A mixture of: 2,2',2'',2'''-(ethylenedinitrilotetrakis-N,N-di(C16)alkylacetamide; 2,2',2'',2'''-(ethylenedinitrilotetrakis-N,N-di(C18)alkylacetamide

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of: 2,2',2'',2'''-(ethylenedinitrilotetrakis-N,N-di(C16)alkylacetamide; 2,2',2'',2'''-(ethylenedinitrilotetrakis-N,N-di(C18)alkylacetamide

EC Number:

406-640-0

EC Name:

A mixture of: 2,2',2'',2'''-(ethylenedinitrilotetrakis-N,N-di(C16)alkylacetamide; 2,2',2'',2'''-(ethylenedinitrilotetrakis-N,N-di(C18)alkylacetamide

Cas Number:

136920-07-5

Molecular formula:

C58H114N4O6 - C154H308N6O4

IUPAC Name:

Reaction mass of 2,2',2'',2'''-(ethylenedinitrilotetrakis-N,N-di(C12-C18)alkylacetamide and {[2-(Carboxymethyl-di(C12-C18)alkylcarbamoylmethyl-amino)-ethyl]-di(C12-C18)alkylcarbamoylmethyl-amino}-acetic acid

Constituent 2

Reference substance name:

Keroflux ES 3241

IUPAC Name:

Keroflux ES 3241

Details on test material:

Batch No.: (T 75492/ST 1414/90) Partie 1
Manufacturing/Sampling Date: 10.10.1990
Appearance: Light beige, solid; liquid at 80°C
Storage: 4-6°C

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH, WIGA, Sulzfeld, D
- Weight at study initiation: ca. 29 g
- Housing: single in Makrolon cages, type I
- Assigned to test groups randomly: yes, under following basis: appropriate computer program
- Diet: Standardized pelleted feed (Kliba Haltungsdiaet, Klingentalmuehle AG, Kaiseraugst, CH; ad libitum
- Water: drinking water; ad libitum
- Acclimation period: ca. 1 week (housing 5 per cage (Makrolon cage, type M III))


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

The dose leveis were set as follows: Due to technical reasons the highest applicable amount was 3000 mg/kg body weight at a temperature of not less than 42°C in order to guarantee that the test substance stayed liquid and did not become solid for administration. Higher doses were only possible at a temperature of > 60°C which could not be applicated any longer. Therefore, a dose of 3000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 1500 mg/kg and 750 mg/kg body weight were administered as further doses.
The substance to be administered per kg body weight was heaten up to 80°C and dissolved in olive oil. Test group 1 received 10 ml olive oil/kg body weight. Test group 2 were given 3000 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of 30 g/100 ml. Test group 3 was given 1500 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of 15 g/100 ml. Test group 4 was given 750 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of 7.5 g/100 ml.

Duration of treatment / exposure:

Sacrifice after 16, 24 or 48 hours

Frequency of treatment:

single administration

Post exposure period:

Sacrifice after 16, 24 or 48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
3000 mg/kg, 1500 mg/kg, 750 mg/kg and 0 mg/kg body weight in a volume of 10 ml/kg body weight in each case
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

As positive control for clastogenicity, 20 mg of cyclophosphamide/kg body weight, dissolved in aqua dest., was administered once orally to male and female animals in a volume of 10 ml/kg body weight.
As positive contral for spindle poison effects, 0.15 mg of vincristine/kg body weight, dissolved in aqua dest., was administered once intraperitoneally to male and female animals in a volume of 10 ml/kg body weight.

Examinations

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

The two femora were prepared from the animals, and all soft tissues were removed. After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 ml/ femur). The suspension was mixed thoroughly with a pipette, centrifuged at 1500 rpm for 5 minutes, the supernatant was removed except for a few drops, and the precipitate was resuspended. One drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.
The slides were stained in eosin and methylene blue solution for 5 minutes, rinsed in aqua dest. and then placed in fresh aqua dest. for 2 or 3 minutes. They were finally stained in Giemsa solution for 12 minutes. After being rinsed twice in aqua dest. and clarified in xylene, the preparations were embedded in Corbit-Balsam.

Evaluation criteria:

In general, 1000 polychromatic erythrocytes from each of the male and female animais of every test group are evaluated and investigated for micronuclei. The normochromatic erythrocytes, which occur, are also scored.

Statistics:

A statistical evaluation was not necessary to perform.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The number of polychromatic micronucleated erythrocytes after test substance treatment was within the range of the actual control value and within the historical control values.

Any other information on results incl. tables

irregular respiration was observed in all treatment groups 15 -30 min after treatment.

Substance	Dose (mg/kg)	Sacrifice interval (h)	PCE with micronuclei (‰)	NCE with micronuclei (‰)	Ratio NCE/PCE
olive oil	solvent	24	1.7	1.54	0.45
test substance	750	24	1.9	1.54	0.39
test substance	1500	24	1.5	1.84	0.44
test substance	3000	16	1.6	0.84	0.48
test substance	3000	24	1.9	0.93	0.43
test substance	3000	48	2.0	1.51	0.46
cyclophosphamide	20	24	10.4	3.03	0.20
vincristine	0.15	24	35.8	2.15	0.33
PCE = polychromatic erythrocyts (10,000 were scored for micronuclei)
NCE = normochromatic erythrocyts
The NCE/PCE ratio is based on the scoring of 10,000 erythrocyts

According to the results of the present study, the single oral administration of Keroflux ES 3241 in doses of 3000 mg/kg, 1500 mg/kg and 750 mg/kg body weight did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always in the same range as that of the negative control in all dose groups and at all sacrifice intervals. No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.