Oligomerisation products of ethylene oxide with reaction products of rape oil and ethanolamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Date started: 5th January 1994. Date completed: 23rd June 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of GLP inspection: 31/01/1994 Date of Signature on GLP certificate: 16/03/1994

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Details on test material:

Chemical name: ethoxylated fatty acid monoethanolamide
Date received: 5 October 1993
Description: straw-coloured viscous liquid
Container: clear glass bottle
Storage conditions: room temperature

Method

Target gene:

Not applicable.

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteers had not been exposed to high levels of radiation, hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). Using the current batch of foetal calf serum the donors used in this study had values for AGT of approximately 13 hours.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver, S9

Test concentrations with justification for top dose:

Experiment 1:
With and without S9-mix: 5000, 2500, 1250, 625, 312.5, 156.25, 78.1 and 39 μg/ml.

Experiment 2:
20-hour harvest with and without S9 mix: 0, 39, 78.1, 156.25, 234.35 and 312.5 μg/ml.
44-hour harvest with and without S9 mix: 0, 156.25, 234.35 and 625 μg/ml.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Aminol N was accurately weighed and dissolved in DMSO and appropriate serial dilutions prepared.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Cyclophosphamide (CP), 25 μg/ml for cultures where S9 was included. It was dissolved in culture medium without serum.

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

EMS, 500 μg/ml for cultures in the absence of metabolising enzymes, It was dissolved in DMSO.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
48 hrs

- Exposure duration:
Experiment 1: 4 hours
Experiment 2: 20 and 44 hours

- Expression time (cells in growth medium):
Experiment 1: 16 hours
Experiment 2: 16 hours and 40 hours.

- Selection time (if incubation with a selection agent):
Not applicable.

- Fixation time (start of exposure up to fixation or harvest of cells):
20 hrs and 44 hrs.


SELECTION AGENT (mutation assays):
No selection agent.

SPINDLE INHIBITOR (cytogenetic assays):
Demecolcine

STAIN (for cytogenetic assays):
When the slides were dry they were stained in 5% Gurrs Giemsa R66 for 5 minutes, rinsedm dried and mounted in Depex mounting medium.


NUMBER OF REPLICATIONS:
Duplicate cultures


NUMBER OF CELLS EVALUATED:
100/culture


DETERMINATION OF CYTOTOXICITY:
- Method:
mitotic index - A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

-Scoring of Chromosome Damage:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, and if the cell had 46 or more chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. All chromosome aberrations were checked by a senior cytogeneticist prior to decoding the slides.

OTHER EXAMINATIONS:
- Determination of polyploidy:
Frequency of polyploid cells


OTHER:
None.

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared with the concurrent vehicle control value using Fisher's Exact test or Chi-squared test.

Results and discussion

Test results

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Refer to information on results and attached tables.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation noted.


RANGE-FINDING/SCREENING STUDIES: Not conducted.


COMPARISON WITH HISTORICAL CONTROL DATA:
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range.


ADDITIONAL INFORMATION ON CYTOTOXICITY:

EXPERIMENT 1:
The doses selected for evaluation in the 20-hour without metabolic activation treatment group were 39, 78.1 and 156.25 μg/ml. This was based on toxicity as there were no scorable metaphases observed at and above 312.5 μg/ml. The doses selected for evaluation and reported in the 20-hour with metabolic activation treatment group were 39, 78.1 and 156.25 μg/ml. The higher dose levels were deemed not suitable for reporting as the response was not reproducible. Thus, scoring metaphase cells were observed at all dose levels and there was no apparent reduction at all dose levels above 156.25 μg/ml. It was considered that a 'plateau' toxic response had been achieved at 156.25 μg/ml.

Both of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range (Table 4).

The positive control treatments gave significant increases in the frequency of cells with aberrations (Table 4), indicating that the metabolic activation system was satisfactory and that the test method was operating as expected. The B culture in the EMS treatment failed and there were only 30 scorable metaphases. However, the expected response was observed in the A culture.

Aminol N was seen to induce no significant increases in the frequency of cells with aberrations either in the presence or absence of S9 (Table 4).

Aminol N did not induce a significant increase in the numbers of polyploid cells at any dose-level in either of the treatment cases (Table 7).

EXPERIMENT 2:
The dose levels selected for evaluation in the 20-hour without -S9 treatment group were 78.1, 156.25 amd 234.35 μg/ml. The dose range was reduced from that used in the first experiment so as to optimise the toxicity dose-response curve.

The dose levels selected for evaluation in the 20-hour with-S9 treatment group were 39, 78.1 and 156.25 μg/ml. There were insufficient or no scorable metaphases at and above 312.5 μg/ml. The observed toxicity was greater than that seen in Experiment 1 and this was considered to be possibly due to different batches of S9 and experimental variation. A third experiment (data not reported) gave a toxicity response curve similar to that of Experiment 2.

The dose level selected for evaluation in the 44-hour treatment group without S9 was 156.25 μg/ml, as there were no scorable metaphases observed at and above 234.35 μg/ml. The maximum dose scored for the with-S9 treatment group was 625 μg/ml.

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range (Tables 5 and 6).

The positive control treatments gave significant increases in the frequency of cells with aberrations (Table 5), indicating that the metabolic activation system was satisfactory and that the test method was operating as expected.

Aminol N was seen to induce no statistically significant increases in the frequency of cells with chromosome aberrations either in the presence or absence of S9-mix in both the 20 and 44-hour treatment groups (Tables 5 and 6).

Aminol N did not induce a significant increase in the numbers of polyploid cells at any dose level in any of the treatment cases (Table 7).

Remarks on result:

other: strain/cell type: lymphocytes

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

For all result tables and figures (referenced above) please refer to the attached background material section.

Tables 1 -3: Mitotic Index

Table 4: Results of Chromosome Aberration Test - Experiment 1

Table 5: Results of Chromosome Aberration Test - Experiment 2 (20 hours)

Table 6: Results of Chromosome Aberration Test - Experiment 2 (44 hours)

Table 7: Incidence of Polyploid Cells

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Aminol N did not induce a significant increase in the frequency of cells with chromosome aberrations or polyploid cells in either the presence or absence of a liver enzyme metabolising system. Aminol N is therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Method:

Human lymphocytes, treated with Aminol N were evaluated for chromosome aberrations at up to three dose levels, in duplicate, together with vehicle and positive controls. Four treatment conditions were used i.e. 4 hours exposure with the addition of an induced rat liver homogenate metabolising system at 10% in standard co-factors with cell harvest after 16 and 40 hour expression periods and 20 and 44 hour continous exposures in the absence of activation. In Experiment 1 the dose range for evaluation was selected from a series of 8 dose levels on the basis of toxicity.

The method used followed that described in the OECD Guidelines for Testing of Chemicals (1981) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commision Directive 92/69/EEC.

Results:

All vehicle controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control treatments gave significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metobolising system.

Aminol N induced no significant increases in the frequency of cells with aberrations or polyploid cells. Aminol N was shown to be non-clastogenic to human lymphocytes in vitro.