Reaction mass of ethoxylated N-oleyl-1,3-propanediamine, hydrofluorides of and ethoxylated N-palmityl-1,3-propanediamine, hydrofluorides of and ethoxylated N-stearyl-1,3-propanediamine, hydrofluorides of and olaflur

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-01-07 to 2002-01-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 960
- Expiration date of the lot/batch: 2002-04

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

mammalian cell line, other: human peripheral lymphocytes

Details on mammalian cell type (if applicable):

human peripheral lymphocytes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Preliminary study (Cytotoxicity study):
- without (24h exposure) and with (4h exposure) metabolic activation:
10, 25, 100, 250, 1000, 2500 and 5000 ug Olaflur/mL medium

Main Study
- without metabolic activation (4 h and 24h exposure):
1.25, 2.5, 5, 10 ug Olaflur/mL medium
- with metabolic activation (4h exposure):
2.5, 5, 10, 20 ug Olaflur/mL medium

Vehicle / solvent:

Olaflur was dissolved and diluted to the appropriate concentrations in aqua ad iniectabilia prior to the treatment of the cells. Aqua ad iniectabilia served as the negative control substance.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

aqua ad iniectabilia

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation (S9 mix)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

aqua ad iniectabilia

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation (S9 mix)

Details on test system and experimental conditions:

Culture establishment
Human peripheral blood was obtained by venipuncture from healthy donors known to be without any medication and collected in heparinised vessels. Small inocula of whole blood (0.5 mL) were added to tubes containing 5 mL of complete culture medium. The tubes were sealed and incubated at 37°C with occasional shaking to prevent clumping.

Study performance
The test samples of Olaflur were assayed in cultures both in the presence and absence of metabolic activation by rat liver post-mitochondrial fraction (S9 mix) from Aroclor-1254 induced animals. Aqua ad iniectabilia served as the vehicle.
The test was carried out employing 2 exposure times without S9 mix: 4 and 24 hours, and 1 exposure time with S9 mix: 4 hours. Harvesting time was 25 hours after start of exposure. The incubation procedure took place in the dark.
The study was conducted in duplicate; the duplicate cultures being derived from different donors.
The concentrations employed were chosen based on the results of a cytotoxicity study.

Culture harvesting
Two hours before termination cell division was arrested by addition of colcemid; after further 2h-incubation the cells were harvested (80 – 100xg), pellets of cells treated according to guideline and slides prepared for evaluation.

Evaluation criteria:

For each treatment and culture 100 metaphases/ plate were examined. Observed aberrations were noted and scored according to J.R.K. Savage (1975).
Metaphases which differed from the normal diploid complement were excluded from evaluation. However, substance-related variations of the normal chromosome number were noted (polyploidy). For examination of the toxicity of Olaflur 1000 cells were scored and the mitotic index was calculated as the percentage of cells in metaphase.

Statistics:

Statistical evaluation by comparison of the number of chromosome aberrations of samples and solvent control by using the exact test of R.A. Fisher (p<= 0.05). Only total numbers of cells with aberrations were analysed - chromatid gaps were exempted as no true chromosomal aberration.

Results and discussion

Test results

Key result

Species / strain:

mammalian cell line, other: human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity investigated in a preliminary study. Based on these results the concentrations for the main study were selected

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

no data

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

no remarks

Applicant's summary and conclusion

Conclusions:

Under the present test conditions Olaflur tested up to cytotoxic concentrations in the absence and presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage. In the same test, mitomycin C and cyclophosphamide induced significant damage.

Executive summary:

The test samples of Olaflur were assayed in an in vitro cytogenetic study using human lymphocyte cultures both in the presence and absence of metabolic activation by rat liver post-mitochondrial fraction (S9 mix) from Aroclor-1254 induced animals. Aqua ad iniectabilia served as the vehicle.

The test was carried out employing 2 exposure times without S9 mix: 4 and 24 hours, and 1 exposure time with S9 mix: 4 hours. Harvesting time was 25 hours after start of exposure. The incubation procedure took place in the dark. The study was conducted in duplicate.

The concentrations employed were chosen based on the results of a cytotoxicity study.

In this preliminary experiment, a pronounced cytotoxicity was noted at the concentration of 10 ug Olaflur/mL medium in the experiment without metabolic activation (24h exposure), a complete cytotoxicity started at 25 ug Olaflur/mL medium.

A pronounced cytotoxicity was noted at a concentration of 25 ug Olaflur/mL medium in the experiment with metabolic activation (4h exposure), a complete cytotoxicity started at a concentration of 100 ug Olaflur /mL medium.

Main Study Concentrations

Hence, the top concentrations employed in the main study were 10 ug Olaflur/mL medium in the experiment without and 20 ug Olaflur/mL medium in the experiment with metabolic activation (4h and 24h exposure, respectively).

Positive Controls

Mitomycin C and cyclophosphamide were employed as positive control chemicals in the absence and presence of metabolic activation, respectively.

Tests without metabolic activation (4h and 24h exposure)

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with Olaflur at concentrations of 1.25, 2.5, 5 of 10 ug Olaflur/mL medium in the absence of metabolic activation ranged from 1.5 % to 3.0 % in the two independent experiments with blood from different donors.

The top concentration of 10 ug Olaflur/mL medium (mitotic index: 0.13) caused a pronounced cytotoxicity after the 24h exposure, exceeding the toxicity range recommended in the respective guidelines for 24h exposure.

Only at the pronounced cytotoxic concentration of 10 ug Olaflur/mL medium a marginal, though not significant, increase was noted in the number of aberrations to 5.4 %, exceeding slightly the toxicity control range.

It is known that high cytotoxicity causes artefacts in form of aberrations in in vitro chromosomal tests. Hence, the increase at the concentration of 10 ug Olaflur/mL medium at 24h only and obtained at very high cytotoxicity is considered as artefact and not test substance-related.

All other results obtained are considered to be within the normal range of the negative control where a mean incidence of chromosomal aberrations (excluding gaps) of 1.5 or 1.0% was observed after a 4 h or 24 h exposure.

Test with metabolic activation (4 h exposure)

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with Olaflur (2.5, 5, 10 or 20 ug Olaflur/mL medium) in the presence of metabolic activation ranged from 0.0 % to 3.0 % in the two independent experiments.

The concentrations of 10 or 20 ug Olaflur/mL medium (mitotic indices: 0.82 and 0.14, respectively) caused a pronounced cytotoxicity after the 4h exposure. The results obtained are considered to be within the normal range of the negative control where the incidence of chromosomal aberrations (excluding gaps) ranged from 0.0% to 5.0 % for the last 30 experiments. In the same test, mitomycin C and cyclophosphamide induced significant damage.