4,4'-Methylene-bis-(3-chloro-2,6-diethylaniline)

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:

September - December 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

402-130-7

EC Name:

-

Cas Number:

106246-33-7

Molecular formula:

C21 H28 Cl2 N2

IUPAC Name:

4-[(4-amino-2-chloro-3,5-diethylphenyl)methyl]-3-chloro-2,6-diethylaniline

Details on test material:

- Name/code : P5367
- Appearance : Off-white to yellowish powder
- Storage : At ambient temperature in the dark

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stable

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, F.R.G., Germany
- Weight at study initiation: body weights of the males ranged from 142 to 162 g and those of the females from 111 to 137 g
- Housing: gang housing (6 animals/sex/group/cage) in polycarbonate cages
- Diet: Standard laboratory diet (RMH-0. pellet diameter 9 mm, Hope Farms, Woerden,
- Water: ad libitum
- Acclimatisation: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 21°C
- Humidity: mean 60-70%
- Lightning: artificial light sequence was 12 hours light (7:00 to 19:00), 12 hours dark (19:00 to 7:00).

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Remarks:

mixed with diet

Details on oral exposure:

Method of administration: feeding test (test item mixed with diet); for diet preparation, see "any other information on materials and methods"

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Based on analysis of the stability of the test substance in the diets of the pilot study, it was decided to prepare the diets for the 28-day study only once, i.e. 1 day prior to study start. The sequence for diet mixing and pelleting was control group (no test substance), followed by low dose, medium dose and high dose group. The weighed out test substance was mixed with a fraction of the diet in a Kenwood mixer for 6 minutes resulting in a 3% premix. In a professional dough mixer the appropriate amount of diet was moistened with 7% (w/w) fresh tap-water and was subsequently supplemented by the premix.

DIET PREPARATIONS:
Dosage (ppm) Diet (kg) Test substance (g)
0 25 0
100 25 2.5
300 25 7.5
1000 25 25.0

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Dosing regime: 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

= 0 mg/kg bw/day nominal in diet

Doses / concentrations 2

Dose / conc.:

100 ppm

Remarks:

= 100 mg/kg bw/day nominal in diet

Doses / concentrations 3

Dose / conc.:

300 ppm

Remarks:

= 300 mg/kg bw/day nominal in diet

Doses / concentrations 4

Dose / conc.:

1 000 ppm

Remarks:

= 1000 mg/kg bw/day nominal in diet

No. of animals per sex per dose:

MALES:
- 6 animals at 0 ppm
- 6 animals at 100 ppm
- 6 animals at 300 ppm
- 6 animals at 1000 ppm

FEMALES:
- 6 animals at 0 ppm
- 6 animals at 100 ppm
- 6 animals at 300 ppm
- 6 animals at 1000 ppm

Control animals:

yes, plain diet

Positive control:

no positive control tested

Examinations

Observations and examinations performed and frequency:

Cage-side observations:
With the exception of days 0, 7, 21 and 28, cage-side observations were performed once daily on working days (beginning on day 1) as well as weekends until terminal sacrifice on day 28. Any deviations from normal were recorded. In particular, attention was paid to changes of skin, fur, eyes, mucous membranes, respiratory system. faeces and general appearance. On working days a mortality check was performed in the eveni

Physical examinations:
Once a week, i.e. on days 0 (prior to dosing), 7, 14, 21 and 28 the animals underwent a physical examination. In addition to the parameters mentioned for cage-side observations, particular attention was paid to ears. mouth, urogenital region, anus, abnormal masses, gait, general state and behaviour.

Body weights and food consumption
Individual body weights were determined immediately prior to the first dosing (day 0), weekly thereafter (days 7, 14, 21 and 27), and on day 28 prior to sacrifice (fasted body weight). Individual food consumption was recorded weekly (days 7, 14, 21 and 27) and was expressed as grams food consumed per animal and as grams food consumed per 100 grams body weight per day.

Haematology and clinical chemistry
Prior to sacrifice on day 28 the animals were anaesthetized with ether and the abdominal cavity was opened. Blood was collected via the abdominal aorta by means of an infusion set provided with a winged needle (Mediwing, Argyle. Sherwood Medical Industries, Tullamore, Ireland). For haematology approximately 1.5 ml of blood were collected directly into vials containing 0.03 ml of di- and tripotassium EDTA (30% (W/V), pH 7.4). Immediately thereafter blood samples were collected in clot tubes for clinical chemistry. The vials intended for haematology were rotated at 90 rpm until transportation to 'Bergschot Centrum voor Onderzoek CBCO)' in Breda. The Netherlands for analysis. The maximum period between blood collection and transport to BCO was 5.5 hours. The approximately 3 ml blood samples intended for clinical chemistry were allowed to clot during 30-60 minutes. The serum was separated by centrifugation and transferred into another tube. Serum samples were kept on ice until analysis by BCO.
The red blood cells of the samples were counted using a "Coulter" blood cell counter, while their size distribution was determined using the "Haemalog 6000" (Technicon). Measurement of haemoglobin concentration, counting of white blood cells and subsets thereof, i.e. lymphocytes, neutrophils, large unstained cells, monocytes, eosinophils and basophils, and the counting of thrombocytes were also performed using the Haemalog 6000. The haematocrit values were determined using a microcentrifuge. All clinical chemical parameters were measured in serum samples using a "Parallel" clinical chemistry analyzer.

Sacrifice and pathology:

On completion of blood collection the animals were sacrificed by exsanguination. Subsequently a full gross necropsy was performed which included examination of the external surface and openings, and the cranial, thoracic and abdominal cavities with their contents. Liver, adrenals, kidneys, spleen and testes were removed and weighed.
The following tissues were removed and preserved in 10% formalin: Liver. spleen, kidneys, adrenals. heart and macroscopically abnormal tissues. Fixed organs and tissues from animals of the control and high dose group were embedded in paraffin, sectioned, stained with haematoxylin and azophloxine, and examined microscopically.

Statistics:

Means with standard deviation were calculated for all quantitative parameters. Statistical procedures were carried out for quantitative data suspected of changes and were based on a one-way analysis of variance supplemented with a t-test (Biodata Handling with Microcomputers, Barlow. 1983). One-way analysis of variance (F-test) was used to analyse the results for overall effects of dosage. Significant differences between control and individual dosages were also assessed by using the F-test where P < 0.05 was accepted as the lowest level of significance . The total food consumption represents the group mean of the sum of the individual food consumption over the entire period (day 7, 14. 21 and 27). whereas the body weight gain was obtained by calculating the group mean of the mean individual body weight gain (obtained from a linear regression analysis for the individual body weight over the entire period, i.e. day 0, 7, 14, 21 and 27).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

With respect to the group mean haematology data no evident test substance related effect was observed, except for a statistically significant (P<0.05) increase of thrombocytes in the high dose group (i.e. 18% and 20% increase for males and females, respectively). The remaining haemometric values were comparable for the control group and the treatment groups. In addition, the obtained values were in general agreement with those reported in the literature (Charles River Laboratories. Technical Bull~tin, 1984). Incidental abnormal values were obtained with the following animals. Animals A4 and C4 showed an increased haematocrit value: the latter animal also had an increased number of erythrocytes. Animal B8 showed an increase of erythrocytes and haemoglobin and decreased mean cellular volume (MCV) and MCV/RBC values. With respect to the group mean clinical chemistry data no
statistically significant test substance related effect was observed in sera of male or female animals, with the exception of the calcium concentration that was increased in female animals of the high dose group (6% increase; P=0.05). The obtained values were in general agreement with those reported in the literature. Serum aspartate aminotransferase and alanine aminotransferase were increased in animals A11 and C10.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative liver and adrenal weights were increased in both sexes in the top dose group only.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic examination of all animals at necropsy revealed no evident test substance related gross abnormalities. with exception of dilated renal pelvis (observed once in the low dose group and twice in the high dose group; considered as a congenital abnormality in this rat strain), petechiae of the stomach (observed once in the control group), enlarged adrenals (observed once in low dose group and twice in the high dose group) and petechiae of the thymus (observed once in the high dose group). With the exception of enlarged adrenals in animals of the high dose group, the above findings were considered incidental and not test substance related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examination of the liver of animals dosed at 1000 ppm revealed hypertrophy of centrilobular liver cells.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

SUMMARY:

CLINICAL OBSERVATIONS:
No deaths and no clinical signs of toxicity.

LABORATORY FINDINGS:
Platelet counts were increased in both sexes in the top dose group and serum calcium was slightly increased in top dose females.

EFFECTS IN ORGANS:
Absolute and relative liver and adrenal weights were increased in both sexes in the top dose group only. The only effect seen microscopically was hypertrophy of the centrilobular cells of the liver.

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

Target system / organ toxicity

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

100 ppm

Organ:

bladder

Applicant's summary and conclusion

Conclusions:

Based on the findings it was concluded that a dietary exposure level of 300 ppm (equivalent to 38 mglkglday for males and females combined) represents the No Effect Level for P5367 in the present study.

Executive summary:

The study was performed 1987 as GLP-test following EC-test method B.7 on Sprague-Dawley rats. Application of test material was via diet, the dose levels were 0, 100, 300 and 1000 ppm. No deaths and no clinical signs of toxicity were noted. Laboratory findings included increased platelet counts in both sexes in the top dose group and slightly increased serum calcium in top dose females. Absolute and relative liver and adrenal weights were increased in both sexes in the top dose group only. The only effect seen microscopically was hypertrophy of the centrilobular cells of the liver. In conclusion, the NOEL was found to be 300 ppm (= 38 mg/kg/day).