Reaction product of 3,5,5-trimethyl-hexanoic acid and 2-methylpropanoic acid and pentaerythritol

Endpoint summary

Description of key information

An assessment of the toxicokinetics of the substance, Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid (test item), in accordance with Annex VIII, Section 8.8.1 of Regulation (EC) No 1907/2006 (REACH) has been conducted.

 

Toxicological Data

 

Acute toxicity in rat: No mortality observed when tested up to 2,000 mg/kg bw in rats following acute oral (OECD 423) and acute dermal (OECD 402) exposure respectively. No overt toxicity, clinical signs, death or abnormality at necropsy was observed amongst animals therefore, the reported LD₅₀ of >2000 mg/kg bw/day concluded under the condition of these studies.

 

Local Toxicity: The substance was not irritating to the eye or skin in the OECD 405 or OECD 404 and 439 studies, respectively. Although conjunctivitis was observed up to 48 hours post exposure, it was reversible and no other overt toxicity signs were observed in the OECD 405 study. In both dermal exposures, there were no signs of systemic toxicity or dermal reactions through the test period. Furthermore, no sensitisation reactions were observed following an exposure via the local lymph node assay (OECD 429).

 

There are several sub-acute and sub-chronic studies conducted on the substance. The long-term systemic hazard assessment is based on a sub-chronic toxicity study conducted on rats in accordance with OECD 408 under GLP. In this study, the substance was administered to rats by oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day without the inclusion of satellite groups. Control groups were included. The substance was well-tolerated by the animals during the in-life phase of the study with no premature deaths and no clinical signs related to treatment. No toxicological effects of importance were observed and no effects on sensory reactivity, grip strength or motor activity were observed. Bodyweight and food consumption were also unaffected and there were no treatment-related ophthalmic lesions reported. However, specific target organ toxicity in the liver, kidney and thyroid was observed in the mid and high dose groups which was considered to be treatment related. These effects included periportal hepatocyte vacuolation (fatty change) characterised by cytoplasmic fat accumulation and centrilobular hepatocyte hypertrophy in the liver and hyaline droplet accumulation in the cortical tubules, accompanied by the accumulation of granular casts in the corticomedullary junction in the kidney. The observed centrilobular hepatocyte hypertrophy was considered an adaptive response since it was associated with hepatic enzyme induction and decrease in thyroid hormones. The substance contribution to the observed periportal hepatocyte vacuolation could not be ruled out since it was also gender specific with observed dose respond effects.This was accompanied bypale liver, increased liver weights, increases in plasma enzyme activities of alkaline phosphatase, alanine aminotransferase and aspartate amino transferase activities and the decrease of glucose or cholesterol levels. The severity of this effect ranged from very slight at the low dose to moderate at high dose group. The hyaline droplet nephropathy was also dose-related; however, it is a common problem observed in rats following exposure to xenobiotic. It is considered that, under the conditions of the study, the no-observed- adverse-effect-level (NOAEL) was 100 mg/kg/day based on the terminal investigations indicating the liver, thyroids and kidneys as target organs which were considered adverse in nature at dose levels of 300 and 1000 mg/kg/day. Based on the observed effects, the substance satisfies the criteria for classification for specific target organ toxicity - repeated exposure (STOT-RE Category 2) in accordance with Regulation (EC) No 1272/2008 (CLP).

 

Other sub-acute/chronic studies were conducted to address the potential reproductive and developmental toxicity effect of the substance in rat following OECD 421, OECD 416 and OECD 414 guidelines. In the OECD 421 screening study, the animals were exposed to the test substance at concentrations of 100, 300 and 1000 mg/kg bw/day without including a recovery group. Non- treatment related mortalities were observed at the mid and high dose groups without any overt toxicity observed at necropsy. There were no effects on reproductive performance, mating, estrous cycle, fertility with all gestation lengths, however, changes of an uncertain relationship to treatment were seen in the ovary of females at all dose groups. Offspring birth weights were significantly lower than control groups at 300 and 1000 mg/kg/day, respectively. It was concluded that the NOAEL was 1000 mg/kg/day for reproductive performance, in the absence of adverse effects in all treatment groups. 100 mg/kg/day was considered to be the overall NOAEL because of the significantly decreased offspring birth weights at 300 and 1000 mg/kg/day. The effects seen on birth weight in OECD 421 was investigated further in a two-generation study (OECD 416) in rats. The animals received oral gavage doses of 30, 100 or 300 mg/kg/day including control groups, receiving vehicle only. There was no evidence of any adverse effect on clinical condition, bodyweight, bodyweight change, food consumption or food conversion efficiency in the F0 or F1 generations at all dose groups. Although liver weight was increase, microscopic examination revealed no adverse effect. As in OECD 408, hyaline droplet nephropathy was present in the kidneys that were examined for macroscopic findings from the F0 and F1males dosed with 100 or 300 mg/kg/day. Reproductive performance of F0 and F1 adults such as oestrous cycles, mating performance, pre-coital interval, gestation length, litter size and subsequent offspring clinical condition and sex ratio were unaffected. The survival of F1 offspring was unaffected but the viability index (95 %) of F2 was statistically significantly lower in the high dose group and a relationship to parental treatment with the substance could not be ruled out. Abnormalities in the form of hyaline droplet nephropathy in the kidneys as previously noted in the OECD 421 were observed in F0 or F1 males in the 100 or 300 mg/kg/day dose groups, however, no any other effects organs were noted. Based on the results of this study it was concluded that the NOAEL for general toxicity and reproductive performance of the F0 and F1 adult animals was 300 mg/kg/ day. The NOAEL for offspring survival and growth is 100 mg/kg/day, this was based on reduced survival of the F2 offspring between Day 1 and 4 of age, and the low birth weights of the offspring in both generations at 300 mg/kg/day. The observed low birth weights of the offspring were in concordance with the observation in the OECD 421 study. An Embryo-Fetal Developmental toxicity study (OECD 414) was conducted to investigate the effects in offspring further. In the study, the substance was administered to rats via oral gavage at dose of 100, 300 and 1000 mg/kg/day. There were no deaths or clinical signs observed in any of the treatment groups throughout the study. No treatment related effects on organ weight or any macroscopic findings at necropsy or effects on maternal bodyweight and bodyweight gain or food consumption were noted. Embyro-fetal survival, growth and development were also unaffected by treatment, however, incidence of major and minor abnormalities and skeletal variants without any relationship to treatment were observed. These included a slightly higher incidence of incompletely ossified/ unossified cranial centres, hyoid, and pelvic bones compared with concurrent controls at 1000 mg/kg/day.

 

NOAEL (general toxicity and reproductive performance of the F0 and F1) = 300 mg/kg bw/day in OECD 416.

NOAEL for offspring survival and growth = 100 mg/kg bw/day based on reduction in survival of F1 and F2 offspring in OECD 421 and 416, respectively.

 

NOAEL (development & teratogenicity i.e. both maternal toxicity and embryo-fetal development) = 1000 mg/kg bw/day, where no adverse effects were observed in the OECD 414 test.

 

Bioaccumulation data: the bio-concentration factor (BCF) of component 1 [i.e. 2,2-bis{[(2- methylpropanoyl)oxy]methyl}propane-1,3-diyl bis(2-methylpropanoate)] in the substance at concentration of 0.15 mg/L and 1.5 mg/L on zebra fish (danio rerio) was determined using the semi-static bioaccumulation method lasting 28 days. Mean BCF values of 0.648 and 3.611 were reported for nominal concentrations 0.15 mg/L and 1.5 mg/L, respectively. Furthermore, the mean BCF on the 28_th day was reported as 0.945. The study concluded that the BCF for component 1 in the substance on zebra fish was in the range of 0.179 – 14.45, this is below the BCF cut off value of 2000 to be considered bioaccumulative and therefore the substance is considered non-bioaccumulative.

 

Absorption

 

The molecular weight of the 5 main components of the UVCB substance are 416.50, 486.63, 556.76, 626.89 and 697.02 g/mol. The vapor pressure and partition co-efficient of the substance is 2 x 10_^-4 Pa at 25 °C and Log Pow of ≥ 4.9, respectively. The water solubility of the lowest molecular weight component is 2.5 mg/l with the remaining components determined to be < 1 mg/l. These physicochemical properties indicate potential absorption by oral route only. Paracellular absorption is not possible as the molecular weight of all constituents is >250 g/mol. However, uptake via intracellular pathway is possible for the components with molecular weight <500, and for the components > 500, active transport (influx/efflux) and intestinal lymphatic route are favored. Intake from the intracellular and active transport (influx) pathway exit through the basolateral membrane into the portal blood and result in first pass metabolism in the liver. This means that the concentration of the parent substance is reduced before reaching systemic circulation. For the higher molecular weight components, active transporter (efflux) and intestinal lymphatic routes play a role thus avoid hepatic first pass effects. This could result in a high plasma half-life of the components since the they would be expected to go through general metabolic pathway. This also means the delivery of the components would be more targeted as noted by the adverse effects observed in the liver, kidneys and thyroids. However, these pathways are very limited due restriction by the barrier function of the intestines resulting to unabsorbed components remaining in the gastrointestinal (GI) track. Although a similar absorption pattern would be expected via the inhalation route, the substance is not volatile, with a reported vapor pressure of 2 x 10_^-4 Pa at 25 °C. Bioavailability via the inhalation route would therefore be limited. Based on the substances’ Log Pow of ≥ 4.9, high molecular weight and not being a surfactant (refer to IUCLID Section 4.10), dermal absorption would be limited since the transfer of the substances between the stratum corneum and the epidermis would be restricted. Therefore, overall uptake via this route would be slightly reduced, as demonstrated by the lack of toxicity observed following exposure viain vivodermal route.

 

Distribution

 

Based on the physicochemical properties, the substance would be expected to be distributed into lymphoid tissues or metabolic organs (kidneys, liver and/or thyroid) and the GI tract. Absorbed substance via the intracellular and active transport (influx) pathway would result in uptake into the liver. Absorption via the active transporter (efflux) and intestinal lymphatic routes result in uptake into lymphoid tissues with any unabsorbed components remaining in the GI tract. This hypothesis is supported by observed effects such as; periportal hepatocyte vacuolation (fatty change), cytoplasmic fat accumulation and centrilobular hepatocyte hypertrophy in the liver; hyaline droplet accumulation in the cortical tubules and accumulation of granular casts in the corticomedullary junction in the kidney, following repeated exposure to the substance.

 

Metabolism

 

Metabolism proceeds through the liver and GI tract via the phase I and II metabolic enzymes. Once absorbed, the substance is expected to undergo metabolism in the liver through hydrolysis of the esters via esterase into carboxylic acid and alcohol derivatives. These chemicals will, in- turn, be conjugated via glucuronosyl transferases (UGTs) and acyl-CoA enzymes into more soluble metabolites. The metabolites will be excreted via urine and bile supported by changes in the kidneys. This hypothesis is supported by the observed centrilobular hepatocyte hypertrophy in the liver which was associated with hepatic enzyme induction and decrease in thyroid hormones following repeated exposure to the substance in rats. Unabsorbed components of the substance in gut will be hydrolysed by gut microflora into carboxylic acid and alcohol derivatives and respective conjugates excreted via faeces as metabolites or parent compound.

 

Excretion

 

Based on the absorption, distribution and potential metabolic pathways highlighted coupled with available systemic data, the substance will most likely be excreted via bile, faeces and urine. The constituents of low – mid molecular weights (i.e. <500 g/mol) absorbed via paracellular and intracellular pathways would most likely be excreted via urine as supported by the changes observed in the kidney - hyaline droplet nephropathy, species specific effect confined in rats following sub/chronic exposure to substance. Components with molecular weight >500 will be taken up via active transport (influx/efflux) and the intestinal lymphatic route will most likely undergo biliary excretion. Since a high proportion of the components of the substance have molecular weights >500, concentration of unabsorbed constituents will most likely remain in the intestinal tract. These components would be excreted via both biliary excretion or faeces.

 

Conclusion

 

Based on the physicochemical properties of this substance the potential for bioaccumulation of the substance is expected. However, accumulation was not observed at the primary site of exposure (the gut) as demonstrated in the long-term studies; OECD 421, OECD 416, OECD 414 and OECD 408. Specific target organ toxicity in the kidney and liver in the OECD 408 study was reported. However, the hyaline droplet accumulation in the cortical tubules, accompanied by the accumulation of granular casts in the corticomedullary junction in the kidney is considered species specific and not relevant to humans. The centrilobular hepatocyte hypertrophy was considered an adaptive response since it was associated with hepatic enzyme induction and decrease in thyroid hormones. Periportal hepatocyte vacuolation observed in the OECD 408 study was considered treatment related as it was associated with pale liver, increased liver weights, increases in plasma enzyme activities of alkaline phosphatase, alanine aminotransferase and aspartate amino transferase activities and the decrease of glucose or cholesterol levels. These enzymes are similar marker used to assess hepatotoxicity profile in human and as such the potential for similar hepatotoxicity profile cannot be rule out in man. However, in other long term studies increase liver weight was observed but without centrilobular hepatocyte hypertrophy, periportal hepatocyte vacuolation or any increase in plasma enzyme activities of alkaline phosphatase, alanine aminotransferase and aspartate amino transferase. It can be concluded that these changes were adaptive responses or incidental and the effect of the substance in terms of human exposure may be negligible. Furthermore, the bioconcentration factor (BCF) in zebra fish was reported as 14.45 L/kg, much lower than the cut of value of 2000 L/kg for substance considered bioaccumulative. In addition to the above, the substance was well tolerated in repeated exposure with no premature death, no clinical signs related to treatment or of toxicological importance. There were also no effects seen on sensory reactivity, grip strength or motor activity. Bodyweight and food consumption were also unaffected and there were no treatment-related ophthalmic lesions reported.

This demonstrates that the bioaccumulation potential of the substance is very low and it can be concluded that the toxicokinetic of the substance does not pose significant toxicological concern.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

100

Absorption rate - inhalation (%):

100