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Diss Factsheets

Administrative data

genetic toxicity in vivo, other
Combined in vivo mammalian erythrocyte micronucleus test in bone marrow and in vivo mammalian comet assay
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: Combined in vivo mammalian erythrocyte micronucleus test in bone marrow and in vivo mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1,4,5,6,7,7-hexachloro-8,9,10-trinorborn-5-ene-2,3-dicarboxylic acid
EC Number:
EC Name:
1,4,5,6,7,7-hexachloro-8,9,10-trinorborn-5-ene-2,3-dicarboxylic acid
Cas Number:
Molecular formula:
1,4,5,6,7,7-hexachlorobicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid
Test material form:

Test animals

Crj: CD(SD)
Details on species / strain selection:
The rat has been selected because there is a large volume of background data in this species and has been specifically requested
Details on test animals or test system and environmental conditions:
- Source:
- Age at study initiation: 7-9 week
- Weight at study initiation: 175-375 g
- Assigned to test groups randomly: yes
- Housing: Wire topped, solid bottom cages
- Diet: 5LF2 EU Rodent Diet ad libitum
- Water: tap water ad libitum
- Acclimation period: At least five days

- Temperature (°C): 19 to 25°C.
- Humidity (%): 40 to 70%
- Air changes (per hr): 15 to 20 air changes/hour
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:
oral: gavage
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: This vehicle has been used in previous in vivo studies with this test article
- Amount of vehicle: 10 mL/kg
Details on exposure:
Formulations were either freshly prepared prior to each dosing occasion in the Range-Finder Experiment or prepared once for the Main Experiment by formulating Chlorendic Acid in corn oil as follows:
The test article was weighed and transferred to a mortar and pestle. A small volume of vehicle was added and mixed to form smooth paste. Further vehicle was added, with mixing, to form a slurry. The mixture was transferred to the formulation bottle and the mortar and pestle rinsed with the vehicle, which was subsequently added (together with any remaining vehicle) to the formulation bottle to achieve the final volume.
Duration of treatment / exposure:
Three days
Frequency of treatment:
Three administrations, at 0, 24 and 45 hours.
Post exposure period:
Not applicable. Animals are sacrificed three hours after the last administration.
Doses / concentrationsopen allclose all
Dose / conc.:
175 mg/kg bw/day (nominal)
Dose / conc.:
350 mg/kg bw/day (nominal)
Dose / conc.:
700 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6 male ratsper dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethyl MethaneSulfonate (EMS) and Vinblastine (VIN)
- Justification for choice of positive controls: In accordance with the OECD TG 474 and 489
- Route of administration: oral gavage (EMS) or Intraperitoneal Injection (VIN)
- Doses / concentrations: 10 mL/kg (EMS), 2.5 or 5 mL/kg (VIN)


Tissues and cell types examined:
Bone marrow (from femur), liver, stomach, duodenum and gonad.
Details of tissue and slide preparation:
Micronucleus Slide Preparation
The isolated femurs were cleaned of adherent tissue and the ends removed from the shanks. Using a syringe and needle, bone marrow was flushed from the marrow cavity with 2 mL foetal bovine serum into appropriately labelled centrifuge tubes. The samples were filtered through cellulose columns, containing 50 mg/mL equal mix of type 50 and α-cellulose. Once the majority of the 2 mL had passed through the column a further 4 mL of serum was added to the sample tubes and loaded onto the columns. Once filtered, the bone marrow cells were pelleted by centrifugation (200 g, 5 minutes, 15-25°C) and the supernatant aspirated and discarded. Cell pellets were gently resuspended in the remaining volume and the cell suspensions from each femur combined per animal. A further 3 mL of foetal bovine serum was added to the tubes. The cells were pelleted again and the supernatant aspirated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube by using a Pasteur pipette, and from each tube one drop of suspension was placed on the end of each of four uniquely labelled slides. A smear was made from the drop by drawing the end of a clean slide along the labelled slide. Slides were air-dried, then fixed for 10 minutes in absolute methanol and rinsed several times in distilled water. Two slides per animal were immediately stained for 5 minutes in 12.5 μg/mL acridine orange made up in 0.1 M phosphate buffer pH 7.4. Slides were rinsed in phosphate buffer, then dried and stored protected from light at room temperature prior to analysis. Unstained slides were air-dried and initially stored at <-10°C with desiccant. Once final results were confirmed the reserve slides were transferred to storage at room temperature.

Preparation of Comet Slides
Three slides, labelled ‘A’, ‘B’ and ‘C’, were prepared per single cell suspension per tissue. Slides were labelled with the study number, appropriate animal tag number and tissue. Slides were dipped in molten normal melting point agarose (NMA) such that all of the clear area of the slide and at least part of the frosted area was coated. The underside of the slides was wiped clean and the slides allowed to dry. 40 μL of each single cell suspension was added to 400 μL of 0.7% low melting point agarose (LMA) at approximately 37°C. 100 μL of cell suspension/agarose mix was placed on to each slide. The slides were then coverslipped and allowed to gel on ice.
Evaluation criteria:
Micronuclei Induction
For valid data, the test article was considered to induce clastogenic / aneugenic damage if:
1. A statistically significant increase in the frequency of MN PCE occurred at one or more dose levels
2. The incidence and distribution of MN PCE exceeded the laboratory’s historical vehicle control data
3. A dose-response trend in the proportion of MN PCE (where more than two dose levels are analysed) was observed.

DNA Damage
For valid data, the test article was considered to induce DNA damage if:
1. A least one of the test doses exhibited a statistically significant increase in tail intensity, in any tissue, compared with the concurrent vehicle control
2. The increase was dose related in any tissue
3. The increase exceeded the laboratory’s historical control data for that tissue.

The test article was considered positive in this study if all the criteria listed for either micronucleus induction and/or DNA damage were met.

The test article was considered negative in this study if for both end points, none of the following criteria were met and target tissue exposure was confirmed.

Results which only partially satisfied the criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example comparison of the response against the historical control data, consistency of response within and between dose levels and any concurrent cytotoxicity information (such as hedgehog assessment, histopathological changes and any clinical chemistry results).
Statistical analysis was conducted.

Results and discussion

Test results
Key result
Vehicle controls validity:
Negative controls validity:
not examined
Positive controls validity:
Additional information on results:
There were no remarkable observations for any animal dosed in the Main Experiment with the exception of mouth rubbing observed immediately after the third dose administration for 1/6 animals at 175 mg/kg/day, 3/6 animals at 350 mg/kg/day and 5/6 animals at 700 mg/kg/day. One animal at 700 mg/kg/day was also noted to exhibit paddling behaviour. There was a decrease in group mean body weight at 700 mg/kg/day between Day 1-Day 3, with percentage change values of -0.1%, compared to +7.3%, +5.3% or +5.6% for the vehicle control group, 175 mg/kg/day group or 350 mg/kg/day group, respectively.

Minimal increases in aspartate transaminase (AST) and alanine transaminase (ALT) activities were recorded for some animals administered 700 mg/kg/day. There were also small increases in chloride, urea and creatinine at 700 mg/kg/day, and small inconsistent (between animals) increases in potassium & calcium (350 and 700 mg/kg/day), phosphate (700 mg/kg/day), urea & creatinine (175 and 350 mg/kg/day).

On microscopic examination, decreased hepatocyte glycogen was noted in the liver of animals administered 700 mg/kg/day. Single cell necrosis was noted in one animal administered 700 mg/kg/day. In the stomach, inflammation and/or decreased mucous were noted in animals administered 350 or 700 mg/kg/day, with a dose relationship.

Applicant's summary and conclusion

Chlorendic Acid did not induce an increase in micronucleated polychromatic erythrocytes of the bone marrow, nor did it induce biologically relevant increases in DNA strand breaks in the liver, stomach, duodenum or gonad of male Sprague Dawley rats when tested up to 700 mg/kg/day (an estimate of the maximum tolerated dose for this study).
Executive summary:

A combined in vivo mammalian erythrocyte micronucleus test in bone marrow and in vivo mammalian comet assay on liver, glandular stomach, duodenum, and gonadal cells was conducted using the degradation product chlorendic acid. The study was GLP-compliant and conducted according to the OECD TG 474 and 489.

A Dose-Range Finding study was conducted, which concluded test concentrations of 175, 350 and 700 mg/kg bw/day shall be used during the main study.
The study was considered as valid based on the criteria laid down in the OECD TG 474 and 489.

Micronucleus Assay
Animals treated with Chlorendic Acid at 175, 350 and 700 mg/kg bw/day exhibited group mean %PCE that were similar to the vehicle control group mean and which fell within the 95% reference range of laboratory’s historical vehicle control data, thus confirming there was no evidence of test article related bone marrow toxicity.
Animals treated with Chlorendic Acid at 175, 350 and 700 mg/kg bw/day exhibited group mean %MN PCE frequencies that were similar to the vehicle control group mean and which fell within the 95% reference range of the laboratory's historical vehicle control data. There were no statistically significant increases in micronucleus frequency for any of the groups receiving the test article, compared to the concurrent vehicle control. In general, individual animal MN PCE frequencies were considered consistent with the laboratory's historical vehicle control animal distribution data.
These data were indicative of a clear negative response and therefore mechanistic analysis was not required.

Comet Assay
There were no marked, dose-related, increases in hedgehogs in liver, stomach, duodenum and gonad, thus demonstrating that treatment with Chlorendic Acid did not cause excessive DNA damage that could have interfered with comet analysis.
However, it was noted that there was an increase in %hedgehogs in the duodenum at 700 mg/kg bw/day that exceeded the 95% reference range of the laboratory’s historical vehicle control range. This increase apparent in all animals in the group was considered as part of data analysis.
Overall these data in the liver, stomach, duodenum and gonad did not meet all of the evaluation criteria in the protocol and the OECD 489 test guideline for a positive result.