Bis(C12-C13)alkyl-2-hydroxybutandioate

Genetic toxicity: in vivo

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

Study period:

March 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): cosmacol EMI
- Substance type: pure active substance
- Physical state: liquid

Test animals

Species:

mouse

Strain:

C57BL

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River - Calco CO, Italy
- Age at study initiation: no data
- Weight at study initiation: 18 - 20 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: in groups of 5 in transparent polycarbonate cages (dimensions 355x235x19 mm)
- Diet (e.g. ad libitum): standard pellet complete diet ad libitum
- Water (e.g. ad libitum): filtered tap water from local network ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
The housing room conditions of temperature and humidity were maintained by the conditioning plant and continuously recorded, no further details mentioned
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: March 18, 1994 To: March 21, 1994

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: sesame seed oil
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: 250 mg/mL
- Amount of vehicle (if gavage or dermal): 50 mL/kg bw
- Lot/batch no. (if required): no data
- Purity: no data

Details on exposure:

A solution was prepared in sesame seed oil at a concentration of 250 mg/mL. Animals were treated in a single dose by intraperitoneal injection.

Duration of treatment / exposure:

24, 48 and 72 hours

Frequency of treatment:

single intraperitoneal injection

Post exposure period:

none

Doses / concentrations

Remarks:

Doses / Concentrations:
12500 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide in physiological saline
- Justification for choice of positive control(s): no data
- Route of administration: intraperitoneal injection
- Doses / concentrations: volume administration: 50 mL/kg bw; dose: 75 mg/kg bw

Examinations

Tissues and cell types examined:

Preparation of the bone marrow were made on slides.
Poly- and normochromatic erythrocytes and micronucleated polychromatic erythrocytes were counted.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: no data

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 24, 48 and 72 hours

DETAILS OF SLIDE PREPARATION: Thigh-bones were removed and cleaned from muscles. The heads of the femur was removed and foetal calf serum was introduced in the femur channel by means of a thin needle. The bone marrow cells coming out from the other side of the thigh-bone were collected in a test tube, then centrifugated for 10 min at 1000 rpm. The supernatant was removed and cells resuspended in 10 µL of foetal calf serum, smeared on slides, air-dried and stained with May Grunland and Giemsa solutions.

METHOD OF ANALYSIS: The slidas wer observed with an aptic microscope, chossing the areas showed the best distribution of cells and a perfect staining. For each animal 1000 polychromatic erythrocytes were counted, and the frequence of normochromatic and of micronucleated polychromatic erythrocytes were recorded.

OTHER:

Evaluation criteria:

The following data were evaluated:
- difference between the number of micronucleated polychromatic erythrocytes in the treatment group and negative control
- difference between the number of micronucleated polychromatic erythrocytes in the treatment group and positive control
- difference between the number of micronucleated polychromatic erythrocytes in the negative control and positive control
- difference between the proportion of polychromatic/normochromatic erythrocytes in the treatment group and negative control
- difference between the proportion of polychromatic/normochromatic erythrocytes in the treatment group and positive control
- difference between the proportion of polychromatic/normochromatic erythrocytes in the negative control and positive control

Statistics:

The data obtained from the proportional relationship between the polychromatic erythrocytes and the normochromatic erythrocytes was statistically evaluated by means of Student's t-test with 95% probability.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The difference between the number of micronucleated polychromatic erythrocytes in the treatment group and negative control is not significant.
The difference between the number of micronucleated polychromatic erythrocytes in the treatment group and positive control is significant.
The difference between the number of micronucleated polychromatic erythrocytes in the negative control and positive control is significant.
The difference between the proportion of polychromatic/normochromatic erythrocytes in the treatment group and negative control is not significant.
The difference between the proportion of polychromatic/normochromatic erythrocytes in the treatment group and positive control is significant.
The difference between the proportion of polychromatic/normochromatic erythrocytes in the negative control and positive control is significant.

Any other information on results incl. tables

Table #1: Mean of micronucleated polychromatic erythrocytes

Concentration [mg/kg bw]

Sampling time

24 h

48 h

72 h

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12500

1.40 ± 1.14

1.60 ± 1.14

1.60 ± 1.14

1.60 ± 1.52

1.60 ± 1.34

2.00 ± 1.22

Positive control

33.00 ± 4.83

35.00 ± 4.5

35.00 ± 3.94

38.00 ± 2.30

*

*

Negative control

1.80 ± 1.30

1.60 ± 1.14

1.60 ± 1.14

2.20 ± 1.48

1.20 ± 1.30

2.20 ± 1.30

Table #2: Ratio polychromatic/normochromatic erythrocytes

Concentration [mg/kg bw]

Sampling time

24 h

48 h

72 h

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12500

2.24 ± 0.24

2.28 ± 0.25

2.32 ± 0.25

2.28 ± 0.23

2.20 ± 0.24

2.28 ± 0.29

Positive control

0.90 ± 0.00

0.88 ± 0.04

0.82 ± 0.04

0.80 ± 0.07

*

*

Negative control

2.40 ± 0.10

2.36 ± 0.26

2.00 ± 0.25

2.28 ± 0.29

2.00 ± 0.21

2.26 ± 0.24

* = only platelets and blasts were observed

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The test material Bis(C12-C13)alkyl-2-hydroxybutandioate did not cause damage to this mitotic system under the conditions of the study.

Executive summary:

In order to verify its possible mutagenic effect, the test material Bis(C12-C13)alkyl-2-hydroxybutandioate was administered to 90 mice C57 (45 males and 45 females) according to EEC Directive 92/69. 30 were treated with the test material (12500 mg/kg), 30 treated with Cyclophosphamide (75 mg/kg bw) as positive control and 30 treated with sesame seed oil (50 mL/kg bw) as negative control. The 90 mice were sacrificed at the following times:

- 30 (10 from each group) 24 hours after ghe beginning of treatment

- 30 (10 from each group) 48 hours after the beginning of treatment

- 30 (10 from each group) 72 hours after teh beginning of treatment.

After the sacrifice, the bone marrow of each animal was extracted, smeared on a slide, stained and observed with a microscope. For each animal 1000 polychromatic erythrocytes were counted and the frequence of normochromatic and micronucleated polychromatic erythrocytes were recorded. The data obtained from the proportional relationship between the polychromatic erythrocytes and the normochromatic erythrocytes was statistically evaluated by means of Student's t-test. The following results 24 and 48 hours after the beginning of the test were obtained:

- the difference between the number of micronucleated polychromatic erythrocytes in the treatment group and negative control is not significant.

- the difference between the number of micronucleated polychromatic erythrocytes in the treatment group and positive control is significant

- the difference between the number of micronucleated polychromatic erythrocytes in the negative control and positive control is significant

- the difference between the proportion of polychromatic/normochromatic erythrocytes in the treatment group and negative control is not significant

- the difference between the proportion of polychromatic/normochromatic erythrocytes in the treatment group and positive control is significant

- the difference between the proportion of polychromatic/normochromatic erythrocytes in the negative control and positive control is significant.

On the slides prepared with the gone marrow of the animals treated with Cyclophosphamide and sacrificed 72 hours after the beginning of treatment, only platelets and blasts were observed and could not be evaluated.

It was concluded that the test material Bis(C12-C13)alkyl-2-hydroxybutandioate did not have a mutagenic effect on this mitotic system under the conditions of the study.