Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Examination of the differential hepatotoxicity of diallyl phthalate in rats and mice
Author:
Eigenberg, DA, Carter, DE, Schram, KH & Sipes, IG
Year:
1986
Bibliographic source:
Toxicology and Applied Pharmacology 86(1):12-21

Materials and methods

Objective of study:
toxicokinetics
Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
not applicable
Remarks:
As stated in OECD guideline 417: Flexibility, taking into consideration the characteristics of the substance being investigated, is needed in the design of toxicokinetic studies.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test Substance:
Labeled: [14C]Diallyl phthalate (labeled on the 2,3 position of the allyl alcohol (AA) moiety
Specific activity: 12.2 mCi/mmol
Supplied by: Midwest Research Institute (Kansas City, Mo.).
Purity: 99% (TLC)
Unlabeled: diallyl phthalate with purity >99% supplied by Pfaltz and Bauer, Stamford, Connecticut
Labeled Allyl alcohol (AA) obtained by heating [14C]DAP with 7 M NaOH at 90˚C for appr. 14 h. Following hydrolysis, the sample was saturated with NaCl and extracted with diethyl ether. Purity was determined >99%.
Radiolabelling:
yes
Remarks:
14C

Test animals

Species:
other: Rats and Mice
Strain:
other: Fischer-344 Rats and B6C3F1 Mice
Sex:
male
Details on test animals and environmental conditions:
Body weights:
Rats: 150-200 g
Mice: 20-25 g
Food and water ad libitum
12 h light-dark cycle

Administration / exposure

Route of administration:
other: oral: gavage and intravenous
Vehicle:
other: Corn oil for oral toxicity studies; water:ethanol:Emulphor EL-620 (3:2:1) for oral distribution studies.
Duration and frequency of treatment / exposure:
Single dosage, animals were anesthesized 24 hours after dosing.
Because AA is the proposed toxic metabolite of DAP, rats and mice were also dosed orally with AA.
Doses / concentrations
Remarks:
Doses / Concentrations:
Dose response toxicity studies of DAP and allyl alcohol:
Rats: 300, 400, 500, 600 mg/kg of DAP and 25, 50, 75 mg/kg of AA
Mice: 500, 600, 700, 900 mg/kg of DAP and 25, 50, 75 mg/kg of AA

Toxicokinetics (excretion and distribution studies in both rats and mice):
Oral: 1, 10, or 100 mg DAP/kg bw (In section "Results" paragraph "Disposition of DAP" erroneously reported as 1, 10 or 200 mg/kg)
Intravenous: 10 mg DAP/kg bw
No. of animals per sex per dose:
At least 4 for Dose response toxicity studies, 3 for toxicokinetics experiment.
Control animals:
other: yes for Dose response toxicity studies
Positive control:
Not applied.
Details on study design:
Dose response toxicity studies:
1. 24 h after dosing blood was taken from the inferior vena cava and analyzed for serum glutamic-pyruvic transaminase (SGPT) activity.
2. Livers were removed and sections were prepared for histopathological evaluation.
Toxicokinetics:
1. Excretion studies: Following dosing, animals were placed in a metabolism cage and 14CO2, volitaile metabolites, urine and faeces were collected for 24 h.
2. Tissue distribution and kinetic studies: i.v. dosing of 10 mg/kg b.w. followed by sampling of blood at various intervals from 0.5 to 24 h; collection of various organs, urine and intestine contents; blood samples were takenat 5, 10, 15, 20 and 30 min after dosing for rats and at 5, 10 and 15 min for mice.
3. Metabolites were identified using references of the expected metabolites and quantified in urine samples.
4. Further analysis included determination of conjugates and determination of concentrations of DAP and monoallyl phthalate (MAP).
Details on dosing and sampling:
-Determination of dose effect on the elimination of DAP:
Rats and mice were dosed orally with 1, 10 or 100 mg/kg bw DAP. Dosing solutions were prepared in water:ethanol:Emulphor EL-620 (3:2:1).
Rats and mice received 1 mL of dosing solution/kg bw (40 or 120 µCi/kg bw, respectively). Following dosing 14CO2, volatile metabolites,urine and feces were collected for 24 h. At termination,selected tissues were removed and oxidized to 14CO2.
-Tissue distribution and pharmacokinetic studies:
Rats and mice were dosed via the tail vein with 10mg/kg bw of [14C]DAP (40 or 120 µCi/kg bw, respectively) in water/ethanol/Emulphor (3:2:1).

Statistics:
ANOVA tests

Results and discussion

Preliminary studies:
Hepatotoxicity:
1. DAP: significant elevation of SGPT in rats at doses > 300 mg/kg (NOAEL); significant elevation of SGPT in mice at doses > 700 mg/kg (NOEL);
2. AA: significant elevation of SGPT in rats at doses > 25 mg/kg (NOAEL); significant elevation of SGPT in mice at doses 25 mg/kg and higher (LOEL);
although statistically significant increases in SGPT activities were observed in both rats and mice at 50 and 75 mg/kg, the magnitude of these increases was considerably greater in rats (up to a factor of 10).
Hispatological evaluation confirmed a marked species difference in DAP- and AA- induced hepatic injury.
In rats, 300 mg/kg of DAP resulted in periportal inflammation in four of four rats and periportal necrosis in two of four rats. At higher doses (400-60 mg/kg), periportal hemorrhagic and coagulative necrosis was present in 80% of the rats treated with DAP.
In mice doses of DAP up to 700 mg/kg did not induce necrosis, although some of the mice had mild periportal inflammation. At 900 mg/kg, two of four mice developped periportal necrosis. Doses of AA (25, 50, 75 mg/kg) were not hepatotoxic in mice. in rats, periportal necrosis was evident in 9 of 12 animals treated with AA.
Main ADME resultsopen allclose all
Type:
distribution
Results:
6 to 7% and 1 to 3% of the dose were found in the tissues of rats and mice respectively, 24 hours after iv administration of DAP.
Type:
metabolism
Results:
Major metabolites: Monoallyl phthalate (MAP), allyl alcohol (AA), 3-hydroxypropylmercapturic acid (HPMA), and an unidentified polar metabolite (PM)
Type:
excretion
Results:
Major routes of elimination of 14C equivalents were via urine and as 14CO2

Toxicokinetic / pharmacokinetic studies

Details on absorption:
DAP is easily adsorbed after oral dosing and rapidly hydrolysed; the resulting AA is further oxidized to acrolein, which is hepatotoxic. Subsequently, acrolein is oxidized to acrylic acid, which is much less toxic. Further oxidation of acrylic acid probably results in CO2.
Details on distribution in tissues:
Major organs and tissues containing detectable DAP levels after dosing are small intestine, muscle, skin, kidney and liver. Following iv administration of DAP, the parent compound was rapidly cleared from the blood of rats and mice. The t½ for elimination from blood was approximately 2 min in both species. No DAP was found in blood, liver, kidney, muscle, skin, or small intestine 30 min after iv administration of DAP.  Semilogarithmic plots of total 14C in tissues vs time showed an exponential decreases down to values near or below 1 % of the original doses. Clearance of 14C appeared to be generally more rapid in mice tissues compared to rat tissues.
Details on excretion:
Following oralor iv administration of DAP to rats and mice, rats eliminated 60% of the dose in the urine and 30% as 14CO2 within 24 hrs,. In this same time interval mice eliminated 91% of the dose in the urine and 8% as 14CO2.
The route of elimination of DAP as a percentage of the dose administred was not significantly different folowing oral doses of 1 to 100 mg/kg to rats, but in mice there was a significant increase in the quantity of 14CO2 excreted at the 100 mg/kg dose (p<0.005)
Toxicokinetic parametersopen allclose all
Test no.:
#1
Toxicokinetic parameters:
other: t1/2 of DAP in blood = 32 min for rats
Test no.:
#2
Toxicokinetic parameters:
other: t1/2 of DAP in blood = 9 min for mice

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
MAP, AA, HPMA, and PM were found in the urine of rat and mice dosed with DAP but no glucoronide or sulfate conjugates were present.
The mean values for all dose groups for mice and rats, respectively, were MAP-39 vs. 33% ; HPMA-28 vs. 17% ; and PM-20 vs. 8 %.

Any other information on results incl. tables

Dose response toxicity studies, histopathology: 
Histopathological evaluation confirmed a marked species difference in DAP- and AA-induced hepatic injury. 
DAP exposure:
In rats, 300 mg/kg of DAP resulted in periportal inflammation in all four rats and periportal necrosis in two out of four rats (LOEL=300 mg/kg). At the higher doses (400-600 mg/kg), periportal hemorrhagic and coagulative necrosis was present in 80% of the rats treated with DAP (four - six rats/dose). 
In mice, doses of DAP up to 700 mg/kg did not produce necrosis, although some of the mice had periportal inflammation (LOEL=700 mg/kg; NOEL=600 mg/kg). At 900 mg/kg, two of four mice developed periportal necrosis. Doses of AA (25, 50 and 75 mg/kg) were not hepatotoxic in mice.

AA exposure: In rats, periportal necrosis was evident in 9 of 12 animals treated with AA. The severity of the injury was greater at the 50- and 75- mg/kg doses than at the 25-mg/kg dose.  Mice given 100-220 mg/kg bw of AA died within 24hr after administration. Therefore in mice it was not possible to identify a non-lethal dose of allyl alcohol that resulted in extensive liver damage.

AA is a product hydrolysed from DAP at the first stage in an animal body. 

(Excretion, tissue distribution, and pharmacokinetic studies). 
Fischer-344 rats and B6C3F1 mice were given [14C]DAP, 1, 10, or 100 mg/kg po or 10 mg/kg iv, and placed in metabolic cages for 24 hr.  In rats, 25-30% of the DAP was excreted as CO2, and 50-70% appeared in urine within 24 hr.

In mice, 6-12% of the DAP was excreted as CO2, and 80-90 % was excreted in the urine within 24 hr. Monoallyl phthalate (MAP), allyl alcohol (AA), 3-hydroxypropylmercapturic acid (HPMA), and an unidentified polar metabolite were found in the urine of rats and mice dosed with DAP. The polar metabolite was present in the urine of rats after administration of DAP or AA, indicating that the compound is a metabolite of AA.

Following iv administration of DAP, the parent compound was rapidly cleared from the blood of rats and mice. The t½ for elimination from blood was approximately 2 min in both species. No DAP was found in blood, liver, kidney, muscle, skin, or small intestine 30 min after iv administration of
DAP. 

Monoallyl phthalate, formed from the metabolism of DAP, had a  half-life in blood of 32 and 9 min in rats and mice, respectively. Within 4 and 2 hr after dosing rats and mice, respectively, with DAP, no MAP was detected in the blood, liver, kidney, skin muscle, or small intestine.

Table: Diallyl Phthalate Metabolites in Rat and Mouse Urine (a,b)

-------------------------------------------------------------------------------------
Species Dose    Allyl    Monoallyl  3-hydroxypropyl   Polar
       (mg/kg)  alcohol phthalate  mercapturic acid  metab(c)
-------------------------------------------------------------------------------------
Rat    1(po)    2.3±0.2  29.0±0.7   13.2±0.4         6.6±0.4
       10(po)    3.0±1.6  32.5±1.7   18.4±5.9         7.8±1.6
      100(po)    3.1±2.3  32.2±0.6   16.5±1.2         7.5±0.7        
       10(iv)    2.9±2.0  38.0±1.0   20.6±1.1         8.3±1.1

Mouse 1(po)    3.6±1.1  39.2±1.9   27.0±1.0         19.1±1.7
       10(po)    4.8±2.3  37.7±4.2   29.7±0.2         19.0±1.4
      100(po)    2.3±0.6  44.7±1.9   21.1±1.9         19.4±1.4        
       10(iv)    7.5±5.2  31.5±5.8   34.4±2.4        20.8±1.5
---------------------------------------------------------------------------------------
(a) Values are calculated as a percentage of the dose of 14-C administered
(b) Each value in the mean of three animals ± SE
(c) An unidentified metabolite which was not extracted by acetonitrile from urine

Simple statistics with the t-test shows significant differences in concentrations of HMA and PC between mice and rats (p<0.05 and p<0.01 resp.).

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
1. The authors postulate that the differential hepatotoxicity of DAP is related to the extent of glutathione conjugation with allyl alcohol or acrolein (the active metabolite of AA).
2. The pesence of significant greater quantities of the unidentified polar metabolite in mice may indicate another route of detoxiification of AA in protecting mice.
Executive summary:

In rats the LOEL for induction periportal inflammation and periportal necrosis after acute DAP exposure was 300 mg/kg, in mice the LOEL was 700 mg/kg and the NOELwas 600 mg/kg.

Within 24 hr after oral or i.v. dosing, rats excreted  25 -30% of the DAP as CO2, and 50-70% in urine, whereas mice excreted 6-12% of the DAP as CO2, and 80-90 % in the urine. Following i.v. administration of DAP, the parent compound was rapidly cleared from the blood of rats and mice. The t½ for elimination from blood was approximately 2 min in both species. No DAP was found in blood, liver, kidney, muscle, skin, or small intestine 30 min after iv administration of DAP. Major metabolites were monoallyl phthalate (MAP), allyl alcohol (AA), 3-hydroxypropylmercapturic acid (HPMA), and an unidentified polar metabolite were found in the urine of rats and mice dosed with DAP. Monoallyl phthalate, formed from the metabolism of DAP, had a half-life in blood of 32 and 9 min in rats and mice, respectively. The polar metabolite was also present in the urine of rats after administration of AA, indicating that the compound is a metabolite of AA. As mice produced more HPMA than rats the differential hepatotoxicity of DAP is related to the extent of gluthathione conjugation with AA or acrolein, the active metabolite of AA.