Tall-oil fatty acids salts of condensation products of tall-oil fatty acids with diethanolamine and triethanolamine

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:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across from GLP-compliant guideline study performed with similar substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

of 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

of 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH (1996) Guideline S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. PAB/PCD Notification No. 444.

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. PMSB/ELD Notification No. 554.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Method

Species / strain

Species / strain / cell type:

lymphocytes: human, cultured in vitro in whole blood culture

Details on mammalian cell type (if applicable):

- Source of lymphocytes: Human blood collected aseptically from two healthy, non-smoking male donors and pooled.
- Type and identity of media:
RPMI 1640 tissue culture medium supplemented with 10% foetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM glutamine.
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from male Sprague Dawley rats treated with phenobarbital and 5,6-benzoflavone for enzyme induction.

Test concentrations with justification for top dose:

EXPERIMENT 1:
Concentrations prepared without and with metabolic activation (S9): 0, 10.08, 16.80, 27.99, 46.66, 77.76, 129.60, 216, 360, 600 and 1000 μg/mL .
Because of low positive control values following metaphase analysis of cultures without S9, an additional test was conducted.
Concentrations prepared without S9 (additional test): 0, 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 μg/mL.
Microscopically examined (metaphase analysis) without S9: 0, 100, 300 and 400 μg/mL.
Microscopically examined (metaphase analysis) with S9: 0, 360, 600 and 1000 μg/mL.

EXPERIMENT 2:
Concentrations prepared without metabolic activation (S9): 0, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 and 700 μg/mL
Because the appropriate toxicity profile was not achieved without S9, an additional test was conducted.
Concentrations prepared without S9 (additional test): 0, 50, 100, 115, 130, 145, 160, 175, 190, 205, 220, 235 and 250 μg/mL
Concentrations prepared with S9: 0, 125, 250, 500, 750 and 1000 μg/mL
Microscopically examined (metaphase analysis) without S9: 0, 220, 235 and 250 μg/mL
Microscopically examined (metaphase analysis) with S9: 0, 125, 500 and 1000 μg/mL

CRITERIA FOR SELECTING APPROPRIATE TEST CONCENTRATIONS FOR METAPHASE ANALYSIS:
The concentration causing a decrease in mitotic index of at least 50% (where possible) of the solvent control value was the highest concentration selected for metaphase analysis. Intermediate and low concentrations were also selected. Where no decrease in mitotic index was observed, the highest three concentrations tested were selected for metaphase analysis.

Vehicle / solvent:

Ethanol

Justification for choice of solvent/vehicle:
No precipitate was observed at the limit concentration of 5000 µg/mL WS400128 in aqueous tissue culture medium when ethanol was used as a vehicle, thus facilitating exposure of the cultures to quite high test substance concentrations. However, at concentrations > 1000 µg/mL fluctuation in osmolality was > 50 mOsm/kg leading to the choice of 1000 µg/mL as the maximum concentration tested.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Mitomycin C tested at 0.2 μg/mL (3 hour treatment) and 0.1 μg/mL (21 hour continuous treatment), vehicle sterile purified water

Migrated to IUCLID6: without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Cyclophosphamide tested at 5 μg/mL (3 hour treatment), vehicle sterile purified water

Migrated to IUCLID6: with metabolic activation

Details on test system and experimental conditions:

CELL DIVISION STIMULANT:
Phytohaemagglutinin

METHOD OF APPLICATION:
in cell culture medium;

DURATION
- Exposure duration:
3 hours in Experiment 1 (without and with metabolic activation) and in Experiment 2 (with metabolic activation).
21 hours in Experiment 2 (without metabolic activation)
[After the 3 h treatment the cells were cultivated with fresh media for 18 h].
- Final concentration of S9 mix:
Experiment 1: 2 % v/v
Experiment 2: 5 % v/v
- Fixation time (start of exposure up to fixation or harvest of cells):
21 hours in each of both experiments

SPINDLE INHIBITOR (cytogenetic assays):
Colcemid® was added to the cultures (0.1 µg/mL culture medium) 19 hours after treatment start.
2 h later, the cells were treated with hypotonic solution (0.075 M KCl) for 10 min at 37 °C. After incubation in the hypotonic solution, the cells were fixed with 3 + 1 methanol + glacial acetic acid.

STAIN (for cytogenetic assays):
After fixation the cells were stained with 10% Giemsa.

NUMBER OF REPLICATIONS:
Duplicate cultures were treated at each concentration.

NUMBER OF CELLS EVALUATED:
100 metaphases per culture, amounting to a total of 200 metaphases per dose concentration, were scored for structural chromosomal aberrations.
This number was reduced In cultures showing a high level of aberrant cells, where 10 cells in 100 metaphases with structural aberrations (excluding gaps) were observed.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (% cells in mitosis) determined by counting the number of mitotic cells in 1000 cells;

Microscopic examination of the metaphases included the recording of the following parameters:
- Aberrant cells (including and excluding gaps),
- Number of gaps,
- Types of aberrations
Chromatid break, Chromosome break, Chromatid gap, Chromatid exchange, Chromosome exchange, Chromosome gap,
Others: Cells with greater than eight aberrations, pulverised cells and pulverised chromosomes

Determination of polyploidy:
- Polyploid and endoreduplicated cells were noted when seen.

Evaluation criteria:

An assay is considered to be acceptable if the vehicle and positive control values lie within the current historical control range.

The test substance is considered to cause a positive response if the following conditions are met:
-Significant increases (P<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) at one or more test concentration.
-The increases exceed the vehicle control range of this laboratory, taken at the 99% confidence limit.
-The increases are reproducible between replicate cultures.
-The increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
-Evidence of a concentration-related response is considered to support the conclusion.

A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any concentration.

Statistics:

One-tailed Fisher exact test (Fisher 1973) for comparison of the number of aberrant metaphase cells in each test substance group with the solvent control value.

In addition, a Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test substance groups. If this is significant at the1% level, the test is reiterated excluding the highest concentration group - this process continues until the trend test is no longer significant.

ARMITAGE, P. (1955) Tests for linear trends in proportions and frequencies. Biometrics, 11, 375-386. (Cochran-Armitage test).
FISHER, R.A. (1973) The Exact Treatment of 2 x 2 Table in: Statistical Methods for Research Workers. Hafner Publishing Company, New York.

Results and discussion

Test results

Test results 1

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

In both experiments, following 3 h treatment

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

With metabolic acitviation (S9 at 5% v/v final concentration) and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

lymphocytes:

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

At one cytotoxic concentration (250 µg/mL), following 21 h treatment

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
At concentrations > 1000 µg/mL fluctuation in osmolality was > 50 mOsm/kg leading to the choice of 1000 µg/mL as the maximum concentration tested.

Remarks on result:

other: strain/cell type: human lymphocytes with 44- 48 chromosomes

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

 

Table 1:    Experiment 1 Chromosome Aberration and Cytotoxicity Results Based on Duplicate Cultures/Concentration
Treatment period	S9 mix	Nominal concentration of WS400128	Cells with aberrations excluding gaps			Cells with aberrations including gaps			Relative mitotic index (%)	Polyploidy mean incidence (%)
(hours)	(v/v)	(µg/mL)	Individual values (%)		Mean (%)	Individual values (%)		Mean (%)
3	-	0 (Ethanol)	0.0	1.0	0.5	0.0	1.0	0.5	100	0.0
		100	1.0	1.0	1.0	2.0	1.0	1.5	99	0.0
		300	2.0	0.0	1.0	2.0	0.0	1.0	64	0.5
		400	0.0	1.0	0.5	1.0	2.0	1.5	46	0.5
		0.2 (Mitomycin C)	27.8	19.2	22.7***	30.6	19.2	23.9***	-	0.0
3	+	0 (Ethanol)	2.0	2.0	2.0	2.0	3.0	2.5	100	0.5
	(2%)	360	1.0	1.0	1.0	2.0	3.0	2.5	115	1.5
		600	1.0	2.0	1.5	2.0	2.0	2.0	106	2.5
		1000	1.0	4.0	2.5	3.0	7.0	5.0	118	3.5
		5 (Cyclophosphamide)	19.2	25.6	22.0***	19.2	28.2	23.1***	-	0.0

 

One-tailed Fisher's exact test

***                 p<0.001

Otherwise         p>0.01

 

 

Table 2:    Experiment 2 Chromosome Aberration and Cytotoxicity Results Based on Duplicate Cultures/Concentration
Treatment period	S9 mix	Nominal concentration of WS400128	Cells with aberrations excluding gaps			Cells with aberrations including gaps			Relative mitotic index (%)	Polyploidy mean incidence (%)
(hours)	(v/v)	(µg/mL)	Individual values (%)		Mean (%)	Individual values (%)		Mean (%)
21	-	0 (Ethanol)	1.0	2.0	1.5	2.0	2.0	2.0	100	0.5
		220	3.0	3.0	3.0	5.0	6.0	5.5	124	0.5
		235	3.0	5.0	4.0	6.0	6.0	6.0	78	2.5
		250	5.0	7.0	6.0**	5.0	9.0	7.0**	52	1.0
		0.1 (Mitomycin C)	13.2	17.2	14.9***	17.1	22.4	19.4***	-	0.0
3	+	0 (Ethanol)	1.0	0.0	0.5	2.0	0.0	1.0	100	0.0
	(5%)	125	0.0	0.0	0.0	0.0	0.0	0.0	92	0.5
		500	3.0	0.0	1.5	4.0	0.0	2.0	76	1.0
		1000	3.0	1.0	2.0	4.0	1.0	2.5	52	0.0
		5 (Cyclophosphamide)	21.7	4.0	9.6***	21.7	5.0	10.3***	-	0.0

 

One-tailed Fisher's exact test

***                  p<0.001

**                   p<0.01

Otherwise         p>0.01

 

As outlined in the „Validity Assessment Report“ for the read-across approach (see IUCLID Section 13) read-across from testing data obtained with the UVCB substance WS400128 is considered appropriate for the safety evaluation as well as classification and labelling of the UVCB substance WS400136 based on the close chemical similarity between the two substances.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative 3 h treatment without and with metabolic activation (S9)
ambiguous without metabolic activation 21 h treatment

The ambiguous result after 21 hour exposure without metabolic activation is considered as artifactual result. Based on the very low water solubility of the test substance it is to be assumed that cells were not exposed to true solutions of the substance but to dispersions. It is known that exposure of cells to precipitates or dispersions, i.e. concentrated test substance, can lead to artifactual, false positive results.

Executive summary:

As outlined in the „Validity Assessment Report“ for the read-across approach (see IUCLID Section 13) read-across from testing data obtained with the UVCB substance WS400128 is considered appropriate for the safety evaluation as well as classification and labelling of the UVCB substance WS400136 based on the close chemical similarity between the two substances.

Like the read-across source substance WS400128 the read-across target substance WS400136 is considered non-clastogenic in the in vitro chromosome aberration test.