Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Although the study by Watanuki and Sakaguchi (1981) is in an in vitro system, the cells used were Fischer rat embryo fibroblasts. The lack of effect on a cell line with embryonic origin provides support that benzaldehyde does not impact normal cellular function of that embryonic cell line.

Data source

Reference
Reference Type:
publication
Title:
Glucose Dependent Growth Inhibition of SV40 Transformed Rat Cells with Benzaldehyde
Author:
Watanuki M, Sakaguchi K
Year:
1981
Bibliographic source:
Agric Biol Chem 45 (1): 319-21

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Experiment 1. - Sugar-dependent Cell Growth Inhibition by Benzaldehyde:
Normal and SV40-transformed cells plated at 0.5 x 10^5 cells/35 mm-dish (Falcon) in 2 ml of Eagle's minimum essential medium containing 10% newborn calf serum and antibiotics (100 units penicillin, 100 microg streptomycin, and 60 microg kanamycin per ml of medium) were allowed to grow for I day at 37° in a CO2 incubator (95% air and 5% CO2), On day 2, the culture medium was replaced with 2ml of glucose free culture medium of which newborn calf serum was dialized against 4-fold volume of phosphate buffer saline 5 times, which contained 5.6mM glucose (A), 5.6mM mannose (B) or 5.6mM fructose (C). One half of the group was treated with benzaldehyde (50 microg/ml) and another half was for control. On day 4, determinations of viable cell number were performed in a haemacytometer in the presence of trypan blue.

Experiment 2. - Inhibition of Cell Growth by Benzaldehyde in Culture Media with Various Energy Sources:
Experimental procedures were almost the same as those described in Experiment 1 except that cells were counted on day 3. The cells were treated with benzaldehyde (50 microg/ml) in glucose- and glutamine-free culture medium supplemented with 5.6 mM glucose and 2 mM glutamine (A), 8 mM glutamine (B) and 50mM glycerol (C) as the energy source(s) for cultured cells.

Experiment 3. - Glucose Dependent Inhibition by Benzaldehyde of Glucose Incorporation into SV40-transformed Cells:
SV 40-transformed cells plated at I x 10^5 cells/35 mm dish were cultured for 2 days. After 2 washes ofmonolayer cells with Hanks' BSS, pH 7.4, the cells were preincubated in 2 ml of Hanks' BSS for 30 min at 37° in a CO2 incubator. Incubation was initiated by replacing the incubation medium with 1 ml of Hanks' BSS containing bezaldehyde (50 microg/ml) or H20 (for control) and [14C]D-glucose (1 microCi/ml) diluted with the desired concentrations of D-glucose. After 30 min, incubation was stopped by chilling the plate on ice, followed by washing the cells 3 times with 2 ml of cold PBS, pH 7.3. The cells were scraped with a rubber policeman into I ml of cold PBS. The scraped cells were transferred onto Whatman GF/C glass microfibre filter with another 2 ml of cold PBS. The filters were dried, and radioactivities were determined with 10 ml of a toluene scintillator solution.
GLP compliance:
not specified
Type of method:
in vitro

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
No data given

Results and discussion

Observed effects

Experiment 1. - Sugar-dependent Cell Growth Inhibition by Benzaldehyde:
When compared to SV-40 transformed cells, the growth of normal rat embryo fibroblasts was not inhibited by 50 microg/ml benzaldehyde in culture. This was true for all three sugar sources evaluated (glucose, mannose and fructose).

Experiment 2. - Inhibition of Cell Growth by Benzaldehyde in Culture Media with Various Energy Sources:
In this experiment, non-sugar energy sources were evaluated. Similarly, the growth of normal rat embryo fibroblasts was not inhibited by 50 microg/ml benzaldehyde in the presence of 8 mM glutamine or 50 mM glycerol.

Experiment 3. - Glucose Dependent Inhibition by Benzaldehyde of Glucose Incorporation into SV40-transformed Cells:
Where benzaldehyde did inhibit the uptake of glucose in the SV-40 transformed cells, no effect of benzaldehyde on the incorporation of glucose was observed for normal rat embryo fibroblasts.

Applicant's summary and conclusion

Conclusions:
Benzaldehyde did not exhibit effects on normal rat embryo fibroblasts, but did affect SV-40 transformed rat embryo fibroblasts.
Executive summary:

Although this is an in vitro study, it demonstrates that benzaldehyde does not affect normal rat embryo fibroblasts in culture. The relevance of this model to the intact animal is not clear, however, it does provide Weight of Evidence support that benzaldehyde may not have effects on developing embryonic tissues.