polymeric zinc propylenebis(dithiocarbamate);propineb (ISO)

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May-2002 to June-2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

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:

SPF-bred, HsdCpb:WU

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks
- Housing: The animals were housed individually in cages Type II A on low-dust wood granulate.
- Diet: Fixed-formula standard diet ad libitum
- Water: Tap water ad libitum
- Acclimation period: At least 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 22±2°C
- Humidity: approx. 55 ± 5%
- Air changes: >=10 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours rhythm

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

The test item, Propineb/LH30/Z, was blended using a mixing granulator with meals at room temperature and maximally used over 8 days. The amount of test substance per concentration was taking into account the actual content of 82.3%.
The test substance, propineb, was administered via diet from the start of the study until either spontaneous death, moribund sacrifice or scheduled sacrifice of the animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical determination of test substance content and homogeneity of food mixes used in this study as well as test substance stability and homogeneity of 2000 ppm food mixes were performed. As known from published data (Merck-Index) Propineb decomposes at strongly acid or basic conditions and at temperatures >160°C. Since these conditions are not given in the standard food used for the test substance formulation, stability of the test substance over the period used was assumed.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 ppm

Remarks:

Equivalent to a mean daily intake of 0.73 mg/kg bw/d for males and 0.89 mg/kg bw/d for females.

Doses / concentrations 2

Dose / conc.:

25 ppm

Remarks:

Equivalent to a mean daily intake of 1.91 mg/kg bw/d for males and 2.42 mg/kg bw/d for females.

Doses / concentrations 3

Dose / conc.:

100 ppm

Remarks:

Equivalent to a mean daily intake of 7.60 mg/kg bw/d for males and 10.25 mg/kg bw/d for females.

Doses / concentrations 4

Dose / conc.:

400 ppm

Remarks:

Equivalent to a mean daily intake of 31.52 mg/kg bw/d for males and 40.61 mg/kg bw/d for females.
Main and recovery groups

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent no treatment

Positive control:

No

Examinations

Observations and examinations performed and frequency:

The experimental animals were inspected at least once a day. Any clinical signs (findings) and abnormalities were recorded. Body surfaces and orifices, posture, general behavior, breathing and excretory products were assessed once weekly. In addition, this included one open field observation (OFO) in a standard arena once before exposure and once weekly thereafter (except in the weeks in which FOB was performed). Findings and abnormalities were recorded either using a coding system or else uncoded. At least once daily, all animals were observed for morbidity and mortality.
If animals became ill, they were set apart, observed more frequently and sacrificed prematurely, if death seemed imminent.

Ophthalmology:
Before the study start all animals (main + recovery groups) and at the end of the treatment (main groups) and the recovery period (recovery groups) rats treated at 0 and 400 ppm were subjected to ophthalmological inspections: the pupillary reflex of both eyes was first tested in a darkened room and the anterior regions of the eye were inspected. After dilating the pupils with Mydriaticum Stulln® drops, the refractive elements of the eye as well as iris and fundus were examined using an indirect ophthalmoscope.
In addition, the optical media were examined with a ZEISS photo-slit lamp. Rats of the recovery, the 100 and 25 ppm groups were examined with the photo-slit lamp only at the end of treatment.

Functional Observation Battery (FOB):
The functional observational battery (FOB) was conducted at four consecutive days in study week 13 (main and recovery groups) and at one day in study week 17 (recovery groups) including handling of the animals, cage-side and open field observations as well as grip strength, reflexes and physiological observations. Scoring
criteria and explicitly defined scales were used to rank the severity of observations that can not readily be quantified.

Motor and Locomotor Activity Assessment (MA and LMA):
The motor and locomotion activity assessment (MA and LMA) was conducted "not blind" at four consecutive days in study week 15 (main and recovery groups) and at two days in study week 18 (recovery groups). The allocation of animals to the test runs was done via ident-lists.

Determination of Body Weight:
The body weights of the individual experimental animals were determined before the beginning of the study and once a week thereafter up to scheduled necropsy.

Determination of Food/Water Consumption:
The food/water intake of each individual rat was determined once a week. These primary data were then used to calculate the means for each feeding period, the consumption per animal per day and per kg body weight per day.

Clinical Laboratory Investigations:
The determinations were performed using standardized procedures subject to continuous quality control. Occasionally it could have happened that the sample quantity was insufficient to permit measurement of all the parameters as planned, or that measurement was not possible for technical reasons. The planned number of measurements per group was, therefore, not necessarily obtained in each case. In general, all measurements were performed using standardized methods subject to regular internal and external quality controls. Additional comments concerning the appearance of the samples that were recorded in the raw data in connection with certain measurements were not included in the report lists when they were considered to have no bearing on the result, i.e. when there was no detectable relationship with treatment and the results showed that measurement had not been affected.

Hematological Investigations:
The following hematological parameters were determined in peripheral blood: differential blood count, morphology of erythrocytes, erythrocytes, hemoglobin, hematocrit, leucocyte count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, thrombocytes, hepato-quick.

Enzymes, Electrolytes, Substrates and Products of Metabolism in the Blood:
The following parameters were determined:
Enzyme activities in plasma: alkaline phosphatase, alanine aminotransferase,, aspartate aminotransferase, glutamate dehydrogenase, gamma-glutamyltransferase, creatine kinase, lactat dehydrogenase.
Substrates/Electrolytes: albumin, protein (total), cholesterol, creatinine, urea, bilirubin (total), sodium, potassium, glucose
Hormones: thyroxine, triiodothyronine, thyroid stimulating hormone.

Determinations in Liver Tissue:
The following activities were determined in liver tissue:
7-ethoxycoumarin deethylation, 7-ethoxyresorufin deethylation, aldrin epoxidation, epoxide hydrolisation, UDP-glucuronyl transferase, glutathione transferase.

Urinalysis:
The following parameters were determined:
Quantitative determinations: Volume, density, protein Semiquantitative determinations: test strips Muhistix or Multistix 10 SG: bilirubin, blood, glucose, ketone bodies, pH-value, urobilinogen, urine sediment (microscopy).

Organ Weights:
The following exsanguinated organs of the animals sacrificed at necropsy were weighed in the unfixed state:
Adrenal glands, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus, thyroids, uterus, ovaries.
The organ weights are specified in both absolute and relative terms. The relative organ weights were calculated by normalization to 100 g body weight (individual organ weight divided by body weight times 100). The body weight used as a basis for this calculation was determined immediately prior to necropsy of the pertinent animal.

Sacrifice and pathology:

Necropsy
Male and female animals were necropsied after exsanguination under deep ether anesthesia.
Animals that died spontaneously or were killed in a moribund state during the study were dissected at the earliest opportunity.

Statistics:

The statistical evaluation of data related to clinical chemistry, hematology, body and organ
weights as well as feed and water intake is performed using SAS® routines.

A. Descriptive analysis: All variables that are not dichotomous are described by sex, dose group and date using appropriate measures of central tendency (mean, median) and general variability (standard deviation, minimum, maximum).
B. Statistical tests:
B.1 Continuous random variables: Dunnett test, Adjusted Welch Test, Kruskal- Wallis Test followed by Adjusted U Tests.
B.2 Discrete random variables:
Kruskal-Wallis test followed by adjusted U tests.
B.3 Pathology data
Data handling and processing of pathology data were carried out using the PATHDATA program.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Up to and including 100 ppm there was no difference in clinical findings between treated and control rats. At 400 ppm the incidence of animals with the following findings were increased: tail held erect (treatment and recovery period, main and recovery males), high stepping gait (treatment and recovery period, main and recovery females), emaciation (treatment and recovery period, main and recovery females),
flaccid and soft abdominal tissue with no retraction of hind limbs upon touch and leaving finger indents (= special finding; treatment and recovery period, main and recovery females) as well as hindlimbs dragging (treatment period, main and recovery females). Hindlimb dragging was seen in all cases only during week 4-6 of treatment.
During the last two recovery weeks the special finding, the erected tail was not observed any more.

Mortality:

no mortality observed

Description (incidence):

All animals survived until scheduled necropsy. There was thus no treatment-related effect on survival.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the treatment period body weight development was comparable to controls in both sexes up to and including 100 ppm. At 400 ppm body weights were significantly reduced up to 10% in males (recovery group) and 25% or 23% in females (main or recovery group). During the recovery period the difference of body weights as compared to controls decreased with a difference at study end of 5% in males and 17% in females.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Treated and control males and females up to and including 100 ppm showed no difference in food consumption during the treatment and recovery period. At 400 ppm main group females had a slightly reduced individual food consumption. During the recovery period food consumption was increased in both sexes (in males significantly).

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Individual water consumption was comparable in both sexes throughout the treatment and recovery period. Related to body weight females treated at 400 ppm consumed cumulatively clearly more water during treatment and recovery period.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At treatment end two 400 ppm males of the main group had findings (opacity of the lens or cornea, and/or vacuoles in the lens). Examination of all recovery animals and those treated at 25 and 100 ppm (both sexes) revealed no findings in recovery and 25 ppm rats, one male with opacity of the lens and one female with opacity of the cornea at 100 ppm. Since there were no dose-related increase in overall incidence (10% affected rats at 100 and 400 ppm) and no findings after the recovery period, this finding is considered as incidental.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Mean erythrocyte count, hemoglobin concentration (HB) and hematocrit (HCT) of treated and control rats were comparable during the treatment and recovery period.
MCV, MCH and MCHC of treated males were not different from those of controls during treatment and recovery. In females the MCHC was slightly but significantly increased at 400 ppm (week 5 and 14) with all individual values within the normal range of variation. The MCV of females was significantly decreased at 100 ppm (week 14) and 400 ppm (week 5 and 14). After the recovery period MCV, MCH and
MCHC were comparable to control values.
Mean thrombocyte count and mean thromboplastin time of all treated rats were comparable to control values in main and recovery group animals.
Leucocyte and differential blood counts were not different from controls in all treated rats during treatment and in all treated females after the recovery period. Slightly but
significantly reduced neutrophils (males week 5) or increased eosinophils (females week 5) at 400 ppm are considered as toxicologically irrelevant. After the recovery period leucocyte and lymphocyte counts in males were significantly reduced as compared with control values.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The plasma activities of ASAT, ALAT APh, GLDH, GOT, LDH and CK of treated and control rats were comparable throughout treatment and, except for GLDH, after the recovery period. After the recovery period GLDH was increased in both sexes,
though not significantly, with two individual values per sex beyond the 2 s range.
Means identified as significantly different from control are not considered as toxicologically relevant since either dose or time dependence were missing or all individual values were within the normal range (2s).
Glucose concentration was reduced in females at 400 ppm, in week 5 significantly.
Cholesterol concentration was slightly increased in males at 400 ppm, in week 5 significantly.
Creatinine concentration was significantly reduced in females at 400 ppm during the treatment and at the end of the recovery period.
Mean urea, protein and albumin plasma concentration of all treated and control rats were comparable throughout the study and, except urea, also at the end of the recovery
period. After the recovery period urea concentration in females was significantly reduced.
Means identified as significantly different but not referred to are considered as incidental and without lexicological relevance. They are either based on individual values from within the normal range or were distributed without dose-relation.

Mean serum electrolyte concentrations of all animal groups were comparable
throughout the study. Means identified as significantly different from control are based on individual values in the 2s range and are thus considered as toxicologically irrelevant.

The determination of LDH isoenzymes did not indicate any damage to a specific organ or tissue system throughout the study. There were no consistent changes between treatment and control groups.
Total bilirubin concentration of all groups were comparable throughout the study.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Mean thyroid stimulating hormone (TSH) concentrations in plasma of treated and untreated rats were comparable during the treatment and after the recovery period. Triiodothyronine (T3) concentrations were comparable with control values in all treated males and in females up to and including 100 ppm. At 400 ppm T3 concentration was slightly but significantly increased in females in week 2, 5 and 9. After the recovery period T3 was slightly and significantly increased in males. Mean thyroxine (T4) values of all treated rats were not different from controls up to and including 100 ppm. At 400 ppm T4 was decreased, significantly at all dates in males and in week 14 in females. After the recovery T4 values of treated and control rats were comparable. Taking into account the lack of histological findings hi the thyroid and of TSH concentration
changes in the treated rats, the slight differences hi peripheral T3 and T4
plasma concentrations are regarded as biologically irrelevant and thus not adverse.

Urinalysis findings:

no effects observed

Description (incidence and severity):

In the whole dose range tested semiquantitative [blood, glucose, bilirubin, urobilinogen concentrations and ketone bodies; occurrence of insoluble salts (triplephosphates and amorphous salts) and cells: erythrocytes (blood), leucocytes, bacteria, mucus, epithelial and cylinder cells)] and quantitative (urine density, pH-value, volume, protein excretion) investigations of urine samples and sediments revealed no differences of treated to control rats of main and recovery groups

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Functional Observation Battery (FOB) and Motor Activity (MA, LMA):
Up to and including 100 ppm no indication of treatment-related effects were recorded. The only statistically significant difference to control was observed in 100 ppm males, which showed 100% normal posture (sitting or lying normally) in contrast to the control and the other treatment groups with up to 4 rats to rear during the observation period. This finding is not considered as toxicologically relevant. It was not seen after the recovery period.
At 400 ppm lower body weights (both sexes) as well as emaciation (females) were determined, correlating with the findings during daily/weekly observations and body weight determinations.
In addition, at treatment end 8/10 females treated at 400 ppm (main group only) showed walking on tip toes. Grip strength of fore- and hindlimbs was significantly reduced in 400 ppm main and recovery group females at treatment end. After the recovery period no gait abnormality was observed, grip strength of treated females was lower than that of control rats, though not statistically significant.
Motor activity investigations indicated no statistically significant differences between treated and control groups. The consistently increased MA and LMA was not seen during weekly open field observation, detailed daily and weekly inspection or FOB. It is thus considered a chance result.

Immunological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Leucocyte and differential blood counts of treated rats were comparable to controls
during the treatment period. After the recovery period, leucocytes and lymphocytes
were significantly reduced in males. Increased spleen and decreased thymus weights after treatment at 400 ppm were without histological findings. These differences are
therefore considered to be a chance result.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Mean absolute and relative weights of the brain, thyroids, heart, testes, epididymides, ovaries and uterus as well as mean absolute weights of adrenals, liver, spleen and kidneys of all main groups were comparable with controls. Differences at 400 ppm identified as significantly increased are seen in the context with the pronounced body weight reduction of these rats or in case of the epididymides weight as minor.
Additional significant increases (males: absolute weight of liver and epididymides, 10 ppm) are of no toxicological relevance since dose correlation is missing.
After treatment at 400 ppm the significantly increased relative weights were determined for the adrenals (females) the liver (both sexes), the spleen (females) and the kidneys (females). Absolute thymus weights were significantly decreased in females at 400 ppm with a similar trend in males (non-significant).
After the recovery period absolute epididymides weights were significantly reduced, while the relative weights of the liver (both sexes) and the kidneys (males) were significantly increased. The significant increases of brain (both sexes), heart (females) and ovary weights are seen in the context of the clearly reduced body weights of these rats.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Regression of skeletal muscle were seen hi all females at 400 ppm (main and recovery
groups).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Corresponding to necropsy findings, skeletal muscle alterations (fiber atrophy, increased fatty tissue, nerve fiber swelling) were seen at 400 ppm in the thigh and, without correlated gross pathological findings, in the skeletal muscle adjacent to the spinal cord, the sternum and in the skin. In general, females were more sensitive with respect to the incidence and severity of these findings of number of affected locations.
These findings were not reversible within the 4-week recovery period.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Determinations in Liver Tissue:
The investigated enzyme activities in liver tissue after the treatment and recovery period showed only marginal, toxicologically irrelevant changes. Thus, slightly decreased activity was measured for GS-T (males 400 ppm), for ECOD (females 10, 100 and 400 ppm) and for GLU-T (females 100 and 400 ppm) at treatment end. After the recovery period mean GLU-T activity in liver tissue was slightly increased in females.

Effect levels

1

Key result

Dose descriptor:

LOAEL

Effect level:

31.5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical signs

histopathology: neoplastic

2

Key result

Dose descriptor:

LOAEL

Effect level:

40.6 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical signs

histopathology: non-neoplastic

serum/plasma hormone analyses

3

Key result

Dose descriptor:

NOAEL

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Remarks on result:

other: corresponding to 7.6 and 10.25 mg/kg bw/day for male and female, respectively

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

400 ppm

System:

musculoskeletal system

Organ:

other: fiber atrophy, increased fatty tissue, nerve fiber swelling

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:

The administration of Propineb over 14 weeks under the conditions described was tolerated without adverse effects in dosages up to and including 100 ppm by male and female Wistar rats.

Executive summary:

Groups of 10 male and 10 female rats of the strain HsdCpb:WU were administered propineb at levels of 0, 10, 25,100 and 400 ppm in food up to 14 weeks.
In addition 10 rats of each sex respectively were treated for the same period at 0 and 400 ppm followed by a 4 week treatment-free period (recovery groups).
There was no treatment-related effect on survival.
At 400 ppm the incidence of animals with the following clinical findings were increased during the treatment and recovery period: tail held erect (males), high stepping gait (females), emaciation (females), flaccid and soft abdominal tissue with no retraction of hind limbs upon touch and leaving finger indents (= special finding; females). In addition, females dragged their hindlimbs during week 4-6 of treatment. During the last two recovery weeks the special finding (see above) and the erected tail was not observed any more.
MA-investigation at the end of the treatment period did not reveal any statistically significant differences between dosage and control groups. During FOB at exposure end, walking on tip toes (main group only) and significantly reduced grip strength of fore- and hindlimbs was observed at 400 ppm in females. After the recovery period no gait abnormality was observed; as a consequence of the skeletal muscle regression at 400 ppm grip strength of treated females was reduced compared to controls.
Food consumption was slightly reduced in main group females at 400 ppm during treatment and increased in both sexes during the recovery period. Water consumption was increased in females at 400 ppm throughout the study, while that of all males was comparable.
Body weights were significantly reduced throughout the study at 400 ppm in both sexes. Gross and histopathological findings were skeletal muscle regression and skeletal muscle alterations (fiber atrophy, increased fatty tissue, nerve fiber swelling) in the thigh and, without correlated gross pathological findings, in the skeletal muscle adjacent to the spinal cord, the sternum and in the skin. Incidence, severity and number of locations affected were higher in females.
Clinico-chemically significantly reduced creatinine concentrations were determined (400 ppm females). LDH plasma activity and proportional isoenzyme concentration in plasma of treated and untreated rats were comparable throughout the study. The histological findings and the reduction of creatinine plasma concentrations were not reversible within the 4-week recovery period. In addition urea plasma concentration was reduced in females after the recovery period.
Hematology and histopathology did not reveal any treatment-related effect on hematopoiesis.
Significantly reduced leucocytes and lymphocyte counts after the recovery period in males were without histopathological findings.
Slight differences at 400 ppm in one or both sexes (reduced glucose concentration, increase of GLDH activity, of cholesterol concentration and of liver weights) are considered as functional but not adverse, because there were no corresponding microscopic findings.
No biologically relevant difEerences of thyroidal parameters (T3, T4, TSH, histopathology) were determined between treated and control rats.
Increased adrenal (main group females) and reduced epididymides weights (recovery males) at 400 ppm were without histological correlates.
Urinalysis as well as histopathology did not reveal any effect on urinary tract function and morphology at treatment end and after the recovery period.
In conclusion, the administration of propineb over 14 weeks under the conditions described was tolerated without adverse effects in dosages up to and including 100 ppm by male and female Wistar rats.