polymeric zinc propylenebis(dithiocarbamate);propineb (ISO)

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29-June-1998 to 14-February-2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

1998

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Version / remarks:

And EPA OPPTS 870.1300

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EU Directive 92/69/EEC

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

polymeric zinc 1,2-propylenebis(dithiocarbamate)

EC Number:

620-365-5

Cas Number:

9016-72-2

IUPAC Name:

polymeric zinc 1,2-propylenebis(dithiocarbamate)

Test material form:

solid

Remarks:

Powder

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Hsd Cpb: WU (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 weeks
- Housing: During the acclimatization and study periods the animals were housed singly in conventional Makrolon® Type II cages.
- Diet: Standard fixed-formula diet
- Water: Drinking quality tap-water ad libitum
- Acclimation period: one and a half weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: approximately 50 %
- Air changes: approximately 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h/12 h; artificial light from 6.00 a.m. to 6.00 p.m.

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

ca. 2.3 µm

Geometric standard deviation (GSD):

2.2

Details on inhalation exposure:

A subacute 4-week inhalation toxicity study with the aerosolized (dust) LH 30/Z 80 VM 00705/1096 (hereafter referred to as test substance) was performed. 10 male and 10 female Wistar rats per group were exposed to the dust using a directed-flow nose-only mode of exposure. Exposure was 6 hrs/day on five days/week for four consecutive weeks.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Nominal concentrations: Nominal concentrations were not calculated since this would have required a derangement of the dust generating system and in doing so this may had caused an increase in the temporal (day-to-day) variability of test concentrations.
Actual concentrations: The test-substance concentration was determined by gravimetrical analysis (filter: Cellulose-Acetate-Filter, Sartorius, Gottingen, Germany; balance: Mettler AE 100). Chamber samples were taken in the vicinity of the breathing zone. The number of samples taken was sufficient to characterize the test atmosphere and was adjusted so as to accommodate the sampling duration and/or the need to confirm specific concentration values.

Duration of treatment / exposure:

6-hrs/day on five days/week for four consecutive weeks

Frequency of treatment:

5 days/week for 4 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

21.95 mg/m³ air

Remarks:

High Dose Group

Doses / concentrations 2

Dose / conc.:

11.2 mg/m³ air

Remarks:

Mid Dose Group

Doses / concentrations 3

Dose / conc.:

3.97 mg/m³ air

Remarks:

Low Dose Group

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes

Details on study design:

At the end of the acclimatization period the rats were assigned to four exposure groups and 10 rats per group and gender were exposed by inhalation (both sexes in one inhalation chamber) to design concentrations of 0 (conditioned air), 4, 10, and 25 mg test substance (dust)/m3 air for 4 weeks (6 hours/day, 5 days/week; starting with day relative 0). One day following the last exposure rats were anesthetized with sodium pentobarbital and exsanguinated as described in the respective section.

Examinations

Observations and examinations performed and frequency:

Body weights:
Body weights of all animals were measured before exposure twice a week on a Fridays and Mondays. Data were collected on-line using a validated computerized system.

Clinical signs:
The appearance and behavior of each rat was examined carefully before and after exposure and at least once daily on exposure-free days. Weekend assessments were made once a day (morning). Assessments from restraining tubes were made only if unequivocal signs occurred (e.g. spasms, abnormal movements, severe respiratory signs).

Clinical laboratory tests:
General clinical chemistry tests were performed at the end of the 4-week exposure period on 10 animals per group and sex. The terminal blood samples for these tests were obtained by cardiac puncture of the deeply anesthetized rats (pentobarbital narcosis). The blood required for the glucose determination was obtained during the next to last study week (after urine collection) from the caudal vein of non-fasted rats.

Urine Metabolites (TTCA):
Urine was sampled from all rats per group and sex toward the end of study period. The rats' urine was individually collected overnight (approximately 4 p.m. to 8 a.m.) from animals in metabolism cages (with watering bottles, pulverized feed ad libitum). The construction of the urine collection equipment did not allow contamination of urine with water or chow. The collection containers were cooled to nominal 12 °C in order to prevent/suppress uncontrolled growth of microorganisms overnight. To allow quantification of the metabolite, TTCA concentrations were reported also relative to creatinine.

Ophthalmological Examination:
Eye examinations were performed prior to the first exposure and towards the end of the exposure period. For examinations, an indirect ophthalmoscope was used. Five to ten minutes prior to the examination, the pupils were dilated with mydriatic. The eyes were examined for changes in the retina, vitreous humor, lens, cornea and external eye surface.

Organ weight determinations:
The following exsanguinated organs were weighed: Adrenals, Brain, Heart, Kidneys, Liver, Lungs, Ovaries, Spleen, Testes, Thymus. The organ-to-body relationships were specified in both absolute and relative terms. The relative organ weights were calculated by normalizing to 100 grams body weight (individual organ weight divided by body weight times 100). The body weight used as a basis for these calculations was determined prior to necropsy of the pertinent animal. A supplementary analysis of the organ-to-brain weight relationship was performed.

Sacrifice and pathology:

Necropsy:
At the end of the exposure period (one day after the last exposure to the test substance) all surviving rats were sacrificed after cardiac exsanguination using sodium pentobarbital (intraperitoneal injection) for euthanasia. All rats were given a gross-pathological examination. Consideration was given to performing a gross necropsy on animals as indicated by the nature of toxic effects, with particular reference to changes related to the respiratory tract. All gross pathological changes were recorded and evaluated.

Other examinations:

Histopathology:
For the histopathological examination, the following organ tissues were fixed: Adrenal glands, Aorta, Brain (Cerebrum, Cerebellum, Pons/Medulla), Epididymides, Esophagus, Eyes, Eyelids, Extraorbital lacrimal glands, Femur (incl. joint), Harderian glands, Head (with nasal and paranasal cavities), Heart, Intestine (Duodenum, Jejunum, lleum, Cecum, Colon, Rectum and remaining intestine), Kidneys, Larynx, Liver, Lung, Lymph nodes (lung-associated, mandibular and mesenteric), Mammary gland, Ovaries, Oviducts, Optical nerve, Pancreas, Pituitary, Prostate, Salivary glands, Sciatic nerve, Seminal vesicles (incl. Coagulating glands), Skeletal muscle
(Thigh), Skin (Mammary region), Skin (Muzzle), Spinal cord (cervical, thoracic, lumbar), Spleen, Sternum, Stomach (Forestomach and Glandular stomach), Testes, Thymus, Thyroid gland with Parathyroid glands, Teeth, Tongue, Trachea, Ureter, Urethra, Urinary bladder, Uterus with uterine cervix, Vagina, and Zymbal gland and all organs or tissues with macroscopic findings. All organs not scheduled for fixation that exhibited gross changes were also fixed if necessary. Bone marrow smears were prepared but not examined.

Statistics:

For the statistical evaluation of samples drawn from continuously distributed random variates three types of statistical tests were used, the choice of the test being a function of prior knowledge obtained in former studies. Provided that the variate in question could be considered approximately normally distributed with equal variances across treatments, the Dunnett test was used, if heteroscedasticity appeared to be more likely a p value adjusted Welch test was applied. If the evidence based on experience with historical data indicated that the assumptions for a parametric
analysis of variance could not be maintained, distribution-free tests in lieu of ANOVA were carried out, i.e. the Kruskal-Wallis test followed by adjusted MWW tests (U tests) where appropriate. Global tests including more than two groups were performed by sex and date, i.e. each sex x date level defined a family of tests in the context of multiple comparison procedures (Miller (1981)). Within such a family, the experiment-wise error was controlled. If not otherwise noted, all pair-wise tests were two-sided comparisons.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The following list of signs is focusing on toxicologically significant signs only.
Group 1 - 4/males and 1 - 2/females: All rats tolerated the exposure without signs.
Group 3/females: Flaccidity and paralysis of hind-limbs, limp, prostration (lying on belly), reduced motility, emaciation, subdue demeanor, nostrils with red encrustations.
Group 4/females: Flaccidity of hind-limbs, limp, reduced motility, emaciation, subdue demeanor, nostrils with red encrustations, piloerection.

Mortality:

no mortality observed

Description (incidence):

The exposures were tolerated without mortality. Two female rats were killed in a moribund condition (related to paralysis of hind-limps).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no toxicologically consistent effect on body weight up to and including high dose group in males whereas in females there was a statistically significant decrease of body weights in the high-level exposure group. Apart from this group there was an apparent increase in body weights during the exposure-free weekend. Furthermore, it should be noted that the changes in the trend in the body weights of females rats were related to the sacrifice of moribund rats.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmological examination performed (prior to the start of the study, towards the end of the study) revealed no conclusive evidence of test substance-induced changes in the dioptric media or in the fundus.

Haematological findings:

no effects observed

Description (incidence and severity):

Conclusive and toxicologically significant effects on the hematological parameters or clotting time (HQUICK) were not observed.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

As far as statistically significant changes were observed they are considered to be incidental and appear not to be indicative of any change of pathodiagnostic relevance. This interpretation is supported by the absence of unequivocal concentration-dependent effects and also in relation to the respective historical data

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Description (incidence and severity):

Experimental data demonstrate that no specific concentration-dependent effects occurred. Moreover, there was no indication of any concentration-dependent effect with regard to cellular or semi-quantitative parameters (e.g. urothelial cells or blood).

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

In the mid (4/10) and high dose (8/10) group females experienced abnormal reflexes (grip strength, tonus, and righting response were decreased).
The examination of reflexes revealed decreased grid-gripping resistance of the animal to pull in group 4 (high dose females) and also in group 3 (mid-dose females only). In some cases this decrease gained statistical significance. Footsplay was statistically significantly decreased in group 4 (females) only.
Signs and observations:
Mid dose group/females: Flaccidity and paralysis of hind-limbs, limp, prostration (lying on belly), reduced motility, emaciation, subdue demeanor, nostrils with red encrustations.
High dose group/females: Flaccidity of hind-limbs, limp, reduced motility, emaciation, subdue demeanor, nostrils with red encrustations, piloerection.

Reflex measurements:
The examination of reflexes revealed decreased grid-gripping resistance of the animal to pull in high dose group (females) and also in mid dose group (females only). In some cases this decrease gained statistical significance. Foot-splay was statistically significantly decreased in high dose group (females) only.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically significant changes in absolute or relative organ weights (vs. brain weight) except the elevated absolute and relative lung weights in the 25 mg/m³ group (high-dose). Changes of organ weights relative to body weights are not considered to be of pathodiagnostic relevance due to the progressive decrease in body weights of female rats.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The gross pathological examination of the rats sacrificed at the end of the exposure period did not reveal any evidence of group-specific or treatment-related organ damage.

Neuropathological findings:

effects observed, treatment-related

Description (incidence and severity):

Female rats exposed to 11.2 and 21.95 mg/m³ experienced clinical signs indicative of neuromuscular effects (flaccidity and paralysis of hind-limbs, limp, reduced motility) and effects considered to be non-specific or secondary, such as emaciation, subdue demeanor, nostrils with red encrustations, and piloerection.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In all dust exposure groups enlarged and/or foamy macrophages and increased
intra-alveolar material, often associated with a focal septal thickening, were
observed. The intensity of response (effect) appears to be causally related with the
intensity of exposure, i.e., particle load rather than ensuing biological response, i.e.,
inflammation. The most salient histopathological findings were related to the skeletal
muscle (atrophy of muscle fibers, increased number of nuclei and focal degeneration)
in females of the 11.2 and 21.95 mg/m3 exposure groups. Although an
additional stain has been used to visualize potential axonopathic effects, there was
no evidence of any damage of axons, i.e., there was no apparent experimental
evidence of an axonopathic mode of action.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Rectal temperature:
In comparison to the concurrent control groups, there was no evidence of a conclusive change on body (rectal) temperature in any group. Additionally, the temperature measurements made on control animals demonstrate clearly that the animal restrainer had no apparent effect on the body temperature.

Protein Electrophoresis:
The protein electrophoresis did not show any effects of pathophysiological relevance. The statistically significant change of albumin is considered to be incidental because of absence of significant effects on concentrations of protein and albumin.

Liver Tissue:
Statistically significant effects on the hepatic triglyceride concentrations or on the hepatic cytochrome P450 activity were not observed.

Metabolites in Urine:
Comparisons between groups revealed that TTCA in urine was increased in a concentration- dependent manner. In female rats the increase in urine-metabolites was higher than in the respective male group.

Effect levels

1

Key result

Dose descriptor:

LOAEC

Remarks:

local

Effect level:

3.97 mg/m³ air

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

3.97 mg/m³ air

Based on:

test mat.

Sex:

female

Basis for effect level:

behaviour (functional findings)

clinical signs

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

3.97 mg/m³ air

System:

musculoskeletal system

Organ:

other: muscle

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Any other information on results incl. tables

Tables of results are provided in the overall remarks section below.

Applicant's summary and conclusion

Conclusions:

This study appears to suggest that the test item has two mode of actions, viz. local effect to the lower respiratory tract of male and female rats at exposure concentrations equal to or exceeding 3.97 mg/m³ air and in female rats a systemic effect on musculature at exposure levels of 11.2 and 21.95 mg/m³. Pulmonary effects did neither demonstrate gender-specific nor consistent concentration-dependent inflammatory effects whilst neuromuscular effects were predominant in females, i.e., the gender experiencing complementary clinical signs. It appears that elevated urinary TTCA-concentrations and increased evidence of neuromuscular effects are associated. Taking all findings into account 3.97 mg/m³ is considered to be a systemic no-effect-level, however, it does not appear to be a noeffect-level in regard to the pulmonary effects observed.

Executive summary:

A subacute 4-week inhalation toxicity study with the aerosolized (dust) test item (hereafter referred to as test substance) was performed. 10 male and 10 female Wistar rats per group were exposed to the dust using a directed-flow nose-only mode of exposure. Exposure was 6-hrs/day on five days/week for four consecutive weeks. The rats were exposed to mean actual concentrations of 0 (conditioned dry air), 3.97, 11.2, and 21.95 mg/m³ air, respectively. In all dust exposure groups, the aerosol was highly respirable to rats, i.e., the average mass median aerodynamic diameter (MMAD) was approximately 2 μm, the geometric standard deviation (GSD) was ca. 2.
During the study, the body weights were determined twice per week (on Fridays and Mondays). Clinical signs were recorded daily before and after exposure and once during the exposure-free weekend. Rectal temperatures and reflexes were examined repetitively during the course of study. Additionally, due to the anticipated mode of action, foot-splay and grip-strength were examined in a quantitative manner. At sacrifice blood was sampled for determinations of clinico-chemical and hematological endpoints. Urinalysis was performed near to the end of the exposure period. Determinations of the metabolite TTCA (2-thiothiazolidine-4-carboxylic acid) in urine were also made near to the end of the exposure period. During sacrifice, gross pathological examination of rats were made and selected organs were collected for gravimetric and histopathological examinations.
Results: Comparisons between groups revealed that TTCA in urine was increased in a concentration-dependent manner. Moreover, in female rats this increase in urine-metabolites was higher than in the respective male groups. It appears that in the female rats exposed to 11.2 and 21.95 mg/m³ this increase was over-proportional
when compared with the low-level exposure group. These findings appear to suggest that the capacity of females to form putatively more toxic species of the parent test substance may be higher than in males, i.e., this appears to be a possible cause of making female rats more susceptible than males.
The concentrations up to and including 21.95 mg/m³ air were tolerated by all rats without mortality, though it should be noticed that one female rat in each of the intermediate and high-level groups was euthanized in a moribund condition. Male rats of these groups did not elaborate any clinical signs nor was there any statistically significant change on body weights. On the other hand, female rats exposed to 11.2 and 21.95 mg/m³ experienced clinical signs indicative of neuro-muscular effects (flaccidity and paralysis of hind-limbs, limp, reduced motility) and effects considered to be non-specific or secondary, such as emaciation, subdue demeanor, nostrils with red encrustations, and piloerection. Quantitative measurements of grip strength and foot splay complement the clinical observations, i.e., they appear to suggest a disturbance in neuromuscular function in the females of the high-and intermediate-level exposure groups. The onset was time-related, i.e., with increasing duration of study the intensity of effects increased. Statistically significantly decreased body weights were observed only in the female rats exposed to 21.95 mg/m³.
There was no evidence of any concentration-dependent clinico chemical, including thyroid-hormones, or hematological effects considered to be of pathodiagnostic relevance. Urinalysis was unobtrusive. Determination of the hepatic cytochrome P450 and hepatic triglycerides did not demonstrate any specific or concentration-dependent effects.
There were no toxicologically significant changes in absolute or relative organ weights except a statistically significantly increased absolute and relative (versus brain weight) lung weight in the 21.95 mg/m³ group.
The histopathological evaluation revealed in all groups exposed to the test substance enlarged and/or foamy macrophages, associated with focal thickening of the alveolar septa and increase of intra-alveolar material. Additional histopathological findings showed effects to the skeletal muscle (atrophy of muscle fibers, increased number of nuclei and focal degeneration) in females of the 11.2 and 21.95 mg/m³ exposure groups.
In summary, this study appears to suggest that the test item has two mode of actions, viz. local effect to the lower respiratory tract of male and female rats at exposure concentrations equal to or exceeding 3.97 mg/m³ air and in female rats a systemic effect on musculature at exposure levels of 11.2 and 21.95 mg/m³. Pulmonary effects did neither demonstrate gender-specific nor consistent concentration-dependent inflammatory effects whilst neuromuscular effects were predominant in females, i.e., the gender experiencing complementary clinical signs. It appears that elevated urinary TTCA-concentrations and increased evidence of neuromuscular effects are associated. Taking all findings into account 3.97 mg/m³ is considered to be a systemic no-effect-level, however, it does not appear to be a noeffect-level in regard to the pulmonary effects observed.