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Toxicological Data

Acute toxicity:  The substance was well tolerated in acute dermal and oral studies in rats with reported LD50 > 2000 mg/kg bw/day in both studies.  No mortality or clinical signs of systemic toxicity were noted during exposure. No abnormalities were found at macroscopic post mortem examination (OECD 425, 2017 & OECD 402, 2018).

Local Toxicity:  The substance was not a corrosive or irritant to skin or the eye in rabbits (OECD 404 & 405, 2016). Although no sensitisation was observed in chemico, the substance showed sensitisation potential following in vitro and in vivo exposure. In an OECD 442C - 2018, the test item showed 10.6% depletion of the Cysteine peptide which places the test item in the reactivity class of “no to minimal” and therefore it is predicted by DPRA to be a non-skin sensitiser. In the OECD 442D - 2018, the substance showed statistically significant luciferase induction >1.5-fold in repetitions 1 and 2 but not in repetition 3. The EC1.5 values for repetitions 1 and 2 were calculated as 21.802 µM, and 15.494 µM with the IMAX values for repetitions 1 and 2 reported at 2.368 and 3.031, respectively. In an OECD 442E – 2018, the substance activated THP-1 cells, with reported relative fluorescence intensity (RFI) in CD86 cells that was equal to or greater than 150 % in at least one concentration of the first run and an RFI in CD86 and CD54 cells that was equal to or greater than 150 % and 200 %, respectively, in at least one concentration of the second independent run. Based on the outcome of the in vitro studies an in vivo local lymph node assay was conducted. In this study the substance showed sensitisation potential with Stimulation Indices of 1.8, 4.0, and 4.5 at test item concentrations of 10, 25, and 50 % (w/w) in dimethylformamide and a reported EC3 value of 18.2 % (w/w). All treated animals survived the scheduled study period and no signs of systemic toxicity were observed from days 2 to 6, some animals showed an erythema of the ear skin (Score 1 to 2). A statistically significant and biologically relevant increase in ear thickness was not observed (OECD 429, 2019).

In (OECD 422, 2019) the systemic toxicity and potential adverse effects was investigated in oral gavage administration of the substance to Wistar Han™:RccHan™:WIST strain rats. The animals were treated for approximately six weeks for male and eight weeks for females (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 350 and 750 mg/kg bw/day.  A control group of twelve males and twelve females were dosed with vehicle alone (Polyethylene Glycol) over the same period.  A further three males were dosed with N-Nitroso-N-methylurea (vehicle: distilled water) for two consecutive days to act as a positive control group for the comet assay.  

Treatment at dosages of 100, 350 and 750 mg/kg bw/day was well tolerated with isolated incidences of adverse effects observed that showed no dose response relationship or apparent related histopathological changes.  The only unscheduled adult death on the study occurred at 750 mg/kg bw/day, when a female was found dead on Day 50.  This animal had previously shown total litter loss post-partum and hunched posture, pallor of the extremities, apparent hypothermia and piloerection had been apparent on the day prior to death.  Necropsy revealed enlarged liver, spleen and right adrenal, a pale area on the liver and thin appearance of the non-glandular region of the stomach and raised limiting ridge and additionally, a pale mass was observed in the right ventricle of the heart during tissue processing.  Histopathology examination revealed abscessation in the lungs and marked inflammatory change in the heart with the presence of bacterial colonies. It was considered that sepsis, as a result of complications of pregnancy, was the underlying cause of death and this isolated atypical occurrence was considered to be unrelated to treatment.

Food consumption was lower than control for treated females from Day 4 of lactation, with differences being most notable at 750 mg/kg bw/day during the second week of lactation; however, this was considered to reflect lower demand from the smaller litters for treated females, in comparison to their control counterparts. Post-dosing salivation was observed for the majority of animals at 750 mg/kg bw/day and to a lesser extend at 350 mg/kg bw/day, which was considered to reflect slight distaste or irritancy of the dosing formulations rather than any systemic effect of treatment. Isolated occasions of noisy respiration were observed at these dose groups which was considered to be influenced by the increased post-dosing salivation.

There was no treatment related adverse effect on blood chemistry parameters at all treatment groups although, for females at 350 or 750 mg/kg bw/day, mean creatinine levels, mean total protein levels, albumin levels and albumin/globulin (A/G) ratio were statistically significantly lower than the control, with individual values below historical ranges. For males at 750 mg/kg bw/day, mean bile acids levels were statistically significantly higher than control, with 3/5 individual values exceeding the historical control range compared to only one for the control group. For males at all dosages, mean inorganic phosphorus levels were statistically significantly higher than control, but all individual values were within the historical control range.  For females at 750 mg/kg bw/day, mean calcium levels were statistically significantly higher than control, but all individual values were within the historical control range. For both sexes at 350 or 750 mg/kg bw/day, mean total cholesterol and mean total bilirubin levels were statistically significantly higher than control, although mean values showed no dosage relationship for females.  The mean levels of thyroxine (T4) in adult males were statistically significant higher then control at 100, 350 and 750 mg/kg bw/day, although mean values showed no dosage-response relationship at the lower dosage levels. The were no statistical significant differences from control for mean level of thyroxine (T4) in offspring of either sex on Day 13 of age at all dose levels.

There was no adverse effect of treatment on hematology parameters at 100, 350 or 750 mg/kg bw/day.  For both sexes at 350 or 750 mg/kg bw/day, mean reticulocyte and mean total leucocyte counts were statistically significantly higher than control but values showed no dose-response relationship. Furthermore, at 750 mg/kg bw/day, mean values for mean corpuscular hemoglobin and mean corpuscular volume were statistically significantly lower than control. These findings, in the absence of any supporting histopathology, were considered to be incidental and unrelated to treatment.

Intergroup differences in organ weights did not indicate any adverse effect of treatment at all dose levels. For females at 100 mg/kg bw/day and for both sexes at 350 and 750 mg/kg bw/day, absolute and body weight-relative liver weights were statistically significantly higher than control.  The increase in liver weight may represent adaptive changes in liver metabolism but as there was no evidence of histopathological change detected for this organ, this finding was considered not to represent an adverse effect.   For females at 350 and 750 mg/kg bw/day absolute and body weight-relative thyroid weights were statistically significantly higher than control with follicular hypertrophy was observed in the thyroid of some females, although mean values showed no dose-response relationship. Whilst a direct stand-alone change in the thyroid gland cannot be ruled out, it is considered to be less likely when occurring only in females. Additionally, it is normal that thyroid activity increases in females that are pregnant/lactating, therefore, this finding, in view of the lack a true dose-response, may represent an uneven incidence of normal variability for these female animals.  In the absence of any dose-repsonse relationship this finding was considered most likely to be incidental and not to represent an adverse effect of treatment. For females at 750 mg/kg bw/day, absolute and body weight-relative adrenal weights were statistically significantly higher than the control, however mean values were adversely influenced by one female that showed an atypically low adrenal weight.  Excluding this animal, all individual absolute and body weight relative weights were within the historical control range and the absence of any histopathological change this finding was considered to be incidental and unrelated to treatment.

For females at all dosages, absolute and body weight-relative ovary weights were statistically significantly higher than control, however, mean values showed no dosage relationship.  At 100 mg/kg bw/day, 2/12 absolute and 3/12 relative weights were below the respective historical control range, at 350 mg/kg bw/day, 1/12 absolute values exceeded and 3/12 relative weights were below the respective historical control range and at 750 mg/kg bw/day, 2/11 absolute and 4/11 relative weights were below the respective historical control range.  For the control group, however, 6/12 absolute and 11/12 relative weights were below the respective historical control range and it is considered that the observed differences in ovary weight represented atypically low values for the control groups and was unrelated to treatment.

For males at 350 and 750 mg/kg bw/day, absolute and body weight-relative seminal vesicles weights were statistically significantly higher than control, although absolute mean values showed no dosage relationship.  Except for one body weight relative value at 750 mg/kg bw/day, all individual seminal vesicle weights at these dosages were within the respective historical control range.  In the absence of any supporting histopathological change, the observed differences in organ weights were considered to be incidental and of no toxicological significance. At 750 mg/kg bw/day, absolute and body weight-relative prostate weights were statistically significantly higher than control.  In the absence of any supporting histopathological change, the observed differences in organ weights were considered to be of no toxicological significance and not representative of an adverse effect. In particular, there were no test item-related microscopic findings in the reproductive tracts following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or the evaluation of the uterus or of follicles and corpora lutea in the ovaries.

There was no effect of treatment on estrous cycles of females, mating performance or gestation length at 100, 350 or 750 mg/kg bw/day.  Although, there was no effect on fertility, as assessed by the number of females that achieved pregnancy at 100, 350 or 750 mg/kg bw/day, the number of implantations was lower than control at 750 mg/kg bw/day.  The number of implantation were also lower than control at 100 and 350 mg/kg bw/day but the differences were slight and probably reflect normal biological variation.  Histopathological evaluations of reproductive tissues at 750 mg/kg bw/day did not reveal any test item-related microscopic findings for the reproductive tissues and, in particular, there were no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle or for follicles and corpora lutea in the ovaries.  The lower number of implantations at 750 mg/kg bw/day is therefore unexplained, and while this may also reflect normal variation, a treatment related effect cannot be discounted.

Subsequent post-implantation and post-natal survival of the offspring was unaffected by treatment at all dosages.  One female at 750 mg/kg bw/day did show total litter loss post-partum but this was considered to reflect a decline in the clinical condition for this animal due to sepsis, as a result of complications of pregnancy, and therefore the high offspring mortality for this particular litter was considered to be unrelated to maternal treatment.  Mean offspring body weight on Day 1 of age was similar to control at all dosages but, body weight gain at 750 mg/kg bw/day was slightly lower than control, despite the lower litter size at this dosage, leading to lower mean offspring body weight at termination on Day 13 of age.  A number of small offspring were noted during clinical observations and at necropsy, consistent with this lower gainlitter weight at 750 mg/kg bw/day was lower than control throughout, initially reflecting the lower litter size and subsequently the lower litter size and weight gain. At 100 or 350 mg/kg bw/day, there was no effect of maternal treatment on offspring body weight gain up to Day 13; litter weights were lower than control throughout, but this reflected the slightly lower litter size at both dosages.

Mean offspring ano-genital distance (actual and normalized) for males at 100 mg/kg bw/day and both sexes appeared longer than control at 100, 350 or 750 mg/kg bw/day; mean values for males at 100 and 350 mg/kg bw/day showed no dose-response relationship.  For female offspring at 100 mg/kg bw/day measured ano-genital distance was longer than the control but was similar to the control when normalized for body weight.  All individual litter values for ano-genital distance, normalized for body weight, were within the historical control range at 100, 350 or 750 mg/kg bw/day.  As no normalized for body weight values for either sexes were outside the normal historic range an effect of treatment on ano-genital distance appeared unlikely and this finding was considered to be incidental and of no toxicological significance.  Evaluation of visible nipple count for male offspring on Day 13 postpartum did not reveal any effect of maternal treatment at 100, 350 or 750 mg/kg bw/day.  

For the additional comet assay investigation for DNA damage, there were no significant increases in the percentage tail intensity or median percentage tail intensity for the jejunum, glandular stomach or liver at 100, 350 or 750 mg/kg bw/day. The test item was, therefore, considered to not induce DNA damage in these tissues under the conditions of the test.

In summary, based on the results of this study the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of α-phenyl-1H-benzimidazole-2-methanol to the adult animal was considered to be 750 mg/kg bw/day. The NOAEL for reproduction and developmental toxicity was 350 mg/kg bw/day based on the lower number of implantations and suppression of offspring growth.

Other data: The substance was mutagenic in the presence and absence of metabolic activation in OECD 471 and OECD 490 studies (2018 & 2016). However, the substance did not induce micronucleus in mammalian cells – OECD 487 (2018) nor was it mutagenic in mammalian alkaline assay (OECD 489) incorporated in the combine repeated dose reproductive and developmental study in rat (OECD 422, 2019).

Absorption

The molecular weight of the substance is considerably low at 224.26 g/mol with solubility of 147 mg/L. The substance is not surface active and is expected to decompose before boiling at 176 °C and as such not considered volatile with reported vapour pressure of < 3.2 x 10-3 Pa at 25 °C.  The n-octanol/water partition coefficient of substance is also considerably low i.e. 1.89. These physicochemical properties are suggestive of favourable absorption via the oral route.  Although water solubility is moderate, the low molecular weight and n-octanol/water partition coefficient properties ensure both transcellular and paracellular absorption of the substance is possible resulting in substance 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 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 make 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 2014 can be applied as conservative approach for risk assessment.

Based on the low vapour pressure of < 3.2 x 10-3 Pa and the fact that the substance is expected to decompose before boiling at 176 °C, uptake via inhalation route is limited.  However, in the absence of absorption data via this route, the conservative default route-to-route extrapolation according to ECHA 2014 can be applied.

Distribution

The distribution of the substance following uptake into the portal vein is evident from the clinical observation noted in the OECD 422 study. 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 the increase in absolute and relative weight of kidney, liver, ovary, thyroid, seminal vesicle and prostrate.

Metabolism

Metabolism following oral absorption of this substance is mainly through phase I and II enzymes via the OH groups producing sulfonic and glucuronic derivatives. This is supported by the increase in absolute and relative liver weight observed in the OECD 422 treated animals at all dose groups. Furthermore, changes in blood chemistry such as increase mean total bilirubin, cholesterol, protein levels with increased albumin levels and albumin/globulin (A/G) ratio coupled with enlarged, mottled liver and thyroid follicular hypertrophy which are all considered adaptive alteration following exposure to xenobiotics. The lack of histopathological changes in these organs is further support for 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 observed in the kidney such as increases in absolute and relative weights and increased mean creatinine and bile acids levels. Although some of the changes were sex specific effects confined in male rats following sub-acute exposure to substance it is nonetheless an indication of kidney performance in the elimination of xenobiotics. Changes in other blood electrolytes in both sexes without any histopathological effects, this finding is therefore considered adaptive response in relation to elimination of the substance.

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 decomposition at 176 °C.  Exposure via inhalation rout is not expected based on the vapour pressure of the substance, However, oral exposure is the most preferred route with limited bioavailability via dermal. This is supported by no mortality an no macroscopic abnormalities or clinical signs of systemic toxicity during acute exposure via the dermal route (OECD 402, 2018). Based on the moderate water solubility paracellular absorption of the substance is the less favourable route of absorption in comparison to the transcellular pathway resulting into first pass metabolism. Metabolism is expected to follow both Phase I and II resulting to sulfonic and glucuronic conjugates as supported by observation in liver weight and blood biochemical changes observed in the sub-acute oral studies (OECD 422, 2019). Based on these metabolites, elimination is expected via bile and urine as supported by the changes in kidney weight and blood electrolytes.

Based on the in vitro & vivo sensitisation evaluation on the substance, it can be concluded that the substance is a skin sensitiser category 1B in accordance with Regulation (EC) No. 1272/2008 (CLP). In the absence of systemic toxicity, it can be concluded that absorption via the dermal route and systemic bioavailability is limited.

Following subacute exposure, the substance is well tolerated with isolated incidences observed without dose response relationship or apparent related histopathological changes. The No Observed Adverse Effect Level (NOAEL) for systemic toxicity for the adult animal was considered to be 750 mg/kg bw/day. However, substance effects on reproduction were noted with lower numbers of implantations and offspring growth, therefore the NOAEL for reproduction and development was considered to be 350 mg/kg bw/day.

Although the substance displayed mutagenic potential in the presence and absence of metabolic activation in OECD 471 and OECD 490 studies (2018 & 2016, respectively), no induction of micronucleus in mammalian cells was noted in an OECD 487 (2018). The substance was not mutagenic in mammalian alkaline assay (OECD 422 & OECD 489, 2019), there were no significant increases in the percentage tail intensity or median percentage tail intensity for all tested tissues in all treatment groups. Furthermore, there were no effects on germ cell tissues such as ovaries, testis and there was no effect on post-implantation. It can be concluded that the substance is not mutagenic on both somatic and germ cells.

It can be concluded based on the available data that the toxicokinetics of the substance demonstrate very low bioaccumulation potential. Systemic effects related to reproduction observed in the OECD 422 study were lower number of implantations at 750 mg/kg bw/day, slightly lower mean offspring weight gain at 750 mg/kg bw/day and a higher incidence of small offspring at 750 mg/kg bw/day.  Although the mean number of implants was lower at 750 mg/kg/day, there was a considerable within-group variation.  Generally, when there is a true reduction in the number of implantations this will be shown by a slight reduction in each female, rather than the very large within-group variation.  For young female rats, the number of implants will generally be lower, because the rat will still on quite a steep growth curve.  As such, although the NOAEL for this endpoint was determined to be 350 mg/kg/ bw/day based on data observations alone, it is quite possible that the effect is merely a chance distribution of the smaller litter to the treated groups in general and to the high dose group in particular.  There is a slightly lower pup weight gain at 750 mg/kg/day, but it is not possible to state with certainty that this is related to treatment.  Again, based on the data alone a conservative NOAEL of 350 mg/kg bw/day was determined.  There is an interaction between litter size and pup weight, but pups of small litters will not necessarily be larger as a consequence of coming from a small litter.  The observed “small offspring” refers to clinical signs, and this observation will merely supplement the data from pup body weights.  It is not appropriate to classify the substance for reproductive toxicity, because the effects are slight and would not necessarily be repeated in a larger study.  The OECD 422 study is a screening study and the available information does not support classification under the CLP criteria.

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).

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