Mecoprop

Toxicity to birds

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to birds: reproduction test

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please see the read-across justification report in Section 13 of the dossier.

Reason / purpose for cross-reference:

read-across source

Duration (if not single dose):

21 wk

Dose descriptor:

NOEC

Effect level:

400 other: ppm a.i.

Conc. / dose based on:

act. ingr.

Basis for effect:

other: No effects were seen

Conclusions:

Under the conditions of this study there were no treatment-related mortalities, overt signs of toxicity or treatment-related effects upon body weight or feed consumption at any of the concentrations tested. Additionally, there were no treatment-related effects upon any of the reproductive parameters measured at the 80, 200 or 400 ppm a.i. test concentrations. The no-observed-effect concentration for mallard exposed to the test material in the diet during the study was 400 ppm a.i. (53.0 mg a.i./kg/day), the highest concentration tested.



Considering the very close structural similarity between the source and target substances (as justified in the read-across report that is attached to section 13 of the dossier), results from the study performed with mecoprop-p can be extrapolated to mecoprop.

Executive summary:

A reproduction study in the mallard was carried out in accordance with the standardised guidelines OECD 206, EPA OPPTS 850.2300 and EPA OPP 71-4 under GLP conditions.

The objective of this study was to evaluate the effects of dietary exposure to the test material upon adult mallard (Anas platyrhynchos) over a five-month period. Effects on adult health, body weight, and feed consumption were evaluated. In addition, the effects of adult exposure to the test material on the number of eggs laid, fertility of the eggs, normal development of eggs including viability and survival of the embryos, hatchability, offspring survival and egg shell thickness were evaluated.

Mallard (72 males and 72 females) were randomly distributed into one control group and three treatment groups. Each treatment and control group contained 18 pairs of birds with one male and one female per pen. Three treatment groups were fed diets containing 80, 200, and 400 ppm a.i. of the test material for 21 weeks. The control group was fed diet comparable to the treatment groups, but without the addition of the test material.

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. Feed consumption was measured weekly throughout the test. At the beginning of Week 10, 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 an incubator configured for hatching and allowed to hatch. Once hatching was completed, hatchlings were removed from the hatcher and the individual body weights of the hatchlings were determined. At 14 days of age, the individual body weight of each surviving offspring was determined. Upon completion of the test, statistical analyses were performed to determine statistically significant differences among groups.

Under the conditions of this study there were no treatment-related mortalities, overt signs of toxicity or treatment-related effects upon body weight or feed consumption at any of the concentrations tested. Additionally, there were no treatment-related effects upon any of the reproductive parameters measured at the 80, 200 or 400 ppm a.i. test concentrations. The no-observed-effect concentration for mallard exposed to the test material in the diet during the study was 400 ppm a.i. (53.0 mg a.i./kg/day), the highest concentration tested.

 

 

 

Considering the very close structural similarity between the source and target substances (as justified in the read-across report that is attached to section 13 of the dossier), results from the study performed with mecoprop-p can be extrapolated to mecoprop.

Reference 2

Endpoint:

long-term toxicity to birds: reproduction test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 July 2016 to 26 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

OECD Guideline 206 (Avian Reproduction Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.2300 (Avian Reproduction Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 71-4 (Avian Reproduction Test)

Deviations:

no

GLP compliance:

yes

Dose method:

feed

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

Corn oil and acetone.

Details on preparation and analysis of diet:

DIET PREPARATION
Test diets were prepared by mixing the test material into a premix that was used for weekly preparation of the final diet. Control diet and each of the three treated diets were prepared weekly beginning on the 18 July 2016 and presented to the birds on Wednesday of each week. Dietary concentrations were adjusted for the purity of the test material and are presented as parts per million active ingredient (ppm a.i.).

Premixes were prepared on 18 July 2016 and 08 August 2016. Nominal preparation was as follows:
Control: 8918.0 g ration + 202 mL corn oil
- 80 ppm a.i.: 23.7908 g test material + 8894.2 g ration + 202 mL corn oil
- 200 ppm a.i.: 59.477 g test material + 8858.5 g ration + 202 mL corn oil
- 400 ppm a.i.: 118.954 g test material + 8799.0 g ration + 202 mL corn oil
For each of the premixes, an appropriate amount of test material was ground with a mortar and pestle. Once the test material was ground the needed amount was weighed in a tared weigh boat on an analytical or top loading balance. Basal ration, 4500.0 g, was weighed into a tared mixing bowl on a top loading balance. An aliquot of the basal ration was held for later use (retained ration).
A portion of the retained basal ration was placed in a blender and the test material was added to the blender. The weigh boat was rinsed three times with some of the retained ration and the rinsate was added to the blender and the contents were blended for at least two minutes. After blending, the contents were transferred to the mixing bowl with the basal ration. The blender was rinsed two times with portions of the retained ration and the rinsate was added to the mixing bowl. Corn oil was measured in a graduated cylinder and added to the basal ration in the mixing bowl. The remaining retained ration was also added to the mixing bowl. The bowl contents were mixed on a stand mixer for approximately 15 minutes. The remaining amount of basal ration needed was weighed on a top-loading balance and added to the mixing bowl. Mixing then continued for an additional 25 minutes (the first premix was mixed for five and 15 minutes; homogeneity results indicated that additional mixing time would be required and subsequent premixes were prepared by mixing for 15 and 25 minutes).
After mixing, 1800.0 gram aliquots of the premix were weighed on a top-loading balance, placed in appropriately labelled plastic bags, reweighed and stored frozen, unless used immediately for preparation of final diet.
As needed, the appropriate premix was incorporated into the final diet as follows:
- 0 ppm a.i.: 1800 g Premix + 50.45 kg ration + 2.750 kg limestone
- 80 ppm a.i.: 1800 g Premix + 50.45 kg ration + 2.750 kg limestone
- 200 ppm a.i.: 1800 g Premix + 50.45 kg ration + 2.750 kg limestone
- 400 ppm a.i.: 1800 g Premix + 50.45 kg ration + 2.750 kg limestone
The diet was mixed for approximately 20 minutes in a Patterson-Kelley® Twin Shell Blender.

Premixes for test material were prepared on 16 August 2016, 18 September 2016, 10 October 2016 and 11 November 2016. Nominal preparation was as follows:
- Control: 8918.0 g ration + 202 mL corn oil + 400 mL acetone
- 80 ppm a.i.: 23.7908 g test material + 8894.2 g ration + 202 mL corn oil + 400 mL acetone
- 200 ppm a.i.: 59.4771 g test material + 8858.5 g ration + 202 mL corn oil + 400 mL acetone
- 400 ppm a.i.: 118.9542 g test material + 8799.0 g ration + 202 mL corn oil + 400 mL acetone
Acetone was added to premixes beginning on 16 August 2016 through the end of the study, to promote a better mix with the test material. An appropriate amount of test material was ground with a mortar and pestle and the amount needed was weighed in a tared weigh boat on an analytical or top loading balance. The test material was transferred to a beaker.
Acetone was measured in a graduated cylinder and was added to the test material with a portion of the acetone used to rinse the weigh boat. The rinse was added to the beaker. A magnetic stir-bar was placed in the beaker and the mixture stirred for at least approximately two minutes on a magnetic stir plate, until the test material dissolved. Corn oil was measured in a graduated cylinder then added to the beaker and the mixture was stirred for at least one additional minute.
Basal ration, 4500.0 g, was weighed into a tared mixing bowl on a top loading balance. The test material mixture was then added to the basal ration in the mixing bowl. The beaker was rinsed with additional acetone measured in a graduated syringe. The rinse was added to the mixing bowl and the contents of the bowl mixed on a stand mixer for approximately 15 minutes. The remainder of the basal ration needed for the premix was then weighed on a top-loading balance and added to the mixing bowl and mixed for an additional 25 minutes.
After mixing, 1800.0 gram aliquots of the premix were weighed on a top-loading balance, placed in appropriately labelled plastic bags, reweighed and stored frozen, unless used immediately for preparation of final diet.
As needed, the appropriate premix was incorporated into the final diet as follows:
- 0 ppm a.i.: 1800 g Premix + 50.45 kg ration + 2.750 kg limestone
- 80 ppm a.i.: 1800 g Premix + 50.45 kg ration + 2.750 kg limestone
- 200 ppm a.i.: 1800 g Premix + 50.45 kg ration + 2.750 kg limestone
- 400 ppm a.i.: 1800 g Premix + 50.45 kg ration + 2.750 kg limestone
The diet was mixed for approximately 20 minutes in a Patterson-Kelley® Twin Shell Blender.

DIET SAMPLING
Homogeneity of the test material in the diet was evaluated by collecting six samples from each of the 80 and 400 ppm a.i. treated diets and one sample from the control diet 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. Control and treatment group diet samples were also collected from the feed troughs on Day 7 of Weeks 1 and 20 to assess stability of the test material under actual test conditions. Additionally, samples were collected from the 200 ppm a.i. treatment group on Day 0 of Week 1 and samples were collected from the control and treatment group diets during Weeks 8, 16 and 20 of the test to measure/verify test concentrations. The diet samples were transferred to the testing facility’s analytical chemistry facility and stored frozen prior to analysis.

ANALYTICAL METHOD
The analysis of test material in avian diet was based upon methodology developed by the testing facility. Samples were extracted with 0.05 M sodium hydroxide in methanol.
- Instrument: Agilent Series 1200 high performance liquid chromatograph (HPLC) equipped with an Agilent Series 1200 variable wavelength detector (VWD)
- Analytical column: Inertsil ODS-2 AM (250 mm x 4.6 mm I.D., 5 μm particle size)
- Flow rate: 1.000 mL/minute
- Column temperature: 40 °C
- Mobile phase: Channel A: 50:50:0.1 (v/v/v) Acetonitrile: HPLC grade water: Phosphoric Acid; Channel B: Acetonitrile
- Gradient profile: At 1 minute: 90 % A and 10 % B; at 7 and 8 minutes: 10 % A and 90 % B; at 8.10 and 12 minutes: 90 % A and 10 % B
- Injection volume: 25.00 µL
- Test material peak retention time: Approximately 6.4 minutes
- Analytical wavelength: 230 nm

Calibration standards of test material, ranging in concentration from 0.500 to 10.0 ppm a.i. were prepared in 40:60 (v/v) methanol: HPLC grade water using a stock solution of test material in methanol.
The stock solution of tets material was prepared by accurately weighing 0.1069 g (corrected for purity) on an analytical balance. The test material was transferred to a 100-mL volumetric flask, and brought to volume using methanol. The primary stock solution (1.00 mg a.i./mL) was diluted in methanol to prepare a 0.100 mg a.i./mL stock solution. The 0.100 mg a.i./mL stock solution was used to prepare the calibration standards in 40:60 (v/v) methanol: HPLC grade water. The set of calibration standards had the following concentrations: 0.500, 1.00, 2.50, 5.00, 7.50 and 10.0 ppm a.i.

Calibration standards were analysed with each sample set. A calibration curve was constructed for each set of analyses. The peak areas and the theoretical concentrations of the calibration standards were fit with least-squares regression analysis to a linear function. The concentration of test material in the samples was determined by substituting the peak area responses of the samples into the applicable linear regression equation. Examples of equations used in calculations are as follows:
The detected concentration of test material in each sample was determined from the slope and intercept of the calibration curve and the peak area response of each sample injected using the following equation:

Test material detected concentration = (ppm a.i.) (Peak area response - y intercept) / Slope

Determination of Sample Concentration: The concentration expressed as ppm a.i. for each sample was determined using the following equation:

Test material analysed sample concentration (ppm a.i.) = (Test material detected concentration (ppm a.i.) x extraction volume (mL) x Final dilution) / Initial sample weight (g)

Fortification Recoveries: The percent recovery of the method at each level of fortification is calculated as follows:

% Recovery = (Analysed sample concentration (ppm a.i.) / Fortified concentration (ppm a.i.)) x 100

The limit of detection (LOD) was defined as the lowest analyte concentration divided by the signal to noise ratio times 3 times the dilution factor of the matrix blank sample. The mean LOD for the study was calculated to be 0.123 ppm a.i. The limit of quantitation (LOQ) was defined as the lowest analyte concentration divided by the signal to noise ratio times 10, times the dilution factor of the matrix blank sample. The mean LOQ for the study was calculated to be 0.41 ppm a.i. Measured values greater than or equal to the LOQ were reported.
Along with the sample analyses, eight matrix blanks were analysed to determine possible interferences. No interferences were observed at or above the LOQ during the sample analyses.
Avian diet samples were fortified at 60.0 and 1000 ppm a.i. using a dry mix technique and analysed concurrently with the samples to determine the mean procedural recovery. The method yielded mean procedural recoveries of 120, 112, 102, 110, 110, 109, 115 and 108 %. These values correspond to each sample set analysed during the definitive study. Sample measured concentrations were not corrected for the mean procedural recoveries from each sample set.

ANALYTICAL RESULTS
Analysis of the control samples did not show any indication of the presence of the test material or of the presence of a co-eluting material at the characteristic retention time of the test material. Diet samples were collected from the 80 and 400 ppm a.i. test concentrations and were analysed to evaluate the homogeneity of the test material in the diet for Week 1 Day 0. Mean concentrations and standard deviations for the two test concentrations were 71.1 ± 16.0 ppm a.i. and 387 ± 148 ppm a.i., respectively. The coefficients of variation were 22.5 and 38.2 %, respectively. Diet samples were collected from the 80 and 400 ppm a.i. test concentrations and were analysed to evaluate the homogeneity of the test material in the diet for Week 4 Day 0. Mean concentrations and standard deviations for the two test concentrations were 86.4 ± 25.1 ppm a.i. and 403 ± 92.1 ppm a.i., respectively. The coefficients of variation were 29.0 and 22.9 %, respectively. Diet samples were collected from the 80 and 400 ppm a.i. test concentrations and were analysed to evaluate the homogeneity of the test material in the diet for Week 5 Day 0. Mean concentrations and standard deviations for the two test concentrations were 81.0 ± 3.86 ppm a.i. and 380 ± 11.2 ppm a.i., respectively. The coefficients of variation were 4.76 and 2.96 %, respectively. Samples collected during the test to verify test material concentrations for the 80, 200 and 400 ppm a.i. diets had mean concentrations and standard deviations of 82.4 ± 4.60 ppm a.i., 198 ± 26.9 ppm a.i. and 412 ± 25.1 ppm a.i., respectively. The coefficients of variation were 5.59, 13.6 and 6.08 %, respectively. These values represented 103, 99.0 and 103 % of nominal concentrations, respectively. Analysis of Week 1 Day 7 diet samples collected from feeders after being held at ambient temperature for 7 days averaged 97.7 86.4 and 76.7 % of the Week 1 Day 0 values for the 80, 200 and 400 ppm a.i. test concentrations, respectively. Analysis of Week 5 Day 7 diet samples collected from feeders after being held at ambient temperature for 7 days averaged 89.6, 85.6 and 93.7 % of the Week 5 Day 0 values for the 80, 200 and 400 ppm a.i. test concentrations, respectively. Analysis of Week 20 Day 7 diet samples collected from feeders after being held at ambient temperature for 7 days averaged 88.9, 91.1 and 80.3 % of the Week 20 Day 0 values for the 80, 200 and 400 ppm a.i. test concentrations, respectively.

Test organisms (species):

Anas platyrhynchos

Details on test organisms:

TEST ORGANISM
- Common name: Mallard duck
- Age at test initiation: All birds were 18 weeks of age at test initiation (first day of exposure to test diet).
- Weight at test initiation: Birds ranged in weight from 840 to 1290 grams.
- Sexes used / mixed or single sex: Male and female.
- Cultural background: Pen-reared. At the start of acclimation, the mallard were apparently healthy and phenotypically indistinguishable from wild type.
- Disease free: Yes
- Kept according to standard practices: Yes. At the start of acclimation, 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. Sex of the birds was determined by a visual examination of the plumage.
- Breeding population: The birds were from the same hatch, approaching their first breeding season and had not been used in any previous testing.
- Feed and water: All adult birds and their offspring were given feed and water ad libitum during acclimation and testing. The basal diet fed to both adults and offspring was formulated to the testing facility’s specifications by Cargill Animal Nutrition, Shippensburg, PA). The basal ration contained at least 27 % protein and 2 % crude fat, and no more than 5 % crude fibre. The basal diet contained approximately 1.12 % calcium, derived from feedstuffs and limestone used in the formulation of the basal diet by Cargill. 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 % 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 material and without the addition of 5 % supplemental limestone. Water was supplied by the town public water supply. Neither the adults nor offspring received any form of medication in their feed during the test. Feed and water were analysed periodically.

Limit test:

no

Total exposure duration (if not single dose):

21 wk

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

18 animals per sex per dose

Control animals:

yes, concurrent vehicle

Nominal and measured doses / concentrations:

- Nominal concentrations: 0, 80, 200 and 400 ppm a.i.

Details on test conditions:

ACCLIMATION
- Acclimation period: 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.

HOUSING AND ENVIRONMENTAL CONDITIONS
The adult birds were housed indoors in batteries of pens, measuring approximately 75 x 90 x 45 cm high. The pens were constructed of vinyl-coated wire mesh. Sisal rope was added to each pen for animal enrichment at the start of photostimulation.
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.
Only birds associated with this study were maintained in the study room in order to avoid excessive disturbances. The average temperature in the adult mallard study room during the course of the test was 21.7 (20.3 – 22.6) ± 0.6 °C (SD) with an average relative humidity of 67 (30 – 86) ± 15 % (SD).
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.
The photoperiod in the adult mallard room was maintained by a time clock. The photoperiod during acclimation and the first nine weeks of the test was eight hours or less of light per day with an average light intensity of approximately 303 lux (~ 28 ft. candles). The photoperiod was increased to 17 hours of light per day at the beginning of Week 11 to induce egg laying and was maintained at that length until the adult birds were euthanised. Measurement of light intensity in the study room following photostimulation was approximately 462 lux (~ 43 ft. candles), and during the egg-laying phase of the test the light intensity was approximately 461 lux (~ 43 ft. candles). Throughout the test illumination was provided by fluorescent lights that closely approximated the colour spectrum of noonday sunlight.

STUDY PHASES
The primary phases of the study and their approximate durations were:
1. Acclimation - 3 weeks.
2. Pre-photostimulation - 10 weeks.
3. Pre-egg laying (with photostimulation) – 2 weeks.
4. Egg laying - 10 weeks.
5. Post-adult termination (final incubation, hatching, and 14-day offspring rearing period) – 6 weeks.

RANGE FINDING STUDY
The test concentrations were selected based upon the results of a pilot reproduction study.

Details on examinations and observations:

MORTALITY / CLINICAL SIGNS
- Time schedule for examinations: During the study, all adult birds were observed daily for signs of toxicity or abnormal behaviour.
- Remarks: Additionally, all offspring were observed daily from hatching until 14 days of age. A record was maintained of all mortalities and clinical observations.

BODY WEIGHT
- Time schedule for examinations: Adult body weights were measured at test initiation, at the end of Weeks 2, 4, 6, 8, and at adult termination.
- Remarks: Body weights were not measured during egg laying because of the possible adverse effects handling may have on egg production. Statistical comparisons were made between the control group and each treatment group at each weighing interval by sex.

FOOD CONSUMPTION
- Time schedule for examinations: Feed consumption for each pen was measured weekly throughout the test.
- Remarks: 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. Statistical comparisons were made between the control and each treatment group.

Estimated test material intakes, daily dietary dose, for mallards were calculated by treatment group for the pre-egg production period, the egg production period and the overall adult period using the following formula:

Daily Dietary Dose (mg a.i./kg body weight/day) = (Test Concentration (mg a.i./kg) x Daily Feed Consumption (g/bird/day)) / Body Weight (g/bird)

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 21 (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.

WATER CONSUMPTION: Not measured

PATHOLOGY
- Dose groups that were examined: All animals
- Remarks: At the conclusion of the exposure period, all adult birds were euthanised by cervical dislocation, necropsied, and disposed of by incineration.

Details on reproductive parameters:

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 (Kuhl Egg Washer) with a chlorine-based detergent (Kuhl Super CD). 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, rinsed with fresh water and allowed to cool to approximately room temperature. The eggs were then stored in a cold room until incubation. The cold room was maintained at a mean temperature of 14.3 ± 0.3 °C (SD) with a mean relative humidity of approximately 82 ± 8 % (SD). All eggs laid in a weekly interval were considered as one lot.

CANDLING AND INCUBATION
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 (Speed King Model No. 32) to detect egg shell cracks or abnormal eggs. Cracked or abnormal eggs were recorded and discarded.
All eggs not discarded or used for egg shell thickness measurements were placed in an incubator (NatureForm Model No. NMC 4000). In the incubator the temperature was maintained at an average of 37.4 ± 0.0 °C (SD) with an average relative humidity of approximately 55 ± 0 % (SD). 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 two hours through Day 24 of incubation. Eggs were candled on Day 13 - 14 of incubation to determine embryo viability and on Day 20 - 21 to determine embryo survival. Eggs that appeared “clear” at the Day 13 - 14 candling were broken out and examined in an effort to determine fertility.

HATCHING AND BROODING
On Day 24 of incubation, the eggs were placed in an incubator configured for hatching (NatureForm Model No. NMC 4000 or Model # 2340) and allowed to hatch. Pedigree baskets constructed of galvanised steel wire mesh were used to keep hatchlings separated by parental pen of origin. Eggs were not rotated in the hatcher. The average temperature in the hatching compartment was 37.3 ± 0.0 °C (SD) with an average relative humidity of 60 ± 0 % (SD).
All hatchlings, unhatched eggs, and egg shells were removed from the hatcher on Day 27 or 28 of incubation. The body weights of surviving hatchlings were recorded and the average body weight by pen was determined. 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. At 14 days of age, the individual body weight of each surviving hatchling was recorded. The ducklings were euthanised with carbon dioxide and disposed of by incineration.
Hatchlings were housed in batteries of brooding pens. Each pen measured approximately 62 x 92 x 25.5 cm high. The walls, floors and ceilings of each pen were constructed of vinyl-coated wire mesh. Thermostats in the brooding compartment of each pen were set to maintain a temperature of 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.
The average ambient room temperature was 22.4 ± 0.9 °C (SD) with an average relative humidity of 40 ± 11 % (SD). The photoperiod for the hatchlings was maintained by a time clock at 16 hours of light per day.

EGG SHELL THICKNESS MEASUREMENTS
Weekly throughout the egg laying period, one egg was collected, when available, from each of the odd-numbered pens during odd numbered weeks (1, 3, 5, 7 & 9) and from each of the even numbered pens during the even numbered weeks (2, 4, 6, 8 & 10). The eggs were opened at the waist, the contents removed, and 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.

STATISTICAL ANALYSES
Each of the following parameters was analysed statistically:
- Eggs/Hen/Day - The number of eggs laid per female divided by the total number of days of egg production.
- Eggs Cracked of Eggs Laid - The number of eggs determined by candling to be cracked divided by the number of eggs laid, per pen.
- Fertile Eggs of Eggs Set – The number of fertile eggs at the Day 14 candling was divided by the number of eggs set, per pen.
- Viable Embryos of Eggs Set - The number of viable embryos at the Day 14 candling was divided by the number of eggs set, per pen.
- Live 3-Week Embryos of Viable Embryos - The number of live embryos at the Day 21 candling was divided by the number of viable embryos, per pen.
- Hatchlings of 3-Week Embryos - The number of hatchlings removed from the hatcher was divided by the number of live 3-week embryos, per pen.
- 14-Day Old Survivors of Hatchlings - The number of 14-day old survivors was divided by the number of hatchlings, per pen.
- Hatchlings of Eggs Set - The number of hatchlings was divided by the number of eggs set, per pen.
- Hatchlings of Fertile Eggs – The number of hatchlings was divided by the number of fertile eggs, per pen.
- 14-Day Old Survivors of Eggs Set - The number of 14-day old survivors was divided by the number of eggs set per pen.
- Hatchlings/Pen/Day - The number of hatchlings per female divided by the total number of days of egg production.
- 14-Day Old Survivors of Eggs Set - The number of 14-day old survivors per pen divided by the total number of days of egg production.
- Egg Shell Thickness - The average egg shell thickness of indiscriminately selected eggs per pen was measured.
- Offspring's Body Weight - The average body weights of surviving hatchlings and 14-day old survivors were measured by parental pen group.

Reference substance (positive control):

no

Key result

Duration (if not single dose):

21 wk

Dose descriptor:

NOEC

Effect level:

400 other: ppm a.i.

Conc. / dose based on:

act. ingr.

Basis for effect:

other: No effects were seen.

Mortality and sub-lethal effects:

MORTALITY
No adult mortalities occurred during the course of the study.

CLINICAL SIGNS
- Results: No overt signs of toxicity were observed at any of the concentrations tested.
- Remarks: Incidental clinical observations noted during the test, included incidental injuries associated with pen wear such as foot lesions, twisted primary feathers, moulting, and an unkempt appearance and were limited to a few birds. With the exception of incidental-injuries, all birds are normal in appearance and behaviour.

BODY WEIGHT
- Results: There were no apparent treatment-related effects upon adult body weight at any of the concentrations tested.
- Remarks: No statistically significant differences between the control group and the 80, 200 or 400 ppm a.i. treatment groups were observed at any of the body weight intervals.

FOOD CONSUMPTION
- Results: There were no apparent treatment-related effects upon feed consumption at the 80, 200 and 400 ppm a.i. test concentrations.
- Remarks: No statistically significant differences between the control group and any of the treatment groups were observed at any of the feed consumption intervals.

PATHOLOGY
- Results: All findings observed were considered unrelated to treatment.

Effects on reproduction:

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 in the 80, 200, 400 ppm a.i. treatment groups.

REPRODUCTIVE RESULTS
There were no treatment-related effects upon reproductive performance at any of the concentrations tested. When compared to the control group, there were no statistically significant differences in any of the reproductive parameters measured in the 80, 200, 400 ppm a.i. treatment groups.

OFFSPRING BODY WEIGHTS
There were no apparent treatment related effects upon offspring body weight 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 the 80, 200, 400 ppm a.i. treatment groups.

Reported statistics and error estimates:

STATISTICAL ANALYSES
Upon completion of the test, an analysis of variance (ANOVA) was performed to determine statistically significant differences among groups. Dunnett's multiple comparison procedure was used to compare the four treatment means with the control group mean and assess the statistical significance of the observed differences. Sample units were the individual pens within each experimental group, except adult body weights where the sample unit was the individual bird. Percentage data were examined using Dunnett's method following arcsine square root transformation for reproductive parameters.
Additional statistical analyses were performed according to the decision tree currently employed by the EPA and using the Comprehensive Environmental Toxicity Information System (CETIS) software.

Estimated Maximum Mean Daily Dietary Dose of Test Material

Test Interval (Test Weeks)	Test Concentration (ppm a.i.)	Mean Body Weight (g)	Mean Feed Consumption (g/bird/day)	Estimated Daily Dietary Dose* (mg a.i./kg/day)
Pre-egg production (weeks 1 – 10)	0	1053	116	0
	80	1052	111	8.42
	200	1062	118	22.2
	400	1050	115	43.9
Egg production (weeks 11 – 21)	0	1117	164	0
	80	1110	161	11.6
	200	1131	165	29.1
	400	1110	165	59.5
Overall (weeks 1 – 21)	0	1070	141	0
	80	1067	137	10.3
	200	1082	142	26.3
	400	1066	141	53.0

*Results were generated using Excel 2010® in full precision mode. Manual calculations may differ slightly.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study there were no treatment-related mortalities, overt signs of toxicity or treatment-related effects upon body weight or feed consumption at any of the concentrations tested. Additionally, there were no treatment-related effects upon any of the reproductive parameters measured at the 80, 200 or 400 ppm a.i. test concentrations. The no-observed-effect concentration for mallard exposed to the test material in the diet during the study was 400 ppm a.i. (53.0 mg a.i./kg/day), the highest concentration tested.

Executive summary:

A reproduction study in the mallard was carried out in accordance with the standardised guidelines OECD 206, EPA OPPTS 850.2300 and EPA OPP 71-4 under GLP conditions.

The objective of this study was to evaluate the effects of dietary exposure to the test material upon adult mallard (Anas platyrhynchos) over a five-month period. Effects on adult health, body weight, and feed consumption were evaluated. In addition, the effects of adult exposure to the test material on the number of eggs laid, fertility of the eggs, normal development of eggs including viability and survival of the embryos, hatchability, offspring survival and egg shell thickness were evaluated.

Mallard (72 males and 72 females) were randomly distributed into one control group and three treatment groups. Each treatment and control group contained 18 pairs of birds with one male and one female per pen. Three treatment groups were fed diets containing 80, 200, and 400 ppm a.i. of the test material for 21 weeks. The control group was fed diet comparable to the treatment groups, but without the addition of the test material.

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. Feed consumption was measured weekly throughout the test. At the beginning of Week 10, 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 an incubator configured for hatching and allowed to hatch. Once hatching was completed, hatchlings were removed from the hatcher and the individual body weights of the hatchlings were determined. At 14 days of age, the individual body weight of each surviving offspring was determined. Upon completion of the test, statistical analyses were performed to determine statistically significant differences among groups.

Under the conditions of this study there were no treatment-related mortalities, overt signs of toxicity or treatment-related effects upon body weight or feed consumption at any of the concentrations tested. Additionally, there were no treatment-related effects upon any of the reproductive parameters measured at the 80, 200 or 400 ppm a.i. test concentrations. The no-observed-effect concentration for mallard exposed to the test material in the diet during the study was 400 ppm a.i. (53.0 mg a.i./kg/day), the highest concentration tested.

Reference 3

Endpoint:

short-term toxicity to birds: dietary toxicity test

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

21 April 1986 to 29 April 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 71-2 (Avian Dietary Toxicity Test)

Version / remarks:

(U.S.) Environmental Protection Agency, Washington, D.C.: "Pesticide Assessment Guidelines, Subdivision E, Hazard Evaluation Wildlife and Aquatic Organisms"; PB 83-153908, Oct. 1982, § 71-2; "Avian dietary LC50 test", pp. 37 ft.

Deviations:

no

GLP compliance:

no

Remarks:

Study pre-dates GLP.

Dose method:

feed

Analytical monitoring:

no

Remarks:

The stability in the diet has been verified by chemical analysis for a period of at least 10 days in a previous feeding study in rats with a diet of similar composition; thus no analytical control was necessary.

Test organisms (species):

Colinus virginianus

Details on test organisms:

TEST ORGANISM
- Common name: Bobwhite quail
- Age at test initiation: 14 days.
- Sexes used / mixed or single sex: The birds were not sexed since the determination of sex is very uncertain and difficult at that age. Thus chicks in an unknown sex ratio were used.
- Cultural background: Bred from test facility own stock. Indistinguishable from wild birds.
- Disease free: Yes. The chicks were treated prophylactically against the common chick diseases with 0.3 g Tiamutin R/L drinking water from the time of their hatching onward for 3 consecutive days.

Limit test:

no

Total exposure duration (if not single dose):

5 d

Post exposure observation period:

Three days diet ad libitum without test material.

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

10 birds per dose

Control animals:

yes, plain diet

Nominal and measured doses / concentrations:

Dietary concentration: 0, 313, 625, 1 250, 2 500, 5 000 mg/kg.

Details on test conditions:

- Hatching conditions: Adequate for this species
- Date of hatching: 07 April 1986
- Bird maintenance: First days: The chicks were placed into a stainless sheet steel cage (1.10 x 0.52 x 0.49 m), the initial 2 days on filter paper and then on mesh wire (stainless steel), about 280 chicks/cage for the initial 3 days and then 140 chicks/cage until randomisation and allotment to the test cages. The temperature was maintained using ceramic radiant heaters (type IOT) at an adequate level until allocation of the chicks to the test cages. The room was air conditioned. 16 hours light and 8 hours darkness. The relative humidity in the cages was sufficient.
Four days prior to the beginning of the administration of the test material the chicks were randomly allocated to the test groups and put into test cages.

ACCLIMATION
- Acclimation period: Four days prior to the beginning of the administration of the test material.
- Acclimation conditions: Same as test.
- Feeding: Ad libitum throughout maintenance before the study and during the test.
Experimental diet, standard diet in meal form for quails with the following composition: 24 % crude protein, 3.5 % crude fat, 3.0 % crude fibre, 7.5 % crude ash, 3.1 % calcium, 0.7 % phosphorus.
Content of additives per kg diet: 15 000 I.U. vitamin A, 1 500 I.U. vitamin D3. Vitamins E, K3, B-spectrum, nicotinic acid, choline chloride, biotin; BHT Date of production: April, 15, 1986.
Non-perishable guarantee: At least 3 months.
Drinking water: Ad libitum throughout bird maintenance and the study.
- Fasting period before study: No.


PEN SIZE AND CONSTRUCTION MATERIALS
- Description: Stainless sheet steel cages (520 x 350 x 490 mm; floor area 0,18 m^2).
- Floor covering: Stainless steel mesh wire bottom (mesh size 5 x 5 mm).
- Health: No medical treatment.
- Caging: Group: 10 birds per cage.


TEST CONDITIONS
- Temperature: About 25 °C in the test room; in addition, an area with a higher temperature was maintained by a ceramic radiant heater (type IOT) above each cage. Thus the chicks could find their optimum conditions themselves.
- Relative humidity (%): Relative humidity: generally about 50 – 60 % in the test room.
- Photoperiod: 16 hours light and 8 hours darkness; warm light fluorescent lamps.
- Ventilation: Air conditioned.


RANGE FINDING STUDY
- Range finding: No preliminary range finding study was performed.
- Reasons for the concentrations chosen: The EPA Protocol, Oct. 1982, requires a minimum of 4, better 5 or 6 concentrations for the calculation of the LC50. The maximum recommended dosage is 5 000 mg/kg diet. In addition, it is desirable to determine the "No Observable Effect Concentration”. In view of the objective, these requirements and as no range finding study had been performed the concentrations, spaced by a factor of 2, were used.


OTHER:
- Procedure for the preparation of the test substance/diet mixtures:
Each concentration was mixed separately by preparing a premix in a mixing bowl. These premixes were then mixed with meal form basal diet in a suitable laboratory mixer to make sufficient quantities of final diet mixtures for each test group for the whole study. The final mixtures were then stored in stainless steel containers at room temperature. The method for the preparation of the sub stance/diet mixtures is stored with the raw data

- Chemical analyses - Stability: The stability in the diet has been verified by chemical analysis for a period of at least 10 days in a previous feeding study in rats with a diet of similar composition; thus no analytical control was necessary.
Homogeneity: The homogeneous distribution of the test substance in the diet has been verified in a previous study in a diet of similar composition. The same mixing procedure was used; thus no analytical control was necessary.
Concentration control: Each of the concentrations was verified analytically (two determinations each). All samples were taken on the day of mixing and were stored at about -20 °C until analysis.

- Randomisation The birds were weighed individually shortly before the beginning of the test and were allocated to the test groups by a randomisation plan on the basis of their body weights closely following the subprogram RANPER taken from: Nijenhuis A. and Wilf H.S., Combinatorial Algorithms, Academic Press, New York, San Francisco, London, 1978, pp. 62 - 64. The plan is stored with the raw data.

Details on examinations and observations:

Mortality: Once daily.

Clinical signs: Signs of toxicity and other abnormal behavior were determined once daily during the whole study (except one day = Sunday; day 6 of the study).

Feed consumption: Mean/bird/day, calculated from the group mean feed consumption/day; means of days 1 to 5 and 6 to 8

Body weight: Birds were weighed individually; the mean body weight was calculated for each cage (= test group) on days 0, 5 and 8.

Post-mortem examination: All birds were examined macroscopically at death (except those which were cadaverous) or termination of the study.

Duration (if not single dose):

5 d

Dose descriptor:

NOEC

Effect level:

2 500 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

other: No test material-related effects observed.

Key result

Duration (if not single dose):

5 d

Dose descriptor:

LC50

Effect level:

ca. 5 000 mg/kg diet

Conc. / dose based on:

test mat.

Basis for effect:

mortality

Mortality and sub-lethal effects:

Mortality: Test material-related mortality only occurred in the highest dose group (5 000 mg/kg). The mortality in the 1 250 mg/kg group (one chick) is due to injuries received by fighting (bleeding beak).
The LC50 is estimated to be about 5 000 mg/kg diet.
Highest concentration tested causing no test material-related mortality: 2 500 mg/kg diet.
Minimum concentration causing 100 % mortality: Greater than 5 000 mg/kg diet.

Clinical signs: Besides sporadically occurring injuries to the beaks (cannibalism, fighting) which are obviously not test material-related no pronounced toxic signs were observed except apathy in the surviving birds in the highest concentration (5 000 mg/kg diet).

Feed consumption: There was a reduction in feed consumption only in the highest dose group (5 000 mg/kg feed) during the test material-feeding period.

Body weight: In general no statistically significant reduction in body weight gain occurred at the end of the test material-feeding period (day 5) and at the end of post-exposure period (day 8) but there seemed to be - although statistically not significant - a slight reduction in body weight gain in the highest, dose group at the end of the test material-feeding period (day 5).

Post-mortem examination All birds were examined macroscopically.
Surviving birds: No test material-related pathological findings.
Birds that had died: No test material-related findings (Birds were sometimes cadaverous due to the high temperature in the test cages which is necessary for the chicks at that age; they therefore could not be examined thoroughly.)

Reported statistics and error estimates:

The statistical evaluation of the body weight was performed by one-way analyses (ANOVA) followed by Dunnet’s test: "A multiple comparison procedure for comparing several treatments with a control", C. W. Dunnet 1955, JASO 50 and "New tables for multiple comparisons with a control", C. W. Dunnet 1964, Biometrics 20. The statistical evaluation was performed with the computer systems of the Department of Toxicology using the INSTEM-Toxicology-data system (programme TOXREP). As the mortality values obtained in this study were not adequate for the calculation of the LC50 value, the LC50 was estimated.

Mortality

Group No.	Concentration (mg/kg diet)	Time of Death (Cumulative Mortality)
		Test Material Feeding Period						Post-Exposure Period
		Day 0	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	Day 7	Day 8
0	0 (untreated control)	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10
1	313	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10
2	625	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10
3	1 250	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	1* / 10	0 / 10
4	2 500	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10
5	5 000	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	2 / 10	5 / 10	5 / 10	5 / 10

*Possibly died from injuries.

 

Clinical Signs

Group No.	Concentration (mg/kg diet)	Time of Death (Cumulative Mortality)
		Test Material Feeding Period						Post-Exposure Period
		Day 0	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	Day 7	Day 8
0	0 (untreated control)	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
1	313	n.d.	n.d.	n.d.	n.d.	I1	I2	-	I2	I1
2	625	n.d.	I1	I1	I1	I2	I2	-	n.d.	n.d.
3	1 250	n.d.	n.d.	I1	I2	I2	I2	-	I1	I1
4	2 500	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	-	n.d.	n.d.
5	5 000	n.d.	n.d.	I2	I3	I3	I2	-	I1 A5	I1 A5

A: Apathy

I: Injured beak. I is obviously not test material-related and is due to mutual aggression.

Number relates to the number of birds affected.

n.d.: No symptoms detectable.

-: Not determined.

 

Feed Consumption

Group Mean Feed Consumption (g/bird/day)	Group No. (Concentration mg/kg)
Day	0 (Untreated Control)	1 (313)	2 (635)	3 (1 250)	4 (2 500)	5 (5 000)
1	3.6	3.7	3.3	3.2	3.1	2.4
2	3.8	3.7	4.0	3.7	3.9	2.3
3	4.9	4.8	4.8	5.0	4.7	3.7
4	5.6	5.2	5.5	5.5	5.7	4.1
5	5.1	4.8	5.3	5.7	5.4	5.2
Mean (Days 1 – 5 Exposure Period)	4.6	4.4	4.6	4.6	4.6	3.5
6	5.2	4.9	5.6	-**
7	6.6	5.0	7.4	-**	11.9***	11.6***
8	6.4	5.8	7.0	5.6	7.7	6.8
Mean (Days 6 – 8) Post-Exposure Period)	6.1	5.2	6.7		6.5	6.1

*The birds that were found dead in their cages on the respective days of the determination of the feed consumption are not included in the calculations and thus in the feed consumption figures.

**Unclear raw data.

***Inadvertently weighed for two days.

 

Concentration Control

Nominal Concentration (mg/kg diet)	Analytically Detected Concentration (mg/kg Diet)	% of Nominal Concentration (Mean)
313	309	98
	303
	Mean: 3.6
625	667	107
	673
	Mean:6 70
1 250	1 246	101
	1 287
	Mean: 1 262
2 500	2 481	101
	2 560
	Mean: 2 521
5 000	5 285	105
	5 230
	Mean: 5 258

 

Validity criteria fulfilled:

not specified

Conclusions:

Under the conditions of the study, the LC50 for the bobwhite quail (chicks) is about 5 000 mg/kg diet. The NOEC was determined to be 2 500 mg/kg diet.

Executive summary:

The toxicity of the test material to bobwhite quail was assessed according to EPA OPP 71-2.

The test material was tested on chicks of the bobwhite quail by administration in the feed for five consecutive days of the concentrations of 0 (control), 313, 625, 1 250, 2 500 and 5 000 mg/kg diet. A post-exposure period of another 3 days followed this treatment.

Mortality: Highest concentration tested causing no test material-related mortality: 2 500 mg/kg diet

Minimum concentration causing 100 % mortality: > 5 000 mg/kg diet

LC50: The LC50 for the bobwhite quail (chicks) is about 5 000 mg/kg diet.

Clinical signs:  Besides sporadically occurring injuries to the beaks (cannibalism and fighting) which are not test material-related no pronounced toxic signs were observed except apathy in the surviving birds in the highest concentration (5 000 mg/kg).

Feed consumption: There was a reduction in feed consumption only in the highest dose group (5 000 mg/kg feed) during the test material-feeding period.

Body weight: There was no statistically significant reduction in body weight gain ,at the end of the exposure period (day 5) and at the end of post-exposure period (day 8).

Post-mortem examination: Surviving birds: No test material-related pathological findings

Birds that had died: No test material-related findings

Under the conditions of the study, the LC50 for the bobwhite quail (chicks) is about 5 000 mg/kg diet. The NOEC was determined to be 2 500 mg/kg diet.

Reference 4

Endpoint:

short-term toxicity to birds: acute oral toxicity test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 March 1986 to 17 March 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 71-1 (Avian Acute Oral Toxicity Test)

Version / remarks:

EPA “pesticide Assessment Guidelines, Subdivision E, Hazard Evaluation Wildlife and Aquatic Organisms": PB 83-153908, October 1982, § 71-1, pp. 33ff., “Avian single-dose oral LD50”.

Deviations:

no

GLP compliance:

no

Remarks:

Study pre-dates GLP.

Dose method:

gavage

Analytical monitoring:

yes

Remarks:

The stability of aqueous solutions was verified for a period of 4 days by analytical determination in a previous acute study on fish (Project No. 14F47/83).

Vehicle:

yes

Remarks:

0.5 % aqueous carboxymethylcellulose

Details on preparation and analysis of diet:

DIET PREPARATION
- Description and nutrient analysis of basal diet provided in study report: Yes

Test organisms (species):

Colinus virginianus

Details on test organisms:

TEST ORGANISM
- Common name: Bobwhite quail
- Age at test initiation: Hatched June 15 to 30, 1985.
- Sexes used / mixed or single sex: Male and female birds; the sex could be determined properly due to the colouration of the animals at that age.

Limit test:

no

Post exposure observation period:

The observation period lasted 14 days.

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

Five per sex per dose.

Control animals:

yes, concurrent vehicle

Nominal and measured doses / concentrations:

125, 250, 500, 1 000, 2 000 mg/kg bw.

Details on test conditions:

ACCLIMATION
- Acclimation period: Adaptation to the housing conditions of a group consisting of a sufficient number of birds in an iron wire pen of sufficient size with a stainless steel wire mesh bottom from Nov. 26, 1985 onward. Adaptation to the test cages from February 20 to March 3, 1986 (beginning of the test).
- Feeding: The birds were offered a commercial poultry diet ad libitum throughout maintenance before the study and during the test with the exception of a fasting period prior to dosing.
Experimental diet, special mixture for quails in pellets with the following analytical data: 24.0 % crude protein, 3.5 % crude fat, 2.75 % crude fibre, 7.0 % crude ash, 1.0 % calcium, 0.8 % phosphorus.
Content of additives per kg diet: 15 000 I.U. vitamin A, 1 500 I.U. vitamin D3
Vitamins E, K3, B-spectrum, nicotinic acid, choline chloride, biotin.
Date of production: January 1986
Nonperishable guarantee: 3 months.
Residues in the diet: The feed used in the study is regularly assayed for· contaminants. On the basis of these findings the study director decides whether the level of contaminants is acceptable. Fed. Reg. Vol. 44, No. 91 of May 9, 1979, page 27 354 (EPA) serves as a guideline for maximum tolerable contaminants.
- Health: After arrival at the laboratory (Nov. 26, 1985) prophylactical treatment against the common chick diseases with 300 mg Tiamutin/L in the drinking water for two periods (Nov. 26 - Nov. 30 and Dec. 9 - Dec. 19, 198 5).

FEED WITHHOLDING PERIOD BEFORE DOSING
- No. of hours: Fasted for about 24 hours before administration.

PEN SIZE AND CONSTRUCTION MATERIALS
- Description: Stainless steel wire mesh cages with wire mesh floors (0.59 x 0.45 x 0.26 m); floor area about 0.26 m^2 for 5 birds each.
- Caging: Group: 5 birds each; males and females are caged separately.

TEST CONDITIONS
- Temperature: During adaptation and test period about 22 °C.
- Relative humidity (%): Adaptation and test period about 50 to 60 %.
- Photoperiod: During adaptation and test period 16 hours light, 8 hours dark; warm-light fluorescent lamps.
- Ventilation: Air-conditioned room.

RANGE FINDING STUDY
- The initial range finding with 5 male birds/dose indicated that the LD50 could be expected at about 1 000 mg/kg and a NOEL lower 500 mg/kg body weight.

OTHER:
- For each dose group 70 g of a dispersion in 0.5 % aqueous carboxymethylcellulose (CMC; bidistilled water was used) were prepared separately. The concentrations of the test compound were 20, 10, 5, 2.5, or 1.25 % (w/w). The compound was mixed into the CMC preparation with an ultrasonic stirrer. The mixing procedure was then continued on a magnetic stirrer during the drawing of samples for the administration.
- Concentration: 1.25, 2.5, 5.0, 10, 20 g/100g.
- Dosing: The birds were administered the test substance once by gavage into the crop in a total amount of 10 g of the respective preparation per kg body weight.
Volume administered: 10 g/kg bw.
- The birds were weighed individually shortly before the beginning of the test and were allocated to the test groups by a randomisation plan on the basis of their body weights prepared by following a standard laboratory method.
- Birds were used before their first egg-laying season; visually indistinguishable from wild birds.
- Drinking water: Municipal water ad libitum throughout bird maintenance and the study
- Analyses of drinking water: The drinking water is regularly assayed for contaminants by the municipal authorities of Frankenthal and the Technical Services of BASF Aktiengesellschaft. In view of the aim and duration of the study there are no special requirements exceeding the specifications of the drinking water.

Details on examinations and observations:

Mortality: Twice on day zero, daily thereafter.
Clinical signs: Twice on day zero, daily at least on work days thereafter.
Feed consumption: Mean feed consumption (g/bird/day), calculated from the feed consumption/cage separately for male and female birds. The feed consumption was determined for days zero to 3 on day 3, days 4 to 7 on day 7 and ·days 8 to 14 (mean of two determinations on days 10 and 14).
Body weight: Individual and group mean body weights determined separately for male and female birds on day zero, 7 and 14 of the study.
Gross post-mortem examination: All sporadic deaths; all remaining birds at the termination of the study.

Key result

Dose descriptor:

NOEL

Effect level:

125 mg/kg bw/day (actual dose received)

Conc. / dose based on:

test mat.

Basis for effect:

other: No test material-related effects observed.

Remarks on result:

other: Single dose

Key result

Dose descriptor:

LC50

Effect level:

> 500 - < 1 000 mg/kg bw/day (actual dose received)

Conc. / dose based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Single dose

Mortality and sub-lethal effects:

Mortality: No deaths occurred in the control group or in the three lowest dose groups (125, 250 and 500 mg/kg).
Highest dose tested causing no mortality was 500 mg/kg body weight (males plus females).
Minimum dose tested causing 100 % mortality: 1 000 mg/kg body weight.
The LD50 is > 500 and < 1 000 mg/kg body weight for male plus female birds.

Clinical signs: In the control group and in the 125 or 250 mg/kg group all birds were in good health during the whole study (except very slight diarrhoea in the 250 mg/kg group on day one of the study). In the higher dose groups severe toxic signs such as apathy, side- and prone position, ruffled feathers, difficulties in walking (ataxia) and diarrhoea were seen. The survivors had completely recovered from day 3 onward.

Feed consumption: The mean total feed consumption/bird/day during the 14-day observation period was in the same order of magnitude in all test groups with surviving birds and in the control group. The initial feed consumption/bird/day was reduced - dose dependent - in the males and females in the 250 and 500 mg/kg groups for the period of the days zero to three. Nearly no feed was taken up by the male and female birds of the two highest test groups (1 000 and 2 000 mg/kg body weight).

Body weight: There was no statistically significant reduction in body weight gain in the surviving birds on day 7 and at the end of the study. The body weights of the birds that had died during the study are not reported as they are considered not to be relevant for the results of the study.

Gross post-mortem examination: All birds were examined macroscopically.
Surviving birds: No test material related effects
Birds that had died (1 000 and 2 000 mg/kg): General congestive hyperemia.

Reported statistics and error estimates:

The statistical evaluation of the body weights was performed by one-way analyses (ANOVA) followed by Dunnet’s test. Dunnet’s test: "A multiple comparison procedure for comparing several treatments with a control", C. W. Dunnet 1955, JASO 50 and "New tables for multiple comparisons with a control”, C. W. Dunnet 1964, Biometrics 20. The statistical evaluation was performed with the computer systems of the Department of Toxicology using the INSTEM-Toxicology-data system (program TOXREP). The LD50 values were estimated as the results obtained were not adequate for calculating an LD50.

Concentration Control

An analytical concentration control was performed for all dose levels as they had to be prepared separately.

Nominal Concentration (mg/kg)	Analytically Detected Concentration (mg/kg)	% of Nominal Concentration (Mean)
12 500	12 034	94.6
	11 604
	Mean: 11 819
25 000	23 577	93.2
	23 019
	Mean: 23 298
50 000	49 690	99.6
	49 915
	Mean: 49 803
100 000	101 043	101.8
	102 500
	Mean: 101 772
200 000	200 795	101.0
	203 115
	Mean: 201 955

 

Cumulative Mortality

5 male sand 5 female birds/test group.

Test Group	Dose Level (mg/kg)	Day of the Study
		0*		0**		1		2		3		4		5		6
		M	F	M	F	M	F	M	F	M	F	M	F	M	F	M	F
0	0 (carrier control)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
1	125	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
2	250	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
3	500	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
4	1 000	0	0	0	0	0	0	2	3	4	4	5	5	5	5	5	5
5	2 000	0	0	0	0	2	4	5	5	5	5	5	5	5	5	5	5

* Shortly after administration.

** Approximately 1 to 2 h after administration.

 

Test Group	Dose Level (mg/kg)	Day of the Study
		7		8		9		10		11		12		13		14
		M	F	M	F	M	F	M	F	M	F	M	F	M	F	M	F
0	0 (carrier control)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
1	125	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
2	250	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
3	500	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
4	1 000	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
5	2 000	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5

Validity criteria fulfilled:

not specified

Conclusions:

Under the conditions of the study, the acute oral LD50 of the test material in bobwhite quail was > 500 mg/kg bw/day and < 1 000 mg/kg bw/day. The NOEL was 125 mg/kg body weight.

Executive summary:

The acute toxicity of the test material to bobwhite quail according to EPA OPP 71-1.

The test material (aqueous preparation with 0.5 % CMC) was tested for its acute toxicity to the bobwhite quail by gavage of single doses of 0, 125, 250, 500, 1 000 and 2 000 mg/kg body weight followed by a 14-day observation period.

No deaths occurred in the control group or in the three lowest dose groups (125, 250 and 500 mg/kg). Highest dose tested causing no mortality: 500 mg/kg body weight (males plus females).

Minimum dose tested causing 100 % mortality: 1 000 mg/kg body weight.

The LD50 for the bobwhite quail is > 500 and < 1 000 mg/kg body weight.

In the control group and in the 125 or 250 mg/kg groups all birds were in good health (except very slight diarrhoea in the 250 mg/kg group on day one of the study). In the higher dose groups severe toxic signs such as apathy, side- and prone position, ruffled feathers, ataxia and diarrhoea were diagnosed. The survivors had completely recovered from day 3 onward.

Feed consumption: The mean total feed consumption/bird/day during the 14-day observation period was in the same order of magnitude in all test groups with surviving birds and in the control group. The initial feed consumption/bird/day was reduced - dose dependent - in the males and the females in the 250 and 500 mg/kg body weight groups. Nearly no feed was taken up by the male and female birds of the two highest test groups (1 000 and 2 000 mg/kg body weight).

Body weight: There was no statistically significant reduction in body weight gain in the surviving birds on day 7 and at the end of the study.

Gross post-mortem examination of surviving birds: No substance related effects.

Birds that had died (1 000 and 2 000 mg/kg): General congestive hyperemia.

Under the conditions of the study the NOEL was 125 mg/kg body weight. The LD50 for the bobwhite quail is > 500 and < 1 000 mg/kg wt.

Reference 5

Endpoint:

short-term toxicity to birds: dietary toxicity test

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

04 June 1986 to 12 June 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Section 71-2 of the Environmental Protection Agency Registration Guidelines Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ASTM Standard E857-81 “Standard Practice for Conducting Subacute Dietary Toxicity Tests with Avian Species”.

Deviations:

no

GLP compliance:

yes

Dose method:

feed

Analytical monitoring:

no

Vehicle:

yes

Remarks:

Corn oil

Details on preparation and analysis of diet:

- The dietary concentrations were established based upon known toxicity data.
- During the exposure period the control group received an amount of the carrier in their diet equivalent to the greatest amount used in the treated diets.

DIET PREPARATION
- Preparation of doses: Mixing of the test diet was done with a Hobart (Model Number AS200T) mixer. Diets were prepared on the day of study initiation. An amount of diet sufficient to last the five day exposure period was presented to the birds at initiation of the study.
The dietary concentrations were not adjusted for purity of the test material. Therefore all dietary concentrations and the LC50 value are reported as parts per million of the test material as received.
- Type, identity and function of solvent/vehicle: The test diets were prepared by mixing the test material into the diet with corn oil.
- Amount of vehicle: The concentration of corn oil in the treated and control diets was 2 %

The diet was prepared as follows:
562 ppm: 3.3721 g test material + 130 mL corn oil + 5 876.6 g game bird ration.
1 000 ppm: 6.0001 g test material + 130 mL corn oil + 5 874.0 g game bird ration.
1 780 ppm: 10.6800 g test material + 130 mL corn oil + 5 869.3 g game bird ration.
3 160 ppm: 18.9600 g test material + 130 mL corn oil + 5 861.0 g game bird ration.
5 620 ppm: 32.7199 g test material + 130 mL corn oil + 5 846.3 g game bird ration.
For each concentration, the ration was weighed on Scale #2, and approximately one-half was placed in a Hobart mixer with the corn oil. The experimental material was weighed on Scale #8 on tared weigh paper. The experimental material was ground into a fine powder with a small portion of feed and placed in the mixer. The mortar was rinsed with the feed. The remaining portion of the ration was added and the diet was mixed for approximately 10 minutes.

Test organisms (species):

Anas platyrhynchos

Details on test organisms:

TEST ORGANISM
- Common name: Mallard
- Age at test initiation: 9 days of age
- Sexes: The birds used in this study were too immature to differentiate by sex.
- Cultural background: Birds were hatched on 26 May 1986 and received at Wildlife International Ltd. on 27 May 1986. All birds were pen-reared and phenotypically indistinguishable from wild birds.

Limit test:

no

Total exposure duration (if not single dose):

5 d

Post exposure observation period:

Following the five day exposure period all groups were given untreated feed for three days.

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

10 per group.

Control animals:

yes

Nominal and measured doses / concentrations:

Nominal dietary concentrations used in this study were 562, 1 000, 1 780, 3 160 and 5 620 ppm.

Details on test conditions:

ACCLIMATION
- Acclimation period: Birds were acclimated from the day they were received until test initiation.
- Feeding: Throughout acclimation and testing all test birds were fed a game bird ration formulated to the test facility's specifications. Water from a 400 foot well on the Wildlife International Ltd. site and feed were provided ad libitum during acclimation and during the test.
- Health: All birds appeared to be in good health at initiation of the study. The birds received no form of antibiotic medication during acclimation or the study.

PEN SIZE AND CONSTRUCTION MATERIALS
- Description: During acclimation and testing, all birds were housed indoors by test group in batteries of brooding pens manufactured by Beacon Steel Products Co. (Model No. B735). Birds were assigned to pens by random draw. Each pen had floor space that measured approximately 72 x 90 cm. Ceiling height was approximately 24 cm. External walls, ceilings and floors were constructed of galvanised steel wire and sheeting.
- Compliant to good husbandry practices: Yes. Housing and husbandry practices were based upon the "Guide for the Care and Use of Laboratory Animals," 1978 DHEW Publications No. (NIH) 78-23.
- Caging: Group: Each test or control group was assigned a pen that contained ten ducklings.

TEST CONDITIONS
- Room temperature: Average ambient room temperature for this study was 82 ± 3 °F (SD)
- Relative humidity: 72 %
- Photoperiod: The photoperiod (maintained by a time clock) was seventeen hours of light per day during acclimation and throughout the study. The light source was Chroma 50 fluorescent lights which closely approximate noon-day sunlight (noon-day sun - 4870 ° Kelvin, Chroma 50 - 5 000 ° Kelvin). The birds received approximately twelve footcandles of illumination.

Details on examinations and observations:

MORTALITY / CLINICAL SIGNS
- Time schedule for examinations: During acclimation all birds were observed daily. Birds exhibiting abnormal behaviour or physical injury were not used. Following test initiation and continuing until termination, all birds were observed at least twice daily.
- Remarks: A record was maintained of all mortality, signs of toxicity, or abnormal behaviour.

BODY WEIGHT
- Time schedule for examinations: Body weights by group were measured at initiation of the study, on Day 5, and at termination of the test on Day 8.

FOOD CONSUMPTION
- Time schedule for examinations: Average estimated feed consumption was determined for each test concentration group and control group for the exposure period, Days 0 - 5, and for the observation period, Days 6 - 8.
- Remarks: Feed consumption was measured accurately, but is presented as an estimate due to the unavoidable wastage by the birds.

Key result

Duration (if not single dose):

5 d

Dose descriptor:

LD50

Effect level:

> 5 620 other: ppm

Conc. / dose based on:

test mat.

Basis for effect:

other: The highest concentration tested.

Duration (if not single dose):

5 d

Dose descriptor:

NOEL

Effect level:

1 780 other: ppm

Conc. / dose based on:

test mat.

Basis for effect:

body weight

Remarks:

Based on the reduction in body weight gain at the 3 160 ppm.

Mortality and sub-lethal effects:

MORTALITY
- Controls: There were no mortalities in the control group.
- Test material: There were no mortalities or overt signs of toxicity throughout the test period

CLINICAL SIGNS
- Test material: All birds, at all concentrations, were normal in appearance and behaviour.
- Controls: All birds were normal in appearance and behaviour throughout the test period.

ABNORMAL BEHAVIOUR
- Test material: All birds, at all concentrations, were normal in appearance and behaviour.
- Controls: All birds were normal in appearance and behaviour throughout the test period.

BODY WEIGHT
- Test material: When compared with the control group, there was no apparent effect on body weight gain at the 562 ppm, 1 000 ppm and 1 780 ppm concentrations. A reduction in body weight gain was noted at the 3 160 ppm concentration during the exposure period (Days 0 - 5).
When compared with the control group, a marked reduction in body weight gain was noted at the 5 620 ppm concentration during the exposure period (Days 0 - 5).

FOOD CONSUMPTION
- Test material: When compared with the control group, there was no apparent effect on feed consumption at the 562 ppm, 1 000 ppm and 1 780 ppm concentrations. No apparent effect
on feed consumption was noted at the 3 160 ppm concentration. When compared with the control group, a marked reduction in feed consumption was noted at the 5 620 ppm concentration during the exposure period (Days 0 - 5).

Reported statistics and error estimates:

The mortality pattern in this study was not conducive to calculating the LC50 value. Therefore, an estimation of the LC50 value was made by a visual inspection of the mortality data.

Cumulative Mortalities of Control Mallards

Concentration (ppm)	Number Dead / Number Exposed
	Day of Study
	0	1	2	3	4	5	6	7	8
0	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10
0	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10
0	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10

 

Cumulative Mortalities of Mallards Exposed to the Test Material for Five Days

Concentration (ppm)	Number Dead / Number Exposed
	Day of Study
	0	1	2	3	4	5	6	7	8
562	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10
1 000	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10
1 780	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10
3 160	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10
5 620	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10

 

Validity criteria fulfilled:

not specified

Conclusions:

Under the conditions of the study the dietary LC50 value of the test material in the mallard was determined to be > 5 620 ppm, the highest concentration tested. The no-observed-effect concentration was determined to be 1 780 ppm.

Executive summary:

The toxicity of the test material to birds was assessed according to Section 71-2 of the Environmental Protection Agency Registration Guidelines Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms. and ASTM Standard E857-81 “Standard Practice for Conducting Subacute Dietary Toxicity Tests with Avian Species” and according to the principles of GLP.

Groups of ten mallard ducklings were assigned to each of the treatment and control groups by random draw were acclimated from the day they were received until test initiation. The test consisted of a geometric series of five test concentrations and three control groups. Nominal dietary concentrations used in this study were 562, 1 000, 1 780, 3 160 and 5 620 ppm.

Under the conditions of the study the dietary LC50 value of the test material in the mallard was determined to be > 5 620 ppm, the highest concentration tested.  The no-observed-effect concentration was determined to be 1 780 ppm.

Reference 6

Endpoint:

short-term toxicity to birds: acute oral toxicity test

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

21 April 1986 to 05 May 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Section 71-1 of the Environmental Protection Agency Registration Guidelines Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms

Deviations:

no

GLP compliance:

yes

Dose method:

gavage

Analytical monitoring:

no

Vehicle:

yes

Remarks:

Corn oil

Test organisms (species):

Anas platyrhynchos

Details on test organisms:

TEST ORGANISM
- Common name: Mallard
- Age at test initiation: 30 weeks of age.
- Weight at test initiation: Mallards ranged in weight from 856 g to 1 259 g at study initiation.
- Sexes used: Male and female
- Disease free: Yes. All mallards appeared to be in good health at initiation of the study.
- Breeding population: All birds were pen-reared and phenotypically indistinguishable from wild birds.

Limit test:

no

Post exposure observation period:

14 days

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

Five males and five females per treatment dose.

Control animals:

yes, concurrent vehicle

Nominal and measured doses / concentrations:

Nominal dosages used in this study were 292, 486, 810, 1 350 and 2 250 mg test material /kg body weight.

Details on test conditions:

ACCLIMATION
- Acclimation period: Birds were acclimated for a minimum of fourteen days prior to test initiation.
- Feeding: Throughout acclimation and testing all test birds were fed a game bird ration formulated to the test facility's specifications. Water from a 400 foot well on the Wildlife International Ltd. site and feed were provided ad libitum during acclimation and during the test.
- Health : All mallards appeared to be in good health at initiation of the study. The birds received no form of antibiotic medication during the study.

FEED WITHHOLDING PERIOD BEFORE DOSING
- No. of hours: The birds were fasted for at least 15 hours prior to dosing.

PEN SIZE AND CONSTRUCTION MATERIALS
- Description: Each pen had floor space that measured approximately 75 x 90 cm. Ceiling height was approximately 45 cm. External walls, ceilings and floors were constructed of galvanised wire.
- Compliant to good husbandry practices: Yes. Housing and husbandry practices were based upon the 11 Guide for the Care and Use of Laboratory Animals, 11 1978 DHEW Publications No. (NIH) 78 - 23.
- Caging: Group. Test birds were housed indoors by dosage group in batteries of pens manufactured by Safeguard Products, Inc. (Model No. H5355). Birds were assigned to pens by random draw. Each dosage group was assigned two pens. One pen contained five males and the other five females.

TEST CONDITIONS
- Temperature: Birds were maintained at ambient room temperature. Average temperature for this study was 66 ± 3 °F (SD).
- Relative humidity (%): Relative humidity of 80 %.
- Photoperiod: The photoperiod (maintained by a time clock) was eight hours of light per day during acclimation and throughout the study. The light source was Chroma 50 fluorescent lights which closely approximate noon-day sunlight (noon-day sun - 4870° Kelvin, Chroma 50 - 5 000 ° Kelvin). The birds received approximately twelve footcandles of illumination.

OTHER
- The dosages were established based upon known toxicity data.
- Animals were assigned to each of the treatment groups and the control group by random draw.
At initiation of the test, a single dose of the test material in diluent was orally intubated directly into the crop or proventriculus of each bird using a stainless steel catheter. Each bird was
individually weighed and dosed on the basis of mg of test material per kg of body weight.
- All treatment and control birds received a constant dosage volume of 6 mL/kg of body weight.
- The test material was dispersed in corn oil. The concentration of the test material in the diluent was adjusted to provide a constant volume to body weight dosage for all treatment birds.
- The dosages and LD50 value reported were not corrected for purity of the test material.

Details on examinations and observations:

MORTALITY / CLINICAL SIGNS
- Time schedule for examinations: During acclimation all birds were observed daily. Birds exhibiting abnormal behaviour or physical injury were not used. Following test initiation until termination all birds were observed at least twice daily.
- Remarks: A record was maintained of all mortality, signs of toxicity, or abnormal behaviour.

BODY WEIGHT
- Time schedule for examinations: Individual body weights were measured at initiation of the test and by group on Days 3 - 7 and 14 of the test.

FOOD CONSUMPTION
- Time schedule for examinations: Average estimated feed consumption was determined for each dosage group and the control for Days 0 - 3, 4 - 7 and 8 - 14.
- Remarks: Feed consumption was measured accurately, but is presented as an estimate due to the unavoidable wastage by the birds.

Key result

Dose descriptor:

LD50

Effect level:

> 486 mg/kg bw/day (actual dose received)

Conc. / dose based on:

test mat.

Basis for effect:

other: The highest dosage at which no significant regurgitation was observed.

Dose descriptor:

NOEL

Effect level:

< 292 mg/kg bw/day (actual dose received)

Conc. / dose based on:

test mat.

Basis for effect:

other: The lowest dosage tested, based on overt signs of toxicity (regurgitation).

Mortality and sub-lethal effects:

MORTALITY
Control group: There were no mortalities in the control group.
Test material: There were no mortalities at dosages of 292, 486, 810 and 1 350 mg/kg. There was 10 % mortality (1 of 10) at 2 250 mg/kg.

CLINICAL SIGNS
- Results: At the 292 mg/kg dosage, regurgitation was noted in one female approximately one-half hour after dosing. However no regurgitation was noted at the 486 mg/kg dosage. No other overt signs of toxicity were noted during the study at the 292 mg/kg and 486 mg/kg dosages. At the 810 mg/kg dosage, five males and three females were noted to regurgitate after dosing. Lethargy was noted approximately two hours after dosing; however, all birds had recovered approximately five hours after dosing and were normal in appearance and behaviour until study termination.
All birds except one male were observed regurgitating after dosing at the 1 350 mg/kg dosage and all except one female at the 2 250 mg/kg dosage. Overt signs of toxicity were noted within one-half hour of dosing at the 1 350 mg/kg and 2 250 mg/kg dosages. By the morning of Day 3, all birds at the 1 350 mg/kg dosage had recovered. All survivors at the 2 250 mg/kg dosage had recovered by the morning of Day 2.
Overt signs of toxicity typical of intoxication with the test material included depression or lethargy, a ruffled appearance, reduced reaction to external stimuli (sound and movement), wing droop, loss of coordination, lower limb weakness, prostrate posture, convulsions and coma.
Control group: All birds were normal in appearance and behaviour throughout the test period.

BODY WEIGHT
- Results: From Day 0 through Day 3, changes in body weight were variable. However there appeared to be a reduction in body weight gain among males at 810 mg/kg and 1 350 mg/kg dosages when compared to the controls and a body weight loss among females at the 2 250 mg/kg dosage.

Reported statistics and error estimates:

Statistical Calculations: The mortality pattern in this study was not conducive to calculating the LD50 value. Therefore, an estimation of the LD50 value was made by a visual inspection of the mortality data.

Cumulative Mortality of Mallards Gavaged with the Test Material by Sex

Dosage (mg/kg)

Number Dead / Number Exposed

Day of Study

Total

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Control Males

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 10

Control Females

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

292 Males

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 10

292 Females

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

486 Males

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 10

486 Females

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

810 Males

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 10

810 Females

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

1 350 Males

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 10

1 350 Females

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

2 250 Males

0 / 5

0 / 5

1 / 5

1 / 5

1 / 5

1 / 5

1 / 5

1 / 5

1 / 5

1 / 5

1 / 5

1 / 5

1 / 5

1 / 5

1 / 10

2 250 Females

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

0 / 5

 

Validity criteria fulfilled:

not specified

Conclusions:

Under the conditions of the study the mallard acute oral LD50 value for the test material was determined to be > 486 mg/kg, the highest dosage at which no significant regurgitation was observed. The no-observed-effect dosage was < 292 mg/kg, the lowest dosage tested, based on overt signs of toxicity (regurgitation).

Executive summary:

The acute oral toxicity of the test material to birds was assessed according to Section 71-1 of the Environmental Protection Agency Registration Guidelines Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms and according to the principles of GLP.

Groups of ten mallards, five males and five females, were assigned to each of the treatment groups and the control group by random draw. The test consisted of a geometric series of five dosage groups and a control group. Nominal dosages used in this study were 292, 486, 810, 1 350 and 2 250 milligrams of the test material/ kg of body weight. The control group was dosed with diluent only.

There were no mortalities at dosages of 292, 486, 810 and 1 350 mg/kg. There was 10 % mortality (1 of 10) at 2 250 mg/kg.

Under the conditions of the study the mallard acute oral LD50 value for the test material was determined to be > 486 mg/kg, the highest dosage at which no significant regurgitation was observed. The no-observed-effect dosage was < 292 mg/kg, the lowest dosage tested, based on overt signs of toxicity (regurgitation).

Description of key information

SHORT TERM

 

Key Acute Toxicity Study: Munk (1986)

Under the conditions of the study the NOEL was 125 mg/kg body weight. The LD50 for the bobwhite quail is > 500 and < 1 000 mg/kg wt.

 

Supprting Acute Toxicity Study: Grimes (1986)

Under the conditions of the study the mallard acute oral LD50 value for the test material was determined to be > 486 mg/kg, the highest dosage at which no significant regurgitation was observed. The no-observed-effect dosage was < 292 mg/kg, the lowest dosage tested, based on overt signs of toxicity (regurgitation).

 

Supporting Dietary Study: Grimes (1986)

Under the conditions of the study the dietary LC50 value of the test material in the mallard was determined to be > 5 620 ppm, the highest concentration tested.  The no-observed-effect concentration was determined to be 1 780 ppm.

 

Supporting Dietary Study: Munk (1986)

Under the conditions of the study, the LC50 for the bobwhite quail (chicks) is about 5 000 mg/kg diet. The NOEC was determined to be 2 500 mg/kg diet.

 

 

 

LONG TERM

 

Key study: Long-Term Toxicity to Birds: Pagano et al. (2017) - Read across (MCPP-P)

 

Under the conditions of this study there were no treatment-related mortalities, overt signs of toxicity or treatment-related effects upon body weight or feed consumption at any of the concentrations tested. Additionally, there were no treatment-related effects upon any of the reproductive parameters measured at the 80, 200 or 400 ppm a.i. test concentrations. The no-observed-effect concentration for mallard exposed to the test material in the diet during the study was 400 ppm a.i. (53.0 mg a.i./kg/day), the highest concentration tested.

Key value for chemical safety assessment

Short-term EC50 or LC50 for birds:

500 mg/kg bw

Additional information

SHORT TERM

 

 

Key Acute Toxicity Study: Munk (1986)

The acute toxicity of the test material to bobwhite quail according to EPA OPP 71-1. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material (aqueous preparation with 0.5 % CMC) was tested for its acute toxicity to the bobwhite quail by gavage of single doses of 0, 125, 250, 500, 1 000 and 2 000 mg/kg body weight followed by a 14-day observation period.

No deaths occurred in the control group or in the three lowest dose groups (125, 250 and 500 mg/kg). Highest dose tested causing no mortality: 500 mg/kg body weight (males plus females).

Minimum dose tested causing 100 % mortality: 1 000 mg/kg body weight.

The LD50 for the bobwhite quail is > 500 and < 1 000 mg/kg body weight.

In the control group and in the 125 or 250 mg/kg groups all birds were in good health (except very slight diarrhoea in the 250 mg/kg group on day one of the study). In the higher dose groups severe toxic signs such as apathy, side- and prone position, ruffled feathers, ataxia and diarrhoea were diagnosed. The survivors had completely recovered from day 3 onward.

Feed consumption: The mean total feed consumption/bird/day during the 14-day observation period was in the same order of magnitude in all test groups with surviving birds and in the control group. The initial feed consumption/bird/day was reduced - dose dependent - in the males and the females in the 250 and 500 mg/kg body weight groups. Nearly no feed was taken up by the male and female birds of the two highest test groups (1 000 and 2 000 mg/kg body weight).

Body weight: There was no statistically significant reduction in body weight gain in the surviving birds on day 7 and at the end of the study.

Gross post-mortem examination of surviving birds: No test material related effects.

Birds that had died (1 000 and 2 000 mg/kg): General congestive hyperemia.

Under the conditions of the study the NOEL was 125 mg/kg body weight. The LD50 for the bobwhite quail is > 500 and < 1 000 mg/kg wt.

 

Supporting Acute Toxicity Study: Grimes (1986)

The acute oral toxicity of the test material to birds was assessed according to Section 71-1 of the Environmental Protection Agency Registration Guidelines Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms and according to the principles of GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Groups of ten mallards, five males and five females, were assigned to each of the treatment groups and the control group by random draw. The test consisted of a geometric series of five dosage groups and a control group. Nominal dosages used in this study were 292, 486, 810, 1 350 and 2 250 mg of the test material/ kg of body weight. The control group was dosed with diluent only.

There were no mortalities at dosages of 292, 486, 810 and 1 350 mg/kg. There was 10 % mortality (1 of 10) at 2 250 mg/kg.

Under the conditions of the study the mallard acute oral LD50 value for the test material was determined to be > 486 mg/kg, the highest dosage at which no significant regurgitation was observed. The no-observed-effect dosage was < 292 mg/kg, the lowest dosage tested, based on overt signs of toxicity (regurgitation).

 

Supporting Dietary Study: Grimes (1986)

The toxicity of the test material to birds was assessed according to Section 71-2 of the Environmental Protection Agency Registration Guidelines Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms. and ASTM Standard E857-81 “Standard Practice for Conducting Subacute Dietary Toxicity Tests with Avian Species” and according to the principles of GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Groups of ten mallard ducklings were assigned to each of the treatment and control groups by random draw were acclimated from the day they were received until test initiation.  The test consisted of a geometric series of five test concentrations and three control groups. Nominal dietary concentrations used in this study were 562, 1 000, 1 780, 3 160 and 5 620 ppm.

Under the conditions of the study the dietary LC50 value of the test material in the mallard was determined to be > 5 620 ppm, the highest concentration tested.  The no-observed-effect concentration was determined to be 1 780 ppm.

 

Supporting Dietary Study: Munk (1986)

The toxicity of the test material to bobwhite quail was assessed according to EPA OPP 71-2. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was tested on chicks of the bobwhite quail by administration in the feed for five consecutive days of the concentrations of 0 (control), 313, 625, 1 250, 2 500 and 5 000 mg/kg diet. A post-exposure period of another 3 days followed this treatment.

Mortality: Highest concentration tested causing no test material-related mortality: 2 500 mg/kg diet

Minimum concentration causing 100 % mortality: > 5 000 mg/kg diet

LC50: The LC50 for the bobwhite quail (chicks) is about 5 000 mg/kg diet.

Clinical signs:  Besides sporadically occurring injuries to the beaks (cannibalism and fighting) which are not test material-related no pronounced toxic signs were observed except apathy in the surviving birds in the highest concentration (5 000 mg/kg).

Feed consumption: There was a reduction in feed consumption only in the highest dose group (5 000 mg/kg feed) during the test material-feeding period.

Body weight: There was no statistically significant reduction in body weight gain ,at the end of the exposure period (day 5) and at the end of post-exposure period (day 8).

Post-mortem examination: Surviving birds: No test material-related pathological findings

Birds that had died: No test material-related findings

Under the conditions of the study, the LC50 for the bobwhite quail (chicks) is about 5 000 mg/kg diet. The NOEC was determined to be 2 500 mg/kg diet.

 

 

 

 

LONG TERM

 

Key study: Long-Term Toxicity to Birds: Pagano et al. (2017) - Read across (MCPP-P)

A reproduction study in the mallard was carried out in accordance with the standardised guidelines OECD 206, EPA OPPTS 850.2300 and EPA OPP 71-4 under GLP conditions.The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of this study was to evaluate the effects of dietary exposure to the test material upon adult mallard (Anas platyrhynchos) over a five-month period. Effects on adult health, body weight, and feed consumption were evaluated. In addition, the effects of adult exposure to the test material on the number of eggs laid, fertility of the eggs, normal development of eggs including viability and survival of the embryos, hatchability, offspring survival and egg shell thickness were evaluated.

Mallard (72 males and 72 females) were randomly distributed into one control group and three treatment groups. Each treatment and control group contained 18 pairs of birds with one male and one female per pen. Three treatment groups were fed diets containing 80, 200, and 400 ppm a.i. of the test material for 21 weeks. The control group was fed diet comparable to the treatment groups, but without the addition of the test material.

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. Feed consumption was measured weekly throughout the test. At the beginning of Week 10, 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 an incubator configured for hatching and allowed to hatch. Once hatching was completed, hatchlings were removed from the hatcher and the individual body weights of the hatchlings were determined. At 14 days of age, the individual body weight of each surviving offspring was determined. Upon completion of the test, statistical analyses were performed to determine statistically significant differences among groups.

Under the conditions of this study there were no treatment-related mortalities, overt signs of toxicity or treatment-related effects upon body weight or feed consumption at any of the concentrations tested. Additionally, there were no treatment-related effects upon any of the reproductive parameters measured at the 80, 200 or 400 ppm a.i. test concentrations. The no-observed-effect concentration for mallard exposed to the test material in the diet during the study was 400 ppm a.i. (53.0 mg a.i./kg/day), the highest concentration tested.

 

 

 

 

Considering the very close structural similarity between the source and target substances (as justified in the read-across report that is attached to section 13 of the dossier), results from the study performed with mecoprop-p can be extrapolated to mecoprop.