polymeric zinc propylenebis(dithiocarbamate);propineb (ISO)

Neurotoxicity

Description of key information

The key studies for neurotoxicity are as follows:

90-day oral neurotoxicity study in rats (OECD 424, GLP, RL1), NOAEL (male) = 17.11 mg/kg bw/day (highest dose), NOAEL (female) = 9.36 mg/kg bw/day (M-066913-01-2, Gilmore, 2004);

Developmental Neurotoxicity study in rats (OECD 426, GLP, RL1), NOAEL (maternal) = 12.36 mg/kg bw/day (highest dose), NOAEL (developmental) = 12.36 mg/kg bw/day (highest dose) (M-370251-01-1, Gilmore, 2010).

For this endpoint the neuromuscular findings in the available repeated dose studies were taken into account (summarized in Section 7.5).

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

neurotoxicity: oral

Remarks:

Developmental neurotoxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2009/01/12 to 2009/04/24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 426 (Developmental Neurotoxicity Study)

Deviations:

no

Remarks:

The exception is that the homogeneity and stability of the test substance in the diet were verified after the study was completed, due to unanticipated challenges associated with developing the analytical method. This is not believed to have had an affect

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.6300 (Developmental Neurotoxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Health Canada PMRA DACO No. 4.5.14

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Rat / Approximately 120 male and 120 female (nulliparous and nonpregnant) rats were placed on study to provide a minimum of 20 litters per dietary level. At least 12 (females) and 15 (males) weeks of age at co-housing (based on calculated birth date provided by vendor). 191.1 – 248.3 g range for females (± 20% weight determination); Males had no specified weight requirements. Suspended stainless steel cages; Individually, except with one male each during co-habitation with deotized cage board in the bedding tray. Individually in plastic cages with corn cob bedding during gestation and lactation. Each cage contained a feeder and a source of water (pressure-activated water lixits). Purina Mills Certified Rodent Diet 5002 in meal form provided for ad libitum consumption during the acclimation period and throughout the study except during neurobehavioral testing. Tap water (Kansas City Missouri Municipal Water) was provided ad libitum except during neurobehavioral testing.

Environmental conditions:
Temperature: Daily average within the range of 18-26°C.
Humidity: Daily average within the range of 30-70% (Relative).
Air changes: Minimum daily average of 10.75 air changes per hour.
Photoperiod: 12 hr of light alternating with 12 hr of darkness; lights toggled off during ophthalmic examinations.
Acclimation period: 7 days: 1/5/09 (receipt) - 1/12/09 (released for study)

Route of administration:

oral: feed

Vehicle:

acetone

Remarks:

Acetone served as a solvent in the diet preparation process and was allowed to evaporate prior to administration.

Details on exposure:

Dietary levels were selected based primarily on the results of a subchronic dietary toxicity study utilizing doses of 10, 25, 100 and 400 ppm, a subchronic neurotoxicity screening study utilizing doses of 30, 150, and 300 ppm and a three-generation reproduction study utilizing doses of 20, 60, 200 and 600 ppm. In the subchronic dietary study, effects were only observed in the animals at 400 ppm.

Formulations were prepared weekly by mixing appropriate amounts of the test substance in the diet Purina Mills Certified Rodent Diet 5002 in meal form and were stored at freezer (approximately -23.0°C) conditions.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations of the test substance in the diet were measured using an LC-MS/MS method that measured the amount of propylenediamine (PDA) in the feed for all five batches of feed that were used in this study. The stability (at room temperature and freezer conditions) and homogeneity of the test substance in the feed were verified at dietary concentrations (15 and 180 ppm) that bracket those used in the present study.

Duration of treatment / exposure:

Dietary levels during
gestation were 0, 30, 60 or 180 ppm and were then reduced during weeks 1-3 of lactation by
50% to offset the substantial increase in food consumption that is normally observed in dams
during lactation (dietary levels during lacation were 0, 15, 30 and 90 ppm). The treated feed was
provided for consumption beginning on GD 6 and continuing through lactation Day 21 with
fresh feed provided every seven days. A sample of each batch of feed mixed was taken and
retained in the freezer until the study was complete and the analytical data deemed satisfactory.

After Day 21 of postnatal development, untreated feed was provided for consumption to all F1-
generation animals that were retained on study. A given batch of feed was available for ad
libitum consumption for a period of one or two (post-weaning) weeks prior to changing, at which time any uneaten feed was collected and disposed of.

Frequency of treatment:

Test substance was administered via the diet from gestation Day 6 through Day 21 of lactation / postnatal development. On postnatal Day 21, pups were weaned and maternal animals were sacrificed. The offspring (F1-generation) that remained on study after weaning were sacrificed at study termination on PND 75 (± 5 days).

Dose / conc.:

30 ppm

Remarks:

Low dose group, equivalent to a mean daily intake of 2.3 mg/kg bw/d

Dose / conc.:

60 ppm

Remarks:

Mid dose group, equivalent to a mean daily intake of 4.4 mg/kg bw/d

Dose / conc.:

180 ppm

Remarks:

High dose group, equivalent to a mean daily intake of 12.3 mg/kg bw/d

No. of animals per sex per dose:

One male and/or female per litter (approximately 16 - 20 (minimum 10)/sex/dietary level, representing at least 20 litters per level)

Control animals:

yes

Details on study design:

See table one in extra methods information

Observations and clinical examinations performed and frequency:

Upon receipt, P-generation females were examined and those considered acceptable were placed into individual cages and acclimated to their ambient laboratory conditions for at least six days prior to being placed on study. For the holding period, animal care personnel observed the animals at least once daily for moribundity and mortality. All planned or unplanned activities associated with either the animals or their room, as well as changes in the status of either the animals or their room, were documented.

Upon arrival, male animals were randomly assigned an identification number as they were
arbitrarily selected and removed from the shipping crates and placed into individual cages.
Following at least six days of acclimation, P-generation females were weighed and those with a
body weight more or less than 20% of the mean weight were rejected. The remaining
females were assigned to the control or an exposure group in sequence, as they were determined to be inseminated.

Parental (P)-generation males and females were identified by cage card and tail mark (males) or tail tattoo (females). F1-generation animals that were born alive were identified by tattoo; pups that were found dead were identified with a marking pen.

Neurobehavioural examinations performed and frequency:

At approximately 50-60 days of age, randomly selected animals (a minimum of 10/sex/dietary
level, representing at least 20 litters per level) from Sets A, B and C were subjected to an
ophthalmologic examination. At termination (PND 75 (± 5 days)), these animals were
anesthetized and sacrificed by perfusion, with neural and muscle tissues collected for
microscopic examination. Also at termination on PND 75 (± 5 days), brains were collected from
additional randomly selected animals (10/sex/dietary level; representing 20 litters per level).
These brains were weighed (fresh tissue weight) and discarded.

Sacrifice and (histo)pathology:

The whole brain was collected from a separate group of randomly selected offspring (Set D; 10/sex/dietary level; representing 20 litters per level) for micropathologic examination and morphometric analysis.

Positive control:

The control diet was prepared the same way, excluding the test substance.
Dietary concentrations were not adjusted to correct for purity (percent active ingredient) in the
test substance but were adjusted (reduced by 50%) during lactation, to maintain a more constant level of exposure (mg/kg/day) throughout the period of exposure.

Statistics:

See other information on materials for more details.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related clinical signs at any dietary level. Findings considered
incidental and unrelated to the test substance included red vaginal discharge on GD 13 and bend in tail (1 high-dose dam each), missing digits (1 mid-dose dam) and hair loss (1 to 3 dams from each dose group, including controls), which is a common finding associated with nest-building behavior in pregnant rats.

There were no test substance-related clinical signs during lactation at any dietary level. Findings
that are considered incidental and unrelated to the test substance included red vaginal discharge
(1 high-dose dam on LD 1), lacrimal stain (1 high-dose dam on LD 15 and 16), nasal stain and
dehydration (1 high-dose dam on LD 18) and areas of hair loss (2-3 dams at all dietary levels,
including control). None of these findings are considered to be related to the test substance since
incidence was generally low (only seen in 1 animal on 1 or 2 days) and findings are seen in
controls as well as treated animals.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No P-generation females were found dead during gestation or lactation. There were also no P-generation males found moribund or dead after initiation of the study (males did not receive the test substance).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects on body weight, body weight gain during gestation at any dietary level. Body weight during lactation was not different from controls at any dietary level. Body weight was not affected by the test substance at any dietary level in either sex. Body
weight was statistically increased in low-dose males and females on PND 0 (7% in both sexes)
and 4 (7-8% in males pre- and post culling and 7% in females pre-culling). In addition, body
weight was statistically increased in low-dose males on PND 21 (5%). For all remaining time
points, body weights in low-dose males and females were non-statistically increased (4% in
males and 4-7% in females).
Body weight gain was not affected by the test substance at any dietary level in either sex. Body
weight gain for PND 11-21 was statistically increased in low-dose males (7%) and males and
females, combined (6%). There was a similar non-statistical trend in body weight gain for PND
43.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no effects on food consumption during gestation at any dietary level. Food consumption during lactation was not different from controls at any dietary level.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related lesions in males or females at any dietary level. The
corneal opacity in males and females from various dietary levels (including control) was
considered to be incidental and unrelated to the test substance, due to lack of dose response (in
females), consistency by gender and/or because the incidence was within the range of historical
control.

Haematological findings:

not examined

Clinical biochemistry findings:

not specified

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no test substance-related findings in dams at any dietary level. Findings that are
considered incidental and unrelated to the test substance included red vaginal discharge (1 mid and high-dose dam, each on GD 13 and one low-dose dam on LD 21) and areas of hair loss
described as alopecia at various time points (1-2 dams at all dietary levels, including control).
There were no test substance-related findings in males or females at any dietary level.
There was a statistical increase in the mean number of rears in the open field, compared to
controls for low-dose females on PND 45 (6.9 versus 4.3 for controls). This difference from
control is not thought to be test substance-related since the incidence was not dose related and the mean average for rears in the control animals (4.3) is below the range for historical controls in the last ten studies conducted in this laboratory.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Absolute and relative fixed brain weights for perfused Day 21 males and females were not different from control at any dietary level. There were no test substance-related effects on absolute and relative fixed brain. There were no test substance-related microscopic lesions evident in any tissue from the
termination high-dose males or females. There were a few microscopic observations of which
“axonal degeneration” of individual nerve fibers in spinal cords, nerves, and dorsal root
ganglions were the most common. Also, focal retinal dysplasia occurred in two 180 ppm male
rats.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross observations considered test substance-related at any dietary level in either
sex for perfused Day 21 or termination animals or in non-perfused termination animals. One
high-dose male was sacrificed due to injuries that occurred from teeth caught on either the feeder or on the cage. This animal was selected for perfusion but was replaced with another animal.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There was no effect on reproduction parameters at any dietary level. There were no test substance-related clinical signs during lactation in males or females at any dietary level. One mid- and high-dose pup each had extra digits. This finding was not considered to be related to treatment since this finding was not seen in the multigeneration study conducted at much higher dietary levels or in the two-generation
reproduction study conducted in this laboratory at the same dietary levels. Additional
incidental findings that were evident on occasion in a few (1-3) individuals from various dose
groups, including controls, were bruising (including at time of delivery), wound or cuts, scab and ocular opacity. These findings are common in such young rats and did not occur in a pattern (e.g., dose-related or sustained) to indicate a relationship with exposure to the test substance.

Key result

Dose descriptor:

NOAEL

Effect level:

12.3 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other:

Remarks on result:

other: Equivalent to 180 ppm

Key result

Critical effects observed:

no

Table three

Achieved intake

Concentrations (ppm)	30 ppm	60  ppm	180 ppm
GD 6-13	2.1	3.7	10.5
GD 13-20	2.0	3.7	10.5
LD 0-7	1.9	4.0	10.3
LD 7-14	2.7	5.0	13.9
LD 14-21	3.0	5.7	16.5

Based on these results, the average daily intake of active ingredient during gestation and lactation was 0, 2.3, 4.4 and 12.3 mg/kg/day.

 

Table four

Brain measurements (mm), body and brain weight on offspring

Parameter	Dose (ppm in diet)
	Control	30 PPM	60 PPM	180 PPM
Males
Gross Measurements
Day 21
Ant/Post Cerebrum Length (mm)	13.67±0.27 (10)	13.86±0.28 (10)	13.70±0.32 (10)	13.68±0.14 (10)
Ant/Post Cerebellum (mm)	7.39±0.48 (10)	7.31±0.48 (10)	7.31±0.17 (10)	7.32±0.30 (10)
PND 75 (±5) (Termination - Perfused)
Ant/Post Cerebrum Length (mm)	14.69±0.23 (10)	14.90±0.26 (10)	14.80±0.33 (10)	14.55±0.39 (10)
Ant/Post Cerebellum (mm)	8.19±0.27 (10)	8.35±0.30 (10)	8.14±0.56 (10)	8.10±0.34 (10)
Microscopic Measurements
PND 21
Frontal Cortex (mm)	1.832±0.010 (10)	--	--	1.804±0.005 (9)
Parietal Cortex (mm)	1.880±0.011 (10)	--	--	1.905±0.006 (9)
Caudate Putamen (mm)	2.704±0.059 (10)	--	--	2.700±0.108 (9)
Hippocampal Gyrus (mm)	1.701±0.007 (10)	--	--	1.719±0.012 (10)
Cerebellum (mm)	5.007±0.020 (10)	--	--	4.970±0.230 (10)
PND 75 (±5) (Termination - Perfused)
Frontal Cortex (mm)	1.799±0.014 (9)	--	--	1.803±0.015 (9)
Parietal Cortex (mm)	1.985±0.015 (9)	--	--	2.000±0.014 (9)
Caudate Putamen (mm)	3.266±0.029 (9)	--	--	3.216±0.039 (9)
Hippocampal Gyrus (mm)	1.948±0.014 (9)	--	--	1.975±0.019 (10)
Cerebellum (mm)	5.575±0.144 (10)	--	--	5.576±0.088 (10)
Females
Gross Measurements
PND 21
Ant/Post Cerebrum Length (mm)	13.50±0.23 (10)	13.63±0.21 (10)	13.63±0.24 (10)	13.69±0.15 (10)
Ant/Post Cerebellum (mm)	7.15±0.28 (10)	7.27±0.43 (10)	7.25±0.26 (10)	7.09±0.28 (10)
PND 75 (±5) (Termination - Perfused)
Ant/Post Cerebrum Length (mm)	14.07±0.34 (10)	14.42±0.35 (10)	14.36±0.42 (10)	14.42±0.32 (10)
Ant/Post Cerebellum (mm)	8.15±0.40 (10)	8.28±0.28 (10)	8.02±0.31 (10)	8.25±0.30 (10)
Microscopic Measurements
PND 21
Frontal Cortex (mm)	1.636±0.003 (10)	--	--	1.807±0.007* (9)
Parietal Cortex (mm)	1.702±0.003 (10)	--	--	1.906±0.005* (9)
Caudate Putamen (mm)	2.539±0.039 (10)	--	--	2.805±0.031* (9)
Hippocampal Gyrus (mm)	1.580±0.008 (10)	--	--	1.621±0.016 (10)
Cerebellum (mm)	4.995±0.118 (10)	--	--	4.900±0.142 (10)

Cerebellum (mm)	4.995±0.118 (10)	--	--	4.900±0.142 (10)
PND 75 (±5) (Termination - Perfused)
Frontal Cortex (mm)	1.821±0.002 (10)	--	--	1.860±0.011 (9)
Parietal Cortex (mm)	1.955±0.003 (10)	--	--	1.948±0.014 (10)
Caudate Putamen (mm)	3.227±0.022 (10)	--	--	3.312±0.033 (10)
Hippocampal Gyrus (mm)	1.821±0.021 (10)	--	--	1.778±0.014 (9)
Cerebellum (mm)	5.073±0.068 (10)	--	--	4.958±0.176 (10)

 

Conclusions:

Technical grade propineb was administered via the diet from gestation Day (GD) 6 through lactation Day (LD) 21 to mated female Wistar rats at nominal concentrations of 0, 30, 60 or 180 ppm with adjustment during lactation to maintain a more consistent dosage throughout the period of exposure. There were no treatment-related effects in the dams at any dose level.
There were no test substance-related findings in the offspring at any dose level.
The maternal and developmental NOAELs were 180 ppm (equivalent to 12.3 mg/kg bw/day).

Executive summary:

Technical grade propineb was administered via the diet from gestation Day (GD) 6 through lactation Day (LD) 21 to mated female Wistar rats at nominal concentrations of 0, 30, 60 or 180 ppm with adjustment during lactation to maintain a more consistent dosage throughout the period of exposure. The average mean daily intake of the test substance (mg propineb/kg body wt/day) based on the average dietary consumption for the last two weeks of gestation and three weeks of lactation at nominal dietary concentrations of 30, 60 or 180 ppm, respectively, was 0, 2.3, 4.4 and 12.3 mg/kg/day.

 

All test diets (including control) were provided for ad libitum consumption throughout the study, except during neurobehavioral testing. The Parental (P)-generation females were evaluated by cage-side and detailed clinical observations, body weight, food consumption and reproductive endpoints. On postnatal Day (PND) 4, litters with a minimum of seven pups, including at least three per sex, were culled to yield, as closely as possible, four males and four females.

 

Subsets of surviving offspring, representing 19 - 20 litters per dietary level, were subjected to evaluation using the following observations and measurements - detailed clinical observations and a detailed observational battery, pupil response, surface righting, preputial separation or vaginal patency, body weight, food consumption, automated measures of activity (figure-eight maze), auditory startle habituation, learning and memory (passive avoidance after weaning and a water maze task beginning on PND 60±2 days) and an ophthalmic examination.

 

Neural tissues were collected from 10 rats/sex/dietary level (representing 20 litters) on PND 21 (brain only) and at study termination (approximately 75 days of age) for microscopic examination and morphometry. There were no treatment-related effects in the dams at any dose level. There were no test substance-related findings in the offspring at any dose level. The maternal and developmental NOAELs were 180 ppm (equivalent to 12.3 mg/kg bw/day).

 

Reference 2

Endpoint:

neurotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2002/09/16 to 2002/12/18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Those animals considered acceptable were then placed into individual cages and acclimated to their ambient laboratory conditions (temperature 19°-25°C, relative humidity 30-70%, 12- hr light/dark cycle and a minimum daily average of 10 air changes per hour) for at least six days prior to placement on the study. Purina Mills Rodent Lab Chow 5002 in meal form and tap water were provided for consumption during the acclimation period and throughout the study except during neurobehavioral testing.

The room, cages and cage racks were thoroughly cleaned and disinfected before arrival of the animals. Deotized Animal Cage Board was used in the bedding trays and changed at least three times weekly. The cages and racks were replaced at least once every two weeks with clean, disinfected cages and racks. The room was disinfected at least once every two weeks and feeders were changed weekly.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

The room, cages and cage racks were thoroughly cleaned and disinfected before arrival of the animals. Deotized Animal Cage Board was used in the bedding trays and changed at least three times weekly. The cages and racks were replaced at least once every two weeks with clean, disinfected cages and racks. The room was disinfected at least once every two weeks and feeders were changed weekly.

Analytical verification of doses or concentrations:

yes

Remarks:

Based on analytical results, Propineb was homogeneous in the ration at dietary concentrations used in this study and the actual mean concentrations of Propineb in the diet were 0, 24.4, 128 and 258 ppm for males and females.

Details on analytical verification of doses or concentrations:

nominal concentrations of 0, 30, 150 and 300 ppm for males and females.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily administered in diet

Dose / conc.:

0 ppm

Dose / conc.:

24.4 ppm

Remarks:

corresponds to 1.45 and 1.90 mg/kg bw/day for male and female, respectively

Dose / conc.:

128 ppm

Remarks:

corresponds to 7.63 and 9.36 mg/kg bw/day for male and female, respectively

Dose / conc.:

258 ppm

Remarks:

corresponds to 17.11 and 21.21 mg/kg bw/day for male and female, respectively

No. of animals per sex per dose:

12/sex/dose

Control animals:

yes, plain diet

Details on study design:

Each rat was identified by cage card and tail tattoo with a number that did not reveal the animal's treatment group. In addition, animals were assigned an identification number that specified the rat's sex, treatment group, cage number and identified it with the study. Randomization procedures utilized software from INSTEM Computer Systems. Following acclimation, the animals were weighed and those with body weights that were more or less than 20% of the mean weight for each sex were rejected. The remaining animals were randomly assigned to a control group or one of three dietary levels in order that, for each sex, groups had comparable body weights when treatment was initiated. This was done to facilitate comparisons following treatment. The order of testing and assignment of animals to mazes were done in a semi-random manner, such that groups were balanced across test times and test devices, and no animal would be tested more than once in the same maze.

The oral route of exposure was employed in accordance with the test guideline. Four dose groups (12 rats/sex/dietary level) were administered the test substance in the diet at nominal concentrations of 0, 30, 150 or 300 ppm. All twelve rats/sex/dietary level were used for neurobehavioral testing, with half used for micropathology. A sample of each batch of feed mixed was taken and retained in the freezer until the study was complete and the analytical data deemed satisfactory. A given batch of feed was available for consumption for a period of one week prior to changing, at which time any uneaten feed was collected and disposed of by incineration.

Cage-side observations were conducted twice daily (once daily on holidays and weekends) for mortality or clinical signs of moribundity. Detailed physical examinations for clinical signs of toxicity were carried out and recorded once each week. Individual body weight and food consumption determinations were made weekly. Perfused animals were also weighed on the day sacrificed for terminal body weight measurement.

Observations and clinical examinations performed and frequency:

For the holding period, animal care personnel observed the animals at least once daily for moribundity and mortality. Ten animals/sex were subjected to a gross necropsy and serological examination for sentinel animal/vendor surveillance evaluation (five/sex within the day following the arrival of the animal shipment and the remaining five/sex approximately five weeks later). Upon completion of the acclimation period, a veterinarian reviewed the status of the animals prior to their release for study.

Cage-side observations were conducted twice daily (once daily on holidays and weekends) for mortality or clinical signs of moribundity. Detailed physical examinations for clinical signs of toxicity were carried out and recorded once each week. Individual body weight and food consumption determinations were made weekly. Perfused animals were also weighed on the day sacrificed for terminal body weight measurement.

Neurobehavioural examinations performed and frequency:

Motor activity was measured by testing animals individually for 60 minutes in one of eight figure-eight mazes . The figure-eight maze was selected as an established and widely-used automated activity-measuring device that can be used to detect both increases and decreases in activity. Each maze consists of a series of inter-connected alleys (approximately 10 x 10 cm in crosssection) converging on a central arena and covered by transparent plastic. Each maze has eight infrared emitter/ detector pairs (three in each of the figure-eight alleys and one in each of the blind alleys) to measure activity; each time a beam is interrupted, an activity count is registered. The floor of each maze rested above absorbent paper which was changed at the end of each day. A Columbus Instruments (Columbus, OH) Universal Maze Monitoring System and a personal computer were used for automated data collection.

Broad-spectrum background noise (approximately 74dB(A)) was provided throughout the test to minimize acoustical variations during testing. A white noise generator (Model S81-02, Coulbourn Instruments) was used to produce the background noise which was amplified by an audio-mixer amplifier (Model S82-24, Coulbourn Instruments) and presented through four speakers (Realistic Minimus-7, Radio Shack) that were positioned around the test area. The uniformity of light intensity (100+70 lux) over each of the mazes was verified daily using a digital light meter. Studies with untreated animals and with rats treated with reference substances that increase (triadimefon) and decrease (chlorpromazine) motor activity have established the sensitivity, reliability and validity of these test procedures.

Motor and locomotor activity were examined for the 60-minute session and during each ten-minute interval. Motor activity was measured as the number of beam interruptions that occurred during the test session. Locomotor activity was measured by eliminating consecutive counts for a given beam. Thus, for locomotor activity, only one interruption of a given beam was counted until the rat relocated in the maze and interrupted one of the other beams. Habituation was evaluated as a decrement in activity during the test session.

All animals that were assigned to the study were tested using the FOB and motor activity on five occasions - once during the week prior to initiating the exposure and again during weeks 2, 4, 8 and 13. On the day prior to each test day, the appropriate animals were placed in the correct sequence that had been established for testing on that day. Animals were then transferred to the room where testing took place and allowed to acclimate with minimal disturbance until testing on the following day. The dose group identification was concealed prior to testing to ensure that testing would be conducted without knowledge of the group assignment.

The test room was a standard animal room that was maintained on the same light:dark cycle and settings for temperature and relative humidity as the animal room, with tests conducted during the light phase. Sets of eight animals (maximum) were evaluated individually using the FOB and then, approximately 30 minutes after the last animal in the set had finished being tested in the FOB, all eight rats were placed individually into the mazes to measure activity. Each week, testing was staggered over two days for each sex to accommodate the schedule for behavioral testing. Males and females were tested on separate days, with the open field and mazes cleaned during the ensuing interval to reduce the residual scent from the other sex.

Sacrifice and (histo)pathology:

Hematologic and serum chemistry evaluations were performed on all surviving animals (six/sex/dietary level) on the day of sacrifice. Blood samples were collected via the orbital plexis and animals were fasted overnight before collecting samples.

All animals placed on study were subjected to a complete gross necropsy. The necropsy involved an examination of all organs, body cavities, cut surfaces, external orifices and surfaces. The first six males and six females at each dietary level were selected for perfusion and collection of tissues, with replacement, as necessary, if the perfusion was considered inadequate. These animals were deeply anesthetized using an intraperitoneal dose (50 mg/kg) of pentobarbital and then perfused via the left ventricle with a sodium nitrite (in phosphate buffer) flush followed by Universal fixative (1% (w/v) glutaraldehyde and 4% (w/v) EM-grade formaldehyde) in phosphate buffer.

The entire brain and spinal cord, both eyes (with optic nerves) and selected (bilateral) peripheral nerves (sciatic, tibial and sural from the hind leg-both legs of males and females and radial, median, and cutaneous from the fore legright leg females only), the gasserian ganglion, gastrocnemius muscle from hind leg, ulnar flexor/deep flexor muscles of front leg, both forelimbs, gross lesions in neural tissues or skeletal muscle and physical identifier were dissected from each animal and post-fixed in 10% buffered formalin. The brain was weighed upon removal from the skull, prior to placement into formalin, and the brain:body weight ratio was calculated. Other animals that survived to term were sacrificed by CO2 asphyxiation without perfusion and thyroids were collected, weighed and preserved in 10% phosphate buffered formalin for microscopic examination.

The spinal cord, dorsal root ganglia, and spinal nerve roots in intermediate-dose animals undergoing perfusion fixation were collected at necropsy but were not routinely dissected from the vertebral column and examined grossly unless they were processed for micropathology. These tissues are nevertheless listed as within normal limits on the necropsy summary table to indicate that they were collected.

The following tissues from control and high-dose animals, and any gross lesions collected at necropsy, were further processed for microscopic examination. Eight coronal sections of the brain and sections from three levels of the spinal cord (cervical, thoracic, lumbar) and the cauda equina were embedded in paraffin and examined utilizing hematoxylin and eosin (H&E). Dorsal root ganglia (including dorsal and ventral root fibers) from the cervical and lumbar swellings and gasserian ganglion were embedded in glycol methacrylate (GMA). Eyes, optic nerves and gastrocnemius muscle were embedded in paraffin and stained using H&E.

Peripheral nerve tissues (sciatic, tibial and sural nerves) were embedded in GMA cut in cross/transverse-section as well as longitudinal section. Thyroids from control and high-dose non-perfused animals were embedded in paraffin and examined utilizing H&E stain. In addition, the following tissues were collected from the right forelimb of all females (radial, median and cutaneous nerves (longitudinal and cross-sections) and skeletal muscle (muscles from forearm/antebrachium, which may include the ulnar and/or deep digital flexors). These tissues were examined microscopically for lesions.

Other examinations:

The tissues from the high-dose males and females were examined relative to those from the control groups. Based on abnormal findings in skeletal muscle of the hindlimbs, in-life observations and findings from past studies indicating abnormal use in rear limbs in several high-dose females, the sciatic, sural and tibial nerves and gastrocnemius muscle from both rear legs and skeletal muscle from the right hind leg from low- and mid-dose females were processed for microscopic examination, according to procedures used for control and high-dose females. Previous studies performed at Bayer's Toxicology laboratory with trimethyltin and acrylamide have established the sensitivity and reliability of these procedures for detecting lesions in peripheral nerves and the central nervous system.

The brains were trimmed in a standard manner using a metal rodent brain matrix (RBM-4000C,
Activational Systems, Inc., Warren, Ml). Each of the resulting eight standard brain levels was surveyed for any histopathologic alteration. The microscopic evaluation included the following major brain regions: olfactory bulbs (Level 1); cerebral cortex (Levels 2 to 6); caudate-putamen/globus pallidus (Levels 2 to 5); hippocampus (Levels 5 to 6); thalamus (Levels 4 to 5); hypothalamus (Levels 4 to 5); midbrain including tectum, tegmentum, and cerebral peduncles (Level 6); cerebellum (Levels 7 to 8); and medulla oblongata (Levels 7 to 8). Individual animal data were entered into a computerized laboratory data system (DATATOX, Instem Computer Systems, Stone, Staffordshire, England) for subsequent presentation and analysis.

Statistics:

See other information for methods for more details

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no deaths before terminal sacrifice. Compound-related signs were evident in high-dose females, but not at lower dietary levels nor in males at any dietary level. Compound-related signs in high-dose females consisted of ataxia (one female, week 4 and weeks 9 through 12) and a gait abnormality (flat-footed, with short steps) affecting both hindlimbs (observed in 8 females, weeks 2 through 13). In addition, four females had red nasal stain (weeks 4 through 13), and one female had red nasal discharge (week 4) and urine stain (week 10).

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No deaths occurred at any dietary level prior to scheduled terminal sacrifice.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight was significantly reduced, relative to controls, in high-dose females only and was not affected in males at any dietary level. The difference in high-dose females was statistically significant from week 3 through the end of the study, with differences from control of 11-20%.
Based on these results, the NOAEL for body weight is 300 ppm for males and 150 ppm for females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was significantly reduced, relative to controls, for high-dose females but was not affected in females at lower dietary levels nor in males at any level of exposure. For high-dose females, the difference from control was statistically significant (in the range of 11-13%) during days 7-14 through days 35-42 and only minimally reduced (7-11%) during days 42-49 and continuing through the end of the study. Mid-dose females also appeared to consume slightly less food than controls during some weeks, however there were no occasions when the difference was statistically significant and there was no associated effect on body weight at anytime. Therefore, this is not considered an effect associated with exposure. Based on these results, the NOAEL for food consumption is 300 ppm for males and 150 ppm for females.

The average consumption of propineb test item for males and females that received diets containing analytically determined concentrations of 0, 24.4, 128 and 258 ppm Propineb was as follows:
0, 1.45, 7.63 and 17.11 mg/kg/day, respectively, for males and
0, 1.90, 9.36 and 21.21 mg/kg/day, respectively, for females.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ophthalmology findings in the rats of this study that were considered to be compound related.

Haematological findings:

no effects observed

Description (incidence and severity):

The only statistically significant changes for hematology parameters occurred in 300 ppm males - red cell distribution width (ROW) and hemoglobin distribution width (HDW) were both increased. None of these changes were thought to be compound-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no compound-related findings in males and females at any dietary level. There were
statistically significant differences from control in non-thyroid clinical chemistry parameters in males
including chloride (increased), uric acid (decreased), creatine kinase (decreased), lactate
dehydrogenase (decreased), and aspartate aminotransferase (decreased) and in females including
potassium (decreased). These differences were not considered to be biologically significant or
compound-related.
Of the thyroid parameters [thyroxine (T4), triiodothyronine (T3), and thyroid stimulating hormone (TSH)],
only male TSH was decreased (non-statistically) relative to controls. This change was not considered to be related to compound-administration.

Endocrine findings:

no effects observed

Description (incidence and severity):

Slight diffuse thyroid follicular cell hypertrophy was evident in one 300 ppm male. Since the thyroid is considered a target organ for this compound (hyperplasia and/or adenoma), this change could indicate a possible compound-related affect. However, since thyroid organ weights and thyroid clinical chemistry parameters (T4, T3, and TSH) were not affected by compound administration and there were no micropathology changes in the thyroid in males at lower dietary levels, the thyroid follicle cell hypertrophy in the one 300 ppm male was not considered to be compound-related in this study.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

An examination of inherent variability, using the average pretreatment values among the four groups of males and females provides a measure of the magnitude of the difference that should be considered biologically significant. For motor activity, the pretreatment values for groups that later received the test substance averaged from 5% to 23% lower than controls for males and from 11% lower to 15% higher than controls for females. For locomotor activity, the pretreatment values for groups that later received the test substance averaged from 4% to 18% lower than controls for males and from 18% lower to 15% higher than controls for females. As a general guide, these results confirm that differences of + 20% are within the range of normal variability in this laboratory for groups of 10-12 rats/sex/dietary level and, therefore, are not biologically significant.
For the overall 60-minute test session, there were treatment-related differences in motor and locomotor activity for high-dose females but not at lower doses nor in males at any dietary level. Motor and locomotor activity were reduced in high-dose females during weeks 2 (40% and 46%, respectively) and 4 (31% and 37%, respectively), and locomotor activity was slightly lower (28%) than controls during week 8. These differences from control were not statistically significant but are attributed to treatment due to the magnitude of change, and relationship with treatment; occurring in high-dose animals following the initiation of treatment. There were a few (six) occasions where measures of motor activity were very slightly outside of the standard (+20%) range of normal variability. In addition, measures of locomotor activity were also slightly outside of the standard (+20%) range of normal variability for males during weeks 2 (22% lower in low-level) and 8 (21% lower in high-level) and in low-dose females during weeks 4, 8 and 13 (21-43% higher). These minimal differences in activity were considered incidental and unrelated to treatment, since there was no relationship with dietary level and included both higher and lower levels of activity, relative to control.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Male terminal body weights at all dietary levels were not affected by compound administration. Female terminal body weights at the highest dietary level were significantly different (decreased) from controls. This statistical difference in female terminal body weight was thought to be compound-related. Male fixed brain (absolute and relative) weights were not affected by compound administration. Female fixed relative brain weight was statistically increased over controls. This statistical difference in fixed brain (relative) weight was due to a decreased compound-related terminal body weight. Male and female fixed thyroid (absolute and relative) weights were not affected by compound administration.

Gross pathological findings:

no effects observed

Description (incidence and severity):

All gross observations were considered to be incidental and not related to compound administration.

Neuropathological findings:

effects observed, treatment-related

Description (incidence and severity):

Nervous system tissues from all animals were evaluated microscopically due to in-life observations, abnormal findings in skeletal muscle of the hindlimbs, and findings in past studies.
Compound-related lesions were limited to atrophy of the skeletal muscle (both fore leg and hind leg) in females at the 300 ppm dietary level.
Minimal to moderate, focal to multifocal "atrophy" of the skeletal muscle occurred in both the hind leg and fore leg of 300 ppm females and was considered to be compound-related. In general, the myofiber size within specific muscle bundles was reduced with clustering of the cells (sarcolemmel nuclei and/or myosatellite cells). There was little to no evidence of tissue debris indicative of individual myofiber necrosis or of swollen or vacuolated myofibers indicative of myodegeneration. Some atrophied muscle bundles were scattered throughout the section, laying next to normal-appearing muscle bundles.
Atrophy of the skeletal muscle (foreleg and hindleg) was not observed in the 30 or 150 ppm females.
All other lesions were considered to be incidental or background and not related to compound
administration. Several incidental microscopic changes unrelated to treatment were present in control and/or treated animals. These alterations were generally minimal and included but were not limited to "axonaldegeneration" of scattered individual nerve fibers in the peripheral nerves and/or spinal cord, "atrophy" of optic nerve, "cystic follicles" and "mineralization" in the thyroid, and "cornealor limbus mineralization" and "retinal dysplasia" in the eye.

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

9.36 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

neuropathology

other:

Key result

Dose descriptor:

NOAEL

Effect level:

17.11 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

neuropathology

other:

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 ppm

System:

peripheral nervous system

Organ:

myofibres

myofilaments

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Conclusions:

Technical grade Propineb was administered in the diet for 13 weeks to young-adult Wistar rats
(12/sex/dietary level), using nominal concentrations of 0, 30, 150 and 300 ppm for males and females. Based on analytical results, Propineb was homogeneous in the ration at dietary concentrations used in this study and the actual mean concentrations of Propineb in the diet were 0, 24.4, 128 and 258 ppm for males and females. In summary, the present study established an overall NOAEL of 300 ppm for males and 150 ppm in females, based on decreased body weight and food consumption, clinical signs, decreased motor activity and micropathology in females at the 300 ppm dietary level.

Executive summary:

Technical grade Propineb was administered in the diet for 13 weeks to young-adult Wistar rats (12/sex/dietary level), using nominal concentrations of 0, 30, 150 and 300 ppm for males and females. All 12 rats/sex/dietary level were used for neurobehavioral evaluation, with six/sex/dose used for micropathology. The following observations and measurements were included in the study: clinical observations, mortality, body weight, food consumption, automated measurements of activity (figureeight maze), functional observational battery, ophthalmology, hematology and serum chemistry, brain weight, thyroid weight and microscopic evaluation and gross necropsy.

 

Skeletal muscle, peripheral nerves, eyes (with optic nerves) and tissues from the central nervous system were also examined microscopically for lesions. Based on analytical results, Propineb was homogeneous in the ration at dietary concentrations used in this study and the actual mean concentrations of Propineb in the diet were 0, 24.4, 128 and 258 ppm for males and females. Since the method used to measure propineb in the diet involved quantification of zinc (a component of the parent molecule), rather than the entire molecule (a method for propineb is not available because it exists as polymer chains that vary in length) it was not possible to verify stability in the diet. To avoid confusion between nominal and analytically confirmed doses in the report text and tables, dietary concentrations are referred to throughout this report on the basis of nominal concentration.

No deaths occurred at any dietary level prior to scheduled terminal sacrifice. Clinical signs associated with treatment were evident by cage-side observation in females that received the 300 ppm dietary level, but not at lower dietary levels nor in males at any level.Compound-related signs included a flat-footed, short-stepped gait affecting the hind limbs, ataxia, red nasal discharge, red nasal stain and urine stain. Body weight and food consumption were reduced, relative to controls, at the 300 ppm dietary level for females but not at lower levels of exposure and neither measure was affected in males at any dietary level. Body weight was reduced, relative to controls, for high-dose females in the range of 11 % to 20% (beginning at week 3 and continuing through the end of the study), while food consumption was reduced as much as 13% beginning the first week of exposure.

Based on body weight, food consumption and diet analysis data, the average daily intake of active ingredient was as follows for nominal levels of 0, 30, 150 or 300 ppm: 0, 1.45, 7.63 and 17.11 mg/kg/day, respectively, for males and 0, 1.90, 9.36 and 21.21 mg/kg/day, respectively, for females. For the functional observational battery (FOB), compound-related effects were apparent in high-dose females but not at lower levels nor in males at any dietary level. Effects in high-dose females consisted of a flat-footed, short stepped gait affecting the hind limbs, uncoordinated gait and righting response,red nasal stain, a decrease in the number of rearing events in the open field and decreased forelimb and hindlimb grip strength.

 

`Compound-related effects on motor and locomotor activity in the figure-eight maze were only evident in high-dose females. Motor and locomotor activity were reduced as much as 40% and 46%, respectively, relative to control, during weeks 2 and 4. In addition, locomotor activity was as much as 28% less than control during week 8. Habituation was not affected by treatment at any dose. There were no compound-related ophthalmic findings.

 

There were no compound-related clinical pathology findings (i.e., clinical chemistry and hematology). There were no compound-related gross lesions in males or females at terminal sacrifice. Terminal body weight was significantly reduced relative to controls in females at the 300 ppm dietary level but not at lower levels nor in males at any dietary level. There was no compound-related effect on brain weight (absolute) in either sex at any dietary level.

 

Relative brain weight was significantly increased relative to controls in high-dose females due to decreased body weight. There were no other treatment-related organ weight (i.e., thyroid) findings in either sex at any dietary level. Compound-related microscopic lesions were evident in high-dose females but not at lower dietary levels nor in males at any dietary level. Findings consisted of skeletal muscle atrophy in both the fore leg and hind leg in females. Based on these findings, the NOAEL for microscopic lesions is 300 ppm for males and 150 ppm for females. In summary, the present study established an overall NOAEL of 300 ppm for males and 150 ppm in females, based on decreased body weight and food consumption, clinical signs, decreased motor activity and micropathology in females at the 300 ppm dietary level.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

9.36 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Reliable studies according to GLP and guideline are provided.

Effect on neurotoxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In the subchronic neurotoxicity study propineb was administered in the diet for 13 weeks to young-adult Wistar rats (12/sex/dietary level), using nominal concentrations of 0, 30, 150 and 300 ppm (equivalent to 0, 1.45, 7.63 or 17.11 and 0, 1.90, 9.36 and 21.21 mg/kg bw/day in males and females, respectively). Dietary administration of propineb up to the highest dose level of 300 ppm did not provoke any effect in the male rats. In females, compound-related signs included clinical signs (flat-footed, short-stepped gait affecting the hind limbs), reduced motor activity and reduced body weight. Histopathology examination showed skeletal muscle atrophy in both the forelimbs and hind limbs in females receiving 300 ppm. The study NOAEL was 300 ppm for males (equivalent to 17.11mg/kg bw/day) and 150 ppm for females (equivalent to 9.36 mg/kg bw/day).

Technical grade propineb was administered via the diet from gestation Day (GD) 6 through lactation Day (LD) 21 to mated female Wistar rats at nominal concentrations of 0, 30, 60 or 180 ppm (equivalent to approximately 0, 2.3, 4.4 and 12.3 mg/kg/day) with adjustment of test material concentration during lactation to maintain a more consistent propineb intake throughout the period of exposure. No effects were observed in the developmental neurotoxicity study up to the high dose level tested. The dose of 180 ppm (equivalent to 12.3 mg/kg bw/day) was the NOAEL for both maternal and offspring toxicity.

 

It is of note that in repeated dose toxicity studies neuromuscular effects (flaccidity and paralysis of hind legs, reduced motility and grip strength, muscle atrophy and nerve fiber swelling) were observed (summarized in detail in Section 7.5). The overall NOAEL for dogs was 4.3 mg/kg bw per day on the basis of neurological signs in the
90-day study. Based on the effects observed after repeated exposure, propineb has a harmonised classification STOT RE cat 2 (H373, may cause damage to organs (thyroid, peripheral nervous system) through prolonged or repeated exposure if inhaled or swallowed).

 

Justification for classification or non-classification

Based on the effects observed after repeated exposure in the neuromuscular system 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.