Formaldehyde

Toxicity to reproduction: other studies

Administrative data

Endpoint:

toxicity to reproduction: other studies

Type of information:

experimental study

Adequacy of study:

other information

Study period:

not specified

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: very high concentrations leading to stress-related secondary effects

Data source

Reference

Reference Type:

publication

Title:

Effect of formaldehyde inhalation on Hsp70 in seminiferous tubules of rat testes: an immunohistochemical study

Author:

Özen OA, Akpolat N, Songur A, Kuş I, Zararsiz I, Ozaçmak VH, Sarsilmaz M

Year:

2005

Bibliographic source:

Toxicol Ind Health. 2005 Nov;21(10):249-54

Materials and methods

Test guideline

Qualifier:

no guideline required

Principles of method if other than guideline:

Immunohistochemical investigation of the effect of formaldehyde inhalation on changes in Hsp70 content in testicular tissue following subchronic exposure at cytotoxic concentrations.

GLP compliance:

not specified

Type of method:

in vivo

Test material

Test material information

Constituent 1

Reference substance name:

Formaldehyde

EC Number:

200-001-8

EC Name:

Formaldehyde

Cas Number:

50-00-0

Molecular formula:

CH2O

IUPAC Name:

formaldehyde

Details on test material:

- Name of test material (as cited in study report): formaldehyde

Specific details on test material used for the study:

- Formaldehyde gas was generated from paraformaldehyde (Merck KgaA, 64271 Darmstadt, Germany) by thermal depolymerization

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 215 - 225 g
- Housing:
- Diet: Kavimix VM 23-Z open formula diet, Elazig Yem Fabrikasi, Elazig, Turkey, ad libitum except while exposure
- Water: tap water, ad libitum except while exposure
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 30
- Humidity (%): 47 - 55
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

inhalation: gas

Type of inhalation exposure (if applicable):

not specified

Vehicle:

not specified

Details on exposure:

- Rats were exposed to cytotoxic doses of formaldehyde in glass chambers, where the temperature was maintained at 20 - 30°C, humidity at 47 - 55 % and airflow was adjusted to 10 L/min

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Formaldehyde level of the test atmosphere was monitored continually by a monitor (Environmental Sensors Co., Boca Raton, FL, USA) maintained within the exposure chamber
- The monitor was calibrated with a formaldehyde permeation tube (Vici Metronics, Santa Clara, CA, USA), at a permeation rate of about 60 ng/min per centimeter
- The actual permeation rates were confirmed periodically by using the NIOSH 3500 method

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

8 hours/day, 5 days/week

Duration of test:

not specified

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 ppm

Doses / concentrations 2

Dose / conc.:

10 ppm

No. of animals per sex per dose:

6

Control animals:

yes

Details on study design:

- At the end of the experiments, the animals were decapitated under deep ether anesthesia and testicular tissue and blood samples were obtained
- Blood was collected and serum was separated and stored at -20°C for analysis
- Serum testosterone levels were measured by Chemiluminescent Enzyme Immunoassay (Immulite Testosterone, Immulite Model 2000, DPC, Los Angeles, CA, USA)
- Testicular tissues from each group were fixed in Bouin solution, dehydrated and then embedded in paraffin blocks
- Sections of 5 mm thickness were obtained using a microtome, samples were stained with Hematoxylin-Eozine
- Preparations were then studied using a search microscopy (Olympus BH-50)
- In order to see any effect on seminiferous tubules, diameters of 100 tubules from each group were randomly chosen and measured with an ocular micrometer adjusted to the microscopy
- For each tubule, the diameter was measured four times; then a mean value was calculated
- Immunohistochemical Hsp70 staining of the seminiferous tubules was evaluated semi-quantitatively
- Cytoplasmic intensity of immunostaining of spermatocytes and spermatides in the adluminal compartment of the seminiferous epithelium were primary criterion for semi-quantitative evaluation. Immunostaining was graded on a relative scale of no staining (0), minimal (1+), low (2+), moderate (3+), strong (4+) and heavy (5+)

Statistics:

not specified

Results and discussion

Effect levels

Dose descriptor:

LOAEL

Effect level:

5 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

food consumption and compound intake

histopathology: non-neoplastic

water consumption and compound intake

Observed effects

CLINICAL FINDINGS
The fur of rats exposed to 5 ppm of FA turned yellow on the fifteenth day of exposure, and the fur of rats exposed to 10 ppm of FA turned yellow on the tenth day of exposure. In addition, unsteady breathing, an increase in nose cleaning, excessive licking, frequent sneezes and hemorrhage in nasal mucosa were observed because of FA irritation. In addition, food and water consumption was relatively decreased in these groups. There was no mortality in the groups.

SERUM TESTOSTERON LEVELS
A significant decrease (P < 0.0001) was observed in the serum testosterone levels in both test groups in comparison with the control. On the other hand, it was very striking that the difference between the serum testosterone values of both test groups was significant (P < 0.0001).

HISTOLOGICAL FINDINGS
Diameters of the seminiferous tubules in both test groups were decreased compared to those of the control group (P < 0.001). In addition, diameters in the high dose group were lower compared to the low dose group, but the difference was not statistically significant.

IMMUNHISTOCHEMICAL FINDINGS
The evaluation of immunohistochemical staining was carried out based on the density of staining. Thus, the more immunoreaction of Hsp70 in tissue or in the cell, the more binding will occur, resulting in darker staining. Hsp70 immunolocalization in both cytoplasms of spermatocytes and spermatides in the adluminal compartment of the seminiferous epithelium were evaluated separately. The staining was observed in stages I/VI and stages XII/XIV. It was highest in stages XII/XIV. Therefore, Hsp70 staining was determined and graded in stages XII/XIV. No staining was observed in the Sertoli cells. When studying spermatogenetic cells of the control group, we did not observe Hsp70 staining in mitotic spermatogonias (0), which are lined on the basal lamina as relatively small and spheric cells, with nuclei stained pale with hematoxylene. On the other hand, an immunoreaction of Hsp70 that was considered as (2+) with low density was detected, especially in the cytoplasms of spermatocytes from the control group. In addition, Hsp70 immunolocalization with low density (2+) was observed in the spermatides in the adluminal compartment of the seminiferous epithelium. Besides staining of the spermatogonias of animals exposed to formaldehyde, an increase in staining was observed in particular in the cytoplasms of spermatocytes and spermatides. Once the density of stainings among groups subjected to formaldehyde exposure were compared, the low dose group showed minimal density of staining in spermatogonias (1+), while having strong staining in the cytoplasms of spermatocytes and spermatides in the adluminal compartment of the seminiferous epithelium (4+). In the high dose group, spermatogonias were stained with low density (2+) and the cytoplasms of spermatocytes and spermatides had a significantly heavier density of Hsp70 immunoreaction (5+). Comparing the densities of staining in the experimental groups to those in the control group, the staining increased proportionally to the concentrations of formaldehyde.

Applicant's summary and conclusion

Conclusions:

Formaldehyde gas may damage spermatogenetic cells and increase Hsp70 synthesis.

Executive summary:

In a non-reliable study (very high concentrations leading to stress-related secondary effects) the effect of formaldehyde inhalation on changes in Hsp70 content in testicular tissue following subchronic exposure at cytotoxic concentrations was investigated immunihistochemically. One parameter which might provide an insight into the underlying mechanism of the effect of formaldehyde inhalation on testicular tissue, is the assessment of heat shock protein 70 (Hsp70), which increases promptly in cells exposed to stress caused by chemical toxicity. Thus, following subchronic exposure at cytotoxic concentrations, the immunohistochemical effect of formaldehyde inhalation on changes in Hsp70 content in testicular tissue was investigated. Eightteen albino Wistar rats were divided into three groups, exposed to 0 (control), 5 and 10 ppm formaldehyde gas for a total of 91 days, 8h/day, five days a week. Serum testosterone levels were determined using a chemiluminescent enzyme immunoassay. Testicular tissues were stained with Hematoxylin-Eosine and Hsp70 immunohistochemically performed.

Diameters of seminiferous tubules and serum testosterone levels in animals inhaling FA were significantly decreased. In seminiferous epithelium stained for Hsp70, compared to those in the control group, the spermatogenetic cells in the experimental groups demonstrated an obvious increase in immunoreaction spermatides in the adluminal region and especially in the cytoplasm of spermatocytes. Immunoreaction of Hsp70 was detected in the spermatogonias of animals exposed to formaldehyde inhalation as opposed to those of the control group. Compared to the control, there was a significant increase in the immunoreactions observed not only in the cytoplasm of primary spermatocytes, but also spermatides in the adluminal region of the seminiferous tubules.

In conclusion, formaldehyde gas may damage spermatogenetic cells and increase Hsp70 synthesis.