cyproconazole (ISO); (2RS,3RS;2RS,3SR)-2-(4-chlorophenyl)-3-cyclopropyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol

Toxicity to birds

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to birds: reproduction test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Dec 2006 to 25 Oct 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 206 (Avian Reproduction Test)

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 71-4 (Avian Reproduction Test)

Version / remarks:

1982

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.2300 (Avian Reproduction Test)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes

Dose method:

feed

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

feed

Details on preparation and analysis of diet:

DIET PREPARATION
- Description and nutrient analysis of basal diet provided in study report: The basal ration contained at least 27% protein and 2.5% fat and no more than 3 8% crude fiber. The basal diet contained approximately 1.0% calcium derived from feedstuffs and the 0.6% limestone used in the formulation of the basal diet. While this level of calcium is sufficient for growth and maintenance rations. Additional calcium is required in the ration of breeding birds for egg shell formation. Therefore, an additional 5% (w/w) of limestone (approximately 38.5% Ca) was added to the basal diet for the adults. This raised the calcium level in the diet for the breeding birds to approximately 3%, slightly above the minimum recommended for mallard (2.75%). Offspring received basal diet without test substance and without the addition of 5% supplemental limestone.

- Preparation of doses: Test diets were prepared by mixing the test substance into a premix that was used for weekly preparation of the final diet. Control diet and each of the four treated diets were prepared weekly beginning on December 13, 2006 and presented to the birds on Wednesday of each week. 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
- Diet sampling: Homogeneity of the test substance in the diet was evaluated by collecting six samples each from the 8 and 27 ppm a.i. treated diets on Day 0 of Week 1. Samples were collected from the top, middle and bottom of the left and right sections of the mixing vessel. A sample of the control diet and samples from the 12 and 18 ppm a.i treated diets were collected on Day 0 of Week 1. Control and treatment group diet samples also were collected from the bin feeders on Day 7 of Week 1 to assess stability of the test substance under actual test conditions Additionally, samples were collected from the control and treatment group diets during Weeks 4, 8. 12. 16 and 20 of the test to measure/verify test concentrations. The diet samples were stored frozen prior to analysis.

- Analytical method: Samples were extracted with toluene. Concentrations of the test substance in extracts of the samples were determined by gas chromatography (GC) equipped with a nitrogen/phosphorous detector (NPD).

Calibration standards of the test substance ranging m concentration from 0.250 to 3.00 µg a.i./mL were analysed with each sample set. Linear regression equations were generated using the peak area responses versus the respective concentrations of the calibration standards. The concentration of test substance in the samples was determined by substituting the peak area responses into the applicable linear regression equation. The instrument limit of detection (LOO) was set based upon the injection volume (4 µL) and the lowest standard concentration 0.250 µg a.i./mL. The LOD was set at 100 ng on-column. The method limit of quantitation (LOQ) for these analyses was set at 5 ppm a.i. based upon the lowest matrix fortification level analysed concurrently with the samples

GC PARAMETERS
- Analytical column: DB-5 (30m x 0.53 mm x 1.5 µm. film thickness)
- Injector temperature: 250 °C
- Oven initial temperature: 180 °C
- Oven initial hold time: 1.00 minute
- Oven ramp: 30 °C/minute
- Final temperature: 240 °C
- Final hold time: 7.00 minute
- Detector temperature: 275 °C

Test organisms (species):

Anas platyrhynchos

Details on test organisms:

TEST ORGANISM
- Common name: Mallard duck
- Age at test initiation: 20 weeks old
- Weight at test initiation: 802 - 1279 g
- Other: The birds were from the same hatch, approaching their first breeding season and had not been used in any previous testing. Sex of the birds were determined by a visual examination of the plumage.

Limit test:

no

Total exposure duration (if not single dose):

20 wk

No. of animals per sex per dose and/or stage:

1 male and 1 female animal per pen, and 16 pens per dose

Control animals:

yes, plain diet

Nominal and measured doses / concentrations:

Nominal concentrations: 0 (negative control), 8, 12, 18 and 27 ppm a.i.
Only 8 and 27 ppm a.i. nominal concentrations were measured, the measured concentrations were 6.87 ± 0.214 ppm a.i. and 24 8 ± 1.23 ppm a.i., respectively.

Details on test conditions:

ACCLIMATION
- Period: 4 weeks
- Health condition: At the start of acclimation. the mallard were apparently healthy and phenotypically indistinguishable from wild type. A random number generating function in a spreadsheet program was used to randomise pen assignment for each bird. Immediately prior to test initiation, all potential study birds were examined for physical injuries and general health. Birds that did not appear healthy, either due to injury or inability to acclimate to laboratory conditions, or were outside the weight range for the test. were excluded from the study.
- Feeding: All adult birds and their offspring were given feed and water ad libitum during acclimation and testing.

PEN SIZE AND CONSTRUCTION MATERIALS (adult)
- Description: Approximately 75 x 90 x 45 cm high.
- Feed and water supply: The pens were constructed of vinyl-coated wire mesh. Each pen was equipped with a bin feeder. Weekly, sufficient feed for the feeding period was placed in the bin feeder for each pen and presented to the birds. During the feeding period additional feed was weighed and added to the bin feeders as needed. Water was supplied by nipple-type waterers.
- Compliant to good husbandry practices: Yes
- Suitable to avoid crowding stress: Yes (Only birds associated with this study were maintained in the study room in order to avoid excessive disturbances.)

NO. OF BIRDS PER STAGE OR REPLICATE
- For negative control: 2 (1 male and 1 female)
- For treated: (1 male and 1 female)

NO. OF REPLICATES PER GROUP
- For negative control: 16
- For treated: 16

TEST CONDITIONS (adult)
- Room temperature: 20.9 ± 1.2 °C
- Relative humidity (%): 46 ± 10%
- Photoperiod: The photoperiod in the adult mallard room was maintained by a time clock. The photoperiod during acclimation and the first eight weeks of the test was eight hours of light per day. The photoperiod was increased to 17 hours of light per day at the beginning of week 9 to induce egg laying and was maintained at that length until the adult birds were euthanised. Throughout the test, the birds received a mean of approximately 322 lux ( about 30 ft candles) of illumination provided by fluorescent lights that closely approximated noonday sunlight.
- Ventilation: The air handling system in the study room was designed to vent up to 15 room air volumes every hour and replace them with fresh air.
- Egg Collection and Storage: Eggs were collected daily from all pens, when available. Eggs to be incubated were washed to reduce the possibility of pathogen contamination before storing them in the cold room Eggs were washed in a commercial egg washer with a chlorine-based detergent. Water in the washer was warmed to approximately 45 °C. The eggs were placed in the wash water and soaked for approximately 15 seconds. The washer's circulation motor was then turned on for approximately three minutes. The eggs were removed from the washer allowed to cool to approximately room temperature and rinsed with fresh water. The eggs were then stored in a cold room until incubation. The cold room was maintained at a mean temperature of 14.5 ± 0.1 °C with a mean relative humidity of approximately 74 6%. Groups of eggs were identified by an alphabetic lot code. All eggs laid in a weekly interval were considered as one lot.
- Candling and incubation: All eggs not discarded or used for egg shell thickness measurements were placed in an incubator/a hatcher. In the incubator the temperature was maintained at an average 37.4 ± 0.0°C with an average relative humidity of approximately 54 ± 0%.
The incubator was equipped with a pulsator fan and blades that produced a mild breathing air movement designed to eliminate intracabinet temperature and humidity variation during incubation. In order to prevent adhesion of the embryo to the shell membrane, the incubator was also equipped with an automatic egg rotation device designed to rotate the eggs from 45° off of vertical in one direction to 45° off of vertical in the opposite direction (total arc of rotation was 90°) every hour through Day 24 of incubation.

PEN SIZE AND CONSTRUCTION MATERIALS (hatching)
- Description: Approximately 62 x 92 x 25.5 cm high.
- Floor covering: The walls, floors and ceilings of each pen were constructed of vinyl-coated wire mesh.

TEST CONDITIONS (hatching)
- Hatching and Brooding: On Day 24 of incubation, the eggs were placed in an incubator/a hatcher and allowed to hatch. Pedigree baskets constructed of galvanised steel wire mesh were used to keep hatchings separated by parental pen of origin. Eggs were not rotated in the hatcher. The average temperature in the hatching compartment was 37.3 ± 0°C with an average relative humidity of 58 ± 2%.
Hatchlings were wing banded for identification by pen of origin and then routinely housed according to the appropriate parental concentration grouping in brooding pens until 14 days of age. The hatchlings were fed untreated diet without the addition of 5% supplemental limestone. The ducklings were euthanised with carbon dioxide after 14 days of age and disposed of by incineration.
- Temperature of brooding compartment: Approximately 38° C from the time of hatching until the birds were five to seven days of age, when the temperature was adjusted to maintain a temperature of approximately 29 °C
- Average ambient room temperature: 24.1 ± 1 0°C
- Average relative humidity: 53 ± 14%
- Photoperiod: Maintained by a time clock at 16 hours of light per day

Details on examinations and observations:

- Observations: The test birds were acclimated to the facilities and study pens for four weeks prior to initiation of the test. During acclimation, all birds were observed daily. Birds exhibiting abnormal behaviour or debilitating physical injuries were not used for the test. During the study, all adult birds were observed daily for signs of toxicity or abnormal behaviour. Additionally, all offspring were observed daily from hatching until 14 days of age. A record was maintained of all mortalities and clinical observations

- Necropsy: Adult birds that died or were euthanized during the course of the study were subjected to a gross necropsy. At the conclusion of the exposure period, all surviving adult birds were euthanized by cervical dislocation, necropsied, and disposed of by incineration.

- Adult Body Weight: Adult body weights were measured at test initiation, at the end of Weeks 2, 4. 6, 8, and at adult termination. Body weights were not measured during egg laying because of the possible adverse effects handling may have on egg production.

- Feed Consumption: Feed consumption for each pen was measured weekly throughout the test 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.

Details on reproductive parameters:

- Egg shell thickness: At the end of the weekly interval all eggs were removed from the cold room counted and eggs selected by indiscriminate draw for egg shell thickness measurement. The remaining eggs were candled with an egg-candling lamp to detect egg shell cracks or abnormal eggs. Cracked or abnormal eggs were recorded and discarded.
Weekly throughout the egg laying period, one egg was collected, when available, from each of the odd numbered pens during odd numbered weeks and from each of the even numbered pens during the even numbered weeks. The eggs were opened at the waist the contents removed. The shells thoroughly rinsed with water. The shells were then allowed to air dry for at least one week at room temperature. The average thickness of the dried shell plus the membrane was determined by measuring five points around the waist of the egg using a micrometre. Measurements were made to the nearest 0.002 mm.

- Embryo viability and survival: Eggs were candled on Day 14 of incubation to determine embryo viability and on Day 21 to determine embryo survival.

- Body weight of hatching: All hatchlings, unhatched eggs and egg shells were removed from the hatcher on Day 27 or 28 of incubation. The group body weight of the surviving hatchlings by pen was determined. At 14 days of age, the average body weight by parental pen of all surviving ducklings was determined.

Reference substance (positive control):

no

Key result

Duration (if not single dose):

20 wk

Dose descriptor:

NOEL

Effect level:

18 mg/kg diet

Conc. / dose based on:

act. ingr.

Basis for effect:

reproductive parameters

Mortality and sub-lethal effects:

An overview of the results is provided in Table 1 - Table 6 in 'Any other information on results inlc. tables'

- Mortality: There were no treatment-related mortalities at any of the concentrations tested. While no mortalities occurred in the control group or in either the 12 or 18 ppm a.i. treatment groups, one incidental mortality occurred in both the 8 and 27 ppm ai treatment groups.
The single mortality m the 8 ppm a.i. treatment group (the male in Pen 524) was found dead on Day 5 of Week 14. Prior to being found dead, the bird was observed with foot lesions. At necropsy the bird weighed 1237 g. Externally, foot lesions were noted. Internally, there were petechial hemorrhages noted on the heart, the spleen was enlarged (about 3 x 4 cm), the kidneys were pale, and the liver was firm with a greyish-coloured lesion (about 1 x 2 cm) on the right lobe. Additionally, there was clotted blood along the intestines and fluid in the abdominal cavity (around 30 mL of a bloody fluid, and around 70 mL of an amber-coloured fluid) Necropsy of the male’s pen-mate was unremarkable.
The single mortality in the 27 ppm a.i. treatment group (the female in Pen 570) was found dead on
Day 4 of Week 20 without any prior clinical signs observed. At necropsy, the bird was noted as emaciated, with a loss of muscle mass, a prominent keel, and a body weight of 725 g. Externally, the bird had feather loss around the eyes. Internally, the spleen was pale, there were yellowish plaques on the air sacs, the bottom margins of the liver were dark in colour, and there were extensive lesions consistent with egg-yolk peritonitis in the abdominal cavity Additionally. the ovary was regressing, there was autolysis of the reproductive tract and a developed egg in the tract. Necropsy of the female's pen-mate was unremarkable.
No other mortalities occurred during the course of the study. Due to the nature of the lesions observed at necropsy. the mortalities that occurred were not considered to be related to treatment.


- Clinical Observations: No overt signs of toxicity were observed at any of the concentrations tested Incidental clinical observations noted during the test included those that normally arc associated with injuries and pen-wear. Such signs included head and foot lesions, unilateral wing droop, a leg fracture, leg swelling, a prominent keel, and molting and feather loss. In addition, lower limb weakness and lameness also were observed, and were typically associated with the incidental injuries. Except for these incidental findings, all birds appeared normal throughout the study.

- Gross Necropsy: All surviving adults were subjected to gross necropsy following adult termination. All findings observed were considered unrelated to treatment.

- Adult Body Weight: There were no apparent treatment-related effects upon adult body weight at any of the concentrations tested. No statistically significant differences between the control group and the 8, 12, 18 or 27 ppm a.i. treatment groups were observed at any of the body weight intervals.

- Adult Feed Consumption: There were no apparent treatment-related effects upon feed consumption at the 8, 12, or 18 ppm a.i. test concentrations and no statistically significant differences between the control group and the 8, 12, or 18 ppm a.i. treatment groups were observed at any of the feed consumption intervals. At the 27 ppm a.i. test concentration, there was a slight reduction in feed consumption during Week 1 that was statistically different from the control group at p < 0.05 and may have represented a reluctance to consume treated diet. During Week 15 there was also a slight reduction in feed consumption that was statistically different from the control group at p < 0.05. However, since the decrease in feed consumption observed during Week 15 was slight, transient, and not associated with an impact upon body weight. The difference was not considered to be biologically meaningful.
The mean body weight value is the mean of both male and female body weights. For the pre-egg production interval, the body weights were averaged over Weeks 0, 2, 4. 6 and 8 For the egg-production interval body weights were averaged over Weeks 8 and 20 (adult termination). The accuracy of the estimated mean daily dietary dose may be impacted by differences in individual feed consumption, both within and between pens, and feed wastage.

Effects on reproduction:

An overview of the results is provided in Table 1 - Table 6 in 'Any other information on results inlc. tables'

- Reproductive Results: There were no treatment-related effects upon reproductive performance in the 8, 12 or 18 ppm a.i. treatment groups and any differences in mean values were not significantly different from control values for any of the reproductive parameters measured. However, at the 27 ppm a.i. test concentration, there was a treatment-related reduction in embryo survival that was statistically significant from the control group. The number of live 3-week embryos, expressed as a percentage of viable embryos, was significantly different from the control at p < 0.01. Additionally, while not significantly different, the reduction in embryo survival resulted in reductions in the numbers of hatchlings and 14-day old survivors as a percentage of eggs set for incubation.

- Egg Shell Thickness: There were no apparent 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 m the 8, 12. 18 or 27 ppm a.i. treatment groups.

- Offspring Body Weights: There were no apparent treatment related effects upon offspring body weights at any of the concentrations tested When compared to the control group, there were no statistically significant differences in d1e body weight of hatchlings or 14-day old survivors from the 8, 12. 18 or 27 ppm a.i. treatment groups.

Reported statistics and error estimates:

See Statistical analysis in 'Any other information on materials and methods incl. tables'

Table 1. Mean Adult Body Weight (g)1

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   

Experimental group (mg a.i./kg)	Sex	Week 0	Change Week 0 – 2	Week 2	Change Week 2 – 4	Week 4	Change Week 4 – 6	Week 6	Change Week 6 – 8	Week 8	Change Week 8 - Term.	Test Term.	Total change
Control	Male	1097	-19	1078	-17	1060	23	1084	4	1088	51	1139	42
	Female	952	-15	937	-15	922	16	938	10	948	156	1101	152
6	Male	1106	-30	1075	-18	1057	10	1067	18	1084	66	1157	54
	Female	967	-13	955	-8	946	8	955	29	983	168	1149	182
12	Male	1108	-46	1062	-5	1057	8	1065	7	1072	45	1117	9
	Female	953	-20	934	-11	923	12	935	10	946	149	1095	142
18	Male	1088	-19	1069	-25	1044	21	1065	13	1078	75	1153	65
	Female	914	-18	925	-8	918	24	942	13	955	172	1127	183
27	Male	1082	-36	1046	-11	1035	4	1038	20	1058	51	1105	30
	Female	948	-25	923	-2	921	16	937	18	955	157	1115	167

The means for body weights and body weight changes are calculated and rounded separately.

Differences between control and each treatment group were not significant (p>0.05).

1. Only surviving birds were included in the calculations for each body weight interval.

Table 2. Mean Feed Consumption (g/bird/day)

Experimental group (mg a.i./kg)	Weeks
	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20
Control	109	110	109	105	102	120	120	125	119	145	167	173	184	182	179	180	189	177	182	163
8	96	101	109	95	99	109	120	124	115	149	175	174	168	177	167	186	209	186	200	166
12	18	106	110	103	105	113	116	124	116	147	164	187	171	190	184	188	191	184	199	173
18	102	100	104	90	105	99	109	115	103	133	160	173	159	172	157	100	187	166	181	156
27	87*	97	111	96	98	103	117	122	119	140	171	177	163	173	139*	158	189	154	185	150

* Significantly different from the control at p < 0.05

Table 3. Summary of Reproduction and Normalized as Percentages(%)¹

Reproduction parameter	Experimental group (mg a.i./kg)
	Control	8	12	18	27
Nr. of replicate	16	15	16	16	15
Total eggs laid^2	680	717	786	717	688
Eggs cracked	3	5	4	6	8
Egg set	608	642	694	637	609
Viable embryos	478	607	638	587	565
Live 3-week embryos	470	591	612	552	528
Hatchings	355	434	411	403	311
14-Day old survivors	348	425	408	399	304
Eggs Laid/Hen	43	48	49	45	46
Eggs Laid/Hen/Day^3	0.50	0.56	0.58	0.53	0.54
14-day Old Survivors/Hen	22	28	26	25	20
Eggs Laid/Maximum Laid (%)	56	63	65	59	60
Eggs cracked/Egg laid (%)	1	4	1	1	1
Viable embryos/Egg set (%)	81	94	92	93	94
Live 3-week embryos/ Viable embryos (%)	99	98	96	93	92**
Hatchings/ Live 3-week embryos (%)	75	75	65	68	58
14-Day old survivors/Hatchings (%)	98	98	99	99	98
Hatchings/Eggs set (%)	60	70	58	60	51
14-Day old survivors/Eggs set (%)	59	68	56	60	50
Hatchings/Maximum set (%)	32	42	37	37	30
14-Day old survivors/Maximum set (%)	31	41	37	36	29

** Significantly different from the control at p < 0.01

1. Values represent pen means for experimental group.

2. Represents the total number of eggs laid in each group.

3. Based on 85 days of eggs production.

Table 4. Mean Eggshell Thickness Measurements (mm)

                                                                                                                                                                                                                                                                                                                                                                                                                                               

Experimental group (mg a.i./kg)	No eggs measured	Shell thickness mean±SD
Control	65	0.385 ± 0.016
8	60	0.382 ± 0.033
1.2	71	0.377 ± 0.022
18	65	0.371 ± 0.024
27	65	0.382 ± 0.021

Differences between the control and each treatment groups were not significant (p > 0.05).

Table 5. Mean Body Weight (g) of Hatchlings and 14-Day Old Survivors

Experimental group (mg a.i./kg)	Hatchings		14-Day survivors
	Number	Mean±SD	Number	Mean±SD
Control	350	32± 3	348	268± 27
8	434	33± 2	425	277± 21
12	411	33± 3	408	269± 30
18	403	34± 3	399	269± 30
27	311	33± 3	304	273± 25

The number of hatchlings weighed may differ from the total number of hatchlings since those hatchlings found dead were not weighed

Differences between the control and each treatment group were not significant (p > 0.05).

  Table 6. Summary of Gross Pathological Observations (Adult Birds Euthanized at Termination of the Test)

	Males- Treatment Group (mg a.i./kg)					Females- Treatment Group (mg a.i./kg)
	Control	8	12	18	27	Control	8	12	18	27
Number of birds	16	15	16	16	15	16	15	16	16	15
External - feather loss	0	2	1	0	0	1	3	5	2	4
External - head lesion	0	0	0	0	0	1	0	0	0	1
External - neck lesion	0	0	0	0	0	1	0	0	0	1
External - foot lesions	6	4	3	3	5	10	13	15	11	9
External - swelling in leg	0	0	0	0	n	0	0	0	0	1
Liver - pale	0	0	0	0	0	0	0	1	0	0
Spleen - enlarged	0	0	0	()	0	0	0	0	1	0
Spleen - mottled	0	0	0	0	0	0	(0	0	2	1
Respiratory - yellow abscess on lower margin of lung	1	0	0	0	0	0	0	0	0	0
Kidneys- pale	0	1	0	0	0	0	0	0	0	0
Abdominal cavity - yolk remnants	-	-	-	-	-	0	1	0	0	0
Abdominal cavity - egg yolk peritonitis	-	-	-	-	-	4	4	8	4	5
Reproductive - persistent right oviduct	-	-	-	-	-	0	1	1	1	0
Reproductive - cystic follicles	-	-	-	-	-	2	1	0	2	2
Reproductive - egg remnants in reproductive tract	-	-	-	-	-	0	1	0	0	0
Reproductive - hemorrhagic follicle	-	-	-	-	-	0	1	1	0	0
Reproductive - ovary regressing	-	-	-	-	-	1	3	3	4	6
Reproductive - ovary regressed	-	-	-	-	-	4	3	1	4	0
Reproductive - testes small,≤3.25 cm	1	1	2	1	1	-	-	-	-	-
Not remarkable	10	8	10	12	9	1	0	0	2	2

 

Validity criteria fulfilled:

yes

Conclusions:

There were no treatment-related mortalities, overt signs of toxicity or treatment-related effects upon body weight at the 8, 12. 18 or 27 mg a.i/kg diet or feed consumption at the 8, 12 or 18 mg a.i/kg diet. Additionally, there were no treatment-related effects upon any of the reproductive parameters measured at the 8. 12 or 18 mg a.i/kg diet. However, at the 27 mg a.i/kg diet, there was an initial transient reduction in feed consumption and a treatment-related reduction in embryo survival that was significantly different from the control. Based upon the effect observed in the 27 mg a.i/kg diet treatment group, the NOEL for mallard exposed to the test substance in the diet during the study was 18 mg a.i/kg diet.

Executive summary:

The impact of the test substance on the Mallard (Anas platyrhynchos) was investigated by following OECD TG 206, EPA OPP 71-4 and EPA OPPTS 850.2300 test guidelines. The study was also in compliance with GLP criteria. Mallard ducks were randomly distributed into one control group and four treatment groups (16 pairs/treatment, 1 male and 1 female/pair). Four treatment groups were fed diets containing 8, 12. 18 or 27 mg a.i. test substance/kg diet for approximately 20 weeks. The control group was fed diet comparable to the treatment groups. but without the addition of the test substance. All adult birds were observed daily throughout the test for signs of toxicity or abnormal behaviour. Adult body weights were measured at test initiation, at the end of Weeks 2. 4, 6. 8, and at adult termination, and feed consumption was measured weekly throughout the test. At the beginning of week 9, the photoperiod was increased to induce egg production. Following the start of egg production, eggs were set weekly for incubation. Weekly, eggs were selected by indiscriminate draw for egg shell thickness measurement and all remaining eggs were candled prior to incubation to detect egg shell cracks or abnormal eggs. Eggs were also candled twice during incubation to detect infertile eggs or embryo mortality. On Day 24 of incubation, the eggs were placed in a hatcher and allowed to hatch. Once hatching was completed, hatchlings were removed from the hatcher and the group body weight of the hatchlings by pen was determined. At 14 days of age, the average body weight by parental pen of all surviving offspring was determined. Upon completion of the test, statistical analyses were performed to determine statistically significant differences between groups.

The results showed that there were no treatment-related mortalities at any of the concentrations tested. One incidental mortality occurred in both the 8 and 27 mg a.i./kg diet treatment groups, and no other mortalities occurred during the course of the study. No overt signs of toxicity were observed at any of the concentrations tested. There were no apparent treatment-related effects upon adult body weight or gross necropsy findings of surviving adults at any of the concentrations tested. There were no statistically significant differences between at the control, 8, 12, or 18 mg a.i./kg diet treatment groups at any of the feed consumption intervals. At the 27 mg a.i./kg diet test group, the feed consumption during Week 15 was statistically different from the control group. However, since the decrease in feed consumption was slight, transient, and not associated with an impact upon body weight, the difference was not considered to be biologically meaningful. 

There were no treatment-related effects upon reproductive performance in the 8, 12 or 18 mg a.i./kg diet treatment groups and any differences in mean values were not significantly different from control values for any of the reproductive parameters measured. However, at the 27 mg a.i./kg diet test group, there was a treatment-related reduction in 3-weeks embryo survival that was statistically significant from the control group. There were no apparent treatment related effects upon egg shell thickness at any of the concentrations tested. Additionally, there were no statistically significant differences in egg shell thickness between treatment groups. There were no apparent treatment related effects upon offspring body weights at any of the concentrations tested. When compared to the control group, there were no statistically significant differences in the body weight of hatchlings or 14-day old survivors from any test substance treatment group. Based on the findings, the NOEL was determined to be 18 mg a.i./kg diet. 

 

Description of key information

All available data was assessed and the study representing the most accurate NOEC value is included here as key study. The key study is selected for the CSA. The other studies are included as supporting information.

20-wk NOEL = 18 mg/kg diet, Anas platyrhynchos, reproduction parameters, OECD TG 206, Frey et al. 2007

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for birds:

18 mg/kg food

Additional information

There are six standard guideline followed and GLP compliant studies available for this endpoint, i.e. three long-term and three short-term studies all with dietary exposure. The long-term study on the reproduction of mallard duck (Anas Platyrhynchos) is selected to be the key study, because it represents the most accurate NOEC value. In this study, mallard ducks were randomly distributed into one control group and four treatment groups (16 pairs/treatment, 1 male and 1 female/pair). Four treatment groups were fed diets containing 8, 12, 18 or 27 mg test substance/kg diet for approximately 20 weeks. All adult birds were observed daily throughout the test for signs of toxicity or abnormal behaviour. Adult body weights were measured at test initiation, at the end of Weeks 2. 4, 6. 8, and at adult termination, and feed consumption was measured weekly throughout the test. At the beginning of week 9, the photoperiod was increased to induce egg production. Following the start of egg production, eggs were set weekly for incubation. Weekly, eggs were selected by indiscriminate draw for egg shell thickness measurement and all remaining eggs were candled prior to incubation to detect egg shell cracks or abnormal eggs. Eggs were also candled twice during incubation to detect infertile eggs or embryo mortality. On Day 24 of incubation, the eggs were placed in a hatcher and allowed to hatch. Once hatching was completed, hatchlings were removed from the hatcher and the group body weight of the hatchlings by pen was determined. At 14 days of age, the average body weight by parental pen of all surviving offspring was determined.

The results showed that there were no treatment-related mortalities at any of the concentrations tested. No overt signs of toxicity were observed at any of the concentrations tested. There were no apparent treatment-related effects upon adult body weight or gross necropsy findings of surviving adults at any of the concentrations tested. There were no statistically significant differences between at the control, 8, 12, or 18 mg/kg diet treatment groups at any of the feed consumption intervals. At the 27 mg/kg diet test group, the feed consumption during Week 15 was statistically different from the control group.

There were no treatment-related effects upon reproductive performance in the 8, 12 or 18 mg/kg diet treatment groups However, at the 27 mg/kg diet test group, there was a treatment-related reduction in 3-weeks embryo survival that was statistically significant from the control group. There were no apparent treatment related effects upon egg shell thickness at any of the concentrations tested. Additionally, there were no statistically significant differences in egg shell thickness between treatment groups. There were no apparent treatment related effects upon offspring body weights at any of the concentrations tested. When compared to the control group, there were no statistically significant differences in the body weight of hatchlings or 14-day old survivors from any test substance treatment group. Based on the findings, the NOEL was determined to be 18 mg/kg diet (Frey 2007, OECD TG 206, Reliability 1).

 

The other two long-term studies on reproduction and the three short-term studies on mortality are supporting studies. The first long-term supporting study was a 22-wk test performed with mallard duck (Anas platyrhynchos). Four groups of adult birds received the test substance at concentrations of 0 (carrier control), 10, 50 and 250 mg/kg feed (mean measured: < 5.00, 10.2, 50.9 and 259 mg/kg feed) ad libitum. Based on nominal concentrations, the 22-week NOEC for reproduction was 10 mg/kg feed (Beavers et al 1993, OECD TG 206, Reliability 1). Considering the dose rates applied in this study compared to the key study, the NOEC determined in the key study is more accurate as dose levels were closer together. The second-long term supporting study was a 22-wk test performed with bobwhite quail (Colinus virginianus). Four groups of adult birds received the test substance at concentrations of 0 (carrier control), 10, 50 and 250 mg/kg feed (mean measured: < 5.00, 10.2, 50.9 and 259 mg/kg feed) ad libitum. Based on nominal concentrations, the 22-week NOEC for  reproduction was 50 mg/kg feed (Beavers at al 1993, OECD TG 206, Reliability 1).

The first short-term supporting study was an 8-d test performed with mallard duck (Anas platyrhynchos). Ten young birds (9 days old, undetermined sex) per dose level were used with 5 test concentrations and 3 control groups, and exposed for five consecutive days followed by an additional three-day observation period. Based on nominal concentrations, the 8-day LC50 was 1197 mg/kg feed (Beavers 1985, FIFRA 71-2, Reliability 1). The second short-term supporting study was an 8-d test performed with bobwhite quail (Colinus virginianus). Ten chicks (11 days old, undetermined sex) per dose level were used with 5 test concentrations and 4 control groups, and exposed for five consecutive days followed by an additional three-day observation period. Based on nominal concentrations, the 8-day LC50 was 816 mg/kg feed (Beavers 1985, FIFRA 71-2, Reliability 1). The third short-term supporting study was an 11-d test performed with bobwhite quail (Colinus virginianus). Ten chicks (11 days old, undetermined sex) per dose level were used with 6 test concentrations and 4 control groups, and exposed for five consecutive days followed by an additional six-day observation period. Based on nominal concentrations, the 11-day LC50 was 1292 mg/kg feed (Beavers et al 1991, FIFRA 71-2, Reliability 1).