polymeric zinc propylenebis(dithiocarbamate);propineb (ISO)

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

A reliable two-generation study in rats is available showing no effects of propineb on reproductive performance.

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008/6/26 to 2010/6/04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Principles of method if other than guideline:

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 effect on the outcome or interpretation of the study, since the results verified the homogeneity and stability of the test substance in the feed, under the conditions that were used in this study.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han CRL

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. Raleigh, North Carolina
- Age at study initiation: (P) 8-9 weeks old
- Weight at study initiation: (P) Males: mean value range: 214.2 – 258.7 g; Females: mean value range: 127.4 – 175.4 g;
- Housing: Individual hanging stainless steel cages with deionized cage board in the bedding tray. During the gestation and lactation phases, individual dams and their litters, and F1 and F2 pups were housed in polycarbonate cages with ground corn cob bedding (Bed-OCOBs).
- Diet: Purina Mills Certified Rodent Diet 5002 meal, St. Louis, MO (USA)
- Water: Water from the municipal supply, ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26°C
- Humidity (%): 30-70%
- Air changes (per hr): at least 10.67 air changes per hour
- Photoperiod (hrs dark / hrs light): Alternating 12-hour light and dark cycles
IN-LIFE DATES: The study was initiated on July 7th 2008 and the in-life phase was completed on April 30th 2009.

Route of administration:

oral: feed

Vehicle:

acetone

Details on exposure:

Diet preparation and analysis:
The test substance was dissolved in acetone and then mixed with the feed. Treated diet was mixed at room temperature; aliquots of the chemical were taken from the original test batch and transferred to the mixing area. The control test diet was prepared in the same manner as chemically-treated test diet, excluding only the test substance. 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. Replacement admixtures for each treatment group were prepared weekly (or at greater intervals depending on freezer stability) and stored under freezer conditions until presented to the animals the following week (or weeks). The concentration of the test substance in the feed for the females only was adjusted during the lactation period (Days 0-21) by 50%.

Details on mating procedure:

Four groups of 30 male and 30 female rats each were given 0, 30, 60, and 180 ppm of propineb in the diet seven days/week throughout the entire study. These rats were designated the P-Generation. After 10 weeks, each male was cohabited with a female in the same group, the females were allowed to litter, and wean their offspring. The offspring were designated the F1 Generation.
After weaning, F1-pups were maintained for approximately six weeks prior to initiation of the second generation. 30 male and 30 female rats from each group were selected for growth and subsequent mating to produce the F2 Generation. F2-pups were sacrificed at weaning on lactation day 21.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of Propineb in the various test diets was verified for batches intended for weeks 1, 2, 3, and at monthly intervals thereafter. Test diets intended for the first week of lactation were also analyzed. The homogeneity and stability of Propineb when mixed in the rodent feed was characterized.
Mean analytical concentrations for each dose group were 24.7, 49.1, and 153 ppm, ranging from 82-85% of the corresponding nominal concentrations of 30, 60, and 180 ppm, respectively. During lactation, the concentration of the test substance in the feed for the females was adjusted by 50%. Mean analytical concentrations for each dose group during lactation were 13.0, 25.9, and 78.9 ppm, ranging from 86-88% of the corresponding nominal concentrations of 15, 30, and 90 ppm, respectively. The AI of the test substance was not detected in the control diet. Mean recovery was 77% and ranged from 72-86% for rodent ration spiked with 14.6 ppm of Propineb and
mean recovery was 102% and ranged from 92 110% for rodent ration spiked with 180 ppm of Propineb. The mean concentrations of Propineb in the feed, sampled from three distinct layers in the mixing bowl and containing a nominal concentration of either 15- or 180- ppm, were determined to be 13.3 ppm (range 12.3-14.3 ppm; %RSD = 5.3) and 150 ppm (range 141-156 ppm; %RSD = 3.0), respectively. Based on a %RSD < to10%, Propineb was judged to be homogeneously distributed in the feed over a concentration range of 15-180 ppm. Following 7 days of room temperature storage, the analytically-determined concentration of the AI of the test substance in the 15- or 180-ppm admixture was determined to be 12.7 ppm (13.6 ppm on Day 0) and 155 ppm (146 ppm on Day 0), respectively. Following 28 days of freezer storage, the analytically-determined concentration of the AI of the test substance in the 15- and 180-ppm admixtures was determined to be 13.4 ppm (13.3 on Day 0) and 161 ppm (150 on Day 0), respectively. Propineb mixed in rodent ration was judged to be stable at room temperature for at least seven days and following freezer storage for a minimum of 28 days, over a concentration range of 15-180 ppm.

Duration of treatment / exposure:

continuously

Frequency of treatment:

Seven days/week throughout the entire study.

Details on study schedule:

- F1 parental animals not mated until 6 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 6 weeks

Dose / conc.:

180 ppm

Remarks:

High dose group, equivalent to a mean daily intake of 10.0 and 9.5 mg/kg bw/d for (P0-gen)-males and (P1-gen)- males respectively and 12.5 and 11.9 mg/kg bw/d for (P0-gen)-females and (P1-gen)-females respectively.

Dose / conc.:

60 ppm

Remarks:

Mid dose group, equivalent to a mean daily intake of 3.2 and 3.0 mg/kg bw/d for (P0-gen)-males and (P1-gen)- males respectively and 4.0 and 3.6 mg/kg bw/d for (P0-gen)-females and (P1-gen)-females respectively.

Dose / conc.:

30 ppm

Remarks:

Low dose group, equivalent to a mean daily intake of 1.6 mg/kg bw/d for (P0-gen)-males and (P1-gen)- males and 2.0 and 1.9 mg/kg bw/d for (P0-gen)-females and (P1-gen)-females respectively.

No. of animals per sex per dose:

30 rats/sex/group; four groups (control, 30, 60, and 180 ppm Propineb)

Control animals:

yes, concurrent no treatment

Parental animals: Observations and examinations:

Females and males were observed (cage side) for clinical signs twice daily during the working week and at least once on weekends and holidays. Cage side observations, mortality, moribundity, behavioural changes, signs of difficult or prolonged delivery, and overt toxicity by viewing the animal in the cage were conducted. A detailed evaluation of clinical signs, and a physical examination was conducted once per week.

Body weight:
Parental animals (P and F1) body weights were recorded weekly for both males and females during the premating period. During the mating period and until sacrificed, body weights for the males were recorded once per week. During gestation, dam body weights were recorded on Days 0, 6, 13, and 20 and during lactation, on Days 0, 4, 7, 14, and 21.

Food consumption and compound intake:
Food consumption was recorded once per week for both males and females parental animals (P and F1) during the premating periods. During gestation, dam food consumption was recorded on Days 0, 6, 13, and 20 and on lactation days 0, 4, 7, 14, and 21.

Urine collection:
Prior to sacrifice, urine was collected from 10 control and 10 high dose adult males. Urine was not collected from the females. Males were individually housed throughout the day in cages fitted with urine collection trays, with food and water available. Urine was collected on ice over a one to six hour period and samples were transferred to the ultralow freezer (~ -80°C) as soon as possible after collection. After urine collection, males were transferred back to their appropriate gang cage.

Oestrous cyclicity (parental animals):

The oestrus cycle was determined by examining daily vaginal smears over a three-week period prior to mating of the P- and F1-Generation females, immediately prior to the cohabitation period. Additionally, the estrous cycle stage was determined for all females just prior to termination.

Sperm parameters (parental animals):

Sperm was collected from one testis and one epididymis for enumeration of homogenization-resistant spermatids and cauda epididymal sperm reserves, respectively at sacrifice for all P- and F1-Generation males.
In addition, an evaluation of the morphology and motility was performed on sperm sampled from the distal portion (closest to the urethra) of the vas deferens on males of the control and high dose groups of both generations. Sperm motility and counts were conducted using the Integrated Visual Operating System (IVOS,
Hamilton-Thorne Research, 1998).

Litter observations:

The PND 21 pup urine was collected overnight with up to 2 pups per sex if available from the control and high dose groups. Pups were housed throughout the day in cages fitted with urine collection trays, with food and water available. Urine was not collected on ice for the weanlings. After urine collection, pups were transferred back to their appropriate nesting cage.
The size of each litter was adjusted on lactation Day 4 to yield, as closely as possible, four males and four females per litter. If the number of male or female pups was less than four, a partial adjustment was made (e.g., three females and five males). No adjustment was made for litters of fewer than eight pups. Adjustments were made by random selection of the pups using software provided by SAS.10. Grossly abnormal pups underwent a
gross internal and external examination, and all culled pups were discarded.
The F1- and F2-pups not culled on lactation Day 4 were maintained with the dam until weaning on lactation Day 21. On lactation Day 21, a sufficient number of randomly selected F1- pups/sex/litter were maintained to produce the next generation. F1-pups not selected to become parents of the next generation were sacrificed, examined macroscopically and had organs weighed. One randomly selected pup/sex/litter for each generation had tissues collected and evaluated for any structural abnormalities or pathological changes.
Random selection of pups for selection to go to next generation and those for organ weight collection was performed using software provided by SAS

Postmortem examinations (parental animals):

All surviving parental males were sacrificed as soon as possible after the last litters were produced. Maternal animals were sacrificed following the weaning of their respective litters (lactation Day 21). F1 adult males were sacrificed after the beginning of the delivery phase for the F1-females. The animals were subjected to postmortem examinations as follows: Males were euthanized by carbon dioxide asphyxiation and a gross external examination was performed. Terminal body weights were measured and the recording of all gross pathologic alterations, weighing designated organs, and saving all gross lesions was conducted on all males.
Each dam (both P- and F1-generations) was euthanized by carbon dioxide asphyxiation and a gross external examination was performed. Terminal body weights were measured and the recording of all gross pathologic alterations, weighing designated organs, and saving all gross lesions was conducted on all females. The uterus was excised and the implantation sites, if present, were counted.
Females that were sperm positive and/or had an internal vaginal plug but did not deliver were sacrificed and necropsied after gestation Day 24. Females that were never observed as being inseminated and/or with an internal vaginal plug and did not deliver at least 24 days after the completion of the mating phase, were sacrificed and necropsied. A gross necropsy was performed on these animals as described above. In addition, patency of the cervical/uterine os in these females was examined via flushing of the uterine horns with 10% buffered formalin. The following tissues were collected (X), collected and weighed (XX), and micropathology was performed on those tissues designated with an (O) (see table 1).
Animals found moribund while on study were sacrificed and a gross necropsy performed.
Animals found dead were necropsied as soon as possible. Necropsy examinations included those parameters previously described. Pups found dead, stillborn or terminated in a moribund condition underwent a gross necropsy for possible defects and/or to determine the cause of death.

Postmortem examinations (offspring):

The F1-offspring not selected as parental animals and all F2-offspring were sacrificed at 21 days
of age. These animals were subjected to postmortem examinations (macroscopic and/or
microscopic examination) as follows.
The following tissues from 21-day weanlings were collected (X), collected and weighed (XX),
and micropathology was performed on those tissues designated with an (O) (see table 2).
Pups found dead or terminated in a moribund condition underwent a gross necropsy for possible
defects and/or cause of death.

Statistics:

Parametric data (including body weight gain and food consumption) were analyzed using a univariate Analysis of Variance (ANOVA), and if significant differences were observed, a Dunnett's Test was performed. Nonparametric data (e.g., number of estrous cycles, litter size, and number of implantation sites) were first analyzed by the Kruskal-Wallis test and then subjected to Dunn's Test if significant differences were identified. Nonparametric dichotomous data (e.g. fertility and gestation indices) were initially analyzed by the Chi-Square Test and if significance was observed between groups then by the Fisher's Exact Test with the Bonferroni adjustment. To the extent possible, the frequency of gross lesions were first examined visually, then, in the event of questionable distribution, by statistical analysis using the Chi-square and Fisher's exact tests. Sperm parameters were analyzed using ANOVA, single factor. Differences between the control and test compoundtreated
groups were considered statistically significant when p < 0.05 or p < 0.01.

Reproductive indices:

Reproductive indices: The following reproductive indices were calculated from breeding and parturition records of animals in the study:
Mating Index (%) = (# inseminated females(a) x 100)/(# of females co-housed)
Fertility Index (%) = (# of pregnant females(b) x 100)/(# of inseminated females)
Gestation Index (%) = (# of females with live pups x 100)/(# of pregnant females)
(a): Includes pregnant females not observed sperm positive or with an internal vaginal plug.
(b): Includes females which did not deliver, but had implantation sites.

Offspring viability indices:

Offspring viability indices: The following viability indices were calculated from lactation records of litters in the study:
Birth Index (%) = (total # of pups born/litter x 100)/(total # of implantation sites/litter)
Livebirth Index (%) = (# of live pups born/litter x 100)/(total # of pups/litter)
Viability Index (%) = (# of live pups/litter on day 4 (pre-culling) x 100)/(# of live pups born/litter)
Lactation Index (%) = (# of live pups/litter on day 21 x 100)/(# of live pups/litter on day 4 (post-culling))
Gestation Length = Number of whole days from day in which insemination is observed in the vaginal smear (designated Day 0 of gestation) to Lactation Day 0 (delivery of pups and entry in computer system).

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substance-related mortalities or clinical observations observed during the course of this study at any dietary level tested in either generation.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No test substance-related findings were observed on body weight and body weight gain during the study for the P0-generation males at any dietary level tested.
The P0-generation females of the 180 ppm dose group exhibited significant body weight declines for weeks 5, 9, and 10 of the premating phase. At week 10, body weights were declined 7.3%, relative to controls. A decline in body weight gain was also observed in the 180 ppm dose group (declined 32.6%, relative to controls). No test substance-related findings were observed on body weight or body weight gain during the 10-week premating phase at any other dietary level tested.
Gestation:
In the 180 ppm dose group, significant declines in body weight (mean decline Days 0-20 of 6.5%) were observed. There were no test substance-related findings observed on absolute body weight at any other dietary level tested. There were no effects observed on body weight gain.
Lactation:
There were no significant body weight declines during lactation at any dietary level tested.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test substance-related findings were observed on food consumption during the study for the P0-generation males and females at any dietary level tested.
Gestation:
There were no effects observed on food consumption at any dietary level tested.
Lactation:
There were no effects on food consumption considered to be test substance-related at any dietary level tested.

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:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test substance-related microscopic findings at any dietary level tested.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no test item-related effects on oestrus cycle length

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

There were no effects considered to be test-substance-related on any sperm parameter evaluated at any dietary level tested for either generation.

Reproductive performance:

no effects observed

Description (incidence and severity):

Overall reproductive performance was not affected for any parameter (e.g., mating, fertility or gestation indices, days to insemination, gestation length, or the median number of implants) in either generation at any dietary level tested.

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive

Effect level:

12.5 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

mortality

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

4 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substance-related mortalities or clinical observations observed during the course of this study at any dietary level tested in either generation.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The P1-generation males of the 180 ppm dose group exhibited slight declines in body weight throughout premating (overall mean decline of 5.0%) with significance observed weeks 7 and 8 (mean decline for these two weeks of 6.0%, relative to controls). No effect on body weight gain was observed in this dose group.
No test substance-related findings were observed on body weight or body weight gain during the study for the P1-generation males at any other dietary level tested. Incidental body weight declines were observed in the 30 ppm dose group and is not considered to be test substance-related due to a lack of a dose response relationship. The P1-generation females of the 180 ppm dose group exhibited significant body weight declines for weeks 2 through 10 of the premating phase. At week 10, body weights were declined 11.0%, relative to controls. A decline in body weight gain was also observed in the 180 ppm dose group (declined 46.7%, relative to controls). No test substance-related findings were observed on body weight or body weight gain during the 10-week premating phase at any other dietary level tested.
Gestation:
In the 180 ppm dose group, significant declines in body weight (mean decline Days 0-20 of 9.3%) was observed. There were no test substance-related findings observed on absolute body weight at any other dietary level tested. There were no effects observed on body weight gain at any dietary level tested.
Lactation:
In the 180 ppm dose group, significant declines in body weight (mean decline Days 0-21 of 5.5%) was observed. Body weight effects were not observed at any other dietary level tested.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Premating: Food consumption on a g/kg/day basis was decreased during the first week of premating in the 60 and 180 ppm dose groups in males but by the second week was comparable to controls. Food consumption was unaffected by treatment at any dietary level tested in females.
Gestation:
A slight increase in food consumption on a g/kg/day basis was observed in the females of the 180 ppm dose group (significant week 13- 20). Test substance-related effects on food consumption were not observed at any other dietary level tested.

Lactation:
There were no effects on food consumption considered to be test substance-related at any dietary level tested.

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:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no terminal body weight or organ weight effects in males observed at any dietary level tested.
A decline in terminal body weight was observed in the females of the 180 ppm dose group, declined 5.4% relative to controls. Test substance-related organ weight effects were not observed at any dietary level tested.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no test article-related effects on oestrus cycle length

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

There were no effects considered to be test-substance-related on any sperm parameter evaluated at any dietary level tested for either generation. Epididymal counts for the P1-males of the 180 ppm dietary group appear low compared to the controls but are not considered to be a result of treatment with the test substance based on the following. There is a wide variability with epididymal counts, there was no reproductive consequence of this finding, and micropathology evaluation did not show any effect on the epididymis.

Reproductive performance:

no effects observed

Description (incidence and severity):

Overall reproductive performance was not affected for any parameter (e.g., mating, fertility or gestation indices, days to insemination, gestation length, or the median number of implants) in either generation at any dietary level tested.

There were no test substance-related effects observed on the mean primordial (preantral) follicles, antral follicles, or corpora luteal counts for the F1-females at any dietary level tested.

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction

Effect level:

12.5 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction

Effect level:

9.5 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive performance

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

3.6 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

9.5 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

mortality

body weight and weight gain

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substance-related clinical observations observed in either generation at any dietary level tested.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no test substance-related effects observed on the viability of the pups at any dietary level tested.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Pup body weights at birth for all three treated groups were comparable to the control group.
There were no test substance-related effects on pup body weight or body weight gain observed during the lactation period at any dietary level tested.

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

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

There were no effects observed on either vaginal patency or balanopreputial separation for the F1-pups at any dietary level tested.

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

13.8 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

body weight and weight gain

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substance-related clinical observations observed in either generation at any dietary level tested.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no test substance-related effects observed on the viability of the pups at any dietary level tested.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Pup body weights at birth for all three treated groups were comparable to the control group.
There were no test substance-related effects on pup body weight or body weight gain observed during the lactation period at any dietary level tested.

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

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

13.8 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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

Conclusions:

The parental male systemic NOAEL is 180 ppm (10.0 mg Propineb/kg bw/day).
The parental female systemic NOAEL is 60 ppm (4.0 mg Propineb/kg bw/day), based on decreased body weight and/or body weight gain during premating (P and F1), gestation (P and F1) and lactation (F1) at 180 ppm (11.9 mg Propineb/kg bw/day).
The reproductive NOAEL is 180 ppm in both the males and females (10.0 mg Propineb/kg bw/day for males and 12.5 mg Propineb/kg bw/day for females) based on no test-substance- related reproductive findings observed at the highest dose tested.
The offspring NOAEL is 180 ppm (13.8 mg Propineb/kg bw/day) based on no test substance-related findings observed in the pups.

Executive summary:

In a two generation-reproduction study, Propineb was administered continuously in the feed to the Wistar rat (30 animals/dose/sex) at nominal dietary concentrations of 0, 30, 60 and 180 ppm. All test diets (including control) were available for ad libitum consumption; the homogeneity and stability of Propineb as a dietary admixture was confirmed. Body weight and food consumption determinations and detailed clinical examinations of each animal were conducted weekly throughout the study, as well as, an evaluation of multiple reproductive parameters. All animals placed on study were subject to a post-mortem examination, which included recording all gross lesions, weighing designated organs and collecting representative tissue specimens for histopathological evaluation.

 

The mean daily intake of the test substance (mg propineb/kg bw/day) throughout this two-generation reproduction study at nominal dietary concentrations of 0, 30, 60 or 180 ppm, respectively, were 1.6, 3.0-3.2 and 9.5-10.0 in the males and between 1.7 -2.1, 3.6-4.1 and 11.6-13.8 in the females. In the parent and the adult of F1 generation the main effects consisted of decreased body weight and bodyweight gain in the females after 5 week of exposure. There were no toxicological effects in the offspring. There were no reproductive effects up to highest dose level.

 

The dose level of 60 ppm (equivalent to 4.0 mg/kg bw/day) in the females and 180 ppm (equivalent to 10.0 mg/kg bw/day) in the males. The dose level of 180 ppm (equivalent to 13.8 mg/kg bw/day) was also the NOAEL for the offspring and for reproductive toxicity.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subchronic

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable 2-generation study, and is thus sufficient to fulfill the standard information requirements set out in Annexes VII-X, 8.7, of Regulation (EC) No 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Reliable studies of developmental toxicity study in rat and rabbit are available.  In the developmental toxicity study in rats an overall evidence of delayed ossification suggestive of a slight delayed development secondary to maternal toxicity was observed.  In the rabbit study, increased post-implantation loss was apparent at maternally toxic dose levels.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 August to 10 October 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

LH 30/Z [Propineb], purity 83.9%, batch number 231 501 115

Species:

rabbit

Strain:

Chinchilla

Remarks:

Kfm: CHIN, Chinchilla/rabbit hybrid

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: KFH
- Age at study initiation: 4-7 months
- Weight at study initiation: 2630 - 4227 g
- Fasting period before study: none
- Housing: individually in stainless steel cages
- Diet: ad libitum pelleted standard diet (Kliba 341)
- Water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26 Aug 1986 To: 10 Oct 1986

Route of administration:

oral: gavage

Vehicle:

other: distilled water with 0.5% Cremophor EL

Details on exposure:

The test material was dissolved in distilled water with 0.5% Cremophor EL and administered orally by gavage, once daily in the morning from GD6-18. All groups received a dose volume of 4 mL/kg bw with a daily adjustment of individual volume to the actual body weight. Control animals were similarly dosed with the vehicle alone.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test material/vehicle mixtures were prepared daily prior to administration. Stability and homogeneity of the test material/vehicle mixtures were determined prior to the first administration. Samples were taken immediately after preparation. and again after 2 hours. During the treatment period, additional samples for confirmation of homogeneity, concentration and stability were taken once.

Details on mating procedure:

After at least seven days of acclimatisation, the females were housed with males (1:1) until mating has been observed. After mating, the females were removed and caged individually. The day of mating was recorded as Gestation Day (GD) 0.

Duration of treatment / exposure:

13 days (GD6-18)

Frequency of treatment:

Daily

Duration of test:

Rabbits were gavaged on GD6-18 and terminated on GD28

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control (0.5% aqueous Cremophor EL; 4 mL/kg bw); daily adjustment of individual volume to the actual body weight

Dose / conc.:

10 mg/kg bw/day

Remarks:

Daily adjustment of individual volume to the actual body weight

Dose / conc.:

30 mg/kg bw/day

Remarks:

Daily adjustment of individual volume to the actual body weight

Dose / conc.:

100 mg/kg bw/day

Remarks:

Daily adjustment of individual volume to the actual body weight

No. of animals per sex per dose:

16

Control animals:

yes, concurrent vehicle

Details on study design:

Mated female rabbits were dose by gavage on GD6-18. Dams were sacrificed and fetuses removed by caesarean section on GD28.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were observed twice daily for mortality and clinical signs

BODY WEIGHT: Yes
- Time schedule for examinations: bodyweights were recorded daily from GD0-28

FOOD CONSUMPTION: Yes
- Time schedule for examinations: food consumption was recorded on GD 6, 11, 15, 19, 24 and 28

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Gestation Day 28
- Organs examined: gross macroscopic examination of all internal organs

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 100 mg/kg bw/d, treatment of mated female rabbits caused severe signs of maternal toxicity.
Symptoms were noted in 5 of the 16 animals. In three females, one of which died prior to the last dose, dyspnoea, ventrolateral recumbency, inability to sit or stand, abnormal head position (opisthonoid) and inability to move the extremities (cataleptoid) were observed. The symptoms were first observed on GD15, 17 and 21, respectively, and continued until death (one female died on GD18 post coitum) or until termination of the study in the surviving animals. In the other two females, signs of abortion were noted on GD21 and 27, respectively.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

One rabbit at 100 mg/kg bw/d died prior to the last dose

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight gain showed a dose-related reduction at 30 and 100 mg/kg bw/d but differences from the control value only attained statistical significance at 100 mg/kg bw/d.
Additionally, calculation of body weight gain corrected for uterus weight showed treatment -related statistically significant reductions in dams at 100 mg/kg bw/d.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Evaluation of food consumption data showed dose-related and statistically significant reductions at 30 and 100 mg/kg bw/d. At 30 mg/kg bw/d, statistical significance was attained between GD15-24 and at 100 mg/kg bw/d, statistical significance was attained between GD6-19.

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:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Gravid uterus weight was unaffected by treatment

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

Signs of abortion were noted on GD 21 and 27 in two dams at 100 mg/kg bw/d

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

Evaluation of the reproduction parameters showed dose-related increased post-implantation losses at 30 and 100 mg/kg bw/d compared with the vehicle control group. At 30 mg/kg bw/d, the losses were caused by total resorption in two females and the incidences of 9.9% were
within the laboratory historical control data range (0.7-10.4%; covering studies carried out from 1984-1986). At 100 mg/kg bw/d, the increased incidence of post-implantation loss was caused by total resorption or abortion in six females and resulted in a significantly reduced number of live fetuses in this group.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

Total resorption was noted for four dams at 100 mg/kg bw/d

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

Total resorption was noted for four dams at 100 mg/kg bw/d

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

pre and post implantation loss

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

At 100 mg/kg bw/d, the increased incidence of post -implantation loss caused by total resorption or abortion in six females resulted in a significantly reduced number of live fetuses in this group.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Fetal parameters (except for the number of live fetuses in the 100 mg/kg bw/d group), as assessed by sex ratios, mean fetal body weights, external and visceral fetal examinations, examination of fetal heads and skeletal examination were not adversely affected by treatment with propineb.

Implantation losses (9.9%) observed at 30 mg/kg bw/d are not considered to be relevant because the incidence was still well within the background incidence (Laboratory historical control data from 1984 to 1986) which were included in the study report: the range of implantation
losses was between 0.7 to 10.4%, with values above 10% observed in the controls from 3 studies). As there were no adverse effects in the developmental parameters at this dose level, the NOAEL for developmental toxicity in the rabbit is considered to be 30 mg/kg bw/d. However, the PPR Meeting 146 concluded that as the incidences of post-implantation loss were borderline with the upper range of historical control data from a limited number of studies, the developmental NOAEL is 10 mg/kg bw/d.

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

yes

Maternal findings

	0 mg/kg bw/d	10 mg/kg bw/d	30 mg/kg bw/d	100 mg/kg bw/d
Mated (#)	16	16	16	16
Pregnant (#)	16	14	16	15
Mortality (#)	-	-	-	1
Abortion (#)	-	-	-	2
Implantation sites or resorbed fetuses only (#)	-	-	2	4
Litters (#)	16	14	14	8
Weight gain (g) GD 6-28	401	384	279	167
Weight gain (%) GD 6-28	+12.7	+11.8	+8.8	+5.4
Corrected weight gain (%)	+0.5	-1.8	+5.0	-7.6

Litter parameters

	0 mg/kg bw/d	10 mg/kg bw/d	30 mg/kg bw/d	100 mg/kg bw/d
Pre-implantation loss (#)	2	3	-	5
Pre-implantation loss (%)	1.7	2.4	0.0	4.5
Post-implantation loss (#)	5	7	13	47
Post-implantation loss (%)	4.4	5.8	9.9	44.8
Live fetuses (#)	109	113	118	58

 

Conclusions:

The results of this study indicate that the oral administration of two mated female rabbits causes dose-related toxic effects at dose levels of 30 and 100 mg/kg bw/d. At these dose levels, treatment caused reduced food consumption and body weight gain and increased post-implantation loss. Additionally, one out of five females at 100 mg/kg bw/d with severe symptoms died. There was no evidence of teratogenicity in this study.

Executive summary:

In a pre-natal developmental toxicity study, groups of 16 mated female Chinchilla rabbits were administered propineb (83.9% purity) by oral gavage at dose levels of 0, 10, 30 or 100 mg/kg bw/d from day 6-18 of gestation.  Signs of severe maternal toxicity were seen at 100 mg/kg bw/d, including reduced weight gain and food intake in comparison with controls, dyspnoea and ventro-lateral recumbency, and signs of abortion in two females.  One dam died.  Body-weight gain and food intake were also reduced at 30 mg/kg bw/d.  Evaluation of reproduction parameters revealed a dose-related increase in post-implantation loss at 30 and 100 mg/kg bw/d.  Fetal parameters, including external, skeletal and visceral examinations, revealed no effects associated with treatment with propineb.  A maternal NOAEL of 10 mg/kg bw/d can be determined for this study based on clinical signs, reduced weight gain and food consumption.  A developmental NOAEL of 10 mg/kg bw/d can be determined for this study, based on increased post-implantation loss.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May-2013 to February-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

January, 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

April, 2004

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD (SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: female: 11-13 weeks
- Weight at study initiation: female: 247 - 329 g
- Housing: Individual housing of pregnant females in suspended stainless steel wire mesh cages.
- Diet: A04C-10 pelleted rodent diet from S.A.F.E., ad libitum.
- Water: Filtered and softened tap water from the municipal water supply, ad libitum.
- Acclimation period: At least 10 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24 °C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 10 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h dark/ 12 h light (7 am- 7 pm)

IN-LIFE DATES: From: 23 May 2013 To: 4 July 2013

Route of administration:

oral: gavage

Vehicle:

other: Methylcellulose 400

Details on exposure:

The appropriate amount of test item was suspended (w/w) extemporaneously each day in an aqueous solution of methylcellulose 400 (Fluka, Mulhouse, France) at 0.5%, protected from light and stored at ambient temperature until use. The suspensions were mixed continuously before and during dosing with an electromagnetic stirrer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity of the suspensions was checked on the second and sixteenth formulations (F2 and F16) for all concentrations. The mean values obtained from the homogeneity check were used as measured concentrations of the second and sixteenth formulations. In addition, concentrations of the tenth and twenty-third formulations (F10 and F23) used in the study were checked. Data were recorded and analyzed using Empower 3 (Build 3471).
Since test item suspensions were prepared extemporaneously, no analysis for stability was performed.
The mean concentrations of the 4 formulations checked ranged between 91 and 96% of nominal concentrations, which was within the in-house target range of 90 to 110% of nominal concentration.
Individual checks for homogeneity ranged between 67 and 102% of nominal concentrations. Concentrations below the in-house target range of 90 to 110% were noted in formulation F2 at the three dose levels. As this was observed only for the formulation F2 and as the mean concentrations of the 4
formulations checked were within the in-house target range of 90 to 110%, it was considered to have no impact on the reliability of the study.

Details on mating procedure:

One hundred and twenty adult nulliparous female rats were obtained from the supplier. Females were mated on a one-to-one basis with stock males of the same strain and same supplier. Each morning following pairing, those female rats showing spermatozoa in a vaginal smear or sperm plug in situ were considered as pregnant animals. The day where evidence of mating was found, was designated as gestation Day 0 (GD 0).

Duration of treatment / exposure:

GD 6 to 20

Frequency of treatment:

Once daily

Duration of test:

GD 0 to 21

Dose / conc.:

48 mg/kg bw/day

Remarks:

High dose group

Dose / conc.:

12 mg/kg bw/day

Remarks:

Mid dose group

Dose / conc.:

3 mg/kg bw/day

Remarks:

Low dose group

No. of animals per sex per dose:

23 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Animal assignment and treatment:
The females were assigned to control and treated groups using a body weight procedure for each day of pairing, a computerized randomization procedure (Pristima, version 6.3.2 build 17, Xybion Corp.) for each day of pairing. If possible, those females having been paired with the same male were not allocated to the same group. The acceptable body weight was ±20% of the mean body weight on the day of randomization. Body weight means were checked after the mating period to ensure similar means among all groups. Permanent identification numbers were assigned to animals within each group. Each animal was identified by a cage card and an ear tag bearing a unique number.
The range of doses has been selected in agreement with the Sponsor Representative and based on results obtained in a range-finding study where pregnant rats received Propineb at 0, 10 and 40 mg/kg bw/day from GD 6 to 20. In this study, a dose level of 40 mg/kg bw/day caused slight maternal toxicity as evidenced by reduced mean maternal body weight gain between GD 6 and 8 (-57% compared to controls, p.0.05) and overall mean maternal body weight gain between GD 6 and 21 (-9% compared to controls, not statistically significant). Mean food consumption was also slightly decreased between GD 6 and 8 (-12% compared to the controls, p.0.05). At this dose level, no treatment-related changes were noted for litter parameters or at the external and skeletal fetal examinations. A dose level of 10 mg/kg bw/day did not induce any treatment-related maternal or fetal effects.
Dosage administration:
All doses were administered once daily, from GD 6 to 20 inclusive, orally, by gavage, in a volume of 10 mL/kg body weight/day. Dose volumes were calculated on the basis of the animal's most recently recorded body weight. Control animals received an equivalent volume of vehicle alone (aqueous solution of 0.5% methylcellulose 400).
The suspensions were mixed continuously before and during treatment with an electromagnetic stirrer. They were stored at approximately 5°C (±3°C) when not in use.

Maternal examinations:

Observations:
All clinical signs were recorded for individual animals. All animals were examined daily from GD 0 throughGD 21.
All cages were checked for dead or moribund animals twice daily, once in the morning and again in themafternoon (except at weekends and public holidays when checking was carried out once daily).

Body weight and food consumption:
Body weights were recorded on GD: 0, 6, 8, 10, 12, 14, 16, 18 and 21.
Full feeder weights were measured on GD: 1, 6, 8, 10, 12, 14, 16 and 18.
Empty feeder weights were measured on GD: 6, 8, 10, 12, 14, 16, 18 and 21.
From these records the mean daily consumption was calculated. Food spillage was also noted.

Ovaries and uterine content:

Caesarean sections
Scheduled sacrifice:
On GD 21, all surviving females were sacrificed by inhalation of carbon dioxide, for examination of uterine content. Each female was first subjected to macroscopic examination of the visceral organs. The liver of all pregnant females was weighed.
All maternal, litter and fetal data were recorded without knowledge of treatment group.
The reproductive tract was weighed (gravid uterine weight), except for animals showing total litter resorption, dissected out and the following parameters recorded:
1. Number of corpora lutea, 2. Number of implantations, 3. Number of resorptions (classified as early and late), 4. Number of live and dead fetuses, 5. Sex of live fetuses, 6. Individual weights of live fetuses.
Uterine horn(s) without visible implantations were immersed in a 10% solution of ammonium sulfide according to the Salewski method (1964), in order to visualize any sites which were not apparent. Then, tissues and carcass of dams were discarded.
Intra-uterine death was classified according to Gleich and Frohberg (1977) as:
- Early resorptions: macroscopic discrimination between fetal residues and placental material not possible.
- Late resorptions: distinct macroscopic discrimination between fetal and placental remains possible.
Dead fetuses: defined as dead conceptuses showing distinct digits on fore and hind-paws.

Blood sampling:

- Plasma: No
- Serum: No

Fetal examinations:

All fetal examinations were recorded without knowledge of treatment group.
All the live fetuses were sacrificed by subcutaneous injection (0.02 mL/fetus) of Dolethal® (18.22 g/100 mL, sodium pentobarbital) and subjected to an external examination. Approximately half of the live fetuses from each litter were immersed in Bouin's fluid for subsequent internal examination following free-hand sectioning. The remaining half were skinned, eviscerated and then placed in absolute ethanol before staining with alizarin red S and alcian blue according to a modified staining technique for skeletal examination of bones and cartilages. All specimens were archived.

Structural deviations were classified as follows:
Malformations:
A permanent structural change that is likely to adversely affect the survival or health.
Variations:
A change that occurs within the normal population under investigation and is unlikely to adversely
affect survival or health (this might include a delay in growth or morphogenesis that has otherwise followed a normal pattern of development). Where appropriate color photographs were prepared showing a representative sample of fetal findings and are retained in the study file.

Statistics:

see below under "Any other information on materials and methods incl. tables

Indices:

see below under "Any other information on materials and methods incl. tables

Historical control data:

Historical control data from studies conducted in-house were referred to in order to allow comparison with concurrent controls

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related clinical signs during the study.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no treatment-related mortalities during the study.
One low dose female was prematurely sacrificed on GD 12 due to markedly reduced body weight
and food consumption which were attributed to a dental abnormality.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 48 mg/kg bw/day, mean body weight gain all pregnant females surviving to terminal sacrifice was reduced by 51% between GD 6 and 8 (p=0.01) and by 12% between GD 18 and 21 (p=0.05), when compared to controls.
Throughout other intervals, mean maternal body weight gain was similar to controls. Overall between GD 6 and 21, mean maternal body weight gain was reduced by 10% (not statistically significant) compared to the control group. Mean body weight was marginally affected with a maximum decrease of 4% on GD 21 compared to controls. Moreover, mean maternal corrected body weight change (maternal body weight change between GD 0 and 21 independent of the uterine weight at caesarean section) was reduced by 20% (p=0.05) compared to the control group.
At 12 mg/kg/day, mean maternal body weight parameters were unaffected by treatment. The decrease in body weight change noted between GD 6 and 8 (-39% compared to controls) was considered not to be a treatment-related effect, as it was not statistically significant and because the value remained within the range of in house historical control data (HCD).
There was no effect on mean maternal body weight parameters at 3 mg/kg bw/day. The slight decrease in mean body weight gain noted between GD 14 and 18 (12%, p=0.05) in comparison to the control group was considered to be incidental as it was observed in isolation and with no dose-relationship.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect on mean food consumption at any dose level.

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:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There was no effect on mean maternal liver weight at 48, 12 or 3 mg/kg/day.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At necropsy of dams, there were no treatment-related macroscopic findings. The macroscopic findings observed occurred in one animal only or with no dose-relationship and were thus considered to be incidental.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The pregnancy rate was 96% in the control and low dose groups, and 100% in the mid and high dose group.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

CESAREAN SECTION DATA
The pregnancy rate was 96% in the control and low dose groups, and 100% in the mid and high dose group.
At 48 mg/kg/day, mean fetal body weight (combined and per sex) was slightly reduced by 3% compared to controls (not statistically significant), although mean values remained within the range of in-house HCD.
Other litter parameters including the number of live fetuses, number of implant sites per dam, percentages of pre- and post-implantation losses, number of early and late resorptions, fetal death status, percentage of male fetuses and fetal body weight for combined and separate sexes were unaffected by treatment.
At 12 and 3 mg/kg bw/day, litter parameters were unaffected by treatment. The statistically significant decrease in the number of implantation sites and increase in pre-implantation loss noted in the low dose group in comparison to controls was incidental (mainly attributable to a unilateral pregnancy for 2 dams).

Key result

Dose descriptor:

LOAEL

Effect level:

48 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOEL

Effect level:

12 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related malformations noted at the fetal examination at the
three dose levels.
Nine fetuses were observed with malformations. Since they were evenly distributed between the
groups and as the malformations occurred as isolated cases in each treated group (the 2 cases of retinal fold were observed in the controls), they were considered to have occurred spontaneously.
Variations:
There were no treatment-related variations noted at the external fetal examination at the three dose levels.
The external variation noted in one mid dose fetus (subcutaneous edema on abdomen and neck)
occurred as an isolated finding, was observed with no dose-relationship and its incidence at the fetal and litter levels was within the range of in-house HCD. It was thus considered to have occurred spontaneously.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment-related malformations noted at the fetal examination at the
three dose levels.
Nine fetuses were observed with malformations. Since they were evenly distributed between the
groups and as the malformations occurred as isolated cases in each treated group (the 2 cases of retinal fold were observed in the controls), they were considered to have occurred spontaneously.

Variations:
An overall evidence of delayed ossification was observed only in the high dose group.
At 48 mg/kg bw/day, treatment-related changes consisted of an increased incidence of 4 spontaneous variations at the fetal and litter levels, which were observed in a dose-related manner and for which the difference from controls was statistically significant at the fetal level. In addition, the incidence was outside the range of in-house HCD at the fetal and litter levels for two of these variations (“Frontal (uni/bi) and/or parietal (uni/bi) and/or interparietal: incomplete ossification” and “Forepaw(s): 3rd and/or 4th proximal phalanx: unossified”).
At 12 mg/kg bw/day, there were no treatment-related variations. The incidence of the variation “Frontal (uni/bi) and/or parietal (uni/bi) and/or interparietal: incomplete” was slightly higher than the controls at the fetal and litter levels and was outside the HCD. However, as this change was observed in isolation at this dose level and because the increased incidence at the fetal and litter levels was of low magnitude and was not statistically significantly different from the controls, it was considered to be incidental.
At 3 mg/kg bw/day, there were no treatment-related variations. The incidence of the variation “1st metatarsal: unossified” was statistically significantly higher than the controls at the litter and fetal levels. However, as this finding was observed with no dose-relationship and as the incidence at the fetal and litter levels remained within the range of in-house HCD, it was considered to be incidental.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related malformations noted at the fetal examination at the
three dose levels.
Nine fetuses were observed with malformations. Since they were evenly distributed between the
groups and as the malformations occurred as isolated cases in each treated group (the 2 cases of retinal fold were observed in the controls), they were considered to have occurred spontaneously.

Variations:
The incidence of remnant thymic present (uni/bilateral) was higher than the controls both at the fetal and litter levels in the mid and low dose groups. As the increased incidences were observed with no dose-relationship and as the incidences at the fetal and litter levels remained within the range of in-house HCD, this variation was considered not to be treatment-related.

Key result

Dose descriptor:

LOAEL

Effect level:

48 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Key result

Dose descriptor:

NOEL

Effect level:

12 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: sceletal: delayed ossification

Description (incidence and severity):

At the skeletal fetal examination, an overall evidence of delayed ossification suggestive of a slight delayed development secondary to maternal toxicity was observed.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

48 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

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

Conclusions:

In conclusion, a dose level of 48 mg/kg bw/day Propineb administered to the pregnant Sprague- Dawley rat by oral gavage was considered to be a Lowest Observed Adverse Effect Level (LOAEL) in terms of maternal toxicity, as evidenced by effects on mean body weight parameters and fetal weights, and in terms of fetal toxicity as a slight retarded ossification was noted for a few elements of the skeleton. A dose level of 12 mg/kg bw/day was considered to be a No Observed Effect Level (NOEL) in terms of maternal toxicity and in terms of fetal toxicity.

Executive summary:

In this study, groups of 23 sperm-positive female Sprague-Dawley rats were exposed to Propineb, by oral gavage from gestation day (GD) 6 to 20. The sperm-positive day was GD 0. The doses given were 0, 3, 12 and 48 mg/kg body weight/day in suspension in aqueous solution of 0.5% methylcellulose 400. The volume of administration was 10 mL/kg bw based on the most recent body weight recorded.
Clinical observations were recorded daily. Maternal body weights were recorded for all females on GD 0, 6, 8, 10, 12, 14, 16, 18 and 21. Food consumption was also measured for all the females during the intervals GD 1-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18 and 18-21. At scheduled sacrifice, on GD 21, a macroscopic examination of the visceral organs was performed, the gravid uterine weight was recorded and the dams were evaluated for number of corpora lutea, number and status of implantations (resorptions, dead and live fetuses). In addition, the liver was weighed at scheduled sacrifice for all pregnant females. Live fetuses were removed from the uterus, counted, weighed, sexed and examined externally. Approximately half of the live fetuses from each litter were fixed in Bouin’s solution and subsequently dissected for internal examination. The remaining half were eviscerated, skinned, fixed in absolute ethanol and stained according to a modification of the TYL and MARR technique (1977), (1), for skeletal examination of bone and cartilage.
Pregnancy rate was unaffected by treatment. There were no treatment-related mortalities, clinical signs or changes in mean food consumption throughout the study in dams. At necropsy, there were no treatment-related macroscopic findings in dams and mean liver weight was unaffected by treatment. At caesarean section, the following litter parameters were unaffected by treatment: number of live fetuses, number of implant sites per dam, percentages of pre- and post-implantation losses, number of early and late resorptions, fetal death status and percentage of male fetuses. There were no treatment-related malformations at the external, visceral and skeletal fetal examination.

At 48 mg/kg bw/day, there was evidence of toxic effects on maternal body weight parameters. Mean body weight gain of all pregnant females surviving to terminal sacrifice was reduced by 51% between GD 6 and 8 (p≤0.01) and by 12% between GD 18 and 21 (p≤0.05), when compared to controls. Throughout other intervals, mean maternal body weight gain was similar to controls. Overall between GD 6 and 21, mean maternal body weight gain was reduced by 10% (not statistically significant) compared to the control group. Mean maternal corrected body weight change (maternal body weight change between GD 0 and 21 independent of the uterine weight at caesarean section) was reduced by 20% (p≤0.05) compared to the control group.
At caesarean section, a 3% reduction in mean fetal body weight (combined and per sex), compared to controls (not statistically significant), although means values remained within the range of in-house historical control data.
At the external and visceral fetal examinations, there were no treatment-related variations.
At the skeletal fetal examination, an overall evidence of delayed ossification suggestive of a slight delayed development secondary to maternal toxicity was observed. The changes consisted of an increased incidence of 4 variations at the fetal and litter levels: “Frontal (uni/bi) and/or parietal (uni/bi) and/or interparietal: incomplete ossification”, “7th cervical centrum: unossified”, “Forepaw(s): 3rd and/or 4th proximal phalanx: unossified”
and “1st metatarsal: unossified”. In addition, the incidence was outside the range of in-house HCD at the fetal and litter levels for two of these variations (“Frontal (uni/bi) and/or parietal (uni/bi) and/or interparietal: incomplete ossification” and “Forepaw(s): 3rd and/or 4th proximal phalanx: unossified”).
At 12 and 3 mg/kg bw/day, there was no treatment-related effect on any of the parameters assayed.
In conclusion, the dose level of 12 mg/kg bw/day was considered to be a No Observed Effect Level (NOEL) in terms of maternal toxicity and in terms of fetal toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

168

Species:

rabbit

Quality of whole database:

The available information comprises adequate and reliable studies in the rat and rabbit, and is thus sufficient to fulfil the standard information requirements set out in Annexes VII-X, 8.7, of Regulation (EC) No 1907/2006.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on toxicity to reproduction does not meet the criteria for classification according to Regulation (EC) 1272/2008, and is therefore conclusive but not sufficient for classification.