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Description of key information

Physico-chemical properties,particularly water solubility and octanol-water partition coefficient and experimental data indicate bioavailability of cyclohexylidenebis[tert-butyl] peroxide via oral and to a lesser extend also via inhalation and dermal route. Intracellular concentration is likely to be higher than extracellular due to the lipophilicity of cyclohexylidenebis[tert-butyl] peroxide. Hydrolytic and metabolic conversion into tert-butyl hydroperoxide and cyclohexanone is expected and hydroxylation/conjugation of Phase I-metabolites may further increase hydrophilicity. Metabolites of cyclohexylidenebis[tert-butyl] peroxide are not considered to be more toxic than cyclohexylidenebis[tert-butyl] peroxide itself. Excretion via urine is assumed to be the main excretion pathway of metabolites formed due to their molecular weight ( 300 g/mol in rat) and water solubility. Bioaccumulation of cyclohexylidenebis[tert-butyl] peroxide itself and its hydrolysis products is not likely to occur due to hydrolytic degradation and the physico-chemical properties of the hydrolysis products. 

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

General

The test item is produced at different EU manufacturing sites. The substance represents a basic industrial chemical used as initiator to start chain reactions in the synthesis of polymers.

Toxicological profile of Cyclohexylidenebis[tert-butyl] peroxide (CH)

An acute oral toxicity study conducted with cyclohexylidenebis[tert-butyl] peroxide with a purity of 65 % using rats revealed a LD50 value of 16653 mg/kg bw. No acute inhalation toxicity study is available as the vapour pressure of the substance is low. In an acute dermal toxicity study with rats a LD50 of > 2000 mg/kg bw was determined for cyclohexylidenebis[tert-butyl] peroxide with a purity of 50 %. In an in vivo skin irritation and corrosion study, cyclohexylidenebis[tert-butyl] peroxide with a purity of 65 % did not cause any skin irritation/corrosion effects when applied to rabbit skin. An eye irritation test performed with 65 % cyclohexylidenebis[tert-butyl] peroxide 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 Buehler test with guinea pigs revealed that cyclohexylidenebis[tert-butyl] peroxide did not cause skin sensitisation. Cyclohexylidenebis[tert-butyl] peroxide did not induce a mutagenic response both, in a bacterial reverse mutation test (Ames test) and in an in vitro mammalian cell gene mutation test (HPRT assay) performed on CHO-cells both in the absence and presence of metabolic activation. No induction of chromosome aberration was observed in the in vitro cytogenetic assay on human lymphocytes. A 14 day dose range finding study using oral administration of cyclohexylidenebis[tert-butyl] peroxide was performed in male and female Wistar rats in order to obtain first information on the toxic potential of the test item after long-term administration 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), 100, 300 and 1000 mg/kg bw/day. No mortality was observed through this study. Salivation was observed in male and female animals of the high and mid dose group. There were no test item-related effects on body weight and food consumption in all dose groups in comparison to control group. An increased platelet (thrombocyte) count was noted at 1000 mg/kg bw/day only for the male species. The test substance caused slightly higher mean concentrations of serum cholesterol in female animals of the high and mid dose group and slightly higher total protein level in male and female animals in the high dose group only. Elevated liver weights at 1000, 300 and 100 mg/kg bw/day and kidney weights at 1000 mg/kg bw/day compared to control group referred to a test item influence in male and female animals. 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: 40, 200 and 600 mg/kg bw/day.

The main study (OECD guideline 422 study) revealed no mortality of male and female animals/dams exposed to cyclohexylidenebis[tert-butyl] peroxide.Test item related salivation was observed in all male and female animals at 600 mg/kg bw/day and in 16/17 males and 15/17 females at 200 mg/kg bw/day from Days 9 and 10 up to the end of the treatment period. No clinical signs and no changes in behaviour and physical condition were noted in any dosage group. At 600 mg/kg bw/day the mean body weight gain was depressed in male animals in comparison to the control group. No effect on body weight and body weight gain was noted for female animals of the same dose group. There were no test item related changes in the examined hematological or blood coagulation parameters in male or female animals at any dose level (600, 200 or 40 mg/kg bw/day) compared to control. A slight elevation of cholesterol level was detected in male and female animals at 600 mg/kg bw/day and was considered to be test item-related. Changes in organ pathology such as pale kidneys and higher kidney weights compared to control with indications of hyaline droplet nephropathy were observed in male animals of the high dose group. Increased liver weights and discoloration of the liver were recorded for male and female animals of the high and mid dose group in comparison to control. Histopathological examination of reproductive organs of both male and female animals revealed no test item-related effect. There were no differences between the control and test item treated groups in the reproductive performance of male and female animals and in delivery data of dams. No test item-related effects were observed on clinical and pathological parameters and on mean litter weight and weight gain of the offspring. No structural or visceral malformations were noted in the offspring at any dosage level. Based on these observations the respective NOAEL for male and female rats was set to 200 mg/kg bw/day. The NOAEL for reproductive performance of the male and female rats was evaluated to be 600 mg/kg bw/day and the NOAEL for the offspring was determined to be 600 mg/kg bw/day.

The embryonic and foetal development toxicity following repeated administration by gavage to the pregnant female during the period of organogenesis was tested in a read across study with Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide (CAS 6731-36-8) over a period of 20 days (OECD guideline 414 study). The test item was administered to four groups each of twenty-four time mated Sprague-Dawley rats, between Days 5 and 19 of gestation inclusive at dose levels 30, 100, 300 and 1000 mg/kg bw/day. No unscheduled deaths were observed during the study. Females treated with 1000 and 300 mg/kg bw/day showed incidences of increased salivation. No such effects were detected in females treated with 100 or 30 mg/kg bw/day. No treatment related effects in body weight development were detected. No toxicologically significant effects were detected in food consumption. No adverse effect on water consumption was detected. Females treated with 1000, 300 and 100 mg/kg bw/day had an enlarged liver at necropsy. One female treated with 1000 mg/kg bw/day also had discolouration on the right lobe of the liver. No such effects were detected in females treated with 30 mg/kg bw/day. Females from all treatment groups showed an increase in liver weight both absolute and relative to terminal body weight. The following treatment related microscopic effects were detected: Liver: Minimal to moderate centrilobular hypertrophy of hepatocytes was evident in females from all treatment groups. No treatment-related effects were detected in the uterine parameters examined, in foetal viability or in growth and development. No treatment-related effects were detected on skeletal development or in the type and incidence of skeletal or visceral findings. The oral administration of Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide (CAS 6731-36-8) to pregnant rats by oral gavage during organogenesis at dose levels of 30, 100, 300 and 1000 mg/kg/day resulted in adaptive liver effects in parental females. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore, considered to be 1000 mg/kg/day. No toxicological significant changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg/day.

Possible health hazards after repeated exposure with the test item were determined in a 90 days study followed by a 28-day recovery period in the high dose and control animals in order to assess reversibility, persistence or delayed occurrence of potential toxicological effects. The test item was administered orally (by gavage) to male and female Wistar rats at 0 (vehicle control), 450, 150 and 40 mg/kg bw/day doses corresponding to concentrations of 0, 225 mg/mL, 75 mg/mL and 20 mg/mL. 5 animals/ sex in the control and high dose groups assigned to the recovery groups were treated identically up to Day 89 then they were observed without administration for four weeks (recovery observations). No animals died during the course of the study. Toxic signs related to the test item were not detected at any dose level (450, 150 or 40 mg/kg bw/day) at the daily and detailed weekly clinical observations and in the course of the functional observation battery. The body weight development of male and female animals was not affected in the course of the entire study (450, 150 or 40 mg/kg bw/day). No test item-related body weight or body weight gain changes were observed with respect to controls at any dose level during the treatment or recovery periods. The mean daily food consumption was comparable in animals of the control and test item treated groups (450, 150 or 40 mg/kg bw/day). There were no abnormalities in the eyes of animals in the high dose group at termination of the treatment (male and female at 450 mg/kg bw/day). A test item influence on the estrous cycle was not detected (450, 150 or 40 mg/kg bw/day). Slight differences between the control and high dose treated female animals in the percentage of animals with irregular cycle, mean number of days in estrous or diestrus were considered to be indicative of biological variation and not related to the test item. In accordance with this finding, histopathological examinations did not reveal changes in the morphology of uterus or ovaries in this study. Moreover, the same compound did not adversely influence the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in female animals in a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD no.422; Study no. 552.439.3249). Dose related elevation of the protein, ketone and leucocytes content of the urine was observed in male animals at 450 and 150 mg/kg bw/day at termination of the treatment. The change in protein content of the urine was not fully reversible because slightly elevated levels were also detected at the end of the recovery period in male animals at 450 mg/kg bw/day. Hematology examinations did not reveal test item related adverse changes in the evaluated parameters (450, 150 or 40 mg/kg bw/day). Adverse changes were not detected at the evaluation of clinical chemistry parameters in male or female animals at 450, 150 or 40 mg/kg bw/day. Specific macroscopic alterations related to treatment with the test item were not observed at the terminal necropsy or at the end of the recovery period. A test item related elevation was detected on the kidney weights in male animals at 450 mg/kg bw/day in accordance with histopathological observations. A test item influence on the hepatic and renal functions, due to changes in weights of liver in male and female animals and of kidneys in female animals, could not be excluded but the effect was regarded as not adverse as the test item did not induce histopathological changes in these organs. Sperm analysis did not reveal any test item influence on the sperm parameters (count, motility and morphology) at 450 mg/kg bw/day. The test item did not cause increased α2µ-globulin levels in male rats. The values obtained by image analysis using previously a monoclonal antibody were generally lower as compared with control animals. Histopathology examinations revealed hyaline-like droplets in the epithel cells of some proximal convoluted tubules in the kidneys of male animals at 450 mg/kg bw/day. These findings were not seen in the kidneys of male animals in the middle dose group as well as in high dose female animals. Hyaline droplet nephropathy was not associated with interference to α2μ-globulin according to the results of the immunohistochemistry investigation. Under the conditions of the present study, the test item caused salivation (male and female) changes in some urine parameters (protein, ketone, leucocytes) in male animals and in organ weights (kidney and liver weights – male and female), hyaline-like droplets in the epithel cells of proximal convoluted tubules (male) following an oral administration at 450 mg/kg bw/day dose to Hsd.Han:Wistar rats for consecutive 90 days. The changes in the urine parameters (protein levels) were not fully reversible in male animals. At 150 mg/kg bw/day, salivation and slight changes in urine parameters and in liver weights were detected. However, no effect on liver function neither any histopathological effects were noted, and the observed weight changes remained within the range of the historical control data. Therefore these effects were judged as not adverse. There were no toxicologically relevant changes in the examined parameters in male or female animals at 40 mg/kg bw/day. Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows: NOAEL: 150 mg/kg bw/day for male and female Hsd.Han:Wistar rats.

Toxicokinetic analysis of Cyclohexylidenebis[tert-butyl] peroxide (CH)

Cyclohexylidenebis[tert-butyl] peroxide is a colourless liquid at room temperature with a molecular weight of 260.3697 g/mol. The water solubility of the substance is low (1.07 mg/L at 20°C). The log Pow of cyclohexylidenebis[tert-butyl] peroxide was measured and determined to be 7.2 at 25°C. Based on this log Pow, a BCF of 10620 L/kg wet-wt was calculated. The vapour pressure of cyclohexylidenebis[tert-butyl] peroxide is 12.3 Pa at 25°C. In an aqueous solution, cyclohexylidenebis[tert-butyl] peroxide is degraded hydrolytically to tert-butyl hydroperoxide and cyclohexanone. The half-lives of cyclohexylidenebis[tert-butyl] peroxide in an aqueous solution at 25°C at a pH of 4, 7 and 9 are 27 hours, 64 hours and 101 hours, respectively. Both hydrolysis substances have a lower log Pow value than cyclohexylidenebis[tert-butyl] peroxide itself (approximately 0.846 for tert-butyl hydroperoxide and 0.86 for cyclohexanone). Also the BCF values are lower as compared to cyclohexylidenebis[tert-butyl] peroxide (approximately 3.16 L/kg wet-wt for both hydrolysis products). Water solubility of both hydrolysis products is higher than of cyclohexylidenebis[tert-butyl] peroxide itself (> 691 g/L for tert-butyl hydroperoxide and 86 g/L for cyclohexanone).

Absorption

Generally, oral absorption is favoured for molecular weights below 500 g/mol. Since dissolution of cyclohexylidenebis[tert-butyl] peroxide in the gastro-intestinal fluids is limited by its lipophilic properties (log Pow > 4 and water solubility 1mg/L) absorption through the mucosal surface is estimated to be slow. Administered without a vehicle in an acute oral toxicity study performed on rats, cyclohexylidenebis[tert-butyl] peroxide (65 % solution) lead to a LD50 of 16653 mg/kg bw/day. Furthermore, long-term administration of cyclohexylidenebis[tert-butyl] peroxide in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening study (OECD guideline 422) indicate that the compound, and to a lower amount, its hydrolysis products became bioavailable. This is supported by the result of the developmental toxicity study (OECD guideline 414) and the subchronic repeated dose toxicity study (OECD guideline 408). Changes in organ weight (kidney and liver) were observed in both studies. Dose related elevation of the protein, ketone and leucocytes content of the urine observed in the 90 day study substantiate the fact that the substance influence the activity of the kidneys and therefore becomes bioavailable.

As indicated by the half-life values from the hydrolysis test, a certain proportion of cyclohexylidenebis[tert-butyl] peroxide will hydrolyze to tert-butyl hydroperoxide and cyclohexanone following oral administration which is indicated by the 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 slightly alkaline milieu (~ pH 5 to 8). Due to the lower log Pow values of the hydrolysis products, readily absorption through the GIT epithelium is assumed. Furthermore, molecular weights of the hydrolysis products combined with their 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.

Based on the vapour pressure of 12.3 Pa at 25 °C cyclohexylidenebis[tert-butyl] peroxide might become available for inhalation only to a lower extend. If the substance would reach the lungs in its vapour or gaseous state, slow absorption directly across the respiratory tract epithelium by passive diffusion is likely to occur due to its log Pow value and water solubility. Since specific effects of systemic toxicity were observed after oral administration systemic availability is expected also after inhalation exposure.

Cyclohexylidenebis[tert-butyl] peroxide is unlikely to penetrate through the skin as the log Pow value and water solubility limit dermal penetration. It is general accepted that if a compound’s water solubility is low (1 mg/L), absorption can be anticipated to be low. Moreover, for substances with a log Pow above 4, both penetration into stratum corneum and partition into the epidermis are unlikely to occur. These assumptions based on the physico-chemical properties of cyclohexylidenebis[tert-butyl] peroxide are further supported by the results achieved from an acute dermal toxicity study performed on rabbits. During this study no test item related mortality and no specific effects of systemic toxicity were observed. The LD50 was > 2000 mg/kg bw. However, cyclohexylidenebis[tert-butyl] peroxide caused slight skin irritation which in turn may favour direct absorption into the systemic circulation. Taken together, physico-chemical properties and experimental data indicate bioavailability of cyclohexylidenebis[tert-butyl] peroxide via oral and to a lesser extend also via inhalation and dermal route.

Distribution

Assuming that cyclohexylidenebis[tert-butyl] peroxide 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, slow hydrolysis of cyclohexylidenebis[tert-butyl] peroxide into tert-butyl hydroperoxide and cycohexanone is likely to occur. As mentioned above, the physico-chemical properties, especially the lower molecular weight and relatively high water solubility of the hydrolysis products favour systemic 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 chemical. Elevated liver weight at 1000, 300, and 100 mg/kg bw/day and kidney weights at 1000 mg/kg bw/day compared to control group were observed. Similar results were observed in the read across study (OECD guideline 414) determining the developmental toxicity over a period of 20 days. Females had an enlarged liver at necropsy at doses of 1000, 300 and 100 mg/kg bw/day. Also minimal to moderate centribular hypertrophy of hepatocytes was evident in females from all treatment groups. Further evidence can be found in the 90 days repeated dose toxicity study, showing changes in some urine parameters, in male animals and in organ weights (kidney and liver weights – male and female). Also occurance of hyaline-like droplets in the epithel cells of proximal convoluted tubules of males was observed.

Metabolism

Based on the structure of the molecule, cyclohexylidenebis[tert-butyl] peroxide may be hydrolysed after being in contact with an aqueous solution as well as enzymatically. The first degradation product tert-butyl hydroperoxide may be converted by glutathione peroxidase into tert-butanol which in turn could be conjugated with glucuronic acid or sulfate to increase the compound’s hydrophilicity (Chance, B. et al. 1979). Oxidation of tert-butanol by alcohol dehydrogenases (ADH) and aldehyde dehydrogenases (AlDH) is an alternative metabolic pathway resulting in 2,2-dimethyl-propionic acid (pivalic acid). Pivalic acid is estimated to be conjugated with glucuronic acid or amino-acids like glutamine in order to ultimately facilitate excretion. The second degradation product, cyclohexanone, was shown to be reduced to cyclohexanol and subsequently converted to 1,2- and 1,4-cyclohexanol (Mraz et al., 1994). Thus, metabolites are not assumed to be more toxic than the parent compound which is further supported by the results obtained in the in vitro mutation and cytogenetic assays in the presence of a metabolic activation system.

Excretion

As discussed above, cyclohexylidenebis[tert-butyl] peroxide will be hydrolysed both after being in contact with an aqueous solution or enzymatically and will probably not be excreted in its unhydrolysed form. The degradation products tert-butyl hydroperoxide and cyclohexanone have a low molecular weight (90.12 g/mol and 98.15 g/mol, respectively), are miscible in water and thus may directly be excreted by urine. Cyclohexanone was shown to be converted to 1,2- and 1,4-cyclohexanol and excreted via urine in its unconjugated or conjugated form (Mraz et al., 1994).

Summary

Physico-chemical properties,particularly water solubility and octanol-water partition coefficient and experimental data indicate bioavailability of cyclohexylidenebis[tert-butyl] peroxide via oral and to a lesser extend also via inhalation and dermal route. Intracellular concentration is likely to be higher than extracellular due to the lipophilicity of cyclohexylidenebis[tert-butyl] peroxide. Hydrolytic and metabolic conversion into tert-butyl hydroperoxide and cyclohexanone is expected and hydroxylation/conjugation of Phase I-metabolites may further increase hydrophilicity. Metabolites of cyclohexylidenebis[tert-butyl] peroxide are not considered to be more toxic than cyclohexylidenebis[tert-butyl] peroxide itself. Excretion via urine is assumed to be the main excretion pathway of metabolites formed due to their molecular weight ( 300 g/mol in rat) and water solubility. Bioaccumulation of cyclohexylidenebis[tert-butyl] peroxide itself and its hydrolysis products is not likely to occur due to hydrolytic degradation and the physico-chemical properties of the hydrolysis products.

References

ECHA (2014), Guidance on information requirements and chemical safety assessment, Chapter R.7c: Endpoint specific guidance

Marquardt H., et al., (1999). Toxicology. Academic Press, 1999

Mutschler E., et al., (2001).Arzneimittelwirkungen. Lehrbuch der Pharmakologie und Toxikologie. Wissenschaftliche Verlagsgesellschaft,, 2001

Zeiger, Errol (1993) United States Department of Health and Human Services; NIH publication 93-3341, 1993