Registration Dossier

Administrative data

Link to relevant study record(s)

Description of key information

Key value determined using OECD Guideline 417.

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

This assessment of the toxicokinetic properties of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline is based on the results obtained for the toxicological end‑points listed below with simultaneous reference to relevant physico-chemical data:

 

-        Acute oral toxicity

-        Acute dermal toxicity

-        Skin irritation

-        Eye irritation

-        Skin sensitisation

-        Subacute (28 day) oral toxicity

-        Reproduction/Developmental Toxicity Screening test 

-        Bacterial reverse mutation test

-        In vitro chromosome aberration test

-        In vitro mammalian gene mutation test

Toxicological Profile

 

4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline is amono constituent substance (origin: organic). The substance has a molecular weight of 405.0, a water solubility of <0.0067 mg/L at 20 °C and an estimated log Kow in the range of 7.90 ± 1.22

 

The substance was tested for acute oral toxicity in male and female rats (Wistar strain).After single oral administration at a dose level of 2000 mg/kg body weight neither deaths nor adverse symptoms occurred in male and female rats. No clinical signs of toxicity were observed and there were no treatment-related changes in body weight. No abnormalities or gross lesions were observed at necropsy.

 

Similarly, single dermal application of >2000 mg/kg body weight of the substance, ontomale and female rats (Wistar strain)produced no deaths or symptoms of systemic toxicity. Well defined or slight erythema was observed at the site of application; this subsequently healed within the study period. No other symptoms were noted.  

 

In a skin irritation test in albino rabbits, dermal application of the substance had minimal effects.Thesingle exposure determined in this study was PII = 0.4. According to this value the test article belongs to response category “negligible”. The administration of the substance into the conjunctival sac of rabbit eyes had negligible effects on the cornea, iris orconjunctiva. Some blood vessels were hyperacmic (injected) in conjunctivae of all rabbits 1 hour after application. This change had continued in one rabbit (No.2) 24 hours after application and lids of rabbits No.1 and 3 had swollen above normal in the same time. All changes had value of 1 grad. Eyes of all rabbits were normal 48 hours after application of test article. Consequently, the test substance is not irritating to the skin or the eyes.

 

A Local Lymph Node Assay was performed ito assess Skin Sensitisation. Application of three concentrations (25%, 50% and 75%) resulted in positive response at concentration 50% with SI value 3.05. Calculated EC3 value was 49.36%. Based on the results, EC3 value and human potency estimation classification, the substance was classified as a rare allergen. This indicates that some skin absorption occurs, which is confirmed by the skin and dermal toxicity effects noted.

A 28 day repeated dose toxicity study by the oral route was conducted on the substance. The test article did not cause treatment-related deaths of animals.  Frequent incidence of smooth stools, decrease of creatinine and increase of total bilirubin, increase of triacylglycerols and induced retarded decrease of total cholesterol in males of dose group 80mg/kg was noted. The test article caused an increase of Quick test values in females of dose group 80mg/kg, and an increase of ALP in males and females in dose group 80mg/kg. The substance also produced moderate degenerative changes “nephrosis” in proximal tubules females’ kidneys of all dose groups. It is proposed that the effects noted are due to adaptive changes, rather than actual toxicity, particularly as the effects noted do not seem to be dose dependant. The effects may also be specific to the rat strain (Wistar) used in the study. It is well-known from the literature (Barthold, 1979; Gray et al., 1982) that chronic progressive nephropathy (CPN) is a common disease occurring spontaneously in various strains of laboratory rats. The NOAEL was deemed to be 40 mg/kg/day.

 

The results of the reproduction/developmental screening study indicated that per os application of the test article Dusnatox 86 didn’t show any significant changes in all used doses in comparison with the control animals. Three groups of Wistar Rats received the test article test substance in graduated doses of 5, 25 and 50 mg.kg-1. There were no test article-related deaths of animal during the study. There were no visible signs of intoxication in all animals during clinical observation. The incidence of smooth stool in one animal of dose group 50mg.kg-1males and diarrhoea in one male of dose group 25mg.kg-1were observed. The incidence of alopecia in one female dose group of 50mg.kg-1was observed. These incidences were temporary and may not be indicate relation to the test application. The bodyweight of all animals moderately increased during the study. There were no significant differences between all dose groups and the control groups. The number of alive pups in all females of the control and all dose groups was stable. The relative weight of kidneys and reproduction organs in both sexes were without statistical differences. Number of corpora lutea and count of implantation sites in females of all dose groups and in the control group were without statistical differences. Detailed histological examination was performed on reproduction organs and kidneys (target organs) of the males and female of the control and the highest dose group. Macroscopical and histopathological findings showed that test substancedid not cause pathological lesions in organs of treated animals. The sporadic presence of lesions in the animals of the high dose groups was similar to control groups.

In the Ames test, with use of five strains of Salmonella typhimurium (TA 97, TA 98, TA 100, TA 102 and TA 1535) the substance produced neither a statistically significant dose-related increase in the number of revertants nor a statistically significant and reproducible positive response at any one of the test points and according to these results it is considered non mutagenic in the system. The substance at concentrations of 100 – 1000 μg. ml-1in the absence or presence of metabolic activation system (S9) after 3-h exposure in Chinese Hamster lung (V79) cells did not induce significant increases in cells with structural chromosomal aberrations. Extended exposure for 24h without metabolic activation at concentrations up to 1000 μg. ml-1also resulted in a negative response. The results of this study demonstrate that the substance is not genotoxic under the conditions of the in vitro chromosomal aberration assays in V79 cells. In addition, the substance in the mammalian cell gene mutation (V79/HPRT) test in the absence of metabolic activation at five tested doses from 100g/mlto 1000g/mldid not induced increase in the mutation frequency more than 3 times of the negative control value. The results demonstrated that the substance did not induce a concentration-related increase in mutation frequency at the HPR T locus in V79 cells, both in the absence and presence of metabolic activation.

  

Finally, a toxicokinetic study via oral administration provided the following results:

 

Absorption

The typical radioactivity profile in plasma increased progressively peaking at the same time (Tmax = 6.8h) for both doses with a maximal plasma concentration Cmax of 1.59 μg/ml at low and 10.81 μg/ml at high dose. The concentrations in plasma time-dependent declined in an exponential manner resulting in an AUC0-oo of 11.52 μg/h/ml at low dose and which approximately 10-fold increased at high dose to value 122.13 μg/h/ml. The terminal elimination half-life (t1/2el) of test substance was 12.87h at low dose. It was eliminated from plasma with constant of elimination kel of 0.054 h-1. Mean residence time (MRT) was 16h. The total body clearance (CL) was 154ml/h. The apparent volume of distribution for elimination (Vel) and at steady state (Vss) was very similar (2.9L and 2.5L).

 

The plasmatic levels of test substanceexpressed in μg per ml plasma demonstrated marked dose-dependent differences. However, 14C-test substancederived radioactivity (%AA/ml) was without significant changes. Following a single oral administration of high dose, the elimination half-life was approximately two-fold longer t1/2el = 20.61 h; the same as mean residence time MRT = 28.62h. Total body clearance was CL = 116ml/h and constant of elimination kel = 0.034h-1. Compared to low dose the volumes of distribution Vel and Vss were logically higher and analogically similar (3.3 – 3.5L).

The concentration of the radioactivity after oral administration demonstrated a clear dose-dependence on the time-period of determination, but the difference of plasmatic levels gradually decreased after 24h.A default absorption rate of 100% for the oral, inhalation and dermal routes is proposed to be taken for risk characterisation purposes as a worst case.

 

Distribution

At low dose, the radioactivity in most gastrointestinal tissues, such as stomach and intestine, was highest at the first and second sampling point of 2h (0.8 – 0.9%AA/g) and 8h (app. 0.6%AA/g). It declined as a function of time during 48h (from 0.2 to 0.05-0.1%AA/g). Significant amount of radioactivity was sequestered in the liver between 8 – 24h, when the radioactivity declines with time from 0.2 to 0.1%AA/g. The significant amount of radioactivity (0.35%AA/g) was observed in the spleen at 8h. The organs and tissues such as heart, skin, colon and lung, respectively maximally contained ~0.05-0.08% of the administered dose per g tissue during the first 24h. The radioactivity levels in other tested organs and tissues, including kidney, muscle and adipose fat were lower than 0.05%AA/g. It was noted that brain and testes did not accumulate radioactivity during all time of the experiment; there were observed very low concentrations 0.01-0.02%AA/g.

 

There appeared to be no gross difference in the tissue levels of radioactivity between doses; however, the concentrations of test substance expressed in μg of compound at dose of 80mg/kg are approximately 8-fold higher.

 

At the beginning of the experiment (2h post dosing), the GIT tissues (stomach and intestine) analysis showed the high radioactivity (0.5 - 0.9%AA/g). Liver contents also high radioactivity (0.3 – 0.4% AA/g) at the same time intervals than at low dose (between 8 – 24h). Also the high radioactivity was observed in spleen at 8h (0.25%AA/g). Similarly than at low dose, heart, skin, colon and lung, contented approximately 0.05 – 0.13%AA/g, whereas in kidney, muscle and adipose fat, respectively, was detected maximally 0.06%AA/g. Analogically, low concentrations at high dose were observed in brain and testes (0.01 – 0.02%AA/g) too.

 

The radioactivity in the organs and tissues, did not peak until as late as 48h post-dosing. No retention of radioactivity in tissues other than liver, spleen, stomach and intestine was noted.

Metabolism and elimination

Excretion of 14C-test substance in the urine was low; only 0.4% of the dose was recovered in the urine as total radioactivity after oral administration of a 10 ng per kg dose of [14C]-test substance to rats. Mean recoveries of test substance at high dose of 80 mg/kg accounted for 0.3%AA.

 

Administration of 14C-test substance to rats revealed that the compound is predominantly eliminated via the faecal route, accounting for 79-97% of the administered radioactive dose. The maximal amount of radioactivity 86% of the low administered dose was eliminated in the first 24 hours. Faecal elimination of total radioactivity in rats was 96.8 ± 11.2%AA during 4 days. The time-dependent excretion by the faeces at low dose over 96h was similar to that at high dose. Of this, the bulk was eliminated in the faeces at high dose, and the majority (~69%AA) of this was within the first 24h; and summary 79.3 ± 9.4% of dose after four days.

Intraduodenal dosing of [14C]-test substance to bile-duct cannulated rats showed no extensive biliary secretion of radioactive material, with approximately 0.04% of the dose eliminated by this route at low dose and the same amount excreted at high dose during 6h post-dosing.

The radioactivity remained in the organism in carcass on day 4 was about 2.5 – 2.7% of administered dose at both cases.

 

Total recovery of administered radioactivity calculated from urine, cage washing, faeces and carcass was 99.6 ± 11.3%AA at low dose and only 82.3 ± 9.6%AA at high dose during 96h.

Conclusion

The Toxicokinetic of test substance was studied in male Wistar rats at two oral dose levels 10 and 80 mg/kg.

Maximum plasmatic levels of test substance generally occurred to 7h after dosing, consistent with no high or rapid adsorption. Pharmacokinetic parameters for the elimination of test article from the plasma of rats show that its terminal elimination half-life is approximately 2-fold at high dose. The high apparent elimination and the steady-state volume of distribution indicated low concentration of test substance in plasma and pronounced tissue accumulation.

 

The results from the distribution do not suggest a potential for accumulation in any tested organs and tissues, apart from liver. Spleen and liver content a maximum level around 0.2-0.35% of dose per g tissue at 8h and approximated to 0.05% AA/g 72h post-dosing. The GIT tissues, such as stomach and intestine had the highest concentrations at the beginning of experiment (0.8-0.9% AA/g).

 

Elimination of test substance is predominantly via the faeces (≥80% AA) and small extent via the urine (0.3-0.4% AA), suggesting a significant non-renal pathway. The relative contributions of these routes were dose dependent. The excretion by faeces at low dose was higher and faster than at high dose (97 vs. 79% AA). In all cases, approximately 2.5-2.7% of the dose remained in the carcass of rats at 96h. The total recovery of radioactivity accounted for 82% of the high dose and more than 99% of the low dose. Elimination via the bile ranged from less than 0.05% AA at the both intraduodenal doses.

 

The LC/UV/MS profile of the plasma extracts showed a single major peak and one minor peak. Major peak was identified as unchanged compound on the basis of standard test substance and eluted at approximately tR6 min. There was detected also one metabolite with tR2.7 min and molecular weight of 421g/mol. On the basis of molecular weight it is probably a hydroxyderivate of the test substance. Analyses identified either parent compound or metabolites in urine or bile. In summary, test substance is excreted by the faeces mostly as unchanged compound and its two probably hydroxyl- and dihydroxy- metabolites, first with the same retention time and molecular weight as metabolite in plasma and second metabolite with tR1.5 ming and M.w. 437g/mol.

It can be concluded that test substance is not rapidly and mightily metabolized in rats.