicate that the prepared formulations were within ± 5% of the nominal concentration.

Details on mating procedure:
Details on mating procedure not included in report. The test item was administered to mated female rats.
Duration of treatment / exposure:
Mated females were dosed once daily by gavage, from Day 3 (prior to implantation) to Day 19 of gestation (day prior to expected parturition).
Frequency of treatment:
Once daily.
Duration of test:
All females were terminated on Day 20 of gestation.
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
24 females per dose group (including control).
Control animals:
yes, concurrent vehicle
Details on study design:
Four dose groups (control, low, intermediate and high) each comprising twenty four mated females (to ensure as far as possible twenty pregnant females per group) were used. Dose levels of 100, 300 and 1000 mg/kg bw/day were chosen in collaboration with the sponsor based on available toxicity data including a rat OECD 421 toxicity study. In the OECD 421 study a dosage of 1000 mg/kg bw/day was generally well tolerated by females, both prior to and during pregnancy.

As no effect of treatment was observed for pre-implantation loss in a previous rat OECD 421 toxicity study, mated females were dosed once daily by gavage, from Day 3 (prior to implantation) to Day 19 of gestation (day prior to expected parturition). Control animals were treated in an identical manner with the vehicle alone.
Maternal examinations:
CLINICAL OBSERVATIONS:
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. All observations were recorded.

BODY WEIGHT:
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 4, 5, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).

FOOD CONSUMPTION:
Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

WATER CONSUMPTION:
Water intake was observed daily by visual inspection of the water bottles for any overt changes.

POST MORTEM:
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded.










Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes; The ovaries and uteri of pregnant females were removed, examined and the following data recorded:
i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight

The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.

Implantation types were divided into:

Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes

All implantations and viable fetuses were numbered according to their intrauterine position.







Fetal examinations:
The fetuses were killed by subcutaneous injection of sodium pentobarbitone. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were transferred to 90% industrial methylated spirits (IMS) in distilled water and examined for visceral anomalies under a low power binocular microscope. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed in 70% IMS in distilled water. The fetuses were eviscerated, processed and the skeletons stained with alizarin red S. The fetuses were examined for skeletal development and anomalies. Following examination fetuses that were examined for skeletal development were placed in 50% glycerol.
Statistics:
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:

Body weight and body weight change (including adjustment for the contribution of the gravid uterus), food consumption, gravid uterus weight, absolute and body weight relative organ weights, litter data and fetal litter and placental weights: Bartlett’s test for homogeneity of variance. Where the data were shown to be homogeneous one way analysis of variance and, if significant, Dunnett’s multiple comparison test was employed; where the data were found to be non-homogeneous Kruskal-Wallis and, if significant, pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test was employed.

Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney ‘U’ test.

Probability values (p) are presented as follows:

p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: Body weight

Details on maternal toxic effects:
Adult Responses:

A summary of female performance is given in Table 1 (see attached background material).

Mortality:
There were no unscheduled deaths.

Clinical Observations:
A summary incidence of daily clinical observations is given in Table 2 (see attached background material).

At 1000 mg/kg bw/day, the majority of females showed incidences of increased post-dosing salivation between Day 10 and Day 19 of gestation. At 300 mg/kg bw/day, increased post-dosing salivation was restricted to just one female on Day 10 and two females on Day 19 of gestation. Increased post-dosing salivation is commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and, in isolation, is considered to be of no toxicological importance.

There were no other clinical signs apparent during the study.

Body Weight:
Group mean body weights and standard deviations are given in Table 3 and presented graphically in Figure 1. Group mean body weight gains and adjusted body weights and standard deviations are given in Table 4 and Table 5 (see attached background material).

At 1000 mg/kg bw/day, body weight gain was generally lower than control throughout gestation, with differences frequently attaining statistical significance and this lower weight gain was still apparent after values were adjusted for the contribution of the gravid uterus.

Body weight gain during gestation, included where adjusted for the contribution of the gravid uterus, was considered to be unaffected by treatment at 100 and 300 mg/kg bw/day.

Food Consumption:
Group mean food consumptions are given in Table 6 and presented graphically in Figure 2 (see attached background material).

At 1000 mg/kg bw/day, food consumption was generally lower than control between Day 3 and Day 8 of gestation, with differences attaining statistical significance. Thereafter, although food intake remained lower than control, differences failed to attain statistical significance and the magnitude of the differences observed was such that they were considered to represent normal biological variation.

Food consumption appeared unaffected by treatment at 100 and 300 mg/kg bw/day.

Water Consumption:
Daily visual inspection of water bottles did not reveal any overt intergroup differences.

Post Mortem Studies:
A summary incidence of female necropsy findings is given in Table 7 (see attached background material).

No macroscopic abnormalities were detected for adult animals at Day 20 of gestation.














Key result
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Litter Responses:
Litter Data:
Summary fetal data is given in Table 8 (see attached background material).

There was no effect of maternal treatment on litter data as assessed by numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size, sex ratio and pre and post-implantation losses at 100, 300 and 1000 mg/kg bw/day.

At 300 and 1000 mg/kg bw/day, lower pre-implantation losses attained statistical significance when compared with control; these differences were considered to reflect normal biological variation particularly as an improvement in survival is unlikely to represent an adverse effect of treatment.

Litter Placental and Fetal Weights:
Group mean litter data values are given in Table 8 (see attached background material).

At 1000 mg/kg bw/day, mean fetal weight for both sexes was lower than control, resulting in lower mean litter weights; differences from control attaining statistical significance. There was considered to be no effect of treatment on placental weights.

There were no effects of maternal treatment on mean fetal, litter or placental weights at 100 or 300 mg/kg bw/day.

External Fetal Examinations:
Summary fetal external findings are given in Table 9 (see attached background material).

At 1000 mg/kg bw/day, there was a higher incidence of fetuses that were small in appearance compared with control; this finding was consistent with the lower fetal weight observed at this dosage.

At 100 and 300 mg/kg bw/day external fetal findings were unremarkable and did not indicate any effect of maternal treatment.

Detailed Visceral Fetal Examinations:
Summary fetal visceral findings are given in Table 10 (see attached background material).

At 1000 mg/kg bw/day, there was an increased incidence of fetuses/litters showing non-uniform patterning of the rugae, kinked/dilated ureter(s), increased renal pelvic cavitation, absent renal papila and partially undescended thymus lobe, with the percentage incidence of these findings frequently attaining statistical significance. The increased incidence of these findings was principally responsible for a statistically significant increased incidence in the overall number of fetuses showing visceral findings.

At 300 mg/kg bw/day, there was an increased incidence of fetuses/litters showing kinked/dilated ureter(s) compared with control; although the percentage incidence of these findings did not attain statistical significance when compared with control, it was higher than the recent background control incidence. The overall percentage incidence of visceral findings was not greatly increased in comparison to the control and failed to attain statistical significance.

At 100 mg/kg bw/day, the incidence and type of visceral findings observed did not indicate any effect of maternal treatment.

Detailed Skeletal Fetal Examinations:
Summary fetal skeletal findings are given in Table 11 (see attached background material).

At 1000 mg/kg bw/day, there was clear effect of treatment on a large number of ossification parameters affecting most regions of the skeleton, with the number of fetuses/litters affected being increased compared with control and differences frequently attaining statistical significance. These parameters included unossified hyoid and incomplete ossification of the nasal, frontal, parietal, interparietal, occipital, squamosal, premaxilla, presphenoid bones of the skull, incomplete ossification of the cervical arch, thoracic centrum and sacral arch, unossified thoracic centrum and sacral (neural) arch, less than 4 caudal vertebrae ossified, unossified/ incomplete ossification of the sternebra, incomplete ossification of the ischium, unossified/incomplete ossification of the pubis, unossified/incomplete ossification of the metacarpals and metatarsals and incomplete ossification of the humerus and femur. There was also a lower incidence of fetuses showing ossification of the ventral arch of the first cervical vertebra (vertebra 1).

At 100 and 300 mg/kg bw/day, the incidence and type of skeletal findings observed did not indicate any effect of maternal treatment. The percentage incidence of occasional skeletal parameters did attained statistical significance but these differences were considered to reflect normal biological variation.








Key result
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Discussion

Treatment at 1000 mg/kg bw/day was associated with lower maternal body weight gain during gestation and an initial effect on food consumption. While part of the lower overall weight gain observed was attributable to lower litter weight due to reduced fetal weight, an underlying effect of the pregnant dam was still present when body weight gain was adjusted for the contribution of the gravid uterus.

In-utero survival of the developing conceptus appeared unaffected by maternal treatment at 1000 mg/kg bw/day with both pre and post-implantation losses being lower than control. This was despite a clear reduction in fetal weight which resulted in lower litter weight at this dosage. The lower foetal weight at 1000 mg/kg bw/day is suggestive of a retardation of fetal growth at this dosage and this was supported by subsequent findings observed at fetal examination. Externally many of the fetuses appeared small and there was a plethora of skeletal findings indicating incomplete ossification or no ossification for many regions of the skeleton. Visceral findings included non-uniform patterning of the rugae, kinked/dilated ureter(s), increased renal pelvic cavitation, absent renal papila and partially undescended thymus lobe. These visceral findings were also considered to indicate a retardation of fetal growth. 

While there is no doubt that there has been an effect of maternal treatment on the fetuses, there was no evidence of any structural defects that would indicate any adverse effect on development per se and fetal survival was clearly unaffected. The examination of the fetuses on Day 20 is a window in time and the fetuses will continue to grow and develop after this time. 

For females at 300 mg/kg bw/day, clinical signs, body weight performance, food consumption and macroscopic necropsy examinations did not indicate any obvious effect of treatment and this dosage is considered to represent a No Observed Effect Level (NOEL) for the pregnant female. Litter data, fetal, litter and placental weights, external fetal appearance and detailed skeletal fetal evaluation did not indicate any obvious effect of maternal treatment on the developing conceptus. However, visceral examination of the fetuses showed an increased incidence of fetuses/litters with kinked/dilated ureter(s) compared with control. Although the incidence was much lower than observed at 1000 mg/kg bw/day and failed to attain statistical significance, it was higher than the recent background control incidence and an association with maternal treatment cannot be discounted. These visceral findings may be the most sensitive indicator of the retardation of fetal growth demonstrated at the high dosage and therefore the classification of this dosage as a No Observed Effect Level (NOEL) for the developing conceptus is equivocal. However, in view of the findings apparent at 1000 mg/kg bw/day, these findings at 300 mg/kg bw/day are considered not to indicate any effect on the structural development of the fetuses at 300 mg/kg bw/day.

A dosage of 100 mg/kg bw/day is therefore considered to be a clear No Observed Effect Level (NOEL) for the developing conceptus.

Kinked/dilated uretus are considered considered reversible variations (Solecki, R. et al. Reproductive Toxicilogy 17 (2003) 635 -637) and therefore not considered adverse. The NOAEL was therefore 300 mg/kg bw/day.

Conclusions:
Treatment at 1000 mg/kg bw/day was associated with lower maternal body weight gain during gestation and an initial effect on food consumption. No similar effects were apparent at 300 mg/kg bw/day and this dosage is considered to represent the No Observed Effect Level (NOEL) for the pregnant female. Kinked/dilated uretus are considered considered reversible variations (Solecki, R. et al. Reproductive Toxicilogy 17 (2003) 635 -637) and therefore not considered adverse. The NOAEL was therefore 300 mg/kg bw/day.

In-utero survival of the developing conceptus was unaffected by maternal treatment at 1000 mg/g bw/day although reduced fetal weight and external, visceral and skeletal findings indicated an adverse effect on fetal growth. The absence any structural defects indicated that development per se was unaffected at this dosage. Only an equivocal increase in the incidence of fetuses/litter showing kinked/dilated ureter(s) prevented 300 mg/kg bw/day being classified as a fetal No Observed Effect Level and a dosage of 100 mg/kg bw/day is therefore considered to be a clear No Observed Effect Level (NOEL) for the developing conceptus.
Executive summary:

Introduction

The study was designed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female from Day 3 to Day 19 of gestation (and including the period of organogenesis). The results of the study are believed to be of value in predicting the toxicity of the test item during pregnancy, and the estimation of both a maternal and embryofetal ‘No Observed Effect Level’ (NOEL).

 

The study was designed to comply with the following guidelines:

- US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)

- Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)

- OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

Methods

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD (SD) IGS BR strain rats, between Days 3 and 19 of gestation inclusive at dose levels 100, 300, and 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Corn Oil) to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study. 

 

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were preserved in 70% Industrial Methylated Spirit (IMS) and then following examination for skeletal development transferred into 50% glycerol. The remaining half were preserved in Bouin’s solution and transferred to 90% IMS in distilled water and the viscera were examined.

Results…….

Mortality

There were no unscheduled deaths.

 

Clinical Observations

At 1000 mg/kg bw/day, the majority of females showed incidences of increased post-dosing salivation between Day 10 and Day 19 of gestation. At 300 mg/kg bw/day, increased post-dosing salivation was restricted to just three females on isolated days on the study.

 

Body Weight

At 1000 mg/kg bw/day, body weight gain was lower than control throughout gestation and this lower weight gain was still apparent after values were adjusted for the contribution of the gravid uterus.

 

Food Consumption

At 1000 mg/kg bw/day, food consumption was lower than control between Day 3 and Day 8 of gestation.

 

Water Consumption

No treatment related effects were apparent during the study.

 

Post Mortem Studies

No macroscopic abnormalities were detected during adult necropsy.

 

Litter Responses

Litter Data

There was no effect of maternal treatment on litter data at dosages up to 1000 mg/kg bw/day.

 

Litter Placental and Fetal Weights

At 1000 mg/kg bw/day, mean fetal weights for both sexes was lower than control, resulting in lower mean litter weights; placental weights were unaffected.

 

Fetal Examination

External Examinations

At 1000 mg/kg bw/day, there was a higher incidence of fetuses that were small in appearance compared with control.

 

Detailed Visceral Examinations

At 1000 mg/kg bw/day, there was an increased incidence of fetuses/litters showing non-uniform patterning of the rugae, kinked/dilated ureter(s), increased renal pelvic cavitation, absent renal papila and partially undescended thymus lobe, leading to an increased incidence in the overall number of fetuses showing visceral findings.

 

At 300 mg/kg bw/day, there was an equivocal increased incidence of fetuses/litters showing kinked/dilated ureter(s) compared with control.    

 

Detailed Skeletal Examinations

At 1000 mg/kg bw/day, there was clear effect of treatment on a large number of ossification parameters affecting most regions of the skeleton, with the number of fetuses/litters affected being increased compared with control.

 

Conclusion

Treatment at 1000 mg/kg bw/day was associated with lower maternal body weight gain during gestation and an initial effect on food consumption. No similar effects were apparent at 300 mg/kg bw/day and this dosage is considered to represent the No Observed Effect Level (NOEL) for the pregnant female.    

 

In-utero survival of the developing conceptus was unaffected by maternal treatment at 1000 mg/kg bw/day although reduced fetal weight and external, visceral and skeletal findings indicated an adverse effect on fetal growth. The absence any structural defects indicated that development per se was unaffected at this dosage. Only an equivocal increase in the incidence of fetuses/litter showing kinked/dilated ureter(s) prevented 300 mg/kg bw/day being classified as a fetal No Observed Effect Level and a dosage of 100 mg/kg bw/day is therefore considered to be a clear No Observed Effect Level (NOEL) for the developing conceptus.

Kinked/dilated uretus are considered considered reversible variations (Solecki, R. et al. Reproductive Toxicilogy 17 (2003) 635 -637) and therefore not considered adverse. The NOAEL was therefore 300 mg/kg bw/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 19 November 2018. Experimental completion date: 25 January 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Test item: tert-butyl perbenzoate (CAS # 614-45-9).
Test item identity (including alternative names): Trigonox C (Trade name) tert-butyl peroxybenzoate
CAS number: 614-45-9.
Intended use: Industrial chemical - polymerization initiator.
Appearance: Colorless to slightly yellow liquid.
Storage conditions: At ambient temperature (15 to 25 °C) protected from light.
Supplier: Sponsor.
Batch number: 1802465400.
Expiry date: 1 April 2028.
Purity: 98.8%.
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Animals
Strain/Species: New Zealand White rabbit.
Supplier: Envigo RMS Limited (UK).
Number of animals ordered: 96 time mated females (delivered as four batches of 24 females).
Duration of acclimatization: Five days before commencement of treatment.
Age of the animals at the start of the study (Day 1 of gestation): 18 to 22 weeks old.
Weight range of the animals at the start of the study (Day 1 of gestation): 2.44 to 4.16 kg.

Animal Care and Husbandry
Environmental Control
Multispecies facility: Partial barrier with limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity: Monitored and maintained within the range of 15-21°C and 45-70%. There were no deviations from these ranges.
Lighting: Artificial lighting, 14 hours light : 10 hours dark.
Alarm systems: Activated on ventilation failure and when temperature/humidity limits exceeded.
Electricity supply: Public supply with automatic stand-by generators.

Animal Accommodation
Cages: Suspended cages fitted with perforated floor panels and resting platforms, mounted in batteries. Undertrays lined with absorbent paper were changed at least three times a week.
Cage distribution: The cages constituting each group were blocked by group and mounted in batteries.
Number of animals per cage: One female.

Environmental Enrichment
Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study and replaced when necessary.
Stainless steel key ring: Attached to the cage.
Cage paper: Provided to each cage from Day 20 after mating to allow expression of nesting behaviour and replaced as necessary.

Diet Supply
Diet: Teklad 2930, pelleted Diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability: 200 g/animal/day
If individual showed a significant non-treatment related reduced food consumption, moistened diet (50 g pelleted diet moistened with 50 mL of water) was offered and the consumption recorded; this data has not been reported and is retained in the raw data. In addition to this diet, a small supplement of autoclaved hay was given on a daily basis to promote gastric motility and a small amount of chopped fresh vegetables were given twice weekly. Consumption of hay and vegetables were monitored qualitatively but not quantitatively.

Water Supply
Supply: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability: Non-restricted.
Route of administration:
oral: gavage
Vehicle:
other: Aqueous 1% methylcellulose
Details on exposure:
The oral (gavage) route of administration was chosen to simulate the conditions of possible human exposure.

Test Item Preparation and Analysis
Correction factor None.
Vehicle Aqueous 1% methylcellulose
Method of preparation: The required amount of test material was weighed out into a suitable container. Approximately 40% of the final volume of vehicle was added to the test material and mixed with a magnetic stirrer until homogeneous. The solution was then made up to required volume with vehicle. The formulation was then mixed using a high sheer homogeniszer.
A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item.
Frequency of preparation: Weekly
Storage of formulation: Refrigerated (2 to 8°C).
Test item accounting: Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Administration
Route: Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at: Constant doses in mg/kg/day.
Volume dose: 5 mL/kg body weight.
Individual dose volume: Calculated from the most recently recorded scheduled body weight.
Formulation A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found. Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1 and 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.
Achieved concentration: Samples of each of the first and last formulation prepared were analyzed for achieved concentration of the test item.

Test Item Preparation and Analysis
Apparatus
Balances fitted with printers: Capability of weighing to 5 or 6 decimal places
General laboratory apparatus and glassware.
Reagents
Control vehicle: Aqueous 1% methylcellulose
Acetonitrile (ACN): Gradient HPLC grade
Water: Reverse osmosis
ortho-phosphoric acid (o-H3PO4): Analytical reagent

Preparation of Calibration Standards
A primary standard solution (1000 μg/mL) was prepared by dissolving an accurately weighed quantity (ca. 50 mg) of Tert-butyl perbenzoate (CAS # 614-45-9) in ACN/Water 60/40 v/v (50 mL). A secondary standard solution (100 μg/mL) was prepared by appropriate dilution of the primary standard (2 mL) using diluent (20 mL).
Solutions for instrument calibration were prepared by appropriate dilution of the secondary standard using ACN/Water 60/40 v/v and contained Tert-butyl perbenzoate (CAS # 614-45-9) at nominal concentrations of 1 μg/mL, 2 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL and 30 μg/mL.
Calibration solutions were injected onto the UPLC, at the beginning and end of each sample analysis sequence as a minimum, using the conditions detailed in the chromatographic section.

Preparation of Test Samples
A representative sample of test formulation (1 mL, accurately weighed) was dissolved using vigorous shaking and swirling in a suitable volume of ACN/Water 60/40 v/v. The extract was diluted using ACN/Water 60/40 v/v, to provide a solution containing Tert-butyl perbenzoate (CAS # 614-45-9) at an expected concentration within the range 2 μg/mL to 20 μg/mL. The concentration of Tert-butyl perbenzoate (CAS # 614-45-9) in the final solution was quantified by UPLC using UV detection as detailed in the chromatographic section.

Preparation of Recovery Samples
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (aqueous 1% methylcellulose) with known amounts of Tert-butyl perbenzoate (CAS # 614-45-9). The prepared procedural recoveries were analyzed in accordance with the analytical procedure.

Instrumentation Parameters
Ultra performance liquid chromatograph (UPLC): Acquity ultra performance LC (including binary solvent manager, sample manager, column manager and TUV detector)
Column: Waters Acquity BEH C18, 1.7 pm, 2.1 x 50 mm
Column temperature: 50°C
Sample temperature: Ambient
Mobile Phase A: Acetonitrile/Water/ortho-phosphoric acid 10/90/0.1 v/v/v
Mobile Phase B: Acetonitrile/Water/ortho-phosphoric acid 90/10/0.1 v/v/v
Gradient: Time (min) % A %B
0.00 60 40
1.80 10 90
2.20 10 90
2.30 60 40
4.00 60 40
Flow rate: 0.6 mL/min
Weak rinse solvent: ACN/Water 50/50 v/v, 600 μL
Strong rinse solvent: ACN 100%, 200 μL
Detector wavelength: UV, 210 nm
Injection volume: 5 μL
Run time: 4.0 minutes
Approximate retention time: 0.9 minutes

Validation of the Analytical Procedure
The analytical procedure was validated by determining the following parameters:
The specificity of the chromatographic analysis in control sample chromatograms.
The limit of detection and quantification was estimated by examination of control vehicle chromatograms in order to calculate a test item concentration based on a peak height response equivalent to three times baseline noise and ten times baseline noise respectively.
The linearity of detector response over the calibration standard concentration range.
The repeatability of the lowest and highest concentration calibration standards.
The method accuracy and precision, by determining five procedural recoveries at nominal concentrations of 1 mg/mL and 200 mg/mL during the method validation.

Homogeneity and Stability in Aqueous 1% Methylcellulose Formulations
The homogeneity and stability of Tert-butyl perbenzoate (CAS # 614-45-9) in aqueous 1% methylcellulose formulations was assessed at nominal concentrations of 1 mg/mL and 200 mg/mL, during ambient and refrigerated storage. Freshly prepared specimen formulations (300 mL) were equally sub divided into four amber glass screw top bottles by Pharmacy personnel and submitted for analysis.

Ambient Temperature Storage (15 to 25ºC)
On receipt, the contents of one bottle of each formulation were mixed by 20-fold inversion followed by magnetic stirring. After stirring for a minimum of 20 minutes (representing 0 hour) and 4 hours, single samples (nominally 1 mL) were removed for analysis from the top, middle and bottom of the continuously stirred formulation.
The remainder of the bottle was stored at ambient temperature and after storage for 5 days; the contents were remixed and sampled as detailed above.

Refrigerated Storage (2 to 8ºC)
The remaining bottles were refrigerated on receipt and on Day 5 and Day 15. The appropriate bottle was removed from storage and equilibrated to ambient temperature. The contents of the bottle were mixed by 20-fold inversion followed by magnetic stirring for a minimum of 20 minutes and single samples (nominally 1 mL) were removed for analysis from the top, middle and bottom of the stirred formulation.

Concentration of Dose Formulations
For the First formulation and Last formulation, freshly prepared test formulations were sampled (4 × 1 mL, accurately weighed) by Pharmacy personnel and submitted for analysis. Two samples were analyzed in accordance with the analytical procedure, and the remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved.

RESULTS
Method Validation
The analytical procedure was successfully validated for Tert-butyl perbenzoate (CAS # 614-45-9) in aqueous 1% methylcellulose with respect to the specificity of chromatographic analysis, limit of detection and quantification, the linearity of detector response, repeatability, method accuracy and precision, calibration standard stability and extract stability. Results are summarized below:
The specificity of the UPLC assay was demonstrated by the absence of a peak at the characteristic retention time for Tert-butyl perbenzoate (CAS # 614-45-9) in the control sample chromatogram.
The limits of detection and quantification were estimated as 0.0129 μg/mL and 0.0431 μg/mL respectively.
Linearity was confirmed over the nominal concentration range 1 μg/mL to 30 μg/mL with a coefficient of determination >0.999.
The repeatability was <1% for six replicate injections of standard solutions containing Tert-butyl perbenzoate (CAS # 614-45-9) at nominal concentrations of 1 μg/mL and 30 μg/mL.
Method accuracy and precision were confirmed: a mean procedural recovery value of 97.2% (CV=0.17%, n=5) was obtained for 1 mg/mL and 101.0% (CV=0.14%, n=5) was obtained for 200 mg/mL.
Calibration standard stability was confirmed after refrigerated storage for 13 days.
Extract stability was confirmed after refrigerated storage for 13 days.

Homogeneity and Stability of Dose Formulations
The homogeneity and stability of Tert-butyl perbenzoate (CAS # 614-45-9) in aqueous 1% methylcellulose formulations was assessed with respect to the level of concentration at nominal concentrations of 1 mg/mL and 200 mg/mL.
Homogeneity and stability was confirmed during distribution between the bottles, during magnetic stirring for 4 hours, and on re-suspension following storage at ambient temperature for 5 days and refrigeration for up to 15 days. At each time-point, the mean analyzed concentration for the three samples remained within 7% of the initial time zero value and the coefficient of variation was less than 3%.
Recovery results during the trial remained within ±7.5% of the mean recovery found during validation showing the continued accuracy of the method.

Concentration of Dose Formulations
The mean concentrations were within 4% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 1%, confirming precise analysis.

CONCLUSION
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limits of detection and quantification, linearity of detector response, repeatability, method accuracy and precision, calibration standard stability and extract stability.
The homogeneity and stability was confirmed for Tert-butyl perbenzoate (CAS # 614-45-9) in aqueous 1% methylcellulose formulations at nominal concentrations of 1 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 4 hours, ambient temperature storage (15 to 25ºC) for 5 days and refrigerated storage (2 to 8ºC) for up to 15 days.
The mean concentrations of Tert-butyl perbenzoate (CAS # 614-45-9) in test formulations analyzed for the study were within ±4% of nominal concentrations, confirming accurate formulation. The difference from mean remained within 1%, confirming precise analysis.
Details on mating procedure:
Method: Natural mating with New Zealand white bucks of established fertility at the supplier’s facility. Males and females not closely related.
After mating: Each female was injected intravenously with 25 i.u. luteinizing hormone.
Delivery to Envigo: Day 1 after mating.
Duration of treatment / exposure:
Females were treated from Day 6 to Day 28 (inclusive) after mating
Frequency of treatment:
Once daily at approximately the same time each day.
Duration of test:
Mating on day 0. Necropsy day 29
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
80 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Animal Model
The rabbit was chosen as the test species because it is accepted by regulatory agencies. The New Zealand White strain was used because of the historical control data available in this laboratory.

Rationale for Dose Level Selection
Dose levels of 0, 25, 80 and 250 mg/kg/day were selected in conjunction with the Sponsor based on findings from the preliminary embryo-fetal development study (Envigo Study No. MN05DC).
In the preliminary embryo-fetal development study (MN05DC) animals received doses of 100, 200 or 300 mg/kg/day during Gestation Days (GD) 6-28. The primary effect was restricted to low food consumption at 200 and 300 mg/kg/day. The effect on food consumption at 200 mg/kg/day was minimal, however at 300 mg/kg/day one female was killed for welfare reasons after a period of marked inappetence with associated clinical signs and body weight loss. For the surviving females there was no adverse effect on maternal clinical condition or bodyweight and the embryo-fetal survival and development was unaffected by treatment.
In view of the mortality a dose of 250 mg/kg/day was considered suitable as the high dose on this study with low and intermediate dose levels of 25 and 80 mg/kg/day to provide approximate 3-fold dose intervals.

Allocation and Identification
Allocation: On arrival (Day 1 after mating).
Method: Allocation was controlled to prevent any stock male from providing more than one mated female in each treated group and to prevent more than one sibling female in each group, where possible.
Identification of animals: Each animal was assigned a number and identified uniquely within the study using a microchip.
Identification of cages: Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant.
Maternal examinations:
Serial Observations

Mortality
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.
A complete necropsy was performed in all cases

Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
Signs Associated with Dosing
Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration:
Pre-dose observation
One to two hours after completion of dosing
As late as possible in the working day.
Clinical Signs
A detailed physical examination was performed on each animal on arrival (Day 1 after mating) and on Days 6, 12, 18, 24 and 29 after mating to monitor general health.

Body weight
The weight of each adult was recorded on arrival (Day 1 after matting) and on Days 3 and 6-29 after mating.

Food Consumption
The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily from Day 2 after mating.

Terminal Investigation
Method of Kill
Method of kill for all adult animals Intravenous injection of sodium pentobarbitone.
Method of kill for fetuses Subcutaneous injection of sodium pentobarbitone.

Necropsy
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preservedin appropriate fixative.
Schedule: Animals surviving until the end of the scheduled study period were killed on Day 29 after mating.
Sequence: To allow satisfactory inter-group comparison.
Ovaries and uterine content:
Reproductive Assessment
For females surviving to term, the following was recorded:
Uterus: Gravid uterine weight (including cervix and ovaries).
The following were recorded for all animals (including those prematurely sacrificed, where possible):
For each ovary/uterine horn
Number of: Corpora lutea. Implantation sites. Resorption sites (classified as early or late). Fetuses (live and dead).
Apparently non-pregnant animals and for apparently empty uterine horns: The absence or number of uterine implantation sites was confirmed.
Fetal examinations:
Fetal Examination and Processing
Examination of all viable fetuses and placentae: Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded, sampled as appropriate and retained in appropriate fixative. All fetuses were subject to a gross internal examination of the viscera of the neck, thorax and abdominal cavities and the sex of each fetus was also recorded.
Fixation: Half (50%) of the fetuses were decapitated and the heads initially stored in Bouin’s fluid. Remaining fetuses and torsos were eviscerated and fixed in Industrial Methylated Spirit.
Processing: Bouin’s fixed fetal heads were subject to free-hand serial sectioning. Industrial Methylated Spirit fixed fetuses and torsos were processed and stained with Alizarin Red.

Fetal Pathology Examination
Bouin’s fixed heads: Serial sections were examined for soft tissue abnormalities.
Alizarin Red stained fetuses and torsos: Assessed for skeletal development and abnormalities.
Statistics:
Please see any other information on materials and methods incl. tables.
Clinical signs:
no effects observed
Description (incidence and severity):
The clinical condition of the animals at dose levels up to and including 250 mg/kg/day was considered to be unaffected by treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female (no. 26) receiving 25 mg/kg/day was killed for reasons of animal welfare on GD15. Terminal signs included inappetance, including reduced hay consumption, body weight loss (390g), decreased fecal output and dry rales. Macroscopic examination revealed dark red area in the glandular mucosa of the stomach and uterine examination confirmed that the animal was pregnant. The signs exhibited by this low dose female were atypical for the group and the macropathology was not apparent in other females at this dose level or at higher dose levels. This welfare kill was therefore considered to be unrelated to treatment.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
From the onset of treatment on GD6 females at 250 mg/kg/day showed low body weight gain up to GD14, thereafter weight gain was similar to Controls and overall (GD6-29) there was no adverse effect of treatment. Body weight gain for females receiving 25 or 80 mg/kg/day was unaffected by treatment.
Maternal body weight gain following adjustment for the gravid uterine weight showed no adverse effects of treatment at dose levels up to and including 250 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
From GD6 animals receiving 250 mg/kg/day showed slightly reduced food consumption compared to pretreatment values and from GD7/8 food consumption was slightly low when compared with concurrent Controls with the difference attaining statistical significance on GD9-16. From GD17 food consumption at 250 mg/kg/day was similar to the Controls.
At 80 mg/kg/day mean food consumption was slightly low on GD13-15, but overall food consumption at 25 or 80 mg/kg/day showed no adverse effects of treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of females on GD29 did not reveal any abnormality.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Litter data as assessed by the extent of pre- and post-implantation loss was unaffected by maternal treatment at dose levels up to and including 250 mg/kg/day.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Litter data as assessed by the number of resorptions was unaffected by maternal treatment at dose levels up to and including 250 mg/kg/day.
Early or late resorptions:
no effects observed
Description (incidence and severity):
Litter data as assessed by the number of resorptions was unaffected by maternal treatment at dose levels up to and including 250 mg/kg/day.
Dead fetuses:
no effects observed
Description (incidence and severity):
Litter data as assessed by the number live young was unaffected by maternal treatment.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Litter data as assessed by the number of implantations was unaffected by maternal treatment at dose levels up to and including 250 mg/kg/day.
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
gross pathology
maternal abnormalities
pre and post implantation loss
total litter losses by resorption
early or late resorptions
dead fetuses
changes in number of pregnant
necropsy findings
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Fetal and placental weight was unaffected by maternal treatment.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Overall fetal pathology did not show any adverse effects of maternal treatment on embryo-fetal development.
At 25 and 250 mg/kg/day there were a small number of unrelated major abnormalities, but as they were of differing aetiology and were not apparent at 80 mg/kg/day there are considered to be unrelated to maternal treatment.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Overall fetal pathology did not show any adverse effects of maternal treatment on embryo-fetal development.
At 25 and 250 mg/kg/day there were a small number of unrelated major abnormalities, but as they were of differing aetiology and were not apparent at 80 mg/kg/day there are considered to be unrelated to maternal treatment.
Within all groups there were also a number of minor structural vertebral abnormalities affecting all areas of the vertebral column (cervical, thoracic, lumbar and caudal), some causing scoliosis or a minimal curvature in the vertebral column. However, the fetal/litter incidence of these findings did not show any relationship to treatment.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Overall fetal pathology did not show any adverse effects of maternal treatment on embryo-fetal development.
At 25 and 250 mg/kg/day there were a small number of unrelated major abnormalities, but as they were of differing aetiology and were not apparent at 80 mg/kg/day there are considered to be unrelated to maternal treatment.
Other effects:
effects observed, non-treatment-related
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
fetal/pup body weight changes
external malformations
skeletal malformations
visceral malformations
Developmental effects observed:
no
Conclusions:
Oral administration of tert-butyl perbenzoate to pregnant New Zealand White rabbits during organogenesis and the fetal growth phase at 25, 80 and 250 mg/kg/day was well tolerated with no adverse effects on general condition, maternal body weight, food consumption, macropathology or embryo-fetal survival and development. Effects were limited to slight but transient effects on maternal food consumption and body weight.
It is therefore concluded that the no observed adverse effect level (NOAEL) in this study for both maternal toxicity and embryo-fetal survival/development is 250 mg/kg/day.
Executive summary:

The purpose of this study was to assess the influence of tert-butyl perbenzoate (CAS # 614-45-9), a polymerization initiator, on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the New Zealand White rabbit.

Four groups of 24 females received tert-butyl perbenzoate (CAS # 614-45-9) at doses of 25, 80 or 250 mg/kg/day by oral gavage administration, from Day 6 to 28 after mating. A similarly constituted Control group received the vehicle, aqueous 1% methylcellulose at the same volume dose as treated groups. Animals were killed on Day 29 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 29 after mating and the gravid uterine weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

Results and Conclusion

Oral administration of tert-butyl perbenzoate to pregnant New Zealand White rabbits during organogenesis and the fetal growth phase at 25, 80 and 250 mg/kg/day was well tolerated with no adverse effects on maternal condition, body weight, food consumption and macropathology or on embryo-fetal survival and development. Effects were limited to slight but transient effects on maternal food consumption and body weight.

It is therefore concluded that the no observed adverse effect level (NOAEL) in this study for both maternal toxicity and embryo-fetal survival/development is 250 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Species:
rat
Quality of whole database:
The study is reliable without restrictions.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

There were no reproductive effects in an OECD 421 study. For reproduction toxicity the NOEL (No Observed Effect Level) was at 1000 mg/kg/day for females (the highest dose administered) and at 750 mg/kg for males (the highest dose administered).

Due to lower mean pup weight at 1000 mg/kg, in an OECD 421 study, the NOEL for developmental toxicity was considered to be 300 mg/kg bw/day.

The only notable effect in an OECD 414 (rat) study was delayed developmental effects in the presence of reduced maternal weight gain and food consumption. Based on this the NOAEL for development in rats is set at 300 mg/kg bw/day. In an OECD 414 study in rabbits no adverse effecte on development were observed up to the highest dose test; 250 mg/kg bw/day.

Based on these results the substance is not classified for effects on fertility or development.


Route: .live2