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Repeated dose toxicity: inhalation

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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:
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

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
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 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 / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 20, 150, 1000 mg/m3
Basis:
nominal conc.
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

open allclose all
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
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.
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
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)