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Toxicological information

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:
other information
Study period:
1989-01-10 to 1989-02-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
Cited as Directive 84/449/EEC, B.8
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): o-Chloraniline
- Physical state: yellow-orange, clear liquid
- Analytical purity: 99.6 % (IR spectroscopy)
- Impurities (identity and concentrations):
<0.01 % cyclohexylamine
<0.01 % N-isopropylaniline
<0.01 % 2-chloro-N-Isopropylanilin
0.08% aniline
<0.01% 2-Isopropylaniline
<0.01% o-nitrochlorobenzene
0.24% p-chloroaniline
<0.01% m-chloroaniline
<0.01% 2,4-dichloroaniline
<0.01% 2,5-dichloroaniline
<0.01% 2,3-dichloroaniline
<0.01% 2,3-dichloroaniline
<0.01% o-phenylendiamine
0.03 % unknown impurities
- Purity test date: 1 Feb 1988
- Lot/batch No.: Lagerkessel 15/ 594 488
- Stability under test conditions: stable throughout the test period
- Storage condition of test material: 3-7°C, dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Winkelmann, Borchen
- Age at study initiation: 2-3 months
- Weight at study initiation: 165-206 g
- Fasting period before study:
- Housing: 5 (randomized) per macrolon III cage, changed every week, animals marked individually,
- Bedding: wood chips Type S8/15 Ssniff
- Diet: ad libitum; Altromin 1324
- Water: ad libitum; tap water
- Acclimation period: ~14 d


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 50
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12, artificial 14 W/m²

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose/head only
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: not applicable, vapour was tested
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: from Rhema Labortechnik, Hofheim, Germany
- Exposure chamber volume: 7 L
- Method of holding animals in test chamber: animals were held in place in special test tubes
- Source and rate of air: 10 L/ min; about 85 air changes /h
- Method of conditioning air: air was led through a bottle with prewarmed test substance
- Method of particle size determination: aerodynamic particle sizer with laser-velocimeter (TSI-APS 3300)
- Treatment of exhaust air: 70 % of the incoming air were withdrawn as exhaust air
- Temperature, humidity, pressure in air chamber:

vapour: T= ~23°C
Humidity= ~30 %

TEST ATMOSPHERE (vapour)
- Brief description of analytical method used: Ratfisch RS 55 gas chromatograph with flame ionisation detector
- Samples taken from breathing zone: yes, at the beginning, in the middle and towards the end of each test, 0.5 L/min 10-18 L per test sample

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical verifiction of the applied dose was realised with a gas chromatograph equipped with a flame ionisation detector
Duration of treatment / exposure:
4 w
Frequency of treatment:
6 h/d, 5 d/w
Doses / concentrations
Remarks:
Doses / Concentrations:
39, 217 and 886 mg/m³ (7, 41 and 169 ppm)
Basis:
analytical conc.
No. of animals per sex per dose:
10 per sex and dose
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: none
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes / No / No data
- Time schedule: twice a day (also on weekends)

DETAILED CLINICAL OBSERVATIONS: Yes, observed were changes in the skin and fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behaviour pattern, not adequately
performable inside the test tube

BODY WEIGHT: Yes
- Time schedule for examinations: application day 1, 8, 15, 22 and day 29

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: Yes, retina, vitreous, lens, cornea, conjunctivae, eyelids
- Time schedule for examinations: day 0 and week 4
- Dose groups that were examined: 5 animals per dose and sex


HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4th exposure day and week 2
- Anaesthetic used for blood collection: No
- Animals fasted: general no ( for glucose measurement in week 3; yes)
- How many animals: 5 per dose per sex
- Parameters checked: leucocyte count, hematocrit, hemoglobin, erythrocyte count, MCV, MCHC, MCH, reticolocyte count, thrombocyte count, Heinz bodies, methemoglobin, clotting potential

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at time of dissection (after 4 week study), heart puncture ( bleeding to death),
- Anaesthetic used for blood collection: Yes, diethyl ether
- Animals fasted: No data
- How many animals: 40
- Parameters checked:
enzymes: GOT, GPT, AP, GLDH, LDH, ChE, CK,
substrates: glucose, Urea, bilirubin, creatinin, total protein, albumin, triglycerides, cholesterol,
protein electrophorese: globulines, albumin
electrolytes: Na, K, Mg, Ca, anorganic P, Cl

URINALYSIS: Yes / No / No data
- Time schedule for collection of urine: over night for 17 h in week 3
- Metabolism cages used for collection of urine: Yes , animals received 5 mL of water by oral gavage prior to sampling
- Animals fasted: Yes
- Parameters checked:
quantitative: volume, weight, pH, protein
semiquantitative: protein, blood, glucose, bilirubin, urobilinogen, ketone bodies
sediment: leucocytes, erythrocytes, epithelial cells, bacteria, amorphous salts, tripel phosphate, calcium oxalate

NEUROBEHAVIOURAL EXAMINATION: Yes / No / No data
- Time schedule for examinations: day 14 and 25 after exposure
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:
HISTOPATHOLOGY: Yes: aorta, eye, eyelid, gut, extraorbital lacrimal, femur, brain, harder gland, skin, heart, testicles, epididymides, urinary bladder, hypophsis, bone marrow, coagulation gland, head, larynx, liver, lymph knots, stomach, mamma, spleen, muscle, adrenal, nervus ichiadicus, kidneys, esophagus, ovaries, pancreas, pharynx, prostata, medulla, seminal vesicle, thyroid, parathyroid, salivary gland, sternum, thymus, trachea, uterus, tongue
Other examinations:
relative (per 100 body weight) and absolut organ weight: brain, heart, testicles, liver, lung, spleen, adrenal, kidneys, ovaries, thymus, thyroid,
Statistics:
randomized with IBM-programm RANDU, arithmetic mean and simple standard deviations were calculated for: substance turn over, analytical concentration, temperatur, humidity, body weight gain

the statistics for the body weights were done by U-test (Mann and Whitney 1947) with modifications (Walter 1851) with alpha= 1% or 5%
ANOVA and Box-test

clinical findings: arithmetic mean and simple standard deviation, U-Test
dissection: Chi-square and Fisher-test

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
effects observed, treatment-related
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY: Clinical symptoms (cyanosis, tremor, impairment of motions and of some reflexes) were observed in the 
mid-dose group (female) and, more severe, the high-dose groups (both sexes, stronger in females).
No mortality was observed during the study.
BODY WEIGHT AND WEIGHT GAIN: Reduced body weight gains in the male high-dose group only.

OPHTHALMOSCOPIC EXAMINATION: One female in the high dose group displayed an ambilateral subcapsular cataract

HAEMATOLOGY:   Decrease in erythrocytes, hemoglobin level and hematocrit; increased methemoglobin 
(max. 3.3-11.7% in week 1), Heinz bodies (max. 0.1-0.7% in week 1) and reticulocytes; 
observed in all treated female groups and both higher-dose male groups, dose-dependant.

CLINICAL CHEMISTRY: total and urinary bilirubin increased; triglycerides, cholesterol and serum cholinesterase reduced; 
enhanced activity of liver monooxygenase, observed partly in mid-dose groups and generally in 
high-dose groups, females being affected more strongly.

URINALYSIS: Females in the high dose group displayed higher bilirubin and protein excretion and a lower pH


NEUROBEHAVIOUR: two females in the high dose group lost their myotatic reflex in week 3 and one of those was less
sensible to external stimuli, no other influence was observed


ORGAN WEIGHTS: dose-dependant increase of spleen weight (low-dose/both); 


GROSS PATHOLOGY / dark-red coloured spleen (mid-dose/both); deposition of hemosiderin in spleen (mid-dose/male); 
HISTOPATHOLOGY positive hemosiderin test (all groups); dose dependant increase of macroblasts and normoblasts 
in the bone marrow. The major influence seems to be a damage of the erythrocytes; no cumulative 
effects were noted.

Effect levels

Dose descriptor:
NOEC
Effect level:
ca. 6.4 mg/m³ air
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

no further data

Applicant's summary and conclusion

Executive summary:

Märtins, T (1992)

The subacute inhalative toxicity of o-chloraniline vapours was tested with a head/nose exposure apparatus in male and female Wistar rats according to OECD 412 and EU method B.8 for 6 h/day during 28 d. Three dose groups and one concurrent control group with 10 animals per sex were established. The concentrations were analytical verified and were 0, 39, 217 and 886 mg o-chloroaniline /m³. No mortality was observed. Clinical symptoms mainly cyanosis were observed in females of the mid-dose group and males and females of the high-dose group . Only the males in the high-dose group displayed a reduced weight gain. Dose-dependent hematological changes were evident in all treated females (varying degree) and in the male rats of the mid- and high-dose group. They included a reduction in erythrocyte count as well as hemoglobin and an increase in methemoglobin, heinz-bodies and reticulocytes. In the high-dose group liver-monooxygenase activity was increased and the excretion of bilirubin and protein via urine was elevated.The spleen weight increased dose-dependently. The liver weight was increased in the high-dose group. The spleen was of a dark-red colour in the mid- and high-dose group with hemosiderin accumulation and higher blood volume. The bone marrow of all exposed groups displayed an dose-dependent increase in normo- and macroblasts.

The study clearly shows, that the destruction of erythrocytes is the primary toxic effect of o-chloraniline. As this effect is already evident (increased spleen weight, and changes in blood parameters and bone marrow composition) in the lowest tested dose of 39 mg/m³ a NOEC of 6 mg/m³ was extrapolated.