Aniline

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Oct 1999 - 02 Aug 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions (only males treated, examinations on clinical chemistry not included, organ weight determination and histopathology were limited to possible target organs).

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Reference 2

Reference Type:

publication

Title:

Aniline: early indicators of toxicity in male rats and their relevance to spleen carcinogenicity

Author:

Mellert W, Deckardt K, Gembardt C, Zwirner-Baier I, Jäckh R, van Ravenzwaay B

Year:

2004

Bibliographic source:

Hum. Exp. Toxicol. 23, 379-389

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

reduced group size, males only, no clinical chemistry, histopathology in selected organs only.

Principles of method if other than guideline:

Subacute study was designed to study the mode of action of aniline toxicity.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Anilinium chloride

EC Number:

205-519-8

EC Name:

Anilinium chloride

Cas Number:

142-04-1

IUPAC Name:

anilinium chloride

Constituent 2

Reference substance name:

aniline, hydrochloride

IUPAC Name:

aniline, hydrochloride

Details on test material:

- Name of test material (as cited in study report): aniline hydrochloride
- Physical state: solid, grey
- Analytical purity: 100 %
- Lot/batch No.: 99-048
- Stability under test conditions: 7 days
- Storage condition of test material: room temperature, dark, stored under nitrogen
- Other:
Date of production: Jul 15, 19, 20 and 21, 1999
Homogeneity: homogeneous

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 11 weeks (28 days of administration) or 14 weeks (7 days of administration)
- Weight at study initiation: ca. 240 g
- Housing: Animals were housed singly in type DK III stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area
about 800 cm2). Underneath the cages, waste trays were fixed containing absorbent material (type 3/4 dustfree embedding, supplied by SSNIFF,
Soest, Germany). The animals were housed in a fully air-conditioned room.
- Diet (e.g. ad libitum): ad libitum; 9433 LL meal supplied by Eberle Nafag AG Gossau, Switzerland
- Water (e.g. ad libitum): ad libitum; tap water
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70%
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 28 Sep 1999 To: 03 Nov 1999

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: diet

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:


DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): The test substance was weighed out and thoroughly mixed with a small amount of food (9433
LL meal). Then corresponding amounts of food, depending on the dose group, were added to this premix in order to obtain the desired
concentrations, and mixing was carried out for about 10 minutes in a GEBR. LÖDIGE laboratory mixer.
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical investigations of the test substance preparations were carried out at the Bioanalytical Laboratory of the Experimental Toxicology and Ecology of BASF Aktiengesellschaft, Ludwigshafen, Germany. The stability of the test substance was tested over 7 days at room temperature. Homogeneity analyses of the test substance preparations were performed in samples of the high and low dose group at the start of the administration period. These samples also served for concentration control analyses. Additional concentration control analyses were performed with samples drawn from the mid concentration at the start of the administration period and in all concentrations at the end of the administration period. The food used in the study was assayed for chemical and microbiological contaminants. The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and the Technical Services of BASF Aktiengesellschaft as well as for the presence of microorganisms by a contract laboratory.

Duration of treatment / exposure:

7 and 28 days

Frequency of treatment:

continuously

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 10, 30 and 100 mg aniline hydrochloride/kg bw/d
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 4, 12 and 41 mg aniline/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

12 per time point

Control animals:

yes, plain diet

Details on study design:

The animals were randomly distributed according to weight among the individual test groups. The list of randomization instructions was compiled with a computer.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily from Mondays to Fridays and once a day on Saturdays, Sundays and public holidays.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily during the administration period


BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was determined before the start of the administration period in order to randomize the animals.
During the administration period body weight was determined 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: 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: Yes


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was observed daily by visual inspection of the water bottles for any overt changes in volume.


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 29
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: 6 per dose group
The following parameter were determined: leikocytes, erythrocyts, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration,platelets, differential blood count, reticulocytes, Heinz bodies, methemoglobin (using a hemoximeter), hemoglobin adducts


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 29
- Animals fasted: Yes
- How many animals: 6 per dose group
The following parameters were examined: transferrin, iron and total iron-binding capacity


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The animals were sacrificed by decapitation under CO2 anesthesia. The exsanguinated animals were necropsied and subjected to a gross-pathological assessment. The following weight parameters from all animals sacrificed as scheduled were determined: anesthetized animals, liver, kidneys and spleen.
The following organs or tissues were fixed in 4% formaldehyde solution: all gross lesions, target organs, brain, pituitary gland, thyroid glands/parathyroid glands, thymus, trachea, lungs, heart, aorta, salivary glands (Glandula mandibularis and Glandula sublingualis), liver, spleen, kidneys, adrenal glands, pancreas, testes, epididymides, prostate gland and seminal vesicles, skin, esophagus, stomach (glandular and non-glandular stomach), duodenum, jejunum, ileum, colon, rectum, urinary bladder, lymph nodes (Ln. mandibularis and Ln. mesenterialis), skeletal muscle, sciatic nerve, sternum with marrow, bone marrow (femur), eyes, femur with knee joint, spinal cord (cervical, thoracic and lumbar cords), extraorbital lacrimal glands. Parts of the liver of the animals of all groups were fixed in Carnoy's solution for routine investigation. After the organs were fixed, histotechnical processing, the examination by light microscopy, immunohistology and the evaluation of findings were performed.

HISTOPATHOLOGY: Yes
Examination of spleen, bone marrow, liver, kidneys, manibular and mesenteric lymphnodes. Immunohistochemistry (Perl's iron reaction, elastic-van-Gieson, actin and desmin, vascular endothelial growth factor (VEGF), BrdU for S phase response, vimentin) and immunohistology (same parameter as for immunihistochemistry) of the spleen. Lung tissue, skeletal muscle and jejunum served as positive controls.

Statistics:

Clinical examinations: A comparision of each group with the control group was performed using the DUNNETT's test (two-sided) for the hypothesis ofequal means.
Clinical pathology: Non parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using MANN-WHITNEY U-test (two sided) for the equal medians.
Pathology/weights: Non parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using the WILCOXON test for the hypothesis of equal medians.
Pathology/immunohistochemistry: Pairwise comparison of each dose group with the control group using the WICOXON test for the hypothesis of equal medians.

Results and discussion

Effect levels

Dose descriptor:

LOAEL

Effect level:

4 other: mg aniline/kg bw/d acutal ingested

Sex:

male

Basis for effect level:

other: erythrotoxicity, effects in the spleen (vascular congestion)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

CLINICAL CHEMISTRY

Both, at the 1- and 4-week interval significantly increased transferrin concentrations and total iron binding capacity (both up to 9.5% increase) were detected in the sera of the high dose animals. No changes were observed in serum iron concentrations.

ORGAN WEIGHTS

1-week administration: The mean absolute spleen weights were increased in the mid (107% of control) and high dose (164% of control) groups: Only the significantly increased value in the high dose group was regarded treatment-related. The other mean absolute weight parameters did not differ significantly as compared to the control group. The mean relative spleen weights were significantly increased in all dose groups (106%, 121% and 186% of control), however, again only in the mid and high dose groups the values were significantly increased. Only the significantly increased values were regarded treatment-related. The other mean relative weight parameters did not differ significantly as compared to the control group.

4-week administration: The mean absolute spleen weights were increased in all dose groups (104, 119 and 207%). However, only in the mid and high dose groups the values were significantly increased. Only the significantly increased values were regarded treatment-related. The other mean absolute weight parameters did not differ significantly as compared to the control group. Comparably to the absolute weights, the mean relative spleen weights were also increased in all dose groups (104, 119 and 204%), however, again only in the mid and high dose groups the values were significantly increased. Only the significantly increased values were regarded treatment-related. The other mean relative weight parameters did not differ significantly as compared to the control group.

HAEMATOLOGY

1-week administration:

100 mg: increases in white blood cells, polymorphonuclear neutrophils, platelets, reticulocytes, Heinz bodies (6/6 animals), methemoglobin and normoblasts and high incidence of anisocytosis (6/6 animals), polychromasia and hypochromasia (6/6 animals) in the peripheral blood; decreased red blood cell count and hematocrit

30 mg: increased platelets and Heinz bodies (6/6 animals); significantly decreased hemoglobin concentrations and mean corpuscular hemoglobin concentration; increased incidence of hypochromasia (2/6 animals)

10 mg: increased haemoglobin adducts and Heinz bodies in 2/6 animals

4-week administration:

100 mg: increased white blood cells, mean corpuscular volume, mean corpuscular hemoglobin, reticulocytes, Heinz bodies (6/6 animals) and methemoglobin and high incidence of polychromasia, hypochromasia (6/6 animals), anisochromasia (6/6 animals) and normoblasts; decreased red blood cells, hemoglobin, hematocrit and mean corpuscular haemoglobin

30 mg: mean corpuscular volume, mean corpuscular hemoglobin, Heinz bodies (6/6 animals) and reticulocytes were increased; red blood cells and hemoglobin were reduced; elevated incidence of hypochromasia (3/6 animals)

10 mg: increased Heinz bodies (4/6 animals); increased formation of hemoglobin adducts, the hemoglobin adducts did not increase linearly with the dose. This could be explained by a saturated process in the formation of the hemoglobin adducts.

GROSS PATHOLOGY

1-week administration:

100 mg: enlarged spleen in 6/6 animals

4-week administration:

100 mg: enlarged spleen in 6/6 animals

HISTOPATHOLOGY: NON-NEOPLASTIC

1-week administration:

100 mg: moderate vascular congestion in the spleen in 6/6 animals; focal perisplenitis in 1/6 animals; hemosiderin deposition in the spleen in 6/6 animals which, in this case, was comparable to control animals

30 mg: vascular congestion in the spleen in 6/6 animals

4-week administration:

100 mg: moderate vascular congestion in the spleen in 6/6 animals; slight to moderate focal perisplenitis in 6/6 animals; in the liver hemosiderin deposition in 6/6 animals and extramedullary hematopoiesis in 1/6 animals; hypercellularity of the femural bone marrow in 6/6 animals

30 mg: slight vascular congestion in the spleen in 6/6 animals

10 mg: minimal vascular congestion in the spleen in 4/6 animals

OTHER FINDINGS

Immunohistology of the spleen: No treatment related effects on the content and distribution of actin, desmin, vimentin and VEGF.

Cell replication in the spleen:

100 mg after 1- and 4-week administration: lymphoid, polymorphnuclear or solitary fibroblastic cells invading the perisplenic foci positive for BrdU