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Description of key information

In accordance with REACH Annex VIII, Section 8.8.1, an assessment of the toxicokinetic behaviour of the substance to the extent that can be derived from the relevant available information has been conducted.

Toxicological Information

Acute Toxicity: the substance is classified as acutely toxic (Category 4) to rats via the oral and inhalation routes, in accordance with regulation No 1272/2008 (GHS & CLP). During an acute oral toxicity test (OECD 420) and an acute inhalation toxicity test (OECD 436) the reported LD50 and LC50 of 300 – 2000 mg/kg bw/day and > 1.0 – 5.0 mg/L, respectively. In both studies, mortality was observed at dose/concentration of 2000 mg/kg bw/day and 5.0 mg/L, respectively. Macroscopic abnormalities reported in the lungs included hemorrhagic, pale and/or abnormally red, pale, dark patches, abnormally red, abnormally dark.

Local Toxicity: the substance is non-corrosive to the skin in vitro(OECD 431) but irritative to skin (OECD 439) and eye in vitro(OECD 437, 492). The substance caused sensitisation effect in vivo(LLNA – OECD 429) with reported EC3value of 10.7%. No local effects were observed and no signs of systemic toxicity or abnormal observation at necropsy were reported.Ear thickness was within the normal range. There were no clinical abnormalities of the surrounding area noted for any of the animals and no mortality reported in the main test. There were no abnormal changes to the body weight of all main test animals.

Sub-Acute Toxicity: in a dose range finder for a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422), rats were exposed to dose levels between 50 and 300 mg/kg/day. In dosing phase one, groups of rats (5/sex) were administered 300 mg/kg bw/day substance, due to two deaths observed, dosing was stopped allowing the substance formulations to wash out from the remaining animals, phase two dosing was then launched at doses of 50 and 250 mg/kg/day. Dosing stopped after Day 2 at 250 mg/kg bw/day, following the deaths of all Group 3 males. Females were retained on the study, and dosing recommenced at a lower dose level of 175 mg/kg bw/day from Day 21 until Day 34 of the dosing phase. Group 5 animals were dosed at the same dose level, using the 3 remaining spare males.

Clinical observations for animals administered 250 or 300 mg/kg/day included one or more of the following: prostrate, twitching, convulsions, tremors, subdued/ sluggish (slow/deliberate movements), unresponsive, semi-closed to closed eyes; and/or irregular, rapid, or laboured respiration. In addition, these animals showed body weight loss and low food or water consumption.

All animals administered 50, 125, or 175 mg/kg bw/day survived to their scheduled sacrifice. Post-dose observations for animals administered 50, 125, or 175 mg/kg bw/day included mouth rubbing, (occasionally) salivation, and paddling. For one female administered 175 mg/kg bw/day, minimal staggering, subdued/sluggish behaviour, twitching, and decreased respiration were observed on one occasion. Reduced activity was observed on one occasion for two females administered 175 mg/kg bw/day. Other clinical observations included vocalisation or red staining of the neck for animals administered 50 or 175 mg/kg bw/day; these observations were transient and considered not adverse. Test article-related body weight loss was observed throughout the dosing phase for males administered 175 mg/kg bw/day and during the first week of dosing for females administered 175 mg/kg bw/day. During the second week of dosing, body weight gain was comparable with controls for females administered 175 mg/kg bw/day.

For animals administered 125 mg/kg bw/day, test article-related body weight loss was observed during the first week of dosing, followed by reduced body weight gain during the second week of dosing.

Macroscopic observations were recorded in one or two animals from each test article-treated group (excluding controls). These consisted of changes in the stomach (abnormal contents), jejunum (distended), liver (pale and mottled), mandibular lymph nodes (red), seminal vesicles (large), thymus (red), epididymis (small), and testes (small). In the absence of a dose-response, the relationship to test article was uncertain.

One daily oral gavage administration of 50, 125, 175, 250, or 300 mg/kg bw/day substance to rats was not well tolerated which also resulted in mortality at does 250 or 300 mg/kg bw/day. Dose levels of 125 or 175 mg/kg/day resulted in test article-related effects including body weight losses and reduced food and water consumption, which would not be tolerated over longer dosing duration.

 

Therefore, it was concluded that administration of test item dose at up 100 mg/kg bw/day would be better tolerated in the main OECD 422 study.

 

OECD 422 (combined with micronucleus testing): Four groups of 10 male and 10 female sexually mature Crl:WI(Han) rats were administered 0 (control article [vehicle: corn oil]), 30, 50, or 100 mg/kg/day of the substance once daily by oral gavage . Males were dosed for 42 consecutive days (2 weeks prior to pairing, during pairing, and approximately 3 weeks post-pairing) and were sent to necropsy on Day 43. Females were dosed for up to 57 days (2 weeks prior to pairing, during pairing, throughout gestation, and up to lactation day (LD) 13 and were sent to necropsy on LD 14.

Micronuclei (MN) in the polychromatic erythrocytes (PCE) of the bone marrow were not induced following test article administration at any dose level.

No treatment related deaths occurred, however, three mortality were reported (one control male, one female administered 50 mg/kg/day, and one male administered 100 mg/kg/day).

Following 100 mg/kg/day in males, initial body weight loss, lower body weight gains and reduced food consumption were observed. Furthermore, raised hair and shorter hind limb foot splay, with reduced activity and reduced rearing observed during the weekly behavioural assessments. However, locomotor activities were unaffected and in the absence of any overt pathology observation, these findings were not considered as an indication of neurotoxicity. The group mean kidney weights (absolute and body weight-relative) were higher correlating with presence of hyaline droplets; this is a common response in the male rat to xenobiotics and represents accumulations of α2u globulin, a naturally occurring male rat protein. Chemicals that bind to α2u globulin form a complex that is more resistant to catabolism and will result in accumulations of hyaline droplets. This male rat-specific finding is of little relevance to risk assessment in humans.

For females administered 100 mg/kg/day; body weight gain was reduced over the duration of the study, mean absolute liver weights and body weight relative weight ratios were higher with elevated alkaline phosphatase activity, total protein and albumin:globulin (A:G) ratios. Furthermore, lower total protein and globulin levels were observed. In the absence of any overt clinical or pathological findings, these observations were considered to represent adaptive responses to administration of a xenobiotic. In addition to the above, rearing was reduced but locomotive activities were not affected.

Following 50 mg/kg/day administration Initial body weight losses, reduced body weight gains, and lower food consumptions were observed in both sexes. For females, initial reduction in lower food consumption was evident with reduced rearing during lactation. Finally, mean thymus weights (absolute and body weight relative) were lower for females administered at all dose levels without any correlating haematological or microscopic abnormalities. In the absence of any associated decline in physical health, clinical pathology or microscopic changes to indicate an adverse effect, these findings were not considered treatment related.

Reproductive performance was unaffected; the test article did not affect estrous cycles, mating, fertility, pregnancy or parturition. The number and length of estrous cycles was unaffected by test article administration. Mating performance was unaffected by test article administration; all animals mated within 7 days of pairing. Ten, nine, nine, or ten females administered control article (vehicle) or 30, 50, or 100 mg/kg/day, respectively, achieved pregnancy. Nine, nine, seven or ten dams administered control article (vehicle) or 30, 50, or 100 mg/kg/day successfully reared a live litter to LD 13. One control female (Animal R0410) had no viable fetuses (100% in utero litter losses).

No treatment related effects were noted on mean gestation lengths or the mean number of implantation sites. A slightly smaller litter size was noted following administration of 100 mg/kg/day, compared with controls (-13%). No treatment related effect on ano-genital distance, no nipples/areolae were present for male offspring, no effects on thyroid weight or thyroid hormone levels were observed in dose groups.

Test item related offspring effects following maternal exposure of 100 mg/kg/day were confined to slightly smaller litter sizes, compared with controls, and lower mean offspring weights in litters of the groups administered 50 or 100 mg/kg/day, although mean values were ± 10% of controls. In the absence of any test article-related offspring mortality noted at this dose level, these findings were considered as non-adverse. A medulloblastoma was present in one male administered 100 mg/kg/day (decedent Animal R0306). This tumor has been observed as a background finding in short term studies, although it is rare. As only one animal was affected, a relationship to the test article was considered unlikely. Microscopic findings in other tissues were generally infrequent, of a minor nature, and consistent with the usual pattern of findings in rats of this strain and age.

It was concluded that once daily oral gavage administration of 30, 50, or 100 mg/kg/day Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine to male rats for 42 consecutive days and to female rats for up to 58 days (pre-pairing, throughout gestation, and during the first 2 weeks of lactation) resulted in test item related findings at all dose levels which were considered none adverse. The no observed adverse effect level (NOAEL) for male and female systemic toxicity was considered as 100 mg/kg/day. A no observed effect level (NOEL) was considered as 30 mg/kg/day for the males, although a NOEL for females could not be established, due to the lower thymus weights observed in all dose groups. Reproductive performance and offspring development were unaffected as such, the no observed adverse effect level (NOEL) and NOAEL for reproductive toxicity was considered as 100 mg/kg/day.

Absorption

The molecular weight of constituent I is 270 and 129 g/mol, for constituent II, both considerably low. Both constituent vapour pressure of 9.0 Pa and surface tension of 57.2 nM/m. The water solubility of constituent I is  report at 81. mg/L and for constituent II a value of 1490 mg/L, considerable very high.  The have n-octanol/water partition coefficient of substance is 5.7 for constituent I and 2.8 for constituent II. These physicochemical properties are suggestive of favourable absorption via the oral and inhalation routes. Although these properties coupled with the irritation potential of the substance makes uptake from the dermal route possible, based on the surface tension of the substance (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.This is demonstrated by the lack of significant systemic and local toxicity from in vivo sensitisation study on the substance. For absorption calculation, a default value of 100 % skin absorption according to ECHA 20141can be applied as conservative approach for risk assessment.

Absorption from the gastro-intestinal tract: based on the molecular weight of the constituents <300 g/mol and the water solubility, intracellular and paracellular absorption of the substance is possible resulting in substance delivery into the liver via portal circulation i.e. first pass metabolism. For constituent II, intracellular absorption is the main route for systemic bioavailability; this is based on the n-octanol/water partition coefficient of 5.7, low water solubility of 18.8 mg/Land molecular weight of 270 g/mol.Based on the low molecular weight and high water solubility of constituent II, it is expected to undergo paracellular absorption. Furthermore, the irritation potential of the substance may also aid absorption by causing damage to the membrane resulting to high systemic bioavailability of the substance.This is supported by the overt clinical toxicities and mortality observed in acute and sub-acute oral and inhalation studies in the rat as an indication of the systemic availability of the substance. Based on the vapour pressure of 9.0 Pa, exposure via inhalation route is possible as demonstrated by macroscopic clinical abnormalities reported in the lungs and mortality following inhalation study in rats exposed to the substance. Upon inhalation, absorption into pulmonary circulation follow similar route to oral uptake. However, a high plasma concentration of the substance as well as plasma half-life is expected since the rate fast pass metabolism in the lung is slower than in the liver. For absorption calculation, a default route-to-route extrapolation according to ECHA 2014 can be applied as conservative approach for risk assessment.

 Distribution

The substance has physicochemical properties (mainly molecular weight and its high water solubility) means upon oral and inhalation exposure, the substance can easily pass through aqueous pores or be carried through the epithelial barrier (paracellular pathway) by the bulk passage of water into the liver. This will limit distribution of the parent compound systemically especially via oral route and therefore resulting into a reduced half-life of the parent compound in blood plasma. However, based on the irritative potential of the substance, damage to the lining of the GI track and epithelial lining is possible therefore increasing systemic bioavailability of the substance. For constituent I very low water solubility coupled with high n-octanol/water partition coefficient of 5.7could result in a high plasma half-life. This means the delivery of the components would be more targeted as observed in the liver, kidneys and thyroids following sub-acute oral exposure in rats.Oral absorption of constituent II would result into uptake via the portal vein into first pass metabolism with reduction in overall systemic distribution of parent compound. A wide distribution of Constituent II is expected following inhalation exposure since low molecular weight and high water solubility adding passive absorption therefore increased level into circulatory system as demonstrated by the nephropathy syndrome which is associated with α2u globulin accumulation in hyaline droplets, a condition not relevant to humans. This is supported by the clinical observations of increased kidney weights. Systemic distribution via  dermal absorption is limited as demonstrated by the lack of systemic toxicity observed during the local lymph node assay in mice.

Metabolism

Amine metabolism is mainly through phase I and II enzymes as demonstrated by increased liver weight and alkaline phosphatase activity in the liver of both sexes following sub-acute exposure to the substance. Constituent I (tertiary amine) is susceptible to two routes of oxidation catalyzed by monoamine oxidase (MAO) and diamine oxidase, i.e. alpha carbon oxidation resulting into carbinolamine which then undergoes spontaneous cleavage to a secondary amine, this is followed by further oxidation to produce the aldehyde derivative. Another route of oxidation would result into formation of dipolar N-oxide derivatives which is further dealkylated to form secondary amine. For constituent II (secondary amine), an oxidation reaction is expected to yield aldehyde derivatives which would undergo further oxidation by alcoholdehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) resulting into hydroxylatedderivatives (hydroxylamines). These metabolites then undergo phase II metabolic pathways forming mainly conjugative derivatives of glucuronic acid and sulfate and to a lesser extendvian-methylation.

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, faces and urine. Both constituents are of low – mid molecular weights absorbed viaparacellularand intracellular pathways would most likely be excreted via urine as supported by the changes observed in the kidney -hyaline droplet nephropathy, spices specific effect confined in rats following sub/chronic exposure to substance.For constituent II the n-octanol/water partition coefficient log Pow of 5.7 and water solubility of 18.8 mg/L are suggestive of potential accumulation of this substance in fatty tissues after absorption from gastro-intestinal, however, this is not expected to be significant as it was no substance accumulation was observed following sub-acute exposure in rats. Furthermore, the irritating tendency means availability of the substance into the circulatory system is rapid making it readily available for biotransformation into conjugated metabolites which are easily eliminated via bile and urine. Unabsorbed constituent will most likely be eliminated via feces. Elimination of constituent II is assumed to be rapid, therefore no potential for bioaccumulation is to be expected, this is because absorption of the substance would result into fast pass metabolism therefore resulting to reduced parent compound. Based on the potential uptake, the substance is expected to be eliminated as conjugates mainly in urine and biliary and to a small via faeces.

 Conclusion

The substance has physicochemical properties which will not lead to potential bioaccumulation. Exposure from oral and inhalation are most preferred routes and with limited bioavailability via dermal exposure. This is supported by the adverse effects observed in the acute oral toxicity testOECD 420 and inhalation toxicity test OECD 436 with no adverse systemic effects observed in the LLNA test OECD 429 study.

Based on the log Pow of 5.7 and low water solubility of constituent I, potential for bioaccumulation was expected to be significant with slow elimination. However, accumulation was not observed at the primary site of exposure as demonstrated in the acute and sub-acute studies; OECD 422, OECD 420, OECD 429 and OECD 436. Furthermore, no site-specific microscopic evidence of irritation at primary exposure site such as GI tract and the lung was observed which demonstrates that there is no irritative potential of the substance at this site when dosed via the aforementioned routes and therefore irritation at the GI tract will not affect absorption which was previously expected. This means that paracellular absorption is more favoured and therefore fast pass metabolism, ensuring rapid biotransformation and elimination. For constituent II, based on the low molecular weight and high water solubility ensure easy absorption from the gut into portal vein resulting into rapid metabolism and elimination. Therefore, for both constituents, no significant bioaccumulation is expected, and the substance is expected to undergo rapid transformation to more polar metabolites resulting into rapid elimination via bile and urine.

Specific substance toxicity noted in the lung, liver and kidney, in the form of hemorrhagic discolouration, increase organ weight and severity of hyaline droplet accumulation in the cortical tubules reported in OECD 436 and OECD 422 study are considered adaptive response and are not treatment related.

At high doses, the substance is not well tolerated as demonstrated by mortalities and overt toxicities noted in OECD 420 and OECD 436, hence the classification of Acute toxicity category 4 according to regulation No 1272/2008 (CLP). However, at lower dose£100 mg/kg bw/day, the substance is well tolerated. In OECD 422 study there were no site-specific accumulation of the substance was noted, no premature death, no clinical signs related to the treatment and no effects of toxicological importance to humans. 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 toxicokinetics 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

Additional information