Registration Dossier

Administrative data

Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
14 and 28 days
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remarks':
Remarks:
Well-documented and corresponded to the criteria set for assessing animal studies in (Klimisch, et. al. 1996)- A Systematic Approach for Evaluating the Quality of Experimental Toxicological and Ecotoxicological Data, giving due consideration to the published data quality criteria in place at the time the study was conducted.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1986

Materials and methods

Principles of method if other than guideline:
The humoral immunity of newborn mice exposed for 28 days to 22.5 mg/m3 aerolized Pb(NO3)2 (Pb28-aero) or of 2-week old mice similarly exposed for 14 days (Pb14-aero) was compared with that of both 2-week-old mice given 125 ug Pn(NO3)2/day by gastric intubation for 14 days (Pb14-oral) and of 4-week-old nonexposed controls. Mice from each group were immunized with with 10to the eighth sheep red blood cells by intravenous (IV), intraperitoneal (ip), or intratracheal (it) routes of immunization.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Specific details on test material used for the study:
Lead Nitrate Pb(NO3)2

Test animals

Species:
mouse
Strain:
Swiss Webster
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River laboratories (Wilmington, Mass.) and maintained at the South Dakota State University Microbiology Department's breeding colony.
- Age at study initiation: newborn-2 weeks old
- Weight at study initiation:
- Fasting period before study:
- Housing:
- Diet (e.g. ad libitum): All mice were given food and water ad libitum throughout each experiment
- Water (e.g. ad libitum): All mice were given food and water ad libitum throughout each experiment
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
clean air
Remarks:
Filtered air mixed with a solution of 1000 ppm Pb(NO3)2
Remarks on MMAD:
MMAD / GSD: Using an Andersen Cascade Impactor (BGI Inc., Waltham, Mass.) it was determined that 81% of the lead entering the chamber had an aerodynamic particle size of less than 2 um which should have allowed for deep penetration of the lung.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: ILE glove box (S. Blickman Inc., Weehawken, NJ) with an exhaust fan.
- Method of holding animals in test chamber:
- Source and rate of air:
- Method of conditioning air:
- System of generating particulates/aerosols: Filtered aiir mixed with a solution of 1000 ppm Pb(NO3)2 was forced through a nebulizer producing a liquid aerosol which was blown into a heated desolvation chamber. The spray entering this chamber was flash evaporqted producing water vapor and particulates composed primarily of lead. The water vapor was removed by a 50-cm Graham condenser cooled to 8 degrees centigrade. Collision with the walss of the condenser spiral also removed the larger dry particles of lead. The smaller-sized particles were able to pass down the spiral and enter the exposure chamber.
- Temperature, humidity, pressure in air chamber:
- Air flow rate:
- Air change rate:
- Method of particle size determination: Using an Andersen Cascade Impactor (BGI Inc., Waltham, Mass.) it was determined that 81% of the lead entering the chamber had an aerodynamic particle size of less than 3 um which should have allowed for deep penetration into the lung.
- Treatment of exhaust air:

TEST ATMOSPHERE
- Brief description of analytical method used:
- Samples taken from breathing zone: yes/no

VEHICLE (if applicable)
- Justification for use and choice of vehicle:
- Composition of vehicle:
- Type and concentration of dispersant aid (if powder):
- Concentration of test material in vehicle: The spray entering this chamber was flash evaporated producing water vapor and particulates composed primarily of lead. The water vapor was removed by a 50-cm Graham condenser cooled to 8 degrees centigrade.
- Lot/batch no. of vehicle (if required):
- Purity of vehicle:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Mice in the chamber were exposed to 2.5 mg Pb(NO3)2/m3 as determined by atomic absorption spectrophotometry. If a minute volume of 1.1 ml/gm body wt (Rosebury, 1947) and total retention of all inhaled metal is assumed, then each mouse received approximately 80 ug Pb/day.
Duration of treatment / exposure:
Five groups consisting of eight randomly distributed mice were used for each study. These consisted of one group exposed to aerolized lead for 2 weeks (Pb14-aer); one group receiving an equivalent amount of orally administered lead for 2 weeks (Pboral); and two control groups, one for each treatment (Caero and Coral). These four groups were two weeks old at the beginning of their treatments. A fifth group of mice (Pb28-aero) was born and reared in the aerosolization chamber for 28 days.

With the exception of the Pb28-aero group, all mice were exposed to lead for 9 days prior to and 5 days after intraperitoneal (ip) or intravenous (iv) immunization and for 7 days prior to and following intratracheal (it) instillation. The Pb28-aero mice were exposed 23 days prior to and 5 days after ip or iv immunization and for21 days prior to and 7 days following intratracheal (it) instillation.
Frequency of treatment:
Mice continuously exposed to aerosolized lead were placed into a chamber similar to that described by Arvik et al. (1973).
Doses / concentrations
Dose / conc.:
80 other: micrograms of lead per day
Remarks:
Basis: analytical conc.
No. of animals per sex per dose:
Five groups consisting of eight randomly distributed mice were used for each study. These consisted of one group exposed to aerolized lead for 2 weeks (Pb14-aer); one group receiving an equivalent amount of orally administered lead for 2 weeks (Pboral); and two control groups, one for each treatment (Caero and Coral). These four groups were two weeks old at the beginning of their treatments. A fifth group of mice (Pb28-aero) was born and reared in the aerosolization chamber for 28 days.
Control animals:
yes, concurrent vehicle
Details on study design:
Five groups consisting of eight randomly distributed mice were used for each study. These consisted of one group exposed to aerolized lead for 2 weeks (Pb14-aero); one group receiving an equivalent amount of orally administered lead for 2 weeks (Pboral); and two control groups, one for each treatment (Caero and Coral). These four groups were two weeks old at the beginning of their treatments. A fifth group of mice (Pb28-aero) was born and reared in the aerosolization chamber for 28 days.
Mice continuously exposed to aerolized lead were placed into a chamber similar to that described by Arvik et al. (1973). The chamber consisted of an ILE glove box (S. Blickmaan., Weehawken, N.J.) with an exhaust fan. Filtered air mixed with a solution of 1000 ppm Pb(NO3)2 was forced through a nebulizer producing a liquid aerosol which was blown into a heated desolvation chamber. The spray entering this chamber was flash evaporated producing water vapor and particulates composed primarily of lead. The water vapor was removed by a 50-cm Graham condenser cooled to 8 degrees centigrade. Collision with the walls of the condenser spiral also removed the larger dry particles of lead. The smaller-sized particles were able to pass down the spiral and enter the exposure chamber. Mice in the chamber were exposed to 2.5 mg Pb(NO3)2/m3 as determined by atomic absorption spectrometry. If a minute volume of 1.1 ml/gm body/wt (Rosebury, 1947) and total retention of all inhaled metal is assumed, then each mouse received approximately 80 ug Pb/day. Using an Andersen Cascade impactor (BGI Inc., Waltham, Mass.) it was determined that 81% of the lead entering the chamber had an aerodynamic particle size of less than 2 um which should have allowed for deep penetration into the lung. Control aerolization mice were housed under the same conditions as the lead group but were exposed to clean air.
Mice in the lead ingestion group were administered an equivalent amount of lead orally via gastric intubation using a balled biomedical animal feeding needle (Popper & Sons, Inc., New Hyde Park, NY). This group received 125ug Pb(NO3)2 in 0.1 cc deionized water daily. Ingestion control mice received an equivalent quantity of deionized water..
With the exception of the Pb28-aero group, all mice were exposed to lead for 9 days prior to and 5 days after intraperitoneal (ip) or intravenous (iv) immunization and for 7 days prior to and following intratracheal (it) instillation. The Pb28-aero mice were exposed 23 days prior to and 5 days after ip or iv immunization and for 21 days prior to and 7 days following its instillation.
Immunizations: All immunizations consisted of the injection or instillation of 10 to the eighth sheep red blood cells (SRBC) which had been previously washed three times in sterile buffered saline. The SRBC used in this study came from a single sheep and were never more than 5 days old when used for immunization. Intraperitoneal and iv tail vein injections were performed by standard techniques using a 0.1 cc sterile SRBC-saline suspension. Intratracheal immunizations were done according to the method of Bice et al (1979) with the exception that the animals were anesthesized by an ip injection of 0.1 mg sodium pentobarbital/g body/wt. For it instillations, the SRBC were suspended in 0.05 cc sterile saline. Prior studies revealed that nonimmunized mice did not have any detectable antibody or antibody-forming cells and therefore such groups were not included in this study.
Lymphoid cell suspensions: To determine their level of immunity, mice were were anesthesized by ether inhalation, exsanguinated, and terminated by cervical dislocation on Day 5 after either ip or iv immunizations and on Day 7 following it immunization. These times were previously determined by our lab to correspond to the day at which the peak immune response occurred for each immunization route. The thoracic lymph nodes (TLN), cervical lymph nodes, and spleen of each experimental animal were removed and placed in RPMI 1640 medium supplemented with 10% fetal calf serum which was heat inactivated at 56 degrees centigrade for 30 minutes and absorbed with SRBC. Cell suspensions of lymph node tissues were made by disrupting the tissues with toothed iris forceps white cell suspensions of the spleeen were made by passing the tissue through size 100 stainless steel mesh screen. Erythrocytes in the spleen suspensions were lysed with Tris-buffered isotonic ammonium chloride. After 15 minutes incubation at 37 degrees centigrade, the spleen suspension was washed two times with the RPMI 1640 medium described above. Cell counts were made with a Model F Coulter Counter. After this procedure cell viabilities were at least 97% as determined by the trypan blue dye exclusion test.
Positive control:
Two control groups: one for each treatment (Caero and Coral).

Examinations

Observations and examinations performed and frequency:
Hemagglutination titers-All animals were bled from the subclavian artery. A sample of blood was allowed to clot and the serum removed after centrifugation and frozen until assayed. Additional blood samples were taken from each animal for total leukocyte counts and differential white blood cell determinations. Hemagglutination titers were performed on the serum using serial two-fold dilutions in V-bottomed Linbro microtiter plates (Flow Laboratories, Rockville, Md.). Titers were determined in the presence and absence of 2-mercaptoethanol. The titer resistant to this reducing agent indicated the presence of immunoglobulin G/(IgG) antibody in the serum.
Hemolytic plaque assay: The number of lymphoid cells producing IgM or IgG SRBC antibody was measured using the Cunningham-Szenberg modification of the in vitro Jerne plaque assay previously described in detail by Bice et al. (1979). The plaques formed after the addition of complement (Grand island Biological Co., Grand Island, NY) were considered to be IgM-producing cells. The increased number of antibody-forming cells (AFC) obtained after the addition of monospecific anti-mouse IgG antisera were counted as IgG AFC. Data collected during this study were tabulated and expressed as the number of IgM or IgG anti-SRBC AFC
Sacrifice and pathology:
The spleen, thymus, liver, aand kidney were excised, weighed, and frozen prior to lead analysis. The lead content in these tissues, as well as in the blood, was determined by atomic absorption spectrophotometry using a modification of the procedure used by Christian (1975).

Statistics:
A statistical comparison of the level of immunity in mice exposed to either aerosolized or orally administered lead was made. Previous studies evaluating the number of AFC indicated that the variance of the data increased linearly with the increase in the level of the immune response. A logarithmic transformation of the data was therefore utilized to stabilize the variance (Gottlieb, 1974). The data were evaluated by one-way analysis of variance using the Dunnett's test to compare each treatment group against a control. Since the control aerosolization group experienced no treatment of any kind, it was used as the control for the Dunnett's test. Differences with P < 0.05 were considered significant.

Results and discussion

Results of examinations

Clinical signs:
not examined
Mortality:
not examined
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Details on results:
Relative organ weights: There were no apparent differences in body weights or the rate of food consumption (data not shown) among any of the treatment groups. All three lead treatment groups experienced a significant (P<0.05) to highly significant (P<0.01) decrease in the relative size of both the spleen and thymus. Only the two groups of mice receiving aerolized lead had significant higher relative lung weight. No differences were seen in the relative kidney weights or adrenal weights.
Tissue lead concentrations: There was no significant increase in the lead concentration of any of the reported organs in the Pb14-oral group. Both inhalation groups had a significant increase in the lead content of the liver, lung, and kidney. Only the Pb28-aero group had significantly elevated lead concentrations in the primary organs of immunological interest. This, as well as the increased lead concentrations in the Pb28-aero group, compared with the Pb14-aero group, demonstrates the ability of lead to accumulate in the body.
Differential and total leukocyte counts: Both the Pb14-oral and Pb28-aero groups had a significant leukopenia. Only the Pb28-aero group had any significant alteration in the various classes of leukocytes with a highly significant decrease in the percentage of neutrophils compensated by an equally significant increase in the percentage of lymphocytes. This group also had a significant decrease in the relative number of monocytes. if the actual numbers of these leukocytes are calculated and compared with the Caero groiup, there was a 23% decrease in lymphocytes and a 58% decrease in both neutrophils and monocytes.
Hemagglutination titers: No detectable antibody was present in any of the groups 7 days after it immunization. There were no significant differences in circulating antibody titers after iv immunization in any of the groups. In contrast, there was a highly significant decrease in the antibody titers of the Pb 28-aero group after ip immunization.
AFC resulting from intraperitoneal immunization: Although cervical lymph nodes as well as spleen and thoracic lymph nodes were assayed, only the last two sources contained AFC. Because of this pattern of AFC distribution, only the AFC in the TLN and spleen were compared. In general the ip immunization route resulted in the greatest number of AFC in both the spleen and TLN. A visually detectable enlargement of the thoracic lymph nodes occurred in all animals after ip or it immunization. A similar hyperplasia had previously been observed in nonimmunized Pbaero mice and was found to increase after either ip of it immunization. A greater number of SRBC-specific AFC/10-6 lymphocytes was usually isolated from the TLN than from the spleen after both ip and it immunization. Comparable numbers of AFC were isolated from the spleens of control groups after both ip and iv immunization; however, relative to these numbers there was approximately a 50-fold decrease in the number of SRBC-specific AFC isolated from the spleen after it instillation. Regardless of the route of administration, oral lead exposure did not appear to affect the number of AFC in either the spleen or TLN. In sharp contrast, there was a highly significant reduction in the number of splenic IgM and IgG AFC in both Pbaero groups. The reduction in the total AFC of the Pb28-aero group appeared to be due to reduced levels of both IgM and IgG AFC; whereas in the Pb14-aero group the reduction was primarily due to decreases in the IgG AFC levels. Only the Pb28-aero treatment group showed any significant reduction in TLN ACF.
AFC resulting from intratracheal immunization: Although circulating antibody to SRBC could not be detected in the serum after it immunizations, antibody-forming cells could be detected in both TLN and spleen samples. Intratracheal instillation of SRBC resulted in substantially fewer antigen-specific splenic AFC than was observed after either ip or iv immunization. Both the iv and it methods of immunization resulted in nearly equivalent numbers of AFC in the TLN: however, in both cases this was approximately one-tenth the number observed in the TLN after ip immunization. There was a highly significant reduction in total AFC in the TLN of both Pbaero groups as well as a significant reduction in IgM AFC in the Pb28-aero group. Because of the suppression in AFC in the ATLN it was surprising to observe a significant increase in the number of total and IgM AFC in the spleens of Pb28-aero mice and a highly significant increase in the number of IgG AFC in the Pb14-aero group.
AFC resulting from intravenous immunization: Unlike the other methods of immunization, the iv route resulted in a greater number of antigen-specific AFC in the spleen than in the TLN. There was no significant decrease in the AFC response of the spleen in any of the lead treatment groups. There was a highly significant decrease of total AFC in the TLN of the Pbaero groups. This decrease appeared to be due to reduced IgG AFC levels.

Effect levels

Dose descriptor:
other: Relative immunotoxicity of subclinical lead exposure by inhaltion with that of ingestion.
Remarks:
The purpose of this investigation was to compare the relative immunotoxicity of subclinical lead exposure by inhaltion with that of ingestion.
Effect level:
ca. 2.5 mg/m³ air (analytical)
Based on:
element
Remarks:
Pb(NO3)2 aerosolized exposure for 28 and 14 days
Sex:
not specified
Basis for effect level:
immunology
other: see 'Remark'
Remarks on result:
other: 2.5 mg/m3 aerosolized Pb(NO3) exposure for 14 or 28 days.

Target system / organ toxicity

Critical effects observed:
not specified
System:
immune system

Applicant's summary and conclusion

Conclusions:
Locally expressed immune repsonses are vital in the defense of the host against antigenic and pathogenic materials deposited in the lung. Any pollutant which can suppress this repsonse may therefore jeopardize the health of the host. Many airborne pollutants, including N2O2 and SO2 have been shown to decrease an animal's resistance to subsequent infection and lung immunity (Schnizzlein et al., 1980; Hillam et al., 1983). The results of this investigation now add aerosolized lead to this growing list.
Executive summary:

The humoral immunity of newborn mice exposed for 28 days to 2.5 mg/m3 aerosolized Pb(NO3)2 (Pb28aero) or of 2 -week-old mice similarly exposed for 14 days (Pb14 -aer) was compared with that of both 2 -week-old mice similarly exposed for 14 days (Pb14 -aero) was compared with that of both 2 -week-old mice given 125ug Pb(NO3)2/day by gastric intubation for 14 days (Pb14 -oral) and of 4 -week-old nonexposed controls. Mice from each group were immunized with 10 to the eighth sheep red blood cells by intravenous (iv), intraperitoneal (ip), or intratracheal (it) routes of immunization. Immunity was assessed by both hemagglutination and the enumeration of antibody-forming cells from the spleen and thoracic lymph nodes. All treatment groups had decreased thymus/body weight and spleen/body weight ratios whereas only Pbaero groups had enlarged livers. The most significant immunosuppression occurred in the ip-immunized Pb28 -aero group. A significant suppression of humoral immunity was also observed in thoracic lymph node samples from Pbaero groups immunized it or iv. There was no apparent immunosuppression in any treatment group after iv immunization. These results indicate that aerosolized lead is more immunosuppressive than equivalent amounts of ingested lead. This is most likely due to the greater absorption of inhaled lead and the subsequent cytotoxicity of lead for cells in the draining lymph nodes.