Hexahydro-1,3,5-trimethyl-1,3,5-triazine

Endpoint summary

Description of key information

An assessment of the toxicokinetic behaviour of the substance to the extent that could be derived from the relevant available information was conducted as defined in Annex VIII, Section 8.8 of the REACH Regulation. Using only the available data, general conclusions can be drawn regarding the toxicokinetics of hexahydro-1,3,5-trimethyl-S-triazine. It is clear that systemic effects are elicited after repeated oral administration and the fact that the substance was shown to be a sensitizer indicates that dermal adsorption can occur. However in the absence of ADME data, no reliable quantitative conclusions can be drawn and therefore adsorption is considered to be 100% for all routes for the purposes of chemical safety assessment.

Key value for chemical safety assessment

Bioaccumulation potential:

low bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

100

Absorption rate - inhalation (%):

100

Additional information

TOXICOKINETIC ASSESSMENT

 

Toxicological Data

Acute toxicity:  

The substance was not well tolerated in acute oral studies in rats with a reported LD50 range of  > 300 - <2000 mg/kg bw/day.  Three mortalities at 2000 mg/kg bw/day and one mortality at 300 mg/kg bw/day with reported hunched posture and/or piloerection were noted among the animals treated at 300 mg/kg between Days 1 and 10. Animals dosed at 2000 mg/kg showed a moribund condition and clonic spasms prior to death. Reddish discolouration of the glandular mucosa of the stomach was found in the animals that died during the study, at macroscopic post mortem examination. Macroscopic examination of the surviving animals at termination did not reveal any abnormalities. (OECD 423, 2009).

 

Local Toxicity:  

The substance was corrosive to rabbit eyes and skin. Corneal opacity, iritis and conjunctival irritation, noted in one eye, persisted to day 21. Skin erythema was moderate to severe at 24 hours and severe (with no eschar formation observed) at 48 and 72 hours. By day 7, the dose site was necrotic with pale areas and necrotic on day 14 (OECD 404 & 405, 2009). The substance was concluded to be a skin sensitiser in both OECD 406 and 429 studies following exposure to concentration ranging from 25% to 100%. In both studies, no mortality was observed. In the local lymph node assay, no systemic toxicity was observed, no effects were noted on body weight and no macroscopic abnormalities were noted. The stimulation index (SI) values calculated for the substance for all tested concentrations were >3, i.e. 28.6. 19.3 and 24.8 at 25, 50 and 100 %, respectively (OECD 406, 2010 and OECD 429, 2007).

 

Repeated Dose Toxicity and Reproductive/Developmental Toxicity:

In a combined 28 -day repeated dose toxicity study with reproductive/developmental screening (OECD 422, 2010) the systemic toxicity and potential adverse effects were investigated via oral gavage administration of the substance to rats at dosage of 10, 30 and 100 mg/kg bw/day.  Food consumption mean body weights and body weight gain and neurobehavior were unaffected in all treated groups.  No mortality occurred during the study period that was considered to be related to treatment with the test substance. One male at 100 mg/kg/day died on the scheduled day of necropsy during the anaesthesia procedure before blood sampling. No cause of death could be established but was, however, not considered treatment related. Also, at this dose group total protein and albumin were slightly decreased for both sexes and decreased haemoglobin and decreased APTT for males and increased APTT for females. Statistically significant changes at 30 mg/kg (bile acids for males) and at 100 mg/kg (chloride for males) were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution or remained within the range considered normal for rats of this age and strain. The increased values for bile acids (not statistically significant) noted for females of all dose groups were considered to have occurred by chance and to be of no toxicological relevance as these changes were caused by individual animals only. No toxicologically significant changes were noted in organ weights and organ to body weight ratios up to 100 mg/kg bw/day. The statistically significant decreased values for testes and epididymides weights (absolute and relative) at the high dose group were not considered toxicologically significant as the changes were very slight, no corroborative findings were noted at microscopic examination, and reproduction was unaffected. A thickened limiting ridge of the stomach was noted in five males and eight females of the high dose group and one female at mid dose. Other incidental findings included soft yellowish or greenish nodule at the epididymides, isolated reddish or dark red foci at the glandular mucosa of the stomach, reddish discolouration of the Peyers patches of the jejunum, reddish discolouration of the thymus or mandibular lymph nodes, exophthalmus of the left eye, red-brown foci at the preputial glands or clitoral glands, watery-clear cyst at the uterus or ovaries, greenish foci at the clitoral glands, alopecia, dark red foci at the thymus, and the uterus containing fluid. At 100 mg/kg bw/day, parental toxicity consisted of clinical signs (salivation, rales and/or piloerection), slightly decreased total protein and albumin, a thickened limiting ridge of the stomach at macroscopic examination and microscopic findings for the stomach (lymphogranulocytic inflammation of the glandular stomach and hyperplasia of the epithelium of the limiting ridge and eyes (females only: degeneration of the retina)). There were possible treatment-related microscopic findings in the eyes of 2/5 females at 100 mg/kg bw/day female rats consisting of bilateral degeneration of the retina (atrophy). Bilateral minimal degeneration of the retina was only recorded in one of these females, while the other case was microscopically interpreted as within the normal limits of a normal rat retina. Because this change was minimal and observed in one single animal, the toxicological significance of this change remained undetermined. All remaining microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar Han rats of this age and strain. No parental toxicity was observed at 10 and 30 mg/kg/day. Based on these results, the No Observed Adverse Effect Level (NOAEL) was derived: Parental NOAEL: 30 mg/kg/day.

 

In a prenatal developmental toxicity study (OECD 414; 2019) time-mated female New Zealand White rabbits were exposed to the substance from Day 6 to 28 post-coitum at dose levels of 0, 2, 8 and 25 mg/kg bw/day. The following parameters and end points were evaluated in this study; F0 generation - mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents.  In addition, the following parameters were determined for F1 generation; the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral and skeletal malformations and developmental variations. The substance was well tolerated in maternal animals in all dose group, no adverse effects were observed in all measured parameters. However, in the F1 generation, a non-adverse increased incidence of metacarpals/metatarsals and tarsals (skeletal variations) occurred (9.5% vs. 2.9% per litter in the control group for metacarpals/metatarsals and 2.5% vs. 0.9% in the control group for tarsals) at the high dose group. No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. litter size, post-implantation loss, sex ratio and fetal body weights). Based on the outcomes of the study, the No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity was 25 mg/kg bw/day.

 

In another developmental toxicity study (OECD 414, 2016) mated female Wistar Han rats were administered the test item once daily by oral gavage from Days 6 to 20 post-coitum at doses of 10, 40 and 120 mg/kg (Groups 2, 3 and 4 respectively). The rats of the control group received the vehicle, propylene glycol, alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. Formulations prepared on two days during treatment were analyzed for accuracy, homogeneity and/or stability. All animals surviving to Day 21 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. Stomach and eyes were collected and fixed from all animals at necropsy. Histopathological examination was performed on the stomach and eyes from 10 selected females per group. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative; these fetuses were dissected and examined for visceral anomalies. The other one-half of the fetuses were processed and stained with Alizarin Red S for skeletal examinations. No maternal toxicity was observed in any dose group. There were treatment-related microscopic findings in the stomach (glandular) at 120 mg/kg. Findings consisted of secretory depletion and inflammatory cell infiltrate, both up to slight degree. No developmental toxicity was observed in any dose group. Based on the results in this study the maternal No Observed Effect Level (NOEL) was established as being 40 mg/kg for local toxicity based on microscopic findings in the stomach. The maternal NOAEL for systemic toxicity was at least 120 mg/kg. The developmental toxicity was established as being over or equal to 120 mg/kg (the highest dose tested).

 

In an OECD 443 study, Wistar Han rats were exposed to the substance at dose levels of 0, 10, 30 and 100 mg/kg bw/day. For the F0-generation, the following parameters and end points were evaluated in this study:  mortality/ moribundity, clinical signs, body weight, food consumption, estrous cycle determination, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis, organ weights and histopathologic examinations.  For the F1-generation, the following parameters and end points were evaluated in this study:  mortality/moribundity, clinical signs, body weight, food consumption, vaginal patency and balanopreputial separation, day of first estrus (Cohort 1A), estrous cycle determination (Cohort 1A), functional observations including acoustic startle response (Cohort 2A), clinical pathology including measurement of thyroid hormones and urinalysis (Cohort 1A), gross necropsy findings, sperm analysis and splenic lymphocyte subpopulation analysis (Cohort 1A), organ weights and histopathologic examinations, neurohistopathological examinations and morphometric analysis (Cohort 2A and 2B). In addition, the following reproduction/developmental parameters were determined for the F0-generation: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, macroscopy and measurement of thyroid hormones (Cohort Surplus)).

No mortality, clinical signs or changes in body weights, body weight gain and food intake occurred that were considered to be related to treatment. One male at 100 mg/kg/day (No. 94) was euthanized in extremis on Day 99 of treatment, due to a swelling and wound (with scabs) at the left inguinal region and pale appearance. Veterinary examination on Day 91 and 95 of treatment confirmed an open, partly movable wound/swelling. Macroscopy confirmed a wound in the left inguinal region, which correlated histopathologically with a basal cell carcinoma of the skin of the left inguinal region.  This neoplastic lesion was considered to be a spontaneous alteration unrelated to the treatment with the test item. One female at 30 mg/kg bw/day (No. 168) was euthanized in extremis on Day 109 (Day 13 of Lactation) due to exophthalmos and enlargement of the left eye. Veterinary examination on Day 12 of lactation additionally showed corneal vascularisation, yellowish contents of the posterior chamber of the eye and vitreous humor, and consistent pupillary constriction.  Necropsy confirmed findings of exophthalmos and enlargement and correlated histopathologically with increased fluid in the anterior and posterior chamber. This early sacrifice was regarded to be unrelated to treatment with the test item. A total 13 F1 animals were found dead or were sacrificed in extremis during the first 4 days of treatment. The mortality incidence recorded over the dose groups did not show a dose-related trend. Based on the nature of the necropsy findings recorded for most of these animals in the respiratory tract (pale discolouration of the lungs, lungs not collapsed, foamy contents of the lungs or trachea), the cause of moribundity/death of animals showing these lesions was considered to be related to the gavage administration procedure, possibly in combination with reflux. None of the F1 animals that survived until their scheduled necropsy showed similar lesions, for example, in the respiratory tract. Treatment resulted in adverse macroscopic alterations in the stomach of both sexes at 30 and 100 mg/kg bw/day with adverse microscopic (histopathological) observation at high dose group. Non-adverse microscopic findings in the adrenal glands of males at 100 mg/kg bw/day were also noted.  The stomach findings consisted of a combination of inflammatory and degenerative findings in the glandular stomach in the F0-animals at 30 and 100 mg/kg bw/day, consisting of an increased incidence and/or severity of lymphogranulocytic infiltrate, hemorrhage, erosion/ulceration, degeneration/regeneration mucosa, edema and/or decreased number of parietal cells. These findings were not considered to be the result of systemic toxicity but local irritating properties of the test item and correlated to necropsy findings in these dose groups (foci in the glandular stomach; thickening of the limiting ridge observed at necropsy with no clear histopathological correlate at 30 mg/kg bw/day). Due to the degenerative nature of these findings, they were considered to be adverse. Haematological changes at 100 mg/kg bw/day consisted of increased absolute neutrophil and white blood cell and reticulocyte count in males, increased absolute eosinophil and red blood cell count in females and lower reticulocyte count in females. Clinical biochemistry changes at 100 mg/kg bw/day consisted of decreased total protein, urea, creatinine and inorganic phosphate level in males, and increased chloride and potassium level in males and females, respectively. Urinary changes consisted of increased urinary volume in males at 30 and 100 mg/kg bw/day with secondary lower specific gravity, however, in absence of corroborative morphological lesions, these were considered non-adverse. Non-adverse changes in organ weights at 100 mg/kg bw/day consisted of higher liver weights for Cohort 1A males and females (absolute and/or relative to body weight, PND 89-100). Mean values remained within the historical control data range and no supportive histopathological lesions were noted. All reproductive and developmental parameters were unaffected in both the F0 and F1 generations. No adverse changes in in-life or post-mortem developmental immunotoxicity or neurotoxicity endpoints were recorded. For Cohort 2A animals, these endpoints consisted of acoustic startle response, detailed clinical observations, rectal temperature, hearing ability, pupillary reflex, foot splay, grip strength and motor activity. For both Cohort 2A and 2B animals, developmental neurotoxicity endpoints consisted of fixed brain weights, brain dimensions (length and width of brain), routine sections of brain or peripheral nervous system and morphometric analysis of the brain. Non-adverse changes at 100 mg/kg bw/day consisted of a lower mean rectal temperature for males and lower motor activity for females. Mean rectal temperature of males was marginally reduced (0.98x). Given the minor degree of this change that occurred in the absence of any other corroborative changes, this was considered not adverse. The lower motor activity for females (both for total movements and ambulations) was not supported by clinical observations or other functional observation tests, remained within the normal range for rats of this age and strain, and had no supportive morphological correlates in examined neuronal tissues. Also, all groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. Overall these changes were considered not to represent an adverse effect on neurobehaviour. Based on conditions of the study and results generated, the following No Observed Adverse Effect Level (NOAEL) was derived: General toxicity for both F0 and F1  NOAEL: 30 mg/kg bw/day (based on adverse lesions in the glandular stomach observe at high dose group) and for developmental and reproductive toxicity NOAEL: 100 mg/kg/day.

 

Genotoxicity:

The substance was mutagenic in the presence and absence of metabolic activation in in vitro mammalian chromosome aberration test (OECD 473; 2010). However, the substance did not induce micronucleus in mammalian cells (OECD 484; 2018) nor was it mutagenic in bacterial reverse mutation assay (OECD 471; 2010).

 

Absorption

The molecular weight of the substance is considered low at 129.20 g/mol with high water solubility of ≥320 g/L. The substance is not surface active with a boiling point of 149 °C and considered volatile with reported vapour pressure of ≥ 279 Pa and predicted pKa of 9.0. The n-octanol/water partition coefficient of substance is also considered to be low i.e. log Pow = 0.76. These physicochemical properties are suggestive of significant absorption via all routes with favourable bioavailability via inhalation and oral routes.  Although based on the pKa value of 7.0 - 9.0 means the majority of the molecule is likely to be ionized under physiological pH such as it could affect absorption in the gastro intestinal (G.I.) tract, the high water solubility, the low molecular weight and n-octanol/water partition coefficient properties ensure both transcellular and paracellular absorption of the substance is possible resulting in the substance’s delivery into the liver via portal circulation i.e. first pass metabolism.  The paracellular absorption of the substance is the less favourable route as absorption is both dose and regional dependent, i.e. the tight junctions between cells becomes tighter with reduced surface area.  Therefore, the transcellular pathway is the major route, starting with penetration of the apical membrane followed by diffusion through the cytoplasm exiting through the basolateral membrane into the portal blood.  Based on these properties, 100 % oral absorption can be assumed. Although these properties coupled with the corrosive nature of the substance make uptake from the dermal route possible, based on the lack of surface activity 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 toxicity and absence of macroscopic abnormalities from an in vivo sensitisation study on the substance.  As a Tier I screen, it is proposed in the above REACH guidance that for chemicals with a molecular weight >500 and log Pow of <-1 or >4, that 10% absorption be assumed and that for chemicals outside this range, 100% absorption occurs. Based on the corrosive properties of hexahydro-1,3,5-trimethyl-S-triazine (which may enhance absorption) it is proposed that 100% adsorption is used for risk assessments (ECHA 2014).

 

Considering the boiling point of 149 °C it is unlikely that the substance will reach the nasopharyngeal region or the tracheobronchial region.  However, with the high vapour pressure of ≥ 279 Pa, the low molecular weight at 129.20 g/mol with the high water solubility of ≥320 g/L and a log Pow of 0.76 enhanced bioavailability of this substance is expected via the inhalation route. The water solubility favours dissolution of the substance in the mucus lining of the respiratory tract and the slight lipophilic character favours enhanced absorption through the aqueous pores (alveolar and capillary membranes). Since first pass metabolism is limited via this route, the systemic half-life of the substance is expected to be higher compared to oral absorption because the substance enters general circulation before reaching metabolic organs. In the absence of absorption data via this route, the conservative default route-to-route extrapolation can be applied with pulmonary absorption set at 100% for risk assessment purpose (ECHA 2014).

 

Distribution

The distribution of the substance following uptake into the portal vein is evident from the clinical observation noted in the sub-acute and chronic studies. Changes in blood chemistry and hematology as well as organs demonstrate wide distribution of the substance systemically.  Based on the unionised and reasonable lipophilicity and molecular weight, the substance is expected to permeate cellular membranes as demonstrated by an increased incidence of vacuolation of the zona glomerulosa (up to slight degree) in the adrenal gland in male’s and higher liver weights for Cohort 1A males and females dosed at 100 mg/kg/day in the OECD 443 study.  Considering the non-adverse effects such as lower mean rectal temperature for males and lower motor activity for females, demonstrates potential distribution of metabolites to the brain. Blood placental barrier crossing of metabolites are expected as demonstrated by the non-adverse increased incidence of metacarpals/metatarsals and tarsals (skeletal variations) in F1 in the OECD 414 study.

 

Metabolism

Metabolism following oral absorption of this substance is mainly through phase I and II enzymes forming demethylated, glutathione and glucuronic derivatives. This is supported by the increase in absolute and relative liver weight and slightly increased incidence of vacuolation of the zona glomerulosa in adrenal gland observed in the OECD 443 treated animals at high dose groups. Furthermore, changes in blood chemistry such as increased bile acid, an indication of high glucuronide conjugates are considered an indication of metabolic activity.

 

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 and urine. Absorption via paracellular and intracellular pathways would most likely be excreted via urine as supported by the changes in urinary parameters consisting of increased urinary volume, decreased specific gravity and higher bile acid levels noted in the OECD 443 study.  Glucuronides conjugates are extensively excreted via bile and while the other conjugates are excreted via urine. Based on this observation it can be assumed that no potential for bioaccumulation is expected because any absorption of the substance would result in first pass metabolism therefore resulting in reduced parent compound in the organism i.e. low retention time.

 

Discussion and Conclusion

The substance has physicochemical properties which will not lead to bioaccumulation. Absorption and distribution of the substance are driven by the low molecular weight, low n-octanol/water partition coefficient and high water solubility.  Although absorption is expected via all routes, bioavailability is most favourable via the inhalation and oral routes with potentially limited bioavailability via the dermal route. This is supported by no mortality or clinical signs of systemic toxicity during acute exposure via the dermal route (OECD 405,406, 2010 & OECD 429, 2007). Metabolism is expected to follow both Phase I and II resulting in the formation of demethylated glutathione and glucuronic derivatives as supported by observed changes in liver weight, adrenal and blood biochemical changes observed in the sub-acute and chronic oral studies (OECD 422, 2010, OECD 414 & 443; 2019). Based on these metabolites, elimination is expected via bile and urine as supported by the changes in urine analysis.

 

Based on the local toxicity evaluation on the substance, it can be concluded that the substance is corrosive to the eyes & skin and is a skin sensitiser in accordance with Regulation (EC) No. 1272/2008 (CLP). In the absence of systemic toxicity from these studies, it can be concluded that absorption via the dermal route and systemic bioavailability is potentially limited (OECD 404 & 405, 2009, OECD 406, 2010 and OECD 429, 2007).

 

Following sub-acute and chronic exposures the substance is not well tolerated with corrosion driving general toxicity. The No Observed Adverse Effect Level (NOAEL) for systemic toxicity for the adult animal was considered to be 30 mg/kg /day. The substance is not considered a reproductive or developmental toxicant with the NOAEL for these endpoints considered to be >=100 mg/kg bw/day, the highest dose tested.

 

Based on in vitro and in vivo mutagenicity data, the substance is not considered a mutagen in accordance with Regulation (EC) No. 1272/2008 (CLP).

 

Based on the available data, the toxicokinetics of the substance is suggestive of no potential for systemic bioaccumulation and toxicity is driven by the corrosive potential of the substance. Based on the physicochemical properties, the substance is expected to cross the blood brain barrier as well as the blood placental barrier as demonstrated by the no observed effects following oral exposure via OECD 442, 414 and 443 studies. Absorption is expected to be rapid via the oral route with first past metabolism and excretion via bile and urine.

 

It can be concluded that the toxicokinetics of the substance does not pose significant toxicological concern and no classification related to systemic toxicity is warranted in accordance with Regulation (EC) No. 1272.2008 (CLP). However, local toxicity remains a concern based on the corrosive properties and sensitisation potential.