pymetrozine (ISO); (E)-4,5-dihydro-6-methyl-4-(3-pyridylmethyleneamino)-1,2,4-triazin-3(2H)-one

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

27 Jan 1992 to 21 Oct 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

May 1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Version / remarks:

1982

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese MAFF 59 NohSan No. 4200

Version / remarks:

January 1985

Deviations:

no

GLP compliance:

yes

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
- Premating exposure duration for parental (P0) animals: 10 weeks
- Premating exposure duration for P1 animals: weaning (indirect) and 10 weeks direct dosing.
- Basis for dose level selection: The dietary dose concentrations were selected for this study based on a previous rangefinding study
- Route of administration : Oral, via feed.

Test material

Test material information

Constituent 1

Reference substance name:

6-methyl-4-[(E)-[(pyridin-3-yl)methylidene]amino]-2,3,4,5-tetrahydro-1,2,4-triazin-3-one

EC Number:

602-927-1

Cas Number:

123312-89-0

Molecular formula:

C10H11N5O

IUPAC Name:

6-methyl-4-[(E)-[(pyridin-3-yl)methylidene]amino]-2,3,4,5-tetrahydro-1,2,4-triazin-3-one

Test material form:

solid: particulate/powder

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

histopathology: non-neoplastic

Remarks on result:

other: equivalent to 13.92 and 15.98 mg/kg bw/day for males and females, respectively

2

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

>= 2 000 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: P1 (second parental generation)

Effect levels (P1)

1

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: equivalent to 13.92 and 15.98 mg/kg bw/day for males and females, respectively

2

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

>= 2 000 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P1)

Critical effects observed:

no

Results: F1 generation

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

other: Delayed eye opening

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

other: Delayed eye opening

Target system / organ toxicity (F2)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

2 000 ppm

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Test article: Analysis of the samples of the test article in the diet were conducted on samples mixed on January 23. The results confirmed the stability of the mixes for at least 4 weeks and the homogeneity of the preparations at all concentrations.

In the F0 generation mean daily test substance intakes were approximately 1 to 4, 10 to 40, and 110 to 440 mg/kg bw/day at the feeding levels of 20, 200, and 2000 ppm, respectively.

F0 - parents: There were no treatment - related mortalities or clinical signs in parent animals. Body weights were about 10 % lower than control at 2000 ppm in both sexes from the second week of treatment onwards. Feed consumption was reduced at 2000 ppm in both sexes except in females during the lactation period.

Table 1. Reproductive performance F0 generation

Feeding level	0 ppm		20 ppm		200 ppm		2000 ppm
Males:
- Used for mating [n] - Mating index^a[%] - Fertility index^b[%]	30	96.7 82.8	30	86.7 80.8	30	90.0 92.6	30	96.7 96.6
Females:
- Used for mating [n]	30 29 24 23   96.7 82.8 95.8 95.8		30 26 21 21   86.7 80.8 100 100		30 27 25 24   90 92.6 96 96		30 29 28 27   96.7 96.6 96.4 96.4
- Mated [n]
- Pregnant [n]
- With live born pups [n]
- Mating index^a[%]
- Fertility index^b[%]
- Gestation index^c[%]
- Parturition index^d[%]
- Gestation length [days]	22.2		22.3		22.4		22.1
	± 0.5		±0.5		±0.5		±0.4
- Precoital interval [days]	3.7		4.2		4.3		4.8
	±3.2		±2.0		±2.7		±3.0
- Implantation sites	14.8		15.4		14.5		13.2
	±3.8		±2.2		±2.9		±2.9

a = Percent animals positively mated (females) or producing positive mating (males)

b = Percent of females pregnant / percent of males producing pregnancy

c = Percentage of females with confirmed pregnancy that resulted in the birth of live pups d

d = Percentage of females with confirmed pregnancy that delivered pups

 

Table 2. Incidences of microscopical lesions in P0 generation

Sex	Males				Females
Feeding level, ppm	0	20	200	2000	0	20	200	2000
Liver / Total examined	30	30	30	30	30	30	30	30
Hypertrophy	0	0	5	27	0	0	0	2
Spleen / Total examined	30	0	30	30	30	0	30	30
Hyperplasia of lymphatic follicles	0	-	0	0	0	0	0	25

 

Mating, gestation, fertility, and parturition indices were not affected by treatment (Table 1). Likewise, numbers of stillborn pups as well as livebirth, viability, and lactation indices were not affected.

At parental necropsy, females at 2000 ppm had higher absolute and relative liver and spleen weights than controls. No treatment-related macroscopical changes were detected.

Microscopic histopathology revealed a minimal hepatocellular hypertrophy in most males and 2 out of 30 females at 2000 ppm and in few males at 200 ppm (Table 2). Additionally, minimal to moderate hyperplasia of lymphatic follicles of splenic white pulp was observed in most females at 2000 ppm.

F1 - pups: No treatment-related clinical signs in the pups were reported (Table 3).

Litter weights of the F1 generation were reduced at 2000 ppm from the second week of lactation onwards.

Eye opening was minimally delayed (by about 0.4 days) at 2000 ppm compared to controls.

 

Table 3. Litter data of the F1 generation

Feeding level	0 ppm	20 ppm	200 ppm	2000 ppm
Parameter
Number of litters	23 f	21	24	27
Total pups born	292 f	299	309	316
Mean per litter	12.7 d	14.2	12.9	11.7
Number of stillborn pups	3 f	2	12*	6+
Sex ratio (% females day 0)	46.7	53.5	52.5	51.0
Mean number of pups alive
- on day 0	12.6 f	14.1	12.4	11.5
- on day 4 (post culling)	7.9 f	8.0	7.9	7.5
- on day 7	7.8 f	8.0	7.9	7.4
- on day 14	7.5 f	7.9	7.7	7.4
- on day 21	7.4 f	7.8	7.6	7.4
Viability Index^aLactation Index^b	97.6 f 93.7 f	98.0 97.6	97.0 96.1	97.1 98.5
Mean pup weight
- on day 0	6.2 d	6.1	6.6	6.6
- on day 4 precull	9.9 d	9.5	10.4	10.0
- on day4 postcull	10.0 d	9.6	10.4	10.1
- on day 7	16.5 d	15.9	16.9	15.9
- on day 14	33.9 d	32.8	33.7	31.1*
- on day 21	57.4 d	56.2	58.9	52.8*

a: % Pups surviving days 0 to 4; b: % Pups surviving days 4 to 21;

d = ANOVA + Dunnett-test; f = chi-square + Fishers exact: * significant at p<0.05; p = 0.508

 

Table 4. Reproductive performance F1 generation

Feeding level	0 ppm	20 ppm	200 ppm	2000 ppm
Males:
- Used for mating [n] - Mating index^a[%] - Fertility index^b[%]	30 80 91.7	29 86.7 92.3	30 83.3 96	30 96.7 100
Females:
- Used for mating [n]	30 24 22 21   80 91.7 95.5 95.5	30 26 24 24   86.7 92.3 100 100	30 25 24 24   83.3 96 100 100	30 29 29 29   96.7 100 100 100
- Mated [n]
- Pregnant [n]
- With live born pups [n]
- Mating index^a[%]
- Fertility index^b[%]
- Gestation index^c[%]
- Parturition index^d[%]
- Gestation length [days]	22.3 ± 0.5	22.2 ±0.4	22.2 ±0.4	22.2 ±0.4
- Precoital interval [days]	6.2 ±3.8	6.0 ±5.5	7.0 ±5.3	5.3 ±4.2
- Implantation sites	16.0 ±2.7	14.8 ±4.7	15.5 ±2.2	14.3 ±3.8

a = Percent animals positively mated (females) or producing positive mating (males)

b = Percent of females pregnant / percent of males producing pregnancy

c = Percentage of females with confirmed pregnancy that resulted in the birth of live pups

d = Percentage of females with confirmed pregnancy that delivered pups

 

F1 - parents: In the F1 generation mean daily test substance intakes were similar to the F0 generation. There were no treatment-related mortalities or clinical signs in parent animals. Body weights were about 15 % lower than control at 2000 ppm in both sexes from the start of treatment onwards. Feed consumption was reduced at 2000 ppm in both sexes.

As shown in Table 4, mating, gestation, fertility, and parturition indices were not affected by treatment. Likewise, numbers of stillborn pups as well as livebirth, viability, and lactation indices were not affected.

At parental necropsy, females at 2000 ppm had higher relative liver and spleen weights than controls. At 200 ppm., relative liver weights were increased by approx. 10 %. No treatment- related macroscopical changes were detected. As shown in Table 5, microscopic histopathology revealed a minimal hepatocellular hypertrophy in males and females at 2000 ppm and minimal to moderate hyperplasia of basophilic cells of the adenohypophysis in males at 2000 ppm. A slight increase of hepatocellular hypertrophy was noted in males treated with 200 ppm.

Table 5. Incidences of microscopical lesions in F1 parents

Sex	Males				Females
Feeding level, ppm	0	20	200	2000	0	20	200	2000
Liver / Total examined	30	30	30	30	30	30	30	30
Hypertrophy	0	0	2	26	0	0	0	10
Pituitary / Total examined	30	30	30	30	30	30	30	30
hypertropy of basophilic cells	7	8	7	17	0	0	0	0

 

F2 - pups: As shown in Table 6, there were no treatment-related clinical signs in the pups. Litter weights of the F1 generation were reduced at 2000 ppm from the end of the first week of lactation onwards. Eye opening was minimally delayed (by about 0.5 days) at 2000 ppm compared to controls.

 

Table 6. Litter data of the F2 generation

Feeding level	0 ppm	20 ppm	200 ppm	2000 ppm
Parameter
Number of litters	21	24	24	29
Total pups born	302	310	355	393
Mean per litter	14.4	12.9	14.8	13.6
Number of stillborn pups	3	4	1	2
Sex ratio (% females day 0)	48.2	50.3	50.6	54.5
Mean number of pups alive
- on day 0	14.2	12.8	14.8	13.5
- on day 4 (post culling)	8.0	7.5	8.0	7.7
- on day 7	8.0	7.5	8.0	7.6
- on day 14	7.9	7.5	7.9	7.4
- on day 21	7.9	7.5	7.9	7.4
Viability Index (a) Lactation Index (b)	95.3 98.8	97.4 98.9	97.2 98.4	98.7 96.9
Mean pup weight
- on day 0	6.1	6.1	6.0	6.3
- on day 4 precull	9.6	9.8	9.1	9.1
- on day 4 postcull	9.7	10	9.3	9.3
- on day 7	16.0	15.5	15.3	14.7*
- on day 14	31.3	30.5	30.5	28.9**
- on day 21	52.2	49.9	49.6	45.7**

(a): % Pups surviving days 0 to 4; (b): % Pups surviving days 4 to 21;

*: significant at p<0.05; **: significant at p<0.01

Applicant's summary and conclusion

Conclusions:

It is concluded that reproductive parameters including gonadal function, mating behaviour, conception, parturition, lactation and weaning, as well as sex organ histopathology were not affected in this study.
The dose of 200 ppm was a NOAEL for parents and offspring. At 2000 ppm, parental and offspring body weights were reduced, and eye opening was slightly delayed in pups, and histopathology of adults revealed changes in liver, spleen, and the pituitary.
At 200 ppm, a marginal increased incidence of minimal liver hypertrophy was observed in few parental males (17 %), but this finding was not correlated with any liver weight increase at this dose level and therefore not considered as toxicological relevant. 

Executive summary:

Ten weeks after initiation of exposure to the test material at dietary levels of 0, 20, 200 or 2000 ppm the Tif:RAIf (SPF) rats (30 animals per sex and dose level) were paired. Parents were mated 1:1 until positive mating occurred or for 19 days, whichever came first. After weaning and a premating period of 10 weeks, F1 animals were mated to produce the F2 generation. The animals were continuously exposed to the test substance admixed to feed in two successive generations (F0 and F1). Dams were allowed to litter and suckle naturally. Litters were culled to 4 male and 4 female pups, where possible, on day 4 post partum. Clinical signs, body weights, feed consumption, mating parameters, gestation and delivery parameters, pup survival, and physical and behavioural development (surface righting and eye opening) were recorded. A gross necropsy examination was performed on all pups not selected for mating. All parental animals were necropsied after weaning of their offspring and subjected to pathological examination. Histopathology was performed on the sexual organs and the apparent target organs liver and spleen (as identified by organ weight changes).

Test article: Analysis of the samples of the test article in the diet were conducted on samples mixed on January 23. The results confirmed the stability of the mixes for at least 4 weeks and the homogeneity of the preparations at all concentrations.

Test article: Analysis of the samples of the test article in the diet were conducted on samples mixed on January 23. The results confirmed the stability of the mixes for at least 4 weeks and the homogeneity of the preparations at all concentrations.

In the F0 generation mean daily test substance intakes were approximately 1 to 4, 10 to 40, and 110 to 440 mg/kg bw/day at the feeding levels of 20, 200, and 2000 ppm, respectively.

F0 - parents: There were no treatment - related mortalities or clinical signs in parent animals. Body weights were about 10 % lower than control at 2000 ppm in both sexes from the second week of treatment onwards. Feed consumption was reduced at 2000 ppm in both sexes except in females during the lactation period.

Table 1. Reproductive performance F0 generation

Feeding level	0 ppm		20 ppm		200 ppm		2000 ppm
Males:
- Used for mating [n] - Mating index^a[%] - Fertility index^b[%]	30	96.7 82.8	30	86.7 80.8	30	90.0 92.6	30	96.7 96.6
Females:
- Used for mating [n]	30 29 24 23   96.7 82.8 95.8 95.8		30 26 21 21   86.7 80.8 100 100		30 27 25 24   90 92.6 96 96		30 29 28 27   96.7 96.6 96.4 96.4
- Mated [n]
- Pregnant [n]
- With live born pups [n]
- Mating index^a[%]
- Fertility index^b[%]
- Gestation index^c[%]
- Parturition index^d[%]
- Gestation length [days]	22.2		22.3		22.4		22.1
	± 0.5		±0.5		±0.5		±0.4
- Precoital interval [days]	3.7		4.2		4.3		4.8
	±3.2		±2.0		±2.7		±3.0
- Implantation sites	14.8		15.4		14.5		13.2
	±3.8		±2.2		±2.9		±2.9

a = Percent animals positively mated (females) or producing positive mating (males)

b = Percent of females pregnant / percent of males producing pregnancy

c = Percentage of females with confirmed pregnancy that resulted in the birth of live pups d

d = Percentage of females with confirmed pregnancy that delivered pups

 

Table 2. Incidences of microscoppical lesions in P0 generation

Sex	Males				Females
Feeding level, ppm	0	20	200	2000	0	20	200	2000
Liver / Total examined	30	30	30	30	30	30	30	30
Hypertrophy	0	0	5	27	0	0	0	2
Spleen / Total examined	30	0	30	30	30	0	30	30
Hyperplasia of lymphatic follicles	0	-	0	0	0	0	0	25

 

Mating, gestation, fertility, and parturition indices were not affected by treatment (Table 1). Likewise, numbers of stillborn pups as well as livebirth, viability, and lactation indices were not affected.

At parental necropsy, females at 2000 ppm had higher absolute and relative liver and spleen weights than controls. No treatment-related macroscopical changes were detected.

Microscopic histopathology revealed a minimal hepatocellular hypertrophy in most males and 2 out of 30 females at 2000 ppm and in few males at 200 ppm (Table 2). Additionally, minimal to moderate hyperplasia of lymphatic follicles of splenic white pulp was observed in most females at 2000 ppm.

F1 - pups: No treatment-related clinical signs in the pups were reported (Table 3).

Litter weights of the F1 generation were reduced at 2000 ppm from the second week of lactation onwards.

Eye opening was minimally delayed (by about 0.4 days) at 2000 ppm compared to controls.

 

Table 3. Litter data of the F1 generation

Feeding level	0 ppm	20 ppm	200 ppm	2000 ppm
Parameter
Number of litters	23 f	21	24	27
Total pups born	292 f	299	309	316
Mean per litter	12.7 d	14.2	12.9	11.7
Number of stillborn pups	3 f	2	12*	6+
Sex ratio (% females day 0)	46.7	53.5	52.5	51.0
Mean number of pups alive
- on day 0	12.6 f	14.1	12.4	11.5
- on day 4 (post culling)	7.9 f	8.0	7.9	7.5
- on day 7	7.8 f	8.0	7.9	7.4
- on day 14	7.5 f	7.9	7.7	7.4
- on day 21	7.4 f	7.8	7.6	7.4
Viability Index^aLactation Index^b	97.6 f 93.7 f	98.0 97.6	97.0 96.1	97.1 98.5
Mean pup weight
- on day 0	6.2 d	6.1	6.6	6.6
- on day 4 precull	9.9 d	9.5	10.4	10.0
- on day4 postcull	10.0 d	9.6	10.4	10.1
- on day 7	16.5 d	15.9	16.9	15.9
- on day 14	33.9 d	32.8	33.7	31.1*
- on day 21	57.4 d	56.2	58.9	52.8*

a: % Pups surviving days 0 to 4; b: % Pups surviving days 4 to 21;

d = ANOVA + Dunnett-test; f = chi-square + Fishers exact: * significant at p<0.05; p = 0.508

 

Table 4. Reproductive performance F1 generation

Feeding level	0 ppm	20 ppm	200 ppm	2000 ppm
Males:
- Used for mating [n] - Mating index^a[%] - Fertility index^b[%]	30 80 91.7	29 86.7 92.3	30 83.3 96	30 96.7 100
Females:
- Used for mating [n]	30 24 22 21   80 91.7 95.5 95.5	30 26 24 24   86.7 92.3 100 100	30 25 24 24   83.3 96 100 100	30 29 29 29   96.7 100 100 100
- Mated [n]
- Pregnant [n]
- With live born pups [n]
- Mating index^a[%]
- Fertility index^b[%]
- Gestation index^c[%]
- Parturition index^d[%]
- Gestation length [days]	22.3 ± 0.5	22.2 ±0.4	22.2 ±0.4	22.2 ±0.4
- Precoital interval [days]	6.2 ±3.8	6.0 ±5.5	7.0 ±5.3	5.3 ±4.2
- Implantation sites	16.0 ±2.7	14.8 ±4.7	15.5 ±2.2	14.3 ±3.8

a = Percent animals positively mated (females) or producing positive mating (males)

b = Percent of females pregnant / percent of males producing pregnancy

c = Percentage of females with confirmed pregnancy that resulted in the birth of live pups

d = Percentage of females with confirmed pregnancy that delivered pups

 

F1 - parents: In the F1 generation mean daily test substance intakes were similar to the F0 generation. There were no treatment-related mortalities or clinical signs in parent animals. Body weights were about 15 % lower than control at 2000 ppm in both sexes from the start of treatment onwards. Feed consumption was reduced at 2000 ppm in both sexes.

As shown in Table 4, mating, gestation, fertility, and parturition indices were not affected by treatment. Likewise, numbers of stillborn pups as well as livebirth, viability, and lactation indices were not affected.

At parental necropsy, females at 2000 ppm had higher relative liver and spleen weights than controls. At 200 ppm., relative liver weights were increased by approx. 10 %. No treatment- related macroscopical changes were detected. As shown in Table 5, microscopic histopathology revealed a minimal hepatocellular hypertrophy in males and females at 2000 ppm and minimal to moderate hyperplasia of basophilic cells of the adenohypophysis in males at 2000 ppm. A slight increase of hepatocellular hypertrophy was noted in males treated with 200 ppm.

Table 5. Incidences of microscopical lesions in F1 parents

Sex	Males				Females
Feeding level, ppm	0	20	200	2000	0	20	200	2000
Liver / Total examined	30	30	30	30	30	30	30	30
Hypertrophy	0	0	2	26	0	0	0	10
Pituitary / Total examined	30	30	30	30	30	30	30	30
hypertropy of basophilic cells	7	8	7	17	0	0	0	0

 

F2 - pups: As shown in Table 6, there were no treatment-related clinical signs in the pups. Litter weights of the F1 generation were reduced at 2000 ppm from the end of the first week of lactation onwards. Eye opening was minimally delayed (by about 0.5 days) at 2000 ppm compared to controls.

 

Table 6. Litter data of the F2 generation

Feeding level	0 ppm	20 ppm	200 ppm	2000 ppm
Parameter
Number of litters	21	24	24	29
Total pups born	302	310	355	393
Mean per litter	14.4	12.9	14.8	13.6
Number of stillborn pups	3	4	1	2
Sex ratio (% females day 0)	48.2	50.3	50.6	54.5
Mean number of pups alive
- on day 0	14.2	12.8	14.8	13.5
- on day 4 (post culling)	8.0	7.5	8.0	7.7
- on day 7	8.0	7.5	8.0	7.6
- on day 14	7.9	7.5	7.9	7.4
- on day 21	7.9	7.5	7.9	7.4
Viability Index (a) Lactation Index (b)	95.3 98.8	97.4 98.9	97.2 98.4	98.7 96.9
Mean pup weight
- on day 0	6.1	6.1	6.0	6.3
- on day 4 precull	9.6	9.8	9.1	9.1
- on day 4 postcull	9.7	10	9.3	9.3
- on day 7	16.0	15.5	15.3	14.7*
- on day 14	31.3	30.5	30.5	28.9**
- on day 21	52.2	49.9	49.6	45.7**

(a): % Pups surviving days 0 to 4; (b): % Pups surviving days 4 to 21;

*: significant at p<0.05; **: significant at p<0.01

  

It is concluded that reproductive parameters including gonadal function, mating behaviour, conception, parturition, lactation and weaning, as well as sex organ histopathology were not affected in this study.

The dose of 200 ppm was a NOAEL for parents and offspring. At 2000 ppm, parental and offspring body weights were reduced, and eye opening was slightly delayed in pups, and histopathology of adults revealed changes in liver, spleen, and the pituitary.

At 200 ppm, a marginal increased incidence of minimal liver hypertrophy was observed in few parental males (17 %), but this finding was not correlated with any liver weight increase at this dose level and therefore not considered as toxicological relevant.