Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study meets generally accepted scientific principles, acceptable for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1963
Report Date:
1963

Materials and methods

Objective of study:
metabolism
toxicokinetics
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
In vitro hydrolysis of the test substance by isolated mammalian liver enzymes.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: solid, not further specified
Details on test material:
- Name of test material (as cited in study report): stearyl erucamide
- Substance type: N-alkyl fatty amide
- Physical state: solid
- Analytical purity: not reported
- Lot/batch No.: 6175
- Stability under test conditions: not applicable
- Other: Supplier: Fine Organics, Inc., Lodi, New Jersey
Radiolabelling:
no

Test animals

Species:
rat
Strain:
not specified
Sex:
not specified

Administration / exposure

Route of administration:
other: not applicable, in vitro
Vehicle:
unchanged (no vehicle)
Details on exposure:
Not applicable, in vitro.
Duration and frequency of treatment / exposure:
0, 1, 2, 4 or 6 hours enzymatic hydrolysis at 37 °C
Doses / concentrations
Remarks:
Doses / Concentrations:
3, 10, 100 mg
No. of animals per sex per dose:
liver homogenate isolated from 2 freshly sacrificed rats
Control animals:
no
Details on study design:
The fatty acid amidases (known as Fatty Acid Amide Hydrolases: FAAH) which occur in rat liver were chosen as typical mammalian amidases and used in hydrolysis investigation. Known quantities of test substance (3, 10 or 100 mg) were added to 250 mg of bile salts (ox bile extract) and 50 mL of simulated intestinal fluid (U.S.P. XVI, page 1073) excluding the pancreatin in 250 mL Erlenmeyer flasks.
Livers from 2 freshly killed exsanguinated rats were homogenised and 2.0 g aliquots of the liver homogenate added to the flasks. The sample flasks were incubated with slow agitation at 37 °C for either 0, 1, 2, 4 or 6 hours, in case of the 100 mg samples, and 6 hours only for the smaller quantities.
Details on dosing and sampling:
After the appropriate time the flasks were submerged in a steam bath to inactivate the enzymes, acidified and extracted with several portions of ethyl ether. The solvent was then evaporated to dryness and 25 mL of standard 0.1 N base added to neutralise the liberated fatty acids (0.01 N base was used for the lower concentrations). Excess base was determined by back-titration with 0.1 N acid (0.01 N where appropriate) to pH 8.5.
The result from the 0-hour digestion was used as a blank, to account for the acidity contributed by the bile salts and the liver homogenate. The degree of enzymatic hydrolysis was determined relative to total available acidity as measured by acid hydrolysis of the sample in 6.0 N hydrochloric acid for 3 hours, followed by acidification, extraction and titration.

Results and discussion

Any other information on results incl. tables

Relative digestion in vitro:

Exposure time (hours)

Sample size (mg)

Relative Digestion (%)

1

100

0

2

100

12.6

4

100

22.9

6

100

37.9

6

10

73

6

3

105

 

The 100 mg samples digested at a smooth rate, reaching a maximum near 40%. Apparently the quantity of the enzyme present in the 2.0 g of liver homogenate is the limiting factor in these digestions, since at progressively lower levels of substrate, the hydrolysis proportionately increases to reach completion for the 3 mg sample.

Conclusion:

The small levels of test substance which could be ingested by transfer from packaging materials to food products will probably be completely hydrolised by normal tissue enzymes.

Applicant's summary and conclusion