polymeric zinc propylenebis(dithiocarbamate);propineb (ISO)

Endpoint summary

Description of key information

The key studies for repeated dose toxicity endpoints are as follows:

90-day oral (dietary) rat study (OECD 408 (1998), GLP, RL1), NOAEL (male) = 7.6 mg/kg bw/day, NOAEL (female) = 10.25 mg/kg bw/day (M-108777-01-1, Wirnitzer, 2003)

15-day dermal rabbit study (no guideline, pre-GLP, RL2), NOAEL = 250 mg/kg bw/day (M-116181-01-1, Mihail, 1979)

20-day inhalation rat study (OECD 412, GLP, RL1), NOAEC systemic = 3.97 mg/m³ (M-023867-01-1, Pauluhn, 2000)

28-day inhalation rat study (OECD 412, GLP, RL1), NOAEC local = 1.12 mg/m³ (M-039913-01-1, Pauluhn, 2001)

The following supporting studies are available:

2-year oral (dietary) rat study (no guideline, pre-GLP, RL2), NOAEL = 0.46 mg/kg bw/day (M-050009-01-1, Loser, 1974)

90-day oral (dietary) dog study (OECD 409 (1981), GLP, RL1), NOAEL = 4.3 mg/kg bw/day (M-009667-01-1, Jones, 1999)

15-day inhalation rat study (no guideline, pre-GLP, RL2), NOAEC = 1.12 mg/m³ (M-062375-01-1, Thyssen, 1979). 

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

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

Study period:

May-2002 to June-2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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

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

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.

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.

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.

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

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.

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.

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

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

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

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

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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

7.6 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises adequate and reliable studies and is thus sufficient to fulfil the standard information requirements set out in Annex VII, 8.6, of Regulation (EC) No 1907/2006.

System:

musculoskeletal system

Organ:

other: paralysis of hindlimbs, flaccidity, reduced motility; fiber atrophy

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Version / remarks:

And EPA OPPTS 870.1300

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: EU Directive 92/69/EEC

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

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.

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

Dose / conc.:

21.95 mg/m³ air

Remarks:

High Dose Group

Dose / conc.:

11.2 mg/m³ air

Remarks:

Mid Dose Group

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.

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.

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.

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

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

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

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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

3.97 mg/m³

Study duration:

subacute

Experimental exposure time per week (hours/week):

30

Species:

rat

Quality of whole database:

The available information comprises adequate and reliable studies and is thus sufficient to fulfil the standard information requirements set out in Annex VII, 8.6, of Regulation (EC) No 1907/2006.

System:

musculoskeletal system

Organ:

other: Paralysis of hindlimbs, flaccidity, reduced motility; fiber atrophy

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

19-August-1999 to 06-March-2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

yes

Principles of method if other than guideline:

This study is a follow up study for the 28-day inhalation study (M-023867-01-1) in order to determine a NOAEL in female rats after inhalation exposure. Therefore, only females were tested with special emphasis on lungs. Eighteen female Wistar rats per group were exposed under dynamic directed-flow nose-only exposure conditions, duration of exposure: 6 hours/day on seven consecutive days for one week followed by a 2-week post-exposure period. The objective of this mechanistic study was to clarify equivocal findings (alveolar macrophages laden with deposits) which were observed in a previous 4-week inhalation study. One additional objective was to assess whether the effects caused were related to the active ingredient or dissociated Zn-ions.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Hsd Cpb: WU (SPF)

Details on species / strain selection:

In this mechanistic study only females were used because this gender was considered to be more susceptible than males (with respect to neuromuscular effects).

Sex:

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, ad libitum
- Water: Drinking quality tap-water ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: approximately 50 %
- Air changes: approximately 10 air changes per hour
- Photoperiod: 12 h dark/12 h light; artificial light

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

ca. 2 µm

Geometric standard deviation (GSD):

2

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Dry conditioned air was used to aerosolize the test substance. The test atmosphere was then forced through openings in the inner concentric cylinder of the chamber, directly towards the rats' breathing zone. This directed-flow arrangement minimized re-breathing of exhaled test atmosphere. The stability of the test atmosphere was monitored continuously using an aerosol real-time device (vide infra) as far as technically feasible. The inhalation chamber used consisted of one segment which was suitable to accommodate 20 rats at the perimeter location.
- Method of conditioning air: Compressed air was conditioned (i.e. freed from water, dust, and oil) automatically by a VIA compressed air dryer. Adequate control devices were employed to control supply pressure.
- System of generating particulates/aerosols: The test substance was aerosolized using a Wright-Dust-Feeder. For powder dispersion, conditioned compressed air (30 liters of air/min) was used.
- Temperature, humidity, pressure in air chamber: Temperature and humidity measurements were performed by the computerized HP 3852A Data Acquisition and Control System using FTF11 sensors.
- Method of particle size distribution: The particle-size distribution was analyzed using a BERNER-TYPE AERAS low-pressure critical orifice cascade impactor. The individual impactor stages had been covered by an aluminum foil which was subjected to gravimetric analysis. An adhesive stage coating (silicone spray) was used to prevent particle bounce and re-entrainment. Gravimetric analyses were made using a digital balance.
- Treatment of exhaust air: The exhaust air was purified via cotton-wool and HEPA filters.

TEST ATMOSPHERE
- Brief description of analytical method used: The stability of the test atmosphere was monitored continuously using an aerosol real-time device (vide infra) as far as technically feasible.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or 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.
The test-substance, propineb, concentration was determined by gravimetric analysis (filter: Cellulose-Acetate-Filter). 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. Optimally, three samples per exposure day were collected. All analytical concentrations reported refer to mg of test substance/m3 air.

Duration of treatment / exposure:

7 days, 6 hours per day

Frequency of treatment:

daily

Dose / conc.:

25.8 mg/m³ air

Remarks:

High dose group

Dose / conc.:

5.52 mg/m³ air

Remarks:

Mid dose group

Dose / conc.:

1.12 mg/m³ air

Remarks:

Low dose group

Dose / conc.:

6.9 mg/m³ air

Remarks:

mg ZnO/m3 group

No. of animals per sex per dose:

18 female rats/dose

Control animals:

yes

Observations and examinations performed and frequency:

Body weights:
Body weights of all animals were measured before exposure twice during the exposure week and once weekly thereafter during the post-exposure period. Body weights were also collected prior to sacrifice. 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. Assessments during the post-exposure period were made once a day. Assessments from restraining tubes were made only if unequivocal signs occurred (e.g. spasms, abnormal movements, severe respiratory signs). Following exposure, observations were made and recorded systematically; individual records of clinical signs were maintained for each animal.
Cage-side observations included, but were not limited to, changes in the skin and fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, and somatomotor activity and behavior pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhea, lethargy, somnolence and prostration. The time of death was recorded as precisely as possible. Since these signs can only be assessed adequately from freely moving animals, no specific assessment was performed during exposure while animals were restrained.

Bronchoalveolar lavage:
Samples of bronchoalveolar lavage fluid were collected from the lungs of rats after the exposure period on post-exposure days 1, 3, and 15. Lungs were analyzed for factors indicative of an inflammatory response and lower respiratory tract damage as well as interactions with pulmonary phospholipids (Henderson, 1988; Henderson, 1989; Henderson and Belinsky, 1993).
Within the acellular supernatant of BAL-fluid (BALF), several indicators of pulmonary damage were assessed: (1) protein and angiotensin-converting enzyme (ACE) to quantitate increased permeability of the bronchoalveolar-capillary barrier, alkaline phosphatase to asses the degree of pneumocyte type II activity; (2) the activity of the lysosomal enzymes acid phosphatase (ACPH), to detect the release of enzymes from activated or lysed phagocytes; (3) the activity of the cytosolic enzyme, LDH, to detect general cytotoxicity; (4) y-glutamyltranspeptidase is considered to be a sensitive marker enzyme for a damage/increased activity of Clara-cells in the lower airways. (5) Phospholipids in intraalveolar cells (BALC) and supernatant (BALF) were determined.

Sacrifice and pathology:

Necropsy:
After complete exsanguination via the aorta abdominalis, body and lung weights were determined. Sodium pentobarbital (Narcoren®; intraperitoneal injection) was used for anesthesia. 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.

Histopathology and histochemistry of Metallothionein:
For the histopathological examination, the following organ tissues were fixed: the lung lobe not lavaged (Lobus dexter caudalis), muscle (M. quadriceps), liver and kidneys were fixed and examined histopathologically using the conventional H&E stain. An immunohistochemical staining for quantification of metallothionein (MT) was made in liver, lung (Lobus dexter caudales) and kidneys.

Statistics:

All variables that were not dichotomous were described by sex, dosage group and date using appropriate measures of central tendency (mean, median) and general variability (standard deviation, in most instances also minimum, maximum).
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. Within such a family, the experiment-wise error was controlled. If not otherwise noted, all pair-wise tests were two-sided comparisons.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Some rats in the high dose group experienced a flaccidity of hind-limbs (muscle weakness).

Mortality:

no mortality observed

Description (incidence):

The exposures were tolerated without mortality.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no statistically significant effect on body weights up to and including mid dose group. In high dose group (25.8 mg Propineb/m³) there was a marked, somewhat statistically significant and prolonged decrease of body weights and/or body weight gains.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

effects observed, treatment-related

Description (incidence and severity):

The results of the immunohistochemical reaction for MT at 1 and 3 days
post exposure clearly demonstrate the concentration dependency of the
MT-content in the alveolar macrophages in the LH 30-exposed rats.
Comparing all dose groups evaluated, a clear difference between 1 and 3
days post exposure concerning the MT-positive macrophages only occured
after zinc oxide (decrease from 2,8 to 1,2). The elimination of MT in
LH 30 exposed rats begins after the interim sacrifice of 3 days post
exposure.
At the end of the study period, MT-positive macrophages were only detectable
in the high concentration of LH 30. According to these results, the
increase of intraalveolar macrophages after exposure to LH 30 is a transient
effect, most likely due to the zinc within the LH 30 molecule.
The MT-containing alveolar macrophages are assessed as a marker of exposure
without any damage of the lung tissue.
The immunohistochemical reaction for MT demonstrated complete reversibility
in the low and the mid concentration of LH 30 and ZnO within 15 days
after exposure, but not in the high LH 30-concentration.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

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.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The gross pathological examination of the rats sacrificed at days 7, 9 and 21 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. 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.

Description (incidence and severity):

The histopathological evaluation revealed in all groups exposed to the test substance, propineb, 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.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Results in Bronchoalveolar Lavage and Tissue:
The results of analysis of various end-points in BALF and BALC demonstrate unequivocal effects in the 25.8 mg/m³ and 6.9 mg ZnO/m³ groups particularly on the first post-exposure day. Despite pronounced changes on post-exposure day 1 most effects subsided remarkably on post-exposure day 3. Taking into account the variability from one time point to the other, including the results of the statistical analysis revealed very mild to equivocal effects in the 5.52 mg/m³ group whilst 1.12 mg/m³ is considered to be the NOEL.

Key result

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

5.52 mg/m³ air

Based on:

test mat.

Sex:

female

Basis for effect level:

neuropathology

Key result

Dose descriptor:

NOAEC

Remarks:

local

Effect level:

1.12 mg/m³ air

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

25.8 mg/m³ air

System:

musculoskeletal system

Organ:

other: muscle

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

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

Conclusions:

Taking all findings into account 1.12 mg/m³ is considered to be a no-observable adverse effect-level, whilst mild effects were observed at the 5.52 mg/m³ level. Thus, with respect to pulmonary effects, <5 mg/m³ appeared to be the minimal effect level. Experimental evidence suggests that pulmonary effects are causally related to dissociated Zn2+.

Executive summary:

A subacute 7-day inhalation toxicity study with the aerosolized (dust) of propineb was performed. 18 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 seven consecutive days. Previous studies have shown that this gender is the most susceptible one with respect to neuromuscular effects. The rats were exposed to the following mean actual concentrations of the test substance 0, 1.12, 5.52, and 25.8 mg/m³ air. To evaluate whether the dissociation of Zn or the putatively 'low soluble particle effect' is of more concern, an additional group of rats was exposed to 6.9 mg ZnO/m³ air. Thus, in the 25.8 mg/m³ and 6.9 mg ZnO/m³ groups the Zn-content was roughly identical (i.e., the respective mean concentrations of Zn were 4.6 and 5.5 mg/m³ air). The solid aerosol was highly respirable to rats. With respect to the particle-size, airborne ZnO particles appear to be insignificantly smaller, however, less polydisperse than the aerosolized testsubstance.
During the study, the body weights were determined repeatedly. Clinical signs were recorded daily before and after exposure and once per week during the exposure free postexposure period. During the serial sacrifices on postexposure days 1, 3 and 15 gross pathological examinations were made and lung weights were determined. Prior to fixation, the lung (except one lobe) was subjected to bronchoalveolar lavage (BAL) and the BAL-fluid was analyzed for markers indicative of injury of the bronchoalveolar region, i.e., angiotensin-converting enzyme (ACE), protein, alkaline phosphatase (AP), lactate dehydrogenase (LDH), phosphatidylcholine and acid phosphatase (ACPH) were determined in BAL-fluid and -cells. Zn was determined in BAL-cells and lung tissue. The total cell count was determined and a cytodifferentiation of cytospins was made. The lung lobe not lavaged (Lobus dexter caudalis), muscle (M. quadriceps), liver and kidneys were fixed and examined histopathologically using the conventional H&E stain. An immunohistochemical staining for quantification of metallothionein (MT) was made in liver, lung (Lobus dexter caudales) and kidneys.

Results: Animals from all groups tolerated the 7-day exposure period without mortality or any clinically relevant effect, except those of the 25.8 mg/m³ group. Some rats exposed to this concentrations experienced clinical signs indicative of neuromuscular effects (flaccidity of hind-limbs). The mean body weights were statistically significantly decreased.

Absolute and relative lung weights were statistically significantly increased in the 25.8 mg/m³ and 6.9 mg ZnO/m³ groups on postexposure days 1 and 3. All changes subsided entirely during the 2-week postexposure period. Increased lung weights and the influx of cells into the bronchoalveolar region appear to parallel in the 25.8 mg/m³ and 6.9 mg ZnO/m³ groups on postexposure days 1 and 3. Minimal increased number of cells in BAL (TCC) was still observed at the end of postexposure period. In these groups an alveolar lipoproteinosis was indicated by increased number of alveolar macrophages with foamy appearance, increased concentration of intracellular phospholipids and polychrome positive alveolar macrophages (for the histochemical demonstration of intracellular polar phospholipids). Most changes gained statistically significant differences in the 25.8 mg/m³ and 6.9 mg ZnO/m³ groups on postexposure days 1 and 3 as compared to the respective air control group. Somewhat residual changes were still observed at the end of the 2-week post-exposure period. However, these changes appeared to be associated with the kinetics of elimination or turn-over of cells containing foreign material rather than with any impairment of the restorative capacity of the lung. The increase of TCC in the 25.8 mg/m³ and 6.9 mg ZnO/m³ groups on postexposure days 1 and 3 was apparently caused by an influx of lymphocytes and especially PMN's. The rapid recovery of the increased influx of PMN's appear to suggest that these cells served more a phagocytic rather than inflammatory role. Rats exposed to ZnO dust demonstrated equal or more pronounced responses than those of the 25.8 mg/m³ group.

Evidence of cell lysis and concomitant cytotoxicity (increased LDH in BALF) was observed in the 25.8 mg/m³ and 6.9 mg ZnO/m³ groups. Changes subsided entirely during the 2-week postexposure period. Particularly for alkaline phosphatase activity in BALF, the magnitude of response decreased remarkably from postexposure day 1 through 3. The relative intensity of LDH was quite comparable to that of protein and ACE, suggesting that the changes observed were apparently related to a transient disturbance/damage of the air/blood barrier function. All effects subsided entirely within the 2-week postexposure period. An appreciable increase of enzymes suggestive of involvement of destructive lysosomal processes could not be ascertained because the increase of lysosomal markers (ACPH and NAG) was mild, transient and unremarkable.
Zinc concentrations in lung tissue and BALC, as markers of exposure rather than markers of effect, were elevated in a concentration-dependent relationship in the 5.52 and 25.8 mg/m³ groups and were indistinguishable from the control by day 21. In the ZnO group the Zn content in lung tissue was not significantly elevated in neither BALC nor in lung tissue as compared to the control.

The findings observed by bronchoalveolar lavage in the 25.8 mg/m³ and 6.9 mg ZnO/m³ groups were complemented by the results of the histopathological examinations. Some activation of type II pneumocytes, including increased recruitment of intraalveolar cells (mostly macrophages) and induction of metallothionein (MT) in alveolar macrophages, in the absence of any evidence of any remarkable lung-tissue response, were observed in these groups. Accordingly, the transient increase of
intraalveolar macrophages, in the absence of any inflammatory tissue response, appears to be consistent with an adaptive rather than adverse response. Moreover, MT-immunohistochemical staining in liver and kidneys were unobtrusive, i.e., there was no evidence of conclusive concentration-dependent effects. Although clear grading differences between the groups could not be ascertained, with respect to the area and intensity of MT-positive staining in the tubules of kidneys the rats exposed
to ZnO displayed a mildly higher response as compared to the remaining exposure groups. The MT-induction in rats exposed to 6.9 mg ZnO/m³ was largely comparable to that observed in the 5.5 mg/m³ and 25.8 mg/m³ groups. The mild induction of MT in kidneys in the ZnO group appears to suggest that ZnO is leached out of the lung faster (and accordingly is causing higher systemic Cmax of Zn) than the test substance.

In summary, this study suggests that local pulmonary effects occur at exposure concentrations equal to or exceeding 5.52 mg/m³ whilst exposure to 1.12 mg/m³ produced no effects considered to be adverse. As far as effects were observed in alveolar macrophages they were seen in context with their biological role, namely to phagocytize, transport and detoxify deposited material. For a Zn-releasing material one major route of detoxification is considered to be associated with MT-induction favoring the sequestration and neutralization of potentially toxic metal ions. This is consistent with the transiently increased loading of alveolar macrophages with phospholipids and MT-Zn complexes. Therefore, the engulfment of such material by alveolar macrophages appears to be better described as 'generic adaptive' rather than 'specific adverse'. The rapid recovery of effects and the lack of experimental evidence that alveolar macrophages elaborate mediators causing a sustained inflammatory response corroborate this interpretation. Taking all findings into account 1.12 mg/m³ is considered to be a no-observable adverse effect-level, whilst mild effects were observed at the 5.52 mg/m³ level. Thus, with respect to pulmonary effects, < 5 mg/m³ appeared to be the minimal effect level. Experimental evidence suggests that pulmonary effects are causally related to dissociated Zn2+. 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

1.12 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises adequate and reliable studies and is thus sufficient to fulfil the standard information requirements set out in Annex VII, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

GLP compliance:

no

Remarks:

Study was performed pre-GLP

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 2.8-3.4 kg
- Housing: The rabbits were housed singly in standard type rabbit cages.
- Diet: Z 222 Pelleted Rabbit Breeding Diet
- Water: Water was provided ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2°C
- Photoperiod: artificially lit daily from 7 a.m. to 7 p.m.

Type of coverage:

open

Vehicle:

other: Cremophor EL (approx. 1.5 % v/v) and distilled water

Details on exposure:

TEST SITE
- Test site preparation: The back and flanks of each rabbit were clipped free of hair 48 hours prior to initiation of test compound application. On 3 males and 3 females of each group, the skin areas to be treated were additionally abraded with sandpaper to induce reddening and slight to
moderate swelling of the skin, 24 hours prior to commencement of treatment.
- Area of exposure: Back and flanks of each rabbit, 5x5 cm
- Type of wrap if used: No wrap used.
- Time intervals for shavings or clippings: New growth of hair in and around the treated skin areas was removed once weekly with clippers.

REMOVAL OF TEST SUBSTANCE
- Washing: The treated skin areas were washed with soap and water
- Time after start of exposure: 7-hours

TEST MATERIAL
- Amount applied : 0.5 mL/kg bw

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

7 hours per day for 15 days

Frequency of treatment:

The emulsion was applied once daily on 15 consecutive work days (5 applications per week)

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control group

Dose / conc.:

50 mg/kg bw/day

Remarks:

Low Dose Group

Dose / conc.:

250 mg/kg bw/day

Remarks:

High Dose Group

No. of animals per sex per dose:

6 animals/sex/group

Control animals:

yes

Details on study design:

Prior to each application, LH 30/Z was emulsified in Cremophor EL (approx. 1.5 % v/v) and distilled water. The emulsion was applied once daily on 15 consecutive work days (5 applications per week) to the clipped skin areas on the back and flanks of each rabbit, for a contact time of 7 hours; during the contact time, the treated skin areas were left uncovered. The emulsion was applied in a volume of 0.5 mL/kg body weight at each dose level. The size of each exposed skin area on which the test compound emulsion was applied was about 5 x 5 cm; a Record syringe fitted with a blunted hypodermic needle was used for applying the emulsion and spreading it evenly on the exposed skin areas.
At the end of each 7-hour contact time, the treated skin areas were washed with soap and water. New growth of hair in and around the treated skin areas was removed once weekly with clippers.
The following doses of LH 30/Z were applied daily in 0.5 mL aqueous emulsion per kg body weight:
0 mg/kg body weight - Control Group
50 mg/kg body weight - Dose Group 1
250 mg/kg body weight - Dose 0roup 2

Observations and examinations performed and frequency:

General checks:
During the study, the rabbits were inspected daily for physical appearance, behavioural patterns, appetite and condition of faeces. They were weighed prior to initiation of test compound application, and at the end of each treatment week.

Examinations for local skin compatibility:
The exposed skin areas were inspected prior to commencement of treatment and at the end of each 7-hour contact time (after removal of the test compound) for signs of inflammation (erythema and edema).

Laboratory tests:
Blood and urine samples from each rabbit were analyzed prior to initiation of test compound application and at the end of the three-week treatment period. The blood required for these analyses was withdrawn from the ear vein of each rabbit. The urine was collected from each rabbit for 16 hours overnight.

Sacrifice and pathology:

Necropsy and organ weight measurement:
Twenty-four to forty- eight hours after the final application, the rabbits were narcotized with 1.0 g Evipansodium (hexobarbital-Na) , sacrificed by exsanguination from the Vena cava caudalis, dissected and grossly examined. Heart, lung, liver, spleen, kidneys, adrenals, testes, ovaries and thyroid were weighed.

Histopathology:
Tissues processed for histopathological examination were obtained from several rabbits of the three groups. From each rabbit, two portions of dorsal skin (one portion from the treated area and one portion from the skin around the treated area) were fixed in Bouin's solution, embedded in Paraplast, sectioned, and stained with hemalum and eosin (HE). The tissues from the rabbits that died intercurrently were fixed in 10 % Formol. Additionally, the following tissues from rabbits of the control group and the highest dose group (250 mg/kg) were similarly processed for histopathological examination: genitals (testes, epidid3nnides resp. ovaries, uterus), heart, spleen, adrenals, kidney, liver, lung and thyroid. Portions of the liver were additionally fixed in Formol-calcium and frozen sections cut at 16 M, were stained for fat with Oil Red 0 (ORO). Kidney sections were additionally stained with PAS.

Other examinations:

Urinalyses
pH, protein, sugar, blood (haemoglobin): semiquantitatively tested with Combi-Uristix reagents
Urobilinogen: Urobilistix reagents. Results are expressed in Ehrlich units/100 mL urine. Sensitivity of 0.1 Ehrlich units/100 mL urine.
Deposits: following 5-minute centrifugation of the urine at 2000 G, the deposit was examined microscopically at x45 magnification.
The deposit constituents (bacteria (B), epithelia (E), erythrocytes (Ery), leucocytes (L), amorphous salts (a.S.), triple phosphates (T), calcium oxalate (Co)) were graded on the following scale:
++++ = signifies very many in all fields
+++ = signifies many in all fields
++ a signifies few in some fields
+ = signifies few in deposit smear

Clinical chemistry (blood):
Aspartate aminotransferase (GOT), Alanine aminotransferase (GPT), Alkaline phosphatase (ALP),
Plasma urea (PUR), Blood sugar (BSU), Bilirubin (BILI), Protein-bound iodine (PBJ).

Haematology
Erythrocyte and leucocyte counts (ERY and LEU), Haemoglobin (RGB), Thrombocyte counts(THROM), Haematocrit (HCT), Calculation of medium cell haemoglobin (Hbg value), Calculation of medium corpuscular volume (MCV), Differential blood count on smears stained by method of WRIGHT.

Statistics:

Not specified.

Clinical signs:

no effects observed

Dermal irritation:

effects observed, non-treatment-related

Description (incidence and severity):

Pimples and a mild bluish-red discoloration extending also beyond the treated area appeared on the dorsal skin after 13 applications in one rabbit and after 10 applications in another one. After 5 to 8 applications, 5 rabbits developed mild rhinitis.
No macroscopic variations from the norm were noted in the rabbits with, intact skin. The skinfold measurements also did not provide any indication of edema due to application of the test compound. No differences were seen between control rabbits and treated rabbits. In all the rabbits with abraded skin, slight to moderate erythema and edema caused by the abrasion appeared before test compound, application was initiated. These inflammations induced by abrasion of the skin began to clear during the first four days of the experiment and healed in Week 2 with formation of crusts.
There was no significant difference among the groups.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Three male rabbits died during the experiment. The gross pathology findings for two of them were indicative of pleuropneumonitis. One rabbit had a severe
pneumonitis. Microbiological examinations of the tissues from two rabbits revealed the presence of Escherichia coli and Staphylococcus aureus.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Comparison of the body weights of the rabbits in the different groups did not reveal any group-specific differences. Slight weight depressions were noted transiently for rabbits of each group. These depressions are attributable to the
effects of manipulation and immobilization and to the withdrawal of food and water during the 7-hour contact times. Under the described experimental conditions, body weight development was thus normal.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No variations from normal were noted, i.e. there were no group-specific alterations considered attributable to application of the test compound.
As expected on the basis of experience, the range of the numbers of thrombocytes counted in rabbit's blood was wider than that for the other haematological parameters. The average thrombocyte counts were all within the range considered normal in the determinations.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Lower treatment-unrelated alkaline phosphatase activities in blood serum were noted in all groups at the terminal analysis as compared with the pre-treatment analysis. The values for protein-bound iodine were comparable in all groups at termination of treatment, and did not provide any indication of possible thyroid hypofunction.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Description (incidence and severity):

No variations from normal and no group- specific differences were seen.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

All rabbits had a normal behaviour.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The absolute and relative organ weights were very largely within the normal range. Large individual variations were noted for the thyroid weights, but no treatment-related, group-specific differences were seen. Significant differences
were noted for the lung weights in some rabbits, attributable to the lung edemas.
Slight increases in absolute and relative liver weights were noted for the treated female rabbits.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Gross pathology did not reveal any group- specific alterations.
The following treatment-unrelated effects were seen in the different groups:
lung edemas, lung partially hardened or nodose, kidney alterations (scarred indurations or cysts), spleen with rough surface, uterus wall thickened, testes atrophied

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No alterations attributable to treatment with the test item were seen.
Very minimal thickening of the epidermis of the abraded skin was seen in one female rabbit. In some rabbits, minimal focal cell infiltrations were seen either in the corium of the treated skin or in the skin adjacent to the treated area.
Effects of a nonspecific inflammation, present mainly in lung, heart, kidney and liver, and final pulmonary edemas were seen in rabbits of both groups. The rabbits that died intercurrently, had severe pneumonitis. In one male rabbit of the control group, liver necroses were also observed.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

mortality

Remarks on result:

other: No adverse effects up to the highest dose tested.

Key result

Critical effects observed:

no

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

Conclusions:

From the results of the 3-week dermal toxicity experiment on rabbits, it is concluded that the test item doses of up to and including 250 mg per kg body weight per day were tolerated by the rabbits without having any damaging effects.

Executive summary:

In a three-week subacute dermal cumulative toxicity study on rabbits, the test item was evaluated for local and systemic compatibility.

The test compound was applied once daily, as an aqueous emulsion, for a 7-hour contact time each day on 15 consecutive work days, to the clipped skin on the back and flanks of rabbits, at the following doses:
0 mg/kg body weight - Control group
50 mg/kg body.weight - Low Dose Group 
250 mg/kg body weight - High Dose Group 
Each test group consisted of 6 male and 6 female rabbits, of which 3 had an intact skin and 3 had an abraded skin.
No alterations attributable to application of the test compound were seen on the treated skin areas.
Clinical observations, haematology, clinical chemistry, organ weight measurements, gross pathology and histopathology provided no indications of any treatment-related systemic tissue damage or harmful effects on body functions. Thyroid function in the rabbits was not affected by treatment with the test compound.
Therefore, in the subacute dermal toxicity study on rabbits, the no-untoward-effect dose was 250 mg test item per kg body weight per day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

35

Species:

rabbit

Quality of whole database:

The available information comprises an adequate and reliable study and is thus sufficient to fulfil the standard information requirements set out in Annex VII, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

GLP compliance:

no

Remarks:

Study was performed pre-GLP

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 2.8-3.4 kg
- Housing: The rabbits were housed singly in standard type rabbit cages.
- Diet: Z 222 Pelleted Rabbit Breeding Diet
- Water: Water was provided ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2°C
- Photoperiod: artificially lit daily from 7 a.m. to 7 p.m.

Type of coverage:

open

Vehicle:

other: Cremophor EL (approx. 1.5 % v/v) and distilled water

Details on exposure:

TEST SITE
- Test site preparation: The back and flanks of each rabbit were clipped free of hair 48 hours prior to initiation of test compound application. On 3 males and 3 females of each group, the skin areas to be treated were additionally abraded with sandpaper to induce reddening and slight to
moderate swelling of the skin, 24 hours prior to commencement of treatment.
- Area of exposure: Back and flanks of each rabbit, 5x5 cm
- Type of wrap if used: No wrap used.
- Time intervals for shavings or clippings: New growth of hair in and around the treated skin areas was removed once weekly with clippers.

REMOVAL OF TEST SUBSTANCE
- Washing: The treated skin areas were washed with soap and water
- Time after start of exposure: 7-hours

TEST MATERIAL
- Amount applied : 0.5 mL/kg bw

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

7 hours per day for 15 days

Frequency of treatment:

The emulsion was applied once daily on 15 consecutive work days (5 applications per week)

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control group

Dose / conc.:

50 mg/kg bw/day

Remarks:

Low Dose Group

Dose / conc.:

250 mg/kg bw/day

Remarks:

High Dose Group

No. of animals per sex per dose:

6 animals/sex/group

Control animals:

yes

Details on study design:

Prior to each application, LH 30/Z was emulsified in Cremophor EL (approx. 1.5 % v/v) and distilled water. The emulsion was applied once daily on 15 consecutive work days (5 applications per week) to the clipped skin areas on the back and flanks of each rabbit, for a contact time of 7 hours; during the contact time, the treated skin areas were left uncovered. The emulsion was applied in a volume of 0.5 mL/kg body weight at each dose level. The size of each exposed skin area on which the test compound emulsion was applied was about 5 x 5 cm; a Record syringe fitted with a blunted hypodermic needle was used for applying the emulsion and spreading it evenly on the exposed skin areas.
At the end of each 7-hour contact time, the treated skin areas were washed with soap and water. New growth of hair in and around the treated skin areas was removed once weekly with clippers.
The following doses of LH 30/Z were applied daily in 0.5 mL aqueous emulsion per kg body weight:
0 mg/kg body weight - Control Group
50 mg/kg body weight - Dose Group 1
250 mg/kg body weight - Dose 0roup 2

Observations and examinations performed and frequency:

General checks:
During the study, the rabbits were inspected daily for physical appearance, behavioural patterns, appetite and condition of faeces. They were weighed prior to initiation of test compound application, and at the end of each treatment week.

Examinations for local skin compatibility:
The exposed skin areas were inspected prior to commencement of treatment and at the end of each 7-hour contact time (after removal of the test compound) for signs of inflammation (erythema and edema).

Laboratory tests:
Blood and urine samples from each rabbit were analyzed prior to initiation of test compound application and at the end of the three-week treatment period. The blood required for these analyses was withdrawn from the ear vein of each rabbit. The urine was collected from each rabbit for 16 hours overnight.

Sacrifice and pathology:

Necropsy and organ weight measurement:
Twenty-four to forty- eight hours after the final application, the rabbits were narcotized with 1.0 g Evipansodium (hexobarbital-Na) , sacrificed by exsanguination from the Vena cava caudalis, dissected and grossly examined. Heart, lung, liver, spleen, kidneys, adrenals, testes, ovaries and thyroid were weighed.

Histopathology:
Tissues processed for histopathological examination were obtained from several rabbits of the three groups. From each rabbit, two portions of dorsal skin (one portion from the treated area and one portion from the skin around the treated area) were fixed in Bouin's solution, embedded in Paraplast, sectioned, and stained with hemalum and eosin (HE). The tissues from the rabbits that died intercurrently were fixed in 10 % Formol. Additionally, the following tissues from rabbits of the control group and the highest dose group (250 mg/kg) were similarly processed for histopathological examination: genitals (testes, epidid3nnides resp. ovaries, uterus), heart, spleen, adrenals, kidney, liver, lung and thyroid. Portions of the liver were additionally fixed in Formol-calcium and frozen sections cut at 16 M, were stained for fat with Oil Red 0 (ORO). Kidney sections were additionally stained with PAS.

Other examinations:

Urinalyses
pH, protein, sugar, blood (haemoglobin): semiquantitatively tested with Combi-Uristix reagents
Urobilinogen: Urobilistix reagents. Results are expressed in Ehrlich units/100 mL urine. Sensitivity of 0.1 Ehrlich units/100 mL urine.
Deposits: following 5-minute centrifugation of the urine at 2000 G, the deposit was examined microscopically at x45 magnification.
The deposit constituents (bacteria (B), epithelia (E), erythrocytes (Ery), leucocytes (L), amorphous salts (a.S.), triple phosphates (T), calcium oxalate (Co)) were graded on the following scale:
++++ = signifies very many in all fields
+++ = signifies many in all fields
++ a signifies few in some fields
+ = signifies few in deposit smear

Clinical chemistry (blood):
Aspartate aminotransferase (GOT), Alanine aminotransferase (GPT), Alkaline phosphatase (ALP),
Plasma urea (PUR), Blood sugar (BSU), Bilirubin (BILI), Protein-bound iodine (PBJ).

Haematology
Erythrocyte and leucocyte counts (ERY and LEU), Haemoglobin (RGB), Thrombocyte counts(THROM), Haematocrit (HCT), Calculation of medium cell haemoglobin (Hbg value), Calculation of medium corpuscular volume (MCV), Differential blood count on smears stained by method of WRIGHT.

Statistics:

Not specified.

Clinical signs:

no effects observed

Dermal irritation:

effects observed, non-treatment-related

Description (incidence and severity):

Pimples and a mild bluish-red discoloration extending also beyond the treated area appeared on the dorsal skin after 13 applications in one rabbit and after 10 applications in another one. After 5 to 8 applications, 5 rabbits developed mild rhinitis.
No macroscopic variations from the norm were noted in the rabbits with, intact skin. The skinfold measurements also did not provide any indication of edema due to application of the test compound. No differences were seen between control rabbits and treated rabbits. In all the rabbits with abraded skin, slight to moderate erythema and edema caused by the abrasion appeared before test compound, application was initiated. These inflammations induced by abrasion of the skin began to clear during the first four days of the experiment and healed in Week 2 with formation of crusts.
There was no significant difference among the groups.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Three male rabbits died during the experiment. The gross pathology findings for two of them were indicative of pleuropneumonitis. One rabbit had a severe
pneumonitis. Microbiological examinations of the tissues from two rabbits revealed the presence of Escherichia coli and Staphylococcus aureus.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Comparison of the body weights of the rabbits in the different groups did not reveal any group-specific differences. Slight weight depressions were noted transiently for rabbits of each group. These depressions are attributable to the
effects of manipulation and immobilization and to the withdrawal of food and water during the 7-hour contact times. Under the described experimental conditions, body weight development was thus normal.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No variations from normal were noted, i.e. there were no group-specific alterations considered attributable to application of the test compound.
As expected on the basis of experience, the range of the numbers of thrombocytes counted in rabbit's blood was wider than that for the other haematological parameters. The average thrombocyte counts were all within the range considered normal in the determinations.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Lower treatment-unrelated alkaline phosphatase activities in blood serum were noted in all groups at the terminal analysis as compared with the pre-treatment analysis. The values for protein-bound iodine were comparable in all groups at termination of treatment, and did not provide any indication of possible thyroid hypofunction.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Description (incidence and severity):

No variations from normal and no group- specific differences were seen.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

All rabbits had a normal behaviour.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The absolute and relative organ weights were very largely within the normal range. Large individual variations were noted for the thyroid weights, but no treatment-related, group-specific differences were seen. Significant differences
were noted for the lung weights in some rabbits, attributable to the lung edemas.
Slight increases in absolute and relative liver weights were noted for the treated female rabbits.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Gross pathology did not reveal any group- specific alterations.
The following treatment-unrelated effects were seen in the different groups:
lung edemas, lung partially hardened or nodose, kidney alterations (scarred indurations or cysts), spleen with rough surface, uterus wall thickened, testes atrophied

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No alterations attributable to treatment with the test item were seen.
Very minimal thickening of the epidermis of the abraded skin was seen in one female rabbit. In some rabbits, minimal focal cell infiltrations were seen either in the corium of the treated skin or in the skin adjacent to the treated area.
Effects of a nonspecific inflammation, present mainly in lung, heart, kidney and liver, and final pulmonary edemas were seen in rabbits of both groups. The rabbits that died intercurrently, had severe pneumonitis. In one male rabbit of the control group, liver necroses were also observed.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

mortality

Remarks on result:

other: No adverse effects up to the highest dose tested.

Key result

Critical effects observed:

no

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

Conclusions:

From the results of the 3-week dermal toxicity experiment on rabbits, it is concluded that the test item doses of up to and including 250 mg per kg body weight per day were tolerated by the rabbits without having any damaging effects.

Executive summary:

In a three-week subacute dermal cumulative toxicity study on rabbits, the test item was evaluated for local and systemic compatibility.

The test compound was applied once daily, as an aqueous emulsion, for a 7-hour contact time each day on 15 consecutive work days, to the clipped skin on the back and flanks of rabbits, at the following doses:
0 mg/kg body weight - Control group
50 mg/kg body.weight - Low Dose Group 
250 mg/kg body weight - High Dose Group 
Each test group consisted of 6 male and 6 female rabbits, of which 3 had an intact skin and 3 had an abraded skin.
No alterations attributable to application of the test compound were seen on the treated skin areas.
Clinical observations, haematology, clinical chemistry, organ weight measurements, gross pathology and histopathology provided no indications of any treatment-related systemic tissue damage or harmful effects on body functions. Thyroid function in the rabbits was not affected by treatment with the test compound.
Therefore, in the subacute dermal toxicity study on rabbits, the no-untoward-effect dose was 250 mg test item per kg body weight per day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rabbit

Quality of whole database:

The available information comprises an adequate and reliable study and is thus sufficient to fulfil the standard information requirements set out in Annex VII, 8.6, of Regulation (EC) No 1907/2006.

Additional information

Oral:

A key oral repeated dose toxicity study in rats following the OECD 408 guidelines and in compliance with GLP is available (M-108777-01-1, 2003). Group of Wistar rats (10 animals/sex/dose groups) received propineb at dietary levels of 0, 10, 25, 100 or 400 ppm (equivalent to 0, 0.73, 1.91, 7.60 and 31.52 mg/kg bw/day and 0, 0.89, 2.42, 10.25 and 40.61 mg/kg bw/day for males and females respectively) over 14 weeks. In addition 10 rats of each sex were treated for the same period at 0 and 400 ppm followed by a 4 week treatment-free period (recovery groups).
Dietary administration of propineb via the diet at 400 ppm provoked effects on the skeletal muscle of hind limbs in both sexes, females being affected more severely. Observed clinical signs (stepping gait, slow hind limb retraction and dragged hind limbs) were correlated with gross and histopathology findings of the skeletal muscle in the thigh (fibre atrophy, increased fatty tissue, nerve fibre swelling) and in the skeletal muscle adjacent to the spinal cord, the sternum and in the skin.
In this study the effects of Propineb on thyroid hormones were measured using well described methods and at 400 ppm (31, 52 mg/kg bw/day) there was a decrease of T4 in males and females. There were no effects on thyroid weight and morphology at any dose levels.
The study NOAEL was 100 ppm, equivalent to 7.60 and 10.25 mg/kg bw/day in males and females, respectively and this is considered to be overall NOAEL for the subchronic toxicity of propineb in the rats.

A supporting 90-day repeated dose toxicity study in dogs according to OECD 409 and GLP is available (M009667-01-1). In this study propineb was administered to groups of Beagle dogs (four animals/sex/dose group) at 0, 150, 1500 and 5000 ppm (equivalent 0, 4.6, 46.4, 150.4 and 0, 4.3, 41.4 and 149.8 mg/kg bw/day for males and females, respectively). Additionally three males per treatment level were used as recovery animals. Body weight was affected at the two top dose levels, partly due to decreased food consumption. Neurological findings like proprioceptive deficits and hind-limbs wheel-barrowing were observed at the two higher dose levels in both sexes. However, neurological clinical signs were not accompanied by histopathological changes in the skeletal muscle or nervous fibres. The slight variation of thyroid hormones levels at doses higher than 1500 ppm was accompanied by increased of relative thyroid weights but not with histopathological changes. Similarly, the observed increase of liver enzyme activities and cholesterol levels at the top dose levels was accompanied by increased liver weight, but there were no microscopic changes in the liver. The NOAEL was set at 150 ppm (4.6 and 4.3 mg/kg bw/day in males and females, respectively).

A chronic oral repeated dose toxicity study in rats pre-guideline and pre-GLP is available (M-050009-01-1, 1974). Twenty-five animals/sex dose groups were given Propineb in the diet at 1, 10, 100, 1000, 2000 and 8000 ppm. After only 5 days of feeding, the rats of the three highest dose levels of 1000, 2000, 8000 ppm, the showed severe myasthenia in the hind-limbs which gradually lead immobility and also affected the fore extremities of the animals in the 2000 and 8000 ppm groups. These symptoms developed earlier and more massively in the female rats than in the males. Therefore the achieved test material intake in expressed in mg/kg bw/day could be determined for males up to 2000 ppm (i.e. approximately 0.05, 0.48, 5.03, 57.8 and 120 mg/kg bw/day) and for females up to 1000 ppm (i.e. approximately 0.04, 0.46, 4.5 and 58 mg/kg bw/day). A further consequence of this muscular debility was that the affected rats had considerable difficulty in eating and drinking, with consequent less food consumption, decreased in weight (from 15% in males of the 1000 ppm groups to 50%) and increased mortality. Thyroid weights were increased in males from the 100 ppm dose levels and in surviving females of the 1000 ppm group. Histopathology revealed degenerative changes of skeletal muscle (atrophy and replacement with adipose tissue) and increased incidence of thyroid tumours in males (6/25) at 1000 ppm, a dose level that exceed the MTD. The NOAEL was 10 ppm (approximately 0.5 mg/kg bw/day) based on the effect on thyroid weight at 100 ppm (approximately 4.5 mg/kg bw/day).

Taken together, the leading hazard is the effect on muscle degeneration observed in all studies. Effects on thyroid are reversible and thus considered less relevant. Therefore, the NOAEL from the 90-day rat study is considered to be the most reliable for DNEL derivation. 

Inhalation:

A key inhalation repeated dose toxicity study in rats following the OECD 412 guideline and in compliance with GLP is available (M023867-01-1, 2000). As in this study no NOAEC for local effects could be established a follow up study was performed (M039913-01-1, 2001). Effects on the skeletal muscle (flaccidity and paralysis of hind legs, reduced motility and grip strength and atrophy of muscle fibers) were also observed following the inhalation route of exposure in rats. Mortality occurred in animals that, due to severe effects on motility, had no access to feeder and water. In addition to the effects on the skeletal muscle, exposure via the inhalation route provoked local pulmonary irritation due to deposition of particle and Zn. A systemic NOAEC of 3.97 mg/m³ and a local NOAEC of 1.12 mg/m³ were determined.

Dermal:

A key dermal repeated dose toxicity study in rats similar the OECD 410 guideline and not in compliance with GLP (pre-GLP) is available (M116181-01-1, 1979). Following the dermal route no treatment related effects were observed in the rabbit after daily dermal application (7-hour/day) of up to 250 mg propineb/kg bw/day for 15 consecutive working days.

Justification for classification or non-classification

Based on the effects observed after repeated exposure in the neuromuscular system and in the thyroid Propineb has been classified for specific target organ toxicity after repeated exposure – STOT-RE category 2, H373: May cause damage to organs (peripheral nervous system) through prolonged or repeated exposure if inhaled or swallowed.