Registration Dossier

Diss Factsheets

Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.

REACH

Butanedioic acid, 2,3-dihydroxy- [R-(R,R)]-,C12-13-branched alkyl esters

EC number: 947-004-7 | CAS number: -

Life Cycle description
Uses advised against

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:: in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:: experimental study
Adequacy of study:: key study
Study period:: July 1993
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Justification for type of information:: Please see Analogue Approach

Cross-reference

Reason / purpose for cross-reference:: read-across source

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Please see Analogue Approach

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

C57BL

Remarks:

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 355x235x190 mm)
- Diet (e.g. ad libitum): standard pellet complete diet ad libitum supplied by the authorized breeder Nossan
- Water (e.g. ad libitum): filtered tap water from local network ad libitum
- Acclimation period: 5 days (during this persiod they were observed daily)

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: May 10, 1993 To: May 13, 1993

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

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

Remarks:

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

No. of animals per sex per dose:

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

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

Sacrifice:
Animals were sacrified, by displacement of cervical vertebran.

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.

Smears dyeing:
Once dried, slides were dyed as follows:
immersion in May Grunland solution for 3 minutes;
wash in distilled water;
immersion in Giemsa solution, diluted 1:6 in distilled
water for 10 minutes;
washing in distilled water;
air drying

METHOD OF ANALYSIS: The slidas wer observed with an optic microscope, choosing 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.

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.

Key result

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.

Table #1: Mean of micronucleated polychromatic erythrocytes

Concentration
[mg/kg bw]

Sampling time

24 h

48 h

72 h

male

female

male

female

male

female

12500

1.40 ± 1.14

1.40 ± 0.89

1.20 ± 0.83

1.20 ± 0.44

1.00 ± 0.71

0.80 ± 0.45

Positive control

61.2 ± 6.06

59.4 ± 2.61

62.2 ± 6.22

61.8 ± 3.35

64.4 ± 5.86

63.4 ± 2.61

Negative control

1.60 ± 1.51

1.80 ± 0.83

1.20 ± 1.09

2.00 ± 1.41

0.40 ± 0.89

Table #2: Ratio polychromatic/normochromatic erythrocytes

Concentration
[mg/kg bw]

Sampling time

24 h

48 h

72 h

male

female

male

female

male

female

12500

2.30 ± 0.28

2.47 ± 0.30

2.16 ± 0.41

2.41 ± 0.39

2.20 ± 0.31

2.35 ± 0.41

Positive control

0.99 ± 0.10

0.96 ± 0.08

0.98 ± 0.18

0.94 ± 0.12

0.85 ± 0.11

0.81 ± 0.07

Negative control

2.20 ± 0.40

2.30 ± 0.34

2.23 ± 0.23

2.38 ± 0.32

2.23 ± 0.33

2.26 ± 0.43

* = only platelets and blasts were observed

Conclusions:

Interpretation of results: negative
The test material Butanedioic acid, 2,3-dihydroxy-di-C14-C15 alkyl esters 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 Butanedioic acid, 2,3-dihydroxy-di-C14-C15 alkyl esters 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 the beginning of treatment

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

- 30 (10 from each group) 72 hours after the 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.

It was concluded that the test material Butanedioic acid, 2,3-dihydroxy-di-C14-C15 alkyl esters did not have a mutagenic effect on this mitotic system under the conditions of the study.

Data source

Materials and methods

Test material

Test material information

Test material form:: liquid
Details on test material:: product

Administration / exposure

Route of administration:: intraperitoneal

No. of animals per sex per dose:: 5

Examinations

Tissues and cell types examined:: P

Results and discussion

Test results

: Key result
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

male

female

male

female

male

female

12500

1.40 ± 1.14

1.40 ± 0.89

1.20 ± 0.83

1.20 ± 0.44

1.00 ± 0.71

0.80 ± 0.45

Positive control

61.2 ± 6.06

59.4 ± 2.61

62.2 ± 6.22

61.8 ± 3.35

64.4 ± 5.86

63.4 ± 2.61

Negative control

1.60 ± 1.51

1.80 ± 0.83

1.20 ± 1.09

2.00 ± 1.41

0.40 ± 0.89

Table #2: Ratio polychromatic/normochromatic erythrocytes

Concentration
[mg/kg bw]

Sampling time

24 h

48 h

72 h

male

female

male

female

male

female

12500

2.30 ± 0.28

2.47 ± 0.30

2.16 ± 0.41

2.41 ± 0.39

2.20 ± 0.31

2.35 ± 0.41

Positive control

0.99 ± 0.10

0.96 ± 0.08

0.98 ± 0.18

0.94 ± 0.12

0.85 ± 0.11

0.81 ± 0.07

Negative control

2.20 ± 0.40

2.30 ± 0.34

2.23 ± 0.23

2.38 ± 0.32

2.23 ± 0.33

2.26 ± 0.43

* = only platelets and blasts were observed

Applicant's summary and conclusion