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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 October 2017 - 18 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid - liquid: aqueous solution
Details on test material:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lot # 5314-007-001
- Expiration date of the lot/batch: 04 July 2019


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15-25°C, protected from light
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lot # 5314-007-001
- Expiration date of the lot/batch: 04 July 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15-25°C, protected from light

Method

Species / strain
Species / strain / cell type:
lymphocytes: human
Remarks:
peripheral blood lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
S-9 prepared from male Sprague-Dawley rats induced with Aroclor 1254.
Test concentrations with justification for top dose:
Range-Finder: 18.14 - 5000 μg/mL
Micronucleus Experiment:
3+21 hrs, -S-9: 10-100 μg/mL
3+21 hrs, +S-9: 15-100 μg/mL
24+24 hrs, -S-9: 5-50 μg/mL
A maximum concentration of 5000 μg/mL was selected for the cytotoxicity Range-Finder Experiment, in order that treatments were performed up to the maximum recommended concentration, for test articles of this type, according to current regulatory test guidelines. Concentrations selected for the Micronucleus Experiment were based on the results of this cytotoxicity Range-Finder Experiment.
Test concentrations are based on the active content.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The test material is manufactured as an approximately 30% aqueous solution, therefore further dilution with water, as necessary, is appropriate.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Sterile purified water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: vinblastine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 3 or 24 hours (-S9); 3 hours (+S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 72 or 96 hours (-S9); 72 hours (+S9)

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B; 6 μg/mL

STAIN (for cytogenetic assays): Acridine Orange in phosphate buffered saline; 12.5 μg/mL

NUMBER OF REPLICATIONS: Duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Lymphocytes were kept in fixative at 2-8°C prior to slide preparation for a minimum
of 3 hours to ensure that cells were adequately fixed. Cells were centrifuged (approximately 1250 g, 2-3 minutes) and resuspended in a minimal amount of fresh fixative (if required) to give a milky suspension. Several drops of cell suspension were gently spread onto multiple clean, dry microscope slides. Slides were air-dried and stored protected from light at room temperature prior to staining. Slides were stained by immersion in 12.5 μg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 minutes and washed with PBS (with agitation) for a few seconds. The quality of the staining was checked. Slides were air-dried and stored protected from light at room temperature. Immediately prior to analysis 1-2 drops of PBS were added to the slides before mounting with glass coverslips.

NUMBER OF CELLS EVALUATED: 1000 binucleate cells from each culture (2000 per concentration) were analysed for micronuclei, where possible.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Scoring was carried out using fluorescence microscopy. Binucleate cells were only included in the analysis if all of the following criteria were met:
1. The cytoplasm remained essentially intact, and
2. The daughter nuclei were of approximately equal size.
A micronucleus was only recorded if it met the following criteria:
1. The micronucleus had the same staining characteristics and a similar morphology to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching a main nucleus, and
3. Micronuclei were smooth edged and smaller than approximately one third the diameter of the main nuclei.

DETERMINATION OF CYTOTOXICITY
- Method: Replication index
- Any supplementary information relevant to cytotoxicity: Slides from the cytotoxicity Range-Finder Experiment were examined, uncoded, for proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per concentration. From these data the replication index (RI) was determined.
RI, which indicates the relative number of nuclei compared to vehicle controls was determined using the formula as follows:
RI = (number binucleate cells + 2 (number multinucleate cells))/total number of cells in treated cultures
Relative RI (expressed in terms of percentage) for each treated culture was calculated as follows:
Relative RI (%) = (RI of treated cultures/ RI of vehicle controls) x100
Cytotoxicity (%) is expressed as (100 – Relative RI).
A selection of random fields was observed from enough treatments to determine whether chemically induced cell cycle delay or cytotoxicity had occurred.

OTHER EXAMINATIONS:
- Determination of polyploidy: Not applicable
- Determination of endoreplication: Not applicable
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): Not applicable
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed
3. A concentration-related increase in the proportion of MNBN cells was observed (positive trend test).
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result. Biological relevance was taken into account, for example consistency of response within and between
concentrations, or effects occurring only at very toxic concentrations.
Statistics:
The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.
The proportions of MNBN cells for each treatment condition were compared with the proportion in vehicle controls by using Fisher's exact test.
A Cochran-Armitage trend test was applied to each treatment condition. Probability values of p≤0.05 were accepted as significant.

Results and discussion

Test results
Key result
Species / strain:
other: human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: no
- Precipitation: no
- Definition of acceptable cells for analysis: no
- Other confounding effects: none reported

RANGE-FINDING/SCREENING STUDIES:

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: refer to table below
- Negative (solvent/vehicle) historical control data: refer to table below

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cytotoxicity (%) is expressed as (100 – Relative RI).

Any other information on results incl. tables

Summary of the findings

Treatment

Concentration (µg/mL)

Cytotoxicity $ (%)

Mean MNBN Cell Frequency (%)

Historical Control Range # (%)

Statistical Significance

3+21 hour -S-9

aVehicle

-

0.28

0.20 – 1.00

-

50

7

0.3

NS

80

30

0.35

NS

90

45

0.35

NS

*MMC, 0.30

27

7.1

p≤0.001

3+21 hour +S-9

aVehicle

-

0.35

0.20 – 1.07

-

60

11

0.45

NS

85

36

0.3

NS

100

58

0.53

NS

*CPA, 2.00

2

1.3

p≤0.001

*CPA, 3.00

10

0.85

p≤0.01

24+24 hour -S-9

aVehicle

-

0.15

0.10 – 0.90

-

5

8

0.4

NS

12.5

21

0.35

NS

20

44

0.6

p ≤0.05

25

55

0.25

NS

*VIN, 0.04

55

3.45

p ≤0.001

a

Vehicle control was water

*

Positive control

#

95thpercentile of the observed range

$

Based on replication index

NS

Not significant 

Applicant's summary and conclusion

Conclusions:
It is concluded that the test item did not induce micronuclei in human peripheral blood lymphocytes when tested up to toxic concentrations for 3+21 hours in the absence and presence of a rat liver metabolic activation system (S-9) and for 24+24 hours in the absence of S-9 under the experimental conditions described.
Executive summary:

Amines, C12-14 (even numbered)-alkyldimethyl, N-oxides was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test article was formulated in water (purified water). The highest concentrations analysed in the Micronucleus Experiment were limited by toxicity and were determined following a preliminary cytotoxicity Range-Finder Experiment.

Treatments were conducted 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect of Amines, C12-14 (even numbered)-alkyldimethyl, N-oxides on the replication index (RI). Micronuclei were analysed at three or four concentrations. Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate (MNBN) cells in the cultures fell within the 95th percentile of the current observed historical vehicle control (normal) ranges. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of rat liver S-9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S-9. Cells receiving these were sampled in the Micronucleus Experiment at 24 hours (CPA, MMC) or 48 hours (VIN) after the start of treatment. One concentration of all positive control compounds induced statistically significant increases in the proportion of cells with micronuclei.

All acceptance criteria were considered met and the study was accepted as valid.

Treatment of cells with Amines, C12-14 (even numbered)-alkyldimethyl, N-oxides for 3+21 hours in the absence and presence of S-9 and for 24+24 hours in the absence of S-9 resulted in frequencies of MNBN cells that were similar to those observed in the concurrent vehicle controls and which fell within the normal ranges at all concentrations analysed under each treatment condition. A statistically significant increase in MNBN cell frequency (p≤0.05) was observed at an intermediate concentration of 20 μg/mL for the 24+24 hour treatment in the absence of S-9 but the MNBN cell frequency values for both replicates at this concentration were within the normal range and there was no statistically significant linear trend, therefore this observation was considered not biologically relevant.

It is concluded that Amines, C12-14 (even numbered)-alkyldimethyl, N-oxides did not induce micronuclei in human peripheral blood lymphocytes when tested up to toxic concentrations for 3+21 hours in the absence and presence of a rat liver metabolic activation system (S-9) and for 24+24 hours in the absence of S-9 under the experimental conditions described.