2,6-dibromo-4-cyanophenyl heptanoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Jun - 19 Sep 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 2018

Deviations:

yes

Remarks:

Mainly missing examination, please refer to "Principles of methods if other than guideline".

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 1981

Deviations:

yes

Remarks:

No guideline was mentioned.

Principles of method if other than guideline:

No functional observation battery performed, no lipid analysis in blood, no ophthalmological examination, no blood clotting parameter measured, no TSH measured, no sperm morphology investigated, the brain was not sectioned for histopathology, endocrine parameters and terminal vaginal cytology were not investigated, the estrus cycle was not determined at necropsy.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,5-dibromo-4-hydroxybenzonitrile

EC Number:

216-882-7

EC Name:

3,5-dibromo-4-hydroxybenzonitrile

Cas Number:

1689-84-5

Molecular formula:

C7H3Br2NO

IUPAC Name:

3,5-dibromo-4-hydroxybenzonitrile

Test animals

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

Commonly used for toxicology testing and recommended by the guideline.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, UK Ltd., UK
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 5 - 6 weeks
- Weight at study initiation: 103 - 113 g (males), 89 - 100 g (females)
- Housing: in groups of up to 5 in modified Type RC1 cages (high density polypropylene body measuring 56x38x18 cm, with stainless mesh floor and lid)
- Diet: laboratory animal diet (Altromin 1321 N), ad libitum
- Water: sterilized tap water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 30 - 80
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 Jun 1985 To: 19 Sep 1985

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): The diet was prepared by mixing 7.56 g of the test substance in about 0.5 kg of powdered basal diet for 10 min in a Kenwood Chef mixer. The mixture was made up to 5 and 10 kg with basal diet, each time mixing for 10 min in a Hobart vertical mixture. This mix served as the 800 ppm diet, lower concentrations were obtained by dilution of the mix.
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity and stability of the test substance in the dietary mixtures were determined from a trial mix before commencement of the study. When mixtures containing 50, 500 and 1000 ppm of the test substance were analyzed, 85.8 - 87.5% recovery was achieved. Homogeneity was established by analysis of six spaced samples from each dose. Stability was determined by reanalysis of the samples from the homogeneity assay 7 and 14 days after preparation. At the end of the study Week 13, content check assays were carried out and the technical grade of the material was assayed in order to establish its active ingredient. The analysis by HPLC showed the folowing concentrations of the test substance in the diet (means from date of mixture - Day 14 and Week 13 in brackets):
10 ppm: 8.72 - 8.92 ppm (10.6 ppm)
50 ppm: 41.2 - 43.5 ppm (42.4 ppm)
400 ppm: 320 - 370 ppm (366 ppm)
800 ppm: 626 - 745 ppm (731 ppm)

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

continously via the diet

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 ppm

Remarks:

corresponding to an actual ingested dose of 0.9 and 0.95 mg/kg bw/day for males and females, respectively.

Doses / concentrations 2

Dose / conc.:

50 ppm

Remarks:

corresponding to an actual ingested dose of 4.4 and 4.9 mg/kg bw/day for males and females, respectively.

Doses / concentrations 3

Dose / conc.:

400 ppm

Remarks:

corresponding to an actual ingested dose of 39.4 and 41.6 mg/kg bw/day for males and females, respectively.

Doses / concentrations 4

Dose / conc.:

800 ppm

Remarks:

corresponding to an actual ingested dose of 91.6 and 92.4 mg/kg bw/day for males and females, respectively.

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily (once daily at weekends and on public holidays)
- Cage side observations: Animals were checked for signs of ill-health, reaction to treatment, morbidity and death.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: each animal was handled and closely examined at least once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: at the first day of treatment, weekly thereafter and before sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each group 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: 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: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: The weight of water consumed by each cage of rats was recorded weekly and the group mean intake was calculated.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: blood was sampled after 11 weeks
- Anesthetic used for blood collection: Yes (under ether anesthesia)
- Animals fasted: Yes (time not specified)
- How many animals: all animals
- Parameters checked: microhematocrit, hemoglobin concentration (Hb), erythrocytes (RBC), reticulocytes, platelet count, differential leucocyte count (WBC, including neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B), monocytes (M)), mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: blood was sampled after 11 weeks
- Animals fasted: Not specified
- How many animals: all animals
- Parameters checked: urea nitrogen, blood creatinine, glucose, creatinine phosphokinase (CPK), alanine aminotransferase (ALT), alkaline phosphatase (ALPH), total bilirubin, total protein, calcium (Ca), phosphorus (P), total thyroxine (T4) and total triiodothyronine (T3).

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The post mortem examination procedure entailed studying all external surfaces, body orifices and the contents of the cranial, thoracic and abdominal cavities.

Organs that were weighed: adrenal glands, brain, heart, kidneys, liver, ovaries or testes, spleen, thyroids (for all animals)

HISTOPATHOLOGY: Yes
Tissues fixed: adrenal glands, bone (sternum incl. marrow and tibia femoral joint), heart, kidneys, liver, lungs, ovaries or testes, spleen, thyroids, uterus and all abnormalities (for all animals).
- Embedding media: Paraffin wax
- Section thickness: 4 -5 µm
- Staining: Hematoxylin and eosin
- examined in all animals: all abnormalities, kidney, liver, lungs
- examined in all animals of the control and high dose group: adrenal glands, bone (sternum incl. marrow and tibia femoral joint), heart, ovaries or testes, spleen, thyroids and uterus.

Any organ showing signs of reaction to treatment in the high dosage group was examined in the low, low intermediate and high intermediate dosage groups.

Statistics:

The statistical significance of differences between treated groups and controls in body weight change, clinical pathology parameters or organ weight was assessed by Student’s ’t’-test, using pooled within-group error variance should the Bartlett's Chi-square statistic test confirm homogeneity of variances. If the variances were non-homogeneous, the Behrens-Fisher test was applied. Analysis of incidences of pathological change was performed by the two-tailed Fisher’s exact test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

All clincial signs observed were incidental and occurred at a frequency commonly seen in normal Fischer rats maintained at these laboratories.

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

Mortality:

no mortality observed

Description (incidence):

No premature death occurred during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 10 and 50 ppm: There were no effects of treatment with the test substance on body weights observed up to and including 50 ppm in males and females.
- 400 ppm: The body weight gain was slightly but statistically significantly reduced during the early part of the study (Week 1, 2 and 4 for males, up to -5.9% and Week 1, 2 and 10 in females, up to -6.3%) but animals subsequently attained similar body weights to those of the controls.
- 800 ppm: statistically significantly reduced body weight gain was observed throughout the study in males and females. The terminal body weight was reduced by -12.4% in males and by -12.6% in females.

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

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- 10 and 50 ppm: There were no effects of treatment with the test substance on food consumption observed up to and including 50 ppm in males and females.
- 400 ppm: The food consumption was slightly increased in males compared to controls; statistically significant in Week 3, 4, and 9 (up to +10.6%). No effect was observed in females.
- 800 ppm: An elevated food consumption was observed in males compared to controls; statistically significant compared to controls in Week 7 - 9 (up to +14.9%). In females, the food consumption was also increased in several weeks compared to controls but the difference did not reach statistical significance.

Other statistically significant differences in the 50 ppm group (males and females in Week 9) were not considered toxicologically relevant since these were isolated findings and the difference was small.

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

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

- 10, 50 and 400 ppm: There were no effects of treatment with the test substance on food efficiency observed up to and including 400 ppm in males and females.
- 800 ppm: The food conversion ratio was markedly lower compared to controls during the first two weeks of treatment. In males, the food conversion was 0.01 and 0.012 in Week 1 and 2, respectively (control: 0.3 and 0.25 for Week 1 and Week 2, respectively) and in females -0.02 and -0.07 in Week 1 and Week 2, respectively (control: 0.23 and 0.14 in Week 1 and 2, respectively).

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

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

no effects observed

Description (incidence and severity):

No differences between control and treatment groups regarding water consumption were observed.

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant treatment-related findings were observed in males and females of treatment groups up to the highest dose level.

Not treatment related results:
- 10 ppm: Statistically significant differences were increase in the mean cell hemoglobin concentration in males (-4.5%) compared to controls. Mean cell erythrocyte volume was statistically increased in males (+4.2%) and decreased in females (-2.2%) compared to controls. Since these findings did not show dose-dependency, they were not considered toxicologically relevant.
- 50 ppm: Statistically significant differences were increased packed cell volume (+3%), hemoglobin concentration (+2.5%) and total erythrocyte count (+2.3%) in females. Since these findings were slight and did not show dose-dependency, they were not considered toxicologically relevant.
- 800 ppm: Statistically significant differences were increased packed cell volume and hemoglobin concentration in males (+4.3 and +5.0%, respectively) and females (+5.0 and +6.2%, respectively), increased total erythrocyte count in females (+5.0%), increased mean cell erythrocyte volume in males (+3.5%), increased mean cell hemoglobin concentration in females (+1.4%), reduced number of platelets in females (-12.5%). However, these changes were minor (mainly below 10%) and therefore not considered treatment related.

The increase in mean cell erythrocyte volume in males explains the lack of increase in the erythrocyte count in spite of the elevated packed cell volume.

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

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- 10 and 50 ppm: No toxicologically relevant and/or treatment-related findings regarding clinical chemistry were observed in males and females of treatment groups up to and including 50 ppm.
- 400 ppm: Statistically significant differences were decreased T4 in males (-81.2%) and females (-45.2%), increased ALPH in females (+13.9%), increased creatinine in females (+8.0%) all compared to controls.
- 800 ppm: Statistically significant differences were decreased T4 in males (-79.7%) and females (-32.3%), decreased T3 in males (-23.1%) and females (-17.6%), increased ALPH in males (+15.9%) and females (+43.8%), increased urea in males (+11.5%) and females (+22.1%) and creatinine in females (+6.8%), all compared to control. In males, urea levels were slightly raised along with a small but definite increase in the creatinine level but did not reach statistical significance.

Not considered treatment related:
- 10 ppm: Statistically significant differences were decreased T4 in males (-62.5%) and females (-51.6%), decreased T3 in females (-19.0%), all compared to controls.
- 50 ppm: decreased T4 in males (-75%) and females (-54.8%), decreased T3 in males (-29.5%), increased ALPH in females (+19%), increased fasted glucose in females (+8.4%), all compared to controls.
These effects were considered minor and non-dose related as there were no concurrent gross pathological or histopathological findings and therefore not toxicologically significant.

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

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

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):

- 10 and 50 ppm: No toxicologically relevant and/or treatment-related findings regarding organ weights were observed in males and females of treatment groups up to and including 50 ppm.
- 400 ppm: The absolute (17.4%) and relative (+19.6%) liver weight was statistically significantly increased in females, absolute (-13.9%) and relative (-12.2%) thyroid weight was statistically significantly reduced in females, all compared to the control group.
- 800 ppm: The absolute (+15.0%)and relative (+28.9%) liver weight was statistically significantly increased in females and the relative liver weight was statistically significantly increased in males (+12.3%). The absolute thyroid weight (but not the relative weight) was statistically significantly reduced in females (-14.6%), all compared to the control group.

Not considered toxicologically relevant:
- 10 ppm: The absolute (-12.6%) and relative (-13.4%) thyroid weight was statistically significantly reduced in females compared to the control group.
- 50 ppm: The absolute (-12.6%) and relative (-14.1%) thyroid weight was statistically significantly reduced in females compared to the control group.
These changes were seen as an adaptive response and therefore not considered toxicologically relevant.

All other statistically significant differences between treated groups and the controls may be considered to be a result of the intergroup disparities in body weight or to be incidental.

Summarized results can be found in Attachment 7 in the attached background material.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- 10, 50 and 400 ppm: There were no effects of treatment with the test substance observed up to and including 400 ppm in males and females.
- 800 ppm: reduced abdominal fat pads were observed in 4/10 females compared to the control group, no treatmen-related findings were observed in males.

Not treatment-related:
Other findings such as lobar herniation in the liver, uterine distension and para-ovarian cysts are commonly seen in Fischer rats of this age and were therefore not related to treatment.

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver:
- 10 and 50 ppm: No treatment related changes were observed up to and including 50 ppm.
- 400 ppm: A decrease in cytoplasmic basophilic granularity associated with an increase in cytoplasmic eosinophilic fine granular material and cell volume was noted in males and females.
- 800 ppm: A decrease in cytoplasmic basophilic granularity associated with an increase in cytoplasmic eosinophilic fine granular material and cell volume was noted in males and females.

Uterus:
- 10 and 50 ppm: No treatment-related changes were observed up to and including 50 ppm.
- 400 ppm: atrophy as manifested by reduced stromal ground substance with closely packed stromal cells was noted in females compared to the control group.
- 800 ppm: atrophy as manifested by reduced stromal ground substance with closely packed stromal cells was noted in females compared to the control group.

Thyroids:
- 10, 50, 400 ppm: No treatment-related changes were observed up to and including 400 ppm.
- 800 ppm: An absence of follicles lined by flattened epithelium (males) was observed in most male animals. In these animals, all follicles were lined by cuboidal to columnar epithelium. No such signs were found in females.

Two animals of the control and 400 ppm dose groups showed a similar epithelial pattern in which all follicles were lined by cuboidal to columnar epithelium.

Bone marrow:
- 10 and 50 ppm: No treatment-related changes were observed up to and including 50 ppm. One animal of the control group showed a slight reduction in bone marrow cellularity.
- 400 ppm: 3/10 females showed slight to moderate reduced cellularity, no treatment-related findings were found in male animals.
- 800 ppm: 9/10 females showed slight to moderate reduced cellularity, 5/10 males showed a slight reduction in bone marrow cellularity.

Not considered toxicologically relevant:
Liver:
- 50 ppm: A decrease in cytoplasmic basophilic granularity associated with an increase in cytoplasmic eosinophilic fine granular material and cell volume was observed in males and females. However, since there were no other effects observed at 50 ppm indicating a hepatotoxic effect, this was not considered toxicologically relevant.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

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

Remarks on result:

other: corresponding to 4.4 and 4.9 mg/kg bw/day in males and females, repectively.

2

Key result

Dose descriptor:

LOAEL

Effect level:

400 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: corresponding to 39.4 and 41.6 mg/kg bw/day in males and females, repectively.

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

400 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

All reported results were considered treatment related and toxicological relevant, if not stated otherwise.

Applicant's summary and conclusion

Conclusions:

In this study, the toxicity of the test substance was investigated in rats during and after oral administration of dietary concentrations of the test substance of 10, 50, 400 and 800 ppm for 90 days. The study was not conducted according to any guideline and no GLP compliance was mentioned. However, it was similar to OECD guideline 408 (1981) and is considered valid.
Body weight, food consumption, hematological and clinical chemistry parameters were altered after treatment with the test substance at 400 ppm or above. Treatment-related effects were observed in the liver, bone marrow, uterus and thyroid. Therefore, the NOAEL was set at 50 ppm, corresponding to 4.4 and 4.9 mg/kg bw/day in males and females, repectively. The LOAEL was set at 400 ppm (39.4 mg/kg bw/day for females and 41.6 mg/kg bw/day for males).