Fatty acids, tall-oil, reaction products with 2-[(2-aminoethyl)amino]ethanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-03-27 to 2012-06-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD guideline 471 and EU method B.13/14 in a GLP certified testing facility.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Five genetically manipulated strains of Salmonella typhimurium (TA 97a, TA 98, TA 100, TA 102 and TA 1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (S9) for 48 hours, using the plate incorporation method.

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

mutations: hisG46, uvrB, rfa

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

mutations: hisG428, pKM 101, rfa

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

mutations: hisG46, uvrB, pKM 101, rfa

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

mutations: hisD3052, uvrB, pKM 101, rfa

Species / strain / cell type:

S. typhimurium, other: 97a

Details on mammalian cell type (if applicable):

mutations: hisD6610, uvrB, pKM 101, rfa

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

1st experiment: 5031 / 1509 / 503 / 151 / 50 µg/plate
2nd experiment: 61 / 18 / 6 / 1.8 / 0.6 µg/plate
3rd experiment: 51 / 26 / 13 / 6.4 / 3.2 / 1.6 / 0.8 µg/plate

Vehicle / solvent:

ethanol

Negative solvent / vehicle controls:

yes

Remarks:

H2O, DMSO, ethanol

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

concentration per plate: 20 µg; solvent: DMSO; strains: TA 98; with S9

Positive controls:

yes

Positive control substance:

other: 2-Amino-anthracene

Remarks:

concentration per plate: 1 µg; solvent: DMSO; strains: TA 97a, TA 100, TA 102, TA 1535; with S9

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

concentration per plate: 1 µg; solvent: H2O; strains: TA 100, TA 1535; without S9

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylene diamine

Remarks:

concentration per plate: 20 µg; solvent: DMSO; strains: TA 97a, TA 98, TA 102; without S9

Details on test system and experimental conditions:

First Experiment
Concentrations tested: 5031 / 1509 / 503 / 151 / 50 µg/plate
Incubation time: 48 hours
Incubation temperature: 37 °C
Tester strains: TA97a, TA98, TA100, TA102, TA1535
Method: plate incorporation method

Second Experiment
Concentrations tested 61 / 18 / 6 / 1.8 / 0.6 µg/plate
Incubation time 48 hours
Incubation temperature 37 °C
Tester strains TA97a, TA98, TA100, TA102, TA1535
Method plate incorporation method

Third Experiment
Concentrations tested 51 / 26 / 13 / 6.4 / 3.2 / 1.6 / 0.8 µg/plate
Incubation time 48 hours
Incubation temperature 37 °C
Tester strains TA97a, TA98, TA100, TA102, TA1535
Method pre-incubation method

Key result

Species / strain:

S. typhimurium, other: 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 151 µg/plate and higher

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 151 µg/plate and higher

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 151 µg/plate and higher

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 151 µg/plate and higher

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 151 µg/plate and higher

Vehicle controls validity:

valid

Positive controls validity:

valid

Confirmation of the Criteria and Validity

The treatments for the sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (historical data of the laboratory). All positive controls (diagnostic mutagenes) showed mutagenic effects with and without metabolic activation.

Solubility and Toxicity

The test item was dissolved in ethanol. A stock solution containing 50 g/L was prepared.

Signs of toxicity towards the tested strains were observed in the first experiment in the four highest concentrations (5031, 1509, 503 and 151 µg/plate): No growth was found on these plates.

In the second and third experiment no signs of toxicity towards the tested strains were observed. The background lawn was visible and the number of revertant colonies was not significantly reduced.

 

Mutagenicity

No significant increase in the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase was found over the tested concentration range.

As only one concentration in the first experiment could be evaluated due to the test item’s toxicity, the test was repeated using a lower concentration range.  

The test item was considered as not mutagenic under the test conditions.

A third experiment was performed using the pre-incubation method which verifies this result.

Table 1:           Mean Revertants in the First Experiment (colonies per plate)

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H2O	Mean	115	117	13	14	112	118	155	134	15	15
	sd	3.6	2.2	3.3	1.9	7.8	2.2	20.1	11.0	1.7	2.5
DMSO	Mean	105	112	16	14	106	101	151	139	16	15
	sd	4.8	2.2	2.5	1.0	6.8	10.2	2.6	6.4	1.0	3.3
Ethanol	Mean	105	113	15	15	93	82	141	140	15	16
	sd	9.1	6.6	2.5	2.6	9.9	6.6	6.7	3.6	1.9	1.0
Positive Controls*	Mean	594	590	231	224	467	572	576	574	224	206
	sd	36	30	4	5	35	23	89	56	7	11
	f(I)	5.66	5.27	14.44	16.00	4.17	5.66	3.81	4.13	14.93	13.73
5031 µg/pl.	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0	0	0	0	0	0	0	0	0	0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
1509 µg/pl.	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0	0	0	0	0	0	0	0	0	0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
503 µg/pl.	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0	0	0	0	0	0	0	0	0	0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
151 µg/pl.	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0	0	0	0	0	0	0	0	0	0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
50 µg/pl.	Mean	90	103	14	14	77	80	137	137	13	12
	sd	9	5	3	1	7	8	2	5	3	2
	f(I)	0.86	0.91	0.93	0.93	0.83	0.98	0.97	0.98	0.87	0.75

f(I) = increase factor

* Different positive controls were used

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test item 400112 is considered not mutagenic under the test conditions.

Executive summary:

The mutagenic potential of 400112 has been tested according to OECD guideline 471 and EU method B.13/14 in a GLP certified testing facility.

The test item 400112 is considered not mutagenic under the test conditions.

The test item showed cytotoxicity towards the bacteria in the first experiment in the four highest concentrations (5030.8, 1509.2, 503 and 151 µg/plate). Therefore, a second experiment using the plate incorporation method was performed with lower concentrations (0.6 – 61 µg/plate). No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed. No concentration-related increase over the tested range was found. Therefore, the test item was considered as not mutagenic under the test conditions.

On the base of the results of this second experiment, a third experiment was performed with the pre-incubation method to verify this result.

The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value. The numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory and were definitely increased in comparison to the negative controls, as well as showing mutagenic potential of the diagnostic mutagens.

Spontaneous revertants were within the normal range in comparison with the historical data of LAUS GmbH.

For these reasons, the result of the test is considered valid.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-03-30 to 2012-08-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD guideline 487 in a GLP certified testing facility.

Qualifier:

according to guideline

Guideline:

other: OECD 487

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human peripheral lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

6.25 / 12.5 / 25 / 50 / 100

Vehicle / solvent:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation; 10 and 20 µg/mL

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation; 0.1 and 0.2 µg/mL

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Details on test system and experimental conditions:

Procedure / Treatment Schedule
Cells were sampled at a time equivalent to about 1.5 – 2.0 times the normal (i.e. untreated) cell cycle length either after the beginning or at the end of treatment. Sampling or recovery times might be extended if it is known or suspected that the test item affects the cell cycling time (e.g. when testing nucleoside analogues). Because of the potential cytotoxicity of S9 preparations for cultured mammalian cells, an extended exposure treatment of 1.5 – 2.0 normal cell cycles was used only in the absence of S9.
All treatments commenced and ended while the cells were growing exponentially.

Cell treatment and harvest times for the used human peripheral lymphocytes:
--> Treatment with cytoB +S9: treatment for 4 hrs in the presence of S9, remove the S9 and treatment medium, add fresh medium and cytoB, harvest 1.5 – 2.0 normal cell cycles later.
--> Treatment with cytoB –S9 (short exposure): treatment for 4 hrs, remove the treatment medium, add fresh medium and cytoB, harvest 1.5 – 2.0 normal cell cycles later.
--> Treatment with cytoB –S9 (extended exposure): treatment for 1.5 – 2.0 normal cell cycles, remove the test item, add fresh medium and cytoB, harvest 1.5 – 2.0 normal cell cycles later.

As both initial tests of the short 4-h treatment are negative or equivocal, a subsequent, extended exposure treatment without S9 was used.
Duplicate cultures were used for each test item concentration and for the solvent control cultures.
Each culture was harvested and processed separately. High-quality cell preparations for scoring were obtained. Cell cytoplasm was retained to allow the detection of micronuclei and (in the cytokinesis-block method) reliable identification of binucleate cells. The slides were stained using Giemsa.

Evaluation criteria:

1000 binucleated cells per duplicate cell culture were scored with a phase contrast microscope to assess the frequency of cells with one, two, or more than two micronuclei. Additionally, the cells were classified as mononucleates, binucleates or multinucleates to estimate the proliferation index as a measure of toxicity. The evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI).

CPBI = (MONC + BNC x 2 + MUNC x 3)/n
MONC = mononucleated cells
BNC = binucleated cells
MUNC = Multinucleate cells
n= total number of cells
Cytotoxicity was calculated as % cytostasis. A CBPI of 1 (all cells are mononucleated) is equivalent to 100 % cytostasis.

Cytostasis = 100 - RI
RI = ((BNCt + MUNCt x 2) / nt) / ((BNCc + MUNCc x 2) / nc)
t = treated cultures
c = control cultures

The micronucleus frequencies were analysed in at least 2000 binucleated cells per concentration (at least 1000 binucleated cells per culture; two cultures per concentration). If substantially fewer than 1000 binucleate cells per culture are available for scoring at each concentration, and if a significant increase in micronuclei is not detected, the test would be repeated using more cells, or at less toxic concentrations, whichever is appropriate.

Statistics:

Only the frequencies of binucleate cells with micronuclei (independent of the number of micronuclei per cell) were used in the evaluation of micronucleus induction. Concurrent measures of cytotoxicity and/or cytostasis for all treated and solvent control cultures were determined. Individual culture data were provided.

Positive result: a test item induces a concentration-related increase or a statistical significant and reproducible increase in the number of cells containing micronuclei.
The assessment was carried out by a comparison of the samples with the positive and the vehicle control, using a chi-square test corrected for continuity according to YATES (COLQUHOUN, 1971) as recommended by the UKEMS guidelines (The United Kingdom Branch of the European Environmental Mutagen Society: Report of the UKEMS subcommittee on guidelines for mutagenicity testing, part III, 1989: Statistical evaluation of mutagenicity data).

Negative result: under the used test conditions, the test substance does not induce chromosome breaks and/or gain or loss in cultured mammalian cells.

Key result

Species / strain:

other: human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

up to concentrations of 100 µg/mL without metabolic activation and up to 50 µg/mL with metabolic activation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 50 and 100 µg/mL without and with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation cytotoxicity and test item precipitation were noted starting at a concentration of 100 μg 400112/mL. Hence, 100 μg/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation.

In the main study cytotoxicity in form of haemolysis was noted at concentrations of 50 and 100 μg of 400112/mL in the experiments without and with metabolic activation. Mitomycin C and cyclophosphamide were employed as positive controls in the absence and presence of metabolic activation, respectively.

 

Tests without metabolic activation (4- and 20-hour exposure)

The micronucleus frequencies of cultures treated with 400112 at concentrations from 6.25 to 100 or 50 μg/mL medium (4-h and 20-h exposure, respectively) in the absence of metabolic activation ranged from 1.5 to 8.5 micronuclei per 1000 binucleated cells. Negative controls should give reproducibly low and consistent micronuclei frequencies, typically 5 - 25 micronuclei per 1000 cells according to OECD 487. The vehicle control in this test gave 5.0 or 9.0 micronuclei per 1000 binucleated cells and the untreated controls 8.0 or 5.5 micronuclei per 1000 binucleated cells (4-hour and 20-hour exposure, respectively).

 

Test with metabolic activation (4-hour exposure)

The micronucleus frequencies of cultures treated with 400112 at concentrations from 6.25 to 50 μg/mL medium in the presence of metabolic activation ranged from 2.0 to 9.5 micronuclei per 1000 binucleated cells. Negative controls should give reproducibly low and consistent micronuclei frequencies, typically 5 - 25 micronuclei per 1000 cells according to OECD guideline 487. The vehicle control in this test gave 6.0 or 5.5 micronuclei per 1000 binucleated cells and the untreated controls 8.0 or 4.0 micronuclei per 1000 binucleated cells.

No test item-related increase in micronucleus frequencies (significant at p≤0.05) was noted and no concentration relationship was noted.

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

Under the present test conditions, 400112 tested up to cytotoxic concentrations, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of mutagenic properties in the in vitro micronucleus test.

Executive summary:

Test samples of 400112 were assayed in an in vitro micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals.

The test was carried out employing 2 exposure times without S9 mix: 4 and 20 hours, and 1 exposure time with S9 mix: 4 hours. The experiment with S9 mix was carried out twice. The harvesting time was 20 hours after the end of exposure. The study was conducted in duplicate.

400112 was completely dissolved in dimethyl sulfoxide (DMSO).

Under the present test conditions, 400112 tested up to cytotoxic concentrations, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of mutagenic properties in the in vitro micronucleus test.

No test item-related increase in micronucleus frequencies (significant at p≤0.05) was noted and no concentration relationship was noted.

In the same test, the positive controls mitomycin C and cyclophosphamide induced significant damage.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-08-16 to 2013-01-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD guideline 476 and EU method B.17 in a GLP certified testing facility.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Supplier: ATCC (American Type Culture Collection), 0801 University Blvd., Manassas, VA 20110-2209, USA
The particular clone (3.7.2C) of L5178Y (TK+/-) cells was used in this assay.
- Treatment media: RPMI 1640 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced rats

Test concentrations with justification for top dose:

experiment with/without metabolic activation: 0.625 / 1.25 / 2.5 / 5.0 / 10.0 µg/mL (3 h exposure)
experiment without metabolic activation: 0.313 / 0.63 / 1.25 / 2.5 / 5.0 µg/mL (24 h exposure)

Vehicle / solvent:

- Vehicle/solvent used: acetone
- Justification for choice of solvent/vehicle: 400112 was completely soluble in acetone

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

2.5 µL and 4.0 µL, for studies with metabolic activation

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

10 µL and 15 µL; for studies without metabolic activation

Details on test system and experimental conditions:

DETERMINATION OF CYTOTOXICITY
- test item concentrations: 10, 25, 100, 250, 1000, 2500 and 5000 µg 400112/mL
- without and with S9 metabolic activation
After an exposure time of 3 hours at approx. 37°C on a roller drum at 10 - 15 rpm, the cells were washed and resuspended in growth medium. The cells were then adjusted to 8 cells/mL and for each concentration 0.2 mL was plated into 32 microtiter wells (average 1.6 cells/well). The plates were incubated at 37°C in a humidified incubator gassed with 5% CO2 in air for 7 days. Wells containing viable clones were identified under a microscope and counted.

MUTAGENICITY TESTING
Without metabolic activation
The assay procedure used is based on that reported by COLE et al. (1990). Single cultures were used for each test item concentration level and reference item. The results from the initial mutation assay were confirmed by performing an independent repeat mutation assay.
The cells for the first and second experiments were obtained from logarithmically growing laboratory stock cultures of 7.0 or 7.55 x 10^6 cells/mL and were seeded into a series of tubes, diluted to 5 x 10^5 cells/mL per tube. The cells were pelleted by centrifugation, the culture medium was removed, and the cells were resuspended in treatment medium that contained 5% heat inactivated fetal bovine serum and the corresponding concentration of test substance. The dosed tubes were closed, vortexed and placed on a roller drum at approx. 37°C at 10 - 15 rpm for an exposure period of 3 hours. Thereafter the cells were washed and resuspended in growth medium.
Cell densities were adjusted to 2 x 10^5/mL and the cells were plated for survival and incubated for the expression period in parallel, i.e. an aliquot of the cells was diluted to 8 cells/mL and 0.2 mL of each culture were placed in two 96 well microtiter plates (= 192 wells, averaging 1.6 cells/well) and incubated for 1 week whereas the rest of the cells was incubated for 2 days for the expression period (plating efficiency step 1).
The cells for the plating of survival were counted after 1 week and the number of viable clones was recorded. The cells incubated for the expression period were maintained below 106 cells per mL and a minimum of 4 concentration levels plus positive and negative control was selected for 5-trifluoro-thymidine (TFT) resistance. At the end of the second expression period the selected cultures were diluted to 1 x 10^4 cells/mL and plated for survival (plating efficiency step 2) and TFT resistance in parallel (plating efficiency step 2). The plating for survival was identical to the above described method (plating efficiency step 1 in 192 wells with average 1.6 cells/well). For the plating for TFT resistance 3 µg/mL TFT (final concentration) was added to the cultures and 0.2 mL of each suspensions placed into four 96-well microtiter plates (= 384 wells, averaging 2 x 10^3 cells/well). The plates were incubated for 11 to 14 days and wells containing clones were identified microscopically and counted.
In addition, the number of large and small colonies was recorded with an automated colony counter that can detect colony diameters equal to or greater than 0.2 to 0.3 mm. Large colonies are defined as ≥ 1/4 and small colonies < 1/4 of the well diameter of 6 mm.

Assay with metabolic activation
The activation assay is often run concurrently with the non-activation assay; however, it is an independent assay performed with its own set of solvent and positive controls. In this assay the above-described activation system was added to the treatment medium together with the test item.

Repeat experiment
The non-activation and activation experiment were repeated in an independent experiment. An exposure time of 24 hours and a concentration-range of 0.313, 0.625, 1.25, 2.5 and 5.0 µg/mL were used for the repeat experiment without metabolic activation (non-activation experiment). The exposure time was again 3 hours for the activation experiment and the experiments were conducted with the same concentrations as in the first experiment.

Evaluation criteria:

The minimum criterion to demonstrate mutagenesis is a mutant frequency that is ≥ 2 times the concurrent background mutant frequency.
The following test results must be obtained for activation or non-activation conditions:
- A concentration-related or toxicity-related increase in mutant frequency should be observed.
- If the mutant frequency obtained for a single concentration at or near the highest testable toxicity is about four times the concurrent background mutant frequency or greater, the test item will be considered mutagenic in a single trial.
- Applied concentration or toxicity (% relative growth), can be used to establish whether the increase in mutant frequency is related to an increase in effective treatment.
- Treatments that induce less than 10% relative growth are included in the assay, but are not used as primary evidence for mutagenicity.
- The ratio of small:large colonies will be calculated from the results of the determination of small to large colonies. If the test item is positive, the ratio of small to large colonies for the test item will be compared with the corresponding ratios of the positive and negative controls.
- A test item is evaluated as non-mutagenic only if the minimum increase in mutant frequency is not observed for a range of applied concentrations that extends to toxicity causing 10% to 20% relative growth or a range of applied concentrations extending to at least twice the solubility limit in culture media.
Evaluation criteria according to the Mouse Lymphoma Workgroup, Aberdeen, 2003:
For microwell assays, the global evaluation factor GEF is defined as 126 mutants per 106 viable cells. For valid data, the test item is considered to be mutagenic if the mutant frequency MF of any test concentration exceeded the sum of the mean control mutant frequency plus GEF and the linear trend test is positive. The test item is considered as positive if both of the above criteria are met and negative if neither of the above criteria are met

Statistics:

Calculated parameters:
- % relative survival
- Mutant frequency
- Number of wells containing large colonies and number of wells containing small colonies in the negative control, the positive controls and the different test item concentrations
- Percentage of large colonies and of small colonies, and the small : large ratio
- Relative Total Growth
- Relative suspension growth
- Cloning efficiency
- Suspension growth

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 10 µg/mL at 3 h with and without metabolic activation and at 5 µg/mL at 24 h without metabolic activation

Vehicle controls validity:

valid

Positive controls validity:

valid

In the preliminary experiment without and with metabolic activation cytotoxicity was noted starting at concentrations of 10 µg/mL.

Hence, in the main study the concentration-range of 0.625 to 10.0 µg/mL was used in the experiments without and with metabolic activation with a 3-hour exposure time. A concentration-range of 0.313 to 5.0 µg/mL was used in the second experiment without metabolic activation and a prolonged exposure (24-hour).

In the main study, cytotoxicity was noted immediately after treatment (plating efficiency step 1) and in the following plating for 5-trifluoro-thymidine (TFT) resistance (plating efficiency step 2) in the absence and presence of metabolic activation at the top concentration of 10.0 or 5.0 µg/mL, respectively, in the absence and presence of metabolic activation after a 3-hour exposure and in the second experiment without metabolic activation (24-hour exposure), respectively.

The values of mutation frequencies of the negative controls ranged from 71.42 to 75.89 per 10^6 clonable cells in the experiments without metabolic activation and from 58.51 to 102.88 per 10^6 clonable cells in the experiments with metabolic activation and, hence, were well within the historical data-range.

The mutation frequencies of the cultures treated with 400112 ranged from 52.06 to 85.37 per 10^6 clonable cells (3 hours exposure) and from 61.58 to 114.10 per 10^6 clonable cells (24 hours exposure) in the experiments without metabolic activation and from 56.84 to 79.39 per 10^6 clonable cells (3 hours exposure, first assay) and from 54.90 to 82.41 per 10^6 clonable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 10^6viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation.

In addition, no change was observed in the ratio of small to large mutant colonies, ranging from 0.38 to 1.33 for 400112 treated cells and from 0.54 to 1.11 for the negative controls.

The plating efficiency (PE step 1 and step 2) of the negative control was ≥ 50%, the mean cloning efficiencies (CE) within the range of 65% to 120% two days after treatment, and the mean suspension growth (SG) within the range of 8 to 32 following 3‑hour treatments and between 32 and 180 following 24-hour treatments. Hence, the acceptance criteria were met.

The positive controls methylmethanesulfonate (MMS) and 3 -methylcholanthrene (3-MC) caused pronounced increases in the mutation frequency ranging from 465.09 to 945.73 per 10^6 clonable cells in the case of MMS, and ranging from 766.13 to 1735.09 per 10^6 clonable cells in the case of 3-MC.

In addition, the colony size ratio was moderately shifted towards an increase in small colonies, ranging from 1.65 to 2.24 in the case of MMS.

As the absolute increase in the mean total mutation frequency of at least 300 x 10^-6 (at least 40% small colony MF) and the mean relative total growth (RTG) for the positive controls was greater than 10%, the acceptance criteria were met.

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

Under the present test conditions, 400112, tested up to cytotoxic concentrations, in the absence and presence of metabolic activation in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. The tested concentration range was 0.625 – 10.0 µg/mL in the first experiment and 0.313 – 5.0 µg/mL for the 24-hour exposure without metabolic activation in the second experiment. Under these conditions the positive controls exerted potent mutagenic effects and demonstrated the sensitivity of the test system and conditions.
In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, 400112 also did not exhibit clastogenic potential at the concentration-range investigated.
According to the evaluation criteria for this assay, these findings indicate that 400112, tested up to cytotoxic concentrations, in the absence and presence of metabolic activation, did neither induce mutations nor have any chromosomal aberration potential.

Executive summary:

400112 was assayed in a gene mutation assay in cultured mammalian cells (L5178Y TK+/-) with and without metabolic activation (S9 mix). The test was carried out employing two exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours; the experiment with S9 mix was carried out in two independent assays.

In the preliminary experiment without and with metabolic activation cytotoxicity (decreased survival) was noted starting at concentrations of 10 µg/mL.

In the main study the concentration-range of 0.625 to 10.0 µg/mL was used in the experiments without and with metabolic activation with a 3-hour exposure time. A concentration-range of 0.313 to 5.0 µg/mL was used in the second experiment without metabolic activation (24-hour exposure).

Methylmethanesulfonate (at 10 or 15 µL/mL) was employed as a positive control without metabolic activation and 3-Methylcholanthrene (at 2.5 or 4.0 µg/mL) with metabolic activation.

The mutation frequencies of the cultures treated with 400112 were within the range of the negative control values (acetone) and the normal range of 50 to 170 mutants per 10^6 viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation.

The plating efficiency (PE step 1 and step 2) of the negative control was ≥ 50%, the mean cloning efficiencies (CE) within the range of 65% to 120% two days after treatment, and the mean suspension growth (SG) within the range of 8 to 32 following 3 hour treatments and between 32 and 180 following 24-hour treatments. Hence, the acceptance criteria were met.

The positive controls methylmethanesulfonate (MMS) and 3-methylcholanthrene (3-MC) caused pronounced increases in the mutation frequency ranging from 465.09 to 945.73 per 10^6 clonable cells in the case of MMS, and from 766.13 to 1735.09 per 10^6 clonable cells in the case of 3-MC.

In addition, the colony size ratio was moderately shifted towards an increase in small colonies, ranging from 1.65 to 2.24 for MMS.

As the absolute increase in the mean total mutation frequency of at least 300 x 10-6 (at least 40% small colony MF) and the mean relative total growth (RTG) for the positive controls was greater than 10%, the acceptance criteria were met.

Under the present test conditions, 400112, studied up to cytotoxic concentrations, in the absence and presence of metabolic activation in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames Test

This study was performed according to test guidelines OECD guideline 471 and EU B.13/14 for bacterial reverse gene mutation data in a GLP certified testing facility.

In a reverse gene mutation assay in bacteria, strains TA 97, TA 98a, TA 100, TA 102, TA 1535 of Salmonella typhimurium were exposed to 400112 in ethanol at concentrations of 5031 / 1509 / 503 / 151 / 50 µg/plate in the plate incorporation method. The test item showed cytotoxicity towards the bacteria in the four highest concentrations. Therefore, a second experiment using the plate incorporation method was performed with lower concentrations (0.6 – 61 µg/plate). No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed. No concentration-related increase over the tested range was found.

To verify this result, a third experiment was performed at concentrations of 51 / 26 / 13 / 6.4 / 3.2 / 1.6 / 0.8 µg/plate with the pre-incubation method.

The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value. The numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory and were definitely increased in comparison to the negative controls, as well as showing mutagenic potential of the diagnostic mutagens.

Spontaneous revertants were within the normal range in comparison with the historical data.

For these reasons, the result of the test is considered valid.

The test item was considered as not mutagenic under the test conditions.

Micronucleus test

The genotoxic potential of the test item, 400112 was evaluated in an in vitro micronucleus assay in human peripheral blood lymphocytes, both in the presence and in the absence of metabolic activation by rat S9 mix. The assay was performed according to OECD guideline No. 487 and under GLP compliance.

The test was carried out employing 2 exposure times without S9 mix: 4 and 20 hours, and 1 exposure time with S9 mix: 4 hours. The experiment with S9 mix was carried out twice. The harvesting time was 20 hours after the end of exposure. The study was conducted in duplicate. The following concentrations were tested dissolved in dimethyl sulfoxide (DMSO): 100 / 50 / 25 / 12.5 /6.25 µg/mL.

Under the present test conditions, 400112 tested up to cytotoxic concentrations, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of mutagenic properties in the in vitro micronucleus test.

No test item-related increase in micronucleus frequencies (significant at p≤0.05) was noted and no concentration relationship was noted.

In the same test, the positive controls mitomycin C and cyclophosphamide induced significant damage.

Mouse lymphoma test

The study was performed to investigate the potential of 400112 to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y TK+/-. The test was carried out employing two exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours; the experiment with S9 mix was carried out in two independent assays.

In the preliminary experiment without and with metabolic activation cytotoxicity (decreased survival) was noted starting at concentrations of 10 µg/mL. Hence, in the main study the concentration-range of 0.625 to 10.0 µg/mL was used in the experiments without and with metabolic activation with a 3-hour exposure time. A concentration-range of 0.313 to 5.0 µg/mL was used in the second experiment without metabolic activation (24-hour exposure).

Methylmethanesulfonate (at 10 or 15 µL/mL) was employed as a positive control without metabolic activation and 3 -methylcholanthrene (at 2.5 or 4.0 µg/mL) with metabolic activation.

It can be stated that under the experimental conditions reported, the test item 400112 did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. All acceptance criteria were met.

 

Short description of key information:
AMES TEST
400112 was tested in an Ames test according to OECD guideline 471 an EU B.13/14 in the tester strains TA 97a, TA 98, TA 100, TA 102, TA 1535 of Salmonella typhimurium with and without metabolic activation. No mutagenicity was observed.

MICRONUCLEUS TEST
400112 was tested in an in vitro micronucleus assay according to OECD guideline 487 in human peripheral blood lymophocytes with and without metabolic activation. No genotoxicity was observed.

MOUSE LYMPHOMA
400112 was tested in an in vitro mammalian cell gene mutation test according to OECD guideline 476 and EU B.17 using the cell line L5178Y. No mutagenicity was observed.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Due to the negative results of three in vitro genotoxicity assays, it can be concluded that 400112 is not genotoxic.