Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998

Materials and methods

Objective of study:
metabolism
Principles of method if other than guideline:
Assay for formaldehyde-formation out of butandiolformal under the conditions of an in vitro cytogenicity study.
Additionally, the fate of formaldehyde under cell culture conditions was investigated in order to estimate the true amount of formaldehyde formation. Both formaldehyde and butandiolformal resulted in clastogenic results in in vitro cytogenicity studies.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test substance No.: 97/352-1
Batch No.: BU17'
Chemical purity: 99.7 %
Appearance : liquid, colourless
Radiolabelling:
no

Administration / exposure

Doses / concentrations
Remarks:
Doses / Concentrations:
duplicates of 1.25, 2.5 and 5 mg/ml
Details on study design:
Formation of formaldehyde out of 1,4-Butandiolformal:
Incubates were prepared in Quadriperm dishes and MEM (Minimal Essential Medium incl. glutamin) was used as medium. Supplementation with 10% FCS in all incubations without S9-mix. All incubations were done in duplicates.
The concentrations of the test substance in the incubates were 1.25, 2.5 and 5 mg/ml.
Samples were taken at 0, 4, 8, and 18 hours after starting (- S9-mix) or 0, 1, 2, and 4 hours after starting (+ S9-mix).

Fate of formaldehyde under cell culture conditions:
see above, with two exceptions: no FCS-supplementation and the test substance used was formaldehyde (37% formaldehyde solution GR).
The initial concentrations of formaldehyde were 1.0, 10 and 20 µg/ml.
Samples were taken at 0, 1, 2 and 4 hours after starting.

Analytical determination of formaldehyde:
photometrically with the MBTH-test.

Results and discussion

Any other information on results incl. tables

Formation of formaldehyde out of 1,4-butandiolformal under cell culture conditions:

Table1: Results of the formaldehyde analyses in the incubations without S9-mix

 butandiolformal [mg/ml]       µg formaldehyde at time following incubation
   0h  4h  8h  18h
 1.25  0.06  0.13  0.11   not detectable
 2.5  0.09  0.07  not detectable   not detectable
 5  0.26  0.05   not detectable   not detectable

Irrespective of concentration and duration, no formaldehyde formation occurred.

Table2: Results of the formaldehyde analyses in the incubations with S9-mix

 butandiolformal [mg/ml]       µg formaldehyde at time following incubation
   0h  1h  2h  4h
 1.25  0.76  0.95  1   1.09
 2.5  1.06  1.34  1.53   1.89
 5  1.5  1.76   2.16   2.42

Formaldehyde formation in the incubates with metabolic activation.

Table3: Formaldehyde concentrations corrected by the concentrations at the beginning of the incubations.

 butandiolformal [mg/ml]       µg formaldehyde at time following incubation
   0h  1h  2h  4h
 1.25  -  0.19  0.24   0.33
 2.5  -  0.28  0.47   0.83
 5  -  0.26  0.66   0.92

Formaldehyde concentrations increased with increasing incubation period. However, formation rates tend to decrease with increasing incubation period witch may be due to an exhaust of the metabolizing system.

Doubling of the test substance concentration from 1.25 to 2.5 mg/ml leads to a doubling of the formation of formaldehyde. However, further doubling to 5 mg/ml did only result in a very small increase in formation of formaldehyde. This indicates saturation of the metabolizing system.

Fate of formaldehyde under cell culture conditions:

Table4: formaldehyde metabolization without metabolic activation.

 formaldehyde [µg/ml]       µg formaldehyde/ml at time following incubation (% of initial value)
   0h  1h  2h  4h
 1  0.85  0.56 (66%)  0.58 (68%)   0.9 (106)
 10  7.43  8.27 (111%)  8.35 (112%)   7.97 (107)
 20  14.71  15.81 (108%)  15.91 (108%)   15.76 (107)

Formaldehyde is stable in cell culture medium in the absence of S9-mix.

Table5: formaldehyde metabolization with metabolic activation.

 formaldehyde [µg/ml]       µg formaldehyde/ml at time following incubation (% of initial value)
   0h  1h  2h  4h
 1  0.94  0.94 (100%)  0.52 (55%)   0.27 (29)
 10  6.8  5.32 (78%)  4.94 (73%)   4 (59)
 20  11.77  10.8 (92%)  9.76 (83%)   8.27 (70)

In the presence of S9-mix the formaldehyde concentration decreased with time. The percental decrease was more pronounced the lower the initial formaldehyde concentration was. The decline of formaldehyde is most probably due to metabolic elimination by the S9-mix .

Applicant's summary and conclusion