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Description of key information

There is no toxicokinetic data for isobutyric anhydride. Toxicity data has been read across primarily from isobutyric acid, which is the hydrolysis product of isobutyric anhydride. The hydrolysis is rapid, and thus the read-across is valid. A hydrolyis study is presented in section 5.1.2 of this dossier, and illustrates that at physiological pH the half life is between 5 and 10 minutes. This indicates that read-across to isobutyric acid is appropriate for all studies where water is present, as the anhydride will quickly hydrolyze to the acid.

Key value for chemical safety assessment

Additional information

Isobutyric anhydride, just like other short-chain aliphatic anhydrides undergoes rapid hydrolysis in the presence of water. This hydrolysis makes read-across to butyric acid a relevant approach for every toxicological test, as water is present in all of them. Hydrolysis testing for isobutyric anhydride is presented in section 5.1.2 of this document.

Toxicokinetics, metabolism and distribution of isobutyric acid

Information about ADME of isobutyric acid is available from several studies.

 

DiVincenzo (1979)

 

The metabolic fate of isobutyric acid (IBA) was investigated by oral (gavage) administration of radiolabelled [1-14C]isobutyric acid to groups of 4 male CD rats at doses of 4, 40, and 400 mg/kg bw and to 4 female CD rats at 400 mg/kg bw. IBA was eliminated rapidly in the breath of the dosed animals as expired 14CO2. At 4 hr 67 to 83% and at 48 hr 85 to 90% of the dose was eliminated in the breath. There were no differences between sexes.

 

Urinary radioactivity averaged 3.5% of the dose with about 2/3 of the radioactivity present as urea.

Faecal radioactivity was less than 1% of the dose.

 

Isobutyric acid disappeared rapidly from the plasma of rats dosed by gavage with 400 mg/kg bw. Plasma levels peaked between 0.5 and 1 hr (11.4 ± 2.4 µg/ml), decreased to 3.3 µg/mL at 1 hr and were below the limit of detection at 4 hr (DiVicenzo, 1979).

 

Mahadevan (1969)

 

In fasted human subjects, the natural background levels of isobutyric acid in serum was 30.7 ± 7.9 µg/100mL and in plasma 143 ± 30 µg/100 mL (mean ± S.D., n= 6)

 

Thomas (1958)

 

Unlabelled isobutyric acid was orally administered for a period of 6 days in diet to 10 male rats individually housed in metabolic cages. The dose administered was 1 g/day and rat. During application of the test substance and 6 days prior and 6 days following application, urine of the rats was continuously collected with a sampling period of 48 hrs. The amount of methylmalonic acid and succinic acid excreted in urine was determined by a paper chromatographic method.

 

In the period prior to test substance administration, the background excretion of methylmalonic acid and succinic acid was determined to be between 0.33 to 1.76 mg/day and 4.1 to 7.0 mg/day respectively. During administration, the amounts of acids excreted increased to maximum values of 17.9 g/day for methylmalonic acid during the third sampling period (day 5 and 6 of administration) and of 73.7 g/day for succinic acid during the second sampling period (day 3 and 4 of administration). After termination of isobutyric acid administration, the excretion declined during the 6 day observation period to values close to normal (methylmalonic acid 2.56 mg/day; succinic acid 9.6 mg/day) (Thomas, 1958).

 

Winstead (1973)

 

11C-labelled sodium isobutyrate was synthesized with high specific activity by Grignard reaction of 11C-carbondioxid with the appropriate Grignard reagent. 11C-Carbondioxide was created by transformation of B2O3 (diboron trioxide) to CO by particle beam bombardment (deuterons or protons) followed by oxidation over CuO.

 

The freshly prepared 11C-isobutyrate was administered by i.v. injection into a dog. Radioactivity was rapidly homogeneously distributed over the whole body within 30 min. At 2 - 4 min., some greater activity was observed in heart blood pool, liver, and throughout abdomen. The observations indicate metabolism in liver and participation in general lipid metabolism pathways.

 

Semino (1998)

 

In general, branched-chain aliphatic acyclic alcohols are rapidly absorbed from the gastrointestinal tract.

 

Acids with a methyl substituent located at an even-numbered carbon are extensively metabolized to CO2 via ß-oxidative cleavage in the fatty acid pathway. They also are substrates for ß-oxidation and cleavage in the amino acid and fatty acid pathways. If the methyl group is located at the 3-position, ß-oxidation is inhibited and omega-oxidation predominates.

 

The metabolism of isobutyric acid has been established in rodents. Rats fed isobutyric acid excreted elevated levels of 2-methylmalonic acid, which is an intermediate in the conversion of propionyl CoA to succinyl CoA.

 

Isobutyric acid occurs endogenously as intermediate in the human metabolism of the amino acid valine.

 

Conclusion

 

Isobutyric acid is rapidly absorbed after administration. Peak plasma levels are reached after 30 to 60 min following oral application. Elimination from blood is also fast. In rats, plasma levels fall below the limit of detection 4 hours after dosing (DiVincenzo, 1979).

 

In normal human blood of fasted subjects, isobutyric acid has been determined as natural constituent at a level of ca. 143 µg/100 mL (determination in plasma).

 

As demonstrated by experiments with radioactively labeled test substance, radioactivity from isobutyric acid is rapidly distributed over the whole body and 85 to 90% of the administered dose are finally excreted as CO2 within 48 hours (Winstead, 1973; DiVincenzo 1979).

 

Urinary excretion of methylmalonic acid and succinic acid is enhanced after repeated oral administration of high doses of isobutyric acid demonstrating ß-oxidation under these conditions.

 

These findings are consistent with the structure of isobutyric acid. With a methyl substituent at position 2, it undergoes excessive ß-oxidation, enters intermediary metabolic pathways, and is finally excreted as CO2 (Semino, 1998). The formation and excretion of methylmalonic acid after high doses verifies the ß-oxidation process. At lower doses of isobutyric acid, the greater fraction of methlymalonic acid can be introduced into intermediary metabolic pathways and less methylmalonic acid is excreted.

Information on toxicokinetics and metabolism of supporting substances isobutanol and isobutyl isobutyrate

 

To verify the rapid metabolism of isobutanol and isobutyl isobutyrate to intermediate isobutyric acid, information on toxicokinetics and metabolism of these two substances are presented below. The information is taken from the endpoint summary of the OXEA IUCLID 5 dataset of isobutanol (2009) and from the OECD SIDS/SIAR of isobutyl isobutyrate (last update 2005-09-08). Detailed Information on references can be found in these sources.

 

Isobutanol

 

Isobutanol is readily absorbed from the gastrointestinal as well as from the respiratory tract. Maximum blood levels are reached within 15 minutes (rat, respiration; Poet and Corley (OPP/ACC) 2004), 45 to 60 minutes (human, rabbit, oral application; Bilzer 1990, Saito 1975) or 90 minutes (rat, oral application; Gaillard 1965). Elimination from blood is considerably fast as well. In humans, an elimination half-life of 1.45 h was determined (Bilzer 1990). In rabbits, isobutanol could not be detected any more 6 hours after application (Saito 1975).

 

Isobutanol is metabolized in the liver and it undergoes rapid oxidation (Hedlund 1969, Saito 1975, Ehrig 1988, Sinclair 1990). Isobutyraldehyde and subsequently isobutyric acid are formed (Saito 1975, Poet and Corley (OPP/ACC) 2004) by oxidation with alcohol dehydrogenase and aldehyde dehydrogenase respectively (Hedlund 1969, Saito 1975, Rüdell 1983), Beta-oxidation and decarboxylation of isobutyric acid results in propionyl-coenzyme A (Saito 1975) which can enter the tricarbxylic acid cycle. Accordingly, propionaldehyde, propionic acid, and succinic acid were identified as additional metabolites (Rüdell 1983).

 

Only minor amounts of applied isobutanol doses are excreted unchanged (rabbit 0.54 %, Saito 1975; rat 0.27%, Gaillard 1965). Small amounts have been found as glucuronides in the urine of rabbits (4.4 %, Kamil 1953).

 

In expired air, no metabolites (aldehyde, ketone) could be detected (Kamil 1953).

 

In conclusion, isobutanol is rapidly metabolized to isobutyraldehyde and further on to isobutyric acid with higher levels of isobutyric acid detectable in blood.

 

Isobutyl isobutyrate

 

Metabolism/toxicokinetic studies have been conducted in male rats with isobutyl isobutyrate using intravenous injections (femoral vein) with simultaneous intravenous (jugular vein) sampling (Deisinger, unpublished, 2003). Isobutyl isobutyrate levels within the first 15 seconds had a mean value of 1045 μM and rapidly decreased thereafter and being undetected at 166 seconds. The calculated T1/2 by one-compartment modeling was 11.1 seconds. Isobutanol and isobutyric acid levels increased rapidly up to peak levels of 218 and 304 μM, respectively at 31-45 seconds. Isobutyric acid levels were consistently higher than isobutanol levels, suggesting further metabolism of the isobutanol metabolite to isobutyric acid (see above). Both isobutanol and isobutyric acid levels remained increased throughout the 240 second sampling period.

 

In conclusion, isobutyl isobutyrate is rapidly metabolized to isobutanol and isobutyric acid in rats.

 

Justification for the selection of isobutanol and isobutyl isobutyrate as supporting substances for the systemic toxicity of isobutyric acid.

 

Data from toxicity studies of isobutanol and isobutyl isobutyrate have been included in Section 7 to assess the hazards associated with the systemic toxicity of isobutyric acid. Following exposure, both substances are metabolized rapidly and extensively in vivo to result eventually in isobutyric acid (see above). Thus upon exposure to isobutanol and isobutyl isobutyrate, the rapid appearance of isobutyric acid in the systemic circulation is to be expected. The administration of either substance will result in systemic exposure levels of isobutyric acid. Therefore, systemic toxicity data, obtained from studies with direct administration of isobutanol or isobutyl isobutyrate as test substances, provide valuable information and can be used to assess the systemic toxicity of isobutyric acid.

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