Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics
Type of information:
other: Expert statement
Adequacy of study:
key study
Study period:
2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented expert statement according to standard scientific principles

Data source

Reference
Reference Type:
other: Expert statement
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Objective of study:
toxicokinetics
Test guideline
Qualifier:
no guideline required
Principles of method if other than guideline:
No ADME studies are available for FAT 40045/Z. Therefore, the toxicokinetic assessment of FAT 40045/Z is predicted based on its physico-chemical properties and available toxicological study data. The OECD QSAR application toolbox v3.2 was also utilized to make a qualitative prediction of metabolites formed in the liver and skin.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Oral route:
A combined repeated dose oral toxicity study with the reproduction/ developmental toxicity screening test was performed in Wistar rats. No treatment related mortality occurred in the control or any of the dose groups during the study period (one death occurred which was considered to be related to aspiration of the test item after regurgitation) and there were no adverse effects of toxicological relevance on any of the parameters investigated (clinical signs, body weight, food consumption, organ weights, histopathology, reproduction, development of the pups, macroscopic findings).
Although there were no signs of toxicity exhibited during the study, there was an obvious discolouration of the organs investigated indicating that absorption had occurred. This discolouration was not observed in the control animals. The skin of all male and female animals of the high dose supplementary group was observed to be discoloured blue from the 7th day of treatment until the end of the study. Blue coloured faeces were noted in both males and females of the low dose and the middle dose group from the 4th day of treatment and in both males and females of the high dose supplementary group from the 3rd day of treatment until the end of the study, respectively. Discolouration of the skin and the faeces was considered to be based on the blue colour of FAT 40045 Z. There were no macroscopic findings of toxicological relevance in any of the groups. However, at necropsy, a treatment-related macroscopic finding consisted of green, greenish or blue discolouration of several organs in males and females from all dose groups was found. These results indicate that all or part of FAT 40045 Z has been absorbed. However, it should be noted that ‘digestion’ of FAT 40045 Z in the GI tract (i.e. processes other than phase I and II transformations) could produce smaller blue/green-coloured molecules, especially if these molecules still contain copper. Therefore, it should not be assumed that blue/green discolouration of tissue is (entirely) due to absorption of the whole molecule. The lack of oral toxicity seen in the repeated dose study is supported by the acute oral toxicity study.
The OECD QSAR application toolbox was used to apply Lipinski's Rule of Five. FAT 40045 Z was predicted to be not orally bioavailable based on these rules. The toolbox is also designed to predict possible metabolites that can be produced by phase 1 (e.g. oxidation/reduction) and phase 2 (e.g. conjugation) biotransformations in the liver, based on the structure of the parent molecule. This biotransformation can occur during the first pass effect, but can also occur if the unmetabolised molecule passes into the systemic circulation and returns through the liver.


Inhalation route:
There is no information available regarding the absorption or toxicity of FAT 40045 Z via inhalation. The vapour pressure of FAT 40045 Z is predicted to be very low indicating that inhalation exposure from volatilisation is unlikely to be a potential route of exposure. The particle size distribution (PSD) (L50D = 18.6 µm, L10D = 9.50 µm) of the test material indicates the presence of inhalable and respirable particles. REACH endpoint specific guidance (R.7c) states that respirable particles are <15 µm. However, according to other REACH guidance (chapter R.14) respirable particles are regarded as being < 10 µm. Therefore, assessment of potential respiration will consider both these limits. The study indicated that 12 % of particles were <10 µm (additional information indicated 2 % of particles were <5 µm).
Respirable particles may reach the alveolar region of the respiratory tract where potential dissolution in the alveolar fluid could occur followed by absorption. Inhalable particles are likely to be cleared from the lungs by the mucociliary escalator, but then swallowed making them potentially available for absorption via the GI tract. In conclusion, the PSD indicates a low to very low number of inhaled particles could reach the alveoli. It is expected that absorption of any particles reaching the alveoli would be high, based on the fact that absorption is observed via the oral route, but overall uptake is expected to be very low.

Dermal route:
There are no true dermal toxicity studies available to provide information regarding the dermal absorption or toxicity of FAT 40045/Z. However, a local lymph node assay was performed to assess the sensitisation potential of FAT 40045/Z. Although all animals survived the test period without showing any clinical signs of toxicity, the excised lymph nodes of the animals treated with the three concentrations of the test item were discoloured blue and enlarged. Furthermore, a blue discolouration of the urine and faeces of the animals treated with the highest concentration of the test item was observed.

These results indicate that FAT 40045/Z has undergone some dermal absorption.

However, the degree of absorption is unknown. Based on the high molecular weight of FAT 40045 Z (a MW >500 lowers the ability to be absorbed through skin), it is possible that only a very small amount of the substance was absorbed but sufficient to lead to a discolouration of the lymph nodes, urine and faeces. Alternatively, it may be possible that metabolism in the skin has fragmented the parent to smaller coloured molecules allowing more to get into the systemic circulation. This result is very interesting because the parent molecule would not be considered favourable for urinary excretion on account of its molecular weight being much higher than 500 (actually 1200!). It is also worth noting that simple inorganic salts of copper, such as copper sulphate and copper chloride are blue-coloured, and it is also possible that metabolism to smaller copper-containing molecules would still retain some blue colour due to the copper.
Details on distribution in tissues:
It is possible that FAT 40045/Z could bioaccumulate in fat based on its high log Pow. In the repeated dose oral toxicity study, macroscopic examination indicated that most of the organs studied in many of the animals were discoloured blue or green. This indicates that FAT 40045/Z and/or its metabolites/ breakdown products are distributed extensively throughout the body. The extent to which this absorption and distribution occurs is unknown as it is possible that only a very small amount of the test substance or metabolite (which is still blue/green-coloured) can lead to the appearance of blue or green coloured tissue.

According to the guidance, highly lipophilic substances (log Pow 4-6) such as FAT 40045/Z that come into contact with the skin can rapidly penetrate the lipid rich stratum corneum but are not well absorbed systemically because the rate of transfer from the stratum corneum to the epidermis will be limited. Although they may persist in the stratum corneum, they will be eventually cleared as the stratum corneum is sloughed off.
Details on excretion:
The repeated dose toxicity study reports that the faeces were stained a blue colour. This indicates several possibilities; either the molecule was absorbed then passed through the biliary duct as unmetabolised parent or it could indicate that the blue colour is a metabolite that still maintains the conjugation of the parent and was then excreted via biliary excretion. The blue faeces could also indicate the presence of unabsorbed FAT 40045/Z. There is also the possibility that the blue colour could be due to both unabsorbed parent and excreted parent/metabolite(s). Certainly, the molecular weight of the molecule is much higher than 300 which would increase the likelihood of excretion via the biliary duct rather than via urine. No blue colouration of the urine was reported which provides further evidence regarding the lack of urinary excretion. However, the repeated dose study did not indicate that urine colour was one of its observations. The study did indicate only one test animal in the highest dose had a green bladder, which does suggest that for the oral route excretion via urine did not occur. Contrast this with indications from the local lymph node study which did see blue colour in the urine at the highest concentration applied, but via the dermal route. This indicates that there is a different process occurring for dermal route compared to oral route. It is known that absorption occurs for the oral route, but no excretion occurs via urine. Given the large molecular weight, this would be expected and any excretion would be predicted to occur via the biliary duct. Blue colouration was observed throughout the body tissues. If it is assumed that this is due to the parent, then the only possible way that urine could be blue from the dermal route is if metabolism has occurred via skin that has not occurred via liver?
In conclusion, excretion is via biliary duct and GI tract for oral route, but for dermal route it would appear that excretion (possibly of a blue metabolite not formed by oral route) is also possible.

Metabolite characterisation studies

Details on metabolites:
Potential metabolites of FAT 40045/Z in the liver and skin were predicted using the OECD Toolbox. The results of the prediction were that in the liver FAT 40045/Z forms three metabolites (one of which is chlorine) and chlorine ions are the only metabolite predicted for skin. Two of the liver metabolites have structures that are almost identical to FAT 40045/Z and hence will have similar toxicological profiles to the parent. It was not possible to utilise EPIWIN to generate structures based on the SMILES code from the toolbox.

In addition to the prediction from the toolbox, a visual assessment of the structure of the parent indicates there is the possibility of one of the metabolites being a triazine derivative, specifically 2-chloro-4, 6-diamino-S-triazine could form due to reduction processes either in the liver (Phase I) or via microflora in the intestine which are known to reduce nitro and azo groups. A quick search of TOXNET indicated three Ames tests (on TA100, 97 and 98 strains) all with negative results. No other data was indicated.

Applicant's summary and conclusion