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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, OECD 474 Guideline OECD

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Micronucleus assay in bone marrow

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test compound Dimethylsulfoxyde
CAS no.: 67-68-5
Source: Aldrich Chemical Co.
Batch : 02758HI
Purity : 99.99%

Test animals

Species:
rat
Strain:
other: Han Wistar
Sex:
male/female
Details on test animals and environmental conditions:
ANIMALS
- Strain : out-bred Crl:HanWist (Glx:BRL) BR
- Breeder: Charles River UK Ltd, Margate, UK
- Age on first day of assay: approximately 7-8 weeks old
- Weight at the beginning of the treatment period: males 185-223g, females 163-194g
- Acclimation: no data
- Housing: in groups of no more than three animals of the same sex in appropriate cages
- Food: Special Diets Services Ltd, RM1.(E).SQC. ad libitum
- Water: Bottled water (public supply) ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature : 20-22 °C
- Relative humidity : 47-60%
- Light/dark cycle : 12h/12h
- Ventilation : about 15 cycles/hour of filtered, non-recycled air.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: water for injection (purified water)
Details on exposure:
The test article preparations were stored at room temperature, protected from light, between dosing occasions and used within five days of initial formulation as follows:
Experiment Concentration of dosing preparation Dose administered¶
(mg/mL) (mg/kg)¶
Range-finder 500.0 5000
Main study 20.0 200
100.0 1000
500.0 5000
Prepared on the first day of dosing and administered on five consecutive days
Duration of treatment / exposure:
5 administrations at 24-hour interval (5 consecutive days)
Frequency of treatment:
daily
Post exposure period:
24 hours then sacrifice
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 200, 1000 and 5000 mg/kg/d
Basis:

No. of animals per sex per dose:
6 males and 6 females
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: intraperitoneal
- Doses / concentrations: 20 mg/kg

Examinations

Tissues and cell types examined:
Polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) in bone marrow
Details of tissue and slide preparation:
- Necropsy and slide preparation
Test article and vehicle treated rats were killed in groups, 24 hours after the final administration; CPA-treated rats were killed 24 hours after the single dose. Rats were killed by asphyxiation with carbon dioxide (subsequently ensured by cervical dislocation) in the same order as they were dosed.
A single femur from each animal was exposed, removed, cleaned of adherent tissue and the ends removed from the shank. Using a syringe and needle, bone marrows were flushed from the marrow cavity with 2 mL foetal bovine serum into appropriately labelled centrifuge tubes (one per animal).
The tubes were centrifuged (1250 x 'g', 2-3 minutes) and the serum was aspirated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube and from each tube a small volume of suspension was placed on the end of each of two slides labelled with the appropriate study number, sampling time, sex, date of preparation and tag number. The latter served as a code so analysis could be conducted "blind". A smear was made from the drop by drawing the end of a clean slide along the labelled slide.
Slides were allowed to air-dry and then fixed for 10 minutes in absolute methanol, followed by rinsing several times in distilled water. One slide from each set of two was taken (the other kept in reserve) and, after a second fixation for 10 minutes in absolute methanol, were stained for 4 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 and allowed to dry. When dry, slides were mounted with coverslips.

- Scoring
Initially the relative proportions of polychromatic erythrocytes (PCE), seen as bright orange enucleate cells, and normochromatic erythrocytes (NCE), seen as smaller dark green enucleate cells, were determined until a total of at least 1000 cells (PCE plus NCE) had been analysed.
Counting continued (but of PCE only) until at least 2000 PCE had been observed. All PCE containing micronuclei observed during these two phases of counting were recorded. The vernier coordinates of all cells containing micronuclei were recorded to a maximum of six per 2000 cells scored (although for animal 222 only 5 out of 10 possible coordinates were noted).
Evaluation criteria:
A test article is considered as positive in this assay if:
1. a statistically significant increase in the frequency of micronucleated PCE occurs at least at one dose, and
2. the frequency of micronucleated PCE at such a point exceeds the historical vehicle control range.
Statistics:
For each group, inter-individual variation in the numbers of micronucleated PCE was estimated by means of a heterogeneity ki²
test. The numbers of micronucleated PCE in each treated group were then compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine ki² probability values of p < 0.05 were to be accepted as significant.
A further statistical test (for linear trend) was used to evaluate possible doseresponse relationships.
If the heterogeneity ki² test provides evidence of significant (p < 0.05) variability between animals within at least one group, non-parametric analysis is more appropriate. Provision was made to use the Wilcoxon rank sum test under these circumstances.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary study:
All animals survived to the end of the dosing regimen with clinical signs of lethargy and/or abnormal breathing noted on all dosing occasions. This preliminary study was considered to confirm the acceptability of 5000 mg/kg/day as the maximum test dose for the main study.

Main study:
No clinical signs were observed in any animal administered with the test article although two female animals showed limited signs of irritation at the injection site immediately after dosing.
Negative (vehicle) control rats exhibited normal group mean ratios of PCE (polychromatic erythrocytes) to NCE (normochromatic erythrocytes) and normal frequencies of micronucleated PCE within historical negative control (normal) ranges.
Positive control animals exhibited increased numbers of micronucleated PCE such that the micronucleus frequency in the positive control group was significantly greater than in concurrent controls. The study was therefore accepted as valid.
In general rats treated with Dimethyl sulphoxide at all doses exhibited group mean ratios of PCE to NCE and frequencies of micronucleated PCE which were similar to or lower than the values for the vehicle control group. The data from female animals administered the test article, demonstrated group mean frequencies of micronucleated PCE that were higher than the vehicle control group. However, there were no instances of statistically significant increases in micronucleus frequency for any of the male or female groups receiving the test article.

Any other information on results incl. tables

Treatment group (mg/kg(d)

Kill time (hours)

Mean ration PCE/NCE
(per 1000 cells) (+/-SD)

Groupe mean frequency of micronucleated PCE

 

 

 

 

 

 

Males

 

 

 

 

 

Vehicle Control

24

1,34

0,58

±

0,38

200

24

1,19

0,17

±

0,41

1000

24

0,95

0,17

±

0,26

5000

24

1,42

0,25

±

0,27

CPA, 20+

24

0,81

5,50

±

2,07

 

 

 

 

 

 

Females

24

 

 

 

 

Vehicle Control

24

2,06

0,17

±

0,41

200

24

1,14

0,67

±

0,61

1000

24

1,06

0,50

±

0,45

5000

24

1,12

0,33

±

0,26

CPA, 20+

24

0,84

6,50

±

3,87

 

 

 

 

 

 

+= Administered as a single dose

 

 

 

SD=Standard deviation

 

 

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Dimethyl sulphoxide did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male and female Han Wistar rats treated at dose levels up to 5000 mg/kg/day for five consecutive days.
Executive summary:

The potential of Dimethylsulfoxide (DMSO) to induce micronucleus assay in Han Wistar rat bone marrow was evaluated in accordance with the international guidelines (OECD 474) and in compliance with the Principles of Good Laboratory Practice.

6 males and 6 females rat were treated intraperitoneally with DMSO (Batch: 02758HI, Purity: 99.99%.) at doses of 0, 200, 1000 and 5000 mg/kg/d for 5 consecutive days. Bone marrow cells were harvested at (list hours) post-treatment. 24 hours after the last administration, animals were sacrificed

No clinical signs were observed in any animal administered with the test article although two female animals showed limited signs of irritation at the injection site immediately after dosing.

In general rats treated with DMSO at all doses exhibited group mean ratios of PCE to NCE and frequencies of micronucleated PCE which were similar to or lower than the values for the vehicle control group. The data from female animals administered the test article, demonstrated group mean frequencies of micronucleated PCE that were higher than the vehicle control group.

However, there were no instances of statistically significant increases in micronucleus frequency for any of the male or female groups receiving the test article.

Dimethyl sulphoxide did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male and female Han Wistar rats treated at dose levels up to 5000 mg/kg/day for five consecutive days and is considered as genotoxic in vivo.