Registration Dossier

Administrative data

Endpoint:
immunotoxicity: short-term oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Glycidol modulation of the immune responses in female B6C3F1 mice
Author:
Guo, T.L. et al.
Year:
2000
Bibliographic source:
Drug and chemical toxicology, 23(3), 433-457

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
The immunotoxic potential of glycidol was investigated in female B6C3F1 mice using a battery of functional assays and 3 host resistance models.
Immunological studies:
- Flow Cytometric Analysis of T Cells, T Subsets and B Cells
- Spleen IgM Antibody Response to the T-Dependent Antigen, sRBC
- Spleen Cell Proliferative Responses to Con A, LPS, Anti-IgM F(ab')2 and/or IL-4
- Mixed Leukocyte Reaction (MLR) of Splenocytes to DBA/2 Spleen Cells
- Cytotoxic T Lymphocyte (CTL) Activity
- Resident Macrophage Activity
- Natural Killer Cell Activity
Host resistance studies:
- Host Resistance to the B16F10 Melanoma Tumor Model
- Host Resistance to Listeria monocytogenes
- Host Resistance to Streptococcus pneumoniae
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): glycidol
- Appearance: colorless, viscous liquid
- Source: Sigma Chemical Company, St. Louis, MO, USA

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, MI, USA)
- Age at study initiation: 8-10 weeks
- Weight on arrival: 16-20 g
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Glycidol was administered by gastric intubation using an 18 gauge gavage tube (Fisher Scientific, International, Hampton, NH). Glycidol solutions (2.5, 12.5 and 25.0 mg/ml) were prepared in sterile distilled water. Mice received a volume of 0.1 ml glycidol or sterile water per 10 g of body weight.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
14 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 2.5, 12.5 and 25 mg/mL
Basis:
nominal in water
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle

Examinations

Observations and clinical examinations performed and frequency:
Mice were observed at the time of dosing and throughout the experimental period for pharmacotoxicological signs.
Animals were weighed on the first day of exposure and on days 8 and 15 of the study.
At sacrifice blood was collected by retro-orbital bleed into EDTA tubes. A blood smear was prepared at the time of blood collection and, after dehydration with methanol, stained with Wright-Giemsa (Fisher Scientific, Pittsburgh, PA). Hematological parameters assessed: the number of reticulocytes, erythrocyte and leukocytes; hemoglobin, hematocrit; mean corpuscular volume (MCV); mean cell hemoglobin (MCH); and mean cell hemoglobin concentration (MCHC).
Sacrifice and pathology:
On day 15, mice were weighed and sacrificed by CO2 inhalation or cervical dislocation. Animals were also examined for gross pathology. The following organs were removed, cleaned of connective tissue and weighed: thymus, lungs, liver, spleen and kidneys with adrenals.
Other functional activity assays:
IMMUNOLOGICAL STUDIES:

1) Flow Cytometric Analysis of T Cells, T Subsets and B Cells
- Method: according to Cao et al. (Cao, W., Sikorski, E. E., Fuchs, B. A., Stern, M. L., Luster, M. I. and Munson, A. E., The B Lymphocyte is the Immune Cell Target for 2',3'-dideoxyadenosine. Toxicol Appl Pharmacol, 105: 492-502, 1990.)

2) Spleen IgM Antibody Response to the T-Dependent Antigen, sRBC:
- Method: assay of Jerne and Nordin (Jerne, N. K. and Nordin, A. A., Plaque Formation in Agar by Single Antibody-Producing Cells. Science, 140: 405-406, 1963.)

3) Spleen Cell Proliferative Responses to Con A, LPS, Anti-IgM F(ab')2 and/or IL-4:
- Method: according to Holsapple et al. (Holsapple, M. P., Munson, A. E., Munson, J. A. and Bick, P. H., Suppression of Cell-mediated Immunocompetence After Subchronic Exposure to Diethylstilbestrol in Female B6C3F1 Mice. J Pharmacol Exp Ther, 227: 130-138, 1983.)

4) Mixed Leukocyte Reaction (MLR) of Splenocytes to DBA/2 Spleen Cells:
- Method: according to Holsapple et al. (see above)

5) Cytotoxic T Lymphocyte (CTL) Activity:
- Method: according to Germolec et al. (Germolec, D. R., Maronpot, R. R., Ackermann, M. F., Vore, S. J., Dittrich, K., Rosenthal, G. J. and Luster, M. I., Lack of a Relationship Between Immune Function and Chemically Induced Hepatocarcinogenesis in B6C3F1 Mice. Cancer Immunol Immunother, 27: 121-127, 1988.)

6) Resident Macrophage Activity:
- Method: according to Geissler et al. (Geissler, K., Tricot, G., Leemhuis, T., Walker, E. and Broxmeyer, H. E., Differentiation-inducing Effect of Recombinant Human Tumor Necrosis Factor Alpha and Gamma-interferon In Vitro on Blast Cells From Patients With Acute Myeloid Leukemia and Myeloid Blast Crisis of Chronic Myeloid Leukemia. Cancer Res, 49: 3057-3062, 1989.)

7) Natural Killer Cell Activity
-Method: according to Reynolds and Herberman (Reynolds, C. W. and Herberman, R. B., In Vitro Augmentation of Rat Natural Killer (NK) Cell Activity. J Immunol, 126: 1581-1585, 1981.)
Other examinations:
HOST RESISTANCE STUDIES:

1) Host Resistance to the B16F10 Melanoma Tumor Model:
- Method: method by Murray at al. (Murray, M. J., Kerkvliet, N. I., Ward, E. C. and Dean, J. H., Models for the Evaluation of Tumor Resistance Following Chemical or Drug Exposure. In Immunotoxicology and Immunopharmacology (J. H. Dean, M. I. Luster, A. E. Munson, and H. Amos, Eds.), pp. 113-122. Raven Press, New York, 1985.)

2) Host Resistance to Listeria monocytogenes:
- Method: A stock of Listeria monocytogenes (Strain MCV 19303) of approximately 108 colony-forming units (CFU) per milliliter was maintained as
described by Morahan et al., 1981. Twenty-four hours after the last exposure to glycidol, mice were challenged with Listeria monocytogenes by the intravenous route. Three challenge levels were used in the study. To determine the viable count of the Listeria monocytogenes inoculum, a colony count on tryptose (+ 1% glucose) agar plates was conducted for the challenge suspensions. The challenge levels were 1104, 2104 and 4104 CFU/mouse. For the positive control group, CPS (200 mg/kg) was administered (i.p.) only on day 14. Morbidity was recorded daily for 14 days.

3) Host Resistance to Streptococcus pneumoniae:
- Method: Stock preparations of Streptococcus pneumoniae (ATCC 6314) were maintained at -70°C in defibrinated rabbit blood. A 50-l aliquot of the stock preparation was used to inoculate 50 ml of BHI broth, and incubated overnight at 37°C. The turbidity of the overnight culture was determined using the Abbott Bichromatic analyzer and diluted with fresh BHI broth to yield an absorbency difference (Ad) of 0.020-0.025. The turbidity of the subculture was monitored periodically and, when the optical density reached Ad of 0.080, the subculture was rapidly cooled in an ice bath and diluted to the desired inoculum level. A sample of the subculture was serially diluted and plated on blood agar plates to determine the number of colony-forming units administered to the animals. Mice were challenged with the Streptococcus pneumoniae by the intraperitoneal route using a modification of the method of White et al, 1986. Three challenge levels were employed: 1.38xE10+7, 2.76xE10+7 and 5.52xE10+7 CFU/mouse. Morbidity was recorded twice daily for 7 days. For the positive control group, cobra venom factor was administered intravenously one day prior to challenge.
Positive control:
The positive control animals received 25mg of cyclophosphamide (CPS, Sigma) per kg of body weight on days 11 through 14 by intraperitoneal (i.p.) injection.
Statistics:
The Bartlett's Test for homogeneity was used to select the type of analysis to be conducted. Homogeneous data were analyzed using a one-
way analysis of variance, and the Dunnett's T test was used to determine differences between the experimental and the control group. For non-
homogeneous data, a non-parametric analysis of variance was used. The differences between the control group and the experimental groups were
determined using the Wilcoxon Rank Test. The Student's t Test was used to compare the vehicle and the positive control group. Data for host resistance assays to S. pneumoniae were analyzed by Fisher Exact 2 test. P values of 0.05 or less were considered statistically significant.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A decrease (5% at the highest dose) in hematocrit in glycidol-exposed animals was observed.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The weights of the brain, spleen, lungs and thymus were not altered following glycidol exposure. However, a dose-related increase in liver weight (9% at the highest dose) and kidney weight (13% at the highest dose) was observed.
Details on results:
IMMUNOLOGICAL STUDIES

1) Flow Cytometric Analysis of T Cells, T Subsets and B Cells
The total number of splenocytes was 18.7x10E+7 in the vehicle control animals, which was decreased after exposure to glycidol and reached the level of statistical significance in the high dose group. The percentage of B cells, T cells, CD4+ T cells, CD8+ T cells and CD4+CD8+ T cells in vehicle control animals were 51.4%, 53.3%, 26.8%, 16.3%, and 2.0%, respectively.
A decrease was observed in the number and percentage of B cells in the glycidol-exposed animals with the maximal decrease (23% and 4.2%, respectively) observed in the high dose group. A decrease was also observed in the absolute number of CD4+ T cells in the glycidol-exposed animals, which reached the level of statistical significance in the high dose group where a 9% decrease was observed. A slight increase (4.6%) in the percentage of total T cells was observed in the animals exposed to the high dose of glycidol.

2) Spleen IgM Antibody Response to the T-Dependent Antigen, sRBC:
The spleen IgM AFC response was measured at day 4 after antigen administration, which is the peak day for the IgM response. When the data were presented as AFC/106 spleen cells, there were 1927 +/- 133 antibody-forming cells in the vehicle control animals, which was decreased after exposure to glycidol and reached the level of significant difference at the high dose level. The percentage of reduction was 31%. When the data were expressed as AFCx10E+3/spleen , there were 449 +/- 38x10E+3 antibody-forming cells in the vehicle control group, which was decreased to 321 +/- 34x10E+3 and 263 +/- 29x10E+3 antibody-forming cells in the middle and high dose groups. The percent decrease was 29% and 41%, respectively. The positive control, cyclophosphamide, produced a 99% decrease in AFC/106 spleen cells (specific activity) and a 99% decrease in AFC/spleen (total spleen activity), all of which were significantly different from the vehicle control values.

3) Spleen Cell Proliferative Responses to Con A, LPS, Anti-IgM F(ab')2 and/or IL-4:
Exposure to glycidol at the dose levels of 125 and 250 mg/kg decreased the T cell proliferative response at the ConA concentration of 10 mg/ml. The positive control, cyclophosphamide, significantly decreased the proliferative responses at all the tested concentrations of ConA.
IL-4-induced B cell proliferative response in the vehicle control was 7301 cpm/2105 cells, which was decreased after exposure to glycidol and reached the level of significance in the middle dose level (13% decrease). The F(ab´)2-induced B cell proliferative response in the vehicle control was 15444 cpm/2105 cells, which was decreased after exposure to glycidol and reached the level of statistical significance in the middle and high dose levels. The percent reduction was 30% and 32%, respectively. There was a synergism between IL-4 and F(ab´)2 in inducing B cell proliferation. Exposure to glycidol decreased the response of B cells to IL-4 plus F(ab´)2 and reached the level of statistical significance at the middle dose level (16% decrease).

4) Mixed Leukocyte Reaction (MLR) of Splenocytes to DBA/2 Spleen Cells:
Exposure to glycidol had no effect on the proliferative response of the responder cells alone. While a 25% decrease was observed in the culture
of responder plus stimulator cells at the middle dose of glycidol, no significant difference was observed at the low and high dose levels.

5) Cytotoxic T Lymphocyte (CTL) Activity:
The CTL-specific lysis of 51Cr-labeled P815 cells at the effector:target ratio of 0.75:1, 1.5:1, 3:1, 6:1, 12.5:1 and 25:1 was 8.8%, 12.8%, 20.5%, 31.2%, 50.8% and 53.8%, respectively. Although significant dierences were observed in the low and middle dose levels of glycidol at a few effector:target ratios, no effect was observed at the high dose level for any ratio. CPS, the positive control, consistently produced a decrease of CTL activity.

6) Resident Macrophage Activity:
Although a slight increase of the cytotoxicity was observed at the dose of 25 mg/kg, no significant changes were observed at the middle and high dose levels of glycidol no matter whether the stimulators were present or not. Exposure to glycidol did not affect the number of peritoneal exudate cells obtained from the animals.

7) Natural Killer Cell Activity
At the effector:target ratio of 100:1, the percentage of cytotoxicity was 6.2% in the vehicle control group. This was decreased to 4.3% and 4.4% in animals exposed to glycidol at the dose levels of 125 mg/kg and 250 mg/kg, respectively, which was 31% and 29% reduction when compared to the vehicle control. At the effector:target ratio of 50:1, the percentage of cytotoxicity was 4.4% in the vehicle control group, which was decreased to 2.8% in animals exposed to glycidol at the dose of 250 mg/kg. No suppressive effect was observed for glycidol at the effector:target ratio of
25:1.


HOST RESISTANCE STUDIES:

1) Host Resistance to the B16F10 Melanoma Tumor Model:
Exposure to glycidol resulted in a dose-related increase in the pulmonary tumor formation. Thus, the animals receiving glycidol at the dose of 125 mg/kg had 44 nodules/lung, which is a 96% increase compared to the control. Exposure to the high dose of glycidol (250 mg/kg) resulted in an increase of tumor formation with 51 nodules/lung, which is a 129% increase compared to the control.

2) Host Resistance to Listeria monocytogenes:
No significant differences were observed in morbidity of the glycidol-exposed animals compared to the vehicle controls.

3) Host Resistance to Streptococcus pneumoniae:
No significant alterations in the susceptibility to Streptococcus pneumoniae in glycidol-exposed animals were observed at the two low
challenge levels (A and B). However, at the challenge level C, a slightly increased host resistance to Streptococcus pneumoniae was observed in
the 250-mg/kg glycidol-exposed animals.

Applicant's summary and conclusion

Conclusions:
Overall, it can be concluded that glycidol is an immunosuppressive agent in female B6C3F1 mice.