2,6-dibromo-4-cyanophenyl octanoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 Sep - 10 Dec 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Version / remarks:

adopted 1998

Deviations:

yes

Remarks:

only 2 dogs per sex per dose used, no detailed clinical observations noted, liver was weighed without gall bladder, ornithite decarboxylase was not determined, microscopic exmination of limited organs/tissues of control animals

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,6-dibromo-4-cyanophenyl octanoate

EC Number:

216-885-3

EC Name:

2,6-dibromo-4-cyanophenyl octanoate

Cas Number:

1689-99-2

Molecular formula:

C15H17Br2NO2

IUPAC Name:

2,6-dibromo-4-cyanophenyl octanoate

Test animals

Species:

dog

Strain:

Beagle

Details on species / strain selection:

The beagle dog is a commonly used non-rodent species for toxicity studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Interfauna UK Ltd., Huntingdon, UK
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 19 - 21 weeks
- Weight at study initiation: 6.4 - 10.0 kg
- Fasting period before study: no
- Housing: kennels, floor area of 4.5 m², with graded whitewood sawdust used as litter, up to 2 animals per kennel (same sex and dose group)
- Diet: standard dry diet (Diet A: Special Diets Services Ltd.), 400 g per animal per day
- Water: tap water, ad libitum
- Acclimation period: at least 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 24
- Humidity (%): not reported
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 9 Sep 1992 To: 10 Dec 1992

Administration / exposure

Route of administration:

oral: capsule

Vehicle:

other: gelatine capsules

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Once each week, after the animals had been weighed the dose to be given to individual animals was calculated according to body weight. The required quantities of test substance were weighed out, and inserted into gelatine capsules, (Parke-Davis, profit gelatine capsules size 000) for the next week. Animals of the control group received gelatine capsules without the test substance.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.43 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

1.43 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

7.14 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

2

Control animals:

yes

Details on study design:

- Dose selection rationale: The dosages were chosen by the Sponsor with reference to available toxicological information on the test susbtance and with reference to previous studies with the read across source substance.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: regularly throughout the day

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: prior to the beginning of treatment, once weekly during the treatment period and before termination

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the start of treatment and during Week 13
- Dose groups that were examined: all animals
- Procedure: the eyes of all animals were examined by means of a Keeler indirect ophthalmoscope. Prior to examination the pupils of each animal were dilated using a Tropicamide ophthalmic solution ("Mydriacyl").

HAEMATOLOGY: Yes
- Time schedule for collection of blood: once before dosing commenced and then during Weeks 6 and 13.
- Anesthetic used for blood collection: No
- Animals fasted: Yes (overnight)
- How many animals: all animals of all groups
- Parameters checked: Packed cell volume (PCV), hemoglobin (Hb), red cell count (RBC), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), total white cell count (WBC Total), platelet count (Plts), reticulocyte count (Retic), differential WBC count (including counts of neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B), monocytes (M)), methemoglobin (MetHb), prothrombin time (PT), and activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once before dosing commenced and then during Weeks 6 and 13.
- Animals fasted: Yes (overnight)
- How many animals: all animals of all groups
- Parameters checked: total protein, albumin (Alb), globulin (Glob), urea nitrogen (Urea nitr), creatinine (Creat), sodium (Na), potassium (K), calcium (Ca), inorganic phosphorus (P), chloride (Cl), glucose, alkaline phosphatase (AP), alanine aminotransferase (ALAT, in tables GPT), aspartate aminotransferase (ASAT, in tables GOT), total bilirubin, and cholesterol (Chol).

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes
- Time schedule for collection of urine: once before dosing commenced and then during Weeks 6 and 13.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes (food overnight, water 5 h prior to the start of collection)
- Parameters checked: Volume (Vol), pH, specific gravity (SG), protein (Prot), glucose, ketones, bile pigments, urobilinogen, hem pigments, microscopic examinations (epithelial cells (E), polymorphonuclear leucocytes (P), mononuclear leucocytes (M), erythrocytes (R), organisms (O), renal tube casts (C), and other abnormal constituents (A)).

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER:
Rectal temperatures
- Time schedule: twice before dosing commenced and then at 2, 4, 6 and 24 h after dosing on Days 1, 2 and 3 of dosing and on a suitable day during Weeks 5, 9 and 13 of dosing.

Bone marrow:
Prior to autopsy, bone marrow was obtained from each animal where practicable by sternal puncture and a smear prepared for qualitative examination.

Sacrifice and pathology:

Surviving animals were sacrificed by exsanguination under pentobarbitone anesthesia. The animals were starved overnight prior to post mortem examination.

GROSS PATHOLOGY: Yes
Organs weighed (paired organs being weighed separately): adrenals, brain, heart, kidneys, liver, lungs, pancreas, pituitary, spleen, testes (with epididymides) or ovaries, thymus, thyroids, and uterus or prostate.

HISTOPATHOLOGY: Yes
- fixed tissues: adrenals, alimentary tract (esophagus, stomach body and antrum, duodenum, jejunum, ileum, cecum, colon, rectum), aorta (arch and abdominal), brain (cerebral cortex, thalamic nuclei, mid-brain, medulla and cerebellum), eyes, femur (with articular surface), gall bladder, heart, kidneys, liver, lungs (with bronchi), lymph nodes (cervical and mesenteric), mammary gland, ovaries, pancreas, pituitary, prostate, salivary gland (submandibular), sciatic nerve, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar regions), spleen, sternum (with marrow), testes (and epididymides), thymus, thyroids (and parathyroids), tongue, trachea, urinary bladder, uterus, vagina, and macroscopically abnormal tissues.
Tissues were fixed in 10% buffered formalin. The eyes were preserved in Davidson's fixative whilst additional pieces of liver and kidney were placed in formol calcium. A small piece of the liver was fresh frozen in hexane cooled in an acetone bath containing solid carbon dioxide.
- embedding media: paraffin wax
- thickness of sections: 4 µm
- staining: hematoxylin and eosin (all tissues), Periodic acid Schiff (PAS, fresh frozen liver sections to analyze glycogen)

For cryocuts:
- tissues: liver fixed in formol calcium
- thickness of sections: 12 µm
- staining: Oil red O

Animals investigated:
- 7.14 mg/kg bw/day: all tissues listed above
- all animals of all other dose groups and the control: kidneys, liver, thyroids (and parathyroids) and macroscopically abnormal tissues.

Optional endpoint(s):

None

Other examinations:

None

Statistics:

Data from male and female animals were analyzed both together and separately.

If the data consisted predominantly of one particular value (relative frequency of the mode exceeded 95%), the proportion of animals with values different from the mode were analyzed by Fisher's exact test followed by Mantel's test for a trend with proportion, if appropriate.

Otherwise the Bartlett's test was used to test for heterogeneity of variance between treatments. Where significant (at the 1% level) heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.

If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out. If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks was used.

For pre-dose data, analyses of variance were followed by Student's t-test. For data from the dosing
period, Williams' test for a dose-related response was used. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the t-test and Williams' test (Shirley's test).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were recorded that could be attributable to treatment. A low incidence of liquid feces seen in treated animals was similar to that in the controls and was therefore considered not relevant.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 0.43 and 1.43 mg/kg bw/day: No difference regarding body weight and body weight gain between control and treatment groups up to and including 1.43 mg/kg bw/day.
- 7.14 mg/kg bw/day: statistically significantly decreased mean body weight gain from Week 10 onwards compared to animals of the control group. The terminal body weight was also statistically significantly decreased compared to the control group.

Observations that were not statistically significant:
Mean body weight gain at Week 13 for animals receiving 0.43 and 1.43 mg/kg bw/day were lower than for the controls, the differences were not statistically significant and, at 0.43 mg/kg bw/day the reduced mean body weight gain was due to a single animal.

Summarized results can be found in Attachment 1 in the attached background material.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment-related differences regarding food consumption were observed between control and treatment groups.

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related differences regarding ophthalmological findings were observed between control and treatment groups. Incidental findings observed were consistent with the age and strain of animal used.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- 0.43 and 1.43 mg/kg bw/day: No treatment-related differences regarding hematology were observed between control and treatment groups up to and including 1.43 mg/kg bw/day.
- 7.14 mg/kg bw/day: mean values for red cell parameters (PCV, Hb and RBC) were decreased in Weeks 6 and 13 but the difference did not reach statistical significance.

Summarized results can be found in Attachment 2 in the attached background material.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant differences regarding clinical chemistry were observed between control and treatment groups. In Week 6 and 13, mean phosphorus value were decreased in the mid and high dose group, reaching statistical significance in Week 6. However, since the differences were only slight and as individual values were within the background range this finding was considered of no toxicological importance. Other statistical significant intergroup differences were altered glucose and calcium levels at Week 13, but the changes were either slight or reflected trends already present pre-dose and these findings were considered to be of no toxicological importance.

Summarized results can be found in Attachment 3 in the attached background material.

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

no effects observed

Description (incidence and severity):

No treatment-related differences regarding urinalysis were observed between control and treatment groups.

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- 0.43 and 1.43 mg/kg bw/day: No treatment-related differences regarding organ weights were observed between control and treatment groups up to and including 1.43 mg/kg bw/day.
- 7.14 mg/kg bw/day: statistically significantly increased absolute liver weight, individual relative liver weights for all animals were in excess of the concurrent control range, and for 3 of the animals were near to or in excess of the laboratory's normal upper limit of 4% body weight.

Not considered treatment-related:
- 0.43 mg/kg bw/day: statistically significant increase in relative brain weight compared to the control group (considered an anomaly and in part due to lower body weights)
- 1.45 mg/kg bw/day: mean relative liver weight was statistically significantly increased compared to the control group (not considered treatment-related since there was no dosage relationship, and no histopathological lesions or blood biochemical changes to account for this weight difference), statistically significant increase in relative brain weight compared to the control group (considered an anomaly and in part due to lower body weights).
- 7.14 mg/kg bw/day: mean relative kidney weight was statistically significantly increased compared to the control group (not considered treatment related since there was no dose-relationship, and no histopathological lesions or blood biochemical changes to account for this weight difference), statistically significant increase in relative brain weight compared to the control group (considered an anomaly and in part due to lower body weights).

Summarized results can be found in Attachment 4 in the attached background material.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic findings which could be attributed to treatment.

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- 0.43 and 1.43 mg/kg bw/day: No treatment-related differences regarding histopathology were observed between control and treatment groups up to and including 1.43 mg/kg bw/day.
- 7.14 mg/kg bw/day: Minimal centrilobular hepatocyte enlargement (1/2 male dogs).

The minimal spermatogenesis in the testes and corresponding reduced spermatozoa in the epididymides (2/2 male of the high dose group) were considered to represent incomplete normal sexual maturation and to be consistent with the age of the dogs.

Summarized results can be found in Attachment 5 in the attached background material.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

no effects observed

Description (incidence and severity):

Rectal temperature
No treatment-related differences regarding rectal temperature were observed between control and treatment groups. In the high dose group, statistically significant increase in rectal temperature was observed in Week 5 compared to the control group but this could not be attributed to the test substance. Additionally, no change of rectal temperature was observed at any other time point.

Summarized results can be found in Attachment 6 in the attached background material.

Bone marrow smears
There were no abnormalities of cellularity, distribution or morphology in any animal.

Details on results:

Dose Groups as described in some of the tables:
Group 1: control
Group 2: 0.43 mg/kg bw/day
Group 3: 1.43 mg/kg bw/day
Group 4: 7.14 mg/kg bw/day

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

1.43 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed at this dose level.

2

Key result

Dose descriptor:

LOAEL

Effect level:

7.14 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

7.14 mg/kg bw/day (actual dose received)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

The present study was conducted to assess the effects of the test substance on Beagle dogs when given for a time frame of approximately 13 weeks. The study was similar to the OECD guideline 409 (dated 1981) and was performed under GLP conditions. Beagle dogs received the test substance via gelatin capsules at doses of 0.43, 1.43 and 7.14 mg/kg bw/day.

Under the conditions of the test, the test substance caused reduced body weight gain and changes in hematology in treated animals compared to controls at 7.14 mg/kg bw/day. Increased liver weights were observed in the 7.14 mg/kg bw/day group and minimal centrilobular hepatocyte enlargement in a single animal of this group.
Based on the described effects the NOAEL could be set at 1.43 mg/kg bw/day for male and female Beagle dogs.