2,6-dibromo-4-cyanophenyl heptanoate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Jun - 26 Jul 1991

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:

1991

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 1983

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 2016

Deviations:

yes

Remarks:

No justification for route, duration and choice of vehicle provided, historical control data given without standard deviations, highest dose did not produce toxicity in bone marrow, only 1000 instead of 4000 immature erythrocytes per animal scored

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

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:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: supplied by Charles River Limited, Kent, UK
- Age at study initiation: 7 - 8 weeks
- Weight at study initiation: 25 - 36 g males and 21 - 29 g females
- Assigned to test groups randomly: yes, computer-generated randomization was used for the micronucleus study
- Fasting period before study: none
- Housing: individually in polypropylene and stainless steel cages measuring 48 cm x 15 cm x 13 cm
- Diet: SDS Rat and Mouse Maintenance Diet No. 1 (Special Diet Services Limited, Essex, UK), ad libitum
- Water: ad libitum
- Acclimation period: at least 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 21
- Humidity (%): 37 - 65
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle/solvent used: Corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Immediately prior to dosing, the test compound was suspended in corn oil. The dose volume used for the control and test compound treated animals was a constant 10 mL/kg bw.

Duration of treatment / exposure:

Single treatment

Frequency of treatment:

Once

Post exposure period:

Test substance:
- 35 mg/kg bw: 24 h
- 70 mg/kg bw: 24 h
- 105 mg/kg bw: 24, 48, 72 h
Control animals: 24, 48, 72 h
Positive control: 24, 48, 72 h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

35 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

70 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

105 mg/kg bw (total dose)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: gavage
- Doses / concentrations: 80 mg/kg bw
Cyclophosphamide was prepared fresh as an 8 mg/mL solution in distilled water and administered to the positive control animals in standard dose volumes of 10 mL/kg bw.

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The dose selection was based on a dose range-finding study. 5 groups of 2 male and female CD-1 mice were dosed orally with 100, 250, 400, 550 and 700 mg/kg bw of the test substance. These dose level were chosen based on acute oral LD50 information in rats. The mice were observed for clinical signs or mortality at frequent intervals (1 min, 0.5 h, 1 h, 2 h and 4 h) post dosing, then daily until the end of the observation period. Surviving animals were killed 14 days after dosing, by C02 asphyxiation. Gross pathology was performed on all animals, following compound-induced death or scheduled kill. Based on the results, animals in the main toxicity test were dosed with 50, 125, 200 and 275 mg/kg bw of the test substance. In the main toxicity test, groups of 5 male and 5 female mice were orally treated on a single occasion. Following a similar test procedure as in the range-finding test, animals were observed at the time of dosing and at 1 min, 0.5 h, 1 h, 2 h and 4 h after treatment, then daily until the end of the observation period for clinical signs or mortality. Surviving animals were killed 14 days after dosing by C02 asphyxiation. Based on the results of this study, the doses for the micronucleus test were selected.

TREATMENT AND SAMPLING TIMES:
In the micronucleus test, 5 groups of male and female CD-1 mice were dosed once at 0 h with test or control agents, then marrow samples taken at time intervals of 24 h. In addition, bone marrow was also prepared after 48 and 72 h from animals treated with the positive and vehicle control and 105 mg/kg bw of the test substance. Mice were killed by cervical dislocation.

DETAILS OF SLIDE PREPARATION:
The femora were dissected out and freed of adherent tissue. A small hole was made in the neck of one femur and the marrow flushed, using a 1 mL syringe fitted with a gauge 25 needle, into a heparinized centrifuge tube containing 3 mL of a 1:1 mixture of fetal calf serum and 0.8% trisodium citrate in Sorenson's buffer, pH 6.8. This mildly hypotonic treatment served to make the micronuclei clearly visible and to distinguish them from surrounding artefacts. The contents of the tubes were briefly agitated on a vortex mixer to allow separation of the cells. The tubes were centrifuged to pellet the cells. All but a few drops of supernatant fluid was discarded. The cells were then resuspended on a vortex mixer in this residual amount of supernatant liquid. A drop of the suspension was placed at one end of the slide and a smear made by drawing the top of a Pasteur pipette horizontally along the slide. Two slides were prepared from each tube and animal. The smear was left to air dry, fixed in methanol and stained with 1% May-Grunwald in methanol and Sorenson's buffer and counterstained in 15% Giemsa (Gurr) in Sorenson's buffer. The stained smears were rinsed in 3 changes of distilled water and air dried. Finally, the smears were cleared in Histo-clear and permanent slide preparations obtained by sealing glass coverslips onto the microscopic slides using DPX mountant.

METHOD OF ANALYSIS:
Micronuclei were analyzed with a light microscope. 1000 polychromatic erythrocytes (PCE) were counted per animal. The number of micronucleated normochromatic erythrocytes (NCE) was also recorded. The PCE/NCE ratio was determined for each animal by counting the number of immature (PCE) per mature (NCE) erythrocytes in a minimum total of 500 cells (PCE + NCE).

Evaluation criteria:

Evaluation of the results were based on Salamone et al. 1980 and Tamura et al. 1990 and laboratory historical control data. Based on a mean frequency of micronucleated PCE of 0.128% per mouse (Salamone et al. 1980a) and 0.122% in CD-1 mice for both sexes combined (Tamura et al. 1990), a positive response was suspected if the total numbers of micronuclei within any one sample group of a given number of mice exceeded the criteria adopted from Salamone et al. (1980) or the corresponding value of the historical control data (shown in attachment 1).
A micronucleus within a polychromatic or normochromatic erythrocyte was defined as a distinct Giemsa-positive body of evident chromosomal origin and enclosed by the cytoplasm of the harboring cell.

Statistics:

Statistical analysis was not performed.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

One male treated with 105 mg/kg bw died, but no clinical signs or changes in the PCE/NCE ratios were observed.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
The dose selection was based on a dose range-finding study. 5 groups of 2 male and female CD-1 mice were dosed orally with 100, 250, 400, 550 and 700 mg/kg bw of the test substance. Treatment-related deaths were observed at all doses examined. No abnormal clinical signs were observed prior to death. Gross pathology revealed no compound related findings except autolysis in one female and bright red lungs in two females. Based on the results, animals in the main toxicity test were dosed with 50, 125, 200 and 275 mg/kg bw of the test substance.

RESULTS OF THE MAIN TOXICITY TEST
In the main toxicity test, groups of 5 male and 5 female mice were orally treated on a single occasion. No deaths occurred at 50 mg/kg bw, whereas deaths were observed in the higher dose groups as follows:
125 mg/kg bw: 1/5 males and 1/5 females
200 mg/kg bw: 4/5 males and 3/5 females
275 mg/kg bw: 5/5 males and 3/5 females
These deaths were preceded by dose-related clinical signs comprising reduced activity, piloerection, subdued behavior and hunched appearance (please also refer to the attachment 2). However, no changes were observed at the post mortem examination (14 days after treatment). Based on these results, the LD50 values were calculated for males (157 mg/kg bw) and for females (205 mg/kg bw). Therefore, the highest dose used in the micronucleus test was 105 mg/kg bw. For details, please refer to the attachment 2.

RESULTS OF THE MICRONUCLEUS TEST
- Clinical signs: No adverse reactions were observed following treatment.
- Mortality: One male died following exposure to 105 mg/kg bw of the test substance.
- Body weight: There was no evidence of body weight loss in the treated mice compared to the control animals.
- Induction of micronuclei: No differences were observed between control and treatment groups.
- Ratio of PCE/NCE: No differences were observed between control and treatment groups.
- Positive control: Substantial induction of bone marrow micronuclei at all 3 sampling times were observed in animals treated with 80 mg/kg bw cyclophosphamide. The positive control also caused a decline in PCE/NCE ratios and therefore affected normal cellular proliferation.
- Vehicle control: The numbers of micronucleated bone marrow polychromatic erythrocytes (MN-PCE) in the control mice were within the in-house historical control range for negative control exposed mice at all experimental points.
For details, please refer to the attachment 3.

Applicant's summary and conclusion

Conclusions:

The study was performed according to OECD guideline 474 (1983) and compliant with GLP. Despite a number of deviations in regard to the current version of the OECD guideline 474 (2016), the study is considered acceptable. Under the conditions of this study, the test substance was found to be devoid of micronucleus inducing potential when tested to lethal doses in male and female CD-1 mice using a single oral exposure and multiple sampling times protocol.