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

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan 1985 - Mar 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

some raw data of histopathological examinations is not included, which does not influence the scientific validity of the report

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:

- Analytical purity: 98.4%
- Lot/batch No.: 232114080
- Stability under test conditions: substance is stable for the course of the study (at room temperature)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: young adults
- Weight at study initiation: weighing approx. 160 – 200 g
- Housing: mice were kept under conventional conditions in Makrolon cages type III, 5 mice per cage
- Diet: Altromin 1324 diet for rats and mice
- Water: tap water ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 40 - 70%
- Air changes (per hr): ten times per hour
- Photoperiod (hrs dark / hrs light): 12 h dark/ 12 h light

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

other: 1:1 mixture of polyethylene glycol 400 and ethanol

Remarks on MMAD:

MMAD / GSD: MMAD (mass median aerodynamic diameter) + GSD (geometric standard deviation): approx. 2..2 to 2.4 + 1.9 µm
Mean 88 % of relative mass had MMAD = 5 µm (range: 60 – 100 % respirable fraction)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: cylindrical inhalation chamber
- Method of holding animals in test chamber: animals were exposed in tubes
- Method of conditioning air: compressed air was fully automatically conditioned by a coupled VIA compressed air dryer type 110
- System of generating particulates/aerosols: by means of a jet (binary) and compressed air 200 µl per minute vehicle mixture was vaporised
into the inhalation chamber
- Temperature, humidity, pressure in air chamber: 23 ± 1 °C, 42 ± 4.5%, ~ 600 kPa
- Air flow rate: 10 L/min
- Air change rate: 15 changes per hour
- Method of particle size determination: analyses were made with an aerodynamic particle sizer with laser velocimeter.
Furthermore particle spot analyses were made with a Berner cascade impactor
- Treatment of exhaust air: cleaned with an absolute filter

TEST ATMOSPHERE
- Brief description of analytical method used:
nominal Diuron concentration in the test atmosphere was calculated from the quotients of the total Diuron mass (mg) fed into the chamber and the
total air supply (m3) per exposure. Determination was applied by spectral photometry at 250 nm, layer thickness 1 cm. Glass tubes filled with cotton wool were used to adsorb the diuron aerosol. The test substance could be quantitatively eluted from this using methanol as solvent.
Per concentration three analyses of the test atmosphere were made per week on several exposure days.

VEHICLE (if applicable)
- Composition of vehicle: 1:1 mixture of Lutrol (polyethylene glycol E 400) and ethanol
- Concentration of test material in vehicle: 0.1, 0.75, and 5% solutions
(5% is the maximum technically producible concentration due to solubility of Diuron in the vehicle)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The total diuron concentration in the test atmosphere was calculated from the quotients of the total diuron mass (mg) fed into the chamber and the total air supply (m3) per exposure. The actual active ingredient concentration in the rats` nose area was determined by spectral photometry at 250 nm, layer thickness 1 cm. Glass tubes filled with cotton wool were used to adsorb the diuron aereosol. The active ingredient could be quantitatively eluted from this using methanol as solvent. Per concentration three analyses of the test atmosphere were made per week on several exposure days.

Duration of treatment / exposure:

4 or 8 weeks

Frequency of treatment:

6 h per day, 5 days per week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 20, 150, 1000 mg/m3
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
4.1, 37.4, 268.1 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: concentrations of doses were based on a previous study

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: several times daily (except during exposure)
- mortality was checked daily

BODY WEIGHT: Yes
- Time schedule for examinations: before exposure, then weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at autopsy
- How many animals: all
- Parameters checked: Haematocrit, haemoglobin concentration, erythrocyte count, differential leukocyte count, platelet count, MCV, MCHC, MCH,
reticulocytes, Heinz bodies, methaemoglobin, sulf-haemoglobin and thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at autopsy
- How many animals: all
- Parameters checked: glucose (at the end of second week), urea, total bilirubin, creatinine, total protein, total cholesterol, albumin,
blood urea nitrogen, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, LDH, glutamate dehydrogenase, methaemoglobin, thyroxine binding capacity, T3, T4

URINALYSIS: Yes
Time schedule for collection of urine: end of the study
- How many animals: all
- Parameters checked: pH, protein, glucose, blood, ketone bodies, bilirubin, urobilinogen, sodium, potassium, calcium, chloride, sediment

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- all groups were examined
- The following organs of all animals were weight: liver, lungs, kidneys, adrenals, testes, thyroid, ovaries, thymus, spleen, heart
HISTOPATHOLOGY: Yes
- the following organs were fixed in 10 % formaldehyde solution: eyes, femur and sternum, lung, hilus lymph nodes, testes, ovaries, heart,
head (nose-throat area), liver, stomach, spleen, adrenals, kidneys, oesophagus, trachea, larynx, thyroid, uterus, urinary bladder and ureter

Other examinations:

Liver enzymes: mixed function oxidase (CYP 450, N- and O-demethylase)

Statistics:

Arithmetically group means were calculated, significance of variations between groups was analysed by methods of Mann, Whitney or Wilcoxon

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
- All animals of the top dose group had ungroomed fur in the last third of the study onwards following exposure lasting to the next exposure.
- No mortalities occurred.

BODY WEIGHT AND WEIGHT GAIN
- All groups had a low group mean body weight gain, which is due to stress by exposure in tubes.

HAEMATOLOGY
- Heinz bodies and reticulocytes were significantly increased, erythrocytes and haemoglobin decreased in female rats of the mid dose group and
males and females of the top dose group. No differences in differential blood counts were noted.

CLINICAL CHEMISTRY
- A slight reduction of thyroid function in high dose animals was indicated by slightly decreased T3 and T4 values in combination with an
increased thyroxin binding capacity, particularly in males.
Plasma protein and albumin levels were decreased in top dose animals.

URINALYSIS
- There were no toxicologically relevant findings in the results of urinalysis.

ORGAN WEIGHTS
- A significantly increased spleen and liver weight was found in animals treated with doses of 150 and 1000 mg/m³.
However, no trend in organ weight changes was found.

GROSS PATHOLOGY + HISTOPATHOLOGY: NON-NEOPLASTIC
- Necropsy revealed increased incidences of dark and swollen spleens in males and females exposed to mid and top dose.
Moreover, some animals of all groups exhibited dilated hearts without dose-response correlation.

OTHER FINDINGS
- Enzyme induction was evidenced by increased activity of O-demethylase in males and females of the high dose group especially after 8 weeks.

Effect levels

1

Dose descriptor:

NOAEC

Effect level:

37.4 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse and treatment-related effects were observed at 37.4 mg/m³

2

Dose descriptor:

LOAEC

Effect level:

268.1 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based on adverse effects on haematologic parameters and dark, enlarged spleens at higher dose levels.

3

Dose descriptor:

NOAEC

Effect level:

4.1 mg/m³ air

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse and treatment-related effects were observed at 4.1 mg/m³

4

Dose descriptor:

LOAEC

Effect level:

37.4 mg/m³ air

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Based on adverse effects on haematologic parameters and dark, enlarged spleens at higher dose levels.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Results of a subacute inhalation toxicity study for 4 weeks with Diuron in rats

Sex	Males				Females
Nominal dose /measured concentration [mg/m³]	0	20/ 4.1	150/ 37.4	1000/ 268.1	0	20/ 4.1	150/ 37.4	1000/ 268.1
No. of animals examined	5	5	5	5	5	5	5	5
Mortalities	0	0	0	0	0	0	0	0
Clinical signs*	0	0	0	5	0	0	0	5
Body weight [g] at start	182	182	185	183	171	177	171	175
Body weight** [g] at end	236	240	237	227	176	181	172	175

* ungroomed fur following exposure in the third part of the study

** low weight gain due to treatment-related stress (tube exposure)

Table 2: Necropsy findings from a 4 -week subacute inhalation toxicity study with Diuron

Sex	Males					Females
Nominal dose /measured concentration [mg/m³]	0	20/ 6.6	150/ 47.6	1000/ 311		0	20/ 6.6	150/ 47.6	1000/ 311
Gross pathology
Heart, contracted	0	0	0	0		0	0	0	0
Heart, dilated	0	0	5	5		0	0	1	5
Kidney, mottled	0	1	0	5		0	1	0	0
Lung, distended	1	2	2	2		1	4	1	1
Testicles, small	0	0	1	0		0	0	0	0
Spleen dark/enlarged	0	0	0	5		0	0	5	5
Mean absolute organ weights [mg]
Liver	9715	9036	8472**		8992	6349	6672	5547	5849
Spleen	514	525	462		778**	384	400	401	624**

* p = 0.05, ** p = 0.01

Table 3: Results of a subacute inhalation toxicity study for 8 weeks with Diuron in rats

Sex	Males				Females
Nominal dose /measured concentration [mg/m³]	0	20/ 4.1	150/ 37.4	1000/ 268.1	0	20/ 4.1	150/ 37.4	1000/ 268.1
No. of animals examined	5	5	5	5	5	5	5	5
Mortalities	0	0	0	0	0	0	0	0
Clinical signs*	0	0	0	5	0	0	0	5
Mean body weight [g] at start	182	182	185	183	171	177	171	175
Mean body weight** [g] at end	249	259	250	238	184	186	173	183

* ungroomed fur following exposure in the third part of the study

** low weight gain due to treatment-related stress (tube exposure)

Table 4: Necropsy findings from a 8 -week subacute inhalation toxicity study with Diuron in rats

Sex	Males					Females
Nominal dose /measured concentration [mg/m³]	0	20/ 6.6	150/ 47.6	1000/ 311		0	20/ 6.6	150/ 47.6	1000/ 311
Gross pathology
Heart, contracted	0	0	1	3		0	0	0	1
Heart, dilated	0	4	3	0		0	0	1	3
Kidney, mottled	0	0	1	0		0	0	0	0
Lung, distended	0	1	0	1		0	2	0	1
Testicles, small	0	0	4	5		0	0	5	5
Spleen dark/enlarged	2	1	0	0		0	0	0	0
Mean absolute organ weights [mg]
Liver	8242	8385	8246		8240	6611	6604	5891	6603
Spleen	446	490	433		587**	379	378	436	619**

* p = 0.05, ** p = 0.01

Applicant's summary and conclusion

Executive summary:

In a reliable subacute inhalation study comparable to guideline OECD 412 Wistar rats were exposed to nominal doses of 0, 20, 150 and 1000 mg/m3 (anal. conc. achieved: 0, 4.1, 37.4 and 268.1 mg/m³) of an aerosol of Diuron for 6 h per day, 5 days per week for 14 or 28 days. At all rats exposed to concentrations up to 37.4 mg/m³ Diuron did not exhibit prominent test item-related signs of adverse effects. Therefore the LOAEC was determined to be 268.1 mg/m³ in males and 37.4 mg/m³ in females based on significant alterations in haematologic parameters and dark, enlarged spleens at higher dose levels. The NOAEC was assessed to be 37.4 mg/m³ for male and 4.1 mg/m³ for female animals. (Pauluhn 1986b)