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PBT assessment

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PBT assessment: overall result

Reference
Name:
diisotridecyl adipate
Type of composition:
legal entity composition of the substance
State / form:
liquid
Reference substance:
Composition 1
Reference substance:
Composition 1
PBT status:
the substance is not PBT / vPvB
Justification:

The test substance diisotridecyl adipate was demonstrated to be readily biodegradable in three OECD guideline 301-tests.

An OECD 301F-study (Flach, 2014, key study) (guideline suitable for poorly soluble and volatile substances) with the test substance has been conducted, showing that DITA is readily biodegradable (69 % biodegradation based on ThOD after 28 days). The inoculum used in this study was neither adapted, nor pre-exposed (confirmation of the test institute attached). Two more studies (OECD 301B) are available, showing that DITA is readily biodegradable. In the first OECD 301B study, 81 % biodegradation were achieved within 28 d, fulfilling the 10d-window criterion (although not required for an UVCB). A second OECD 301B-test resulted in a biodegradation extent of 69.6 % after 28 days. The other studies reported for DITA clearly indicate the ready biodegradability of the substance, by almost reaching the 60% biodegradation threshold. In the respective study according to OECD guideline 301B, a biodegradation of 56.6% within 28 days was obtained with DITA. Similar biodegradation was found in the other 301F study (58.53% biodegradation within 28 days). Supporting all these findings, the read-across substance diisotridecyl dodecanedioate was found readily biodegradable in an OECD 301B-test. All of the studies mentioned above are considered valid.

Therefore the substance is not considered to be persistent (P).

The bioaccumulation of DITA via aqueous exposure is negligible. The main route of exposure will be via oral uptake. Based on the rapid metabolism of aliphatic esters, it is reasonable to conclude that the high log Kow, which indicates a potential for bioaccumulation, overestimates the true bioaccumulation potential of the substance. Therefore, there remains no uncertainty that bioaccumulation of DITA is unlikely to occur and the Weight of Evidence (WoE) approach is applicable. DITA is considered not to be bioaccumulative (B).

Bioaccumulation via aqueous exposure

DITA is poorly soluble in water (< 0.001 mg/L at 25 °C) and can be judged, in our view, as readily biodegradable but at least as ultimately biodegradable with degradation pattern very similar to that of readily biodegradable substances. According to the Guidance on information requirements and chemical safety assessment, Chapter R.7b, readily biodegradable substances can be expected to undergo rapid and ultimate degradation in most environments, including biological Sewage Treatment Plants (STPs) (ECHA, 2012a). Independent on the final conclusion of ready biodegradability the available biodegradation data indicate that this is also true for DITA. Therefore, after passing through conventional STPs, only a very low concentration of DITA is likely to be (if at all) released into the environment. The Guidance on information requirements and chemical safety assessment, Chapter R.7b (ECHA, 2012a) states that once insoluble chemicals enter a standard STP, they will be extensively removed in the primary settling tank and fat trap and thus, only limited amounts will get in contact with activated sludge organisms. Nevertheless, once this contact takes place, these substances are expected to be removed from the water column to a significant degree by adsorption to sewage sludge based on its high adsorption potential (DITA: log Koc > 7) and the rest will be extensively biodegraded (Guidance on information requirements and chemical safety assessment, Chapter R.7a, (ECHA, 2012b). Considering this one can assume that the availability of the substances in the aquatic environment will be extremely low, which reduces the probability of adsorption and uptake from the surrounding medium into organisms (e.g., see Björk, 1995, Haitzer et al., 1998).

In addition, DITA consists of main components with estimated high partition coefficients (calculated log Kow ≥ 10). Based on the high log Kow and the very low measured water solubility, one can conclude that the substance is hydrophobic and lipophilic (in nature).

If environmental concentrations facilitate exposure, the uptake of DITA from medium into organisms is expected to be very low based on the molecular weight, size and structural complexity of the substance. DITA is an adipic acid ester with two branched side chains of C11 to C13 carbon length. This large and complex structure assumes a high degree of conformational flexibility. Dimitrov et al. (2002) revealed a tendency of decreasing log BCF with an increase in conformational flexibility of molecules, which they assumed to be related to the enhancement of the entropy factor on membrane permeability of chemicals. This concludes a high probability that a substance may encounter the membrane in a conformation which does not enable the substance to permeate. A calculated mean maximum diameter of 25.75 A (lowest Dmax = 18.16 A) using Catalogic clearly supports this assumption.

This interaction between hydrophobicity, bioavailability and membrane permeability is considered to be the main reason why the relationship between the bioaccumulation potential of a substance and its hydrophobicity is commonly found to be described by a relatively steep Gaussian curve with the bioaccumulation peak approximately at log Kow of 6-7 (e.g. see Dimitrov et al., 2002; Nendza & Müller, 2007; Arno and Gobas 2003). Substances with log Kow values above 10, which have been calculated for DITA, are, however, again considered to have a low bioaccumulation potential (e.g. see Nendza & Müller, 2007; 2010). For those substances with a log Kow value > 10 it is recognized by the relevant authorities that it is unlikely that they accomplish the pass level of being bioaccumulative according to OECD criteria for the PBT assessment (log BCF = 2000; ECHA, 2011).

This assumption is also supported by QSAR calculations using BCFBAF v3.01 and Catalogic. BCF values were calculated to be 14.8 (BCFBAF v3.01) using a regression based method and even lower values of 0.982 (BCFBAF v3.01, Arnot-Gobas, upper trophic) and 7.08 (OASIS Catalogic v5.11.9) were calculated if bioavailability and biotransformation processes were taken into account.

Even if based on the high log Kow value the BCFBAF calculation may be rated as outside of the applicability domain the results are congruent with valid calculations for similar fatty acid esters registered under REACH, such as Diisopropyl adipate (CAS 6938-94-9), Dibutyl adipate (CAS 105-99-7), Dihexyl adipate (CAS 110-33-8), Diisopropyl sebacate (CAS 7491-02-3), Dibutyl sebacate (CAs 109-43-3), Decanedioic acid, bis(2-ethylhexyl) ester (CAS 122-62-3) for which BCF and BAF values ranging from <1 to 29 L/kg were calculated.

Taken all these information into account we feel confident that we provide sufficient reliable evidence that bioaccumulation via aqueous exposure is negligible.

We also think that if released into the water phase the substance will to some degree bind to particulate organic matter, and therefore, the main route of exposure for aquatic organisms such as fish will be via food ingestion or contact with suspended solids:

Bioaccumulation via oral uptake

The accumulation of a substance in an organism is determined, not only by uptake, but also by distribution, metabolism and excretion. Accumulation takes place if the uptake rate is faster than the subsequent metabolism and/or excretion.

If taken up by living organisms, aliphatic esters such as DITA will be initially metabolized via enzymatic hydrolysis to the respective dicarboxylic acid and alcohol components as would dietary fats (e.g., Linfield, 1984; Lehninger, 1970; Mattson and Volpenhein, 1972). The hydrolysis is catalyzed by carboxylesterases and esterases, with B-esterases located in hepatocytes of mammals being the most important (e.g., Heymann, 1980). However, carboxylesterase activity has also been reported from a wide variety of tissues in invertebrates and fishes (e.g., Barron et al., 1999; Wheelock et al., 2008). In fish, the high catalytic activity, low substrate specificity and wide distribution of the enzymes in conjunction with a high tissue content lead to a rapid biotransformation of aliphatic esters, which significantly reduces its bioaccumulation potential (Lech & Melancon, 1980; Lech & Bend, 1980).

Alcohols ranging from C11 (Iso-undecanol) to C13 (iso-tridecanol) are the expected hydrolysis products from the enzymatic reaction catalyzed by carboxylesterase. These metabolites exhibit no potential for bioaccumulation (e.g., see published REACH dossiers for isotridecanol [experimental BCF = 2.27/1.41] or adipic acid [valid calculation, BCF = 3.16]). The metabolism of alcohols has been extensively reviewed in the literature (e.g., see Rizzo et al., 1987; Hargrove et al., 2004). The free alcohols can either be esterified to form wax esters (which are similar to triglycerides) or they can be transformed to fatty acids in a two-step enzymatic process catalyzed by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). The responsible enzymes ADH and ALDH are present in a large number of animals including plants, microorganisms and fish (e.g., Sund & Theorell, 1963; Nilsson, 1990; Watabiki et al., 1999; Reimers et al., 2004; Lassen et al., 2005).

The metabolism of alcohols in fish was extensively studied by Reimers et al. (2004). They isolated and characterized two cDNAs from the zebra fish, Danio rerio, encoding ADHs, which showed specific metabolic activity in in-vitro assays with various alcohol components ranging from C4 to C8. The emerging aldehydes were shown to be further oxidized to the corresponding fatty acid by ALDH enzymes. The most effective ALDH2, which is mainly located in the mitochondria of liver cells showed a sequence similarity of 75% to mammalian ALDH2 enzymes and a similar catalytic activity (also see Nilsson, 1988). The same metabolic pathway was shown for longer chain alcohols, such as stearyl and oleyl alcohol in fish (e.g., Sand et al., 1973). Furthermore, cleavage products with high water solubility like adipic acid do not have the potential to accumulate in adipose tissue due to their low log Pow and are thus widely distributed within the body and rapidly eliminated via renal excretion. To a smaller extent the dicarboxylic acids are also metabolised via peroxisomal beta-oxidation.

This assumption about the fate of aliphatic esters such as DITA is confirmed by studies performed with Bis(2-ethylhexyl) adipate (DEHA) (CAS 103-23-1). The potential for accumulation of the poorly soluble, highly lipophilic substance in aquatic organisms was examined in a bioconcentration test with bluegill sunfish (Lepomis macrochirus) using 14C-labelled DEHA (Felder et al., 1986). The test was carried out for 42 days. Concentrations of DEHA in water, whole fish, viscera, and fillet were analyzed at intervals during the test. After the first 35 days of exposure, the remaining fish were exposed to clean water for an additional 14 days and concentrations of DEHA were measured in the fish at intervals. A whole fish bioconcentration factor (BCF) of 27 was reported at day 35. Following exposure to clean water, a depuration rate for DEHA of 0.26/day (t 1/2 = 2.7 days) was determined. The results imply that the accumulation of DEHA is low despite a high log Pow (log Pow = 8.94), most likely due to rapid metabolization. Furthermore, when transferred to freshwater, the substance is apparently rapidly and extensively excreted from the fish. Similar results were observed in monkeys, rats and mice.

Since this experimental results can be easily explained by the general enzymatic processes as mentioned above, which are numerously published in the scientific literature, we can see absolutely no indication that the ADME pattern confirmed for DEHA should not be the same for DITA, a substance which differs to DEHA structurally only slightly in the chain length of the fatty alcohol.

Hence, based on the rapid metabolism we think it is reasonable to conclude that the high log Kow, which indicates a potential for bioaccumulation, overestimates the true bioaccumulation potential of the substance. Therefore, we think that there remains no uncertainty that bioaccumulation of DITA is unlikely to occur.

References

Arnot JA and Gobas FAPC (2003) A generic QSAR for assessing the bioaccumulation potential of organic chemicals in aquatic food webs. QSAR Comb. Sci 22: 337-345

Barron MG et al (1999) Tissue carboxylesterase activity of rainbow trout. Environ Toxicol Chem 18(11): 2506-2511

Björk M (1995) Bioavailability and uptake of hydrophobic organic contaminants in bivalve filter-feeders. Ann Zool Fenn 32(2): 237-245

Dimitrov SD et al (2002) Predicting bioconcentration factors of highly hydrophobic chemicals. Effects of molecular size. Pure Appl Chem 74 (10): 1823-1830

ECHA (2012a) Guidance on information requirements and chemical safety assessment, Chapter R.7b: Endpoint specific guidance, version 2.2 (August 2013), Helsinki, Finland

ECHA (2012b) Guidance on information requirements and chemical safety assessment, Chapter R.7a: Endpoint specific guidance, version 1.2 (November 2012), Helsinki, Finland#

ECHA. (2011) Guidance on information requirements and chemical safety assessment – Part C: PBT assessment, Helsinki, Finland

Heymann E (1980) Carboxylesterases and amidases. Pp 291-316. In: Jakoby WB (ed) Enzymatic basis of detoxification Vol 2. Biochem Pharmacol Toxicol: A series of monographs, Academic Press

Haitzer M et al (1998) Effects of dissolved organic matter (DOM) on the bioconcentration of organic chemicals in aquatic organisms: a review.Chemosphere37(7): 1335-1362

Hargrove JL (2004) Nutritional Significance and Metabolism of Very Long Chain Fatty Alcohols and Acids from Dietary Waxes. Exp Biol Med 229:

Lassen N et al (2005) Molecular cloning, baculovirus expression and tissue distribution of the zebrafish aldehyde dehydrogenase 2. Drug Metabol Disposit 33(5): 649-656

Lech JJ and Bend JR (1980) Relationship Between Biotransformation and the Toxicity and Fate of Xenobiotic Chemicals in Fish. Environmental Health Perspectives 34: 115-131.

Lech JJ and Melancon MJ (1980) Uptake, metabolism, and elimination of 14c‐labeled 1,2,4‐trichlorobenzene in rainbow trout and carp. J Toxicol Environ health 6(3): 645-658

Lehninger AL (1970) Biochemistry. Worth Publishers, Inc.

Linfield WM et al (1984) Enzymatic fat hydrolysis and synthesis. J Am Oil Chem Soc 61(2): 191-195

Mattson FH and Volpenhein RA (1972) Hydrolysis of fully esterified alcohols containing from one to eight hydroxyl groups by the lipolytic enzymes of rat pancreatic juice. J Lip Res 13, 325-328

Nendz, M and Müller M (2007) Literature Study: Effects of Molecular Size and Lipid Solubility on

Bioaccumulation Potential. Testing laboratory: Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg, Germany and Analytisches Laboratorium für Umweltuntersuchungen und Auftragsforschung, Luhnstedt, Germany. Report no.: FKZ 360 01 043. Owner company: Umweltbundesamt, Dessau, Germany. Report date: 2007-02-15.

Nendza M and Müller M (2010) Screening for low aquatic bioaccumulation (1): Lipinski’s “Rule of 5” and molecular size. SAR and QSAR in Environ Res 21(5-6): 495-512

Nilsson GE (1990) Distribution of aldehyde dehydrogenase and alcohol dehydrogenase in summer-acclimatized crucian carp, Carassius carassius L. J Fish Biol 36(2): 175-179

Nilsson GE (1988) A comparative study of aldehyde dehydrogenase and alcohol dehydrogenase activities in crucian carp and three other vertebrates: apparent adaptations to ethanol production. J Comp Physiol 158(4): 479-485

Reimers et al (2004) Two Zebrafish Alcohol Dehydrogenases Share Common Ancestry with Mammalian Class I, II, IV, and V Alcohol Dehydrogenase Genes but Have Distinct Functional Characteristics. J Biol Chem 279: 38303-38312.

Rizzo WB et al (1987) Fatty alcohol metabolism in cultured human fibroblasts. Evidence for a fatty alcohol cycle. J Biol Chem 262: 17412-17419

Sand DM et al (1973) Wax ester in fish: Absorption and metabolism of oleyl alcohol in the gourami (Trichogaster cosby). J Nutr 103(4): 600-607

Sund H and Theorell H (1963) Alcohol dehydrogenases. The Enzymes 7: 25-83

Watabiki T et al (1999) Intralobular Distribution of Class I Alcohol Dehydrogenase and Aldehyde Dehydrogenase 2 Activities in the Hamster Liver. Alc: Clinic Experimental Res 23: 52-55

Wheelock CE et al (2008) Applications of Carboxylesterase Activity in Environmental Monitoring and Toxicity Identification Evaluations (TI Es). Rev Environ Contam Toxicol 195: 117-178

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Toxicity Aquatic toxicity tests do not indicate the substance meets the criteria to be regarded as toxic (T). Furthermore Diisotridecyl adipate is not classified as carcinogenic, germ cell mutagenic, or toxic for reproduction or STOT.

Based on the available chronic Daphnia study with DITA and the chronic aquatic read across data, it can be concluded that DITA does not exhibit long-term aquatic toxicity in the range of its water solubility. In addition to the OECD 211 study with DITA, resulting in a NOEC above the experimentally determined water solubility, data on the read-across substance DEHA and on diisotridecyl dodecanedioate (DITD) show no chronic toxicity in the range of their water solubility as no effects were observed in the existing studies as well. Using the read across data as supporting information for the toxicity assessment of DITA is considered adequate/appropriate, since data are available for a compound with the same acid and a similar alcohol component, as well as for the same alcohol component and a similar acid. Therefore it can be conlcuded that DITA does not exhibit chronic aquatic toxicity to Daphnia up to the limit of water solubility.

DITA Chronic Daphnia study showing non-toxicity

The available long-term toxicity study towards aquatic invertebrates for DITA (Simon, 2016) was conducted by using the column elution method for test medium preparation.Daphnidswere exposed to a steady state column eluate in form of the 100% eluate and 10 % eluate for a period of 21 days in a semi-static test design. No significant differences between the control group and the test groups were found for the following endpoints: Mobility, age at first reproduction, length and intrinsic rate. In the undiluted eluate, a ~27% reduction of mean cumulative offspring per surviving daphnids after 21 days were seen, whereas the exposure to the 10%-dilution of the eluate resulted in no significant effects for this endpoint, as well. The concentrations measured in both, the undiluted eluate and the 10%- eluate tests solution, are above the reported and experimentally determined (e.g. Kotthoff, 2016) water solubility for DITA of ~0.7µg/L. Hence, micelle formation or other processes causing the inhibition cannot be excluded. As seen for the read-across substances (details below) and based on the valid water solubility figure for DITA, this study supports the fact that there is no toxicity in the range of water solubility.

DEHA Chronic Daphnia studies showing non-toxicity

In a valid 21 day reproduction test according to OECD test guideline 202 (1984, part 2), the water flea Daphnia magna was exposed to three different concentrations of bis-(2-ethylhexyl) adipate (nominal concentrations 0.5, 1.0, and 2.0 mg/L, measured concentrations 0.19, 0.39, and 0.77 mg/L). To solubilise the test substance, a vehicle was used (1g/L MARLOWET R 40). During the test, no effects were observed. Based on the reproduction rate, a NOEC of >= 0.77 mg/L was determined (Huels AG, 1996a). This is considered a valid proof of the absence of long-term aquatic toxicity of bis(2-ethylhexyl) adipate to invertebrates in the range of water solubility. This result is confirmed in another study performed by Robillard et al. (2008) (key study). A chronic Daphnia magna limit test was conducted at an average exposure concentration of 4.4 μg/L (measured water solubility = 5.5 µg/L) in laboratory diluent water to avoid insoluble test material and physical entrapment. One hundred percent of the DEHA-treated organisms survived compared to 90% survival in both the controls and solvent controls. Mean neonate reproduction was 152, 137, and 148 and mean dry weight per surviving female was 0.804, 0.779, and 0.742 mg in the treatment, control and solvent control, respectively. No adverse effects were observed. In the third study (Felder et al. (1986), not conducted according to a current guideline and GLP), which is the only one with DEHA detailed discussed in the Substance Evaluation Draft Decision, a significantly reduced yield of young per adult per day at mean measured exposure levels of 0.087 and 0.18 mg/L were found. A MATC (maximum acceptable toxicant concentration) for long-term toxicity to Daphnia magna was calculated to be between 0.024 and 0.052 mg/L based on statistical analyses of adult mean length, survival and young per adult per reproduction day. The geometric mean of the LOEC and NOEC was 0.035 mg/L, which is approximately ten-fold above the solubility limit of bis(2-ethylhexyl) adipate. Acetone was used as a solvent in this study. No adverse effects to survival growth or reproduction were observed even at levels five times the water solubility of DEHA. Effects were reported at concentrations significantly higher than the water solubility of DEHA (0.005 mg/L). Under such conditions it is likely that the observed effects have been caused by physical entrapment rather than chemical interactions (Rhodes et al. 1995). Furthermore, information provided in the results section of the publication is rather scarce. Neither raw data nor details about the extent of impairment daphnids had to face at a given concentration are given, and no information about a dose-response relationship is included. In our opinion also the study of Felder et al. (1986) with the NOEC of 0.024 mg/L shows that no toxicity occurred within the range of water solubility of 0.005-0.0032 mg/L. The lowest measured water solubility value of DEHA is lower than the NOEC. The study supports the fact that there was no toxicity in the range of water solubility. Therefore this value cannot be used for the PNEC-derivation.

Diisotridecyl dodecanedioate Chronic Daphnia study showing non-toxicity

In addition to the presented long-term toxicity studies with Daphnia magna performed with DEHA, a recently (2013) conducted long-term toxicity test with Daphnia magna (OECD 211) on the read-across substance diisotridecyl dodecanedioate is available. The GLP study was performed under semi-static conditions using DMF as a solvent (key study). In the Substance Evaluation Draft Decision it is discussed by the MSCA that the study cannot be considered valid due to uncertainties in the preparation of samples which resulted in declining of 99 % of the test substance in 48 h to below the LOQ. We would like to defend the study and attached a letter with the complete rationale of the responsible test institute Harlan. "The decline in measured test concentrations in the inoculated test samples was due to adsorption of the test item to the algal cells that were present. In the Daphnia magna Reproduction Test, algal cells are added to the test solutions in order to provide a food source for the daphnids, it is therefore considered that the decline in measured concentrations over each test media renewal period in this test was due to adsorption of the test item to the algal cells and not due to instability and / or volatility. As the test item was adsorbed to the algal cells that the daphnids ingest as a food source it can be considered that the daphnids were exposed to the test item over the period of each test media renewal" (please see attached the complete wording of the rationale). Furthermore based on the structure of diisotridecyl dodecanedioate it is expected that this substance is even more water insoluble than DITA (< 0.001 mg/L).The analytics for such water insoluble UVCB-substances are very difficult. The measured concentrations in this study were extremely low. Therefore it is not unlikely that the measured concentrations were inconsistent in such a sensitive study. Analysis of the freshly prepared 100 % v/v solution preparation on days 0, 5, 12, 19 showed measured concentrations ranging from less than the LOQ (assessed as 0.00027 mg/L) to 0.0024 mg/L. Analyses of the old or expired media on days 2, 7, 14, 21 showed measured concentrations ranging from less than the limit of quantification to 0.00093 mg/L. Given to the apparent decline in measured concentration between each period of media renewal, it was considered justifiable to base the results on the mean measured test concentrations of the test media to give a "worst case" analysis of the data. The No Observed Effect Concentration based on the mean measured concentration of the test media was equal to 0.00063 mg/L respectively. The mean measured concentration of 0.00063 mg/L was estimated for the 100 % v/v solution. We provide additional information including a better justification for the choice of test solution preparation used for the diisotridecyl dodecanoate test (Harlan 2013), and why it would achieve saturation for a low solubility UVCB (please see attachment “issue1_OECD211comments2_diisotridecyldodecanoate_studyno41202662.pdf"). In the study of Robillard et al. (2008) the water solubility of 5.5 (± 0.22) μg/L for di(2- ethylhexyl) adipate (DEHA) was measured using the slow-stir method which is in the Registrants’ scientific opinion, one of the most suitable methods for substances with a very low water solubility. Compared to DEHA, the water solubility of DITA and of diisotridecyl dodecanoate can be expected to be lower because of longer C-chains. Therefore, the higher measured water solubility values for DITA and diisotridecyl dodecanoate must be interpreted with caution, since they were determined with the ASTM-E1148 Standard Test Method, a comparably less sensitive method. In general, the determination of a definite value for the water solubility of UVCB substances poses a very complex problem and is by far more complicated than the determination for single substances (monoconstituents). This study shows like the DEHA-studies no toxic effects at the limit of water solubility.

Overall assessment of Chronic Daphnia for DITA

All of these substances, DEHA, DITA and diisotridecyl dodecanedioate, are dialkylesters of dicarboxylic acids, either adipic acid (1,6-hexanedioic acid) or 1,12-dodecandioic acid. Both dicarboxylic acids are linear and have even-numbered carbon chains. The alcohol component of all three substances has branched alkyl groups (C8 or C13). Chronic Daphnia data are available for DITA and the read across substances with the same acid and a similar alcohol component and the same alcohol component and a similar acid.

Thus, on the basis of the available data it can be concluded that DITA is neither acutely nor chronically toxic to aquatic invertebrates up to its limit of water solubility.

 

Hence the substance does not meet the criteria to be regarded as a PBT or vPvB substance.

An emission characterisation for diisotridecyl adipate is not needed because it is not a PBT.