Octadecanoic acid, 12-hydroxy-, reaction products with ethylenediamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The available data from three in vitro assays show that the substance does not have a genotoxic potential.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29 August 2012 to 17 September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, from male Sprague-Dawley rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Plate incorporation assay: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (with and without metabolic activation)
Pre-incubation assay: 50, 150, 500, 1500 and 5000 µg/plate (with and without metabolic activation)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

Sodium azide: (2 µg/plate for strains TA100 and TA1535), 9-aminoacridine: (50 µg/plate for strain TA1537), 2-nitrofluorene: (2 µg/plate for strain TA98) and 4-nitroquinoline-1-oxide: (2 µg/plate for strain WP2 uvrA (pKM101)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

2-Aminoanthracene: 5 µg/plate for strains TA100 and TA1535 and 10 µg/plate for strain WP2 uvrA (pKM101); Benzo[a]pyrene: 5 µg/plate for strains TA98 and TA1537

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Plate incorporation method: (Test 1)
- Aliquots of 0.1 mL of the test substance solutions (at 5, 15, 50, 150, 500, 1500 and 5000 µg/plate), positive control or vehicle control (water + 0.15% agar) were placed in glass tubes. S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10 h bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labeled with a unique code, identifying the contents of the dish. Triplicates were used for each treatment.
- Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37°C for ca 72 h. After the incubation period, the appearance of the background bacterial lawn was examined and revertant colonies were counted using an automated colony counter. Any toxic effects of the test substance were detected by a substantial reduction in mean revertant colony counts or by a sparse or absent background bacterial lawn. In the absence of any toxic effects, the maximum concentration was selected for use in the second test. If toxic effects were observed, a lower concentration would be chosen, ensuring that signs of bacterial inhibition are present at this maximum concentration. Ideally, a minimum of four non-toxic concentrations should be obtained. If precipitate is observed on the plates at the end of the incubation period, at least one precipitating concentration should be included in the second test.

Preincubation method: (Test 2)
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37°C for 30 minutes with shaking before the addition of the agar overlay. The maximum concentration chosen was again 5000 µg/plate, but only five concentrations were used.

Evaluation criteria:

- Test substance is considered to have mutagenic activity: If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.

- Test substance is not considered to have mutagenic activity: If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.

- If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers.

- Test is considered valid: If the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the current historical control range for the laboratory unless otherwise justified by the Study Director. Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537, which have relatively low spontaneous reversion rates) that of the concurrent vehicle controls. Mean viable cell counts in the 10-h bacterial cultures must be at least 10E09/mL.

Statistics:

The statistical procedures used were usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance was considered along with statistical significance.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Test 1:
- No evidence of toxicity was obtained following exposure to the test substance. A maximum exposure concentration of 5000 µg/plate was, therefore, selected for use in the second test.
- No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to test substance at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix.

Test 2:
- No evidence of toxicity was obtained following exposure to the test substance.
- No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to test substance at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix.

- The results in detail obtained with the test substance and positive control compounds are presented in appendix 2 of the attached background material.

 - The absence of colonies on sterility check plates confirmed the absence of microbial  contamination of the S9 mix, buffer and test substance formulation.

- The viability counts confirmed that the viable cell density of the cultures of the individual organisms exceeded 10⁹/mL in all cases, and therefore met the acceptance criteria.

- The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.

-  Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains in all reported tests, confirming sensitivity of the cultures and activity of the S9 mix.

Conclusions:

Under the study conditions, the test substance showed no evidence of mutagenic activity in the bacterial system.

Executive summary:

An in vitro Ames assay was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471, EU Method B.13/14 and EPA OPPTS 870.5100 in compliance with GLP.

Tester strains TA98, TA100, TA1535 and TA1537 of Salmonella typhimurium and strain WP2uvrA of Escherichia coli were evaluated in the presence and absence of metabolic activation. The test was performed in two independent assays using the plate incorporation and the pre-incubation methods. Water containing 0.15% bacteriological agar was selected as the vehicle of choice. In the first test, the dose levels were: 5, 15, 50, 150, 500, 1,500 and 5,000 µg/plate (with and without metabolic activation). In the second test, the dose levels were 50, 150, 500, 1,500 and 5,000 µg/plate (with and without metabolic activation). No positive mutagenic responses were observed with any of the strains in the absence and presence of S9 mix. Neither any precipitate nor appreciable toxicity was seen.

Under the study conditions, the test substance therefore showed no evidence of mutagenic activity in the bacterial system.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 02 October 2012 to 13 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

21st july 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v (for cell culture).
- Properly maintained: Yes, spontaneous thymidine kinase deficient mutants, TK -/-, were eliminated from the cultures by a 24 h incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196°C, in heat-inactivated donor horse serum (HiDHS) containing 10% DMSO. Cultures were used within 10 d of recovery from frozen stock.
- Periodically checked for Mycoplasma contamination: Yes
- Periodically "cleansed" against high spontaneous background: No

Metabolic activation:

with and without

Metabolic activation system:

S9 was prepared from male SD rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver.

Test concentrations with justification for top dose:

Preliminary toxicity test:
Without and with S9-mix, 3 h treatment: 4.88, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1,250 and 2,500 µg/mL
Without S9-mix, 24 h treatment: 4.88, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1,250 and 2,500 µg/mL

Experiment 1 (3 h exposure in the absence and presence of S9 mix):
Without S9-mix: 50, 100, 125, 150, 175, 200, 250, 300 and 350 µg/mL
With S9-mix: 150, 200, 250, 300, 350, 400, 500, 600 and 700 µg/mL

Experiment 2 (24 h exposure in the absence of S9 mix):
Without S9-mix: 100, 150, 200, 250, 300, 350, 400, 500 and 600 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (ACS Spectrophotometric grade). The final concentration of DMSO added to the cultures was 1% v/v

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Remarks:

Methyl methanesulphonate (MMS) at 10 µg/mL (3 h exposure) and 5 µg/mL (24 h exposure); Benzo[a]pyrene (BaP) at 1 µg/mL (3 h exposure)

Details on test system and experimental conditions:

DURATION
- Exposure duration:
- Short-term treatment: With and without S9-mix: 3 h
- Prolonged treatment period: Without S9-mix: 24 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): Day 11 to 14

SELECTION AGENT (mutation assays): 4 µg/mL trifluorothymidine (TFT)


MEDIUM: R10p medium was used for cell culture. R20p medium was used for the cloning efficiency plating. This was prepared by mixing equal volumes of R10p and R30p.
- R0: RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 ¿g/mL gentamicin.
- R10p: R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v.
- R30p: R0, supplemented with 0.02% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 30% v/v.


NUMBER OF REPLICATIONS:
- Preliminary test: Single cultures per test concentration and vehicle controls in duplicate
- Main test: Duplicate cultures per test concentration of test substance and positive control. Quadruplicate cultures for vehicle controls.


NUMBER OF CELLS EVALUATED: 2 x 10E5 cells/mL (for preliminary and main tests)

DETERMINATION OF CYTOTOXICITY
- Method: Relative suspension growth (RSG; preliminary test) and relative total growth (RTG), cloning efficiency (CE) and mutant frequency (MF) (main mutation experiments)

RANGE-FINDING/SCREENING STUDIES:
-The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 h or only 24 h cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose range for the mutagenicity tests

Evaluation criteria:

The following criteria were applied for assessment of individual assay results using data for MF where the RTG normally exceeded 10%:
Definitions:
GEF = Global Evaluation Factor. For microwell assays this is 126 x 10-6.

The assay was considered valid in accordance with the assay acceptance criteria.

The test agent was regarded as negative if:
- The mean MF of all test concentrations was less than the sum of the mean concurrent vehicle control MF and the GEF.
- If the MF of any test concentrations exceeded the sum of the mean concurrent solvent control MF and the GEF, a linear trend test was applied:
- If the linear trend test was negative, the result was regarded as negative.
- If the linear trend test was positive, this indicated a positive, biologically relevant response.

Where appropriate, other factors were considered in the interpretation of the results, for example, the reproducibility within and between tests, the overall number of mutant colonies (as opposed to MF) and the nature of any concentration-related effect(s).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation was observed at concentrations of 300 µg/mL and greater.

RANGE-FINDING/SCREENING STUDIES:
- Precipitate (observed by eye at the end of treatment) was observed at concentrations of 312.5 µg/mL and greater and 78.13 µg/mL and greater in the absence and presence of S9 mix, respectively, following a 3 h exposure.
- Exposure to test substance at concentrations from 4.88 to 2,500 µg/mL in the absence and presence of S9 mix (3 h exposure) resulted in RSG values from 126 to 0% and from 129 to 0% respectively.
- Following a continuous exposure for 24 h, precipitation was observed at concentrations of 312.5 µg/mL and greater. Exposure to concentrations from 4.88 to 2,500 µg/mL resulted in RSG values from 115 to 0%.

MAIN MUTATION TEST
Experiment 1 (3 h exposure):
3 h treatment treatment in the absence of S9 mix (cultures were exposed to the test substance at concentrations from 50 to 350 µg/mL):
- Precipitation was observed at concentrations of 300 µg/mL and greater. Cultures exposed to the test substance at concentrations from 50
to 250 µg/mL were assessed for determination of MF.
- RTG values from 111 to 16% were obtained relative to the vehicle control.
- There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity.
- The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

3 hour treatment in the presence of S9 mix (cultures were exposed to the test substance at concentrations from 150 to 700 µg/mL):
- Precipitation was observed at concentrations of 300 µg/mL and greater. Cultures exposed to the test substance at concentrations from 150 to 500 µg/mL were assessed for determination of MF.
- RTG values from 107 to 15% were obtained relative to the vehicle control.
- There were no increases in the mean MF of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity.
- The positive control, benzo[a]pyrene, induced an acceptable increase in MF and an acceptable increase in the number of small colony mutants.

24 h treatment in the absence of S9 mix (cultures were exposed to the test substance at concentrations from 100 to 600 µg/mL.)
- Precipitation was observed at concentrations of 300 µg/mL and greater. Cultures exposed to the test substance at concentrations from 100
to 300 µg/mL were assessed for determination of MF.
- RTG values from 112 to 20% were obtained relative to the vehicle control.
- There were no increases in the mean MF of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity.
- The positive control, methyl methanesulphonate, induced an acceptable increase in MF and an acceptable increase in the number of small colony mutants.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

For details on result tables refer to the Main test PDF under the attached background material.

Conclusions:

Under the in vitro study conditions, the test substance was not considered to be mutagenic at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells.

Executive summary:

An in vitro mouse lymphoma assay was conducted to evaluate forward mutation induction potential of the test substance at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The study was conducted according to the OECD Guideline 476, EU Method B.17 and EPA OPPTS 870.5300 in compliance with GLP.

Two independent tests were run:

-        Test 1: for 3 h at the dose levels of: 50, 100, 125, 150, 175, 200, 250, 300 and 350 µg/mL (without S9-mix) and 150, 200, 250, 300, 350, 400, 500, 600 and 700 µg/mL (with S9-mix).

-        Test 2: for 24 h at the dose levels of: 100, 150, 200, 250, 300, 350, 400, 500 and 600 µg/mL (without S9-mix).

In both tests, there were no increases in the mean mutant frequencies at any of the concentrations that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. Cytotoxicity was observed at = 300 µg/mL. Further, the RTG (relative total growth) was reduced to 16 and 15% in the absence and presence of S9 mix, respectively, following 3 hours exposure and to 20% in the absence of S9 mix following 24 hours exposure. The positive controls induced an acceptable increase in mutation frequency and in the number of small colony mutants. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Under the study conditions, the test substance was not considered to be mutagenic at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 25 September 2012 to 08 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

21st july 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chromosomal aberration assay

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Human blood was collected aseptically from two healthy, non-smoking, adult donors, pooled and diluted with 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 L-glutamine. Aliquots (0.4 mL blood : 4.5 mL medium : 0.1 mL phytohaemagglutinin) of the cell suspension were placed in sterile universal containers and incubated at 37°C for
approximately 48 hours. The cultures were gently shaken daily to resuspend the cells.

Metabolic activation:

with and without

Metabolic activation system:

The S9 fraction obtained from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver.

Test concentrations with justification for top dose:

Mitotic index analysis:
Test 1 (3 hour treatment): 0, 50, 100, 150, 200, 250, 300 and 350 µg/mL (without activation) and 0, 200, 300, 400, 500, 600, 700, 800 and 900 (with activation 2% v/v)
Test 2 (21 h treatment): 0, 50, 100, 150, 200, 250, 300 and 350 (without activation) and 0, 100, 200, 300, 400, 500, 600, 700 and 800 (with activation 5% v/v)

Metaphase analysis
Test 1 (3 h treatment): 0, 200, 250 and 300 µg/mL (without activation) and 0, 200, 400 and 600 µg/mL (with activation 2% v/v)
Test 2 (21 h treatment): 0, 100, 150 and 200 µg/mL (without activation) and 0, 300, 500 and 700 µg/mL (with activation 5% v/v)

Vehicle / solvent:

- Vehicle: Dimethyl sulfoxide (DMSO)
- The test substance was found to form a dosable suspension in DMSO at 250 mg/mL. On dosing at 1% v/v into aqueous tissue culture medium, giving a final concentration of 2500 µg/mL, precipitate (visible by eye) was observed. Hence, the highest final concentration of the test substance used for subsequent testing was 2,500 µg/mL (obtained by dosing DMSO at 1% v/v into aqueous tissue culture medium). This is based on the maximum achievable concentration of the test substance in DMSO and this maintained visible precipitate in culture medium.
- The osmolality and pH of the test substance in medium was tested at 2,500 µg/mL; no fluctuation in osmolality of more than 50 mOsm/kg and no change in pH of more than 1.0 unit were observed compared with the vehicle control.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

mitomycin C

Remarks:

0.2 µg/mL (3 hour treatment) and 0.1 µg/mL (21 h continuous treatment)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

cyclophosphamide

Remarks:

5 µg/mL (3 hour treatment)

Details on test system and experimental conditions:

TREATMENT OF CELLS WITH THE TEST SUBSTANCE - FIRST TEST
Duplicate vehicle, positive and treatment cultures were prepared throughout for each treatment in the absence and presence of S9 mix. All cultures were centrifuged and resuspended in fresh medium before treatment.
- In the absence of S9 mix, 50 µL aliquots of test substance were added to give final concentrations of 25.19, 41.99, 69.98, 116.64, 194.4, 324, 540, 900, 1500 and 2500 µg/mL.
DMSO was used as the vehicle control, and mitomycin C at a final concentration of 0.2 µg/mL was the positive control.
- In the presence of S9 mix, 1 mL of medium was removed from each culture and discarded. This was replaced with 1 mL of S9 mix (2% v/v final concentration), followed by 50 µL of each test substance dilution (giving the same series of final concentrations as above).
DMSO was used as the vehicle control, and cyclophosphamide at a final concentration of 5 µg/mL was the positive control.

After 3 h treatments precipitate (visible by eye) was present at final concentrations of 900 µg/mL and above, when compared with the vehicle control.

Following 3 h treatment, cultures were centrifuged at 500g for 5 minutes and the supernatant removed. Cultures were then resuspended in saline (5 mL) and centrifuged at 500g for 5 minutes.
The saline was then removed and the cell pellets resuspended in fresh medium. They were then incubated for a further 18 h.
As an appropriate toxicity profile was not achieved, an additional test was conducted to achieve an acceptable concentration range in both the absence and presence of S9 mix.

Concentrations of the test substances were as follows:
In the absence of S9 mix: 50, 100, 150, 200, 250, 300 and 350 µg/mL.
In the presence of S9 mix: 200, 300, 400, 500, 600, 700, 800 and 900 µg/mL.
In the absence of S9 mix, no notable culture medium changes were observed after 3 h treatment, when compared with the vehicle control.
In the presence of S9 mix, precipitate was observed at a final concentration of 900 µg/mL, when compared with the vehicle control.

Harvesting and fixation:
2 h before the cells were harvested, mitotic activity was arrested by addition of Colcemid® to each culture at a final concentration of 0.1 µg/mL. After 2 h incubation, each cell suspension was transferred to a centrifuge tube and centrifuged for 5 minutes at 500g. The cell pellets were treated with a hypotonic solution (0.075M KCl), pre-warmed at 37°C. After a 10 minute period of incubation at 37°C, the suspensions were centrifuged at 500g for 5 minutes and the cell pellets fixed by addition of freshly prepared cold fixative (3 parts methanol : 1 part glacial acetic acid). The fixative was replaced until it was clear.

Slide preparation:
The pellets were resuspended, then centrifuged at 500 g for 5 minutes and finally resuspended in a small volume of fresh fixative. A few drops of the cell suspensions were dropped onto pre-cleaned microscope slides and allowed to air dry. The slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to air-dry and mounted in DPX. The remainder of the cell pellets in fixative was stored at approximately 4°C until slide analysis was completed.

Microscopic examination:
The prepared slides were examined by light microscopy using a low power objective. The proportion of mitotic cells per 1000 cells in each culture was recorded except for positive control treated cultures, or cultures where there were no signs of cytotoxicity. From these results the concentration causing a decrease in mitotic index of at least 50% (where possible) of the vehicle control value was the highest concentration selected for metaphase analysis.
Intermediate and low concentrations were also selected.
The selected slides were then coded. Metaphase cells were identified using a low power objective and examined at a magnification of x1000 using an oil immersion objective.
The incidence of polyploid and endoreduplicated cells (i.e. the ploidy status) was recorded as a percentage of the 100 metaphases analyzed.

TREATMENT OF CELLS WITH THE TEST SUBSTANCE - SECOND TEST
Cultures were initiated and maintained as previously described. In this second test a 21 h continuous treatment was used in the absence of S9 mix. In the presence of S9 mix, a 3 h treatment was used, as in the first test. However, to modify study parameters the final concentration of S9 mix was increased from 2% v/v to 5% v/v. All cultures were centrifuged and resuspended in fresh medium before treatment.

Concentrations of the test substance were as follows:
In the absence of S9 mix: 50, 100, 150, 200, 250, 300 and 350 µg/mL.
In the presence of S9 mix: 100, 200, 300, 400, 500, 600, 700 and 800 µg/mL.

Duplicate cultures were used for each treatment and two cultures were treated with the vehicle control. Positive control cultures were treated as in the first test. mitomycin C, at a final concentration of 0.1 µg/mL, and Cyclophosphamide, at a final concentration of 5 µg/mL, was added to duplicate cultures.
Following 3 h treatment, cultures containing S9 mix were centrifuged. The cell pellets were rinsed and resuspended in fresh medium. They were then incubated for a further 18 h. Cultures treated in the absence of S9 mix were incubated continuously for 21 h.
In the presence of S9 mix, precipitate was observed in the culture medium at final concentrations of 700 and 800 µg/mL, when compared with the vehicle control.
In the absence of S9 mix, no notable culture medium changes were observed after 21 h continuous treatment, when compared with the vehicle control.
All cultures were treated with Colcemid®, at a final concentration of 0.1 µg/mL, 2 h before the end of the incubation period. They were then harvested, fixed and the slides prepared as previously described. The slides were examined microscopically as previously described.

Evaluation criteria:

The test substance is considered to cause a positive response if the following conditions are met:
- Statistically significant increases (p<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed 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.

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

Statistics:

The number of aberrant metaphase cells in each test substance group was compared with the vehicle control value using the one-tailed Fisher exact test.
A Cochran-Armitage test for trend was applied to the control and all test substance groups. If this is significant at the 1% level, the test is reiterated excluding the highest concentration group - this process continues until the trend test is no longer significant.
D20s (the minimum concentration (mg/mL) at which aberrations were found in 20% of metaphases) were estimated using logistic regression on a log (concentration) scale
The data was analysed using the SAFEStat (SAS statistical applications for end users, version 1.1) Chromosome Aberrations application (version 1.0) which was developed in SAS (SAS INSTITUTE 2002).

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF FIRST TEST
Toxicity data:
- In the absence of S9 mix following 3 h treatment, the test substance caused a reduction in the mitotic index to 53% of the vehicle control value at 300 µg/mL. The concentrations selected for metaphase analysis were 200, 250 and 300 µg/mL.
- In the presence of S9 mix (2% v/v final concentration) following 3 hour treatment, a reduction in the mitotic index to 48% of the vehicle control value was seen at 600 µg/mL was observed. The concentrations selected for metaphase analysis were 200, 400 and 600 µg/mL.

Metaphase analysis:
- In both the absence and the presence of S9 mix, the test substance caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any concentration, when compared with the vehicle control.
- All mean values for the vehicle control DMSO, and all treatment concentrations were within laboratory historical control range, when taken at the 99% confidence limit.
- Both positive control compounds, mitomycin C and cyclophosphamide, caused statistically significant increases (p<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.

Polyploid and endoreduplication analysis:
- No statistically significant increases in the proportion of polyploid or endoreduplicated metaphase cells were observed during metaphase analysis, when compared to the vehicle control.

RESULTS OF SECOND TEST
Toxicity data:
- In the absence of S9 mix following 21 h continuous treatment, a reduction in the mitotic index to 50% of the vehicle control value was observed at 200 µg/mL. The concentrations selected for metaphase analysis were 100, 150 and 200 µg/mL.
- In the presence of S9 mix (5% v/v final concentration) following 3 hours of treatment, caused a reduction in the mitotic index to 52% of the vehicle control value at 700 µg/mL. The concentrations selected for metaphase analysis were 300, 500 and
700 µg/mL.

Metaphase analysis:
- In both the absence and the presence of S9 mix, caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any concentration, when compared with the vehicle control.
- All mean values for the vehicle control DMSO, and all treatment concentrations were within the laboratory historical control range, when taken at the 99% confidence limit.
- Both positive control compounds, Mitomycin C and Cyclophosphamide, caused statistically significant increases (p<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.

Polyploid and endoreduplication analysis
- No statistically significant increases in the proportion of polyploid or endoreduplicated metaphase cells were observed during metaphase analysis, when compared to the vehicle control.

Refer to the attached background material for details on summary of results for the test 1 and 2.

Conclusions:

Under the study conditions, the test substance was not considered to be clastogenic to the human lymphocytes in the in vitro chromosomal aberration assay.

Executive summary:

An in vitro chromosomal aberration assay was conducted to evaluate the clastogenic potential of the test substance in human lymphocytes according to the OECD Guideline 473, EU Method B.10 and US EPA OPPTS 870.5375 in compliance with GLP.

Human lymphocytes, in whole blood culture, were exposed to the test substance both in the absence and presence of S9 mix derived from rat livers. Two hours before the end of the incubation period, cell division was arrested using Colcemid®. The cells were then harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage. On the basis of the mitotic index data obtained from a toxicity test, the following concentrations were selected for two independent tests evaluating the metaphase analysis: 

-        Test 1 (at 3 h treatment) at dose levels of: 0, 200, 250 and 300 µg/mL (in the absence of S9 mix) and 0, 200, 400 and 600 µg/mL (in the presence of S9 mix; 2% v/v),

-        Test 2 (at 21 h treatment) at dose levels of: 0, 100, 150 and 200 µg/mL (in the absence of S9 mix) and 0, 300, 500 and 700 µg/mL (in the presence of S9 mix; 5% v/v).

Concurrent solvent and positive controls (mitomycin-C (in the absence of S9 mix) and cyclophosphamide (in the presence of S9 mix)) were also included. In both the tests, the substance caused no statistically significant increases in the proportion of metaphase figures (containing chromosomal aberrations) at any concentration in the absence and presence of S9 mix when compared with the vehicle control. Also, no statistically significant increases in the proportion of polyploid or endoreduplicated metaphase cells were observed during metaphase analysis when compared with the vehicle control. All positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix. Under the study conditions, the test substance was therefore not considered to be clastogenic to human lymphocytes in thein vitrochromosomal aberration assay

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

An in vitro Ames assay was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471, EU Method B.13/14 and EPA OPPTS 870.5100 in compliance with GLP. Tester strains TA98, TA100, TA1535 and TA1537 of Salmonella typhimurium and strain WP2uvrA of Escherichia coli were evaluated in the presence and absence of metabolic activation. The test was performed in two independent assays using the plate incorporation and the pre-incubation methods. Water containing 0.15% bacteriological agar was selected as the vehicle of choice. In the first test, the dose levels were: 5, 15, 50, 150, 500, 1,500 and 5,000 µg/plate (with and without metabolic activation). In the second test, the dose levels were 50, 150, 500, 1,500 and 5,000 µg/plate (with and without metabolic activation). No positive mutagenic responses were observed with any of the strains in the absence and presence of S9 mix. Neither any precipitate nor appreciable toxicity was seen.

Under the study conditions, the test substance therefore showed no evidence of mutagenic activity in the bacterial system (May, 2012).

An in vitro mouse lymphoma assay was conducted to evaluate forward mutation induction potential of the test substance at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The study was conducted according to the OECD Guideline 476, EU Method B.17 and EPA OPPTS 870.5300 in compliance with GLP. Two independent tests were run:

-        Test 1: for 3 h at the dose levels of: 50, 100, 125, 150, 175, 200, 250, 300 and 350 µg/mL (without S9-mix) and 150, 200, 250, 300, 350, 400, 500, 600 and 700 µg/mL (with S9-mix).

-        Test 2: for 24 h at the dose levels of: 100, 150, 200, 250, 300, 350, 400, 500 and 600 µg/mL (without S9-mix).

In both tests, there were no increases in the mean mutant frequencies at any of the concentrations that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. Cytotoxicity was observed at = 300 µg/mL. Further, the RTG (relative total growth) was reduced to 16 and 15% in the absence and presence of S9 mix, respectively, following 3 h exposure and to 20% in the absence of S9 mix following 24 hours of exposure. The positive controls induced an acceptable increase in mutation frequency and in the number of small colony mutants. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

Under the study conditions, the test substance was not considered to be mutagenic at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells (Woods, 2012a).

Finally, an in vitro chromosomal aberration assay was conducted to evaluate the clastogenic potential of the test substance in human lymphocytes according to the OECD Guideline 473, EU Method B.10 and US EPA OPPTS 870.5375 in compliance with GLP. Human lymphocytes, in whole blood culture, were exposed to the test substance both in the absence and presence of S9 mix derived from rat livers. Two hours before the end of the incubation period, cell division was arrested using Colcemid®. The cells were then harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage. On the basis of the mitotic index data obtained from a toxicity test, the following concentrations were selected for two independent tests evaluating the metaphase analysis: 

-        Test 1(at 3 h treatment) at dose levels of: 0, 200, 250 and 300 µg/mL (in the absence of S9 mix) and 0, 200, 400 and 600 µg/mL (in the presence of S9 mix; 2% v/v),

-        Test 2(at 21 h treatment) at dose levels of: 0, 100, 150 and 200 µg/mL (in the absence of S9 mix) and 0, 300, 500 and 700 µg/mL (in the presence of S9 mix; 5% v/v).

Concurrent solvent and positive controls (mitomycin-C (in the absence of S9 mix) and cyclophosphamide (in the presence of S9 mix)) were also included. In both the tests, the substance caused no statistically significant increases in the proportion of metaphase figures (containing chromosomal aberrations) at any concentration in the absence and presence of S9 mix when compared with the vehicle control. Also, no statistically significant increases in the proportion of polyploid or endoreduplicated metaphase cells were observed during metaphase analysis when compared with the vehicle control. All positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix. Under the study conditions, the test substance was therefore not considered to be clastogenic to human lymphocytes in the in vitro chromosomal aberration assay (Woods, 2012b).

Justification for classification or non-classification

Based on the results from three in vitro guideline compliant assays, the substance is not classified for genotoxicityaccording to regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures.