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REACH

Concentrates of lead and zinc compounds with sulfur resulting from hydrometallurgy (hot acid leaching, super-hot acid leaching and flotation)

EC number: 936-276-2 | CAS number: -

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

According to transformation/dissolution study (OECD guidance 29) conducted for the substance, the most critical constituents leachable to water from this UVCB substance are lead and zinc compounds. Therefore, the chemical safety assessment focuses on the properties of constituents and the key values for CSA are selected based on the read-across data on the most bioavailable compounds of Pb and Zn.
Lead
In an animal study following oral administration NOEL for lead was established: 0.0015 mg Pb/kg bw/day (rats). This value is selected as a starting point for long-term systemic DNEL derivation for workers via dermal route. 
Lead has been documented in observational human studies to produce toxicity in multiple organ systems and body function including the haemotopoietic system, kidney function, reproductive function and the central nervous system. Based on the results from reliable and good quality human epidemiological studies the substance is classified in Category 1 for target organ toxicity (repeated exposure), STOT-RE 1 H372. 
Zinc
In animal studies following oral NOAELs for zinc were established: 104 mg Zn/kg bw/day in mice and 53.5 mg/kg bw/day in rats.
Overall, it can be concluded that from studies in which humans were supplemented with zinc (as zinc gluconate), that women are more sensitive to the effects of high zinc intake and that a dose of 50 mg Zn/day is the human NOAEL. This equals a daily exposure of 0.83 mg/kg bw. At the LOAEL of 150 mg Zn/day, clinical signs and indications for disturbance of copper homeostasis have been observed.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:: chronic toxicity: oral
Type of information:: migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:: weight of evidence
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: other: see 'Remark'
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.Read-across justification:Read-across from the most critical and bioavailable constituents (lead and its compounds):Based on the mineralogical composition, lead is the main constituent in the target substance and appears in sulphate form. The target substance is a solid inorganic UVCB substance and insoluble in water. Therefore, the transformation/dissolution study (OECD guidance 29) was conducted for the target substance to focus on the most critical bioavailable constituents of the substance. According to the T/D study results, the readily soluble constituent is lead. Based on the hazard profile of lead it is also the most hazardous constituent of this substance. Therefore, and in order to avoid the unnecessary animal testing, the read-across data from the most critical constituent is used to evaluate the short-term and long-term toxicological adverse effects of the target substance. The read-across data focuses on the properties of lead sulphate and other bioavailable forms of lead.
Principles of method if other than guideline:: The research techniques used in these investigations were developed by Krasovskii et al.(1976): Krasovskii, G.N., Varshavskaya, S.P., and Borisova, A.I. Toxic and gonadotropic effects of cadmium and boron relative to standards for the substance in drinking water. Environ. Health Perspect. 13:69 (1976) using Caremont and leblond "Spermogenesis of man, monkey, ram, and other mammals as shown by the periodic acid-Sciff acid techniques." Amer J. Anat. 96:229 (1955).
GLP compliance:: yes
Species:: rat
Strain:: other: white rats
Sex:: not specified
Details on test animals or test system and environmental conditions:: Details are reported in Krasovskii, G.N., Varshavskaya, S.P., and Borisova, A.I. Toxic and gonadotropic effects of cadmium and boron relative to standards for the substance in drinking water. Environ. Health Perspect. 13:69 (1976) which was unavailble at the time of input from the Journal of Environmental Perspectives.
Route of administration:: oral: unspecified
Vehicle:: not specified
Details on oral exposure:: The metals were administered orally each day to laboratory animals. lead acetate was administered in doses of 0.05, 0.005. and 0.0015mg/kg (based on ions of lead) to white rats; aluminum chloride in doses of 50, 17, and 6mg/kg (based on ions of aluminum) to rats and guinea pigs, 27, 9, and 3mg/kg to rabbits in short-term exposures, and 2.5, 0.25, and 0.0025mg/kg to rats in chronic exposures. These experiments were 0f 20-30 days and 6-12 months' duration.
Analytical verification of doses or concentrations:: not specified
Duration of treatment / exposure:: These experiments were of 20-30 days and 6-12 months' duration.
Frequency of treatment:: The metals (lead acetate and aluminum chloride were administered orally each day to laboratory animals.
Remarks:: Doses / Concentrations:0.05, 0.005, and 0.0015mg/kgBasis:other: based on the ions of lead
Control animals:: yes
Details on study design:: Lead acetate was administered in doses of 0.05, 0.005, and 0.0015 mg/kg )based on the ions of lead) to white rats; aluminum chloride in doese of 50, 17, and 6 mg/kg (based on ions of aluminum) to rats and guinea pigs, 27, 9, and 3 mg/kg to rabbits in short-term exposures, and 2.5, 0.25, and 0.0025 mg/kg to rats in chronic exposures. These experiments were of 20-30 days and 6-12 months duration.
Positive control:: Yes
Observations and examinations performed and frequency:: In the 6-12 month exposures with lead acetate, special attention was paid to the study of the excretion of alpha-aminolevulinic acid and coporphobilinogen in urine. In order to evaluate the effect of lead on the behavioral responses of animals, their motor activity was recorded by a method which tested conditioned reflexes. Also investigated were the functional and morphological conditions of spermatozoa and gonads. The blood chemistry of the animals and histological examination of rat livers and organ weights were observed in the short-term studies.
Body weight and weight changes:: effects observed, treatment-related
Haematological findings:: effects observed, treatment-related
Clinical biochemistry findings:: effects observed, treatment-related
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Details on results:: The toxic effects of lead were observed when administered to rats in doses of 0.05 mg/kg. The activity of aldolase increased and the level of sulfhydryl groups decreased 5-10 days after intoxication (0< 0.01). The effect of the 0.005 mg/kg dose was expressed as a trend in the increase of aldolase in the blood serum. By the 20th day of intoxication the activity of aldolase normalized., and the activity of B-galactosidase, B-glucosidase, and acid phos- of B-galactosidaee, B-glucosidase, and acid phosphatase and cholesterol content of the blood serum did not change. In addition, substantial increases in the weight coefficients of liver and kidneys were observed with the 0.05 mg/kg doses of lead. A histological examination of the rat livers indicated a definite decrease in RNA and glycogen and in the activity of SDH, LDH, and NAD-diaphorase at all doses, and pyknosis of Kupffer cells at doses of 0.05mg/kg. Furthermore, small droplets of lipid inclusions were observed in the cytoplasm of epithelial cells located in the convoluted tubules of the cortical substance liver. The gonado toxic effect of lead was observed in animals which received the maximum dose. The functional conditional condition of spermatozoa changed and the acid phosphatase activity in the gonadal tissue increased being 25 +/- 1.0 for controls, 34.4 +/- 2.3 at 0.05 mg/kg, and 27.8 +/- 1.4 at 0.005 mg/kg; p < 0.05. The weight coefficients of gonads were increased only in animals which were exposed to the dose of 0.005 mg/kg. Small disruptions in the permability of the vessels and dystrophic changes in the Leydig cells were observed in the gonads, and the activity of oxidizing enzymes increased. In animals which were exposed to 0.0015 mg/kg of lead, no deviations in functional state was observed compared to the control group of animals.Long-Term Exposure- In the 6-12 month exposures, special attention was paid to the study of the excretion of Alpha-aminolevulinic acid and coprophobilinogen in urine. The investigations showed that, beginning with the second month of intoxication, the excretion of alpha-aminoevulinic acid and of perphobilinogen gradually increased in animals which received lead in doses of 0.05 and 0.005 mg/kg. In order to evaluate the effect of lead on the behavioral responses of animals, their motor activity was recorded by a method which tested conditioned reflexes. Animals exposed to lead at 0.05 mg/kg and 0.005 mg/kg had disruptions in their conditioned responses (Table 2), and the wavelike motor activity underwent a shift of phase, depending on the season of the year. The investigation of functional and morphological conditions of spermatozoa and gonads indicated the gonadotoxic effect of lead in doses of 0.05 mg/kg (Table 3). The histological study of gonads indicated a swelling of the follicular epithelial cells; the vascular network was full of blood. The RNA content and sulfhydryl group content of some tubules increased, but decreased in the remaining part of the parenchyma. the activity of AIDH, SGH, NAD, and NADPH-diaphorase was substantially depressed in the spermatogenic epithelium and less depressed in the interstices. The indices of acitivity of acid phosphatase and of beta- glycosidase in gonadal tissues exposed to lead in doses of 0.05 mg/kg, 0.005 mg/kg, and 0.0015 mg/kg were, respectively, 58.5 +/- 3.0, 49.3 +/- 1.5, and 36.2 +/- 2.0 (controls 37.4 +/- 1.5) and 38.9 +/- 1.9, 64.0 +/- 5.4., annd 9.04 +/- 4.8 (controls (13.0 +/- 4.5). The histochemical study of the liver at doses of lead of 0.05 mg/kg and 0.005 mg/kg revealed a decrease in glycogen content, RNA, sulfhydryl groups and activity of oxidising enzymes in the central regions of obules and an increase of NAD and NADPH-diaphorase at the periphery of the lobules. In the kidneys the highest tested dose of 0.05 mg/kg only insignificantly increased chromosome abberrations. The 0.0015 mg/kg dose of lead (which corresponds to 0.03 mg lead/l. water) did not cause any changes. It can be considered that this does not have an effect on the organism
Dose descriptor:: LOEL
Effect level:: ca. 0.005 mg/kg bw/day (actual dose received)
Based on:: other: ions of lead
Sex:: not specified
Basis for effect level:: other: The effect of the 0.005 mg/kg dose was expressed as a trend in the increase of aldolase in the blood serum
Dose descriptor:: NOEL
Effect level:: ca. 0.002 mg/kg bw/day (actual dose received)
Based on:: other: based on the ions of lead
Sex:: not specified
Basis for effect level:: other: The 0.0015 mg/kg dose of lead (which corresponds to 0.03 mg lead/l water) did not cause any changes. It can be considered that this does not have an effect on the organism
Critical effects observed:: not specified
Conclusions:: A concentraion i water of 0.03 mg/l of lead can be considered safe for the health of the general public, and these could be recommended for inclusion into the public health standards for drinking water.
Executive summary:: A wide spectrum of biological effects of lead and aluminum ions is noted during short-term and long-term oral administration to laboratory animals. The general toxic and gonadotoxic effects of these metals during a short-term experiment appeared to be identical, and the correlation of these effects was preserved during chronic experiments. lead (0.03 mg/l) and aluminum (0.5 mg/l) concentrations in water may be dangerous to the health of the population, and hygienic standards are recommended for inclusion in the standard for drinking water quality.

Endpoint conclusion

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 0.002 mg/kg bw/day
Study duration:: chronic
Species:: rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:: chronic toxicity: inhalation
Type of information:: migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:: weight of evidence
Study period:: 14 and 28 days
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: other: see 'Remark'
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.Read-across justification:Read-across from the most critical and bioavailable constituents (lead and its compounds):Based on the mineralogical composition, lead is the main constituent in the target substance and appears in sulphate form. The target substance is a solid inorganic UVCB substance and insoluble in water. Therefore, the transformation/dissolution study (OECD guidance 29) was conducted for the target substance to focus on the most critical bioavailable constituents of the substance. According to the T/D study results, the readily soluble constituent is lead. Based on the hazard profile of lead it is also the most hazardous constituent of this substance. Therefore, and in order to avoid the unnecessary animal testing, the read-across data from the most critical constituent is used to evaluate the short-term and long-term toxicological adverse effects of the target substance. The read-across data focuses on the properties of lead sulphate and other bioavailable forms of lead.
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
Species:: mouse
Strain:: Swiss Webster
Sex:: not specified
Details on test animals or test system 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:
Route of administration:: inhalation: aerosol
Type of inhalation exposure:: whole body
Vehicle:: other: 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/noVEHICLE (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).
Remarks:: Doses / Concentrations:80 micrograms of lead per dayBasis: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).
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.
Clinical signs:: not examined
Mortality:: not examined
Body weight and weight changes:: no effects observed
Food consumption and compound intake (if feeding study):: no effects observed
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not specified
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
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Histopathological findings: neoplastic:: not examined
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.
Dose descriptor:: NOAEC
Effect level:: <= 2.5 mg/m³ air
Based on:: element
Remarks:: Pb(NO3)2 aerosolized exposure for 28 and 14 days
Sex:: not specified
Basis for effect level:: other: see 'Remark'
Remarks on result:: not determinable
Critical effects observed:: not specified
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.

Endpoint conclusion

Endpoint conclusion:: adverse effect observed
Dose descriptor:: LOAEC
: 2.5 mg/m³
Study duration:: subacute
Species:: mouse

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:: chronic toxicity: inhalation
Type of information:: migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:: weight of evidence
Study period:: 14 and 28 days
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: other: see 'Remark'
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.Read-across justification:Read-across from the most critical and bioavailable constituents (lead and its compounds):Based on the mineralogical composition, lead is the main constituent in the target substance and appears in sulphate form. The target substance is a solid inorganic UVCB substance and insoluble in water. Therefore, the transformation/dissolution study (OECD guidance 29) was conducted for the target substance to focus on the most critical bioavailable constituents of the substance. According to the T/D study results, the readily soluble constituent is lead. Based on the hazard profile of lead it is also the most hazardous constituent of this substance. Therefore, and in order to avoid the unnecessary animal testing, the read-across data from the most critical constituent is used to evaluate the short-term and long-term toxicological adverse effects of the target substance. The read-across data focuses on the properties of lead sulphate and other bioavailable forms of lead.
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
Species:: mouse
Strain:: Swiss Webster
Sex:: not specified
Details on test animals or test system 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:
Route of administration:: inhalation: aerosol
Type of inhalation exposure:: whole body
Vehicle:: other: 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/noVEHICLE (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).
Remarks:: Doses / Concentrations:80 micrograms of lead per dayBasis: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).
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.
Clinical signs:: not examined
Mortality:: not examined
Body weight and weight changes:: no effects observed
Food consumption and compound intake (if feeding study):: no effects observed
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not specified
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
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Histopathological findings: neoplastic:: not examined
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.
Dose descriptor:: NOAEC
Effect level:: <= 2.5 mg/m³ air
Based on:: element
Remarks:: Pb(NO3)2 aerosolized exposure for 28 and 14 days
Sex:: not specified
Basis for effect level:: other: see 'Remark'
Remarks on result:: not determinable
Critical effects observed:: not specified
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.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed
Study duration:: subacute
Species:: mouse

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:: no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:: no study available

Additional information

The health hazard assessment was conducted based on the most critical constituents of the substance. This substance is an UVCB substance and can be described as a moist solid powder which is insoluble to water. Therefore, the transformation/dissolution study (OECD guidance 29) was conducted for the substance and the results of this study were used for the chemical safety assessment.

According to the chemical composition analysis, the main phases of the substance are lead sulphate and zinc sulphide. The product consists primarily of sulphur (ca. 35 %), lead (ca. 25 %) and zinc (ca. 17 %) together with minor trace elements such as silver, silicon, aluminium, calcium and iron.

The transformation and dissolution study (OECD guidance 29) results indicated that the release at pH 6 was higher for all studied elements compared to release at pH 8. Based on the screening test results (loading rate 100 mg/L), the most critical components for the assessment were lead and zinc, with releases of 8282 µg/L and 75.4 µg/L, respectively. The other minor leachable metals were silver (34.7 µg/L), cadmium (0.48 µg/L) and copper (17.2 µg/L).

Results from the 7 day T/D test (loading rate 100 mg/L, pH 6) showed similar trend in release rates: 12333 µg/L (Pb), 91.4 µg/L (Zn), 15.6 µg/L (Cu), 31.4 µg/L (Ag) and 0.056 µg/L (Cd). In the 28 day test with lower loading rate (1 mg/L, pH 6), only concentrations of Pb (362.4 µg/L) and Zn (3.2 µg/L) were over the detection limits or blank sample values.

According to T/D study results, the most soluble and critical components of this substance are lead and zinc. Therefore, the studies for this endpoint have been selected as a read-across data for the critical constituents. The read-across justification is presented in CSR annex I. All read-across data for toxicology are based on test data using either soluble Pb or Zn salts or measured (dissolved) Pb or Zn concentrations. The weight of evidence approach was used to make conclusions on the key value for CSA. Conclusion for this endpoint is based on read-across data from zinc and lead compounds.

Lead compounds

Non-human information

Two repeated dose toxicity studies from oral exposure and one from inhalation exposure to lead are assessed here.

In the study by Krasovkii (1979) lead acetate was administered orally each day to laboratory animals in doses of 0.05, 0.005 and 0.0015 mg/kg (based on ions of lead) to white rats. These experiments were 20-30 days and 6-12 months' duration. In the 6-12 month exposures with lead acetate, special attention was paid to the study of the excretion of alpha-aminolevulinic acid and coporphobilinogen in urine. In order to evaluate the effect of lead on the behavioural responses of animals, their motor activity was recorded by a method which tested conditioned reflexes. Also investigated were the functional and morphological conditions of spermatozoa and gonads. The blood chemistry of the animals and histological examination of rat livers and organ weights were observed in the short-term studies.

The toxic effects of lead were observed when administered to rats in doses of 0.05 mg/kg. The activity of aldolase increased and the level of sulfhydryl groups decreased 5-10 days after intoxication (0< 0.01). The effect of the 0.005 mg/kg dose was expressed as a trend in the increase of aldolase in the blood serum. By the 20th day of intoxication the activity of aldolase normalized and the activity of B-galactosidase, B-glucosidase, and acid phos- of B-galactosidaee, B-glucosidase, and acid phosphatase and cholesterol content of the blood serum did not change. In addition, substantial increases in the weight coefficients of liver and kidneys were observed with the 0.05 mg/kg doses of lead. A histological examination of the rat livers indicated a definite decrease in RNA and glycogen and in the activity of SDH, LDH, and NAD-diaphorase at all doses, and pyknosis of Kupffer cells at doses of 0.05mg/kg. Furthermore, small droplets of lipid inclusions were observed in the cytoplasm of epithelial cells located in the convoluted tubules of the cortical substance liver. The gonadotoxic effect of lead was observed in animals which received the maximum dose. The functional conditional condition of spermatozoa changed and the acid phosphatase activity in the gonadal tissue increased being 25 +/- 1.0 for controls, 34.4 +/- 2.3 at 0.05 mg/kg, and 27.8 +/- 1.4 at 0.005 mg/kg; p < 0.05. The weight coefficients of gonads were increased only in animals which were exposed to the dose of 0.005 mg/kg. Small disruptions in the permeability of the vessels and dystrophic changes in the Leydig cells were observed in the gonads, and the activity of oxidizing enzymes increased. In animals which were exposed to 0.0015 mg/kg of lead, no deviation in functional state was observed compared to the control group of animals.

In the 6-12 month exposures, special attention was paid to the study of the excretion of Alpha-aminolevulinic acid and coprophobilinogen in urine. The investigations showed that, beginning with the second month of intoxication, the excretion of alpha-aminoevulinic acid and of perphobilinogen gradually increased in animals which received lead in doses of 0.05 and 0.005 mg/kg. In order to evaluate the effect of lead on the behavioural responses of animals, their motor activity was recorded by a method which tested conditioned reflexes. Animals exposed to lead at 0.05 mg/kg and 0.005 mg/kg had disruptions in their conditioned responses, and the wavelike motor activity underwent a shift of phase, depending on the season of the year. The investigation of functional and morphological conditions of spermatozoa and gonads indicated the gonad toxic effect of lead in doses of 0.05 mg/kg. The histological study of gonads indicated a swelling of the follicular epithelial cells; the vascular network was full of blood. The RNA content and sulfhydryl group content of some tubules increased, but decreased in the remaining part of the parenchyma. the activity of AIDH, SGH, NAD, and NADPH-diaphorase was substantially depressed in the spermatogenic epithelium and less depressed in the interstices. The indices of activity of acid phosphatase and of beta- glycosidase in gonadal tissues exposed to lead in doses of 0.05 mg/kg, 0.005 mg/kg, and 0.0015 mg/kg were, respectively, 58.5 +/- 3.0, 49.3 +/- 1.5, and 36.2 +/- 2.0 (controls 37.4 +/- 1.5) and 38.9 +/- 1.9, 64.0 +/- 5.4., and 9.04 +/- 4.8 (controls (13.0 +/- 4.5). The histochemical study of the liver at doses of lead of 0.05 mg/kg and 0.005 mg/kg revealed a decrease in glycogen content, RNA, sulfhydryl groups and activity of oxidising enzymes in the central regions of obules and an increase of NAD and NADPH-diaphorase at the periphery of the lobules. In the kidneys the highest tested dose of 0.05 mg/kg only insignificantly increased chromosome aberrations. The 0.0015 mg/kg dose of lead (which corresponds to 0.03 mg lead/l. water) did not cause any changes. It can be considered that this does not have an effect on the organism.

In conclusion of oral studies, a wide spectrum of biological effects of lead ions is noted during short-term and long-term oral administration to laboratory animals. Lead (0.03 mg/l) concentrations in water may be dangerous to the health of the population, and hygienic standards are recommended for inclusion in the standard for drinking water quality. Based on this study the NOEL of 0.0015 mg lead/kg bw/day was established and used for the derivation of systemic – long-term DNEL for workers via dermal route.

Repeated inhalation study was conducted for the inorganic lead compound. 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 -aero) 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.

Human information

Documentation of repeated dose toxicity from lead exposure is extensive and includes detailed evaluations of effects in humans. The dose response for health effects in human can usually be defined with precision and indexed to internal measures of systemic exposure such as the concentration of lead in blood. Effects assessment indexed to external dose is thus not necessary. Although repeated dose toxicity can be demonstrated in animals, adverse effects have been more precisely characterised in observational human studies and multiple aspects of repeated dose toxicity are described in section 7.10.2 Epidemiological data. These human studies define effects in terms of blood lead levels - an indicator of systemic or internal exposure that represents the integrated total of exposure that results from oral, dermal and inhalation exposure routes. Reliance upon blood lead as an exposure indicator eliminates the need for route specific metrics of toxicity that are by their very nature inherently more imprecise and variable.

In many instances the toxic manifestations of lead are mediated by interaction of the lead ion with cellular proteins (e. g. metalloenzymes) in place of essential trace minerals such as calcium, zinc or iron. As such, the initial effects of lead upon the function of specific metabolic, cellular, or organ system processes will be detectable as increases or decreases in the activity of specific metalloenzymes. The biological and clinical significance of such interactions requires careful assessment of the extent of enzyme activity alteration and whether or not the alteration alters metabolic pathways to an extent that is of health significance. For example, inhibition of an enzyme present in excess may be evident as an effect of lead, but may not be considered as adverse if the affected enzyme is not involved in rate limiting steps of metabolism and the overall function of the metabolic pathway is not adversely impacted.

As the intensity of exposure increases, the magnitude of specific lead induced effects may increase and/or yet additional steps in specific biosynthetic pathways may be altered. An assessment must thus be made when the severity of impacts results in affects that are considered as adverse. Put another way, evaluation of the toxicological data base must attempt to discriminate between “effect levels” and “adverse effect” levels. This can, in some instances result in the identification of No Observable Effect Levels (NOELs), Lowest Observable Effect Levels (LOELs, No Observable Adverse Effect Levels (NOAEL’s) and Lowest Observable Adverse Effect Levels (LOAELs). In some instances the demarcation points between these different effect levels can be defined with both precision and with a high degree of certainty that the mechanistic basis of toxicity is sufficiently well understood.

NOAEL identification can become complicated if the effects under study are sufficiently subtle as to elude detection at the level of the individual. Effects of this nature may have no material impact upon the individual, but may have significance if large numbers of individuals are potentially exposed. This situation is applicable to multiple health endpoints and significantly complicates discrimination between NOEL’s and NOAEL’s. The following effects are generally considered to be the most important health endpoints defined in observational human studies.

Haematological Effects

The effects of lead upon the heme biosynthetic pathway have been extensively studied and provides one of the best illustrations of a graduated continuum of effects that can be defined with both mechanistic certainty and with precision for dose-effect relationships. Effects of lead can be detected at low levels of exposure but are enzymatic or biochemical changes not deemed to be adverse. As exposure intensity increases, the constellation of effects observed becomes increasingly diverse until impacts upon heme synthesis results in an inhibition of haemoglobin production. Decreased haemoglobin production can be observed at blood lead levels above 40 µg/dL in children and above 50 µg/dL in adults. Impacts upon haemoglobin production sufficient to cause anaemia are associated with blood lead levels of 70 µg/dL or higher.

Clinical anaemia is an unambiguous adverse health effect. Impacts upon the haematopoietic system that are clinical precursors to anaemia (diminished haemoglobin production) can be considered as adverse effects. Diminished haemoglobin production thus serves as a conservative indicator of when the impacts of lead upon heme biosynthesis make the transition from non-adverse biochemical effects to adverse effects. NOAEL’s for haematopoietic system function of 40 µg/dL for children and 50 µg/dL for adults are thus suggested by this analysis.

Renal Effects

Numerous studies have evaluated the effects of lead upon renal function under environmental and occupational exposure conditions. A continuum of responses has been observed with enzymatic changes being reported at lower levels of exposure and clinical lead nephropathy at high levels of exposure. Clinical lead nephropathy exhibits slow onset after prolonged high level exposure to lead and is characterised by a progressive decrease in glomerular filtration rate and a subsequent rise in serum creatinine. These functional changes are associated with apparently irreversible degenerative changes in kidney (interstitial fibrosis) affecting the glomerulus. Early mortality resulting from these changes has been documented by several cohort mortality epidemiology studies of occupationally exposed individuals heavily exposed to lead prior to the promulgation of modern occupational exposure standards.

A small number of recent general population studies have observed a correlation between low levels of lead in blood and biomarkers of kidney function such as creatinine clearance. The significance of such correlations is difficult to reconcile with studies of occupationally exposed individuals that have not observed such effects at far higher levels of exposure. Risk of impaired renal function from environmental levels of lead exposure is thus not expected for adults or children.

Occupational studies indicate that individuals with blood lead levels maintained below 60 µg/dL have renal function (e. g. glomerular filtration rates) equal or superior to individuals without occupational exposure. As initially observed by Buchet et al. (1980), and subsequently confirmed multiple studies, maintenance of blood lead levels at or below 60 µg/dL appears to guard against the onset of lead nephropathy. The collective studies indicate a threshold for significant renal effects that is in excess of 60 µg/dL lead in blood and with a requirement for prolonged (five years or more) lead exposure. A NOAEL of 60 µg/dL, combined with five years or more of lead exposure, is thus adopted for adverse effects upon renal function in adults.

Impaired Vitamin D metabolism that may be mediated at the level of the kidney is of potential concern and could influence the growth and development of children. Blood lead levels below 25 μg/dL appear to be without affect, at least in children with adequate nutrition (Koo et al. 1991). The safety of blood lead levels above 25 μg/dL is unclear and, on a precautionary basis, should not be exceeded. Although other health effects manifest at lower levels of exposure in children, 25 µg/dL can be considered as a NOAEL relevant to renal effects in children.

Blood Pressure and Cardiovascular Effects

Reviews and meta-analyses of the current literature on the blood lead/blood pressure relationship indicate that there is at best a weak positive association between blood lead and blood pressure in general population and occupational studies with average blood lead levels below 45 µg/dL. IPCS (1995), in reflecting upon the conclusions of most other meta-analyses) concluded that the magnitude of this association for a twofold increase in blood lead (i. e., from 0.8 to 1.6 µmol/l or 16-32 µg/dL) is a mean 1.0 mm Hg increase in systolic blood pressure and a 0.7 mm Hg increase in diastolic blood pressure. Slightly smaller estimates of the effect of lead upon blood pressure have been calculated in more recent meta-analyses. The increase in blood pressure statistically associated with an increase in blood lead is small and would not result in a meaningful increase in the risk of the individual to cardiovascular disease. However, it can be hypothesised that a modest increase in blood pressure would increase the overall incidence of cardiovascular disease in a large population of individuals. This consideration of “societal risk” as opposed to “individual risk” thus merits careful evaluation.

Evidence for causality in the relationship between blood lead and pressure is primarily derived from animal studies but the doses used in such studies are much higher than typical human exposures and are thus of uncertain relevance. Evidence of cardiovascular disease has not been found in most observational epidemiology studies, leading many to suggest that the association is not causal but rather the effect of residual confounding in statistical analyses of blood lead –blood pressure relationships. In the small number of studies reporting excess cardiovascular disease risk, relationships between blood lead and blood pressure were either absent or not reported. Although it is possible to make quantitative estimates of the impact of blood lead upon blood pressure and subsequent population–based increases in cardiovascular disease risk, in the absence of a consistent relationship between blood lead, blood pressure, and cardiovascular disease the validity of such calculations is not supported by the observational data.

Indeed, the more recently conducted studies have used more sophisticated blood pressure measurement tools and attempted more precise correction for the multiple confounders known to impact upon blood pressure. These most recent studies have generally failed to observe a relationship between blood lead and blood pressure. A meta-analysis conducted for the purpose of this Assessment, restricted to the highest quality recent studies, further observed that the effect of lead upon systolic blood pressure declines from 1.0 to 0.38 mm Hg for a doubling of lead in blood. No statistically significant associations with diastolic pressure was found. Such a significant shift in effect size as a function of study quality indicates that residual confounding, as opposed to causality, is likely responsible for earlier correlations observed in studies of humans. Due to lack of a quantifiable estimate of effect or disease risk, this endpoint not appropriate for derivation of a NOAEL.

Recent studies suggest that lead body burden may play a role in hypertensive nephropathy. However, the association remains hypothetical and needs to be substantiated with a larger number of cases. Cardiovascular effects secondary to renal damage have long been suggested as a consequence of high levels of occupational exposure, but cohort mortality studies do not provide independent evidence of risk for cardiovascular disease or derivation of a NOAEL via this alternate mechanistic pathway.

Neurological Effects in Adults

Prospective and cross-sectional studies, and associated meta-analyses of neurological function and neuropsychological performance suggest that effects of lead in the areas of sensory motor slowing, coupled with difficulties in remembering recently acquired information are the most sensitive indicators of impact upon the nervous system of adults. The severity of the effect observed appears to increase as a function of the intensity and duration of exposure.

Peripheral nervous system effects (as reviewed by Araki et al., 2000) are first detected as subtle decreases in nerve conduction velocity at blood lead levels as low as 30 µg/dL. Effects observed in the blood lead range of 30 – 40 µg/dL are subtle and well within clinical norms – no known decrements in performance (e. g. changes in reaction time, loss of motor coordination or strength) are associated with these changes induced by lead exposure. Effects further appear to be freely reversible upon the cessation of exposure. Effect reversibility, combined with the absence of functional changes and a lack of long-term clinical sequelae indicates that peripheral nervous system effects in this exposure range should not be considered as adverse effects.

As the duration and intensity of lead exposure increases, impacts upon nerve conduction velocity become more severe and begin to manifest as a peripheral neuropathy characterised by muscle weakness in the upper limbs. The precise intensity and duration of lead exposure required to induce lead neuropathy is somewhat unclear in that most cases were observed prior to the 1920’s (Ehle, 1986). This older literature also suggests that elevated lead exposure for a duration of at least one year is required for neuropathy. The exposure intensity required to produce effects was likely significantly in excess of 70 µg/dL. For the purposes of this assessment, peripheral nervous system effects are not considered further since the central nervous system appears to be more sensitive to the effects of lead and thus provides the basis of a NOAEL for neurological effects in adults.

The effects of lead upon the central nervous system have been evaluated in numerous studies, most of which have been comprehensively evaluated in recent meta-analyses by Meyer-Baron and Seeber (2000) and Goodman et al. (2001). These evaluations of neurobehavioral testing have reached disparate conclusions. Whereas Goodman et al (2001) conclude that there is little consistent evidence of lead effects at blood lead levels below 70 µg/dL, Meyer-Baron and Seeber (2000) maintain that effects can be observed in cohorts with average blood lead levels that approach 40 µ/dL. A comparison of the two different analyses permits the following conclusions to be drawn.

1. Meyer-Baron and Seeber (2000) included fewer study results in their analysis (12 studies were included as opposed to 22 included by Goodman et al., 2001), in part reflective of more rigorous inclusion criteria for study quality. Restriction of the analysis to higher quality studies likely contributed to suggestions of an effect at lower blood lead levels.

2. Both analyses were conducted based upon cohort performance as opposed to the performance of individuals. Within each cohort individuals have concurrent and past lead exposures above and below the cohort average at the time of psychometric testing. As a generalisation, effects seen in a given study will be greater in those individuals with blood lead levels above the cohort average.

3. For cohorts with blood lead averages < 70 µg/dL, the 95th percentile confidence interval for “lead effect size” includes “zero” for performance on most neurobehavioral tests. For those tests with effect sizes significantly different from zero, effects generally attenuate as lead exposure decreases and, within individual studies, the performance of lead exposed cohorts with average blood lead levels of 40µg/dL is similar to that of controls.

4. Subtle effects upon cohort performance become evident as average cohort blood lead levels approach 50 µg/dL. Effects observed at this average exposure level may be reversible, have little clinical significance for the individual and are likely largely reflective of effects in individuals with blood lead levels higher than the average (e. g. 60 µg/dL). These effects can be interpreted as the initial manifestations of lead neurotoxicity in the central nervous system and thus can be considered adverse effects.

5. Given the impacts of confounding, small effects sizes and other technical limitations, precise definition of a LOAEL for lead effects upon the adult central nervous system is difficult. Nor is there agreement as to what level of impact can be considered as adverse. However, adverse effects upon the individual likely require exposures in excess of 50 µg/dL.

6. Given the relative absence of effects in cohorts with average blood lead levels of 40 µg/dL or less, despite the presence of individuals with higher blood lead levels, a blood lead level of 40 µg/dL is identified as a NOAEL for nervous system impacts in individual adults. Establishment of 40 µg/dL as the upper limit for individuals further means that occupational cohorts will have average blood lead levels well below 40 µg/dL and thus is the range where no effects can be detected.

Neurological Effects in Children

The impacts of post-natal lead exposure upon the development of children have been studied and multiple endpoints evaluated using psychometric testing tools. Confounding factors that influence complex behavioural endpoints are imperfectly understood, and quantitative assessment of impacts at low blood lead levels is highly imprecise. Given the inherent difficulty of assessing effects upon behavioural endpoints, intelligence (IQ) is the endpoint for which the most robust measurement tools are available and for which confounder correction can be most comprehensively implemented. The underlying mechanisms for lead-induced IQ decrements is not known, nor have epidemiology studies identified a specific “syndrome” of neuropsychological deficits that would permit more precise quantitation of low-level lead effects independent of residual confounding and other sources of uncertainty in effects assessment.

The effects of lead upon IQ in children are thus summarised as follows:

1. Meta-analyses of human observational epidemiology data show a statistical association between post-natal blood lead and IQ that is small and most likely between a one to three IQ point deficit for a change in mean blood lead level from 10 µg/dL to 20 µg/dL. Meta-analyses performed by Pocock et al. (1994) and IPCS (1995) serve as the basis for this estimate of effect size.

2. IQ decrements on the order of 1 – 3 points are smaller than the standard error of measurement of IQ tests. As such they cannot be detected at the level of the individual and have no known functional significance for the individual. Any effects that occur in the blood lead range of 10 – 20 µg/dL are not regarded as adverse for the individual since they cannot be detected or measured. A NOAEL for the effects of lead upon the IQ of the individual can thus be regarded as 10 µg/dL.

3. Based upon an estimated effect size of 1 – 3 IQ points, and an IQ test Standard Error of Measurement of 5 IQ points, effects hypothetically discernible at the level of the individual may not occur until blood lead levels exceed 20 µg/dL. Even though variability in the multiple variables that contribute to IQ would likely preclude detection of an effect of lead upon IQ in the individual, effects that fall within the precision range of existing psychometric measurement tools can be considered as adverse for the individual. Given that effects could, at least in theory, be detected in the individual at a blood lead level of 20 µg/dL, lead effects for the individual can provisionally be regarded to have a LOAEL of 20 µg/dL.

4. Evidence is suggestive of an effect of blood lead upon IQ at blood lead levels less than 10 µg/dL but is difficult to interpret because of limitations in analytical and psychometric measurement techniques. In the absence of adequate data defining the nature and extent of effects at blood lead levels lower than 10 µg/dL, such effects are difficult to apply to risk assessment in a quantitative fashion.

5. Any IQ decrements that might occur at blood lead levels below 10 µg/dL would not be detectable in the individual. Although such effects may lack significance for the individual, an impact upon large numbers of children may have societal significance.

6. Available data also do not permit the identification of a threshold for lead’s effects upon children. Observational data suggests that population effects may occur at blood lead levels as low as 5 µg/dL. This level of exposure also represents a point where current science is not capable of resolving further effects and any effects that might occur are secondary in magnitude relative to other factors that influence child development. A blood lead level of 5 µg/dL can thus be considered as an epistemic threshold that both recognizes the high degree of scientific uncertainty regarding effects at blood lead levels less than 10 µg/dL and at the same time establishes an exposure benchmark goal that is protective of public health

7. Designation of 5 µg/dL as an epistemic threshold and a “societal blood lead target” also dramatically reduce the probability that individual children might exceed a blood lead level of 10 µg/dL. Maintenance of blood lead levels for the majority of the population below 10 µg/dL would require average population blood lead levels less than 5 µg/dL. For purposes of Risk Characterisation, 5 µg/dL is used to minimize the probability that individual blood lead levels will exceed 10 µg/dL. Thus, the NOAEL of 5 µg/dL is used for the derivation of DNEL.

Zinc compounds

Non-human information

The repeated dose toxicity of water soluble zinc sulphate has been examined in a total of three subchronic oral feeding studies.

The study by Maita et al (1981) was conducted to evaluate the subchronic toxicity (13 wk) of the test material in Wister rats. The study followed was equivalent or similar to OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents). Rats of both sexes were fed a diet containing the test material at 0, 300, 3,000 and 30,000 ppm for 13 wk. The clinical signs of the animals, body weight, food, chemical and water intake, food efficiency, haematological, biochemical examination, necropsy and organ weight and histopathological examination were performed. Animals in the 30,000 ppm group showed retarded growth along with low food intake, abnormal values in a few haematological parameters and regressive changes of the pancreatic exocrine gland. There were no remarkable clinical signs in either sex in group's ≤ 3,000 ppm. Under the test conditions, NOAEL of the test material in rats was determined to be 3000 ppm (approximately equivalent to 234 mg/kg/day corresponding 53.5 mg Zn/kg bw/day in male rats and 243 mg/kg/day in female rats).

A third study was conducted to evaluate the subchronic (13 wk) toxicity of zinc sulphate in ICR mice. The study followed was equivalent or similar to OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents). Mice of both sexes were fed a diet containing test substance at 0, 300, 3000 and 30000 ppm for 13 wk. The clinical signs of the animals, body weight, food, chemical and water intake, food efficiency, haematological, biochemical examination, necropsy and organ weight and histopathological examination were performed. Animals in the 30,000 ppm group showed retarded growth along with low food intake, abnormal values in a few haematological parameters, decreased water intake and significant deviations in biochemical parameters. Histopathological lesions included catarrh at the upper intestine, ulcers at the boundary of fore- and glandular stomach, proliferation of erythropoietic immature cells in the splenic red pulp as well as pancreatic lesions. Under the test conditions, NOAEL of zinc sulphate in mice was determined to be 3,000 ppm (approximately equivalent to 458 mg/kg/day corresponding 104 mg Zn/kg bw/day in male mice or 479 mg/kg/day in female mice.

The zinc NOAEL derived from the feeding studies with zinc sulphate was determined to be 104 mg Zn/kg bw/day in mice and approximately 53.5 mg/kg bw/day in rats. At higher doses the most important effects in the rats were the development of hypocupremia, and significant changes in the pancreas (i. e., focal acinar degeneration and necrosis) and a decreased number of pigmented macrophages in spleen.

A subchronic inhalation study was conducted to evaluate the toxic effects of zinc sulphate on cardiac changes in rats. Healthy male Wistar Kyoto rats of 12 wk age were exposed via nose only inhalation at the doses 10, 30 or 100 μg/m3 of aerosolized zinc sulphate, 5 h/day, 3 d/wk for 16 wk. Necropsy was done 48 h after the last exposure to ensure that the effects were due to chronic exposure. No significant changes were observed in neutrophil or macrophage count, total lavageable cells, or enzyme activity levels in bronchoalveolar lavage fluid, indicating minimal pulmonary effect. In the heart, decreased succinate dehydrogenase and cytosolic glutathione peroxidase activity, increased mitochondrial ferritin levels were observed, suggesting a mitochondria-specific effect. Test material induced minimal lung injury as determined by pathology, BALF markers of injury and inflammation. Cardiac gene array analysis indicated small changes in genes involved in cell signalling, a pattern concordant with known zinc effects indicating environmentally relevant levels of test material induced cardiac effects. Subchronic inhalation of the test material at environmentally relevant levels induced cardiac effects, under the test conditions.

No long-term inhalation studies allowing deriving a robust NOAEL for the inhalatory exposure of the respective zinc compounds has been identified. In one subchronic study by Wallenborn et al (2008) Wistar rats were exposed to aerosol concentration of 10, 30, and 100 ug zinc/m3. Study focused on the evaluation of effects of zinc sulphate on cardiac changes. No cardiac pathology, but cardiac gene array analysis indicated small changes in gene expression. No NOAEL identified.

Considering that the dermal absorption of zinc compounds is low (see toxicokinetics section 5.1) and the lack of acute dermal effects (see acute toxicity), this endpoint is considered not to be of concern.

Human information

Upon supplementing men and women with 150 mg Zn/day (as zinc sulphate capsules), women appeared to be more sensitive than men to the effects of high zinc intake: clinical signs such as headache, nausea and gastric discomfort were more frequent among women and women but not men had decreased activities of serum ceruloplasmin and ESOD. In some earlier oral studies in which humans were supplemented with moderately high amounts of zinc (50 mg Zn/day), a reduction in ESOD activity was also observed and again women appeared to be more sensitive to this effect. Hence, a reduction in ESOD was thought to be a sensitive indicator of copper status. However, in more recent and more sophisticated studies using the same dose level, ESOD was only marginally reduced (without a correlation with changes in copper balance), while findings on more specific copper deprivation signs (decreased serum ceruloplasmin and platelet cytochrome c oxidase) indicated that a sub-optimal intake of zinc was more effective than a moderately high intake of zinc in inducing changes associated with a decreased copper status in postmenopausal women. Given this, and the degree of the observed ESOD reduction in comparison to the natural variability in its activity, the zinc-induced decrease in ESOD activity is considered to have marginal biological significance, if any and also because it may not have been caused by an interference with copper metabolism as deep tissue SOD increases as a function of zinc exposure was observed.

Conclusions

The assessment of the toxicity after repeated exposure of the substance was based on the composition of the substance and the bioavailability and the toxicity of the most critical components in the substance (zinc and lead compounds). Based on the hazard assessment the most critical component is lead. Lead has been shown to produce significant toxicity in humans following repeated exposure. For ES and RC the blood concentration of 5µg lead/dL is used for DNEL derivation based on the neurological effects in children which is the most sensitive subpopopulation upon lead exposure.

Lead compounds have harmonised classification entry for repeated dose toxicity (STOT-RE 2). Based on the results from reliable and good quality human epidemiological studies the lead compounds should be classified for STOT-RE 1. Accordingly, the target substance will be classified in Category 1 for target organ toxicity (repeated exposure), STOT-RE 1 H372.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
No study conducted for the target UVCB substance. The selected study is conducted for lead, the most critical component in the substance with respect to chronic oral hazard.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
No study conducted for the substance. The selected study is conducted for lead, the most critical component in the substance with respect to chronic inhalation hazard.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
No study conducted for the substance. The selected study is conducted for lead, the most critical component in the substance with respect to chronic inhalation hazard..

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
No dermal studies available for the target substance itself or the most critical constituents in the target substance i.e. lead and zinc compounds.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
No dermal studies available for the target substance itself or the most critical constituents in the target substance i.e. lead and zinc compounds.

Justification for classification or non-classification

Based upon the overall observations from read across lead and zinc compounds permit the conclusion to be drawn that the substance need to be classified for repeated dose toxicity.

Current harmonized classification for lead compounds is STOT RE2. Based on the read-across data on lead compounds STOT-RE 2 is changed to STOT-RE 1 as human evidence exists for repeat dose effects on CNS, kidney and haematological (blood) systems. Thus, the substance will be classified for STOT-RE 1 H372 according to CLP Regulation 1272/2008 and T; R48/23/24/25 according to the Directive 67/548/EEC.

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Test	Control	0.05 mg/kg	0.005mg/kg	0.0015mg/kg
Manifestation of reflex (number of combinations	6.33 +/- 0.94	17.66 +/- 0.75*	10.16 +/-1.19**	6.83 +/-1.16
Consolidation of reflex (number of combinations)	3.66 +/- 2.21	35.16 +/- 1.53*	31.83 +/- 1.86**	24.16 +/- 2.89
Latent period	1.67 +/- 0.23	2.66 +/- 0.04**	2.03 +/- 0.15	1.69 +/-0.19
Conditioned Reflex	36.99 +/-6.31	2.66 +/- 0.04**	21.65 +/- 4.53	39.16 +/- 5.51
Unconditioned reflex	24.47 +/- 6.17	5.52 =/-3.25**	46.34 +/- 4.17***	25.47 +/- 5.26
Intersignal time	38.65 +/- 3.28	73.36 +/- 6.9**	44.5 +/- 6.20	35.41 +/- 5.0
Decrease of conditioned reflex	23.66 +/- 2.66	41.5 +/- 1.83*	20.5 +/-1.54	26.16 +/- 2.8
Restoration of conditioned reflex	3.83 +/- 0.86	10.66 +/- 0.55*	7.0 +/- 0.72***	3.33 +/- 0.41

Tests	Tests	0.05 mg/kg	0.005 mg/kg	0.005 mg/kg	0.0015 mg/kg
	Spermatogenesis index	3.75 +/- 0.008	3.61 +/- 0.052*	3.72 +/- 0.0013	3.74 +/- 0.015
	Number of tubules with cast-off epithelium	3.88 +/- 0.9	5.5 +/- 2.9	5.88 +/- 1.8	4.0 +/- 1.48
	Average number of spermatogonia	25.51 +/- 0.81	21.64 0.6**	20.84 +/- 1.35**	25.0 +/- 0.9
	Number of tubules with 12th meiosis stage	2.16 +/- 0.26	2.5 +/- 0.5	2.33 +/- 0.2	2.33 +/- 0.2

Test	Control	0.005 mg/kg	0.0015 mg/kg
Number of yellow bodies	11.76 +/- 0.04	11.0 +/- 0.67	10.9 +/- 1.07
Number of live embryos	9.76 +/- 0.6	8.75 +/- 0.54	9.7 +/- 1.0
Number of resorptions	0.58 +/- 0.19	0.69 +/- 0.4	0.62 +/- 0.34
General mortality of embryos	16.8 +/- 1.76	20.6 +/- 0.4*	14.18 +/- 5.7
Preimplantation mortality	0.15 +/- 0.04	0.13 +/- 0.09	0.04 +/- 0.02
Postimplantation mortality	0.04 +/- 0.017	0.08 +/- 0.03	0.06 +/- 0.039
Average weight of embryo, g	2.35 +/- 0.21	2.18 +/- 0.15	2.29 +/- 0.06
Amount of lead in placenta, mg/g	5.33 +/- 0.85	9.47 +/- 2.4	5.4 +/- 1.42
Amount of lead in embryo, mg/g	2.74 +/- 0.62	5.6 +/- 1.1*	2.7 +/- 0.6