Registration Dossier

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-06-06
Reliability:
2 (reliable with restrictions)
Qualifier:
according to
Guideline:
other: Expert statement
Principles of method if other than guideline:
No guideline followed.
GLP compliance:
yes
Details on test animals and environmental conditions:
Not applicable.
Details on exposure:
Not applicable.
Duration and frequency of treatment / exposure:
Not applicable.
Remarks:
Doses / Concentrations:
Not applicable.
No. of animals per sex per dose:
Not applicable.
Positive control:
Not applicable.
Details on study design:
Not applicable.
Details on dosing and sampling:
Not applicable.
Statistics:
Not applicable.
Preliminary studies:
Not applicable.
Details on absorption:
Generally, oral absorption is favoured for molecular weights below 500 g/mol. However, based on the high logPow of 5.1 to 5.4 TBPND can be regarded as lipophilic substance. This characteristic combined with the relatively low water solubility may limit oral absorption by the inability of this substance to dissolve in the gastro-intestinal fluids, which in turn hinders contact with the mucosal surface. On the other hand, absorption of such a lipophilic compound may be facilitated following possible micellular solubilisation by bile salts. With regards to the high lipophilicty and low water solubility, the mechanisms of micellular solubilisation may be of some importance for TBPND, as the substance would otherwise be poorly absorbed. Administered without a vehicle in an acute oral toxicity study performed on rats, TBPND lead to a high LD50 of 8082 mg/kg bw/day. Moreover, no toxic effects relating to a systemic absorption were observable. Based on this results it can be assumed that only limited absorption of the substance itself across the epithelial lining of the gastro intestinal tract will occur when administered orally. However, the results from the 14 day dose range finding study and the combined repeated dose toxicity study with the reproduction/ developmental toxicity screening study, both conducted on male and female Wistar rats, indicate that the compound, or more likely, its hydrolysis products became bioavailable. This conclusion is supported by the results of the OECD guideline 414 study. In this regards, as indicated by the half-life values from the hydrolysis test, a large fraction, if not all, of TBPND will hydrolyze to tert-butanol and isomers of neodecanoic acids following oral administration which is indicated by the relative short half-live in an aqueous solution at acidic to neutral conditions. The results of the hydrolysis tests at a pH range of 4 to 9 are somewhat representative for the conditions found in the GIT with the stomach having an acidic milieu (~ pH 1.4 to 4.5) and the intestine a slightly acidic to slighty alkaline milieu (~ pH 5 to 8). Due to the lower log Pow values of the hydrolyis products, it is assumed that they may be readily absorbed through the GIT epithelium. Furthermore, the low molecular weight of tert-butanol (74.12 g/mol) combined with its relatively high water solubility (> 10 g/L) may allow the direct uptake into the systemic circulation through aqueous pores or via carriage of the molecules across the membrane with the bulk passage of water. Due to the relatively low vapour pressure of TBPND (approximately 50 Pa) and the resulting low volatility, an inhalation exposure of the compound’s vapour phase is rather unlikely. Moreover, if the substance would reach the lungs in its vapour or gaseous state, the lipophilic character of TBPND hinders the direct absorption across the respiratory tract epithelium. An acute inhalation toxicity study performed on rats using TBPND in its aerosol form revealed a relatively high LC50 of 37.50 mg/L. No specific effects of systemic toxicity were observed and these results indicate for low systemic availability after inhalation. Even if bioavailable, systemic toxicity effects may only occur following an unlikely high exposition to the substance via this route of administration. Similarly, based on physico–chemical properties of TBPND the substance is not likely to penetrate skin to a large extent as the high log Pow value and low water solubility do not favour dermal penetration. It is general accepted that if a compound’s water solubility falls between 1-100 mg/L, absorption can be anticipated to be low to moderate. Moreover, for substances with a log Pow between 4 and 6, the rate of penetration is limited by the rate of transfer between the stratum corneum and the epidermis. Only the uptake into the outer, non-viable layer stratum corneum may be high as the underlying viable epidermis is very resistant to penetration by highly lipophilic compounds. These assumptions based on the physico-chemical properties of TBPND are further supported by the results achieved from an acute dermal toxicity study performed on rabbits. During this study no mortality, no test item related changes in the body weight and no systemic toxic effects were observed for the highest dose of TBPND used in the test and the respective LD50 was determined to be greater than 6000 mg/kg bw. Although, application of TBPND to the skin of rabbits caused irritation in form of erythema and edema in two independently performed skin irritation/corrosion studies, no evidence of full thickness destruction of the skin or scar tissue was observed which in turn could have favoured direct absorption into the systemic circulation. When applied topically onto the skin of guinea pigs, sensitising effects were observed following an initial intradermal induction phase. This indicates that at least a small fraction of the substance has become available in the body to initiate the immune response. However, the effects may also be caused by the formation of reaction products between TBPND and molecules present in the skin. Furthermore, it has to be kept in mind that the vehicle propylene glycol which was used to aid the topical application during the challenge phase could have potentially influenced the extent of dermal absorption.
Details on distribution in tissues:
Assuming that TBPND is absorbed into the body following oral intake, it may be distributed into the interior part of cells due to its lipophilic properties and in turn the intracellular concentration may be higher than extracellular concentration particularly in adipose tissues. However, it is expected that TBPND does not reach the blood without starting to hydrolyse into its hydolysis products. As mentioned above, the physicochemical properties of the hydrolysis products favour systemic absorption. Especially the low molecular weight and relatively high water solubility of tert-butanol favours absorption. Direct transport through aqueous pores is likely to be an entry route to the systemic circulation. The results from the combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test indicate that, following absorption, the liver and the kidney are the primary target organs affected by the chemicals. Based on the results observed on the reproduction and developmental performance of the animals it is difficult to judge if the hydrolysis products have the ability to cross the placenta and cause a specific effect on the foetus development. The prenatal developmental toxicity study leads to the conclusion that it is not very probably that the substance can cross the placenta. The results of this study show a reduction of fetal weight but no incidences of morphological variations in the fetuses. In the same time the body weight gains of the dams were slightly to moderately reduced. These results support the conclusion that the effects observed during the pregnancy are related to the chemicals potential to cause toxicity to the dams. Nevertheless it cannot be excluded completely that the substance has the ability to cross the placenta. Based on its BCF value the parent molecule TBPND has a moderate but not negligible potential to bioaccumulate in the human body. However, due to the fast occurring hydrolysis reaction in the body, it is unlikely that TBPND can be bioaccumulated. In this respective, the two degradation products have very low BCF values (0.35 and < 225 L/kg, respectively) and are thus not considered to be bioaccumulative.
Details on excretion:
TBPND will hydrolyse rather rapidly after being in contact with an aqueous solution, and may not be excreted in its unhydrolysed form. The first degradation product tert-butanol has a low molecular weight of 74.12 g/mol, is miscible in water and thus may either directly excreted or further metabolised by Phase II enzymes before excretion. The second hydrolysis product, consisting of isomers of neodecanoic acids, has a molecular weight of 172.26 g/mol and is poorly soluble in water and thus may be predominantly excreted via the faeces.
Metabolites identified:
not measured
Details on metabolites:
Based on the structure of the molecule, TBPND may be metabolized by Phase I enzymes while undergoing functionalizsation reactions aiming to increase the compound’s hydrophilicity. Here, metabolism to more toxic metabolites cannot completely be excluded in the human body. This assumption is somewhat supported by the results obtained in the in vitro bacterial reverse mutation test where an increase in the revertant frequency of TA100 and WP2 uvr A occured only in the presence of metabolic activation.
Furthermore, Phase II conjugation reactions may covalently link an endogenous substrate to the parent compound or the Phase I metabolite in order to ultimately facilitate excretion.

Not applicable.

Conclusions:
Based on physicochemical characteristics, particularly water solubility, octanol-water partition coefficient and vapour pressure, no or only limited absorption by the dermal and inhalation routes is expected. This assumption is further supported by the results of the dermal and inhalation acute toxicity studies. For the oral route, uptake of the hydrolysis products of TBPND is more likely than an uptake of the less water soluble parent molecule. Bioaccumulation of the hydrolysis products is not likely to occur based on their physico-chemical properties. Excretion of the different hydrolysis products is expected to occur via the urine and the faeces depending on the physicochemical characteristics of the hydrolysis products
Executive summary:

 

Toxicological profile oftert-butylperoxyneodecanoate(TBPND)

An acute oral toxicity study conducted with TBPND with a purity of 75 % using rats revealed a LD50-value of 10776 mg/kgbw. For pure TBPND, a LD50-value of 8082 mg/kg bw was determined by extrapolation. An acute inhalation toxicity study conducted with TBPND of a purity of 75 % using rats revealed a LC50-value of 50 mg/L which corresponds to the calculated LC50 of 37.5 mg/L for the chemical in its pure form. In an acute dermal toxicity study with rats a LD50 of > 8000 mg/kg bw was determined for TBPND with a purity of 75 %. This corresponds to a LD50 of > 6000 mg/kg for the pure chemical. In an in vivo skin irritation and corrosion study, TBPND with a purity of 75 % caused skin irritation effects when applied to rabbit skin. No corrosive effects were observed. An eye irritation test performed with 75 % TBPND on rabbits showed that the substance caused only slight effects on the rabbit’s eye and was not considered to be an eye irritant. A guinea pig maximation test revealed that TBPND can cause skin sensitisation. TBPND did induce reverse mutations in a bacterial reverse mutation test (Ames test) with four Salmonella typhimurium strains and one Escherichia coli strain. The mutagenic response was depended on the bacterial strain and on the presence of a metabolic activation system (S9 liver homogenate). The chemical caused an increase in the reverse mutation frequency in S. typhimurium strains TA1537 and TA98 both in the absence and presence of metabolic activation. However, for the S. typhimurium strain TA1535 a mutagenic response was only observed in the absence of metabolic activation. On the other hand, TBPND caused an increase in the revertant frequency of TA100 and E. coli strain WP2 uvr A only in the presence of metabolic activation. The chemical did not induce a mutagenic response in an in vitro mammalian cell gene mutation test (HPRT assay) performed on CHO-K1 cells both in the absence and presence of metabolic activation. In a further in vivo micronucleous test TBPND did not cause an increase in the frequency of micronucleated polychromatic erythrocytes in mice and was therefore considered as not mutagenic in this tests. A 14 day dose range finding study using oral administration of the TBPND was performed in male and female Wistar rats in order to obtain first information on the toxic potential of the test item to allow a dose-setting for a combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test. The chemical was administered orally (by gavage) once a day for a total of 14 days at 0 (vehicle control), 50, 250 and 750 mg/kg bw/day. Although no mortality was observed through this study, the test substance caused a reduced food intake and, in turn, a reduced body weight for the animals treated with the high dose of 750 mg/kg bw/day. Furthermore, a test item influence on renal and/or hepatic function appeared in the high dose group as indicated by a slightly elevated activity of alanine aminotransferase in male and females and elevated concentrations of total bilirubin, creatinine and urea in the male species only. Also, test item related changes in the appearance of the kidneys (paleness) and in seminal vesicles (smaller than normal) were observed for male animals in the high dose group. In accordance with clinical chemistry and necropsy findings, slightly higher organ weights of liver and kidneys reflected a test item influence at 750 and 250 mg/kg bw/day both in male and female. Based on these results the following three doses were selected for the aforementioned combined repeated dose toxicity study with the reproduction/ developmental toxicity screening study conducted on male and female Wistar rats: 60, 200 and 600 mg/kg bw/day. In this study the highest test concentration of 600 mg/kg bw/day TBPND caused salivation, changes in body weight and food consumption. Several clinical pathology parameters were affected including lower hemoglobin concentration and hematocrit value, an elevated percentage of reticulocytes in female animals and higher mean activity of alanine aminotransferase and urea concentration in male and female animals, higher mean serum levels of creatinine in male animals. Furthermore, changes in organ pathology such as enlarged and pale kidneys, higher kidney weights with indications of hyaline droplet nephropathy were observed in male rats. Moreover, higher liver weights were recorded for male and female animals. Following administration of 200 mg/kg bw/day, salivation and reduced body weight development were observed for male and female rats. Changes in clinical pathology parameters such as elevated percentage of reticulocytes, higher mean activity of alanine aminotransferase and higher mean serum levels of urea in females were observed. Also in males a higher activity of alanine aminotransferase was noted. Furthermore, changes in organ pathology such as higher kidney weights and hyaline droplet nephropathy of male rats and higher liver weights in female animals were observed. At the lower dose of 60 mg/kg bw/day, salivation, higher percent of reticulocytes, slightly elevated mean activity of alanine aminotransferase and liver weight in female animals and mild hyaline droplet nephropathy of male rats were detected. At this stage in needs to be highlighted that hyaline droplet nephropathy was associated with interference toα-2µ-globulin. In this case the observed nephropathy is specific to the male rat and has no relevance to humans. In terms of the animal’s reproductive performance, dam’s delivery was affected by the test item at 600 mg/kg bw/day as the number of dams with prolonged pregnancy was higher and consequently the mean duration of pregnancy was longer than in the control group. Also a higher percentage of post-implantation loss and stillborns were observed. Moreover, at the highest concentration the extra uterine mortality of offspring was elevated with respect to the control animals. In relation to the developmental toxicity investigation, the offspring’s body weight development (for litter and pup’s weights) was depressed at 600 and 200 mg/kg bw/day. However, no structural or visceral malformations were observed in the offspring in any dose group. Based on these observations a NOEL for male rats of < 60 mg/kg bw/day was determined based on the observedα-2µ-globulin nephropathy. The respective NOAEL for male and female rats was set to 60 mg/kg bw/day. The NOAEL for reproductive performance of the male and female rats was evaluated to be 200 mg/kg bw/day and the NOAEL for the offspring was determined to be 60 mg/kg bw/day.The test substance was examined for its possible prenatal developmental toxicity. Groups of 22, 22 and 25 sperm-positive female Hsd. Han: Wistar rats were treated with the test substance by oral (gavage) administration daily at three dose levels of 20, 60 and 200 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 25 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 mL/kg bw. Sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The test substance in sunflower oil was stable at room temperature for 4 hours and in a refrigerator (5 ± 3 °C) for 3 days at the concentrations of 1, 10 and 500 mg/mL. Analytical control of dosing solutions was performed on the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 95 and 106 % of nominal concentrations at both analytical occasions confirming proper dosing. During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day when sperm was detected in the vaginal smear was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded. In total, there were 22 evaluated litters each in the control and 60 mg/kg bw/day group, 17 and 21 in the 60 and 200 mg/kg bw/day groups respectively. None of the females died before scheduled necropsy during the study. There were no treatment related clinical signs and necropsy findings observed. The body weight gains (between 17 and 20 as well as for days 0 to 20 including corrected body weight gain) in the 200 mg/kg bw/day group were judged to be moderately decreased by the treatment. At this dose also a slight reduction of the food consumption between gestation days 17 and 20 in the 200 mg/kg bw/day dose group was noted. The mean number of implantations, pre- and post-implantation loss as well as sex distribution of the fetuses were not influenced by the treatment. Fetal weight was slightly lower in the 200 mg/kg bw/day dose group and the incidence of body weight retardation increased moderately at 200 mg/kg bw/day, both were considered to be related to the treatment of the dams. The test item was judged not to influence the incidences of visceral and skeletal variations. The different type of malformations found at the fetal examinations (umbilical hernia, microphthalmia, split xiphoid cartilage in the 200 mg/kg bw/day group each in one fetus, a dumb-bell shaped cartilage of a thoracic centrum in the 60 mg/kg bw/day group as well as hydronephrosis, split sternum and bent scapula in three different fetuses in the 20 mg/kg bw/day group were judged to be incidental according to the experience with this species in this laboratory and in line with historical control data of other Wistar rats as well as due to the lack of a clear dose response-relationship and/or occurrence in the actual control group. Oral treatment of pregnant Hsd. Han: WISTAR rats from gestation day 5 up to day 19 (the day before Caesarean section) with the test substance at the dose levels of 60 and 20 mg/kg bw/day did not cause death, clinical signs and necropsy findings. The body weight gains and food consumption were slightly to moderately reduced in the 200 mg/kg bw/day group from gestation day 17 onwards. The test substance did not reveal any adverse effect on the pre- and postimplantation loss, number of implantation and the sex distribution of the fetuses. The slightly lower fetal weight was observed at 200 mg/kg bw/day at a dose level with slight maternal effects. The test substance did not increase the incidence of visceral and skeletal variations and induced no fetal malformations. Based on these observations the No Observed Adverse Effect Level (NOAELs) were determined as follows: NOAEL (maternal toxicity): 60mg/kg bw/day, NOAEL (developmental toxicity): 60 mg/kg bw/day, NOAEL (teratogenicity): 200 mg/kg bw/day (high dose).

 

Toxicokinetic analysis of tert-butyl peroxyneodecanoate (TBPND)

Tert-butyl peroxyneodecanoate (TBPND) is a colourless liquid at room temperature with a molecular weight of 244.37 g/mol. The substance is only slightly soluble in water (9 mg/L at 0°C). The log Pow of TBPND was measured and determined to be between 5.1 and 5.4 at 25°C. Based on this log Pow, a BCF of 1358 L/kg was calculated. TBPND has a low vapour pressure of approximately 50 Pa at 25°C. In an aqueous solution, TBPND is relatively rapidly degraded hydrolytically to tert-butanol and isomers of neodecanoic acids. The half-life of TBPND in an aqueous solution at 37°C is 5.1 h and 3.3 h at a pH of 4 and 7, respectively. Both hydrolysis substances have a lower log Pow values than TBPND itself (approximately 0.32 for tert-butanol and 2.1 to 3.83 for the isomers of neodecanoic acids). Also the BCF values are lower as compared to TBPND (approximately 3.2 and < 225 for the aforementioned hydrolysis products respectively).

 

Absorption

Generally, oral absorption is favoured for molecular weights below 500 g/mol. However, based on the high logPow of 5.1 to 5.4 TBPND can be regarded as lipophilic substance. This characteristic combined with the relatively low water solubility may limit oral absorption by the inability of this substance to dissolve in the gastro-intestinal fluids, which in turn hinders contact with the mucosal surface. On the other hand, absorption of such a lipophilic compound may be facilitated following possible micellular solubilisation by bile salts. With regards to the high lipophilicty and low water solubility, the mechanisms of micellular solubilisation may be of some importance for TBPND, as the substance would otherwise be poorly absorbed. Administered without a vehicle in an acute oral toxicity study performed on rats, TBPND lead to a high LD50 of 8082 mg/kg bw/day. Moreover, no toxic effects relating to a systemic absorption were observable. Based on this results it can be assumed that only limited absorption of the substance itself across the epithelial lining of the gastro intestinal tract will occur when administered orally. However, the results from the 14 day dose range finding study and the combined repeated dose toxicity study with the reproduction/ developmental toxicity screening study, both conducted on male and female Wistar rats, indicate that the compound, or more likely, its hydrolysis products became bioavailable. This conclusion is supported by the results of the OECD guideline 414 study. In this regards, as indicated by the half-life values from the hydrolysis test, a large fraction, if not all, of TBPND will hydrolyze to tert-butanol and isomers of neodecanoic acids following oral administration which is indicated by the relative short half-live in an aqueous solution at acidic to neutral conditions. The results of the hydrolysis tests at a pH range of 4 to 9 are somewhat representative for the conditions found in the GIT with the stomach having an acidic milieu (~ pH 1.4 to 4.5) and the intestine a slightly acidic to slighty alkaline milieu (~ pH 5 to 8). Due to the lower log Pow values of the hydrolyis products, it is assumed that they may be readily absorbed through the GIT epithelium. Furthermore, the low molecular weight of tert-butanol (74.12 g/mol) combined with its relatively high water solubility (> 10 g/L) may allow the direct uptake into the systemic circulation through aqueous pores or via carriage of the molecules across the membrane with the bulk passage of water. Due to the relatively low vapour pressure of TBPND (approximately 50 Pa) and the resulting low volatility, an inhalation exposure of the compound’s vapour phase is rather unlikely. Moreover, if the substance would reach the lungs in its vapour or gaseous state, the lipophilic character of TBPND hinders the direct absorption across the respiratory tract epithelium. An acute inhalation toxicity study performed on rats using TBPND in its aerosol form revealed a relatively high LC50 of 37.50 mg/L. No specific effects of systemic toxicity were observed and these results indicate for low systemic availability after inhalation. Even if bioavailable, systemic toxicity effects may only occur following an unlikely high exposition to the substance via this route of administration. Similarly, based on physico–chemical properties of TBPND the substance is not likely to penetrate skin to a large extent as the high log Pow value and low water solubility do not favour dermal penetration. It is general accepted that if a compound’s water solubility falls between 1-100 mg/L, absorption can be anticipated to be low to moderate. Moreover, for substances with a log Pow between 4 and 6, the rate of penetration is limited by the rate of transfer between the stratum corneum and the epidermis. Only the uptake into the outer, non-viable layer stratum corneum may be high as the underlying viable epidermis is very resitant to penetration by highly lipophilic compunds. These assumptions based on the physico-chemical properties of TBPND are further supported by the results achieved from an acute dermal toxicity study performed on rabbits. During this study no mortality, no test item related changes in the body weight and no systemic toxic effects were observed for the highest dose of TBPND used in the test and the respective LD50 was determined to be greater than 6000 mg/kg bw. Although, application of TBPND to the skin of rabbits caused irritation in form of erythema and edema in two independently performed skin irritation/corrosion studies, no evidence of full thickness destruction of the skin or scar tissue was observed which in turn could have favoured direct absorption into the systemic circulation. When applied topically onto the skin of guinea pigs, sensitising effects were observed following an initial intradermal induction phase. This indicates that at least a small fraction of the substance has become available in the body to initiate the immune response. However, the effects may also be caused by the formation of reaction products between TBPND and molecules present in the skin. Furthermore, it has to be kept in mind that the vehicle propylene glycol which was used to aid the topical application during the challenge phase could have potentially influenced the extent of dermal absorption.

 

Distribution

Assuming that TBPND is absorbed into the body following oral intake, it may be distributed into the interior part of cells due to its lipophilic properties and in turn the intracellular concentration may be higher than extracellular concentration particularly in adipose tissues. However, it is expected that TBPND does not reach the blood without starting to hydrolyse into its hydolysis products. As mentioned above, the physicochemical properties of the hydrolysis products favour systemic absorption. Especially the low molecular weight and relatively high water solubility of tert-butanol favours absorption. Direct transport through aqueous pores is likely to be an entry route to the systemic circulation. The results from the combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test indicate that, following absorption, the liver and the kidney are the primary target organs affected by the chemicals. Based on the results observed on the reproduction and developmental performance of the animals it is difficult to judge if the hydrolysis products have the ability to cross the placenta and cause a specific effect on the foetus development. The prenatal developmental toxicity study leads to the conclusion that it is not very probably that the substance can cross the placenta. The results of this study show a reduction of fetal weight but no incidences of morphological variations in the fetuses. In the same time the body weight gains of the dams were slightly to moderately reduced. These results support the conclusion that the effects observed during the pregnancy are related to the chemicals potential to cause toxicity to the dams. Nevertheless it cannot be excluded completely that the substance has the ability to cross the placenta. Based on its BCF value the parent molecule TBPND has a moderate but not negligible potential to bioaccumulate in the human body. However, due to the fast occurring hydrolysis reaction in the body, it is unlikely that TBPND can be bioaccumulated. In this respective, the two degradation products have very low BCF values (0.35 and < 225 L/kg, respectively) and are thus not considered to be bioaccumulative.

 

Metabolism

Based on the structure of the molecule, TBPND may be metabolized by Phase I enzymes while undergoing functionalizsation reactions aiming to increase the compound’s hydrophilicity. Here, metabolism to more toxic metabolites cannot completely be excluded in the human body. This assumption is somewhat supported by the results obtained in the in vitro bacterial reverse mutation test where an increase in the revertant frequency of TA100 and WP2 uvr A occured only in the presence of metabolic activation. Furthermore, Phase II conjugation reactions may covalently link an endogenous substrate to the parent compound or the Phase I metabolite in order to ultimately facilitate excretion.

 

Excretion

As discussed above, TBPND will hydrolyse rather rapidly after being in contact with an aqueous solution, and may not be excreted in its unhydrolysed form. The first degradation product tert-butanol has a low molecular weight of 74.12 g/mol, is miscible in water and thus may either directly excreted or further metabolised by Phase II enzymes before excretion. The second hydrolysis product, consisting of isomers of neodecanoic acids, has a molecular weight of 172.26 g/mol and is poorly soluble in water and thus may be predominantly excreted via the faeces.

 

Summary

Based on physicochemical characteristics, particularly water solubility,octanol-water partition coefficient andvapourpressure, no or only limited absorption by the dermal and inhalation routes is expected. This assumption is further supported by the results of the dermal and inhalation acute toxicity studies. For the oral route, uptake of the hydrolysis products of TBPND is more likely than an uptake of the less water soluble parent molecule. Bioaccumulation of the hydrolysis products is not likely to occur based on theirphysico-chemical properties. Excretion of the different hydrolysis products is expected to occur via the urine and thefaecesdepending on the physicochemical characteristics of the hydrolysis products.

Description of key information

Based on physicochemical characteristics, particularly water solubility, octanol-water partition coefficient and vapour pressure, no or only limited absorption by the dermal and inhalation routes is expected. This assumption is further supported by the results of the dermal and inhalation acute toxicity studies. For the oral route, uptake of the hydrolysis products of TBPND is more likely than an uptake of the less water soluble parent molecule. Bioaccumulation of the hydrolysis products is not likely to occur based on their physico-chemical properties. Excretion of the different hydrolysis products is expected to occur via the urine and the faeces depending on the physicochemical characteristics of the hydrolysis products.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

Toxicological profile of tert-butylperoxyneodecanoate (TBPND)

An acute oral toxicity study conducted with TBPND with a purity of 75 % using rats revealed a LD50-value of 10776 mg/kgbw. For pure TBPND, a LD50-value of 8082 mg/kg bw was determined by extrapolation. An acute inhalation toxicity study conducted with TBPND of a purity of 75 % using rats revealed a LC50-value of 50 mg/L which corresponds to the calculated LC50 of 37.5 mg/L for the chemical in its pure form. In an acute dermal toxicity study with rats a LD50 of > 8000 mg/kg bw was determined for TBPND with a purity of 75 %. This corresponds to a LD50 of > 6000 mg/kg for the pure chemical. In an in vivo skin irritation and corrosion study, TBPND with a purity of 75 % caused skin irritation effects when applied to rabbit skin. No corrosive effects were observed. An eye irritation test performed with 75 % TBPND on rabbits showed that the substance caused only slight effects on the rabbit’s eye and was not considered to be an eye irritant. A guinea pig maximation test revealed that TBPND can cause skin sensitisation. TBPND did induce reverse mutations in a bacterial reverse mutation test (Ames test) with four Salmonella typhimurium strains and one Escherichia coli strain. The mutagenic response was depended on the bacterial strain and on the presence of a metabolic activation system (S9 liver homogenate). The chemical caused an increase in the reverse mutation frequency in S. typhimurium strains TA1537 and TA98 both in the absence and presence of metabolic activation. However, for the S. typhimurium strain TA1535 a mutagenic response was only observed in the absence of metabolic activation. On the other hand, TBPND caused an increase in the revertant frequency of TA100 and E. coli strain WP2 uvr A only in the presence of metabolic activation. The chemical did not induce a mutagenic response in an in vitro mammalian cell gene mutation test (HPRT assay) performed on CHO-K1 cells both in the absence and presence of metabolic activation. In a further in vivo micronucleous test TBPND did not cause an increase in the frequency of micronucleated polychromatic erythrocytes in mice and was therefore considered as not mutagenic in this tests. A 14 day dose range finding study using oral administration of the TBPND was performed in male and female Wistar rats in order to obtain first information on the toxic potential of the test item to allow a dose-setting for a combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test. The chemical was administered orally (by gavage) once a day for a total of 14 days at 0 (vehicle control), 50, 250 and 750 mg/kg bw/day. Although no mortality was observed through this study, the test substance caused a reduced food intake and, in turn, a reduced body weight for the animals treated with the high dose of 750 mg/kg bw/day. Furthermore, a test item influence on renal and/or hepatic function appeared in the high dose group as indicated by a slightly elevated activity of alanine aminotransferase in male and females and elevated concentrations of total bilirubin, creatinine and urea in the male species only. Also, test item related changes in the appearance of the kidneys (paleness) and in seminal vesicles (smaller than normal) were observed for male animals in the high dose group. In accordance with clinical chemistry and necropsy findings, slightly higher organ weights of liver and kidneys reflected a test item influence at 750 and 250 mg/kg bw/day both in male and female. Based on these results the following three doses were selected for the aforementioned combined repeated dose toxicity study with the reproduction/ developmental toxicity screening study conducted on male and female Wistar rats: 60, 200 and 600 mg/kg bw/day. In this study the highest test concentration of 600 mg/kg bw/day TBPND caused salivation, changes in body weight and food consumption. Several clinical pathology parameters were affected including lower hemoglobin concentration and hematocrit value, an elevated percentage of reticulocytes in female animals and higher mean activity of alanine aminotransferase and urea concentration in male and female animals, higher mean serum levels of creatinine in male animals. Furthermore, changes in organ pathology such as enlarged and pale kidneys, higher kidney weights with indications of hyaline droplet nephropathy were observed in male rats. Moreover, higher liver weights were recorded for male and female animals. Following administration of 200 mg/kg bw/day, salivation and reduced body weight development were observed for male and female rats. Changes in clinical pathology parameters such as elevated percentage of reticulocytes, higher mean activity of alanine aminotransferase and higher mean serum levels of urea in females were observed. Also in males a higher activity of alanine aminotransferase was noted. Furthermore, changes in organ pathology such as higher kidney weights and hyaline droplet nephropathy of male rats and higher liver weights in female animals were observed. At the lower dose of 60 mg/kg bw/day, salivation, higher percent of reticulocytes, slightly elevated mean activity of alanine aminotransferase and liver weight in female animals and mild hyaline droplet nephropathy of male rats were detected. At this stage in needs to be highlighted that hyaline droplet nephropathy was associated with interference toα-2µ-globulin. In this case the observed nephropathy is specific to the male rat and has no relevance to humans. In terms of the animal’s reproductive performance, dam’s delivery was affected by the test item at 600 mg/kg bw/day as the number of dams with prolonged pregnancy was higher and consequently the mean duration of pregnancy was longer than in the control group. Also a higher percentage of post-implantation loss and stillborns were observed. Moreover, at the highest concentration the extra uterine mortality of offspring was elevated with respect to the control animals. In relation to the developmental toxicity investigation, the offspring’s body weight development (for litter and pup’s weights) was depressed at 600 and 200 mg/kg bw/day. However, no structural or visceral malformations were observed in the offspring in any dose group. Based on these observations a NOEL for male rats of < 60 mg/kg bw/day was determined based on the observedα-2µ-globulin nephropathy. The respective NOAEL for male and female rats was set to 60 mg/kg bw/day. The NOAEL for reproductive performance of the male and female rats was evaluated to be 200 mg/kg bw/day and the NOAEL for the offspring was determined to be 60 mg/kg bw/day.The test substance was examined for its possible prenatal developmental toxicity. Groups of 22, 22 and 25 sperm-positive female Hsd. Han: Wistar rats were treated with the test substance by oral (gavage) administration daily at three dose levels of 20, 60 and 200 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 25 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 mL/kg bw. Sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The test substance in sunflower oil was stable at room temperature for 4 hours and in a refrigerator (5 ± 3 °C) for 3 days at the concentrations of 1, 10 and 500 mg/mL. Analytical control of dosing solutions was performed on the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 95 and 106 % of nominal concentrations at both analytical occasions confirming proper dosing. During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day when sperm was detected in the vaginal smear was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded. In total, there were 22 evaluated litters each in the control and 60 mg/kg bw/day group, 17 and 21 in the 60 and 200 mg/kg bw/day groups respectively. None of the females died before scheduled necropsy during the study. There were no treatment related clinical signs and necropsy findings observed. The body weight gains (between 17 and 20 as well as for days 0 to 20 including corrected body weight gain) in the 200 mg/kg bw/day group were judged to be moderately decreased by the treatment. At this dose also a slight reduction of the food consumption between gestation days 17 and 20 in the 200 mg/kg bw/day dose group was noted. The mean number of implantations, pre- and post-implantation loss as well as sex distribution of the fetuses were not influenced by the treatment. Fetal weight was slightly lower in the 200 mg/kg bw/day dose group and the incidence of body weight retardation increased moderately at 200 mg/kg bw/day, both were considered to be related to the treatment of the dams. The test item was judged not to influence the incidences of visceral and skeletal variations. The different type of malformations found at the fetal examinations (umbilical hernia, microphthalmia, split xiphoid cartilage in the 200 mg/kg bw/day group each in one fetus, a dumb-bell shaped cartilage of a thoracic centrum in the 60 mg/kg bw/day group as well as hydronephrosis, split sternum and bent scapula in three different fetuses in the 20 mg/kg bw/day group were judged to be incidental according to the experience with this species in this laboratory and in line with historical control data of other Wistar rats as well as due to the lack of a clear dose response-relationship and/or occurrence in the actual control group. Oral treatment of pregnant Hsd. Han: WISTAR rats from gestation day 5 up to day 19 (the day before Caesarean section) with the test substance at the dose levels of 60 and 20 mg/kg bw/day did not cause death, clinical signs and necropsy findings. The body weight gains and food consumption were slightly to moderately reduced in the 200 mg/kg bw/day group from gestation day 17 onwards. The test substance did not reveal any adverse effect on the pre- and postimplantation loss, number of implantation and the sex distribution of the fetuses. The slightly lower fetal weight was observed at 200 mg/kg bw/day at a dose level with slight maternal effects. The test substance did not increase the incidence of visceral and skeletal variations and induced no fetal malformations. Based on these observations the No Observed Adverse Effect Level (NOAELs) were determined as follows: NOAEL (maternal toxicity): 60mg/kg bw/day, NOAEL (developmental toxicity): 60 mg/kg bw/day, NOAEL (teratogenicity): 200 mg/kg bw/day (high dose).

 

Toxicokinetic analysis of tert-butyl peroxyneodecanoate (TBPND)

Tert-butyl peroxyneodecanoate (TBPND) is a colourless liquid at room temperature with a molecular weight of 244.37 g/mol. The substance is only slightly soluble in water (9 mg/L at 0°C). The log Pow of TBPND was measured and determined to be between 5.1 and 5.4 at 25°C. Based on this log Pow, a BCF of 925 L/kg was calculated. TBPND has a low vapour pressure of approximately 50 Pa at 25°C. In an aqueous solution, TBPND is relatively rapidly degraded hydrolytically to tert-butanol and isomers of neodecanoic acids. The half-life of TBPND in an aqueous solution at 37°C is 5.1 h and 3.3 h at a pH of 4 and 7, respectively. Both hydrolysis substances have a lower log Pow values than TBPND itself (approximately 0.32 for tert-butanol and 2.1 to 3.83 for the isomers of neodecanoic acids). Also the BCF values are lower as compared to TBPND (approximately 3.2 and < 225 for the aforementioned hydrolysis products respectively).

 

Absorption

Generally, oral absorption is favoured for molecular weights below 500 g/mol. However, based on the high logPow of 5.1 to 5.4 TBPND can be regarded as lipophilic substance. This characteristic combined with the relatively low water solubility may limit oral absorption by the inability of this substance to dissolve in the gastro-intestinal fluids, which in turn hinders contact with the mucosal surface. On the other hand, absorption of such a lipophilic compound may be facilitated following possible micellular solubilisation by bile salts. With regards to the high lipophilicty and low water solubility, the mechanisms of micellular solubilisation may be of some importance for TBPND, as the substance would otherwise be poorly absorbed. Administered without a vehicle in an acute oral toxicity study performed on rats, TBPND lead to a high LD50 of 8082 mg/kg bw/day. Moreover, no toxic effects relating to a systemic absorption were observable. Based on this results it can be assumed that only limited absorption of the substance itself across the epithelial lining of the gastro intestinal tract will occur when administered orally. However, the results from the 14 day dose range finding study and the combined repeated dose toxicity study with the reproduction/ developmental toxicity screening study, both conducted on male and female Wistar rats, indicate that the compound, or more likely, its hydrolysis products became bioavailable. This conclusion is supported by the results of the OECD guideline 414 study. In this regards, as indicated by the half-life values from the hydrolysis test, a large fraction, if not all, of TBPND will hydrolyze to tert-butanol and isomers of neodecanoic acids following oral administration which is indicated by the relative short half-live in an aqueous solution at acidic to neutral conditions. The results of the hydrolysis tests at a pH range of 4 to 9 are somewhat representative for the conditions found in the GIT with the stomach having an acidic milieu (~ pH 1.4 to 4.5) and the intestine a slightly acidic to slighty alkaline milieu (~ pH 5 to 8). Due to the lower log Pow values of the hydrolyis products, it is assumed that they may be readily absorbed through the GIT epithelium. Furthermore, the low molecular weight of tert-butanol (74.12 g/mol) combined with its relatively high water solubility (> 10 g/L) may allow the direct uptake into the systemic circulation through aqueous pores or via carriage of the molecules across the membrane with the bulk passage of water. Due to the relatively low vapour pressure of TBPND (approximately 50 Pa) and the resulting low volatility, an inhalation exposure of the compound’s vapour phase is rather unlikely. Moreover, if the substance would reach the lungs in its vapour or gaseous state, the lipophilic character of TBPND hinders the direct absorption across the respiratory tract epithelium. An acute inhalation toxicity study performed on rats using TBPND in its aerosol form revealed a relatively high LC50 of 37.50 mg/L. No specific effects of systemic toxicity were observed and these results indicate for low systemic availability after inhalation. Even if bioavailable, systemic toxicity effects may only occur following an unlikely high exposition to the substance via this route of administration. Similarly, based on physico–chemical properties of TBPND the substance is not likely to penetrate skin to a large extent as the high log Pow value and low water solubility do not favour dermal penetration. It is general accepted that if a compound’s water solubility falls between 1-100 mg/L, absorption can be anticipated to be low to moderate. Moreover, for substances with a log Pow between 4 and 6, the rate of penetration is limited by the rate of transfer between the stratum corneum and the epidermis. Only the uptake into the outer, non-viable layer stratum corneum may be high as the underlying viable epidermis is very resitant to penetration by highly lipophilic compunds. These assumptions based on the physico-chemical properties of TBPND are further supported by the results achieved from an acute dermal toxicity study performed on rabbits. During this study no mortality, no test item related changes in the body weight and no systemic toxic effects were observed for the highest dose of TBPND used in the test and the respective LD50 was determined to be greater than 6000 mg/kg bw. Although, application of TBPND to the skin of rabbits caused irritation in form of erythema and edema in two independently performed skin irritation/corrosion studies, no evidence of full thickness destruction of the skin or scar tissue was observed which in turn could have favoured direct absorption into the systemic circulation. When applied topically onto the skin of guinea pigs, sensitising effects were observed following an initial intradermal induction phase. This indicates that at least a small fraction of the substance has become available in the body to initiate the immune response. However, the effects may also be caused by the formation of reaction products between TBPND and molecules present in the skin. Furthermore, it has to be kept in mind that the vehicle propylene glycol which was used to aid the topical application during the challenge phase could have potentially influenced the extent of dermal absorption.

 

Distribution

Assuming that TBPND is absorbed into the body following oral intake, it may be distributed into the interior part of cells due to its lipophilic properties and in turn the intracellular concentration may be higher than extracellular concentration particularly in adipose tissues. However, it is expected that TBPND does not reach the blood without starting to hydrolyse into its hydolysis products. As mentioned above, the physicochemical properties of the hydrolysis products favour systemic absorption. Especially the low molecular weight and relatively high water solubility of tert-butanol favours absorption. Direct transport through aqueous pores is likely to be an entry route to the systemic circulation. The results from the combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test indicate that, following absorption, the liver and the kidney are the primary target organs affected by the chemicals. Based on the results observed on the reproduction and developmental performance of the animals it is difficult to judge if the hydrolysis products have the ability to cross the placenta and cause a specific effect on the foetus development. The prenatal developmental toxicity study leads to the conclusion that it is not very probably that the substance can cross the placenta. The results of this study show a reduction of fetal weight but no incidences of morphological variations in the fetuses. In the same time the body weight gains of the dams were slightly to moderately reduced. These results support the conclusion that the effects observed during the pregnancy are related to the chemicals potential to cause toxicity to the dams. Nevertheless it cannot be excluded completely that the substance has the ability to cross the placenta. Based on its BCF value the parent molecule TBPND has a moderate but not negligible potential to bioaccumulate in the human body. However, due to the fast occurring hydrolysis reaction in the body, it is unlikely that TBPND can be bioaccumulated. In this respective, the two degradation products have very low BCF values (0.35 and < 225 L/kg, respectively) and are thus not considered to be bioaccumulative.

 

Metabolism

Based on the structure of the molecule, TBPND may be metabolized by Phase I enzymes while undergoing functionalizsation reactions aiming to increase the compound’s hydrophilicity. Here, metabolism to more toxic metabolites cannot completely be excluded in the human body. This assumption is somewhat supported by the results obtained in the in vitro bacterial reverse mutation test where an increase in the revertant frequency of TA100 and WP2 uvr A occured only in the presence of metabolic activation. Furthermore, Phase II conjugation reactions may covalently link an endogenous substrate to the parent compound or the Phase I metabolite in order to ultimately facilitate excretion.

 

Excretion

As discussed above, TBPND will hydrolyse rather rapidly after being in contact with an aqueous solution, and may not be excreted in its unhydrolysed form. The first degradation product tert-butanol has a low molecular weight of 74.12 g/mol, is miscible in water and thus may either directly excreted or further metabolised by Phase II enzymes before excretion. The second hydrolysis product, consisting of isomers of neodecanoic acids, has a molecular weight of 172.26 g/mol and is poorly soluble in water and thus may be predominantly excreted via the faeces.

 

Summary

Based on physicochemical characteristics, particularly water solubility,octanol-water partition coefficient andvapourpressure, no or only limited absorption by the dermal and inhalation routes is expected. This assumption is further supported by the results of the dermal and inhalation acute toxicity studies. For the oral route, uptake of the hydrolysis products of TBPND is more likely than an uptake of the less water soluble parent molecule. Bioaccumulation of the hydrolysis products is not likely to occur based on theirphysico-chemical properties. Excretion of the different hydrolysis products is expected to occur via the urine and thefaecesdepending on the physicochemical characteristics of the hydrolysis products.