diuron (ISO); 3-(3,4-dichlorophenyl)-1,1-dimethylurea

Toxicity to reproduction

Administrative data

Endpoint:

multi-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Diuron

EC Number:

206-354-4

EC Name:

Diuron

Cas Number:

330-54-1

Molecular formula:

C9H10Cl2N2O

IUPAC Name:

3-(3,4-dichlorophenyl)-1,1-dimethylurea

Details on test material:

- Name of test material (as cited in study report): Diuron
- Analytical purity: 97.1% (Purity was confirmed at the beginning, middle, and end of the study).
- Stability under test conditions: stable for the course of the study

Test animals

Species:

rat

Strain:

other: Crl:CD BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina.
- Age at study initiation: 60 days of age for males and females 57 days old.
- Weight at study initiation: Males: 120 to 163 g; Females: 83-139 g.
- Fasting period before study: No data
- Housing: All rats were individually housed in stainless steel, wire-mesh cages suspended above Upjohn DACB or R-2 Reemay-backed bage boards.
- Diet : During the pretest, all rats were fed irradiated Purina Certified Rodent Chow #5002 meal; during the test period, all rats were fed the diet of thier respective treatment groups (See tabel 1).
- Water : Tap water was provided ad libitum.
- Acclimation period: 3 weeks.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±2
- Humidity (%): 50±10
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle (fluorescent light)

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: Purina Chow #5002

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): All diets were prepared weekly and refrigerated until used.
- Mixing appropriate amounts with (Type of food): Diuron was added to irradiated PCRC and throughly mixed for three minutes in a high-speed mixer.

Details on mating procedure:

- M/F ratio per cage: Each P1 female was housed continually with a randomly selected male for the same dietary concentration group.
- Length of cohabitation: Until evidence of copulation was obtained (intravaginal or extruded copulation plug) or until three weeks elapsed. Upon detection of a copulation plug (designated as day 0 of gestation), the female was transferred back to individual cage housing.
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations of diuron in diets prepared April 5, June 6, and August 29, 1989, and January 9, 1990, were determined by high-performance liquid chromatography (HPLC). The compound was distributed homogeneously in the diet. Recovery of diuron from diet was tested at 10 and 1750 ppm to confirm the analytical method.
Measured concentration of recovery samples were 90% to 115% of nominal at the 10-ppm level and 95 to 108% of nominal at the 1750-ppm level, indicating that the method performed satisfactorily over this concentration range.

Duration of treatment / exposure:

Duration of exposure before mating was 73 days for both sexes.
Males were dosed during mating and females additionally during pregnancy and the weaning period. The first and second generation were dosed during growth, mating, pregnancy until weaning of the second generation (105 days after weaning for F1 generation).

Frequency of treatment:

Once per day.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
Approximately 250 ppm = P1 Male: 14.8 mg/kg bw/day, Female: 18.5 mg/kg bw/day. F1 Male: 18.9 mg/kg bw/day and Female:22.1 mg/kg bw/day.
Basis:

Doses / concentrations 2

Remarks:

Doses / Concentrations:
Approximately 1750ppm = P1 Male: 101 mg/kg bw/day, Female: 123mg/kg bw/day. F1 Male: 139 mg/kg bw/day and Female:116 mg/kg bw/day.
Basis:

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0, 10, 250 and 1750 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

30

Control animals:

yes

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals observed daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Bodyweights of males and females were recorded once weekly for the test period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Weekly intervals


CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily throughout the study.
- Cage side observations checked: Cage-site examinations to detect moribound or dead rats and abnormal behaviour and/or appearance amoung rats.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: All P1 rats were weighed once a week during the premating feeding period. During gestation and lactation, females were weighed on days 0, 7, 14, and 21 of each period. Females without a known start of gestation and all males continued to be weighed on a weekly schedule. Females that copulated and did not deliver a litter continued to be weighed weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Parameters examined in [all] male parental generations: Testis weight, epididymis weight, prostate and seminal vesicles.
Parameters examined in P/F1 male parental generations: testis weight

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in [F1/F2 ] offspring:number and sex of pups, stillbirths, live births, presence of gross anomalies, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Surviving P1 males were sacrificed after 119 to 120 days of feeding.
- Maternal animals: P1 females were sacrificed after 117 to 132 days of feeding.
The method of sacrifice was chlotoform anesthesia and exsanguination.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathological examination of the tissues was conducted only for the control and high-concentration groups of both generations. All gross lesions from the low- and intermediate-concentration groups were also examined histologically.
The following tissues were collected from all parental generation rats that died (tissue integrity premitting), were sacrificed in extremis, or were sacrificed by design:
Male: Testes (weighes), Epididymides, Prostate, Seminal vesicles, Coagulating Gland
Female: Ovaries, Uterus (cervix, uterine horns, and uterine body), vagina
Both sexes: Pituitary, Gross Lesions.

Postmortem examinations (offspring):

SACRIFICE
Parental generation males were sacrificed at the discretion of the study director after siring litters. Parental generation females was sacrificed after weaning litters.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathological examination of the tissues was conducted only for the control and high-concentration groups of both generations. All gross lesions from the low- and intermediate-concentration groups were also examined histologically.
The following tissues were collected from all parental generation rats that died (tissue integrity prmitting), were sacrificed in extremis, or were sacrificed by design:
Male: Testes (weighes), Epididymides, Prostate, Seminal vesicles, Coagulating Gland
Female: Ovaries, Uterus (cervix, uterine horns, and uterine body), vagina
Both sexes: Pituitary, Gross Lesions.

Statistics:

Body weights, body weight gains, food consumption, organ weights, and gestation length will be analysed by a one-way analysis of variance. When the test for differences among test group (F test) is significant, pairwise comparisons between test and control groups will be made with the Dunnett’s tests. The Bartletss’s test for homogeneity of variances will be performed on the organ weight and, if significant (alpha = 0.005), will be followed by nonparametric procedures.
Incidence of clinical observation will be evaluated by the Fisher’s Eact test with a Bonferroni coreection and the Cochran-Armitage test for trend. Measures of reproduction and lactation performance will be evaluated with either the Fisher’s Exact test (Mating, Fertility, and Gestation Indices and Litter Survival) or the Mann-Whitney U test (pup numbers, survival, weights, viability index, and Lactation index).
Except for Bartlett’s test, all other significance will be judged at alpha = 0.05. Other methods will be used, if appropriate, at the time of analyses.

Reproductive indices:

The following indices of reproductive function were calculated for the P1 and F1 adults:
Mating Index (%) = Number copulating(a)/ Number cohoused x 100
Fertility Index (%) = Number bearing litters(b)/ Number copulatinga x 100
Gestation Index (%) = Number of litters with at least one live pup/ Number of pups born x 100
Pups Born Alive (%) c = Number of pups born alive/ Number of pups born x100
Viabiity Index (5) c,d = Number of pups alive day 4 preculling/ Number of pups born alive x 100
Lactation Index (%) c,d = Number of pups alive at weaning (21 postpartum)/ Number of pups alive day 4 postculling x 100
Litter Survival (%) d = Number of litters weaned/ Number of litters delivered x 100

a - Evidence of copulation = copulatory plug, found dead pregnant, or delivery of a litter.
b -Including those found dead pregnant during gestation
c - To be determined for each litter. Mean and standard deviation for each dose level will be calculated.
d -Excluding litters sacrificed due to death of dam during lactation.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

significantly lower at 1750 ppm than in control

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

significantly lower at 1750 ppm than in control

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No treatment-related mortalities or clinical signs were noted.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight , overall body weight gain, food efficiency and food consumption were significantly lower at 1750 ppm than in control.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
No effects observed.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No compound-related effects on fertility index, mating index or length of gestation were observed.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No substance related gross or microscopical observations were made in the parent generations.

OTHER FINDINGS (PARENTAL ANIMALS): During lactation, overall body weight gains in the P1 female 1750 ppm groups were greater than the control group. This finding is common in reproduction studies where a compound - related decrease in body weight has occurred.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

14.8 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: This NOAEL was based on reduced body weight, body weight gain, and food consumption in parental rats in the 1750 ppm dose groups.

2

Dose descriptor:

NOAEL

Effect level:

18.5 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: This NOAEL was based on reduced body weight, body weight gain, and food consumption in parental rats in the 1750 ppm dose groups.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
No treatment-related mortalities or clinical signs were noted.
CLINICAL SIGNS (OFFSPRING)
No treatment-related clinical signs were noted.
BODY WEIGHT (OFFSPRING)
Body weight and overall body weight gain were significantly lower at 1750 ppm than the controls.
SEXUAL MATURATION (OFFSPRING)
No compound-related effects on fertility index, mating index or length of gestation were observed.
GROSS PATHOLOGY (OFFSPRING)
No diuron-related gross observations were made in the F1 parental generations. However, enlarged spleen with corresponding congestion was found in top dose females in the F1 generation.
HISTOPATHOLOGY (OFFSPRING)
No diuron-related microscopical observations were made in the F1 parental generations.
OTHER FINDINGS (OFFSPRING)
There were no effects on litter size or survival rates in either F1 or F2 generations.
During lactation, overall body weight gains in the F1 female 1750 ppm groups were greater than the control group. This finding is common in reproduction studies where a compound - related decrease in body weight has occurred.

Effect levels (F1)

1

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

18.9 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: This NOAEL was based on reduced body weight in the F1/F2 pups in the 1750 ppm dose groups.

2

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

22.1 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: This NOAEL was based on reduced body weight in the F1/F2 pups in the 1750 ppm dose groups.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 3: Summary of reproductive indices

Parameter			Control		10 ppm		250 ppm		1750 ppm
		Generation	m	f	m	f	m	f	m	f
Mortality (not related to treatment)	Incidence	P	0	1	0	1	0	0	0	0
		F1	1	0	0	0	1	0	1	0
Food consumption	Increased/decreased	P	-	-	-	-	-	-	¿	¿
		F1	-	-	-	-	-	-	¿	¿
Body weight		P	-	-	-	-	-	-	¿	¿
		F1	-	-	-	-	-	-	¿	¿
Body weight gain		P	-	-	-	-	-	-	¿	¿
		F1	-	-	-	-	-	¿	¿	¿
Food efficiency		P	-	-	-	-	-	-	¿	¿
		F1	-	-	-	-	-	-	-	-
Clinical Observations (e.g. alopecia, discoloured discharge of eyes, sore)		P F1			No significant changes vs. controls
Pathology  - Enlarged spleen  - Congestion of spleen		F1			No significant changes vs. control				- -	¿ ¿

Table 4.Summary of reproductive indices

 

Group	0 ppm	10 ppm	250 ppm	1750 ppm
P1 generation
Mating index (%) (#copulated/cohoused)	93.3 (28/30)	100.0 (30/30)	96.7 (29/30)	100.0 (30/30)
Fertility index (%) (#delivered/copulated)	18.6 (2/28)	83.3 (25/29)	79.3 (23/29)	96.7 (29/30)
Gestation length (days)	22.9	22.6	22.8	22.6
F1 generation
Mating index (%) (#copulated/cohoused)	96.7 (29/30)	86.7 (26/30)	93.3 (28/30)	93.3 (28/30)
Fertility index (%) (#delivered/copulated)	89.7 (26/29)	76.9 (20/26)	82.1 (23/28)	85.7 (24/28)
Gestation length (days)	22.6	22.7	22.1	22.5

Table 5:Reproductive and fertility effects of Diuron

  

Group	0 ppm	10 ppm	250 ppm	1750 ppm
F1 generation
Mean pup number*	8.0	7.9	7.9	7.9
Mean born alive	14.3	13.9	14.9	14.4
Litter survival rate*	95.5	95.8	100	100
Sex ratio (males)	0.49	0.53	0.58	0.48
Mean pup weight*	58.9	61.3	57.9	48.4**
F2 generation
Mean pup number*	7.2	7.8	7.7	7.9
Mean born alive	13.1	14.0	13.4	12.5
Litter survival rate*	100	100	100	95.8
Sex ratio (males)	0.52	0.49	0.53	0.51
Mean pup weight*	62.7	64.7	61.1	51.1**

*on day 2,. ** p < 0.05

Table 6. Summary of clinical observations

Group	0 ppm	10 ppm	250 ppm	1750 ppm
F1 generation
No. of litters	21	24	23	29
No. of litters affected	4	5	8	7
No. of signs*	7	10	10	13
F2 generation
No. of litters	26	20	23	23
No. of litters affected	5	6	1	6
No. of signs	6	14	1	14

Applicant's summary and conclusion

Executive summary:

A reliable two-generation study in rats was conducted according EPA 83-4, thus in compliance with OECD guideline 416.

Four groups of Wistar rats (30 males and 30 females per group) received Diuron at 0, 10, 250 or 1750 ppm in the diet for < 70 days prior to mating, then during the mating period and finally during pregnancy and weaning. Under the conditions of this study, Diuron exhibited no adverse effects on reproductive parameters. The NOAEL of the parent male and female was 14.8 mg/kg bw/day and 18.5 mg/kg bw/day, respectively. This was based on reduced body weight, body weight gain, and food consumption in parental rats in the 1750 ppm dose. The NOAEL for the F1 males and females were 18.9 mg/kg bw/day and 22.1 mg/kg bw/day, respectively. This was based on reduced body weight in pups in the 1750 ppm dose group. (Cook, 1990)