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Diss Factsheets

Toxicological information

Genetic toxicity: in vivo

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Administrative data

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: other:
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1999-12-01 - 2000-02-02
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
The GLP study was conducted according to an internationally accepted guideline. All study parameters are given in detail. Nevertheless, according to the ECHA's practical guide 6: "How to report read-across and categories" the maximum for read-cross is 2.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
Type of assay:
micronucleus assay

Test material

Constituent 1
Reference substance name:
Test material form:
other: liquid
Details on test material:
Identity: SAS 40 including 3% of CEL 2021P
Appearance: Colourless liquid
Storage conditions: Room temperature in the dark
Batch number: 990323

Test animals

Details on test animals or test system and environmental conditions:
All animals in this study were Specific Pathogen Free CD-I outbred albino mice of Swiss origin. Males weighed between 28 and 30 grams and females weighed between 22 and 24 grams on despatch from Charles River UK Limited, Margate, Kent, England.
On arrival the weight of the animals was checked and found to be acceptable. The animals were randomly assigned to groups and tail marked. Each group was kept, with the sexes separated, in cages and maintained in a controlled environment, with the thermostat set at 22°C and relative humidity set at 50%. The room was illuminated by artificial light for 12 hours per day. All animals were allowed free access to pelleted expanded rat and mouse No. 1 maintenance diet (SQC grade obtained from Special Diets Services Ltd, Witham, Essex, UK) and tap water ad libitum. Food and tap water are routinely analysed for quality at source. Dietary contaminants are not suspected of having any significant effect on parameters measured in this test in this laboratory at any time over the last ten years. All animals were acclimatised for a minimum of 5 days, examined daily and weighed prior to dosing.

Administration / exposure

Route of administration:
corn oil
Details on exposure:
All animals in all groups were dosed with the standard volume of 20 ml/kg bodyweight. The test substance and negative control were dosed by intraperitoneal injection, and mitomycin C, the positive control compound, was administered orally by intragastric gavage.
Duration of treatment / exposure:
single intraperitoneal administration of the test substance
Frequency of treatment:
single dose
Doses / concentrationsopen allclose all
Doses / Concentrations:
250, 500 and 1000 mg/kg bodyweight
nominal conc.
main study
Doses / Concentrations:
250, 500, 1000, 1500, 2000 mg/kg bw
nominal conc.
preliminary toxicity test
No. of animals per sex per dose:
Preliminary toxicity test: 2 males and 2 females per dose

Main study:
vehicle control: 10 males and 10 females
250 mg/kg: 5 males and 5 females
500 mg/kg: 5 males and 5 females
1000 mg/kg: 12 males and 12 females
Positive control: 5 males and 5 females
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin C, obtained from Sigma Chemical Company, batch number 49H2508; was used as the positive control compound. It was prepared as a solution in purified water, at a concentration of 0.6 mg/mI, just prior to administration.


Tissues and cell types examined:
The animals were ldlledby cervical dislocation following carbon dioxide inhalation and both femurs dissected out from each animal. The femurs were cleared of tissue and the proximal epiphysis removed from each bone. The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 2 ml of pre-filtered foetal calf serum using a 2 mJ disposable syringe fitted with a 21 gauge needJe. The cells were sedimented by centrifugation, the supernatant was discarded and the cells were resuspended in a small volurne of fresh serum. A small drop of the cell suspension was transferred to a glass rnicroscope slide and a smear was prepared in the conventional manner (Schrnid 1976). At least three smears were made from each anima!. The prepared smears were fixed in methanol (> 10 minutes). After air-drying tbe smears were stained for 10 rninutes in 10% Giemsa (prepared by 1 : 9 dilution of Gurr's improved R66 Giemsa (BDH) with purified water). Following rinsing in purified water and differentiation in buffered purified water, the smears were rinsed in purified water, air-dried and mounted with coverslips using DPX.
The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per anima!. Usually only one smear per animal was exarnined. The remaining smears were held temporarily in reserve in case of technical problems with the first smear.
Evaluation criteria:
Micronuclei are identified by the following criteria:
• Large enough to discern morphologie al characteristics
• Should possess a generally rounded shape with a clearly defined outline
• Should be deeply stained and similar in colour to the nuclei of other cells -not black
• Should lie in the same focal plane as the cell
• Lack internal structure, ie they are pyknotic
• There should be no micronucleus-like debris in the area surrounding the cel!.

The proportion of immature erythrocytes for each animal was assessed by exarnination of at least
1000 erythrocytes.
A positive response is normaIly inilicated by a statistically significant dose-related increase in the incidence of rnicronucleated immature erythrocytes for the treatment group compared with the concurrent control group (p<Ü.01); individual and/er group mean values should exceed the laboratory historical control range (Morrison and Ashby 1995). A negative result is indicated where individual and group mean incidences of micronucleated immature erythrocytes fer the group treated with the test substance are not significantly greater than incidences fer the concurrent control group (P>0.01) and where these values fall within the historical control range. An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response.
The results for each treatment group were compared with the results for the concurrent contro] group using non-parametric statistics. Non-parametric statistical methods were chosen fer analysis of results because:
• They are suited to analysis of data consisting of discretelinteger va lues with lies such as the incidence of micronucleated immature erythrocytes
• The methods make few assumptions ab out the underlying distribution of data and therefere the values do not require transfonnation to fit a theoretical distribution (where data can be approximately fitted to a normal distribution, the results of nonparametric analysis and classical analysis of variance are very similar)
• 'Outliers' are frequently found in the proportion of immature erythrocytes for both control and treated animals; non-parametric analysis based on rank does not give these values an undue weighting.

For incidences of micronucleated immature erytruocytes, exact one-sided p-values are calculated by permutation (StatXact, CYTEL Software Corporation, Cambridge, Massachusetts). Comparison of several dose levels are made with the concurrent control using the Linear by Linear Association test for trend in a step-down fashion if significance is detected (Agresti et al. 1990); for individual intergroup comparisons (ie the positive control group) this procedure simplifies to a straightforward permutation test (Gibbons 1985). For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores are used, ie exact versions of Wilcoxon's sum of ranks test and Jonckheere's test for trend.

Results and discussion

Test results
no effects
Vehicle controls validity:
Positive controls validity:

Any other information on results incl. tables

Summary of results and statistical analysis:





Incidence mie

Incidence mme






(group mean)

24 Hours

Vehicle control
























Mitomycin C





48 Hours

Vehicle control











TM SAS 40 including 3% of CEL 2021P

% ie/(ie+me) Proportion of immature erythrocytes

mie Number of micranucleated cells observed per 2000 immature erythrocytes examined

mme Number of micranucleated cells calculated per 2000 mature erythrocytes

Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-sided probabilities): *** P < 0.001 (highly significant) ** P < 0.01 (significant) otherwise P > 0.01 (not significant)

Applicant's summary and conclusion

Interpretation of results (migrated information): negative
It is concluded that SAS 40 including 3% of CEL 202lP did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered by intraperitoneal injection in this in vivo test procedure.
Executive summary:

This study was designed to assess the potential induction of micronuclei by SAS 40 including 3% of CEL 2021P in bone marrow cells of mice. Mice were treated with a single intraperitoneal administration of the test substance at dose levels of 250, 500 and 1000 mg/kg bodyweight. A preliminary toxicity test had previously shown that a dose of 1000 mg/kg was expected to be approximately the maximum tolerated; this level was therefore selected as an appropriate maximum for use in the micronucleus test.

The test substance and negative control were administered by intraperitoneal injection. The negative control group received the vehicle, corn oil. A positive control group was dosed orally, by intragastric gavage, with mitomycin C at 12 mg/kg bodyweight.

Bone marrow smears were obtained from five male and five female animals in the negative control, each of the test substance groups and the positive control group 24 hours after dosing. In addition bone marrow smears were obtained from five male and five female animals in the negative control and high level treatment groups 48 hours after dosing. One smear from each animal was examined for the presence of micronuclei in 2000 immature erythrocytes. The proportion of immature erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated mature erythrocytes was also kept.

No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decrease in the proportion of immature erythrocytes were observed in mice treated with SAS 40 including 3% of CEL 2021P and killed 24 or 48 hours later, compared to vehicle control values (P>0.01 in each case).

The positive control compound, mitomycin C, produced large, highly significant increases in the frequency of micronucleated immature erythrocytes.

It is concluded that SAS 40 including 3% of CEL 2021P did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered by intraperitoneal injection in thisin vivotest procedure.