Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2001-06-19 - 2001-09-10
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Rationale for reliability of 2: Guideline study, well-performed and well-documented, read-across Justification of read-across: the registration substance and the read-across supporting substance belong to homolog series of (Polypropylensuccinimido)-caproic acid.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
chromosome aberration assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
Molecular formular: C22H37NO4
Molecular weight: 379 g/mol
Batch number: 137305500
Apperance: clear brown liquid
Purity: 100%
Vapor pressure: 21.6 kPa at 100°C
Solubility in water: < 1g/L

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: male mean =193.6 g; female: mean = 149.3 g
- Assigned to test groups randomly: yes
- Housing: in fully air-conditioned rooms in makrolon cages type 4 (five animals per cage) on soft wood granulate
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days under study conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 20 %
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 hours daily

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: sesame oil
Frequency of treatment:
two applications at an interval of 24 hours
Post exposure period:
24 h
Doses / concentrations
Remarks:
Doses / Concentrations:

Basis:
actual ingested
500, 750, 1000 mg/kg bw
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
positive control: CPA (Endoxan@, batch no. 9M642A) containing cyclophosphamide, dissolved in distilled water
- Route of administration: gavage
- Doses / concentrations: single dose / 20 mg/kg bw

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: In a preliminary dose range finding study, oral administration of 1200 mg Hostacor 4221 per kg body weight resulted in mortality in 1 male and 1 female Sprague Dawley rats. Therefore a highest dose of 1000 mg per kg body weight was selected for the main study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Animals from each group were killed 24 hours after the second treatment.

DETAILS OF SLIDE PREPARATION: After killing, one femur was removed and the bone completely stripped of muscle tissue. After removal of the epiphyses, the bone marrow was flushed out of the diaphysis (if necessary in alternate directions) into a centrifuge tube by means of a syringe containing Hanks solution (approx. 3 ml/femur) at a temperature of 37°C. This suspension was mixed and centrifuged for 10 minutes at 1000 rpm. All but one drop of the supernatant was drawn off.
For hypotonic treatment, approximately 5 ml of approx. 0.075 M potassium chloride solution at 37°C was added and suspended. This suspension was incubated for 10 minutes in a water bath at 37"C. Approximately 1.5 ml fixative (methanol: glacial acetic acid 4 : 1) was then added and the suspension was bubbly mixed by air with a Pasteur pipette.
After re-centrifuging for approx. 10 minutes at 1000 rpm, all but one drop of the supernatant was drawn off. The sediment was carefully covered with a layer composed of approx. 2.5 ml fixative. After at least 20 minutes the fixation was carefully removed (after re-centrifuging, when needed) and the sediment was suspended in approx. 2.5 ml fresh fixative. If needed, the mixture was then centrifuged after another 30 - 60 minutes, after which the liquid was removed and fresh fixative added. The tubes were covered and kept for at least 12 hours (overnight) in a refrigerator at approx. 4°C.
After re-centrifuging (as needed) for 10 minutes at 1000 rpm, all but one drop of the liquid was removed and a new suspension was formed with freshly prepared fixative. A few drops of this suspension were placed with a Pasteur pipette onto cleaned and frozen microscopic slides. The drops were then briefly passed through a Bunsen flame. Afterwards the slides were examined under a microscope. Then the slides were airdried for at least 12 hours.

METHOD OF ANALYSIS: After the slides had been coded, 25 - 100 meta phases per animal were examined. 5 males and 5 females of each dose group were examined. The set of chromosomes was examined for completeness and the various chromosomal aberrations were asessed. Only metaphases with 42 +/- 2 chromosomes are included in the analysis. The metaphases were examined for the following aberrations: chromatid gap, chromosome gap, chromatid break, chromosome break, minute, double minute, chromatid deletion, chromosome deletion, chromatid exchanges including intrachanges, chromosome
exchanges including intrachanges, dicentrics, pulverization and ring formation. Furthermore the incidence of polyploid metaphases was determined in 1000 cells of each animal.
Additionally the mitotic index was determined bv counting the number of cells undergoing mitosis in a total of 1000 cells. The mitotic index is expressed as a percentage. After the metaphases had been evaluated, the code was opened. The values from the control groups were compared with the results from the dose groups and the positive control.
Evaluation criteria:
The evaluation of the results was performed as follows:
The test compound is classified as clastogenic if it induces a statistically significant increase in the number of phases with aberrations (without gaps) at one or more of the concentrations tested as compared with the negative controls
The test compound is classified as clastogenic if there is a concentration-related increase in the number of phases with aberrations (without gaps)
The test compound is classified as non-clastogenic if the tests are negative
Statistics:
The biometry of the results was performed with a one-sided Fisher-Exact test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
slight cytotoxicity in the high dose group (Reduction of the mitotic index)
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
All animals survived after treatment. No signs of toxicity were observed. The dissection of the animals revealed no test substance related macroscopic findings. In all study groups no polyploid metaphases were observed. No significant increase in the aberration rate exclusive and inclusive gaps occurred in all dose groups as compared with the negative control. No increase in the number of chromosome aberrations after administration of the test substance were observed. Marked increases of chromosome aberrat'ons were obtained with the positive control substance Cyclophosphamide in males and females, thus indicating the validity of the assay.

Any other information on results incl. tables

Justification for the read-across approach using (Tetrapropylensuccinimido)-caproic acid as supporting substance:

The registration substance (Pentapropylensuccinimido)-caproic acid and proposed supporting substance (Tetrapropylensuccinimido)-caproic acid are homolog series of (Polypropylensuccinimido)-caproic acid. The only structural difference is that the alkyl moiety of the registratoin substance is composed of five propylen unit, whereas that of the supporting substance is composed of four propylen unit. The given structural difference is not likely to be associated with significantly deviating chemical reactivity, thus is not likely to be associated with significantly deviating reactivity with genetic materials.

Evaluation of the genotoxicity of the registration substance

The read-across supporting substance is not mutagenic in Ames test, not mutagenic in HPRT test. It is however clastogenic in in-vitro chromosome aberration test and not clastogenic in in-vivo chromosome aberration test.

The observed clastogenicity in in-vitro assays may be related to the highly branched alkyl moiety that thus to the ability to form stabilized radical species. This property is certainly linked to the technical profile in that the substance is used as corrosion inhibitor. In the in-vivo chromosome aberration assay no clastogenic acitivity was found in the bone marrow of orally treated animals. The study is valid, since cytoxicity was found in the bone marrow cells, demonstrating the bioavailbility to the bone marrow cells were given. Overall, no significant genotoxicity can be assigned for the read-across supporting substance based on the available data. Likewise no significant genotoxicity property can be derived for the registration substance.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The genotoxicity potential of (Tetrapropylensuccinimido)-caproic acid was investigated according to the OECD Guideline 475. No clastogenic effect was found. The study is valid as cytotoxicity in the bone marrow cells was observed.
Executive summary:

The genotoxicity of (Pentapropylensuccinimido)-caproic acid was assessed based on the toxicity data or the read-across supporting substance (Tetrapropylensuccinimido)-caproic acid.

 

(Tetrapropylensuccinimido)-caproic acid was tested in the in vivo cytogenetic test in bone marrow cells of the Sprague Dawley rat. The test compound was suspended in sesame oil and was given twice at an interval of 24 hours as oral doses of 0 (negative controls), 500, 750 and 1000 mg per kg body weight to male and female Sprague Dawley rats. The negative control group received sesame oil alone.

All animals survived after treatment. No signs of toxicity were observed in the main study.

The animals were killed 24 hours after the second administration for bone marrow preparation.

The dissection of the animals revealed no test substance related macroscopic findings. Evaluation of the slides indicated slight cytotoxicity in the high dose group (mitotic index 6.5%) as compared with the negative controls (mitotic index 8.0%).

No increase occurred in the number of polyploid meta phases.

No significant increase in the aberration rate excluding and including gaps was observed in all dose groups as compared with the negative control.

No increase in the number of chromosome aberrations after administration of the test substance was observed.

In summary, administration of (Tetrapropylensuccinimido)-capronic acid induced cytotoxicity but not chromosome aberrations in bone marrow cells.