Reaction mass of 2-ethylhexyl (3-isocyanato-4-methylphenyl)carbamate and 2-ethylhexyl (5-isocyanato-2-methylphenyl)carbamate and 2-ethylhexyl (3-isocyanato-2-methylphenyl)carbamate

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics, other

Type of information:

other: Written assessment

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A written assessment of toxicokinetic behaviour is considered appropriate for the substance.

Summary and discussion of toxicokinetics

This assessment of the toxicokinetic properties of the substance is based on the results obtained with the test substance and read-across substances for the toxicological end-points listed below and with reference to the relevant physicochemical data.

·           acute oral toxicity

·           acute dermal toxicity

·           skin irritation

·           skin sensitisation

·           combined repeated dose toxicity study with reproductive developmental toxicity screening study

·           in vitro bacterial reverse mutation test

·           in vitro chromosome aberration test

·           in vitro mammalian gene mutation test

The substance is an organic multi-constituent substance which is highly reactive with water, when an isocyanate group reacts with water it will form an amine, which in itself reacts even faster with another isocyanate group to yield urea. This reaction mass of isomers has a molecular weight of 304 g/mol. The substance has an estimated water solubility of 0.13 mg/L at 20 °C and a n-octanol/water partition coefficient (log Kow) ranging between from 3.6 to > 6.5 .

The acute toxicity of the substance was evaluated with read-across substance Reaction mass of Carbamic acid, N-(5-isocyanato-2-methylphenyl)-, 2-ethylhexyl ester and Carbamic acid, N-(3-isocyanato-4-methylphenyl)-, 2-ethylhexyl ester and N,N'-(4-Methyl-1,3-phenylene)bis(carbamic acid) C,C'-bis(2-ethylhexyl) ester (EC: 937-955-6). Male and female rats administered with a single dose of 5 mL/kg bw did not induce mortality, affect body weight measurements, or release gross pathological findings. Whilst yellow mucoid diarrhoea was noted under the 3-hour observation period, this was not observed again in this study. The LD50 value was determined to be LD50 > 5 mL/kg bw.

 

Dermal toxicity was also assessed with the read-across substance Reaction mass of Carbamic acid, N-(5-isocyanato-2-methylphenyl)-, 2-ethylhexyl ester and Carbamic acid, N-(3-isocyanato-4-methylphenyl)-, 2-ethylhexyl ester and N,N'-(4-Methyl-1,3-phenylene)bis(carbamic acid) C,C'-bis(2-ethylhexyl) ester (EC: 937-955-6). Where a single semiocclusive dressing containing 2 mL/kg bw of the test substance was applied to male and female rabbits. The test substance did not cause mortality; however bodyweight was seen to be increased in all of the treated animals. Test substance related clinical signs were again limited to soft stools. The LD50 value was determined to be LD50 > 2 mL/kg bw.

 

The skin corrosion/irritation parameter was assessed using a dermal range finding study on rabbits using Reaction mass of Carbamic acid, N-(5-isocyanato-2-methylphenyl)-, 2-ethylhexyl ester and Carbamic acid, N-(3-isocyanato-4-methylphenyl)-, 2-ethylhexyl ester and N,N'-(4-Methyl-1,3-phenylene)bis(carbamic acid) C,C'-bis(2-ethylhexyl) ester (EC: 937-955-6). Under the conditions of the study the primary dermal irritation index was determined to be 1.2 (slightly irritating, according to Draize dermal irritation scoring) and therefore was not regarded as corrosive or irritating to the skin of rabbits.

 

Eye Irritation was assessed with the use of data from the read-across substance: Reaction mass of Carbamic acid, N-(5-isocyanato-2-methylphenyl)-, 2-ethylhexyl ester and Carbamic acid, N-(3-isocyanato-4-methylphenyl)-, 2-ethylhexyl ester and N,N'-(4-Methyl-1,3-phenylene)bis(carbamic acid) C,C'-bis(2-ethylhexyl) ester (EC: 937-955-6) on rabbit eyes. Administration to the eyes of six animals produced a positive reaction for corneal injury and conjunctival redness in 3/6 of the animals while two eyes exhibited a positive response for iridial injury and another for chemosis. The study concluded that the substance was an eye irritant.

 

Skin sensitisation for the substance was assessed with the use of read-across to a Guinea Pig maximisation test conducted on Reaction mass of Carbamic acid, N-(5-isocyanato-2-methylphenyl)-, 2-ethylhexyl ester and Carbamic acid, N-(3-isocyanato-4-methylphenyl)-, 2-ethylhexyl ester and N,N'-(4-Methyl-1,3-phenylene)bis(carbamic acid) C,C'-bis(2-ethylhexyl) ester (EC: 937-955-6). The study concluded that the substance was deemed to present a contact dermatitis potential to humans based on positive reactions to guinea pigs in the main study as well as in the subsequent rechallenges.

 

The mutagenic potential of the substance was assessed with the in vitro bacterial reverse mutation study using S. typhimurium TA97a, TA98, TA100, TA102 and TA1535, mammalian chromosome aberrations using cultured lympocytes and Hprt locus in Chinese Hamster cells (V79). The results of three mutagenicity studies resulted in negative results both with and without metabolic activation and as such is assumed to not be mutagenic.

 

A 14 day repeat dose toxicity range finding study following oral gavage administration of the substance at, 100, 300, or 1000 mg/kg once daily to male and female sprague dawley rats. Parameters evaluated during the study included mortality, physical examinations, cageside observations, body weights, body weight changes, food consumption, gross pathology findings, and absolute and relative organ weights. Under the conditions of the study there were no test substance related effects noted, therefore the repeated dose NOAEL was determined to be 1000 mg/kg bw/day.

 

A subsequent 28 day repeated dose toxicity study combined with a reproductive/developmental screening study via the oral route was conducted using the substance where doses of 100, 300 and 1000 mg/kg bw/day was administered daily to male and female rats.

Mortality was not affected by administration of the test substance. Two control females and one male and female administered 1000 mg/kg/day were found dead or euthanized in a moribund condition. The cause of death was undetermined based on gross observations and limited microscopic tissue evaluation. However, the deaths were not considered test substance-related because the deaths in the females were considered to be complications of parturition and because of the low incidence of deaths in males.

Test substance-related changes in body weight and food consumption were observed but were not considered adverse. Body weight and food consumption were statistically significantly decreased compared to the control. All groups gained weight and there was no clear dose response; therefore the effect on body weight was not considered adverse.

Test substance-related changes in thyroid hormone levels of F0 male rats were observed. Mean T4 levels of treated groups were significantly lower than the control group in a dose-dependent manner. In the absence of thyroid organ weight and histopathological effects this finding was not considered adverse. Furthermore, no other toxicologically significant observations with respect to survival, clinical observations, body weights, food consumption, and ophthalmology were observed.

Test substance-related changes in clinical pathology parameters were limited to ~2-fold increases in ALTi activities in animals administered 1000 mg/kg/day consistent with minimal hepatocellular injury; and decreases in measures of erythroid mass with increases in reticulocyte counts in males at all doses and females administered ≥300 mg/kg/day consistent with blood loss or erythrocyte injury and a normal erythrocytic regenerative response. No correlating microscopic changes occurred in the liver; however, test substance-related increased erythropoiesis was observed in the spleen of 4 of 5 males and 1 of 5 females administered 1000 mg/kg/day and 3 of 5 females administered 300 mg/kg/day which correlated with the increases in reticulocyte counts. None of these changes were considered adverse, as the effects on erythroid mass were not of a magnitude to affect the overall well-being of the animal.

The only other test substance-related microscopic change occurred in the testes of 3 of 5 males administered 1000 mg/kg/day and consisted of spermatid retention in some, but not all, Stage IX tubules. Notably, spermiation occurred normally within most tubules and no increase in cellular debris or hypospermia was observed in the epididymides of affected animals. Spermatid retention was considered an adverse change due to the potential effects on reproduction.

The test substance did induce developmental toxicity at doses of 1000 mg/kg/day. Offspring toxicity consisted of a reduction in the number of live births and a decreased postnatal survival from PND 0 to 4. Observations on the morphology of the pups were performed from birth to PND 13 and there was no indication that the test substance induced specific malformations. Therefore, it is unknown what resulted in the low offspring survival rate but potentially could be due to maternal or paternal toxicity rather than an assumption of impaired development in utero. However, there is still a potential for impaired development that could not be determined in this study. 

The repeat dose no-observed-adverse-effect level (NOAEL) for male rats was determined to be 300 mg/kg/day due to microscopic findings (spermatid retention). The repeat dose NOAEL for female rats was considered to be 1000 mg/kg/day, the highest dose level tested. The developmental NOAEL was determined to be 300 mg/kg/day dose due to the decreased survival of the offspring at the high dose

 

Toxicokinetic parameters

Absorption

Oral

The acute oral toxicity study conducted on Reaction mass of Carbamic acid, N-(5-isocyanato-2-methylphenyl)-, 2-ethylhexyl ester and Carbamic acid, N-(3-isocyanato-4-methylphenyl)-, 2-ethylhexyl ester and N,N'-(4-Methyl-1,3-phenylene)bis(carbamic acid) C,C'-bis(2-ethylhexyl) ester (EC: 937-955-6) only revealed yellow mucoid diarrhoea which was only noted at the 3-hour observation period. A 14 day repeated dose range finding study with the test substance did not reveal any substances related effects with oral: gavage administration of 1000 mg/kg bw day. Conversely in a repeated dose toxicity study combined with a screening for reproductive/developmental toxicity study using the substance, microscopic changes occurred in the testes of 3 of 5 males administered 1000 mg/kg/day and consisted of spermatid retention in some, but not all, Stage IX tubules. In addition it was noted that the substance also induced developmental toxicity at doses of 1000 mg/kg/day. Pup toxicity consisted of reduction in the number of live births and a decreased postnatal survival from PND 0 to 4. Presumably due to maternal/parental toxicity. The information from these studies indicate that the test substance would be absorbed following oral exposure.

The test substance has a molecular weight of 304 g/mol, an estimated water solubility of 0.13 mg/L at 20 °C and a log Kow value between 3.6 to > 6.5. The absorption of highly lipophilic substances (log Kow ≥ 4) may be limited by the inability of such substances to dissolve in gastrointestinal fluids and therefore make contact with the mucosal surface. However, the absorption of such substances will be increased if they undergo micellular solubilisation by bile salts. As a worst case, for risk assessment purposes the oral absorption of the test substance is set at 100%.

 

Dermal

The results from the dermal studies from the read-across substance; including the dermal toxicity study in vivo skin irritation/sensitisation studies indicates that there is sufficient skin absorption when applied to animal skin. This is based on the slightly irritating effects noted in rabbits in an in vivo skin irritation study and the sensitised guinea pigs from the guinea pig maximisation study.

The log Kow value of the test substance ranges from 3.6 to > 6.5 therefore the dermal absorption of the substance is expected to be limited based on the high log Kow value. At log Kow values above 6, the rate of transfer between the stratum corneum and the epidermis will be slow and will limit absorption across the skin and uptake into the stratum corneum itself may be slow. Whilst values between 1 and 4 would be favourable. Maximum dermal absorption is often associated with values of log Kow between +1 and +2 (ECETOC (European Centre for Ecotoxicology and Toxicology of Chemicals). Monograph No, 20; Percutaneous absorption. August 1993). In addition, the substance must be sufficiently soluble in water to partition from the stratum corneum into the epidermis. The test substance is highly reactive with water, when an isocyanate group reacts with water it will form an amine, which in itself reacts even faster with another isocyanate group to yield urea and has an estimated water solubility of 0.13 mg/L at 20 °C therefore dermal uptake of the substance itself would be expected to be relatively low on this basis.

The vapour pressure of the substance however may potentiate the absorption of the substance given that it contains an isocyanate group (a group known to be sensitising). Substances which have a vapour pressure of < 100 Pa are likely to be well absorbed dermally, possibly more than 10% of the amount that would be absorbed via the inhalatory route. The substance has been experimentally determined to have a vapour pressure of 1.0E-5Pa at 25 °C. The vapour pressure coupled with the damage to the skin surface from the irritating/sensitising effects noted above may enhance dermal penetration.

In conclusion, dermal absorption of the substance based on its reactivity with water, its water solubility data and log Kow would be is expected to be relatively low. However factoring in the irritation/sensitisation effects from the read-across substance, and vapour pressure for the substance, it appears that significant dermal absorption could be anticipated.  Therefore, for risk assessment purposes the dermal absorption of the test substance is set at 100%.

Inhalation

Currently there are no studies associated with the the substance via the inhalation route. The substance has a low vapour pressure (1.0E-5Pa), therefore signification inhalation exposure to vapours would not be expected. Moderate log Kow vapours between -1 and 4 are favourable for absorption directly across the respiratory tract epithelium by passive diffusion. The test substance has a moderate to high log Kow value (3.6 - > 6.5) therefore it may be taken up by micellular solubilisation particularly as the substance is poorly soluble in water (0.13 mg/L at 20 °C). As a worst case, for risk assessment purposes the inhalation absorption of the test substance is set at 100%.

 

Distribution and accumulation.

Information on the distribution from the acute oral toxicity study with the read-across substance and 28 day repeated dose study with the test substance provides limited details with regards to the symptoms expressed by the animals through exposure to the substance, presumably because the study duration is too short for distribution and accumulation of the test substance to exert effects on the treated animals.

The repeated dose toxicity study combined with a screening for reproductive/developmental toxicity study using the substance, decrease in measures of erythroid mass with increases in reticulocyte counts and reported microscopic changes in the male rats testes and also has been shown to induce developmental toxicity affecting survival at high doses. Based on the differences observed from the acute and chronic studies it is apparent that concentration and duration of exposure is a driving factor for the rate and extend of distribution throughout the body.

The test substance is lipophilic therefore it is likely to distribute into cells and the intracellular concentration may be higher than extracellular concentration particularly in fatty tissues. Substances with high log Kow values and low solubility in water tend to have longer half-lives unless their large volume of distribution is counterbalanced by a high clearance. There is the potential for highly lipophilic substances to accumulate in individuals that are frequently exposed to that substance. Once exposure stops, the concentration within the body is proposed to decline at a rate determined by the half-life of the substance.

 

Metabolism and excretion

The ester groups in the test substance are likely to undergo lipase-catalysed hydrolysis in the gastrointestinal tract, releasing short chain fatty acids so only low and transient exposure to the parent compound is expected. Following absorption, the fatty acids are transported to the tissues of the body including the liver where they undergo oxidation in the cells to carbon dioxide, acetate and ketones. Fatty acids are transported across the outer mitochondrial membrane by carnitine acyl transferases. Once inside the mitochondrial matrix, the fatty acyl-carnitine reacts with coenzyme A to release the fatty acid and produce acetyl-CoA. Fatty acids then undergo β-oxidation. During this process, two-carbon molecules acetyl-CoA are repeatedly cleaved from the fatty acid. Acetyl-CoA can then enter the citric acid cycle (Krebs cycle), which produces NADH and FADH2. NADH and FADH2 are subsequently used in the electron transport chain to produce ATP, the energy currency of the cell.

The methyl isocyanate group may conjugate with glutathione, creating a reactive S-(N-methylcarbamoyl)glutathione which has the potential to carbamoylatenucleophilic amino acids.

From the mutagenicity assays it appears that the substance is not metabolised toward genotoxic structures.

Excretion of the test substance or its metabolites is likely to be done so via the urine due to its low molecular weight (304 g/mol) and faeces, given the evidence of coloured diarrhoea reported in the read-across acute toxicity study.

 

Conclusion

In conclusion, there is evidence to suggest that the substance could be absorbed via the oral, dermal and the inhalation route. The results of an oral repeated dose toxicity study indicate that the substance may be absorbed orally in the rat, to exert adverse effects, partially in the testes of males. It is likely that the ester groups in the substance will undergo lipase-catalysed hydrolysis in the gastrointestinal tract, releasing fatty acids. Following absorption, the fatty acids are transported to the tissues of the body including the liver where they undergo oxidation in the cells. The methyl isocyanate group may conjugate with glutathione to create S-(N-methylcarbamoyl)glutathione. Excretion of the substance and its metabolites will likely be via the faeces or urine due to the low molecular weight of the substance and evidence of coloured diarrhoea in the oral acute toxicity study with the read-across substance.

Consequently, the substance itself is considered to have low bioaccumulation potential as the substance itself is highly reactive with water, when an isocyanate group reacts with water it will form an amine, which in itself reacts even faster with another isocyanate group to yield urea. Therefore it is anticipated that the accumulation will be that of urea and any unreacted reaction mass constituents. 

Conclusions:

In conclusion, there is evidence to suggest that the substance could be absorbed via the oral, dermal and the inhalation route. The results of an oral repeated dose toxicity study indicate that the substance may be absorbed orally in the rat, to exert adverse effects, partially in the testes of males. It is likely that the ester groups in the substance will undergo lipase-catalysed hydrolysis in the gastrointestinal tract, releasing fatty acids. Following absorption, the fatty acids are transported to the tissues of the body including the liver where they undergo oxidation in the cells. The methyl isocyanate group may conjugate with glutathione to create S-(N-methylcarbamoyl)glutathione. Excretion of the substance and its metabolites will likely be via the faeces or urine due to the low molecular weight of the substance and evidence of coloured diarrhoea in the oral acute toxicity study with the read-across substance.
Consequently, the substance itself is considered to have low bioaccumulation potential as the substance itself is highly reactive with water, when an isocyanate group reacts with water it will form an amine, which in itself reacts even faster with another isocyanate group to yield urea. Therefore it is anticipated that the accumulation will be that of urea and any unreacted reaction mass constituents.

Description of key information

In conclusion, there is evidence to suggest that Trixene AS could be significantly absorbed via the oral, dermal and the inhalation route. The results of an oral repeated dose toxicity study indicate that the substance may be absorbed orally in the rat, to exert adverse effects, partially in the testes of males. It is likely that the ester groups in the substance will undergo lipase-catalysed hydrolysis in the gastrointestinal tract, releasing fatty acids. Following absorption, the fatty acids are transported to the tissues of the body including the liver where they undergo oxidation in the cells. Whilst the methyl isocyanate group may conjugate with glutathione to create S-(N-methylcarbamoyl)glutathione. Excretion of the substance and its metabolites will likely be via the faeces or urine due to the low molecular weight of the substance and evidence of coloured diarrhoea in the oral acute toxicity study with the read-across substance.

Consequently, the substance itself is considered to have low bioaccumulation potential as the substance itself is highly reactive with water, when an isocyanate group reacts with water it will form an amine, which in itself reacts even faster with another isocyanate group to yield urea. Therefore it is anticipated that the accumulation will be that of urea and any unreacted reaction mass constituents.

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

TRIXENE AS is anticipated to be significantly absorbed via the oral, dermal and inhalatory route, therefore the values used for the chemical safety assessment have been set to 100%