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Description of key information

Two subchronic studies are used for endpoint assessment, one in rats (BASF Farben & Fasern AG, 1976; Report No 622-06329) and one in beagle dogs (BASF Farben & Fasern AG, 1976; Report No 611-06328). Rats and dogs were dosed at 2000, 5000 and 20,000 ppm in the feed in 90 day studies that are similar to OECD guideline 408 and 409, respectively, although ophthalmological examinations were not performed. Lead carbonate was given to the dogs as a positive control substance in concentrations of 100, 300 or 1000 ppm in the diet. In rats, mortality was not significantly elevated and lesions involved mainly relative weight fo the kidneys, however without histopathological changes. In dogs, all but one of the high dose animals died. Lesions involved mainly the kidney and the liver. Hematological changes were also observed in a dose-dependent manner. The mid dose was determined as the NOAEL in the rat study, however, in the dog study, no NOAEL could not be determined. Two additional subchonic studies are also available (Kennedy et al., 1975), also conducted in rats and in Beagle dogs, where lead and lead containing pigments were evaluated. Although the exerted effects were qualitatively similar, they were in general more severe or earlier in appearance with white lead than with the pigments of lower lead content. Medium Chrome Yellow, which was the lowest content of acid soluble lead, was clearly less effective than White Lead or the other pigments of similar lead content but for the group of pigments, the effects could not be quantitatively related to lead or chromium content or to acid soluble lead.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well documented study similar to guideline (non-GLP, no ophthalmological examination, composition of test substance not specified)
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity in Non-Rodents)
Version / remarks:
blood lead level and tissue distribution of lead were also evaluated
Deviations:
yes
Remarks:
no ophthalmological examination, composition of test substance not specified
GLP compliance:
no
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: dogs acquired from own colony and under observation for 2 weeks prior to the start of the investigation
- Age at study initiation: appr. 6 months
- Weight at study initiation: 7.2 kg for males and 6.4 kg for females
- Fasting period before study: no data
- Housing: in kennels equipped with outside runs, 4 dogs of the same sex and group being accommodated in a single kennel.
- Diet (e.g. ad libitum): Purina Dog Chow, Ralston Purina Company, St Louis, Missouri. During the entire study, all diets were made from 1 single lot of stock ration, assayed as containing an average of 2.43±0.09 µg Pb/g of diet.
- Water (e.g. ad libitum): no data
- Acclimation period: not applicable

ENVIRONMENTAL CONDITIONS
no data
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Pre-weighed amounts were distributed into self-feeding units and maintained in excess of the animals' consumption. One such unit was available to the dogs in each kennel for 5 hours each day. During these 5-hour intervals, food was consumed on an ad libitum basis. At the end of each 7-day period, all unconsumed food was collected and weighed. Food consumption was then calculated and recorded.
- Mixing appropriate amounts with (Type of food): At the beginning of each week, the appropriate dietary constituents for each of the groups were thoroughly blended in a Hobart mixer.
- Storage temperature of food: no data

VEHICLE
not applicable
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 2000, 5000 and 20000 ppm in diet (corresponding to appr. 75.4, 179.6 and 287.1 mg/kg bw per day considering mean weekly food consumption of 264, 251.5 and 100.5 g/kg)
Basis:
nominal in diet
No. of animals per sex per dose:
4
Control animals:
yes, plain diet
Details on study design:
Outline of Experiment: see below (Table 1)
Post-exposure period: none
Positive control:
The 3 positive control groups were fed lead carbonate at dietary levels of 100, 300 and 1000 ppm, respectively.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: the dogs were under observation during the investigation and were examined daily for signs of toxicity

BODY WEIGHT: Yes
- Time schedule for examinations: initially, the body weight of each dog in every group was determined and recorded. Thereafter, weighing was conducted weekly for the duration of the test.

FOOD CONSUMPTION, FOOD EFFICIENCY AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, at the beginning of each week, diets in excess of anticipated consumption were distributed into feeding units. At the end of each week unconsumed food was weighed and mean food consumption for males and females of each group was determined.

WATER CONSUMPTION: water was available at all times.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: just prior to the inception of the study and after 30 and 84 days of testing.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- Parameters examined: Total Leukocyte Count, Erythrocyte Count, Hemoglobin Concentration, Hematocrit Value, Cell Indices (mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration), Differential Leukocyte Count, Erythrocyte Morphology

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see above
- Animals fasted: No data
- Animals analysed: Clinical Blood Chemistry Studies (conducted on control and high dose only)
- Parameters examined: Serum Alkaline Phosphatase Activity (SAP), Serum Glutamic-Pyruvic Transaminase Activity (SGPT), Blood Urea Nitrogen Concentration (BUN), Serum Glucose Concentration, Serum Glutamic-Oxalacetic Transaminase Activity (SGOT)

URINALYSIS: Yes
- Time schedule for collection of urine: see above.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: Glucose Concentration, Albumin Concentration, Microscopic Elements Examination (leukocytes, erythrocytes, crystals), pH, Specific Gravity, Aminolevulinic Acid (ALA),

NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, At the conclusion of the investigation, all animals were sacrificed by exsanguination immediately following induction of anesthesia with an intravenous injection of a mixture of Ketaset and Totaltox. A complete gross necropsy examination was conducted upon each dog and the weights of the following organs were obtained: adrenals, brain, gonads, heart, kidneys, liver, spleen, thyroid gland and pituitary gland.
HISTOPATHOLOGY: Yes, at autopsy, representative specimens of the following tissues and organs were fixed in 10% neutral buffered formalin (testes, epididymides fixed in Bouin's solution), embedded in paraffin and stained with Hematoxylin and Eosin: Adrenal Glands, Optic Nerves, Aorta (thoracic), Pancreas, Brain (cerebrum, cerebellum and pons), Parathyroid Glands, Peripheral Nerve (sciatic), Caecum Pituitary Gland, Colon, Prostate Gland, Epididymis, Salivary Gland (submaxillary), Esophagus, Small Intestine (duodenum, jejunum, and ileum), Eyes, Gonads, Spinal Cord, Heart, Spleen, Kidneys, Sternum (bone marrow), Liver, Stomach (cardia, fundus, and pylorus), Lung, Lymph Nodes (cervical and mesenteric), Thyroid Glands, Trachea, Mammary Gland, Urinary Bladder, Muscle and Uterus
Other examinations:
Blood Lead Determinations: conducted just prior to study inception and after 30, 60 and 84 days of testing.
Tissue Analyses: samples of liver, kidney, bone and brain tissues were analyzed for lead and chromiurn content by means of atomic absorption spectrophotornetry.
Statistics:
One-Way Analysis of Variance followed by either the Tukey's (equal population size) or the Scheffels (unequal population size) Multiple Comparison Test or Kruskal-Wallis Statistic test followed by the Kruskal-Wallis Multiple Comparison Test. Historical information developed at these laboratories were also considered. The presence of statistical significance does not lead per se to the conclusion that test material-related effects are occurring.
Details on results:
CLINICAL SIGNS
Emesis was noted among dogs fed 20000 ppm test substance, but only during the first 2 days of testing. The response occurred within the first hour of feeding the test diet.
Lethargy, anorexia, dehydration and emaciation were noted in all dogs which died or were sacrificed in extremis during the study (8/8 at 20000 ppm test substance). These reactions appeared (and became progressively worse) 7 to 10 days prior to sacrifice.
No abnormal behavioral reactions were seen among dogs fed either 2000 or 5000 ppm test substance or among any fed at the 3 levels of lead carbonate.

MORTALITY
Mortality occurred in all dogs at the highest level of test substance. With 1 exception (1 male), all dogs were sacrificed in extremis.

BODY WEIGHT AND WEIGHT GAIN (Table 2)
An Analysis of Variance was conducted first and any significant effects disclosed were further analyzed by either the Tukey's (equal population size) or the Scheffe's (unequal population size) Multiple Comparison Test.
Animals fed 100 ppm lead carbonate showed overall body weight gains comparable to those of the untreated controls.
At 300 ppm lead carbonate, 2 males showed body weight effects: one, a slight weight gain suppression and a second, a slight weight loss. However, the 2 remaining males and all 4 females at this level displayed overall weight gains comparable to those of untreated controls.
At 1000 ppm positive control substance, a slight body weight gain suppression was seen in 1 male. All remaining dogs had overall weight gains comparable to those of untreated controls.
One male at 2000 ppm test substance showed a slightly lower overall body weight gain than seen among untreated males. However the weight gain was considered normal for a dog of this age. The remaining animals at this level had overall body weight gains comparable to those of untreated controls.
At 5000 ppm test substance, 3 males showed slight body weight gain suppression and a female showed a slight weight loss. The remaining dogs at this level showed overall body weight gains either comparable to those of untreated controls or considered normal for their age.
Severe body weight loss was seen in all animals at the 20, 000 ppm level.


FOOD CONSUMPTION AND COMPOUND INTAKE (Table 3)
Food intake at either 100, 300 or 1000 ppm positive control substance did not differ significantly from that of untreated controc4s.
Among dogs fed test substance, food consumption at either 2000 or 5000 ppm was comparable to that of untreated controls but was severely reduced at 20000 ppm.

HAEMATOLOGY
- The following hematologic studies showed no treatment-related effects at any of the levels of either lead carbonate or test substance: total leukocyte count, erythrocyte count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, lymphocytes, monocytes, eosinophils, basophils, erythrocyte shape.
- Treatment-related effects were noted in the following hematologic parameters: hemoglobin (decrease), hematocrit (decrease), mean corpuscular volume (decrease), segmented neutrophils (decrease), banded neutrophils (increase), and erythrocyte morphology (nucleation, size alteration and color alteration)
- When seen among dogs fed lead carbonate, these effects were marginal only, with the exception of banded neutrophils (positive effect all levels) and erythrocyte nucleation and size (positive effect at 1000 ppm only).
- Effects among animals fed test substance were only marginal at 2000 ppm but much more positive at 5000 ppm. At 20000 ppm, mortality precluded a meaningful evaluation.

CLINICAL CHEMISTRY
- Isolated instances of elevated blood urea nitrogen, depressed serum glucose, elevated serum alkaline phosphatase, elevated serum glutamic-oxalacetic transaminase and elevated serum glutamic-pyruvic transaminase were seen at the 2 highest dietary levels of test substance. However, these values were felt to be the result of either the poor nutritional state or moribund condition of these animals.
- Blood chemistry values for dogs fed 2000 ppm test substance and for dogs fed the 3 levels of lead carbonate did not differ significantly from those of the untreated controls.

URINALYSIS
Urinary aminolevulinic acid (ALA) concentrations were elevated at the highest levels of both the positive control and the test substances. At the 2 lower levels of either compound, ALA concentrations were comparable to those of untreated controls.
The remaining urinalyses revealed no treatment-related abnormalities at any of the levels of either compound tested.

NEUROBEHAVIOUR
Not evaluated

GROSS PATHOLOGY
Aside from emaciation and dehydration (20000 ppm test substance), no treatment-related gross pathologic lesions were seen in the tissues and organs of any of the positive control or test animals.

ORGAN WEIGHTS
- With the exception of 1 animal, evaluation of organ to brain weight ratios revealed no direct treatment-related abnormalities in organ weights to any of the levels of either compound. A female fed 5000 ppm test substance showed an elevated liver weight; however, by virtue of this being an isolated finding, it was considered to be of questionable significance.
- Depressed testes weights among males at the highest level of test substance were considered as an indirect treatment-related effect, the result of the physical stress they experienced during the study and their sexual immaturity at time of sacrifice.
- Where elevated organ to body weight ratios were seen in dogs fed the 2 highest levels of test substance, they were attributable to either body weight loss or suppressed body weight gains rather than increased organ weights.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related lesions observed by light microscopy occurred in dogs fed either lead carbonate or test substance. The nature and severity of the lesions varied, but, in most instances, these lesions were related to either the compound fed, dose level, or length of exposure.
- Renal and bone marrow lesions occurred among dogs fed 1000 ppm of lead carbonate (PC-III Group). The kidney lesions consisted of focal degenerative tubular changes which affected all dogs in this group. A slight hypercellularity of erythrocytic precursor cells in the sternal bone marrow was evident in 1 dog of this group that was also anemic. In general, the above lesions were similar to, but less severe than those reported in dogs fed higher levels of lead carbonate in a previous study.
- Dose-related lesions involving either the kidney, bone marrow, and/or liver were present among dogs in all groups fed test substance. There were focal degenerative lesions present involving kidney tubules which affected a few T-I and most T-II and T-III dogs. Hypocellularity of the sternal bone marrow occurred in 2 of the T-III animals. The liver lesion, present in 6 of the 8 T-III animals, was characterized by degeneration and necrosis of hepatocytes located predominantly in the centrilobular region of the liver lobules.
- Testicular lesions consisting of either hypospermatogenesis, focal testicular degeneration, or both, occurred among most of the males of all 3 dose groups of dogs fed lead carbonate (PC Groups I, II, III). No adverse effects were noted in the ovaries and uterus of females after they were fed lead carbonate for 90 days. The exact cause for the gonadal lesions among males fed lead carbonate was not determined. These lesions probably resulted from impairment in their nutritional status with delayed development of puberty. These lesions were regarded as an indirect effect rather than a direct effect of lead carbonate on the testes.
All dogs in the T-1 and T-II groups fed test substance had reached sexual maturity by the end of the study. One T-III female, sacrificed in extremis after 50 days on test, had reached sexual maturity. The remaining 7 dogs of the T-III group (4 males, 3 females) had not reached sexual maturity when sacrificed in extremis prior to the 27th test day. Males of all 3 test groups had focal testicular degeneration which was most severe among the T-III group. The gonadal lesions present among animals fed test substance appeared to be primarily related to the age of animal at time of sacrifice and only indirectly related to ingestion of the test material.
- In the gastrointestinal tract of some of the positive control and test animals, there was increased mitotic activity of the mucosal epithelial cells located in the glandular crypts. This finding was regarded as a regenerative change indicative of an increased rate of proliferation of mucosal cells. It was noted throughout the gastrointestinal tract, but was most pronounced in the distal portion (ileum) of the small intestine.

HISTORICAL CONTROL DATA (if applicable)
were considered for data interpretation
Dose descriptor:
LOAEL
Effect level:
ca. 75.4 other: mg/kg bw/day (actual dose received in diet)
Sex:
male/female
Basis for effect level:
other: hematological changes; lesions involving the kidney, bone marrow, intestines and liver
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
75.4 mg/kg bw/day
Study duration:
subchronic
Species:
dog

Additional information

In a 90 study, rats (20/sex/dose) were fed dietary concentrations of 2,000, 5,000 or 20,000 ppm of the test substance, which corresponds to approximately 160, 400 and 1600 mg test substance/kg bw, respectively. Treatment related mortality did not occur and food consumption as well as body weights were comparable to control animals. Hematology, clinical blood chemistry and gross pathological findings did not differ significantly between dose group and control group animals. Aminolevulinic acid was slightly elevated among males fed 2,000 ppm at examination days 30 and 60 only. Blood lead concentrations were elevated in a dose-related manner. The lead content of the bone and kidney samples was markedly increased among the test animals. The brain lead content (males only) and liver lead content (males and females) were only slightly increased when compared with those of the controls. The chromium content was increased in the kidney and liver tissues among most test animals. Brain chromium content was slightly elevated among a few females fed 2,000 ppm or more and among the males fed 20,000 ppm. Brain chromium content among males fed 2,000 or 5,000 ppm was either less than, or comparable to, that of the controls. Increases in chromium content in bone samples from animals fed either 5,000 or 20,000 ppm were due to detectable amounts in the bone samples from 1 animal from each of these levels . No detectable amount of chromium was found in the bone samples from any of the animals fed 2,000 ppm. The absolute liver and relative kidney and liver weights were significantly elevated in males fed 20,000 ppm, however, treatment-related histopathological changes were not noted upon microscopic examination.

In a further 90 day study, beagle dogs (4/sex/dose) were fed dietary concentrations of either 2,000, 5,000 or 20,000 ppm (ca. 70, 180 and 700 mg/kg bw, respectively) of the test material (groups T-I, T-II and T-III,) or 100, 300 or 1,000 ppm of lead carbonate (groups PC-I, PC-II and PC-III). All but one of the T-III animals were sacrificed in extremis which precluded a meaningful examination of these animals. Body weight development was not affected in animals fed 2,000 ppm of the test material or 100 ppm of lead carbonate. Animals of the other T- or PC-groups showed a dose-related increase in body weight suppression, severely so in T-III animals. Abnormal behavioral reactions were not observed in any of the T-group or PC-group animals. The lead content of liver, kidney, bone and brain tissues was elevated at all levels of either compound. Increases were directly proportional to the length of time on test and/or dietary concentration. In dogs fed the 3 levels of lead carbonate, chromium content in all 4 tissues was comparable to that seen in untreated controls, either below detectable limits or found in trace amounts only. At 2,000 ppm test material, chromium content was elevated in all 4 tissues examined, the liver showing the highest concentration, kidneys the next highest, then bone; brain tissue showed the lowest concentration. At 5,000 ppm, a similar pattern of elevations was seen, with the concentrations in kidney, bone and brain tissues essentially comparable to those found in 2,000 ppm animals. However, in liver tissue, the chromium content in 2,000 ppm animals was considerably higher than that found in 5,000 ppm animals, though both levels were tested for 90 days. At 20,000 ppm, chromium content of liver and kidney tissues was much lower than that found in either 2,000 or 5,000 ppm dogs and in bone, chromium was below detectable limits. These findings correlate with the short time (3 weeks) the animals were on test. In brain tissue, however, chromium content was comparable to concentrations found at the 2 lower levels.

Hematological studies revealed treatement related effects in parameters related to hematopoiesis (hemoglobin, hematocrit: decrease) and additionally, a decrease in the mean corpuscular volume, segmented neturophils, banded neutrophils and changes in the erythrocyte morphology (nucleation, size alteration, color alteration) were observed. Blood chemistry revealed no relevant changes related to the treatment with either lead carbonate or the test substance.

Organ weights were unaffected by the treatment with the test substance. Treatment-related lesions observed by light microscopy occurred in dogs fed either lead carbonate or test substance. The nature and severity of the lesions varied, but, in most instances, these lesions were related to either the compound fed, dose level, or length of exposure. Renal and bone marrow lesions occurred among dogs fed 1,000 ppm of lead carbonate (PC-III group). The kidney lesions consisted of focal degenerative tubular changes which affected all dogs in this group. Dose-related lesions involving either the kidney, bone marrow, and/or liver were present among dogs in all groups fed the test material. There were focal degenerative lesions present involving kidney tubules which affected a few T-I and most T-II and T-III dogs. Hypocellularity of the sternal bone marrow occurred in 2 of the T-III animals. The liver lesion, present in 6 of the 8 T-III animals, was characterized by degeneration and necrosis of hepatocytes located predominantly in the centrilobular region of the liver lobules.

Testicular lesions consisting of either hypospermatogenesis, focal testicular degeneration, or both, occurred among most of the males of all 3 dose groups of dogs fed lead carbonate. No adverse effects were noted in the ovaries and uterus of females after they were fed lead carbonate for 90 days. The exact cause for the gonadal lesions among males fed lead carbonate was not determined. These lesions probably resulted from impairment in their nutritional status with delayed development of puberty. These lesions were regarded as an indirect effect rather than a direct effect of lead carbonate on the testes. Males of all 3 test groups had focal testicular degeneration which was most severe among the T-III group. The gonadal lesions present among animals fed the test substance appeared to be primarily related to the age of animal at time of sacrifice and only indirectly related to ingestion of the test material. In the gastrointestinal tract of some of the positive control and test animals, there was increased mitotic activity of the mucosal epithelial cells located in the glandular crypts. This finding was regarded as a regenerative change indicative of an increased rate of proliferation of mucosal cells. It was noted throughout the gastrointestinal tract, but was most pronounced in the distal portion (ileum) of the small intestine.

In conclusion, the test substance's bioavailability was conclusively shown by findings of lead and chromate ions in several tissues, independent of the species tested. Also, although the dog seems to be the more sensitive species, the target organs (liver and kidney) seem to be identical. Hematopoiesis was affected in the dog and early signs (increase in ) was also seen in the rat. Although neurotoxicity was not specifically assessed, abnormal behavior was not noted in either the rat or the dog.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
This study is the key study for this endpoint with the most critical effect level.

Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: hematopoiesis; digestive: liver; urogenital: kidneys

Justification for classification or non-classification

The accumulation of lead ions in different tissues in a time and dose-dependent manner justifies a classification with R33 according to EU-criteria and STOT repeated exposure Cat. 2. A further classification is not warranted because of the R45 / Carc. Cat. 1A categorization (see carcinogenicity).