2,6-dibromo-4-cyanophenyl octanoate

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:

01 Feb - 11 Apr 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:

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:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Limited, Kent, UK
- Age at study initiation (MNT part): 7 - 8 weeks
- Weight at study initiation (MNT part): 23 - 37 g males and 18 - 31 g females
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: individually in polypropylene and stainless steel cages measuring 48 cm x 15 cm x 13 cm.
- Diet: SOS Rat and Mouse Maintenance Diet No. 1 (Special Diet Services Limited, Essex, UK), ad libitum
- Water: ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 21
- Humidity (%): 37 - 69
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: (MNT part) from: 11 Mar 1991 To: 14 Mar 1991

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle/solvent used: corn oil (Mazzola R)
- Amount of vehicle: 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Immediately prior to dosing, the test compound was dissolved in corn oil to give the required test concentration. All dosing solutions were kept on a magnetic stirrer during the dosing procedures.

Duration of treatment / exposure:

Single treatment

Frequency of treatment:

Once

Post exposure period:

Test substance:
- low dose: 24 h
- mid dose: 24 h
- high dose: 24, 48, 72 h
Control animals: 24, 48, 72 h
Positive control: 24, 48, 72 h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

52 mg/kg bw (total dose)

Remarks:

for male mice

Doses / concentrations 2

Dose / conc.:

105 mg/kg bw (total dose)

Remarks:

for male mice

Doses / concentrations 3

Dose / conc.:

183 mg/kg bw (total dose)

Remarks:

for male mice

Doses / concentrations 4

Dose / conc.:

76 mg/kg bw (total dose)

Remarks:

for female mice

Doses / concentrations 5

Dose / conc.:

153 mg/kg bw (total dose)

Remarks:

for female mice

Doses / concentrations 6

Dose / conc.:

267 mg/kg bw (total dose)

Remarks:

for female mice

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 doses were selected based on a dose range-finding test with 5 groups of 2 male and female CD-1 mice. Mice were orally treated once with doses ranging from 100 to 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. In the main toxicity test, 4 groups of 5 male and 5 female mice were orally treated on a single occasion with doses ranging from 125 to 500 mg/kg bw. Analogously to 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. All animals were examined for gross pathology. 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 the high dose of the test substance. The 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.
The main 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 Brue and Heddle (1979) and laboratory historical control data. Based on a mean frequency of micronucleated PCE of 0.128% per mouse (Salamone et al. 1980a), a positive response was suspected if the total numbers of micronuclei within any one sample group of a given number of mice exceeded 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. A negative response was recorded if the number fell below the corresponding historical control data. The cumulative historical micronucleus incidence in vehicle control dosed mice (distilled water, corn oil or 0.5% carboxymethylcellulose) in this laboratory has been determined as 0.121%, which is in line with the mean frequency of micronucleated PCE of 0.128% per mouse by Salamone et al.

Statistics:

Statistical analysis was not performed

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Two females treated with 267 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
Treatment-related deaths were observed at 250, 400, 550 and 700 mg/kg bw. No abnormal clinical signs were observed prior to deaths. No unusual findings apart from bright red lungs in one male and one female mouse were observed by gross post mortem examination. Based on the results, animals in the main toxicity test were dosed with 125, 250, 375 and 500 mg/kg bw of the test substance.

RESULTS OF THE MAIN CYTOTOXICITY TEST
No deaths occurred at 125 mg/kg bw, whereas deaths were observed in the higher dose groups as follows:
250 mg/kg bw: 1/5 males and 0/5 females
375 mg/kg bw: 5/5 males and 3/5 females
500 mg/kg bw: 5/5 males and 4/5 females
These deaths were preceded by dose-related clinical signs comprising reduced activity, piloerection and subdued behavior (please also refer to the attachment 2). However, no changes were observed at the gross post mortem examination. Based on these results, the LD50 values were calculated for males (262 mg/kg bw) and for females (382 mg/kg bw). Therefore, the highest dose used in the micronucleus test was 183 mg/kg bw for males and 267 mg/kg bw for females (equal to 0.7 x LD50 dosages). For details, please refer to the attachment 2.

RESULTS OF THE MICRONUCLEUS TEST
- Clinical signs: No adverse reactions were observed following treatment.
- Mortality: 2 female mice died following exposure to 267 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 difference were observed between control and treatment groups. The highest frequency of micronucleated polychromatic erythrocytes observed in the test groups was 0.16%, which was identical to the highest vehicle control frequency.
- Ratio of PCE/NCE: No difference were observed between control and treatment groups at any time point.
- Positive control: Substantial induction of bone marrow micronuclei were observed in animals treated with 80 mg/kg bw cyclophosphamide, notably in samples taken 24 and 48 h after dosing. The positive control also caused a decline in PCE/NCE ratios compared to the vehicle control.
- Vehicle control: The numbers of micronucleated bone marrow polychromatic erythrocytes 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 and compliant with GLP. 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 time protocol.