(Z)-3,7-dimethylocta-2,6-dienal

Endpoint summary

Description of key information

Short description of key information on bioaccumulation potential result: 
Absorption from the gastro-intestinal tract is rapid and almost complete (ca. 90-95%). 
First-pass liver metabolism occurs rapidly within a few minutes, no saturation up to the highest investigated doses of 500 mg/kg bw. Metabolism includes formation of 14CO2 by rapid oxidation and decarboxylation of the aldehyde group. More hydrophilic metabolites with additional polar -COOH and -OH groups were identified in urine, and glucuronic acid conjugates in bile. 
14C from [1,2-14C]-citral is distributed throughout the body tissues with no major depot (< 2% of 14C in any tissue at 72 h p.a.).
Excretion of metabolites via the bile results in enterohepatic circulation with further biotransformation leading to urinary rather than fecal excretion.
Excretion kinetics (% of 14C-dose): within 24 h 45% in urine, 6% in faeces, exhalation of 16% as 14CO2 and < 1% as 14C-citral; within 72 h 80% in excreta and 3% in tissues; excretion profiles independent from dosing (single dose 5-500 mg/kg or 10 daily doses of 5 mg/kg) and application mode (oral or i.v.); whole body half-life 8 hrs after i.v. exposure. No indications of bioaccumulation of citral.
Short description of key information on absorption rate: 
A considerable part of a dermal dose may be lost before percutaneous absorption may occur due to the high volatility of citral. Citral remaining on the skin, is rapidly and fairly well absorbed. Taken together in a weight of evidence, for derivation of dermal DNELs, a dermal penetration rate of 50% is assumed on the basis of high volatility of citral.

Additional information

Absorption and distribution

Absorption of citral from the gastro-intestinal tract is rapid and almost complete (Diliberto 1988; Phillips 1976).

In Fischer 344 rats, the appearance of ¹⁴CO₂ and citral-derived radioactivity in the urine of rats within 2 h post oral application of [1,2-¹⁴C]-citral (doses of 5-500 mg/kg) indicated rapid absorption. Comparing the difference between fecal excretion after oral and i.v. exposure (5 mg/kg), 5 to 9% of the oral dose was not absorbed from the gastrointestinal tract and was eliminated via the faeces.

The rapid elimination of more than 75% of ¹⁴C from blood within 2 min after intraveneous application of 5 mg ¹⁴C-citral/kg may indicate that the apparent volume of distribution of citral is in the larger pool including the interstitial water and/or cellular fluids rather than in the vascular tissue. The lipophilicity of citral (log Pow 2.76) that facilitates oral absorption also facilitates its passive diffusion across cell membranes and results in its wide dispersion throughout the body tissues with no evidence of a major depot. In any tissue amounts were below 2% of the applied dosed at 72 h after administration. This residual radioactivity may either represent ¹⁴C associated with the one-carbon pool leading to incorporation in cellular molecules by various anabolic pathways, or distribution of lipophilic citral metabolites. The relative amount in tissue was independent from dose or route of administration. Highest tissue concentrations were observed in liver, muscle, blood, adipose tissue. Multiple dosing had no overall effect on disposition although biliary excretion was increased significantly (Diliberto 1988).

Two to 2.5% of the applied¹⁴C-dose were found in the carcass (excluding the gastro-intestinal tract) of Wistar rats after 24 hrs independent from dose level (single application by gavage at doses of 5, 770, and 960 mg/kg bw). Besides the gastro-intestinal tract (7 to 12.5%), the

radioactivity was mainly associated with the liver (1.5-2%) . In mice, a considerable proportion of the radioactivity was absorbed rapidly after administration and appeared throughout the tissues (Phillips 1976).

Metabolism

In vitro, citral was readily reduced by rat liver ADH (alcohol dehydrogenase) forming the corresponding alcohol. Different affinities of ADH for the two citral stereoisomers, geranial and neral,may result in differential ADH-mediated reduction rates. Reduction by ADH or hydroxylation via cytochrome P-450 may be metabolic steps required for subsequent oxidation by hepatic ALDH (aldehyde dehydrogenase; Boyer 1991).

In vitro, at low concentrations citral was found to be an inhibitor of all three isozymes of aldehyde dehydrogenase (E1, E2, E3)

in hepatic mitochondrial and cytosolic fractions of untreated rats (Boyer 1991; Kikonyogo 1999). However, citral was also a substrate of ALDH as indicated by regain of activity upon long incubation in the presence of NAD⁺, and formation of NADH and geranic acid. The isomer geranial appeared to be metabolized preferentially by ALDH. Enzyme kinetics suggest, that the isozyme E1 may be the enzyme involved in citral metabolism in vivo (Kikonyogo 1999).

In vivo or in cellular systems, citral is an inducer of phase I metabolism by cytochrome P-450 (Roffey 1990).

Citral induced phase II metabolism by glutathione-S-transferase (GST) in a rat liver epithelial cell line in vitro. The isomer geranial had a significant inducibility of GST activity, mainly through the expression of GSTP1 protein, while neral had not. Further, geranial was shown to act as an electrophil which directly conjugates with GSH and which stimulatesGSH synthesis including gamma-glutamylcysteine synthetase activity (Nakamura 2003). However, the metabolite profiles identified by (Diliberto 1990) in vivo indicate that 1,4-addition reactions of the alpha,beta-unsaturated aldehyde structure are not a major path of the metabolism of citral.

In vitro, citral was a substrate for GST transferase isolated from rat olfactory epithelium, from respiratory epithelium of the nose and from lung tissue. GST activity in olfactory epithelium was the highest among all extrahepatic tissues tested withthe model substrate CDNB (46% of liver) . Consequently, inhalative exposure to citral, e.g. during use as odorant is expected to result in phase II metabolism taking place in the cells of the airways before systemic absorption occurs (Ben Arie et al., 1993). The further fate of formed glutathione conjugates, which are practically membrane-impenetrable, has not been investigated.

In rats, gastrointestinal absorption results in first-pass liver metabolism which occurs rapidly within a few minutes. In addition, it is known that intestinal bacteria can metabolise citral. The excretion of metabolites via the bile (about 25% of citral-derived radioactivity in total, 20% within 1 hr p.a., no unchanged citral) into the intestine results in enterohepatic circulation of citral-derived radioactivity. Thus, metabolites of citral are retained in the body for a longer time and may undergo further transformation leading to urinary (48-63%) rather than fecal excretion (7-16%). The overall transport or metabolism of citral was not saturated at doses ranging from 5 to 500 mg/kg bw. The rapid formation of ¹⁴CO₂implies that the aldehyde group of the citral molecule is rapidly oxidized and decarboxylated (Diliberto 1988).

A total of seven metabolites of citral have been characterized in urine and bile after oral exposure:

E-3,7-dimethyl-2,6-octadienoic acid

3,8-dihydroxy-3,7-dimethyl-6-octenoic acid

3,9-dihydroxy-3,7-dimethyl-6-octenoic acid

E-3,7-dimethyl-2,6-octadienedioic acid;

Z-3,7-dimethyl-2,6-octadienedioic acid;

3 -hydroxy-3,7,dimethyl-6-octenedioic acid;

3,7-dimethyl-6-octenedioic acid;

The parent compound, i.e. citral, has not been detected.

The biotransformation of citral includes reduction or hydration of the 2,3-double bond, oxidation of the aldehyde function, and allylic oxidation at C-8, and, possibly, C-9. Enzymes involved in the formation of these metabolites may be aldehyde dehydrogenase, ß-oxidation by aldehyde oxidase, oxygenation at C-8 or C-9 by cytochrome P-450, and alcohol dehydrogenase for oxidation of intermediate alcohols. The molecules formed by these processes are more hydrophilic, since they contain COOH and other polar groups. Changes of the metabolite profile after glucuronidase treatment indicate the presence of glucuronic acid conjugates in bile but not in urine (Diliberto 1990).

Elimination

In Fischer 344 rats, [1,2-¹⁴C]-citral-derived radioactivity was rapidly excreted in urine, faeces, and expired air. Within 24 h after oral uptake of 5 mg/kg, 45% of the ¹⁴C-dose were excreted in urine and 6% in the faeces, 16% were exhaled as ¹⁴CO₂and less than 1% as ¹⁴C-citral. Within 72 h, 80% of the dose was recovered in excreta and 3% in the tissues. Excretion profiles did not change with increasing dose (up to 500 mg/kg) and were similar for oral and i.v. administration.The whole body half-life was 8 hrs after i.v. exposure. However, a small percentage tended to persist with a clearance half-life of 24 hrs (Diliberto 1988). A similar elimination profile was found in the study with gavage application to Wistar rats. The observed rapid exhalation of  ¹⁴CO₂, e.g. 16% of the 5 mg/kg-dose within 6 hrs, suggested that the aldehyde function of the molecule is rapidly oxidized and directly decarboxylated. Radioactivity was retained in the body of the mouse for a longer period than in the rat. In mice, most of the administered radioactivity was excreted within 5 days with the urine as major route. There was no evidence for long-term storage or bioaccumulation of citral in the two species (Phillips 1976).

After multiple dosing of citral (10 times 5 mg/kg) no change was observed in the excretion via urine, feces, and expired ¹⁴CO₂. Because the terminal component of the clearance half-life accounts for such a small amount of the total radioactivity (pool size < 1%), it is unlikely that repeated exposure to citral would result in bioaccumulation of this chemical in the body. In contrast, biliary excretion increased by 34% suggesting induction of some metabolic processes during multiple dosing (Diliberto 1988).

Discussion on absorption rate:

Due to its high volatility a considerable part of an applied dermal dose of citral may be lost before percutaneous absorption may occur. Citral remaining on the skin, is rapidly and fairly well absorbed as indicated by a weight of evidence approach.

About 50% of the applied dose was lost due to evaporation (approx 30% of applied dose) during dermal administration of the test substance (remaining part of the dose defined as initial body burden) and due to absorption to the protective device over the application site (approx. 24% of applied dose) , i.e. a perforated tissue capsule. After the 72 hr observation period recoveries as % of the initial body burden (IBB) were ca. 10% on the treated skin site, ca. 10% in the remaining tissues, ca. 20 or 30% in the excreta (5 or 50 mg/kg-dose groups). Total recoveries accounted to approx. 60-70% (Diliberto 1988).

Within 16 hrs after application to the skin of guinea pigs (1.88 mg/animal), a systemic absorption of at least 20% applied citral was indicated by excretion in urine and faeces (approx. 10% of the initial dose) and presence in deeper skin layers excluding the stratum corneum (approx 10% of the initial dose). Citral in the stratum corneum amounted to approx. 10% of the applied dose. Amounts excreted via exhalation of ¹⁴CO₂or deposited in body tissues were not analyzed in this study (Barbier 1983).

Rapid percutaneous absorption into the systemic circulation was indicated by an immediate acute response (vasoconstriction of the ventral rat prostrate) within 2.5 min after topical application of citral (Scolnik 1994).

An in vitro study confirmed that citral (applied as lemon myrtle oil with 96.6% citral) is absorbed into all layers of freshly excised human skin samples. 1.29% and 1.57% of the administered dose were recovered from full-thickness skin after 1 and 12 hrs of incubation, respectively. Neral and geranial were the only detectable lipophilic components in epidermis, dermis and subcutaneous fat tissue. As exposure time increased, the recovery in the fat tissue increased, while the recovery in epidermis/dermis showed a maximum at 4 hrs p.a. At all timepoints, the recovery in skin layers was higher than in subcutaneous fat (Hayes 2003).

Taken together in a weight of evidence, for derivation of the systemic dermal DNEL, a dermal penetration rate of 50% is assumed on the basis of high volatility of citral.