Registration Dossier

Administrative data

Endpoint:
long-term toxicity to birds: reproduction test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-12-27 - 2012-09-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.2300 (Avian Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Amitrole
EC Number:
200-521-5
EC Name:
Amitrole
Cas Number:
61-82-5
Molecular formula:
C2H4N4
IUPAC Name:
1H-1,2,4-triazol-3-amine
Test material form:
liquid
Details on test material:
-Batch No.: RD-877-142C
-Held under ambient conditions
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Nufarm S.A.S., identified as CA2735 batch nr. RD-877-142C and batch nr. 310123555
- Expiration date of the lot/batch: October 2012
- Purity test date: 22/10/2010

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient conditions in locked storage

Dose method:
feed
Analytical monitoring:
yes
Vehicle:
yes
Remarks:
Acetone (350 ml/ 8100 g diet)
Details on preparation and analysis of diet:
DIET PREPARATION
- Description and nutrient analysis of basal diet provided in study report: yes
- Preparation of doses: test diets were prepared by mixing amitrole into a premix that was used for weekly preparation of the final diet. Dietary concentrations were adjusted for purity of the test substance and are presented as parts per million active ingredient (ppm a.i.)


HOMOGENEITY AND STABILITY OF TEST MATERIAL IN DIET
- How often was homogeneity and stability tested: day 0 and 7 of week1, and week 5, 9, 12, 17, 36, 57 and 63
- When and at what dose levels were samples of treated food analyzed for stability and concentration during the study: See sampling dates above. Samples were collected from the top, middle and bottom of left and right sections of the mixing vessel - 6 samples rom each of the treatment diets and one sample from the control
- Results of homogeneity analysis (range of values): Diet samples were collected from the 95, 156 and 256 ppm a.i. test concentrations, and were analyzed to evaluate the homogeneity of the test substance in the diet. Means and standard deviations for the three test concentrations were 89.3 +/- 2.96 ppm a.i., 144 +/- 5.49 ppm a.i., and 243 +/- 10.8 ppm a.i., respectively. The coefficients of variation were 3.31%, 3.81% and 4.44%, respectively. Samples collected during the test to verify test substance concentrations for the 95, 156 and 256 ppm a.i. diets had means and standard deviations of 91.2 +/- 6.95 ppm a.i., 153 +/- 8.84 ppm a.i. and 238 +/- 10.0 ppm a.i., respectively. The coefficients of variation were 7.62%, 5.78% and 4.20%. These values represented 96%, 98% and 93% of nominal concentrations. Analysis of diet samples collected from feeders after being held at ambient temperature for 7 days averaged 95%, 102%, and 93% of the Day 0 values for the 95, 156, and 256 ppm a.i. test concentrations, respectively.
- Nominal concentration (mg/kg feed): 95, 156, 256
- Concentration analysed (mg/kg feed): see above
- % of nominal: see above
- Mixing procedure adequate and variance between nominal and actual dosage acceptable : yes

Test organisms

Test organisms (species):
Anas platyrhynchos
Details on test organisms:
TEST ORGANISM
- Common name: Mallard duck
- Source: Purchased from Whistling Wings, Inc., 113 Washington street, Hanover, IL 61041-0509, USA, from the same hatch
- Age at test initiation (mean and range, SD): 31 weeks (from the same hatch)
- Weight at test initiation (mean and range, SD): 863-1295 g
- Sexes used / mixed or single sex: mixed (paired)
- Cultural background:
- Disease free: yes
- Kept according to standard practices:yes

Study design

Limit test:
no
Total exposure duration (if not single dose):
24 wk
Remarks:
The mallards were exposed to test itm pulses into the feed: - F0 : 9 wks of exposure - F1 : 8 wks of exposure followed by 14 wks on non-treated diet; then 8 wks of exposure followed by 14 wks on non-treated diet; then 8 wks after paired housing
Post exposure observation period:
6 weeks (final egg incubation, hatching and 14-day off-spring rearing period)
No. of animals per sex per dose and/or stage:
F0 : 10 pairs
Control animals:
yes, concurrent vehicle
Nominal and measured doses / concentrations:
Nominal: 0, 95, 156 and 256 mg/kg
Details on test conditions:
ACCLIMATION
- Acclimation period: 8 wks
- Acclimation conditions (same as test or not): same as test
- Feeding: ad libitum during acclimation and test - basal diet contains at least 27% proteins and 2.5% crude fat, and no more than 3.8% crude fiber, approximately 1.10% calcium
- Health (any disease or mortality observed): birds that did not appear healthy were excluded from the study.
- Fasting period before study: none


PEN SIZE AND CONSTRUCTION MATERIALS
- Description: Batteries of pens measuring approximately 75 x 90x45 cm high, contructed of vinyl-coated wire mesh, equiped with a binfeeder
- Compliant to good husbandry practices: yes
- Suitable to avoid crowding stress: yes

NO. OF BIRDS PER STAGE OR REPLICATE
- For vehicle control: 10 pairs
- For treated: 10 pairs per treatment


TEST CONDITIONS (range, mean, SD as applicable)
- Temperature:
- Room temperature: 20.3 - 24.8°C according to the test phases
- Relative humidity (%): 24-79% according to the test phases
- Photoperiod: 8 or less to 17hours light according to the test period

Examinations

Details on examinations and observations:
MORTALITY / CLINICAL SIGNS
- Time schedule for examinations: daily

BODY WEIGHT
- Time schedule for examinations: Individual F0 adult body weights were measured at test initiation and at primary adult termination. In addition, body weights of withdraw F0W adult birds were measured at the start of the withdraw phase and at adult termination. For all generations, individual body weights of live offspring were collected at Day 0 and Day 14. F1 offspring being grown out were individually weighed at approximately 9, 23, 31 and 45 weeks of age to correspond with transitions to and from treated diets. F1 adults were also weighed at the termination of the egg laying portion of the study. Similarly, body weights of F1W withdraw offspring being grown out were individually weighed at approximately 8, 23, 31 and 45 weeks of age to correspond with diet transitions and at termination of the egg laying portion of the study. Body weights were not measured during egg laying because of the possible adverse effects handling may have had on egg production.


FOOD CONSUMPTION
- Time schedule for examinations: Feed consumption was measured for the adults and offspring for a seven-day period each week throughout the test when treated diet was presented. Feed consumption was determined by weighing the freshly filled feeder on Day 0, recording the amount of any additional diet added during the week and weighing the feeder and remaining feed at the end of the feeding period (Day 7). The amount of feed wasted by the birds was not quantified, since the wasted feed was normally scattered and mixed with water and excreta. Therefore, feed consumption is presented as an estimate of total feed consumption.


PATHOLOGY
- Dose groups that were examined: Adult birds that died or were euthanized during the course of the study were subjected to a gross necropsy. At the conclusion of the F0 and F0W study periods and at the conclusion of the F1 and F1W study periods, all surviving adult birds were euthanized by cervical dislocation and necropsied. Thyroid glands of each bird were collected, fixed in Davidson’s solution and preserved in formalin for potential histopathological evaluation. In addition, up to two offspring (when available) per parental pen of origin from Lot D of the F1 offspring and Lot WC of the WF1 offspring were euthanized at hatch and thyroid glands were collected, fixed and preserved in formalin for potential histopathological evaluation. At 14 days of age, thyroids were collected from up to two additional offspring per pen (when available) from the Lot D, F1 and Lot WC, WF1. Similarly, thyroids of up to two offspring per pen (when available) from the Lot D and E F2 offspring and Lot WD and WE WF2 offspring were collected at hatch and 14-days of age, fixed and preserved for potential evaluation. Young adult mallards that were not used for the F1 or WF1 portion of the test were euthanized by cervical dislocation, necropsied and disposed of by incineration.

Details on reproductive parameters:
The following parameters were examined per parental pen per week:
- Eggs laid
- Eggs cracked; eggs broken
- Eggshell thickness or eggshell strength
- Embryos viable
- 14-day old surviving chicks
- Chick body weight at hatching and 14 days after hatching

Reference substance (positive control):
no

Results and discussion

Effect levelsopen allclose all
Key result
Duration (if not single dose):
24 wk
Dose descriptor:
NOEC
Effect level:
95 mg/kg diet
Conc. / dose based on:
act. ingr.
Basis for effect:
reproductive parameters
Key result
Duration (if not single dose):
24 wk
Dose descriptor:
NOEL
Effect level:
16 mg/kg bw/day (nominal)
Conc. / dose based on:
act. ingr.
Basis for effect:
reproductive parameters
Duration (if not single dose):
24 wk
Dose descriptor:
NOEC
Effect level:
156 mg/kg diet
Conc. / dose based on:
act. ingr.
Basis for effect:
body weight
Repellency factors (if applicable):
Not applicable
Mortality and sub-lethal effects:
No treatment related mortalities were observed during any generation of at any test concentration.
At the 95 ppm a.i. test concentration no overt signs of toxicity were observed and there were no treatment related effects upon body weight or feed consumption.
At the 156 ppm a.i. test concentration there were reductions in F1 14-day old survivor body weight and feed consumption during brooding and early grow out, but subsequent recovery.
At the 256 ppm a.i. test concentration there were impacts upon offspring health during brooding and grow-out and impacts upon body weight and feed consumption for both the F0 and F1 generations.
At all necropsy intervals and across generations, primarily concentration responsive impacts upon thyroid gland size were noted at the 95, 156 and 256 ppm a.i. concentrations.


Effects on reproduction:
At the 95 ppm a.i. test concentration, there were no treatment related effects upon any of the reproductive parameters measured at any generation.
At the 156 ppm a.i. test concentration, there were treatment related effects upon egg production of both the F0 and F1 generations.
At the 256 ppm a.i. test concentration, forthe F0 generation there were marked impacts upon the reproductive parameters measured and an impact upon hatchability for the F1 generation.
Results with reference substance (positive control):
No reference substance
Further details on results:
Clinical observations:
F0: incidental clinical observations noted included those that normally are associated with injuries and pen wear.
F1: Clinical observations during brooding were not considered to be treatment related. While clinical observations noted during rearing at the 95 and 156 ppm a.i. test concentrations were not considered to be reatment related, the increased incidence of small and/or abraded birds in the 256 ppm a.i. test concentration was considered to be treatment related.
F2: Clinical observations noted at the 95 and 156 ppm a.i. test concentrations during brooding were not considered to be treatment related. At the 256 ppm a.i. test concentration, two F2 offspring were noted as wet and weak, while five additional offspring were noted to be weak. While only a few offspring were impacted, the increased numbers of weak ducklings at the 256 ppm a.i. test concentration were considered to be related to treatment. Except for findings noted, all birds appeared normal throughout the study.

Gross necropsy:
All surviving adults were subjected to gross necropsy following adult termination. When compared to the control group, primarily concentration responsive enlarged thyroids were evidenced in the F0 and F1 adult birds of the 95, 156 and 256 ppm a.i. treatment groups. Additionally, during collection of thyroids from the 156 and 256 ppm a.i. F1 and F2 offspring for possible histopathological evaluation, thyroid enlargement was noted.

Body weight measurements:
F0: For the F0 adults there were no apparent treatment-related effects upon adult body weights at the 95 and 156 ppm a.i. test concentrations and no statistically significant differences between the control group and the 95 and 156 ppm a.i. treatment groups were observed. At the 256 ppm a.i. test concentration there were statistically significant (p < 0.01) reductions in weight gain and terminal body weight of F0 hens over the treatment period.
F1: For the F1 offspring (receiving treated diets) there were no apparent treatment related effects upon or statistically significant differences in hatchling (Day 0) body weight any of the concentrations tested or 14-day old survivor body weight at the 95 ppm a.i. test concentration. When compared to the control group, there were statistically significant (p < 0.01) reductions in the body weight of 14-day old survivors from the 156 and 256 ppm a.i. treatment groups. There were no statistically significant differences in body weight at any concentration tested at the Week 21 body weight interval (end of Pulse 1). At the start of Pulse 2, there was a statistically significant increase in the body weight of males from the 256 ppm a.i. treatment group, but no other statistically significant differences between the control group and any treatment group were noted in F1 body weights at any other interval.
F2: For the F2 offspring (receiving untreated diets) there were no apparent treatment related effects upon hatchling or 14-day old survivor body weight any of the concentrations tested. When compared to the control group, there were no statistically significant differences in offspring body weight at the 95, 156 and 256 ppm a.i. treatment groups.

Feed consumption measurements:
F0: During the F0 exposure phase, there were no apparent treatment-related effects upon feed consumption at the 95 and 156 ppm a.i. test concentrations and no statistically significant differences
were observed at any of the feed consumption intervals. At the 256 ppm a.i. test concentration, statistically significant reductions (p < 0.05) in feed consumption were observed during Weeks 7, 8 and 9 (p < 0.05) and Week 10 (p < 0.01).
F1: While F1 birds were housed in groups by treatment during brooding and rearing, a statistical comparison of feed consumption values could not be performed. However, at the 156 and 256 ppm a.i.
treatment groups there was an apparent treatment-related reduction in feed consumption during brooding and at least the first two weeks of grow-out, a time when offspring were exposed to treated diets. There was no apparent treatment related effect upon feed consumption at any test concentration during the second exposure pulse (Weeks 35 through 42) or during the third exposure pulse (Weeks 57 through 63, reproduction). No statistically significant differences were observed between the control group and any treatment group during the third pulse.

Reproductive results:
F0: For the F0 generation, there was no apparent treatment related impact upon reproductive success at the 95 ppm a.i. test concentration and no statistically significant differences were noted for any reproductive parameter measured. At the F0 156 ppm a.i. test concentration, while not statistically significant, there was a reduction in egg production that was considered to be related to treatment. At the F0 256 ppm a.i. test concentration there were profound effects upon reproductive performance, including egg production and hatchability. Statistically significant differences (p < 0.01) were observed in eggs laid of maximum laid, hatchlings of live 3-week embryos and hatchlings and 14-day old survivors of both eggs set and maximum eggs set.
F1: For the F1 generation, there was no apparent treatment related impact upon reproductive success at the 95 ppm a.i. test concentration and no statistically significant differences were noted for any reproductive parameter measured. At the F1 156 ppm a.i. test concentration there was a slight, but statistically significant (p < 0.05) reduction in egg production that was considered to be related to treatment. At the F1 256 ppm a.i. test concentration, while not statistically significant, there was a reduction in hatchability that was considered to be related to treatment.

Egg shell thickness
F0: There were no apparent treatment related effects upon F0 egg shell thickness at the 95 or 156 ppm a.i. test concentrations and, when compared to the control group, there were no statistically significant differences in egg shell thickness in the 95 or 156 ppm a.i. treatment groups. However, at the 256 ppm a.i. test concentration there was a reduction in mean egg shell thickness that was statistically significant at p < 0.05.
F1: For the F1 generation, there were no treatment-related effects upon egg shell thickness at any of the concentrations tested. When compared to the control group, there were no statistically significant differences in egg shell thickness in any treatment group.

Reported statistics and error estimates:
See details on results above

Applicant's summary and conclusion

Validity criteria fulfilled:
not specified
Conclusions:
No impact upon growth or reproductive success was noted at the 95 ppm a.i. test concentration. Multiple, and typically concentration responsive, effects were noted upon growth and reproduction at the 156 and 256 ppm a.i. test concentrations, including the corresponding withdraw groups. At all necropsy intervals, primarily concentration responsive impacts upon thyroid gland size were noted at the 95, 156 and 256 ppm a.i. concentrations. The reproductive no-observed-effect concentration was 95 ppm a.i., approximately equivalent to a calculated average daily dietary dose of 16 mg/kg bw/day during the exposure phases.
Executive summary:

Mallards received amitrole at nominal dietary concentrations of 95, 156 and 256 ppm a.i. at intervals during a two generation reproduction study. The F0 generation was exposed to treated diets from photostimulation through an egg laying cycle of seven weeks and selected F1 offspring were exposed to treated diets for the first eight weeks of rearing. The F1 offspring were exposed to treated diets for an eight-week period during grow-out and were paired and exposed to appropriate treated diets again at approximately 45 weeks of age and monitored through a six week reproduction cycle. F2 hatchlings were reared to 14-days of age on untreated diet. Due to the impact of the test substance upon mallards in the 156 and 256 ppm a.i. treatment groups for the F0 generation, 10 pairs of mallards from each of those groups were transferred to untreated diet for a five week withdrawl (W) phase. Offspring from those birds (F1W) were handled comparably to the F1 exposure phase birds.

No impact upon growth or reproductive success was noted at the 95 ppm a.i. test concentration. Multiple, and typically concentration responsive, effects were noted upon growth and reproduction at the 156 and 256 ppm a.i. test concentrations, including the corresponding withdraw groups. At all necropsy intervals, primarily concentration responsive impacts upon thyroid gland size were noted at the 95, 156 and 256 ppm a.i. concentrations. The reproductive no-observed-effect concentration was 95 ppm a.i., approximately equivalent to a calculated average daily dietary dose of 16 mg/kg bw/day during the exposure phases.