Acrylaldehyde

Toxicity to reproduction: other studies

Administrative data

Endpoint:

toxicity to reproduction: other studies

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: - Acceptable, well-documented publication report which meets basic scientific principles. However, test system used, is not relevant in relation to exposure of humans

Data source

Reference

Reference Type:

publication

Title:

Characterizing the Ovotoxicity of Cyclophosphamide Metabolites on Cultured Mouse Ovaries

Author:

Desmeules P and Devine PJ

Year:

2006

Bibliographic source:

Toxicol. Sci. 90 (2): 500-509

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Intact mouse ovaries (postnatal-day-4) were cuItued in vitro and exposed to multiple breakdown products of CPA on day 0 (d0). Tissues were cultured up to d8, and then foIlicle counts and immunohistochemistry were performed.

GLP compliance:

no

Type of method:

in vitro

Test material

Test material information

Constituent 1

Reference substance name:

Acrylaldehyde

EC Number:

203-453-4

EC Name:

Acrylaldehyde

Cas Number:

107-02-8

Molecular formula:

C3H4O

IUPAC Name:

acrylaldehyde

Details on test material:

Acrolein (no further data)

Administration / exposure

Details on exposure:

Culture of ovaries: Ovaries from PND4 CD-1 mice were placed in culture in 48-well culture plates on top of a small piece of membrane (Millicell·CM filter insert) floating on 250 µl of culture medium, one ovary per well (humidified, 37°C, 5% CO2 in air), culture medium was replaced every 2 days.

Preliminary tests: Preliminary in vitro toxicity studies were performed (from 0.01 to 300 µM) to determine the range of concentrations to be used for each chemical in these studies, based upon survival of exposed tissues. From these results, concentrations of 30 - 100 µM were used for acrolein.

Exposure: Additions of chemicals to medium were made on d0 of culture within 1h after establishment of cultures and were not replaced when culture medium was changed. Comparisons between control und treated ovaries in each set of experiments were necessary due to the inter litter variability in the numbers of follicles per ovary. Since no effect of single exposure to acrolein was observed after 8 days (not shown), acrolein was replaced with each exchange of culture medium. Chemicals were dissolved in culture medium.

Ovarian follicle counts and morphological analysis: Briefly, standard histological processing was performed on ovaries, and every 12th section (5 µm thick) was taken for follicle counts. Healthy/nonatretic follicles were classified according to specific criteria und were only included if follicles had a distinct oocyte nucleus. Primordial follicles contained a single layer of squamous granulosa cells, small (< 20 granulosa cells) or large ( > 20
granulosa ceIls) primary follicles had at least three cuboidal granulosa cells in a single Iayer, und secondary follicles had >/= 2 layers of granulosa cells. Follicles with eosinophilic (pyknotic) oocytes or granulosa cells were considered as degenerating or atretic and were counted separately from healthy follicles.

Immunohistochemistry and analysis of staining: Immunohistochemistry was performed on ovaries fixed in buffered formaldehyde (3.7%) for 2 h. Paraffine embedded tissues were sectioned at 5 µm and at every 12th section, two adjacent sections were used for cleaved caspase-3 staining and terminal UTP nick end labelling (TUNEL), respectively. Microwave antigen retrieval was done, boiling sections for 13 min sodium citrate (1 M, pH 6.1). For active caspase-3 staining, sections were blocked with 5% BSA for 5 min, then incubated with a polyclonal rabbit antibody against cleaved caspase-3 (1:25 dilution in PBS, 1% BSA, and 0.5% Tween 20, pH 7.2) overnight at 4°C (all other steps were performed at room temperature). Sections were blocked
15 min each with streptavidin and then biotin (Vector blocking kit), followed by incubations with a goat anti-rabbit biotin-conjugated secondary antibody (1:75, 1 h) then alexa-fluor-568 conjugated to streptavidin (1:100, 1 h), Nuclei were stained with Hoechst (0.01 mg/ml) and mounted in Vectashield. TUNEL was used to locatize fragmented DNA in situ. TUNEL staining was perforemed according to kit instructions with the exception that antibody incubation was lengthened to 40 min. Nuclei were stained as above. Sections were inspected using a Leica DMRE fluorescent microscope (Deerfield, IL). Images were digitized and merged using ImagePro Plus (version 4.0, Media Cybemetics. Silver Spring. MD), After assembling multiple images per section in Adobe Photoshop Elements (version 2.0) into images of whole sections, follicles containing at least one positively stained cell (oocyte/granulosa cell) were counted manually.

Statistics: For each set of experiments involving concentration-specific follicle counts or immunohistochemistry, data were analyzed by one-way
ANOVA and where appropriate, by the Turkey HSD post-hoc test with JMP® (Version 5.1. SAS Institute Ine, Cary, NC). For time-course experiments,
follicle counts were analyzed by Student's t-test for each concentration and time point separately, because experiments of each concentration were
performed independently. Significance was assigned at p < 0,05 in each case. Follicle counts were performed by two observers blinded to sampIe identity.

Analytical verification of doses or concentrations:

not specified

Results and discussion

Observed effects

Acrolein had no effect on follicle numbers at the concentrations tested (1-100 µM) following single exposures. Under continuous exposures to 30-100 µM acrolein, there were significant decreases in the numbers of primordial follicles, but there was also nonspecifie toxicity in the cortical region of exposed tissues at >/= 60 µM, seen as pyknotic cells.

Applicant's summary and conclusion

Executive summary:

Intact mouse ovaries (postnatal-day-4) were cultured in vitro and exposed to acrolein among other breakdown products of cyclophosphamide on day 0 (d0). Tissues were cultured up to d8, and then follicle counts and immunohistochemistry were performed. Acrolein had no effect on follicle numbers at the concentrations tested (1-100 µM) following single exposures. Under continuous exposures to 30-100 µM acrolein, there were significant decreases in the numbers of primordial follicles, but there was also nonspecifie toxicity in the cortical region of exposed tissues at >/= 60 µM, seen as pyknotic cells.

The reported decrease in the number of primordial follicles occurred together with nonspecific toxicity in the cortical region. Therefore, it is not possible to distinguish ovary specific effects from nonspecific toxicity and this study gives no indication on ovary specific effects of acrolein.

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