Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

Toxicity to birds

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to birds: reproduction test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Jul 2016 to 06 Feb 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 71-4 (Avian Reproduction Test)

Version / remarks:

1982

Qualifier:

according to guideline

Guideline:

OECD Guideline 206 (Avian Reproduction Test)

Version / remarks:

1984

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.2300 (Avian Reproduction Test)

Version / remarks:

1996

GLP compliance:

yes (incl. QA statement)

Dose method:

feed

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

Feed

Details on preparation and analysis of diet:

DIET PREPARATION
Test diets were prepared by mixing the test substance into a premix that was used for weekly preparation ofthe final diet. Control diet and each of the three treated diets were prepared weekly beginning on July 19, 2006 and presented to the birds on Wednesday of each week. Dietary concentrations were adjusted for purity of the test substance and are presented as parts per million active ingredient (ppm).

ANALYTICAL METHOD
Samples were extracted with acetonitrile. Concentrations of the test substance in extracts of the samples were determined by Gas Chromatograph (GC) equipped with a mass selective detector (MSD). Gas chromatographic separations were achieved using a ZB-5 column (30 m x 0.25 mm I.D., 0.25 μm film thickness).
Matrix-matched calibration standards of the test substance, ranging in concentration from 0.250 to 2.50 μg/mL, were analysed with each sample set. Linear regression equations were generated using the sum of the peak area responses of the two isomers, syn-isomer and anti-isomer, versus the respective concentrations of the calibration standards. The concentration of test substance in the samples was determined by substituting the sum of the peak area responses of the two isomers into the applicable linear regression equation.
The instrument limit of detection (LOD) was set based upon the injection volume (2 μL) and the lowest standard concentration 0.250 μg/mL. The LOD was set at 0.500 ng on-column. The method limit of quantitation (LOQ) for these analyses was set at 300 ppm based upon the lowest matrix fortification level analyzed concurrently with the samples. The 0.250 μg/mL standard was equivalent to a calculated value of 125 ppm in the matrix blank extract. Measured values greater than or equal to the ppm a.i. equivalent were reported. Along with the sample analyses, seven matrix blanks were analyzed to determine possible interferences. No interferences were observed at or above the ppm a.i. equivalent of the lowest standard during the sample analyses. Avian diet samples were fortified at 300 and 6000 ppm and analyzed concurrently with the samples to determine the mean procedural recovery. The method yielded mean procedural recoveries of 99%, 91%, 91%, 97%, 95%, 89%, and 102%. These values correspond to each sample set analyzed during the definitive study. Sample measured concentrations were not corrected for the mean procedural recoveries from each sample set.

GS PARAMETERs
- Column: ZB-5 (30 m x 0.25 mm, 0.25 μm film thickness)
- Injection temperature: 275°C
- Oven Initial temperatire: 65°C
- Oven initial hold time: 1.00 minute
- Oven ramp: 30°C/minute
- Oven final temperature: 320°C
- Oven final hold time: 7.50 minutes
- Detector temperature: 280°C
- Injection volume: 2 μL (Split less)

Test organisms (species):

Colinus virginianus

Details on test organisms:

TEST ORGANISM
- Common name: Northern bobwhite
- Age at test initiation: 25 weeks of age
- Weight at test initiation: 184 - 238 g
- Feeding: Diet formulated to the test facility specifications ad libitum during test. The basal ration contained at least 27% protein, 2.5% fat and no more than 3.8% crude fibre.
- Disease free: Yes, at the start of acclimation, the bobwhite were apparently healthy and phenotypically indistinguishable from wild type.
- Others: The birds were from the same hatch, approaching their first breeding season and had not been used in any previous testing.

Limit test:

no

Total exposure duration (if not single dose):

21 wk

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

1 male and 1 female / pen
16 pens per treatment group and control

Control animals:

yes, plain diet

Nominal and measured doses / concentrations:

- Nominal concentrations: 0 (control), 480, 1200 and 3000 mg/kg diet
- Measured concentrations: 476 ± 40.9, 1140 ± 31.5, and 2740 ± 208 mg/kg diet

Details on test conditions:

ACCLIMATION
- Acclimation period: Approximately 8 weeks
- Acclimation conditions: Same as test
- Health: At the start of acclimation, a random number generating function in a spreadsheet program was used to randomize 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.

PEN SIZE AND CONSTRUCTION MATERIALS
- Description: Batteries of pens; approximately 25 X 51 cm; The pens had sloping floors that resulted in ceiling height ranging from 20 to 26 cm. The pens were constructed of galvanized wire mesh and galvanized sheeting. Sisal ropes were added to each pen for animal enrichment.
- Compliant to good husbandry practices: Yes
- Suitable to avoid crowding stress: Yes
- Caging: Group (1 male and 1 female)

NO. OF BIRDS PER REPLICATE
- For negative control: 2
- For treated: 2

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

TEST CONDITIONS
- Room temperature: 22.6 ± 1.9°C
- Relative humidity: 57 ± 14%
- Photoperiod: The photoperiod during acclimation and the first seven weeks of the test was eight hours of light per day. The photoperiod was increased to 17 hours of light per day at the beginning of Week 8 to induce egg laying and was maintained at that length until the adult birds were euthanized.
- Light intensity: Throughout the test, the birds received a mean of approximately 395 lux (~ 37 ft. candles) of illumination provided by fluorescent lights that closely approximated noon-day sunlight.
- Ventilation: Vent up to 15 room air volumes every hour and replace them with fresh air.
- Feed and water: Each pen was equipped with feed and water troughs. Weekly, sufficient feed for the feeding period was placed in the trough for each pen and presented to the birds. During the feeding period additional feed was weighed and added to the troughs as needed. Water troughs were changed and water added as necessary to provide potable water (generally every 2 - 3 days).

Details on examinations and observations:

CLINICAL SIGNS
During the study, all adult birds were observed daily for signs of toxicity or abnormal behavior. 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
Adult body weights were measured at test initiation, at the end of Weeks 2, 4, 6, 8, and at adult termination. Body weights were not measured during egg laying because of the possible adverse effects handling may have on egg production.

FOOD CONSUMPTION
Feed consumption for each pen was measured weekly throughout the test. Feed consumption was determined by weighing the freshly filled feeder on Day 0, recording the amount of any additional diet added during the week and weighing the feeder and remaining feed at the end of the feeding period (Day 7). An attempt was made to minimize feed wastage by the birds by using externally mounted feeders designed with a “feed-saver” lip. Feed wastage was further reduced by placement of a piece of wire grid on the top of the feed. The wire grid allowed to birds to feed unencumbered, but prevented the birds from “scooping” feed out of the feeder. 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.

NECROPSY
Adult birds that died or were euthanized during the course of the study were subjected to a gross necropsy. At the conclusion of the exposure period, all surviving adult birds were euthanized with carbon dioxide gas, necropsied, and disposed of by incineration.

Details on reproductive parameters:

- Egg Collection and Storage: Eggs were collected daily from all pens, when available. The eggs were stored in a cold room until incubation. The cold room was maintained at a mean temperature of 14.1 ± 0.2°C (SD) with a mean relative humidity of approximately 81 ± 8% (SD). Groups of eggs were identified by an alphabetic lot code. All eggs laid in a weekly interval were considered as one lot.

- Shell cracks: 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 a egg-candling lamp to detect egg shell cracks or abnormal eggs. Cracked or abnormal eggs were recorded and discarded. All eggs to be incubated were fumigated with formaldehyde gas in an airtight cabinet with a circulating fan for approximately two hours, to reduce the possibility of pathogen contamination prior to incubation. Formaldehyde gas was generated by combining 20 g of potassium permanganate and 19 ml of 37% commercial grade formalin in a porcelain bowl at the base of the airtight cabinet.

- Incubation: All eggs not discarded or used for egg shell thickness measurements were placed in an incubator. In the incubator the temperature was maintained at an average 37.4 ± 0.1°C (SD) with an average relative humidity of 54 ± 1%. The incubator was equipped with a pulsator fan and blades that produced a mild breathing air movement designed to eliminate intracabinet temperature and humidity variation during incubation. In order to prevent adhesion of the embryo to the shell membrane, the incubator was also equipped with an automatic egg rotation device, designed to rotate the eggs from 45° off of vertical in one direction to 45° off of vertical in the opposite direction (total arc of rotation was 90°) every hour through Day 21 of incubation. Eggs were candled on Day 10 or 11 of incubation to determine embryo viability and on Day 21 to determine embryo survival.

- Hatching and Brooding: On Day 21 of incubation, the eggs were placed in a hatcher and allowed to hatch. Pedigree baskets constructed of galvanized 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 37.2 ± 0°C (SD), and the average wet bulb temperature of 33.3 ± 0°C (SD) (relative humidity of approximately 77%). All hatchlings, unhatched eggs, and egg shells were removed from the hatcher on Day 25 or 26 of incubation. The group body weight of the surviving hatchlings by pen was determined. Hatchlings were leg 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 average body weight by parental pen of all surviving chicks was determined. The chicks were euthanized with carbon dioxide and disposed of by incineration. Hatchlings were housed in batteries of brooding pens.Each pen measured approximately 72 X 90 X 23 cm high. The external walls and ceilings of each pen were constructed of galvanized wire mesh and galvanized sheeting. Floors were of galvanized 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 14 days of age. The average ambient room temperature was 25.7 ± 0.8°C (SD) with an average relative humidity of 28 ± 12% (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, etc.) and from each of the even numbered pens during the even numbered weeks (2,4,6, etc.). 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 micrometer. Measurements were made to the nearest 0.002 mm.

Reference substance (positive control):

no

Key result

Duration (if not single dose):

21 wk

Dose descriptor:

NOEC

Effect level:

480 mg/kg diet

Conc. / dose based on:

act. ingr.

Basis for effect:

reproductive parameters

Remarks on result:

other: equivalent to 32.5 mg/kg bodyweight/day

Mortality and sub-lethal effects:

An overview of the results is provided in Table 1 – Table 2 in ‘Any other information on results incl. tables’
- Mortalities and Necropsy: Four incidental mortalities occurred during the course of the study, one each in both the control group and the 480 ppm treatment group and two in the 1200 ppm treatment group. No mortalities occurred at the 3000 ppm treatment group. The female in Pen 413 of the control group was found dead on Day 6 of Week 18. Prior to being found dead, the bird was normal in appearance and behaviour. At necropsy, the bird had feather loss and bruising on the head, the liver was slightly pale with a hematoma on the left lobe and loose blood was present throughout the abdominal cavity. Necropsy of the bird’s penmate was unremarkable. The single mortality in the 480 ppm treatment group was the female in Pen 422 and was found dead on Day 6 of Week 14. Prior to death the bird was noted to have swelling of the left foot and lameness from Day 6 of Week 3 until death. At necropsy, the bird had a swollen left foot with a missing digit. Internally the heart and kidneys were pale, the liver friable and mottled, and loose blood was noted in the abdominal cavity and lower small intestines. The female’s ovary was developing. Necropsy of the hen’s penmate was unremarkable. The first mortality in the 1200 ppm treatment group was the female in Pen 444 and was found dead on Day 1 of Week 20. Prior to death the bird was noted to have slight wing droop, loss of coordination, reduced reaction to external stimuli, ruffled appearance and excreta that was yellow and watery. At necropsy the bird was thin, with a loss of muscle mass, prominent keel and bruising on the cranium. Internally, the liver was pale with white foci throughout and the abdomen was distended and contained yellow fluid and egg remnants. The female’s ovary was regressed. Necropsy of the bird’s penmate was unremarkable. The second mortality at the 1200 ppm test concentration was the female in Pen 443 and was found dead on Day 2 of Week 20. Prior to being found dead, the bird had foot and leg lesions. At necropsy the bird had feather loss, with feather picked wings and tail and old lesions on the legs and feet. The bird was thin, with a loss of muscle mass and prominent keel. The female’s kidneys were slightly pale. Necropsy of the female’s penmate was unremarkable. No other mortalities occurred during the course of the study. Due to the nature of the lesions observed at necropsy, none of the mortalities that occurred was considered to be related to treatment.

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

- Clinical Observations: No overt signs of toxicity were observed at any of the concentrations tested. Incidental clinical observations noted during the test included those that normally are associated with injuries and penwear. Such signs included feather loss, and lesions (foot, shoulder, head and back). Additional clinical signs observed included lameness, reduced reaction to external stimuli, wing droop, loss of coordination, ruffled appearance, lower limb weakness and lethargy. One female in the 480 ppm treatment group suffered a dislocated leg, which was set and bandaged and one male in the 3000 ppm treatment group had a wing fracture. Except for incidental findings, all birds appeared normal throughout the study.

- Adult Body Weight: There were no apparent treatment-related effects upon adult body weight at any of the concentrations tested. No statistically significant differences between the control group and the 480, 1200 or 3000 ppm treatment groups were observed at any of the body weight intervals.

- Adult Feed Consumption: There were no apparent treatment-related effects upon feed consumption at the 480 ppm test concentration and any differences between the 480 ppm treatment group and the control group were not statistically significant at any of the feed consumption intervals. At both the 1200 and 3000 ppm test concentrations there were slight, transient reductions in feed consumption during the first week of the study. In the 1200 ppm treatment group, the slight reduction during Week 1 was not statistically different from the control group, but the compensatory increase in feed consumption during Week 2 was statistically significant at p < 0.01. The slight reduction in feed consumption during Week 1 at the 3000 ppm test concentration was statistically different from the control group at p < 0.01. No statistically significant differences between the control group and the 1200 or 3000 ppm treatment groups were observed at any of the other feed consumption intervals. Given the transient nature of the differences observed, and their probable association with palatability/avoidance of the treated diet, the slight, transient reductions in feed consumption were not considered to be biologically meaningful.

Effects on reproduction:

An overview of the results is provided in Table 3 – Table 7 in ‘Any other information on results incl. tables’
- Reproductive Results: There were no treatment-related effects upon reproductive performance at the 480 ppm test concentration and differences from the control group were not statistically significant for any of the reproductive parameters measured. However, at the 1200 and 3000 ppm test concentrations, there were treatment related, concentration responsive reductions in 14-day old survivors as percentages of the numbers of hatchlings that were statistically significant at p < 0.05 and p < 0.01, respectively. While not statistically significant, there was an increase in cracked eggs as a percentage of eggs laid at the 3000 ppm test concentration. The increase was particularly notable in four pens (Pens 452, 455, 456 and 462) where the percentages of cracked eggs were 38, 12, 14 and 16%, respectively, and may have been related to treatment.

- Egg Shell Thickness: There were no apparent treatment related effects upon egg shell thickness at the 480 or 1200 ppm test concentrations and, when compared to the control group, no statistically significant differences. While not statistically significant, there was a slight reduction in the mean shell thickness value in the 3000 ppm treatment group. The slight reduction was reflective of a reduction in shell thickness that was confined primarily to four pen (Pens 452, 455, 456 and 462) where the mean shell thickness values were 0.186, 0.189, 0.192 and 0.187 mm, respectively. Given the correlation between shell thickness and the percentage of cracked eggs in those four pens, a treatment related effect could not be precluded.

- Offspring Body Weights: There were no apparent treatment-related effects upon offspring body weight at the 480 ppm test concentration. However, there were treatment-related reductions in both hatchling and 14-day old survivor body weights at the 1200 and 3000 ppm test concentrations that were statistically significant at p < 0.01.

Further details on results:

- Analytical Results: None of the control samples showed any indication of the presence of the test substance or of the presence of a co-eluting substance at the characteristic retention time of the test substance. Diet samples were collected from the 480, 1200 and 3000 ppm test concentrations, and were analysed to evaluate the homogeneity of the test substance in the diet. Means and standard deviations for the three test concentrations were 488 ± 16.0 ppm, 1190 ± 38.2 ppm and 2910 ± 197 ppm, respectively. The coefficients of variation were 3.28%, 3.21% and 6.79%, respectively. Samples collected during the test to verify test substance concentrations for the 480, 1200 and 3000 ppm a.i. diets had means and standard deviations of 476 ± 40.9 ppm 1140 ± 31.5 ppm and 2740 ± 208 ppm, respectively. The coefficients of variation were 8.60%, 2.77% and 7.60%. These values represented 99, 95 and 91% of nominal concentrations. Analysis of diet samples collected from feeders after being held at ambient temperature for 7 days averaged 92%, 97%, and 96% of the Day 0 values for the 480, 1200 and 3000 ppm test concentrations, respectively.

Table 1. Mean Adult Body Weight (g) from a Northern Bobwhite Reproduction Study with the test substance1

Experimental group (mg/kg diet)	sex	Week 0	Change week 0-2	Week 2	Change week 2-4	Week 4	Change week 4-6	Week 6	Change week 6-8	Week 8	Change week 8-Term	Test Term	Total change
Control	Male	207	4	211	6	216	2	218	4	222	0	223	15
	Female	207	5	213	4	217	2	218	1	219	33	252	45
480	Male	208	2	211	5	216	4	219	3	222	1	225	15
	Female	204	3	208	0	207	3	210	-2	209	32	243	40
1200	Male	203	4	207	6	213	1	214	4	218	0	217	14
	Female	209	2	211	5	216	1	217	2	219	13	233	23
3000	Male	206	1	207	4	211	3	214	5	219	2	221	14
	Female	201	1	202	3	206	1	207	3	210	29	239	38

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

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

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

Table 2. Mean Feed Consumption (g/bird/day) from a Northern Bobwhite Reproduction Study with the test substance

Experimental group (mg/kg diet)	Weeks
	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Control	14	14	15	14	15	14	15	14	15	17	17	17	19	21	23	24	24	24	25	25	23
480	14	14	14	13	15	13	15	14	15	16	18	18	19	21	23	24	23	24	25	25	24
1200	13	16**	15	14	15	14	15	14	15	17	17	17	18	21	22	23	24	23	24	25	23
3000	13 **	14	14	14	14	13	16	14	15	17	18	18	20	22	23	24	23	23	24	24	23

**Significantly different from the control at p < 0.01

Table 3. Summary of Reproductive Performance from a Northern Bobwhite Reproduction Study with the test substance

Reproductive parameter	Experimental group (mg/kg diet)
	Control	480	1200	3000
Number of Replicates	15	15	14	16
Total Eggs Laid	740	684	676	767
Eggs Cracked	21	19	20	60
Eggs Set	635	586	590	606
Viable Embryos	590	556	539	557
Live 3-Week Embryos	574	552	538	549
Hatchlings	559	530	514	527
14-Day Old Survivors	547	518	484	483
Eggs Laid/Hen	49	46	48	48
Eggs Laid/Hen/Day1	0.50	0.47	0.49	0.49
14-Day Old Survivors/Hen	36	35	35	30

1. Based on 98 days of eggs production.

Table 4. Summary of Reproductive Performance, Normalized as Percentages(%) from a Northern Bobwhite Reproduction Study with the test substance

Reproductive parameter	Experimental group (mg/kg diet)
	Control	480	1200	3000
Number of Replicates	15	15	14	16
Total Eggs Laid 2	740	684	676	767
Eggs Laid/MaximumLaid (%)	76	70	74	74
Eggs Cracked/Eggs Laid (%)	3	3	3	8
Viable Embryos/Eggs Set (%)	93	93	92	89
Live 3-Week Embryos/Viable Embryos (%)	98	99	100	97
Hatchlings/Live3-Week Embryos (%)	97	95	96	96
14-Day Old Survivors/Hatchlings (%)	98	98	93*	91**
Hatchlings/Eggs Set (%)	89	88	89	83
14-Day Old Survivors/Eggs Set (%)	87	86	83	76
Hatchlings/Maximum Set (%)	63	60	62	56
14-Day Old Survivors/Maximum Set (%)	62	59	59	51

* Significantly different from the control at p < 0.05

* * Significantly different from the control at p < 0.01

1 Values represent pen means for experimental group.

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

  Table 5. Mean Eggshell Thickness Measurements (mm) from a Northern Bobwhite Reproduction Study with the test substance

Experimental group (mg/kg diet)	No. egg measured	Shell thickness Mean±SD
Control	71	0.223 ± 0.015
480	67	0.222 ± 0.013
1200	62	0.222 ± 0.013
3000	74	0.217 ± 0.019

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

 

Table 6.Mean Body Weight (g) of Hatchlings and 14-Day Old Survivors from a Northern Bobwhite Reproduction Study with the test substance

Experimental group (mg/kg diet)	Hatchings		14-day old survivors
	Number	Mean±SD	Number	Mean±SD
Control	557	6.5 ± 0.6	547	31. 6 ± 2.4
480	529	6.2 ± 0.3	518	30.1 ± 2.3
1200	512	6.0** ± 0.3	484	29.0** ± 2.1
3000	523	5.6** ± 0.4	483	27.7** ± 2.3

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

**Significantly different from the control at p < 0.01

Table 7. Summary of Gross Pathological Observations from a Northern Bobwhite Reproduction Study with the test substance (Birds Euthanized at Test Termination)

	Males - Experimental group (mg/kg diet)				Females - Experimental group (mg/kg diet)
	Control	480	1200	3000	Control	480	1200	3000
Number of birds	15	15	14	16	15	15	14	16
External - feather loss	0	0	0	1	0	0	2	I
External - foot or leg lesions	0	0		0	2	0	4	0
External - left leg twisted	0	0	0	0	0	1	0	0
External - lesions on back, rump and/or shoulder	0	0	0	0	0	0	2	0
External - scarring on head	0	0	0	0	0	0	0
External - small growth over left eye	0	0	0	0	0	0	0	1
General condition - emaciated, prominent keel	0	0	0	0	0	0	1	0
General condition - loss of muscle mass	0	0	0	0	0	0	1	0
Musculature/skeletal - fused left knee dislocation	0	0	0	0	0	1	0	0
Liver - friable	0	1	0	0	0	0	1
GI tract - areas of hyperemia in small intestines	1	0		3	0	0	0	0
Reproductive - ovary regressing	-	-	-	-	0	1	0	0
Reproductive - ovary regressed	-	-	-	-	0	0	2	0
Reproductive - testes small, _:s 1.75 cm	1	1	0	1	-	-	-	-
Not remarkable	13	13	12	11	13	13	8	13

Validity criteria fulfilled:

yes

Conclusions:

In a reproduction toxicity study in birds, performed with northern bobwhite and in accordance with OECD TG 206, the 21-wk NOEC was determined to be 480 mg/kg diet, equivalent to a daily dietary dose of 32.5 mg/kg bw/day.

Executive summary:

In this study the effects of the test substance on reproduction in the northern bobwhite (Colinus virginianus) were investigated. The study was performed according to OECD TG 206 and in compliance with GLP criteria. Three treated groups of 16 replicates, each of one male and one female, were offered test diet containing 480, 1200 and 3000 mg/kg diet for 21 weeks. The estimated dietary doses for these test concentrations were 32.5, 83.5 and 207.4 mg/kg bodyweight/day respectively. A similar sized control group was given untreated diet only. The birds (approximately 25 weeks old) were housed in pens. All adult birds and their offspring were given feed and water ad libitum during acclimation and testing. The mean temperature and humidity (measured once daily) in the test room were 22.6 ˚C and 57%, respectively. The photoperiod during acclimation and the first seven weeks of the test was eight hours of light per day. The photoperiod was increased to 17 hours of light per day at the beginning of Week 8 to induce egg laying and was maintained at that length until the adult birds were euthanized. Throughout the test, the birds received a mean of approximately 395 lux (~ 37 ft. candles) of illumination provided by fluorescent lights that closely approximated noon-day sunlight. All birds were observed daily throughout the test for signs of toxicity or abnormal behaviour. Adult body weights were measured at test initiation, at weeks 2, 4, 6 and 8, and at adult termination. Adult feed consumption was measured weekly throughout the test. Eggs were collected daily from pens when available and stored in a cold room until incubation. At the end of the weekly interval, eggs were selected for egg shell thickness measurement. All remaining eggs were candled prior to incubation to detect egg shell cracks or abnormal eggs, which were discarded. Eggs were then placed in an incubator at an average temperature of 37.4 ˚C. Eggs were candled twice during incubation to detect infertile eggs or embryo mortality. On day 21 of incubation, the eggs were placed in a hatcher. Once hatching was completed, hatchlings were removed and the group body weight of the surviving hatchlings by pen was determined. At 14 days of age, the average body weight by parental pen of all surviving offspring was determined. All adult birds were subjected to a gross necropsy. This included birds found dead or euthanized during the study, and all surviving adults sacrificed at test termination.

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 480 mg/kg diet treatment. However, at the 1200 and 3000 mg/kg diet treatments there were treatment-related, concentration responsive, reductions in offspring survival (14-day old survivors as a percentages of hatchlings) and both hatchling and 14-day old survivor body weights. Based on the findings, the 21-wk NOEC was determined to be 480 mg/kg diet, equivalent to a daily dietary dose of 32.5 mg/kg bw/day.

 

Description of key information

All available data was assessed and the study representing the worst-case effect was included as key study. Other studies are included as supporting information. The effect value from the key study was selected for the CSA.

21-wk, NOEC = 480 mg/kg diet (equivalent to 32.5 mg/kg bw/day), Colinus virginianus, reproduction, OECD TG 206, Temple 2007

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for birds:

480 mg/kg food

Additional information

Four acute and two chronic toxicity studies of the test substance on birds are available. They all followed standard test guidelines and complied with GLP. One of the chronic studies on northern bobwhite (Colinus virginianus) was selected as key study and its effect value was used as key value. This study was selected as the key study because a long-term toxicity test is preferred for this endpoint, according to Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7c: Endpoint specific guidance (version June 2017). It represents the worst-case effect (i.e. shows the lowest NOEC value in daily dietary dose). Three treated groups of 16 replicates, each of one male and one female, were offered test diet containing 480, 1200 and 3000 mg/kg diet for 21 weeks. The estimated dietary doses for these test concentrations were 32.5, 83.5 and 207.4 mg/kg bodyweight/day respectively. A similar sized control group was given untreated diet only. The birds (approximately 25 weeks old) were housed in pens. The mean temperature and humidity (measured once daily) in the test room were 22.6 ˚C and 57%, respectively. The photoperiod during acclimation and the first seven weeks of the test was eight hours of light per day. The photoperiod was increased to 17 hours of light per day at the beginning of Week 8 to induce egg laying and was maintained at that length until the adult birds were euthanized. Throughout the test, the birds received a mean of approximately 395 lux (~ 37 ft. candles) of illumination provided by fluorescent lights that closely approximated noon-day sunlight. The results show that 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 480mg/kg diet treatment. Based on the findings, the 21-wk NOEC was determined to be 480 mg/kg diet (equivalent to 32.5 mg/kg bw/day) for reproduction parameters (Temple 2007a).

Another chronic study is a 21 -wk reproduction test on mallard duck (Anas platyrhynchos). The test organisms were exposed to nominal test concentrations of 0, 480, 1 200 and 3 000 mg/kg diet (corresponding to estimated dietary doses of 0, 66, 167.6 and 369 mg/kg bodyweight/day, respectively). The 21-wk NOEC was determined to be 3000 mg/kg diet (equivalent to 369 mg/kg bodyweight/day) for reproduction parameters (Temple 2007b).

Two of the acute toxicity tests were performed on northern bobwhite (Colinus virginianus) and the other two acute tests were performed on mallard duck (Anas platyrhynchos) and chicken (Gallus gallus). The northern bobwhite was exposed to the test substance at a single dose, 2000 mg/kg bodyweight (Gallagher & Beavers 2005) or at nominal dietary concentrations of 0 (control), 562, 1000, 1780, 3160 and 5620 mg/kg diet (Gallagher & Beavers 2006a). The 14-d LD50 was determined to be > 2000 mg/kg diet (Gallagher & Beavers 2005). The 8-d LC50 was > 5620 mg/kg diet (equivalent to > 1310 mg/kg bw/day) (Gallagher & Beavers 2006a). The mallard duck was exposed to the test substance at concentrations of 0 (control), 562, 1000, 1780, 3160 and 5620 mg/kg diet. The 8-dLC50 was > 5620 mg/kg diet (equivalent to > 2435 mg/kg bw/day) (Gallagher & Beavers 2006b). The chicken was exposed to the test substance at a single oral dose of 0 (control) and 2000 mg/kg bw. The 14-d LD50 was determined to be > 2000 mg/kg bw (Hubbard, Davis & Temple 2018).