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Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental phases of the study were performed between 26 January 2011 and 14 March 2011.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted on supporting substance (with carbon range C8-26) in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.11 (Mutagenicity - In Vivo Mammalian Bone-Marrow Chromosome Aberration Test)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.5385 (In Vivo Mammalian Cytogenetics Tests: Bone Marrow Chromosomal Analysis)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
chromosome aberration assay

Test material

Constituent 1
Reference substance name:
-
EC Number:
481-740-5
EC Name:
-
Cas Number:
848301-67-7
Molecular formula:
main general molecular formula: CnH(2n+2)
IUPAC Name:
C8-C26 branched and linear hydrocarbons – Distillates

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
Sufficient Wistar HanTM (RCCHanTMWIST) rats were supplied by a reputable supplier. For full details please see full study report. At the start of the test the rats weighed 186 to 229 g and were approximately seven to twelve weeks old. After a minimum acclimatisation period of at least five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a colour coded cage card.
The animals were housed in groups of up to five by sex in solid-floor polypropylene cages with woodflake bedding (Supplied by a reputable supplier. For full details please see full study report). Free access to mains drinking water and food, Harlan Teklad 2014 Rodent Pelleted Diet supplied by Harlan Laboratories U.K. Ltd., Oxon, UK, was allowed throughout the study. The animals were also provided with environmental enrichment items: wooden chew blocks and cardboard fun tunnels (Supplied by a reputable supplier. For full details please seefull study report).

The temperature and relative humidity were set to achieve limits of 19 to 25ºC and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
The vehicle control was supplied by a reputable supplier as follows:
Supplier's identification: Arachis oil
Harlan serial number: V-4855
Date received: 14 June 2010
Description: Straw-coloured slightly viscous liquid
Expiry date: 31 January 2012
Storage conditions: Room temperature
Details on exposure:
Groups, each of seven rats were dosed once only via the oral route with the test item at 2000, 1000, and 500 mg/kg. One group of rats from each dose level was killed by cervical dislocation approximately 24 hours following treatment and a second group dosed at 2000 mg/kg was killed at approximately 48 hours. In addition, two further groups of rats were included in the study; one group of seven rats was dosed via the oral route with the vehicle alone (arachis oil) and a second group of five rats was dosed orally with Cyclophosphamide.
Cyclophosphamide is a positive control item known to produce chromosome aberrations under the conditions of the test. The vehicle control group and positive control group were killed approximately 24 hours following treatment.
All animals were observed for signs of overt toxicity and death approximately one hour after dosing and then once daily as applicable and immediately prior to termination.

Duration of treatment / exposure:
The chromosome aberration test was conducted using the oral route in groups of seven rats at the maximum recommended dose (MRD) 2000 mg/kg for the 24-hour and 48-hour time points, with 1000 and 500 mg/kg as the lower dose levels. Animals were killed 24 or 48 hours later. The experimental design is summarised as follows:

Treatment Group Dose Level(mg/kg) Concentration (mg/ml) Dose Volume (ml/kg) Kill Time (Hours After Dosing) Animal Numbers
1. Vehicle Control (Arachis oil) 0 0 10 24 1 – 7
2. Positive Control (Cyclophosphamide) 25 2.5 10 24 8 – 12
3. Test item 2000 200 10 48 13 – 19
4. Test item 2000 200 10 24 20 – 26
5. Test item 1000 100 10 24 27 – 33
6. Test item 500 50 10 24 34 – 40
Frequency of treatment:
Groups of rats were dosed once only via the oral route
Post exposure period:
All animals were observed for signs of overt toxicity and death approximately one hour after dosing and then once daily as applicable and immediately prior to termination.
Treatment with Mitotic Inhibitor
Animals were injected via the intraperitoneal route with a solution of Colchicine at 4 mg/kg 2 to 4 hours prior to bone marrow harvest. At thescheduled time, animals were killed by cervical dislocation and one femur was extracted from each animal and cleaned of muscle and connective tissue.
Doses / concentrations
Remarks:
Doses / Concentrations:
Groups, each of seven rats were dosed once only via the oral route with the test item at 2000, 1000, and 500 mg/kg.
Basis:
other: The maximum recommended dose of 2000 mg/kg was used as the maximum dose, substance was formulated in Arachis oil at 50, 100 and 200 mg/ml and dosed at 10 ml/Kg to achieve actual dose levels.
No. of animals per sex per dose:
Groups, each of seven male rats were dosed once only via the oral route with the test item at 2000, 1000, and 500 mg/kg. One group of male rats from each dose level was killed by cervical dislocation approximately 24 hours following treatment and a second group dosed at 2000 mg/kg was killed at approximately 48 hours.
Control animals:
yes, concurrent vehicle
Positive control(s):
Five rats were dosed orally with Cyclophosphamide at a dose of 25 mg/kg. Cyclophosphamide is a positive control item known to produce chromosome aberrations under the conditions of the test.
The positive control item was supplied by Acros Organics, as follows:
Supplier's identification : Cyclophosphamide
Supplier’s lot number : A0277203
Harlan serial number : R-4723
Date received: 04 December 2009
Expiry date : 04 December 2011
Storage conditions: Approximately 4ºC, in the dark

Examinations

Tissues and cell types examined:
The mammalian in vivo chromosome aberration test is used for the detection of structural chromosome aberrations induced by test compounds in
rat bone marrow cells. In addition any increases in polyploidy may indicate the induction of numerical aberrations
Details of tissue and slide preparation:
At the scheduled time, animals were killed by cervical dislocation and one femur was extracted from each animal and cleaned of muscle and connective tissue. The bone marrow was aspirated into 5 ml of warm Hanks buffered salt solution (HBSS) supplemented with demecolcine (Colcemid 0.1 µg/ml) and incubated at approximately 37°C for 30 minutes before being spun down in a centrifuge. The supernatant was removed and the cell pellet re-suspended in 0.075 M potassium chloride (KCl) at 37°C for approximately 15 minutes including centrifugation. The cells were re centrifuged and all but 1 ml of the supernatant removed. After re-suspension of the cell pellet, the cells were fixed by the addition of freshly prepared fixative (methanol:glacial acetic acid, 3:1). The fixative was changed several times and the cells stored at approximately 4ºC for at least 4 hours.
Slide Preparation and Staining
After storage the cell suspensions were centrifuged and the fixative removed to leave a sufficient amount to give a milky suspension on re-suspension of the cell pellet. A few drops of each cell suspension were dropped onto clean, wet slides and air-dried. When completely dry the slides were stained in 5% Giemsa for 10 minutes and rinsed in tap water and distilled water. When the slides were dry a cover slip was applied using a mounting medium.
Evaluation criteria:
Slide Evaluation
The stained slides were coded and examined ‘blind’ using light microscopy at x100 and x1000 magnifications. 100 metaphase cells of adequate quality were scored, if possible, from the slides prepared from each animal for both numerical and structural chromosome aberrations. Except where there were approximately 30 to 50% of cells with aberrations, then slide evaluation was terminated at 50 cells. A mitotic index (MI) value was also obtained for each animal by recording the number of metaphase cells that were associated with 1000 cells.
If the cell had 40 to 44 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the UKEMS Guidelines for Mutagenicity Testing. The details of the classification of chromosome aberrations and the evaluation criteria applied to test data are given in Appendix 1 (see in attached section). A Senior Cytogeneticist checked aberrations recorded by the slide scorers.
Statistics:
Statistical Analysis
Comparisons were made between the vehicle control group and each treatment dose group, with a chi-squared test, using observed numbers of cells with aberrations. Analysis of mitotic index data was performed using a Students T-Test following a v(x+1) transformation.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Mortality Data and Clinical Observations
There were no premature deaths seen in any of the test item dose groups and no clinical signs were observed in animals dosed with the test item at any of the dose levels.

Evaluation of Bone Marrow Slides
A summary of the results of the chromosome aberration test is given in the attached Table 1. Individual and group mean data are presented in he attached Tables 2 to 7. (See in attached section).

No marked decreases in the mitotic index mean value were observed with any of the test item groups or the positive control group when compared to the vehicle control group.

All of the vehicle control animals gave values of chromosome aberrations within the expected range (see table 2 in the attached section).

The positive control group animals showed highly significant increases in the frequency of aberrations (see table 3 in the attached section) indicating that the test method itself was operating as expected. It should be noted that due to the toxic response seen with cyclophosphamide in the bone marrow the quality and morphology of the metaphases was less than perfect. Therefore it was necessary to score more than 100 metaphase cells for three of the five animals to demonstrate the sensitivity of the rats following exposure with cyclophosphamide.
There was no evidence of a statistically significant increase in the incidence of cells with chromosome aberrations excluding gaps in animals dosed with the test item, when the dose groups were compared to the vehicle control group.

The test item did not induce a significant increase in the numbers of polyploid cells in any of the treatment groups.

Any other information on results incl. tables

Due to the nature and format of the tables, please see the attached results tables.

Table 1: Group Mean Results of Chromosome Aberration Test

Table 2: Individual Results of Chromosome Aberration Test - 24-Hour Vehicle Control Group

Table 3: Individual Results of Chromosome Aberration Test - 24-Hour Positive Control Group: Cyclophosphamide 25 mg/kg

Table 4: Individual Results of Chromosome Aberration Test - 48-Hour Test Item Group: 2000 mg/kg

Table 5: Individual Results of Chromosome Aberration Test - 24-Hour Test Item Group: 2000 mg/kg

Table 6: Individual Results of Chromosome Aberration Test - 24-Hour Test Item Group: 1000 mg/kg

Table 7: Individual Results of Chromosome Aberration Test - 24-Hour Test Item Group: 500 mg/kg

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test item did not induce any significant or dose-related increases in the frequency of chromosome aberrations. The test item was considered to be non clastogenic to rat bone marrow cells in vivo.
Executive summary:

Introduction. 

The study was performed to assess the potential of the test item to produce damage to chromosomes or the mitotic apparatus when administered to rats.   The method used is compatible with that described in the revised OECD Guidelines for Testing of Chemicals No. 475 “Mammalian Bone MarrowChromosome Aberration Test”, Method B11 of Commission Regulation (EC) No. 440/2008 of 30 May 2008 andUS, EPA, TSCA and FIFRA guidelines.

Methods. 

A range-finding test was not performed as the test item had been previously investigated at Safepharm Laboratories Ltd*(Project No 2041/0045) at a dose of 5000 mg/kg with no ill effects. Therefore, the maximum recommended dose of 2000 mg/kg was used as the maximum dose and at the request of the Sponsor only male animals were investigated via the oral route.

The chromosome aberration test was conducted using the oral route in groups of seven rats at the maximum recommended dose (MRD) 2000 mg/kg for the 24-hour and 48-hour time points, with 1000 and 500 mg/kg as the lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow was extracted, processed and slide preparations made and stained. Bone marrow cells were scored for the presence of chromosome aberrations.

Further group of rats for the 24-hour time point were given a single oral dose of Arachis oil (seven rats) or dosed orally with Cyclophosphamide (five rats) to serve as vehicle and positive controls respectively.

Results. 

There were no premature deaths seen in any of the test item dose groups. No clinical signs were observed in animals dosed with the test item at any dose level.

No marked decreases in the mitotic index mean value were observed in any of the test item groups when compared to the vehicle control group.

There was no evidence of a statistically significant increase in the incidence of cells with chromosome aberrations excluding gaps in animals dosed with the test item, when the dose groups were compared to the vehicle control group. 

The test item did not induce any statistically significant increases in the numbers of polyploid cells at any dose level in any of the exposure groups.

The positive control item produced a marked increase in the frequency of chromosome aberrations.

Conclusion. 

The test item did not induce any significant or dose-related increases in the frequency of chromosome aberrations. The test item was considered to be non-clastogenic to rat bone marrow cells in vivo.


*On the 9thNovember 2008 Safepharm Laboratories Ltd changed its name to Harlan Laboratories Ltd.