Registration Dossier

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
basic toxicokinetics, other
Type of information:
(Q)SAR
Adequacy of study:
key study
Study period:
2019
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
accepted calculation method
Objective of study:
metabolism
toxicokinetics
Principles of method if other than guideline:
- Software tool(s) used including version:
OASIS TIMES platform for simulating metabolism
OECD QSAR Toolbox
- Model description: see field 'Justification for non-standard information', 'Attached justification' and/or 'Cross-reference'
- Justification of QSAR prediction: see field 'Justification for type of information', 'Attached justification' and/or 'Cross-reference'
- Details on the 3 TIMES simulators:
1- in vitro liver S9 metabolic simulator
The in vitro rodent microsomal/S9 mix metabolic simulator reproduces and predicts the metabolic pathways of xenobiotic chemicals for in vitro experimental systems such as rodent (mostly rat) liver microsomes and S9 fraction. The current in vitro rat liver metabolic simulator represents electronically designed set of 517 structurally generalized, hierarchically arranged biotransformation reactions. The metabolism training set contains experimentally documented in vitro metabolic pathways for 261 parent chemicals of a wide structural diversity, and 978 observed metabolites compiled into a searchable electronic database.
As configured in the TIMES system, options for generation of metabolic maps are set to reproduce no more than three levels or confined by products probability threshold (“weight” ≥ 0.1). This is a practical limitation for confining propagation of simulated metabolism maps based on observation that the documented metabolism usually ends up at third transformation level. Also, the limitation is related with the fact that metabolites generated up to the third level are enough to justify the prediction for in vitro mutagenicity endpoints.

2- In vivo rat (whole organisms) metabolic simulator
The in vivo rodent metabolic simulator reproduces and predicts the metabolic pathways of xenobiotic chemicals in vivo in rodents (mostly rats). The metabolism training set contains experimentally documented in vivo metabolic pathways for 647 structurally different parent chemicals, and 4382 observed metabolites compiled into a searchable electronic database. The current in vivo rat metabolic simulator represents electronically designed set of 622 structurally generalized, hierarchically arranged biotransformation reactions. These molecular transformations describe in vivo metabolism in rats accounting for the whole organism.
As configured in the TIMES system, propagation of in vivo metabolism is confined by a threshold of probabilities to generate metabolites (i.e., probabilities to produce metabolites to be ≥ 0.02).

3- Skin metabolic simulator
The skin metabolism simulator reproduced documented in vitro metabolism of 183 unique parent chemicals having 206 documented metabolic maps. Although in vitro data are used to build this metabolism simulator, given the absence of in vivo metabolism data it is assumed that in vitro data should allow adequately prediction of in vivo metabolism in skin. The current metabolic simulator represents electronically designed set of 276 structurally generalized, hierarchically arranged biotransformation reactions. These molecular transformations describe the metabolism of chemicals in the skin compartment and further used to simulate the enzymatic activation of chemicals when predict skin sensitization.
As configured in the TIMES system, options for generation of metabolic maps are set to reproduce no more than four levels of metabolism or confined by using threshold for probability of generated metabolites (“weight” ≥ 0.003). These limitations are related with the fact that the most probable metabolites associated with skin sensitization effect are generated up to the fourth level of metabolism.
GLP compliance:
no
Specific details on test material used for the study:
The QSAR approah had considered and compared the metabolism of the monoconstituent 3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate (RN CAS 59219-71-5), and of the UVCB branched-nonyl 3,5,5- trimethylhexanoate.
Metabolites identified:
yes

SMILES of 3,5,5 trimethylhexyl 3,5,5 trimethylhexanoate (RN CAS 59219-71-5) (monoconstituent) and SMILES of the representative constituents, with the monoconstituent, of branched 3,5,5- trimethylhexanoate (UVCB) were used.

1.    Metabolism of target chemicals

No documented metabolismof the target chemicals has been found in the scientific literature.

 

2.    Metabolism of structural analogues

Exhaustive search in the literature has been performed to identify analogues of the target chemicals having documented metabolism in in vitro S9, in vivo (whole organism) or skin. As a result, four analogues have been found only being common with the target chemicals with respect to the ester functionality. Structural similarity of the analogues has been ignored to some extent at the cost of the preserving ester functionality which, as shown further, determine the principle molecular transformations of target chemicals. In other words, structural analogues of the target chemicals having documented metabolism have been used to confirm that the main metabolic transformation is ester hydrolysis.      

Documented metabolism of these four aliphatic alkyl esters which are analogues of the target chemicals with respect to the ester functionality has been found in:

Ø In vitro rat liver S9 (one analogue);

Ø In vivo rat (three analogues);

Ø Skin metabolism (one analogue).

The structural analogues with information for availability of their documented metabolism are presented in Table 2:

Table 2.Documentedmetabolismof structural analogues.

##

Analogues

In vitro ratliver S9 documented metabolism

In vivodocumented metabolism

Skin documented metabolism

1

CAS 540-88-5

tert-butyl acetate

N/A

Yes [6]

N/A

2

CAS 103-23-1

Bis (2-ethylhexyl) adipate

Yes [7]

Yes [7]

N/A

3

CAS 123-86-4

butyl acetate

N/A

Yes [8]

N/A

4

CAS 14062-23-8

4-Biphenylacetic acid, ethyl ester

N/A

N/A

Yes [9]

 4-Biphenylacetic acid, ethyl ester (the last analogue in Table 2) is the most dissimilar analogue having commonality to the target compounds only with respect to the presence of an ester group. We have used it in the current analysis, because this is the only aliphatic acid ester analogue for which documented metabolism in skin has been found in literature.

In vitro rat liver S9 metabolism

Simulated in vitro rat liver S9 metabolic map of the structural analogue bis(2 -ethylhexyl)adipate (CAS: 103 -23 -1). The major pathway is ester hydrolysis which is obtained with very high probability (P > 0.9). Ester hydrolysis leads to the formation of two metabolitesalcohol and acid. The mono- and dicarboxylic acids (highlighted in green) are documented and simulated correctly by TIMES. It is important to mention that not all simulated metabolites are documented. However, it does not mean that these metabolites are not formed in reality – they are just not documented. Their adequacy is usually analyzed by evaluation of the simulated metabolic “maps” by third-party experts

§ Documented in vitro metabolites are simulated correctly.

§ The major metabolic pathway is enzymatic ester hydrolysis,which results in the formation of acid and alcohol metabolites. 

§ Simulated are some additional oxidation reactions - the secondary hydrolysis product could be oxidized via aldehyde to the corresponding acid.

§ No Phase II metabolites are generated up to three levels of simulation

 

In vivo rat (whole organism) metabolism

Simulated in vivo metabolic maps of three structural analogues are presented in this section.

 

Analogue # 1 -Chemical ID:Name:tert-butylacetate; CAS # 540-88-5

Analogue # 2 -Chemical ID: Name: bis(2-ethylhexyl) adipate; CAS # 103-23-1

Analogue # 3 -Chemical ID:Name: butyl acetate; CAS # 123-86-4

 

§ Documented in vivo metabolites are simulated correctly.

§ The major simulated metabolic transformation is ester hydrolysis which occurs as a first step (with high probability, P > 0.9) and leads to the formation of acid and alcohol.

§ The alcohol metabolite with a primary alcohol group is further oxidized via aldehyde intermediate to the corresponding carboxylic acid.

§ Significant amount ofPhase II metabolites is obtained in vivo as compared with in vitro metabolism.

Skin metabolism

Documented skin metabolism has been found for one structural analogue (4-Biphenylacetic acid, ethyl ester, CAS # 14062-23-8).

The selected analogue (4-Biphenylacetic acid, ethyl ester) having benzyl-type biphenyl fragment next to the alkyl ester functionality is structurally dissimilar to some extent to the targets. Still, ester hydrolysis occurs as primary metabolic transformation forming an acid and alcohol in skin. This metabolic reaction is more facilitated in the presence of benzyl-type biphenyl fragment due to its slight electron-withdrawing effect. This could increase the partial positive charge on the carbonyl carbon atom and the reactivity of the ester carboxyl group towards the SN2-type hydrolysis.

It should be stressed that carboxylesterase enzymatic system which is responsible for the ester hydrolysis in liver is also expressed in skin

Significant amount of Phase II metabolites is obtained by the skin metabolism simulator. However, one should emphasize that the specific enzymatic activity of Phase II UDP-glucuronosyltransferase in skin is reported to be about 10 – 50 % of that in rat liver microsomes. On the other hand, the same UDP-glucuronosyltransferase is significantly more expressed in vivo than in in vitro S9 due to the fact that the whole organism in vivo is multi-organ system involving not only liver but also bile, GI tract, etc. Hence, Phase II glucuronides of a given substrate should be essentially more abundant in vivo followed by in vitro microsomal/S9 system and, ultimately, skin. Nevertheless, the products of esters hydrolysis in skin – acid and alcohol are directly involved in Phase II metabolic reactions, instead of undergoing Phase I aliphatic C-oxidations which occur with the in vitro microsomal/S9 and in vivo.This could be justified by the fact that Phase I oxidizing enzymes such as cytochromes (CYPs), alcohol/aldehyde dehydrogenase, aldehyde oxidase, etc. show lower specific activities in skin than in microsomes and liver. This explains why oxidative reactions such as aliphatic C-oxidations, occurring mainly in liver and following ester hydrolysis are more pronounced in vivo than in skin. The lower activities of Phase I oxidizing enzymes in skin causes an increase in the relative role of Phase II conjugation reactions, although Phase II enzymatic activities in skin are lower as compared to in vivo and in vitro S9 systems.

Similar result is obtained by the TIMES in vivo metabolic simulator.

 

Conclusions:

§ Documented metabolites are simulated correctly by TIMES.

§ Same enzymes controlling metabolism inin vivorat are involved in the metabolism in skin. However, activity of these enzymes is different in all three metabolic systems which provides some specificity of skin metabolism:      

       Activity of Phase I oxidizing enzymes, such as alcohol/aldehyde dehydrogenase and aldehyde oxidase, is lower in skin as compared to S9 and liver.

       Lower activity of Phase I oxidizing enzymes increases the relative importance of Phase II metabolic reactions (such as glucuronidation) in skin which, otherwise, are less expressed in skin as compared to in vivo and in vitro S9.  

§ 

Provided structural analogue is slightly different from the target chemicals due to the presence of a biphenyl-type fragment.

§ Similarity between skin andin vivo rat metabolism has been found with respect to simulation of Phase II metabolites. Both simulators provide large amount of Phase II metabolites.

§ The major simulated metabolic transformation is enzymatic ester hydrolysis which occurs with probability P > 0.9 and results in the formation of acid and alcohol.

General conclusions on simulated metabolism of the structural analogues:

 

A.   Documented metabolites are simulated correctly by all three (in vitro S9 mix, in vivo whole organism and skin) TIMES metabolic simulators. The correctly simulated ester hydrolysis pathway is a premise for adequate model predictions.

B.   Same pattern is obtained by all three TIMES metabolic simulators:

a)    Ester hydrolysis is first and major transformation obtained with high probability (P > 0.9).

b)    Primary alcohol is documented and simulated. This product of ester hydrolysis is further oxidized via aldehyde intermediate to carboxylic acid.

C. Activity of Phase II enzymes such as UDP-glucuronosyltranferase is more pronounced for:

a) In vitro microsomal/S9 system as compared to skin;

b) In vivo (whole organism) as compared to in vitro.

D. However, the relative amount of Phase II metabolites is higher in skin as compared with S9 and in vivo at the cost of the lower Phase I (oxidative) enzymatic activities.

E. Phase II metabolic transformations are acting as detoxification reactions in vivo by increasing the water solubility (hydrophilicity) of the substrates, and facilitating their renal and biliary excretion via the resulting Phase II conjugates

 

3.    Simulating metabolism of the target chemicals

In vitro rat liver S9 metabolism

All target chemicals, considered as parent compounds, belong 100% to the parametric and above 80% to the structural domain. Hence, the prediction could be considered as reliable at domain threshold > 80%.

All target chemicals have same metabolic pattern which is similar to the S9 metabolic pattern of the analogue:

·      The first and major metabolic transformation is enzymaticester hydrolysisoccurring with very high probability (P > 0.9);

·      Alcohol product is further oxidized via aldehyde intermediates to corresponding carboxylic acids;

·      The other metabolic product - carboxylic acid undergoes directPhase IIglucuronidation.

·      Predictions could be considered as reliable at domain threshold above 80%.

 

In vivo (whole organisms) metabolism

All target chemicals belong 100% to the parametric and above 75% to the structural domain. Hence, the prediction could be considered as reliable at domain threshold > 75%.

in vivo ester hydrolysis leads to the formation of two metabolic productsalcohol and acid. The alcohol is further oxidized via aldehyde intermediate to the corresponding carboxylicacids. This metabolic pathway is common for both in vitro and in vivo metabolism of this metabolic product. However, as light difference with respect to the second metabolic product (the acid) has been found in in vivo,as reported in literature. Concisely, the acid metabolite contains a methyl substituent at β-position which blocks certain steps in the β-oxidation,typical for straight-chain fatty carboxylic acids in vivo. Alternatively, ω-hydroxylation at the terminal C{sp3}carbon atom is more plausible metabolic pathway as shown for some saturated, branched chain carboxylic acids. Example for such ω-hydroxylation reaction is provided for Phytanicacid (CAS: 14721-66-5) as illustrated below.

 

Figure. Metabolic ω-hydroxylation of Phytanic acid

 

ω-Hydroxylation can be further followed by oxidation of the primary alcohol to aldehyde, which is then oxidized to the corresponding carboxylic acid. Ultimately, dicarboxylic acids might be formed.

Hence, the ω-hydroxylation of acids, as ester hydrolysis products, is a slightly different in vivo metabolic pathway as compared to in vitro S9 rat, where the corresponding acid is directly involved in Phase II glucuronidation. Analogically, in skin metabolism (see next section) the acid obtained by the ester hydrolysis is also involved in Phase II reactions.

Conclusions:

Same in vivo metabolic pattern is obtained for the four target chemicals:

•       Ester hydrolysis – first and major metabolic transformation with very high probability (P > 0.9);

•       Two products of ester hydrolysis are obtained – alcohol and acid. Alcohol is further oxidized to corresponding carboxylic acids, whereas acid undergoes hydroxylation at the terminal (ω) carbon, ultimately leading to Phase II conjugates.

•       Phase II reactions are more pronounced in vivo than in in vitro S9.

•       Prediction could be considered as reliable at domain threshold >75%.

 

 

Skin metabolism

The predictions are reliable given the fact that all target chemicals belong 100% to parametric and structural domain.

Conclusions:

•       Simulated skin metabolism is similar to the metabolism obtained by the in vitro S9 and in vivo mammalian metabolic simulators:  

•       Ester hydrolysis is the first and major metabolic transformation which occurs with very high probability (P > 0.9).

•       Both ester hydrolysis products further undergo Phase II conjugation reactions.

•       Same enzymes that control in vitro S9 and in vivo (whole organism) metabolism are involved in skin metabolism with activity  : in vivo > in vitro S9 > skin. The corresponding differences in enzymes activity provide specificity of the skin metabolism:

•       Phase I oxidizing enzymes such as alcohol/aldehyde dehydrogenase and aldehyde oxidase show lower activity in skin as compared to liver.

•       Lower activity of Phase I enzymes in skin increases the relative amount of Phase II metabolites as compared to in vivo (whole organism) and in vitro S9 metabolism.

•       The products (an acid and alcohol) of enzymatic hydrolysis in skin undergo direct Phase II metabolic reactions rather than Phase I aliphatic C-oxidation as in in vitro S9 and in vivo metabolism.

•       Kinetics of biotransformations are not provided in the current TIMES metabolic simulators reproducing metabolic transformation statically – within the time frame of the experimental conditions.  

•       The predictions are considered as reliable as all target chemicals belong 100% to parametric and structural domain.

 

Conclusions:
A. No documented metabolism of the target chemicals has been found.
B. Structural analogues of the target chemicals having documented metabolism have been used to confirm the main metabolic transformation - ester hydrolysis.
C. No difference between simulated metabolism pattern of the target chemicals within each of the analyzed (in vivo, in vitro S9, skin) metabolic systems.
D. Commonality of simulated metabolism of the target chemicals with respect to all three metabolic systems is the first and major metabolic transformation, enzymatic ester hydrolysis, which occurs with very high probability (P > 0.9).
E. Metabolic pattern of the acid hydrolysis product (left branch in the maps) in all three metabolic systems could be summarized as follows:
a. Similar metabolism has been found in in vitro microsomes/S9 and skin, where the acid is directly involved in Phase II conjugation reactions.
b. Slightly different transformation has been found for in vivo metabolism, where the acid undergoes hydroxylation at the terminal (ω) carbon, ultimately leading to Phase II conjugates.
F. Metabolism of the alcohol hydrolysis product (right branch in the maps) in all three metabolic systems could be summarized as follows:
a. Similarity has been found for in vitro microsomes/S9 and in vivo metabolism. The only difference is an additional Phase II glucuronidation reaction in vivo which does not occur in vitro S9.
b. Slightly different transformation has been found in skin metabolism due to the lower activity of Phase I oxidizing enzymes as compared to in vitro S9 and in vivo systems. Lower activity of these oxidizing enzymes in skin increases the relative importance of Phase II metabolic reactions.
G. Kinetics of biotransformations are not provided in the current TIMES metabolic simulators reproducing metabolic transformation statically – within the time frame of the experimental conditions. Nevertheless, the different activity of enzymes in the three metabolic systems is not expected to affect significantly the rate of metabolic reactions.
H. According to our experts, no alerts associated with skin sensitization and genotoxicity could be expected in the target chemicals and the obtained metabolites.
I. All predictions could be considered as reliable given the fact that all target chemicals belong 100% to the parametric domain and above 75% to the structural domain.

Executive summary:

The QSAR Approach developped by the Laboratory of Mathematical Chemistry, Bourgas, Bulgaria, consist in simulating metabolism of aliphatic alkyl esters by TIMES.

4 combined elements were used :

•       OASIS TIMES platform for simulating metabolism; with TIMES metabolic simulators used:

•       in vitro rat liver S9;

•       in vivo rat whole organisms;

•       Skin metabolism.

•       Toolbox 4.2 for searching analogues of the target chemicals;

•       Documented metabolism from research publications and websites;

•       Expert evaluation of the simulated metabolism.

SMILES of target substances were used as input for the model : SMILES of 3,5,5 trimethylhexyl 3,5,5 trimethylhexanoate (RN CAS 59219-71-5) (monoconstituent) and SMILES of the representative constituents, with the monoconstituent, of branched 3,5,5- trimethylhexanoate (UVCB)

3 Simulators were considered :

       In vitro rat liver S9 (one analogue);

       In vivo rat (three analogues);

       Skin metabolism (one analogue).

Documented metabolites are simulated correctly by all three (in vitro S9 mix, in vivo whole organism and skin) TIMES metabolic simulators. The correctly simulated ester hydrolysis pathway is a premise for adequate model predictions.

Same pattern is obtained by all three TIMES metabolic simulators:

a)       Ester hydrolysis is first and major transformation obtained with high probability (P > 0.9).

b)       Primary alcohol is documented and simulated. This product of ester hydrolysis is further oxidized via aldehyde intermediate to carboxylic acid.

Formation of Phase II metabolites is more pronounced in vivo and in skin than in in vitro S9 metabolism.

For the simulating metabolism of the target chemicals, following results were obtained:

A. No documented metabolism of the target chemicals has been found.

B. Structural analogues of the target chemicals having documented metabolism have been used to confirm the main metabolic transformation - ester hydrolysis.

C. No difference between simulated metabolism pattern of the target chemicals within each of the analyzed (in vivo, in vitro S9, skin) metabolic systems.

D. Commonality of simulated metabolism of the target chemicals with respect to all three metabolic systems is the first and major metabolic transformation, enzymatic ester hydrolysis, which occurs with very high probability (P > 0.9).

E. Metabolic pattern of the acid hydrolysis product (left branch in the maps) in all three metabolic systems could be summarized as follows:

a. Similar metabolism has been found in in vitro microsomes/S9 and skin, where the acid is directly involved in Phase II conjugation reactions.

b. Slightly different transformation has been found for in vivo metabolism, where the acid undergoes hydroxylation at the terminal (ω) carbon, ultimately leading to Phase II conjugates.

F. Metabolism of the alcohol hydrolysis product (right branch in the maps) in all three metabolic systems could be summarized as follows:

a. Similarity has been found for in vitro microsomes/S9 and in vivo metabolism. The only difference is an additional Phase II glucuronidation reaction in vivo which does not occur in vitro S9.  

b. Slightly different transformation has been found in skin metabolism due to the lower activity of Phase I oxidizing enzymes as compared to in vitro S9 and in vivo systems. Lower activity of these oxidizing enzymes in skin increases the relative importance of Phase II metabolic reactions.  

G. Kinetics of biotransformations are not provided in the current TIMES metabolic simulators reproducing metabolic transformation statically – within the time frame of the experimental conditions. Nevertheless, the different activity of enzymes in the three metabolic systems is not expected to affect significantly the rate of metabolic reactions.

H. According to our experts, no alerts associated with skin sensitization and genotoxicity could be expected in the target chemicals and the obtained metabolites.  

I. All predictions could be considered as reliable given the fact that all target chemicals belong 100% to the parametric domain and above 75% to the structural domain.  

Description of key information

Key value for chemical safety assessment

Additional information

Basic toxicokinetics

One study is available to estimate the toxicokinetic behaviour,which was completed by data on the source substance 3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate (CAS 59219-71-5) and a bibliographic search :

-  QSAR approach :  Simulating metabolism of aliphatic alkyl esters by TIMES - Laboratory of Mathematical Chemistry, Bourgas, Bulgaria (2019)

In accordance with Annex VIII, Column 1, Item 8.8.1, of Regulation (EC) 1907/2006 and with Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance (ECHA, 2008), assessment of the toxicokinetic behaviour of the substance is conducted to the extent that can be derived from the relevant available information. This comprises a qualitative assessment of  the available substance specific data on physicochemical and toxicological properties according to the relevant Guidance (ECHA, 2008) (ECHA, 2008) and taking into account available information on the analogue substances from which data was used for read-across to cover data gaps.

Isononyl isononanoate is UVCB substance of complex composition derived from 3,5,5 trimethylhexanoic acid and isononyl alcohol, the main components being structural isomers of C18H36O2.

Physico-chemical properties

The molecular weight is about 285 g/mol and the substance is a liquid at 20 °C (Stearinerie Dubois Fils, 2012). The water solubility was measured as < 0.05 mg/L (20 °C) was (Frischmann, 2011). The log Pow of isononyl isononanoate was estimated to be 6.92-7.90 (Müller, 2012) and the vapour pressure was estimated to be 0.004-0.045 Pa at 20 °C (Nagel, 2012).

Absorption

Absorption is a function of the potential for a substance to diffuse across biological membranes. The most useful parameters to provide information on this potential are the molecular weight, octanol/water coefficient (log Pow) value and water solubility (ECHA, 2008). The log Pow value provides information on the relative solubility of the substance in water and lipids (ECHA, 2008).

Oral

The molecular weight of Isononyl Isononanoate is lower than 500 g/mol, indicating that the substance is available for absorption (ECHA, 2008). The high log Pow in combination with the low water solubility suggests that any absorption will likely happen via micellar solubilisation (ECHA, 2008).

The available acute oral toxicity data on analogue substances consistently showed LD50 > 5000 mg/kg bw and no systemic effects (Dufour, 1991; Dufour, 1991; Masson, 1986).

A 28 days repeated dose toxicity study with the test substance monoconstituant Isononyl Isononanoate : 3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate (RN CAS: 59219-71-5) on rats by gavage at 0, 100, 300 and 1000 mg/kg/day, induced systemic effects and exacerbation of effects induced by vehicle corn oil on liver (effect induced by the high fat load in liver by administration of the vehicle and the test item).

It indicated an gastrointestinal absorption, the kinetics of absorption is difficult to estimate. But change in clinical signs appear after 6 days of exposure.

The potential of a substance to be absorbed in the gastrointestinal (GI) tract may be influenced by chemical changes taking place in GI fluids as a result of metabolism by GI flora, by enzymes released into the GI tract or by hydrolysis. These changes will alter the physicochemical characteristics of the substance and hence predictions based upon the physico-chemical characteristics of the parent substance may no longer apply (ECHA, 2008).

In general, alkyl esters are readily hydrolysed in the gastrointestinal tract, blood and liver to the corresponding alcohol and fatty acid by the enzymatic activity of ubiquitous carboxylesterases. There are indications that the hydrolysis rate in the intestine by action of pancreatic lipase is lower for alkyl esters than for triglycerides, the natural substrate of this enzyme. The hydrolysis rate of linear esters increases with increasing chain length of either the alcohol or acid. Branching reduces the ester hydrolysis rate, compared with linear esters. (Mattson and Volpenhein, 1969, 1972; WHO, 1999).

The substance isononyl isononanoate is therefore anticipated to be enzymatically hydrolysed to isononyl alcohol (mainly isoC9) and isononanoic acid (isoC9).

Free fatty acids and alcohols are readily absorbed by the intestinal mucosa. Within the epithelial cells, fatty acids are (re-)esterified with glycerol to triglycerides. In general, short-chain or unsaturated fatty acids are more readily absorbed than long-chain, saturated fatty acids. In rats given a single dose of radiolabelled octadecanol via duodenal cannula, 56.6 ± 14% of the administered material was absorbed within 24 h. As for fatty acids, the rate of absorption is likely to increase with decreasing chain length (Greenberger et al., 1966; IOM, 2005; Mattson and Volpenhein, 1962, 1964; OECD, 2006; Sieber, 1974).

In conclusion, based on the available information, the physicochemical properties and molecular weight of isononyl isononanoate suggest oral absorption. However, the substance is anticipated to undergo enzymatic hydrolysis in the gastrointestinal tract and absorption of the ester hydrolysis products is also relevant. The absorption rate of the hydrolysis products is considered to be high.

Dermal

The dermal uptake of liquids and substances in solution is higher than that of dry particulates, since dry particulates need to dissolve into the surface moisture of the skin before uptake can begin. Molecular weights below 100 g/mol favour dermal uptake, while for those above 500 g/mol the molecule may be too large. Dermal uptake is anticipated to be low, if the water solubility is < 1 mg/L; low to moderate

if it is between 1-100 mg/L; and moderate to high if it is between 100-10000 mg/L. Dermal uptake of substances with a water solubility > 10000 mg/L (and log Pow < 0) will be low, as the substance may be too hydrophilic to cross the stratum corneum. Log Pow values in the range of 1 to 4 (values between 2 and 3 are optimal) are favourable for dermal absorption, in particular if water solubility is high. For substances with a log Pow above 4, the rate of penetration may be limited by the rate of transfer between the stratum corneum and the epidermis, but uptake into the stratum corneum will be high. Log Pow values above 6 reduce the uptake into the stratum corneum and decrease the rate of transfer from the stratum corneum to the epidermis, thus limiting dermal absorption (ECHA, 2008).

The substance isononyl isononanoate is almost insoluble in water, indicating a low dermal absorption potential (ECHA, 2008). The molecular weight of about 285 g/mol indicates a potential for dermal absorption. The log Pow is > 6, which means that the uptake into the stratum corneum is likely to be slow and the rate of transfer between the stratum corneum and the epidermis will be slow (ECHA, 2008).

The dermal permeability coefficient (Kp) can be calculated from log Pow and molecular weight (MW) applying the following equation described in US EPA (2004):

log(Kp) = -2.80 + 0.66 log Pow – 0.0056 MW

The Kp is thus ca. 1.88 – 5.88 cm/h. Considering the water solubility (0.00005 mg/l), the dermal flux is estimated to be ca. 8.94E-05 – 2.76E-04 mg/cm²/h, indicating a medium- to low dermal absorption potential.

If the substance is a skin irritant or corrosive, damage to the skin surface may enhance penetration (ECHA, 2008).

The experimental animal and human data on isononyl isononanoate and the analogue substance 3,5,5- trimethylhexyl 3,5,5-trimethylhexanoate shows that no significant skin irritation occurred, which excludes enhanced penetration of the substance due to local skin damage (Guibaud, 2004; Masson, 1986).

3 studies were available to estimate the repeated dose toxicity study by dermal route. No relevant NOAEL or LOAEL could be defined but similar systemic toxic effects than by oral route were reported plus skin irritation effects that were observed after at least 3 days of repeated dermal exposure. The results indicated a probable hydrolysis of the ester in the skin by the skin esterase. The skin injury observed may be due to the acid generated very slight to moderate erythema, very slight to slight edema, fissuring and desquamation at the dermal application site.

Overall, based on the available information, the dermal absorption potential of isononyl isononanoate is predicted to be low and slow but the injury caused to the skin at high dose level (> 500 mg/kg bw), may enhanced the skin penetration of the substance after repeated exposure.

Inhalation

As the vapour pressure of isononyl isononanoate is very low (0.004-0.045 Pa at 20 °C), the volatility is also low. Therefore, the potential for exposure and subsequent absorption via inhalation during normal use and handling is considered to be negligible.

If the substance is available as an aerosol, the potential for absorption via the inhalation route is increased. While droplets with an aerodynamic diameter < 100 μm can be inhaled, in principle, only droplets with an aerodynamic diameter < 50 μm can reach the bronchi and droplets < 15 μm may enter the alveolar region of the respiratory tract (ECHA, 2008).

As for oral absorption, the molecular weight, log Pow and water solubility are suggestive of absorption across the respiratory tract epithelium either by micellar solubilisation.

Esterases present in the lung lining fluid may also hydrolyse the substance, hence making the resulting alcohol and acid available for inhalative absorption.

Due to the limited information available, absorption via inhalation is assumed to be as high as via the oral route in a worst case approach.

Distribution and Accumulation

Distribution of a compound within the body depends on the physicochemical properties of the substance; especially the molecular weight, the lipophilic character and the water solubility. In general, the smaller the molecule, the wider is the distribution. If the molecule is lipophilic, it is likely to distribute into cells and the intracellular concentration may be higher than extracellular concentration, particularly in fatty tissues (ECHA, 2008).

The substance isononyl isononanoate will mainly be absorbed in the form of the hydrolysis products. The fraction of ester absorbed unchanged will undergo enzymatic hydrolysis by ubiquitous esterases, primarily in the liver (Fukami and Yokoi, 2012). Consequently, the hydrolysis products are the most relevant components to assess. Both hydrolysis products are expected to be distributed widely in the body.

After being absorbed, fatty acids are (re-)esterified along with other fatty acids into triglycerides and released in chylomicrons. Fatty acids of carbon chain length ≤ 12 may be transported as the free acid bound to albumin directly to the liver via the portal vein, instead of being re-esterified. Chylomicrons are transported in the lymph to the thoracic duct and eventually to the venous system. Upon contact with the capillaries, enzymatic hydrolysis of chylomicron triacylglycerol fatty acids by lipoprotein lipase takes place. Most of the resulting fatty acids are taken up by adipose tissue and re-esterified into triglycerides for storage. Triacylglycerol fatty acids are likewise taken up by muscle and oxidised for energy or they are released into the systemic circulation and returned to the liver (IOM, 2005; Johnson, 1990; Johnson, 2001; Lehninger, 1993; Stryer, 1996).

Absorbed alcohols are likewise transported via the lymphatic system. Twenty-four hours after intraduodenal administration of a single dose of radiolabelled octadecanol to rats, the percent absorbed radioactivity in the lymph was 56.6 ± 14. Thereof, more than half (52-73%) was found in the triglyceride fraction, 6-13% as phospholipids, 2-3% as cholesterol esters and 4-10% as unchanged octadecanol. Almost all of the radioactivity recovered in the lymph was localized in the chylomicron fraction. Thus, the alcohol is oxidised to the corresponding fatty acid and esterified in the intestine as described above (Sieber, 1974).

Taken together, the hydrolysis products of isononyl isononanoate are anticipated to distribute systemically. Long-chain alcohols are rapidly converted into the corresponding fatty acids by oxidation and distributed in form of triglycerides, which can be used as energy source or stored in adipose tissue. Stored fatty acids underlie a continuous turnover as they are permanently metabolised for energy and excreted as CO2. Bioaccumulation of fatty acids takes place, if their intake exceeds the caloric requirements of the organism.

Metabolism

The metabolism of isononyl isononanoate initially occurs via enzymatic hydrolysis of the ester resulting in isononyl alcohol (mainly isoC9) and isononanoic acid (isoC9). The esterases catalysing the reaction are present in most tissues and organs, with particularly high concentrations in the GI tract and the liver (Fukami and Yokoi, 2012). Depending on the route of exposure, esterase-catalysed hydrolysis takes place at different places in the body. After oral ingestion, esters of alcohols and fatty acids undergo enzymatic hydrolysis already in the gastrointestinal tract. In contrast, substances which are absorbed through the pulmonary alveolar membrane or through the skin may enter the systemic circulation directly

before entering the liver where hydrolysis will generally take place.

The branched fatty alcohols (mainly isoC9) will mainly be metabolised to the corresponding carboxylic acid via the aldehyde as a transient intermediate (Lehninger, 1993). The stepwise process starts with the oxidation of the alcohol by alcohol dehydrogenase to the corresponding aldehyde, where the rate of oxidation increases with increased chain-length. Subsequently, the aldehyde is oxidised to carboxylic acid, catalysed by aldehyde dehydrogenase. Both the alcohol and the aldehyde may also be conjugated with e.g. glutathione and excreted directly, by passing further metabolism steps (WHO, 1999).

A major metabolic pathway for linear and branched fatty acids is the beta-oxidation for energy generation. In this multi-step process, the fatty acids are at first esterified into acyl-CoA derivatives and subsequently transported into cells and mitochondria by specific transport systems. In the next step, the acyl-CoA derivatives are broken down into acetyl-CoA molecules by sequential removal of 2- the formation of H2O and CO2 (Lehninger, 1993). Branched-chain acids can be metabolised via the same beta-oxidation pathway as linear, depending on the steric position of the branch, but at lower rates (WHO, 1999). The alpha-oxidation pathway is a major metabolic pathway for branched-chain fatty acids where a methyl substituent at the beta-position blocks certain steps in the beta-oxidation (Mukherji, 2003). Generally, a single carbon unit is cleaved off the branched acid in an additional step before the removal of 2-carbon units continues. Alternative pathways for long-chain fatty acids include the omegaoxidation at high dose levels (WHO, 1999). The fatty acid can also be conjugated (by e.g. glucuronides,

sulfates) to more polar products that are excreted in the urine.

The potential metabolites following enzymatic metabolism of the substance were predicted using the QSAR OECD toolbox (OECD, 2011). This QSAR tool predicts which metabolites may result from enzymatic activity in the liver and in the skin, and by intestinal bacteria in the gastrointestinal tract.

Depending on the fatty alcohol moiety, up to 8 hepatic metabolites and up to 12 dermal metabolites were predicted for the substance. Primarily, the ester bond is broken both in the liver and in the skin and the hydrolysis products may be further metabolised. Besides hydrolysis, the resulting liver and skin metabolites are all the product of alpha-, beta- or omega-oxidation (= addition of hydroxyl group). In the case of omega-oxidation, it is followed by further oxidation to the aldehyde, which is then oxidised to the corresponding carboxylic acid. In a few cases the ester bond remains intact, and only fatty acid oxidation products are found, which result in the addition of one hydroxyl group to the molecule. In general, the hydroxyl groups make the substances more water-soluble and susceptible to metabolism by phase II-enzymes. The metabolites formed in the skin are expected to enter the blood circulation and have the same fate as the hepatic metabolites. Depending on the fatty alcohol moiety, up to 99 metabolites were predicted to result from all kinds of microbiological metabolism of the substance. Most of the metabolites were found to be a consequence of fatty acid oxidation and associated chain degradation of the molecule. The results of the OECD Toolbox simulation support the information retrieved in the literature.

In the 28 days repeated dose toxicity study with the test substance monoconstituant Isononyl Isononanoate : 3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate (RN CAS: 59219-71-5) on rats (Manciaux, 2001, GLP, OECD Guideline 407 Method, Klimisch 2), the test substance induced mortality at 300 and 1000 mg/kg/day, signs of kidney and liver steatosis all dose levels (grading slight to severe steatosis) with hepatocellular hypertrophy (grading slight to severe). This hepatic steatosis observed was present in all groups including control group

(5 females) due to high fat load in liver. Indeed the vehicle, corn oil, was used for steatosis model in rats and the effect noted was related to physiological metabolism of fatty acid in liver. This changes in liver became pathological in case of irreversible dysregulation of metabolism seen in higher activity of Alkaline phosphatase, Alanine transferase activities and lower mean value of plasmatic cholesterol (1000 mg/kg/day dose group). This effects was correlated to higher severity grading of steatosis at this two dose levels. In regard with the steatosis induced by control vehicle, the test item exacerbated the changes induced by corn oil. However, this changes was not adverse at control condition and at 100 mg/kg/day, there was no dysregulation of enzymatic activities and no variation of lipidemy in plasma (seen in plasmatic cholosterol and triglyceride concentration) and did not led to mortality (observed at 300 and 1000 mg/day/kg groups). Lower glycemia was observed in all tested animal and was considered as non adverse because values were within the range of historical data of the laboratory.

The No Observed Adverse Effect NOAEL in rats for the test substance in rat treated by oral route was defined at 100 mg/kg/day, the steatosis at this dose was not considered as pathological and adverse, no enzymatic dysregulation was observed, no higher fatty acid in blood was noted, the changes of liver was not considered as irreversible. The results suggested that the test item ester was hydrolysis after oral administration and the effects observed are linked to the metabolism of the fatty acid generated.

Considering the dose effect, the severity of the effects which refers to known mechanism of fatty acid.

The QSAR Approach developped by the Laboratory of Mathematical Chemistry, Bourgas, Bulgaria, consist in simulating metabolism of aliphatic alkyl esters by TIMES, 4 combined elements were used :

•       OASIS TIMES (platform for simulating metabolism; with 3 metabolic simulators : in vitro rat liver S9; in vivo rat whole organisms and Skin metabolism).

•       Toolbox 4.2 for searching analogues of the target chemicals;

•       Documented metabolism from research publications and websites;

•       Expert evaluation of the simulated metabolism.

SMILES of target substances were used as input for the model, they were chosen based on the main and similar constituents:

SMILE of 3,5,5 trimethylhexyl 3,5,5 trimethylhexanoate (RN CAS 59219-71-5) (monoconstituent) and SMILES of the representative constituents, with the monoconstituent, of branched 3,5,5- trimethylhexanoate (UVCB).

3 Simulators were considered :

       In vitro rat liver S9 (one analogue);

       In vivo rat (three analogues);

       Skin metabolism (one analogue).

Documented metabolites are simulated correctly by all three (in vitro S9 mix, in vivo whole organism and skin) TIMES metabolic simulators. The correctly simulated ester hydrolysis pathway is a premise for adequate model predictions.

Same pattern is obtained by all three TIMES metabolic simulators:

a)       Ester hydrolysis is first and major transformation obtained with high probability (P > 0.9).

b)       Primary alcohol is documented and simulated. This product of ester hydrolysis is further oxidized via aldehyde intermediate to carboxylic acid.

Formation of Phase II metabolites is more pronounced in vivo and in skin than in in vitro S9 metabolism.

For the simulating metabolism of the target chemicals, following results were obtained:

A. No documented metabolism of the target chemicals has been found.

B. Structural analogues of the target chemicals having documented metabolism have been used to confirm the main metabolic transformation - ester hydrolysis.

C. No difference between simulated metabolism pattern of the target chemicals within each of the analyzed (in vivo, in vitro S9, skin) metabolic systems.

D. Commonality of simulated metabolism of the target chemicals with respect to all three metabolic systems is the first and major metabolic transformation, enzymatic ester hydrolysis, which occurs with very high probability (P > 0.9).

E. Metabolic pattern of the acid hydrolysis product (left branch in the maps) in all three metabolic systems could be summarized as follows:

a. Similar metabolism has been found in in vitro microsomes/S9 and skin, where the acid is directly involved in Phase II conjugation reactions.

b. Slightly different transformation has been found for in vivo metabolism, where the acid undergoes hydroxylation at the terminal (ω) carbon, ultimately leading to Phase II conjugates.

F. Metabolism of the alcohol hydrolysis product (right branch in the maps) in all three metabolic systems could be summarized as follows:

a. Similarity has been found for in vitro microsomes/S9 and in vivo metabolism. The only difference is an additional Phase II glucuronidation reaction in vivo which does not occur in vitro S9.  

b. Slightly different transformation has been found in skin metabolism due to the lower activity of Phase I oxidizing enzymes as compared to in vitro S9 and in vivo systems. Lower activity of these oxidizing enzymes in skin increases the relative importance of Phase II metabolic reactions.  

G. Kinetics of biotransformations are not provided in the current TIMES metabolic simulators reproducing metabolic transformation statically – within the time frame of the experimental conditions. Nevertheless, the different activity of enzymes in the three metabolic systems is not expected to affect significantly the rate of metabolic reactions.

H. According to our experts, no alerts associated with skin sensitization and genotoxicity could be expected in the target chemicals and the obtained metabolites.  

I. All predictions could be considered as reliable given the fact that all target chemicals belong 100% to the parametric domain and above 75% to the structural domain.  

Excretion

The branched isoC9 fatty acids resulting from the oxidation of the corresponding alcohols and the highly branched isononanoic acid (isoC9) acid resulting from hydrolysis of the ester, are unlikely to be used for energy generation and storage, since saturated aliphatic, branched-chain acids are described to be subjected to omega-oxidation due to steric hindrance by the methyl groups at uneven position, which results in the formation of various diols, hydroxyl acids, ketoacids or dicarbonic acids. In contrast to the products of beta-oxidation, these metabolites may be conjugated to glucuronides or sulphates, which subsequently can be excreted via urine or bile or cleaved in the gut with the possibility of reabsorption (entero-hepatic circulation) (WHO, 1998).

In addition, the alcohol component of the ester may also be conjugated to form a more water-soluble molecule and excreted via the urine (WHO, 1999). In an alternative pathway, the alcohol may be conjugated with e.g. glutathione and excreted directly, by passing further metabolism steps.

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