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Abstract for MG96004

Assessment of the Immunotoxicity of Methoxychlor (Endocrine Disruptor) in Male and Female Sprague-Dawley Rats

CASRN: 72-43-5
Chemical Formula: C16H15Cl3O2
Molecular Weight: 345.651

Abstract

The following abstract presents results of a study conducted by a contract laboratory for the National Toxicology Program. The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by NTP in March 2009. The findings and conclusions for this study should not be construed to represent the views of the NTP or the U.S. Government.

Methoxychlor is a chlorinated hydrocarbon pesticide with proestrogenic activity. Chemicals with estrogenic activity that are present in the environment may be considered potentially hazardous to development and/or reproduction. Release of methoxychlor to the environment occurs due to its use as an insecticide for home and garden applications, livestock and poultry, field produce of alfalfa, soy beans, deciduous fruits, nuts and vegetables. The most probable route of exposure to methoxychlor would be inhalation or dermal contact during application of this insecticide, inhalation of airborne particulate matter or ingestion of contaminated food or water.

Methoxychlor is one of five endocrine disruptor compounds nominated by the National Toxicology Program (NTP) for reproductive assessment studies, neurotoxicological and immunotoxicological studies, and cancer bioassay studies. The title of this project is "Effects of Endocrine Disrupting Chemicals on Fertility and Reproductive Tract Cancers." These studies were carried out as interagency agreement between National Environmental Health Science (NIEHS) and National Center for Toxicology Research (NCTR) with the immunotoxicology component being the only portion conducted outside of the NCTR facility in Jefferson, Arkansas. Thus, the purpose of these reported studies was to determine the potential effects of methoxychlor on the immune system. The methoxychlor/immunotoxicology code identification from NCTR is E2123.14.

The exposure times and the doses, 0, 10, 100 and 1000 ppm, were determined by NCTR. Methoxychlor was procured and administered in feed by personnel at NCTR in Jefferson, Arkansas. The control rats received casein NIH-31C as the feed. The studies were conducted in male and female Sprague Dawley rats. The F0 generation female rats received the test article for 65-72 days beginning 7 days into gestation. The F1 generation male and female rats received the test article in utero for 14 days and continued postpartum until 79-82 days. On the day of sacrifice, whole spleens or bone marrow cells were placed in tubes with the appropriate medium, packed in wet ice, and shipped to Medical College of Virginia Campus/Virginia Commonwealth University (MCV), Richmond, Virginia, for assay evaluation on the following day. The assay days of the study were conducted between September 23, 1997 and October 22, 1997. Data included in the text refer to data generated in the assays conducted at MCV, unless otherwise specified. Data from the pathology studies conducted at NCTR with 6 doses ranging from 0.1 to 1000 ppm are included in the data appendix.

The baseline toxicology studies are summarized in Table ES-1. Exposure to methoxychlor resulted in a statistically significant decrease in terminal mean body weight both in the F1 generation male and female rats as seen in the NCTR pathology study (n= 15). Body weight data are included from the F0 generation female rats. A decrease of 13% and 14% at 1.1 and 1000 ppm, respectively, was observed in the F1 generation male rats. A decrease of 12% at the high dose was observed in the F1 generation female rats as compared to the vehicle control.

Exposure to methoxychlor resulted in a statistically significant decrease in the liver weight and relative liver weight (% body weight) in the NCTR study with the F1 generation male rats. The decrease in absolute liver weight at the high dose, 1000 ppm, was 15% and 18% at the 10 ppm dose as compared to the vehicle control. A statistically significant decrease in spleen weight was observed at 1.1 and 1000 ppm of 21% and 10%, respectively. The thymus weight was also significantly decreased at the 1.1, 100 and 1000 ppm. No alterations in organ weight were observed in the F1 generation female rats. Organ weights were not obtained on the immunotoxicological studies assigned to MCV nor were organ weights received from NCTR for the F0 generation female rats.

The F1 generation male and female rats were evaluated for methoxychlor exposure effects on bone marrow cell number, colony-forming units and DNA synthesis. Although significant alterations were seen in the F1 generation male rat study, emphasis was placed on specific activity with the bone marrow data. A statistically significant increase was observed with the CFU-GM/1x105 cells at the 100 ppm and 1000 ppm closes of 18% and 22%. In F1 generation female rat study, a significant decrease was observed in CFU-M/1x105 at the 1000 ppm dose of 23%.

Table ES-2 summarizes the immunology studies. In the F0 generation female rats, a decrease in the number of splenic B cells by 24% at 1000 ppm dose was the only alteration seen in the splenic surface markers. Exposure to methoxychlor in the F1 generation male rats resulted in a statistically significant decrease in splenic B cells by 30%, T suppressor cells by 29%, and NK cells by 31% at the 1000 ppm dose as compared to the vehicle control. In the F1 generation female study, there were no significant differences seen in surface marker numbers.

In the spleen antibody-forming cell response to T-dependent antigen, sheep erythrocytes, the only significant alteration was observed in the F1 generation male rats with an increased response in IgM AFC/106 spleen cells (99%) and IgM AFC/spleen x 103 (103%) at the high dose. In spleen cells from the F0 generation female rats, a basal proliferative stimulation was seen of 48% at the 1000 ppm dose. A statistically significant dose-dependent increase was observed in the stimulation with the anti-CD3 receptor-mediated proliferation at 100 and 1000 ppm closes of 55% and 149%, respectively. Also seen in this study was a significant decrease in spleen cell number at 100 and 1000 ppm ranging up to 20%.

Exposure to methoxychlor in the F1 generation male rats resulted in a statistically significant dose-dependent increase in basal proliferative stimulation at all closes ranging from 26% to 52%. A significant increase was observed in the stimulation with the anti-CD3 receptor-mediated proliferation at all doses by 23%, 32%, and 43%. Again, a significant decrease in spleen cell number at 1000 ppm (27%) was observed.

Exposure to methoxychlor in the F1 generation female rats resulted in a statistically significant increase in the stimulation with the anti-CD3 receptor-mediated proliferation at the 100 and 1000 ppm doses of 47% and 68%, respectively.

In the F0 generation female rats, statistically significant increases in natural killer cell activity were observed with the effector-to-target ratios of 100:1, 50:1 and 25:1 at 1000 ppm. The observed increases ranged from 43% to 64%.

Exposure to methoxychlor in the F1 generation male rats resulted in statistically significant increases of effector-to-target ratios at 12.5:1, 50:1, and 100:1 at the high dose. Also, lytic units/107 spleen cells were significantly increased at the 100 and 1000 ppm doses by 57% and 105%. No biologically relevant alterations were seen in the F1 generation female rats.

In conclusion, the results from this immunotoxicological evaluation demonstrate that, under the experimental conditions used, exposure to methoxychlor at doses of 10, 100 and 1000 ppm did impact on the immunoalteration of the F0 generation female rats with an increased natural killer cell activity and an increased T cell function for the F1 generation males and females. Also, a decrease in B cell number was seen in the F0 generation female and F1 generation male rats.

Studies

Table ES-1
Summary Table for Toxicology Studies
E2123.14 (Methoxychlor)
Parameter Result Maximum Effect Dose Comment
Body Weights/Male Decrease 14% 1000 ppm
Organ Weights/Male
- Liver
- Spleen
- Thymus
Decrease
Decrease
Decrease
19%
21%
22%
10 ppm
1.1 ppm
1.1 ppm
& Body Wgt
Body Weights/Female Decrease 12% 1000 ppm
Organ Weights/Female
- Liver
- Spleen
- Thymus
No Effect
No Effect
No Effect
Bone Marrow/Male
- Cells/Femur (x106)
- CFU-GM/1x105 cells
- CFU-GM/Femur (x104)
- CFU-M/1x105 cells
- CFU-M/Femur (x104)
- CFU-E/2x105 cells
- CFU-E/Femur (x104)
- DNA Synthesis
Decrease
Increase
Decrease
No Effect
Decrease
No Effect
No Effect
No Effect
42%
22%
43%

43%
1000 ppm
1000 ppm
10 ppm

10 ppm
Bone Marrow/Female
- Cells/Femur (x106)
- CFU-GM/1x105 cells
- CFU-GM/Femur (x104)
- CFU-M/1x105 cells
- CFU-M/Femur (x104)
- DNA Synthesis
No Effect
No Effect
No Effect
Decrease
No Effect
No Effect



23%



1000 ppm



Table ES-2
Summary Table for Immunology Studies
E2123.14 (Methoxychlor)
Parameter Results Maximum Effect Dose Comment
Surface Markers (Absolute Values)
OX-33+
(B cell, CD45+)
Decrease
Decrease
No Effect
24% (F0 F)
30% (F1 M)
F1 F
1000 ppm
1000 ppm
OX-19+
(Pan T cell, CD5+)
No Effect
No Effect
No Effect
F0 F
F1 M
F1 F
OX-38+OX-19+
(C4+, CD5+)
No Effect
No Effect
No Effect
F0 F
F1 M
F1 F
OX-8+OX-19+
(CD8+, CD5+)
No Effect
Decrease
No Effect
F0 F
29% (F1 M)
F1 F

1000 ppm
NKR-P1A+CD-8+
(NK cell)
No Effect
Decrease
Increase
F0 F
31% (F1 M)
44% (F1 F)

1000 ppm
100 ppm
HIS36+
(macrophages)
No Effect
No Effect
No Effect
F0 F
F1 M
F1 F
IgM Humoral Immune Response to Sheep Erythrocytes
IgM AFC/106 Cells No Effect
Increase
No Effect
F0 F
99% (F1 M)
F1 F

1000 ppm
IgM AFC/Spleen No Effect
Increase
No Effect
F0 F
103% (F1 M)
F1 F

1000 ppm
Mixed Leukocyte Response
MLR Responders +
Stimulators
No Effect
Increase
No Effect
F0 F
67% (F1 M)
F1 F

1000 ppm
Anti-CD3 Stimulation
Unstimulated Increase
Increase
No Effect
48% (F0 F)
52% (F1 M)
F1 F
1000 ppm
1000 ppm
Stimulated Increase
Increase
Increase
149% (F0 F)
43% (F1 M)
68% (F1 F)
1000 ppm
1000 ppm
1000 ppm
Spleen Cells x 107 Decrease
Decrease
No Effect
20% (F0 F)
27% (F1 M)
F1 F
1000 ppm
1000 ppm
NK Cell Activity
400:1 No Effect
Not Done
Not Done
F0 F
F1 M
F1 F
200:1 No Effect
No Effect
No Effect
F0 F
F1 M
F1 F
100:1 Increase
Increase
No Effect
47% (F0 F)
46% (F1 M)
F1 F
1000 ppm
1000 ppm
50:1 Increase
Increase
No Effect
43% (F0 F)
67% (F1 M)
F1 F
1000 ppm
1000 ppm
25:1 Increase
No Effect
No Effect
64% (F0 F)
F1 M
F1 F
1000 ppm
12.5:1 No Effect
Increase
Increase
F0 F
56% (F1 M)
158% (F1 F)

1000 ppm
1000 ppm
6.25:1 Not Done
Decrease
Decrease
F0 F
61% (F1 M)
20% (F1 F)

10 ppm
1000 ppm
LU 107 Cells No Effect
Increase
No Effect
F0 F
105% (F1 M)
F1 F

1000 ppm
LU/Spleen No Effect
No Effect
No effect
F0 F
F1 M
F1 F