1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

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:

04 Aug 1981 to 11 Feb 1982

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

6-month-old (approaching first breeding season) Mallard ducks (Anas platyrhynchos) were exposed to the test substance via dietary exposure for 11 weeks followed by 9 weeks of egg collection. After that, the laid eggs were collected and hatched. The mortality, bodyweight, toxic symptoms and reproduction parameters were recorded to study the effects. The NOEC was determined.

GLP compliance:

yes

Dose method:

feed

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

Corn oil

Details on preparation and analysis of diet:

The final diet was prepared as follows:
Concentrations A: 13.6078 grams test substance + 130 mL corn oil + 5866.39 grams Breeder Ration.
Concentrations B: 154.4311 grams test substance + 130 mL corn oil + 5825.57 grams Breeder Ration.
Concentrations C: 163.2933 grams test substance + 130 mL corn oil + 5716.71 grams Breeder Ration.
Concentrations D: 544.3108 grams test substance + 130 mL corn oil + 5335.69 grams Breeder Ration.
For each concentrate, the required amount of the test substance was first weighted on a balance and incorporated into the total amount of corn oil. The appropriate amount of game bird breeder ration was then weighed, and in the Hobart mixer. The test substance in corn oil was then added and the concentrate mixed for 20 minutes. The concentrates were divided into and frozen.
Weekly, the appropriate concentrates were removed from the freezer, and incorporated into the final diet as follows:
25 ppm: 500 qrams Concentrate A (1.3400 grams test substance) Q.S. 45.359 kg Breeder Ration
100 ppm: 500 grams Concentrate B ( 4.5359 grams test substance) Q.S. 45.359 kg Breeder Ration
300 ppm: 500 grams Concentrate C (13.6078 grams test substance) Q.S. 45.359 kg Breeder Ration
1000 ppm: 500 grams Concentrate B (45.3592 grams test substance) Q.S. 45.359 kg Breeder Ration
Each final diet was mixed for fifteen minutes in a blender. The basal diet contains 19.4% protein, 6.7% fat, and 3.8% fiber.
Samples of the control diet and each of the test diets were taken on Day 0 and Day 7 of Weeks 1, 8, and 19 of the study. Samples were frozen immediately after collection and shipped, on dry ice to the sponsor for analysis.

GC was used to analyse the diet samples.
GC Parameters:
- Column: 90-cm glass column
- Injection temperature: 240°C
- Column temperature: 210°C
- Retention time: 2.5 min

Test organisms (species):

Anas platyrhynchos

Details on test organisms:

- Common name: Mallard duck
- Age at initiation of study: 6 months (approaching first breeding season)
- Health condition: Disease-free, previously untreated

Limit test:

no

Total exposure duration (if not single dose):

11 wk

Remarks:

and 9 weeks egg collection

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

1 drake and 1 hen per pen, 12 pens per dose

Control animals:

yes, plain diet

Nominal and measured doses / concentrations:

0 (negative control), 25, 100, 300 and 1000 ppm

Details on test conditions:

PEN SIZE AND CONSTRUCTION MATERIALS
- Description: 72 X 90 X 33 cm in height (External walls were constructed of wire grid, while the ceilings and common walls were contructed of galvanized sheeting.)
- Floor covering: Constructed of 1'' x 1/2'' wire mesh rid coated with plastic to reduce egg breakage
- Caging: Group

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

NO. OF REPLICATES PER GROUP
- For control: 12
- For treated: 12

TEST CONDITIONS
- Temperature: 68 ± 8˚F (adult birds, 20 ± 4.4°C); 99.5 ± 0.1˚F (egg incubation, 37.5 ± 0.05°C)
- Relative humidity: 80% (adult birds); 85% (egg incubation)
- Photoperiod: 8 hours light per day for the first 7 weeks, 17 hours per day after 7 weeks until the end of the study
- Ventilation: Yes

Details on examinations and observations:

All adult birds were observed daily throughout the study, and a record was maintained of all mortalities, signs of toxicity or abnormal behavior. Body weights were recorded at initiation, on weeks 2, 4, 6, 8 and at the end of the study. Body weights were not recorded during egg laying because of the adverse effect handling has on egg production. Feed consumption was measured every two weeks throughout the study.

Details on reproductive parameters:

Eggs were collected daily and marked according to the pen from which they were taken. All eggs were candled before incubation to detect egg shell cracks, on day 14 of incubation to measure embryo viability and to remove any E.coli contamination eggs, and on day 21 to measure embryo survival. On day 25 of incubation, the eggs were placed in a hatcher and allowed to hatch. All hatchlings, unhatched eggs and egg shells were removed from the hatcher on Day 28 incubation, and the average body weight of the hatchling was determined. At 14 days of age, the ducklings were removed from the brooding units and the average body weight determined. Weekly throughout egg laying, when available, one egg from every other pen in each experimental group and the controls was randomly selected from egg weight and eggshell thickness measurement. Eggs were first weighed to the nearest one-tenth of a gram. The eggs were then opened at the waist and the contents were thoroughly washed out. The shells were then dried for one week at room temperature. The average thickness of the dried shell plus the membrane at the waist was determined.

Reference substance (positive control):

no

Key result

Duration (if not single dose):

20 wk

Dose descriptor:

NOEC

Effect level:

300 other: ppm

Conc. / dose based on:

test mat.

Basis for effect:

reproductive parameters

Remarks on result:

other: equivalent to 300 mg/kg diet

Mortality and sub-lethal effects:

An overview of the results is provided in Table 1- 5.
- Mortality: No mortality occurred in the control or any treatment group.
- Observation: No overt symptoms of toxicity were observed in any treatment group. All birds in the control and at all concentrations appeared normal throughout the study.
- Adult body weight and feed consumption: While a statistically significant difference in adult body weight (p <0.05) was observed at the 25 ppm treatment level during weeks 4, 6, and 8, and at the 100 ppm treatment level for all weight intervals, the differences observed do not appear to be biologically relevant. No statistically significant effect was observed on feed consumption at any treatment level.

Effects on reproduction:

An overview of the results is provided in Table 1- 5.
- Reproduction: The test substance cause no significant reproductive impairment at concentration levels up to 1000 ppm. At the 1000 ppm treatment level, there was a 21 percent reduction in the percent of viable embryos of eggs set when compared to the control group. This parameter was also at least twelve percent less than any other treatment group. Though not statistically significant, this effect is profound enough to be considered biologically significant.

Reported statistics and error estimates:

The statistical analysis of the data generated during the course of a One-Generation Reproduction Study conducted in mallard ducks with the test substance was conducted in two different ways. The analysis on the data consisting of body weight and other "measurement variable" is of the traditional analysis of variance type, partitioning variability into different sources. The analysis on the egg data and other "count variables" is based on Cochran's concept of extraneous variability for the binomial distribution. To facilitate the utilization of Cochran's analysis, which is based on percentage, raw numbers, such as the number of eggs laid per pen, must be converted to a percentage. These raw data are divided by a theoretical maximum, which , in the case of the eggs laid, is based on the hypothetical prediction of one egg per hen per day for the duration of the egg laying period.

The data were re-analysed in the report from 2014, to determine the EC10 and EC20 when possible, for the parameters adult bodyweight, adult food consumption, number of eggs laid, eggs cracked as a proportion of eggs laid, eggs set as a proportion of eggs not-cracked, viable embryos as a proportion of eggs set, live 3-week embryos as a proportion of those viable, normal hatchlings as a proportion of live 3-week embryos, 14-d survivors as a proportion of normal hatchlings, initial hatchling body weights, 14-d hatchling survivor body weights, egg shell thickness, and egg weight. However, no conclusively effect of the test substance at 10 and 20% effect levels were observed. Therefore, estimates of the EC10 and EC20 values based on nominal concentrations of the test substance could not be reliably calculated.

Table 1. Results of body weight and feed consumption-adult mallard ducks

	The test substance (ppm)
	Control		25		100		300		1000
Week	B.W.	F.C.	B.W.	F.C.	B.W.	F.C.	B.W.	F.C.	B.W.	F.C.
0	1146	-	1146	-	1116*	-	1143	-	1146	-
2	1114	39	1101	46	1066*	35	1147	48	1125	42
4	1109	69	1069*	80	105/*	75	1150	76	1126	71
5	1107	77	1071*	57	1057	61	1147	63	131	65
8	1124	78	1083*	84	1074*	77	1149	65	1139	76
10	-	75	-	84	-	74	-	68	-	72
12	-	95	-	102	-	114	-	100	-	94
14	-	109	-	114	-	126	-	112	-	119
16	-	147	-	139	-	153	-	147	-	134
18	-	179	-	134	-	163	-	147	-	142
20	1325	171	1303	158	1271*	169	1330	175	1306	153

B.W.: Body weight in grams. The body weight data are presented as a group mean.

F.C.: feed consumption in grams. The feed consumption data are presented as a group mean feed consumption per bird per day.

*Statistically significant difference (P < 0.05)

Table 2 reproduction data

		The test substance (ppm)
	Control	25	100	300	1000
Egg laid	424	308	461	475	392
Number hens laying	12	8	10	12	12
Eggs cracked	16	9	8	10	7
Egg set	369	272	410	421	347
Viable embryos	279	181	386	333	190
Live Three-week embryos	258	166	363	305	186
Normal Hatchlings	184	121	263	204	132
14-day old survivors	175	121	256	195	131
Egg laid/hen in 9 weeks^a	35	26	38	40	33
Egg laid/hen in 9 production	35	39	46	40	33
14-day old survivors/hen	15	10	21	16	11
14-day old survivors/hen in productions	15	15	26	16	11

a.Based on 12 hens

Table 3. Body weight

		The test substance (ppm)
	Control	25	100	300	1000
body weight data-hatching
No. of duckling weighted	183	118	260	198	132
Mean body weight (g)	35	36	36	38	38
bpdy weight data-14 dyas old survivors
No. of duckling weighted	175	121	256	195	131
Mean body weight (g)	226	245	226	229	213

Table 4 Egg weight

		The test substance (ppm)
	Control	25	100	300	1000
No. of eggs weighted	39	27	43	44	38
Mean egg weight (g)	54.8	57.6	58.0	57.8	60.7

Table 5 Eggshell thickness

		The test substance (ppm)
	Control	25	100	300	1000
No. of eggs measured	39	27	43	44	38
Mean shell thickness (mm)	0.376	0.381	0.377	0.373	0.373

 

Validity criteria fulfilled:

not specified

Conclusions:

In a one-generation reproduction study in birds, without following standard guideline but internal protocol, the 20-week NOEC for reproduction of Mallard duck was determined to be 300 ppm (equivalent to 300 mg/kg diet).

Executive summary:

In a GLP study without following standard guidelines but internal protocol, 6-month-old (approaching first breeding season) Mallard ducks (Anas platyrhynchos) were exposed to the test substance at concentrations of 0 (control, plain diet), 25, 100, 300 and 1000 ppm via dietary exposure for 11 weeks. After that, the laid eggs were collected and hatched. The egg collection period lasted for 9 weeks. The test substance was pre-mixed with corn oil and the birds’ diet. During the study, the birds were housed in 72 x 90 x 33 cm in height pens (one drake and one hen per pen, 12 pens per control/treatment group).  The test conditions were: temperature 68 ± 8 ˚F (for adult birds, 20 ± 4.4°C) and 99.5 ± 0.1 ˚F (for egg incubation, 37.5 ± 0.05°C), relative humidity 80% (for adult birds) and 85% (for egg incubation) and photoperiod 8 hours light per day for the first 7 weeks and 17 hours light per day afterwards. All adult birds were observed daily throughout the study, and a record of all mortalities, signs of toxicity or abnormal behaviour was maintained. Body weights were recorded at initiation, on weeks 2, 4, 6, 8 and at the end of the study. Body weights were not recorded during egg laying because of the adverse effect handling has on egg production. Feed consumption was measured every two weeks throughout the study. Eggs were collected daily and marked according to the pen from which they were taken. All eggs were candled before incubation to detect eggshell cracks. On day 25 for incubation, the eggs were placed in a hatcher and allowed to hatch. All hatchings, unhatched eggs and egg shells were removed from the hatcher on Day 28 incubation, and the average body weight of the hatchling was determined. At 14 days of age, the ducklings were removed from the brooding units, and the average body weight was determined. Weekly throughout egg laying, when available, one egg from every other pen in each experimental group and the controls was randomly selected from egg weight and eggshell thickness measurement. Eggs were first weighed to the nearest one-tenth of a gram. The eggs were then opened at the waist and the contents were thoroughly washed out. The shells were then dried for one week at room temperature. The average thickness of the dried shell plus the membrane at the waist was determined.

No mortality of the adult birds occurred in the control or any treatment groups. All adult birds in the control and at all concentrations appeared normal throughout the study. While a statistically significant difference in adult body weight (p <0.05) was observed at the 25 ppm treatment level during Weeks 4, 6, and 8, and at the 100 ppm treatment level for all weight intervals, the differences observed do not appear to be biologically relevant. No statistically significant effect was observed on feed consumption at any treatment level. The test substance causes no significant reproductive impairment at concentration levels up to 1000 ppm. At the 1000 ppm treatment level, there was a 21% reduction in the percent of viable embryos of eggs set when compared to the control group. This parameter was also at least 12% less than any other treatment group. Though not statistically significant, this effect is profound enough to be considered biologically significant. Based on the results, the 20-week NOEC for the reproduction of Mallard duck was determined to be 300 ppm (equivalent to 300 mg/kg diet). The EC10 and EC20 could not be determined.

Description of key information

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

20-wk, NOEC = 300 ppm (equivalent to 300 mg/kg diet), Anas platyrhynchos, reproduction, no standard guideline followed, Beavers 1982a

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for birds:

300 mg/kg food

Additional information

Eight acute and three chronic toxicity studies of the test substance are available for this endpoint. One of the chronic studies (GLP, no standard guideline followed, Reliability 1) on Mallard ducks (Anas platyrhynchos) was selected as the key study because it represents the worst-case effect (i.e. shows the lowest NOEC value). The birds were exposed to the test substance at concentrations of 0 (control, plain diet), 25, 100, 300 and 1000 ppm via dietary exposure for 11 weeks. After that, the laid eggs were collected and hatched. The egg collection period lasted for 9 weeks. Two birds were housed per pen (one drake and one hen, 12 pens per control/treatment group). The test conditions were: temperature 68 ± 8 ˚F (for adult birds, 20 ± 4.4°C ) and 99.5 ± 0.1 ˚F (for egg incubation, 37.5 ± 0.05°C), relative humidity 80% (for adult birds) and 85% (for egg incubation) and photoperiod of 8 hours light per day for the first seven weeks and 17 hours light per day afterwards. The 20-week NOEC for the reproduction of Mallard duck was determined to be 300 ppm (equivalent to 300 mg/kg diet) ), based on a reduction in the percent of viable embryos of eggs set (Beavers & Fink 1982a). Bobwhite quail (Colinus virginianus) showed less sensitivity to the test substance than mallard duck in the other two chronic studies performed without following standard guidelines (GLP, Reliability 1). The NOEC for Bobwhite quail was determined to be equal to or above 1000 mg/kg diet (Beavers & Fink 1982b and Beavers & Fink 1981).

The acute toxicity of the substance to Mallard ducks (Anas platrhynchos) was studied in two GLP studies without following standard guidelines (Reliability 2). It is concluded that the LD50 is above the highest tested concentrations in both studies (Fink & Beavers 1980b and Fink & Beavers 1980c). The same conclusion was found in the acute toxicity studies on Japanese quails and Pecking ducks (Anas domestic) (no standard guideline followed, performed before GLP adaptation, Reliability 2, Ullmann 1978a and Ullmann 1978b). But the concluded LD50 values may be incorrect due to birds partially refusing food in high-concentration treatment groups during the exposure period. Another study on Japanese quails shows that the LD50 was 2223 mg/kg body weight (no standard guidelines followed, not GLP, Reliability 2, Ullmann 1979). The LD50 of the substance for Bobwhite quail (Colinus virginianus) was determined to be 2825 mg/kg body weight and > 5620 mg/kg diet in two GLP studies without following standard guidelines (Fink & Beavers 1980a and Fink & Beavers 1980d, Reliability 2). Zebra finch (Taeniopygia guttata) is the most sensitive species to the acute toxicity of the substance in all tested birds. The LD50 was determined to be 759 mg/kg diet (EPA 850.2200, GLP, Hubbard 2018).