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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 1989 – January 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data

Data source

Referenceopen allclose all

Reference Type:
review article or handbook
Title:
Unnamed
Year:
2011
Reference Type:
review article or handbook
Title:
Unnamed
Year:
1992

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EPA OPP 83-4 (Reproduction and Fertility Effects)
GLP compliance:
yes
Remarks:
A signed Statement of Compliance with EPA GLPs, dated December 13 and 20, 1990, was provided .

Test material

Constituent 1
Reference substance name:
Diuron
EC Number:
206-354-4
EC Name:
Diuron
Cas Number:
330-54-1
Molecular formula:
C9H10Cl2N2O
Test material form:
solid: crystalline

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, Raleigh, NC
- Age at study initiation:
Review for US EPA:
P males--approximately 60 days at start of study
P females--approximately 57 days at start of study
Review Australian Government: approximately 6 weeks at the beginning
- Weight at study initiation:
Review for US EPA:
P males--311.0-394.5 g at start of study
P females--189.3-252.7 g at start of study
Review Australian Government: 120-163 g for males and 83-139 g for females at the beginning
- Acclimation period: 23 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25°C
- Humidity (%): 40-60%.
- Photoperiod (hrs dark / hrs light): 12/12 hour light/dark cycle was maintained.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Remarks:
diet (Purina* Certified Rodent Chow #5002)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared weekly and stored in a refrigerator until used.
- Mixing appropriate amounts with (Type of food): The test diets were adjusted for purity.
The test material was mixed with irradiated rodent chow (Purina* Certified Rodent Chow #5002) for three minutes in a high-speed mixer. The purity of the test material was confirmed three times during the study. Stability, concentration, and homogeneity of the test material in the diet were analyzed prior to start of the study.
Stability was analyzed after storage in a refrigerator for 14 days or at room temperature for 7 and 14 days. In addition, concentration analyses of the test material in the diet at all dosage levels were conducted three times during the study.
- Storage temperature of food: Diets were prepared weekly and stored in a refrigerator until used.

Details on mating procedure:
- M/F ratio per cage:1:1
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After 21 days of unsuccessful pairing replacement of first male by another male with proven fertility.
Sibling matings were avoided.

Analytical verification of doses or concentrations:
yes
Remarks:
Concentration, stability, and homogeneity of the test material in the diet were confirmed to be within ±15% of nominal values.
Details on analytical verification of doses or concentrations:
Stability, concentration, and homogeneity of the test material in the diet were analyzed prior to start of the study.
Stability was analyzed after storage in a refrigerator for 14 days or at room temperature for 7 and 14 days.
In addition, concentration analyses of the test material in the diet at all dosage levels were conducted three times during the study.

The purity of the compound was 95%-100% of target. Concentrations of the test material in the diets were 91%-115% of nomina1 values. Homogeneity analyses revealed concentrations of 95%-105% of nominal values; stability analyses were 85%-103% (after 14days at room temperature) of nominal values.
Duration of treatment / exposure:
P generation: 73 days
F1 generation: 105 days after weaning
Frequency of treatment:
daily in diet
Details on study schedule:
- Selection of parents from F1 generation when pups were at least 105 days after weaning.
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
control
Dose / conc.:
10 ppm
Remarks:
Calculated daily intake on average (P and F1 premating)
Females: 0.8 mg/kg/day
Males: 0.68 mg/kg/day
Dose / conc.:
250 ppm
Remarks:
Calculated daily intake on average (P and F1 premating)
Females: 20.3 mg/kg/day
Males: 16.9 mg/kg/day
Dose / conc.:
1 750 ppm
Remarks:
Calculated daily intake on average (P and F1 premating)
Females: 144 mg/kg/day
Males: 120 mg/kg/day
No. of animals per sex per dose:
30
Control animals:
yes
Details on study design:
- Dose selection rationale: Dosages were selected based upon several previous studies with diuron. Sprague-Dawley rats fed diuron for two years exhibited body weight and hemoglobin decreases at 250 ppm and bladder hyperplasia at 2500 ppm (1963); Sprague-Dawley rats fed diuron for only 90 days exhibited a body weight decrease at 2500 ppm, while decreased red blood cell count and increased bone marrow hyperplasia were evident at 250 and 2500 ppm (1963). A two-year feeding study in Wistar rats showed a decrease in body weight and an increase in bladder neoplasia at 250 ppm and an increase in anemia at 25 ppm (1985)." A one-month pilot feeding study, conducted prior to the present study, showed decreases in body weight and weight gain at 2500 ppm.
- Rationale for animal assignment (if not random): Parental animals were distributed amongst four groups using a computer-generated randomization based on body weight.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day.
- Observations for mortality, moribundity, and overt signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly; for females during gestation and lactation, data were recorded on days 0, 7, 14, and 21. Terminal body weight data were recorded for all animals.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was measured weekly; for females during gestation, it was determined on days 0, 7, and 14 (it was not determined during lactation).

Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible). Pups found dead were necropsied. Thirty male and thirty female F1 pups were randomly selected as F1 parental animals. Twenty pups per sex, group, and generation were randomly selected for a complete necropsy. The remaining pups were sacrificed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
-Number of live and dead pups, pup weight (collectively by sex), sex, and external abnormalities at birth and on lactation days 4, 7, and 14
-Number of live and dead pups, individual pup weight, external abnormalities, and sex at weaning on lactation day 21
-Daily clinical signs

GROSS EXAMINATION OF DEAD PUPS: Offspring that died during the lactation period were necropsied (except 4 pups due to technical error).

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after litter production.
- Maternal animals: All surviving animals after litter production.
P males and females after 119-120 and 117-132 days of feeding, respectively; F1 males and females af ter 158-167 and 151-177 days of feeding, respectively).

GROSS NECROPSY
- Gross necropsy not specified

HISTOPATHOLOGY / ORGAN WEIGHTS
The testes were weighted, and the reproductive tissues (testes, epididymides, prostate, seminal vesicles, coagulating glands, pituitary, and ovaries, uterus, vagina and pituitary) from 0 and 1725 ppm groups, and gross lesions from all groups were collected for examination microscopically.



Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed.
From F1 weanlings not selected to continue to the next generation and from surviving F2 weanlings, approximately 20/sex/group were selected for gross post-mortem examination.

GROSS NECROPSY
- Gross necropsy not specified.
Statistics:
The following analyses were conducted.
-Body weight, body weight gain, food consumption, organ weights and length of gestation—Bartlett’s test for homogeneity of variances, ANOVA, and Dunnett's test for pairwise comparisons between groups
-Incidences of clinical, gross and microscopic observations--Fisher's Exact test with/without Cochran Armitage trend test
-Mating, fertility, and gestation indices and litter survival-Fisher's Exact test
-Pup numbers, survival, and weights and viability and lactation indices--Mann-Whitney U test
-Significance was judged at alpha - 0.05.
Reproductive indices:
Fertility index
Mating index
Offspring viability indices:
Live birth index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No compound-related clinical signs were observed in any sex and generation.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
In the Fo generation, one female from the control group was sacrificed in extremis due to difficulties at the time of delivery.
One female from the 10-ppm group was found dead; necropsy did not reveal cause of death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Compound-related effects in body weight and body weight gain were observed at 1750 ppm in both sexes.
Among males at 1750 ppm body weight decreased significantly by an average of 7% starting on day 7. Body weight gain among males decreased significantly at 250 ppm on days 91-98 (18%) and at 1750 ppm on days 0-14 (21%), 21-28 (15%), 42-49 (28%), 77-84 (53%), 91-98 (90), 0-70 (premating; 16%), 70-112 (mating and postmating; 28%), and 0-112 (entire feeding period;18%).
Among females at 1750 ppm, body weight decreased significantly by an average of 7% starting on day 7 during premating and by 9% during the gestation and lactation periods. Body weight gain among females decreased significantly at 1750 ppm on days 0-7 (48%), 21-28 (69%), and 0-70 (premating; 28%).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Among males at 1750 ppm food consumption (g/animal/day) decreased significantly on days 0-14, 21-28, 42-56, and 0-70.
Among females at 1750 ppm food consumption decreased significantly on days 0-7, 21-28, 35-49, and 0-70. During gestation food consumption decreased consistently at 1750 ppm.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Compound-related effects on food efficiency were observed at 1750 ppm in both sexes.
Food efficiency among males decreased significantly at 10 ppm on days 42-49 and at 1750 ppm on days 0-14,42-49, and 0-70. It increased significantly at 10 ppm on days 63-70; at 250 ppm on days 35-42 and 63-70; and at 1750 ppm on days 63-70.
Food efficiency among females decreased significantly at 1750 ppm on days 0-7, 21-28, and 0-70. It increased significantly at 10 ppm on days 0-7. During gestation no differences between groups were noted in food efficiency.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No compound-related histologic findings were observed for any sex and generation.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Fertility index significantly increased at 1750 ppm due to a low fertility index in the control group.

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
250 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: parental toxicity
Dose descriptor:
LOEL
Effect level:
1 750 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Description (incidence and severity):
No compound-related clinical signs were observed in any sex and generation.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No compound-related mortalities were observed.
In the F1 generation, three males, one each from the control group, 250-ppm group, and 1750-ppm group were found dead. The control animal had kidney cysts; necropsies of the other two males did not reveal cause of death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Compound-related effects in body weight and body weight gain were observed at 1750 ppm in both sexes and generations.
Among males at 1750 ppm body weight decreased significantly by an average of 16% starting on day 0. Body weight gain among males decreased significantly at 250 ppm on days 63-70 (33%) and 147-154 (52%) and at 1750 ppm on days 0-28 (16%), 42-49 (20%), 63-70 (26%), 91-98 (46%), 147-154 (72%), 0-105 (premating; 15%), 105-161 (mating and postmating; 41%), and 0-161 (entire feeding period; 17%).
Among females at 1750 ppm body weight decreased significantly by an average of 16% starting on day 0 during premating; by 14% during the gestation period; and by 19% during the lactation periods. Body weight gain among females decreased significantly at 250 ppm on days 0-7 (11%) and 14-21 (12%) and at 1750 ppm on days 0-14 (17%) and 0-105 (premating; 14%); it increased significantly at 10 ppm on days 70-77. During gestation, body weight gain decreased significantly on days 14-21 (21%) and 0-21 (19%). During the lactation period, significant increases in body weight gain were observed at 1750 ppm on days 0-7, 14-21, and 0-21.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Among males at 1750 ppm food consumption consistently decreased significantly (with the exception of days 77-84). It increased significantly at 10 ppm on days 84-91.
Among females food consumption decreased significantly at 10 ppm on days 91-98; at 250 ppm on days 7-14, 21-35, 56-63, 91-105, and 0-105; and at 1750 ppm during the entire premating. During gestation, food consumption decreased consistently at 1750 ppm
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Compound-related effects on food efficiency were observed at 1750 ppm in F1 females.
Among males food efficiency decreased significantly at 1750 ppm on days 91-98 ; Iamong females food efficiency decreased significantly at 1750 ppm on days 0-7, 21-28, and 0-70 and increased significantly at 10 ppm on days 70-77. During gestation no differences between groups were noted in food efficiency.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Testis weights: No compound-related effects were observed for any generation. At 1750 ppm, relative (percent of body weight) testis weight significant1y increased in both generation males, which was due to decreased body weight rather than caused by the test compound. An incidental significant increase was noted in absolute testis weight at 10 ppm in F1 males.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No compound-related gross findings were observed for any sex and generation.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No compound-related histologic findings were observed for any sex and generation.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Effect levels (P1)

open allclose all
Dose descriptor:
NOEL
Effect level:
250 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: parental NOEL
Dose descriptor:
LOEL
Effect level:
1 750 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No compound-related effects were noted in pup clinical observations.
Mortality / viability:
no mortality observed
Description (incidence and severity):
No compound-related effects were noted in pup survival.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Pup body weight for sexes combined or separated significantly decreased at 1750 ppm on lactation days 7, 14, and 21; this was considered to be a compound-related effect.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In males food consumption decreased significantly.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
In males food consumption decreased significantly in week 91-98.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
No compound-related effects were noted in pup anomalies.
Histopathological findings:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

open allclose all
Dose descriptor:
NOEL
Generation:
F1
Effect level:
250 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: parental NOEL
Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 750 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Description (incidence and severity):
No compound-related effects were noted in pup clinical observations.
Mortality / viability:
no mortality observed
Description (incidence and severity):
No compound-related effects were noted in pup survival.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Compound-related reproductive toxicity was observed at 1750 ppm. It was manifested in pups as significantly decreased body weight in both sexes and generations.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No compound-related effects were noted in pup anomalies.
Histopathological findings:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

open allclose all
Dose descriptor:
NOEL
Generation:
F2
Effect level:
250 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: foetal NOEL
Dose descriptor:
LOEL
Generation:
F2
Effect level:
1 750 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 750 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

Applicant's summary and conclusion

Conclusions:
NOEL for parental toxicity: 250 ppm
LOEL for parental toxicity: 1750 ppm
NOEL for reproductive toxicity: 250 ppm
LOEL for reproductive toxicity: 1750 ppm
Executive summary:

In a two-generation reproduction study, rats (P, 30/sex/dose) received 0, 10, 250 or 1750 ppm of diuron in the daily diet for 73 days, and then bred within the dose groups to produce F1 litters. At weaning on Day 21, 30 F1 rats/sex/dose were randomly selected, and at least 105 days after weaning, the F1 rats were bred to produce F2 litters. For both generations, administration of the test substance continued during breeding, gestation and lactation. During premating the calculated daily intakes of diuron for both generations were as follows: for males an average of 0.68, 16.9, and 120 mg/kg/day and for females an average of 0.80, 20.3, and 144 mg/kg/day.

Compound-related parental toxicity was observed at 1750 ppm as evidenced by decreased body weight, body weight gain, and food consumption in both sexes and generations. The NOEL and LOEL for parental toxicity were 250 and 1750 ppm, respectively.

Compound-related reproductive toxicity was observed at 1750 ppm as evidenced by decreased pup body weight during the lactation period for both sexes and generations. The NOEL and LOEL for reproductive toxicity were 250 and 1750 ppm, respectively.