Dipraseodymium trioxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Chromosome Aberration Test

Under the conditions of the study, the test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations, in either the absence or presence of metabolic activation. The test material was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

V79 HPRT Gene Mutation Assay

Under the conditions of the study, the test material was considered to be non-mutagenic to V79 cells at the HPRT locus.

Ames Test: Read-across performed with structurally similar substance (Praseodymium (III,IV) Oxide)

Under these experimental conditions, the test material did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Ames Test: Read-across performed with structurally similar substance (Praseodymium Tricarbonate)

The test material was considered to be non-mutagenic under the conditions of this test.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 January 2017 to 29 March 2017

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:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environmental (MOE)

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: chromosome aberration in peripheral human lymphocytes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: PR2O3-4/16
- Expiration date of the lot/batch: 21 December 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in the dark over silica gel, the test material was held under argon gas for shipment and batched into individual containers under Nitrogen for use in the study to avoid exposure to the atmosphere. A new sample was used for each experiment.

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection.
- Sex, age and number of blood donors if applicable: males aged 23 and 24
- Whether whole blood or separated lymphocytes were used if applicable: whole blood

MEDIA USED
- Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at approximately 37°C with 5% CO2 in humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

The molecular weight of the test material was given as 329.81, therefore, the maximum dose level was 2000 µg/mL, the maximum recommended dose level.

- Preliminary toxicity test: 0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL
- Main experiment: 0.125, 0.25, 0.5, 1, 2, 4 and 8 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Minimal Essential Medium (MEM)
- Justification for choice of solvent/vehicle:The test material was insoluble in both dimethyl sulphoxide and acetone at 200 mg/mL but was partially soluble/ suspendable in Minimal Essential Medium (MEM) at 20 mg/mL in solubility checks performed in house.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

EXPOSURE GROUPS

4-HOUR EXPOSURE WITH METABOLIC ACTIVATION (S9)
After approximately 48 hours incubation at approximately 37°C, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1.0 mL of the appropriate solution of vehicle control or test material was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1 mL of 20% S9¯mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and Main Experiment. After 4 hours at approximately 37°C, 5% CO2 in humidified air, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37°C in 5% CO2 in humidified air.

4-HOUR EXPOSURE WITHOUT METABOLIC ACTIVATION
After approximately 48 hours incubation at approximately 37°C with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 1.0 mL of the appropriate vehicle control, test material solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.
After 4 hours at approximately 37°C, 5% CO2 in humidified air, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.

24-HOUR EXPOSURE WITHOUT METABOLIC ACTIVATION
As the exposure was continuous the cultures were established, at a nominal volume of 9.0 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1.0 mL of vehicle control, test material or 0.1 mL of positive control. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37°C, 5% CO2 in humidified air for 24 hours.

PRELIMINARY TOXICITY TEST
- The preliminary toxicity test was performed using all three of the exposure conditions as described above but using single cultures only.
- The dose range of the test material used was 7.81 to 2000 µg/mL. Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test material precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
- Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test material toxicity and for selection of the dose levels for the main test.

MAIN EXPERIMENT
The three exposure groups above were used for the main study with the following test material concentrations: 0.125, 0.25, 0.5, 1, 2, 4 and 8 µg/mL. Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test material precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.

CELL HARVEST
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) 2.5 hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075 M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4°C to ensure complete fixation prior to slide preparation.

PREPARATION OF METAPHASE SPREADS AND STAINING
- The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.
- When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

EVALUATION OF RESPONSE
- Qualitative Slide Assessment: The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test material. These observations were used to select the dose levels for mitotic index evaluation.
- Coding: The slides were coded using a computerized random number generator.
- Mitotic Index: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
- Scoring of Chromosome Damage: Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides. In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including the incidence of endoreduplicated cells) was also reported.

Evaluation criteria:

DATA EVALUATION
The following criteria were used to determine a valid assay:
- The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory control data range.
- All the positive control chemicals induced a positive response (p ≤ 0.01) demonstrating the validity of the experiment and the integrity of the S9-mix.
- The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
- The required number of cells and concentrations were analysed.

CRITERIA FOR DETERMINING THE STUDY CONCLUSION
A test material can be considered to be clearly negative if, in any of the experimental conditions examined:
- The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
- No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
- There is no concentration-related increase at any dose level.

A test material can be classified as genotoxic if:
- The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
- At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
- The observed increase in the frequency of cells with structural aberrations is considered to be dose-related.

When all of the above criteria are met, the test material can be considered able to induce chromosomal aberrations in human lymphocytes.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include numerical aberrations in the form of polyploidy and endoreduplicated cells.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
- A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 125 µg/mL, in the absence of S9 and at and above 62.5 µg/mL in the presence of S9. Precipitate was observed in the blood pellet of the cultures at the end of the exposure period at all test material dose levels in all three exposure groups.
- Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 2000 µg/mL in all three exposure groups. Precipitate from the test material was noted on the slides at all test material dose levels and was particularly heavy at and above 500 µg/mL in all three exposure groups. The test material induced no evidence of toxicity in any of the exposure groups.
- The selection of the maximum dose level for the Main Experiment was based on the lowest dose level where precipitate was observed and was 8 µg/mL for the 4(20)-hour exposure groups and for the continuous exposure group.

MAIN EXPERIMENT
- The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 8 µg/mL in all three exposure groups.
- Precipitate observations were made at the end of exposure in the blood-free cultures and precipitate was noted at 8 µg/mL in the presence of S9 only. Precipitate was observed in the cell pellet of the blood cultures at the end of exposure at and above 0.5 µg/mL in the 4(20)-hour exposure groups, and at and above 2 µg/mL in the 24-hour continuous exposure group. These were considered to be the lowest precipitating dose levels.
- The mitotic index data for the Main Experiment confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed in any of the three exposure groups.
- The maximum dose level selected for metaphase analysis was the lowest precipitating dose level seen in the blood cultures at the end of exposure and was 0.5 µg/mL in the 4(20)-hour exposure groups and 2 µg/mL for the 24-hour exposure group.
- The mitotic index and chromosome aberration data are given in Table 1, Table 2 and Table 3.

- The test material did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
- The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups.

Table 1: Results of Chromosome Aberration Test – Main Experiment 4(20)-hour Exposure Without Metabolic Activation (S9)

Treatment group	Replicate	Mitotic index (%)	Number of cells scored	Number of Aberrations						Total number of aberrations		Frequency of aberrant cells (%)
					Chromatid		Chromosome		Others	(+ Gaps)	(- Gaps)	(+ Gaps)	(-Gaps)
				Gaps	Breaks	Exchanges	Breaks	Exchanges	X
Vehicle control (MEM)	A	4.40	150	0	0	0	0	0	0	0	0	0	0
	B	4.60	150	0	1	0	0	0	0	1	1	1	1
	Total	(100)	300	0	1	0	0	0	0	1	1	1 (0.3)	1 (0.3)
0.125µg/mL	A	5.30	150	0	0	0	0	0	0	0	0	0	0
	B	4.50	150	0	0	0	0	0	0	0	0	0	0
	Total	(109)	300	0	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)
0.25 µg/mL	A	2.85	150	0	0	0	0	0	0	0	0	0	0
	B	6.60	150	0	0	0	0	0	0	0	0	0	0
	Total	(105)	300	0	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)
0.5 µg/mL	A	3.25	150	0	0	0	0	0	0	0	0	0	0
	B	5.45	150	0	1	0	0	0	0	1	1	1	1
	Total	(97)	300	0	1	0	0	0	0	1	1	1 (0.3)	1 (0.3)
Positive control (MMC 0.4 µg/mL)	A	4.10	150	0	6	4	1	0	0	11	11	9	9
	B	1.35	83*	1	15	4	1	0	0	21	20	15	15
	Total	(61)	233	1	21	8	2	0	0	32	31	24 (10.3)	24** (15.2)

MMC = Mitomycin C

* = Slide evaluation terminated when at least 15 cells with aberrations (excluding gaps) had been observed

** = P < 0.001

MEM = Minimal Essential Medium

Table 2: Results of Chromosome Aberration Test – Main Experiment 4(20)-hour Exposure With Metabolic Activation (2% S9)

Treatment group	Replicate	Mitotic index (%)	Number of cells scored	Number of Aberrations						Total number of aberrations		Frequency of aberrant cells (%)
					Chromatid		Chromosome		Others	(+ Gaps)	(- Gaps)	(+ Gaps)	(-Gaps)
				Gaps	Breaks	Exchanges	Breaks	Exchanges	X
Vehicle control (MEM)	A	6.45	150	0	0	0	0	0	0	0	0	0	0
	B	3.45	150	0	1	0	0	0	0	1	1	1	1
	Total	(100)	300	0	1	0	0	0	0	1	1	1 (0.3)	1 (0.3)
0.125µg/mL	A	6.95	150	0	3	0	1	0	0	4	4	3	3
	B	3.40	150	0	0	0	1	0	0	1	1	1	1
	Total	(105)	300	0	3	0	2	0	0	5	5	4 (1.3)	4 (1.3)
0.25 µg/mL	A	5.45	150	0	0	0	1	0	0	1	1	1	1
	B	5.80	150	0	0	0	1	0	0	1	1	1	1
	Total	(114)	300	0	0	0	2	0	0	2	2	2 (0.7)	2 (0.7)
0.5 µg/mL	A	4.75	150	0	0	0	0	0	0	0	0	0	0
	B	4.30	150	1	0	0	0	0	0	1	0	1	0
	Total	(91)	300	1	0	0	0	0	0	1	0	1 (0.3)	0 (0.0)
Positive control (CP 2 µg/mL)	A	2.85	129*	1	14	2	1	0	0	18	17	16	15
	B	3.25	135*	4	10	4	4	0	0	22	18	18	15
	Total	(62)	264	5	24	6	5	0	0	40	35	34 (12.9)	30** (11.4)

 

CP = cyclophosphamide

* = Slide evaluation terminated when at least 15 cells with aberrations (excluding gaps) had been observed

** = P < 0.001

MEM = Minimal Essential Medium

Table 3: Results of Chromosome Aberration Test – Main Experiment 24-hour Continuous Exposure Without Metabolic Activation (S9)

Treatment group	Replicate	Mitotic index (%)	Number of cells scored	Number of Aberrations						Total number of aberrations		Frequency of aberrant cells (%)
					Chromatid		Chromosome		Others	(+ Gaps)	(- Gaps)	(+ Gaps)	(-Gaps)
				Gaps	Breaks	Exchanges	Breaks	Exchanges	X
Vehicle control (MEM)	A	9.30	150	1	2	0	0	0	0	3	2	2	2
	B	11.30	150	0	0	0	0	0	0	0	0	0	0
	Total	(100)	300	1	2	0	0	0	0	3	2	2 (0.7)	2 (0.7)
0.25µg/mL	A	7.55	150	0	1	0	0	0	0	1	1	1	1
	B	9.30	150	0	0	0	0	0	0	0	0	0	0
	Total	(82)	300	0	1	0	0	0	0	1	1	1 (0.3)	1 (0.3)
0.5 µg/mL	A	7.45	150	0	0	0	0	0	0	0	0	0	0
	B	7.60	150	0	1	0	0	0	0	1	1	1	1
	Total	(73)	300	0	1	0	0	0	0	1	1	1 (0.3)	1 (0.3)
1 µg/mL	A	6.15	150	1	1	0	1	0	0	3	2	3	2
	B	9.40	150	1	0	0	0	0	0	1	0	1	0
	Total	(75)	300	2	1	0	1	0	0	4	2	4 (1.3)	2 (0.7)
2 µg/mL	A	9.35	150	1	0	0	0	0	0	1	0	1	0
	B	6.50	150	0	0	0	0	0	0	0	0	0	0
	Total	(77)	300	1	0	0	0	0	0	1	0	1 (0.3)	0 (0.0)
Positive control (MMC 0.2 µg/mL)	A	2.90	31*	2	19	6	1	0	0	28	26	18	18
	B	3.65	43*	0	6	7	3	0	0	16	16	15	15
	Total	(32)	74	2	25	13	4	0	0	44	42	33 (44.6)	33** (44.6)

MMC = mitomycin C

* = Slide evaluation terminated when at least 15 cells with aberrations (excluding gaps) had been observed

** = P < 0.001

MEM = Minimal Essential Medium

Conclusions:

Under the conditions of the study, the test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolising system. The test material was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

The potential of the test material to cause chromosomal aberrations in human lymphocytes was determined in accordance with the standardised guidelines OECD 473 and The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environmental (MOE), under GLP conditions.

During the study, duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by precipitate. The dose levels selected for the Main Test were:0.125, 0.25, 0.5, 1, 2, 4, 8 µg/mL.

All vehicle (Minimal Essential Medium) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive controls induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material did not demonstrate any marked toxicity and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level. 

Under the conditions of this study, the test material was considered to be non-clastogenic to human lymphocytes in vitro.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 July 2017 to 20 October 2017

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:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: HPRT Gene Mutation Assay

Target gene:

The hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. The high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) make it an appropriate cell line to use for this study type. The cells have a stable karyotype with a modal chromosome number of 22.
- Laboratory stock cell cultures will be periodically checked for stability and absence of mycoplasma contamination. The stock of cells is stored in liquid nitrogen. For use, a sample of cells will be removed before the start of the study and grown in Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10 % foetal bovine serum (FBS)) at approximately 37 °C with 5 % CO2 in humidified air.
- Cell stocks spontaneously mutate at a low but significant rate. Before a stock of cells is frozen for storage the number of pre-existing HPRT-deficient mutants must be reduced. The cells are cleansed of mutants by culturing in HAT medium for four days. This is MEM growth medium supplemented with Hypoxanthine (13.6 µg/mL, 100 µM). Aminopterin (0.0178 µg/mL, 0.4 µM) and Thymidine (3.85 µg/mL, 16 µM). After four days in medium containing HAT, the cells are passaged into HAT free medium and grown for four to seven days. Bulk frozen stocks of these “HAT” cleansed cells are frozen down prior to use in the mutation studies, with fresh cultures being removed from frozen before each experiment.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

4-hour without S9: 0.03, 0.06, 0.13*, 0.25*, 0.5*, 1*, 2* , 3* µg/mL
4-hour with S9 (2%): 0.03, 0.06, 0.13, 0.25, 0.5*, 1*, 2*, 4* , 8* , 16* µg/mL
(* = plated for cloning efficiency and mutation frequency)

Vehicle / solvent:

- Following solubility checks performed in-house for the Micronucleus Test in Human Lymphocytes in vitro performed on the same test material, the test material was accurately weighed and formulated in MEM medium prior to serial dilutions being prepared. The molecular weight of the test material was 329.81, therefore, the maximum proposed concentration level in the solubility test was set at 2000 µg/mL and no correction for the purity of the test material was applied.
- There was no significant change in pH when the test material was dosed into media and the osmolality did not increase by more than 50 mOsm at the concentration levels investigated.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

MEM

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: Dimethyl benzanthracene (DMBA) in the presence of S9-mix

Details on test system and experimental conditions:

PRELIMINARY CYTOTOXICITY TEST
- Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. The preliminary cytotoxicity test was performed on cell cultures plated out at 1 x 10^7 cells/225 cm^2 flask approximately 24 hours before dosing. This was demonstrated to provide at least 20 x 10^6 available for dosing in each flask using a parallel flask, counted at the time of dosing. On dosing, the growth media was removed and replaced with serum-free Minimal Essential Medium (MEM). One flask per concentration was treated for 4-hours without metabolic activation and for 4-hours with metabolic activation (2 % S9). The concentrations of test material used were 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 µg/mL.
- The dose levels were selected to avoid excessive precipitation and toxicity as indicated in a previous study. The test was repeated, due to excessive heterogeneity using the same dose levels.
- Exposure was for 4 hours at approximately 37 °C with a humidified atmosphere of 5 % CO2 in air, after which the cultures were washed twice with phosphate buffered saline (PBS) before being detached from the flasks using trypsin. Cells from each flask were suspended in MEM with 10 % FBS, a sample was removed from each concentration group and counted using a Coulter counter. For each culture, 200 cells were plated out into three 25 cm^2 flasks with 5 mL of MEM with 10 % FBS and incubated for 6 to 7 days at approximately 37 °C in an incubator with a humidified atmosphere of 5 % CO2 in air. The cells were then fixed and stained and total numbers of colonies in each flask counted to give cloning efficiencies (CE).
- Results from the preliminary cytotoxicity test were used to select the test material concentrations for the mutagenicity experiment.

MUTAGENICITY TEST- MAIN EXPERIMENT
- Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. Cells were seeded at 1 x 10^7 cells/225 cm^2 flask approximately 24 hours being exposed to the test or control materials. This was demonstrated to provide at least 20 x 10^6 available for dosing in each flask using a parallel flask. Duplicate cultures were set up, both in the presence and absence of metabolic activation, with eight test material concentrations, and vehicle and positive controls. Treatment was for 4 hours in serum free media (MEM) at 37 °C in an incubator with a humidified atmosphere of 5 % CO2 in air.
- The concentration range of test material was 0.03 to 3 µg/mL in the 4-hour –S9 exposure. The concentration range of test material was 0.03 to 16 µg/mL in the 4-hour +S9 exposure.
- At the end of the treatment period the flasks were washed twice with PBS, before cells were detached from the flasks with trypsin and then suspended in MEM with 10 % FBS. A sample of each concentration group cell suspension was counted using a Coulter counter. Cultures were plated out at 2 x 10^6 cells/flask in a 225 cm^2 flask to allow growth and expression of induced mutants, and in triplicate in 25 cm^2 flasks at 200 cells/flask to obtain the cloning efficiency, for an estimate of cytotoxicity at the end of the exposure period. Cells were grown in MEM with 10 % FBS and incubated at 37 °C in an incubator with a humidified atmosphere of 5 % CO2 in air.
- Cytotoxicity flasks were incubated for 6 or 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.
- During the 7 Day expression period the cultures were sub-cultured and maintained on days 2 and 5 to maintain logarithmic growth. At the end of the expression period the cell monolayers were detached using trypsin, cell suspensions counted using a Coulter counter and plated out as follows:
i) In triplicate at 200 cells/25 cm^2 flask in 5 mL of MEM with 10 % FBS to determine cloning efficiency. Flasks were incubated for 6 to 7 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.
ii) At 2 x 10^5 cells/petri dish (ten replicates per group) in MEM with 10 % FBS supplemented with 11 µg/mL 6-Thioguanine (6-TG), to determine mutant frequency. The dishes were incubated for 7 days at 37 °C in an incubator with humidified atmosphere of 5 % CO2 in air, then fixed with methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each dish.
- The percentage cloning efficiency and mutation frequency per survivor were calculated for each dose group.
- Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10 % Giemsa solution for 5 minutes.

CALCULATIONS
The cloning efficiency (CE), % control, mutant plate counts, mutant frequency/10^6 (MF10^-6) and mutant frequency/10^6 survival rate (MFSV) were calculated using the following formulae:
CE % = (CE counts/200)x100
% Control = (CE % of Dose IDx/CE% of Dose ID0) x 100
MF 10^-6 for each dose = Total mutant plate counts/2
MFS 10^-6 for each dose = (MF 10^-6/Day 7 CE%) x 100
Where:
Concentration ID0 = Vehicle control values
Concentration IDx = Concentration values
Small errors may occur when calculating mean cell concentrations and volumes for diluting; and in the calculation of means for cloning efficiency and mutant frequency; if these errors are ≤ 5 % they are regarded to be within reasonable experimental error and considered not to affect the integrity of the study.

Evaluation criteria:

ASSAY ACCEPTANCE CRITERIA
- The average absolute cloning efficiency of the Day 7 negative controls should exceed 50%.
- The background (spontaneous) mutant frequency of the vehicle controls is generally within the historical range. The background values for the with and without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays.
- The concurrent positive controls should induce responses that are comparable with those generated in the historical positive control range and produce a statistically significant increase compared with the concurrent negative control.
- The criteria for selection of the maximum concentration have been met. Test materials with little or no mutagenic activity, should include an acceptable assay where concentrations of the test material have reduced the clonal survival to approximately 10 to 15 % of the average of the negative controls, reached the maximum recommended concentration, or include the lowest precipitating concentration. Where a test material is excessively toxic, a concentration that is at least 75 % of the toxic concentration should be used. There is no maximum toxicity requirement for test materials that are clearly mutagenic. Treatments that reduce relative clonal survival to less than ten percent will not be scored for mutant frequency in the assay.
- Adequate numbers of cells and concentrations are analysable. Mutant frequencies are normally derived from sets of ten dishes/flasks for the mutant colony count and three dishes for cloning colony counts. It is acceptable to score a minimum of eight mutant selection dishes and two cloning efficiency flasks.
- Five concentrations of test material, in duplicate, in each assay will normally be assessed for mutant frequency. A minimum of four analysed duplicate concentrations is considered necessary to accept a single assay for evaluation of the test material.

Statistics:

When there is no indication of any marked increases in mutant frequency at any concentration then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual concentration, using Student’s t-test. Other statistical analysis may be used if they are considered to be appropriate.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY CYTOTOXICITY TEST
- A concentration range of 0.5 to 128 µg/mL was used in the preliminary cytotoxicity test. The maximum concentration tested was selected to avoid too many precipitating dose levels. At the end of the exposure period, precipitate of the test material was observed at and above 32 µg/mL in both the absence and presence of metabolic activation. There was evidence of marked or concentration related reductions in cloning efficiency in both the absence and presence of metabolic activation. Maximum exposure occurred around 2 µg/mL in the absence of metabolic activation and around 4 µg/mL in the presence of metabolic activation. Higher dose levels exhibited an increase in cloning efficiency, but this was attributed to interaction with the test material precipitate where the cells were not fully exposed to the culture. The results indicated that it would be very hard to predict the toxicity and precipitate level of the test material in the main test.
- The maximum concentration selected for the main mutagenicity experiment was therefore set at 3 µg/mL in the 4-hour –S9 exposure based on test material toxicity, and 4 µg/mL for the 4-hour +S9 exposure, based on a combination of precipitate and test material induced toxicity.

MUTAGENICITY TEST- MAIN EXPERIMENT
- At the end of the exposure period, precipitate of the test material was not observed at 16 µg/mL in the presence of metabolic activation only.
- There were marked concentration related reductions in the Day 0 cloning efficiency values in the absence of metabolic activation only, where optimum toxicity was achieved at 3 µg/mL. There was no evidence of any marked reductions in the Day 7 cloning efficiencies in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred.
- The test material did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any of the concentration levels in the main test. Both exposure groups met the requirements of the OECD 476 guideline.
- The vehicle control values were all considered to be within an acceptable range, and the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.

Table 1: Summary of the Main Experiment

Dose (µg/mL)	+/- Metabolic activation	Mean % Control		Day 7 Mutant Group Mutant Frequency per Survivor 10^-6
		Day 0 Viability	Day 7 Viability
0	-	100	100	14
0.03		95	Discarded	Discarded
0.06		98	Discarded	Discarded
0.13		102	92	12
0.25		105	103	13
0.5		102	101	12
1		96	99	12
2		83	102	11
3		18	100	16
EMS 750		86	95	449
EMS 500		95	95	268
0	+	100	100	9
0.03		101	Discarded	Discarded
0.06		98	Discarded	Discarded
0.13		98	Discarded	Discarded
0.25		88	Discarded	Discarded
0.5		97	84	19
1		106	93	13
2		101	95	8
4		101	96	15
8		94	84	16
16 P		96	90	13
DMBA 2		93	90	455
DMBA 1		99	100	280

 

Conclusions:

Under the conditions of this study, the test material was considered to be non-mutagenic to V79 cells at the HPRT locus.

Executive summary:

The mutagenicity of the test material was investigated on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line, in accordance with the standardised guideline OECD 476 under GLP conditions.

Chinese hamster (V79) cells were treated with the test material at six concentrations, in duplicate, together with vehicle (MEM medium) and positive controls in both the absence and presence of metabolic activation. 

The concentrations used in the main test were selected using data from the preliminary toxicity study. The maximum dose levels were limited by a combination of test material induced toxicity and precipitate. The concentrations of test material plated for cloning efficiency and expression of mutant colonies were as follows: 4-hour without S9: 0.13, 0.25, 0.5, 1, 2 and 3 µg/mL and 4-hour with S9 (2 %): 1, 2, 4, 8 and 16 µg/mL.

The vehicle (MEM medium) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any of the concentration levels in the main test, in either the absence or presence of metabolic activation. Optimum levels of toxicity were achieved at 3 µg/mL in the 4-hour –S9 exposure. In the 4-hour exposure +S9 the dose level of 16 µg/mL exhibited precipitate of the test material. Both exposure groups met the requirements of the OECD 476 guideline.

Under the conditions of this study, the test material was considered to be non-mutagenic to V79 cells at the HPRT locus.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 May 2007 to 17 December 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Read-across performed with structurally similar substance.

Reason / purpose for cross-reference:

other: read-across target

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:

S. typhimurium: Histidine locus.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other: rfa mutation; uvrB mutation (except TA102); addition of plasmid pKM101 to TA98, TA100 and TA102.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from a liver post-mitochondrial fraction of rats induced with Aroclor 1254

Test concentrations with justification for top dose:

312.5, 625, 1250, 2500 and 5000 µg/plate for the three experiments, with and without S9 mix.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test material is insoluble in most vehicles - formed homogeneous suspension to the naked eye in DMSO. Suspended at the concentration of 100 mg/mL.
- volume of vehicle/solvent in the medium: 0.05 mL per 2.60 mL medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-Anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: All experiments were performed according to the direct plate incorporation method except for the second and third test with S9 mix, which were performed according to the preincubation method.

DURATION
- Preincubation period: 60 minutes, 37 °C
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATES: three plates/dose-level

OTHER: SCORING METHOD: automated

Evaluation criteria:

A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-response was considered as a positive result. Reference to historical data, or other considerations of biological relevance were taken into account in the evaluation of the data obtained.

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels ≥ 312.5 µg/plate (the precipitate did not interfere with the scoring).

RANGE-FINDING/SCREENING STUDIES:
To assess the toxicity of the test material to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA 98, TA 100 and TA 102 strains, with and without S9 mix. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.
A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels ≥ 100 µg/plate. No noteworthy toxicity was noted towards the three strains used, either with or without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: The control data reported in these report are in the range of the historical control data observed in the laboratory. The study was therefore considered valid.

Results
In the first experiment, no noteworthy toxicity was induced in any of the five tester strains with and without S9 mix. In the second experiment with S9 mix (preincubation method) with the TA 98 strain, up to 3.3-fold increase in the number of revertants were noted, without any clear evidence of dose-relationship. A third experiment was performed with this strain under the same experimental conditions in order to check the reliability of these slight increases. No confirmation of the increase in the number of revertants was observed in this third experiment. The test material did not induce any noteworthy increase in the number of revertants, either with or without S9 mix, in any of the remaining tester strains.

Table 2: First experiment (direct plate incorporation) - Mean revertant colony counts

	TA 1535			TA 1537			TA 98
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	28	22	No	5	8	No	25	38	No
312.5	31	29	No (Mp)	7	10	No (Mp)	24	32	No (Mp)
625	38	35	No (Mp)	8	9	No (Mp)	26	25	No (Mp)
1250	32	24	No (Sp/Mp)	9	8	No (Mp)	27	29	No (Mp)
2500	36	15	No (Sp)	10	9	No (Sp)	18	29	No (Sp)
5000	34	19	No (Sp)	4	6	No (Sp)	16	29	No (Sp)
Positive control	668	188	No	352	128	No	161	1287	No

 

	TA 100			TA 102
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	120	138	No	388	480	No
312.5	118	141	No (Mp)	330	405	No (Mp)
625	123	129	No (Mp)	449	378	No (Mp)
1250	104	115	No (Mp)	405	380	No (Mp)
2500	113	123	No (Sp)	422	406	No (Sp)
5000	126	109	No (Sp)	497	208	No (Sp)
Positive control	604	567	No	2129	3775	No

* solvent control with DMSO

Mp : Moderate precipitate

Sp : Strong precipitate

MA : Metabolic activation

 

Table 3: Second experiment (direct plate incorporation without S9 mix and preincubation with S9 mix) - Mean revertant colony count

	TA 1535			TA 1537			TA 98
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	11	7	No	5	7	No	25	21	No
312.5	12	11	No (Mp)	3	6	No (Mp)	29	44	No (Mp)
625	10	17	No (Mp)	5	10	No (Mp)	40	33	No (Mp)
1250	10	18	No (Sp/Mp)	4	10	No (Sp/Mp)	29	39	No (Sp/Mp)
2500	15	13	No (Sp)	8	4	No (Sp)	37	69	No (Sp)
5000	9	13	No (Sp)	5	6	No (Sp)	21	21	No (Sp)
Positive control	593	153	No	1443	148	No	303	1377	No

 

	TA 100			TA 102
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	127	136	No	341	551	No
312.5	123	150	No (Mp)	275	454	No (Mp)
625	140	164	No (Mp)	362	569	No (Mp)
1250	131	135	No (Sp/Mp)	338	508	No (Sp/Mp)
2500	152	108	No (Sp)	344	523	No (Sp)
5000	146	101	No (Sp)	279	233	No (Sp)
Positive control	655	712	No	1907	1682	No

* solvent control with DMSO

Mp : Moderate precipitate

Sp : Strong precipitate

MA : Metabolic activation

Table 4: Third experiment (preincubation with S9 mix) - Mean revertant colony count

	TA 98
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)
0*	-	21	No
312.5	-	16	No (Mp)
625	-	22	No (Mp)
1250	-	21	No (Sp)
2500	-	23	No (Sp)
5000	-	23	No (Sp)
Positive control	-	1342	No

* solvent control with DMSO

Mp : Moderate precipitate

Sp : Strong precipitate

MA : Metabolic activation

 

Conclusions:

Under the experimental conditions, the test material did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Executive summary:

The objective of this study was to evaluate the potential of the test material to induce reverse gene mutations in Salmonella typhimurium in accordance with the standardised guidelines OECD 471 and EU Method B13/14.

The test material was tested with and without a metabolic activation system (S9 mix, prepared from a liver post mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254).

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test material (three plates/dose-level) ranging from 312.5 to 5000 µg/plate. After 48 to 72 hours of incubation at 37 °C, the revertant colonies were scored. Solvent control (DMSO) and positive controls were used.

The numbers of revertants for the vehicle and positive controls were within the ranges specified in the acceptance criteria. The study was therefore considered valid.

A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels ≥ 312.5 µg/plate. No noteworthy toxicity was induced in any of the five tester strains.

The test item did not induce any noteworthy increase in the number of revertants which could be considered as relevant, either with or without S9 mix, in any of the five tester strains.

Under these experimental conditions, the test material did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

Read-across performed with structurally similar substance.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 July 2012 to 29 October 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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: guidelines published by the Japanese Regulatory Authorities, including METI, MHLW and MAFF.

Qualifier:

according to guideline

Guideline:

other: USA, EPA (TSCA) OPPTS harmonised guidelines.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine requirement in the Salmonella typhimurium strains.
Tryptophan requirement in the Escherichia coli strain.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

- Type and identity of media: Stock cultures were prepared in Oxoid nutrient broth.
- Properly maintained: yes. Stored at approximately -196 °C in a liquid nitrogen freezer. Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Additional strain / cell type characteristics:

other: S. typhimurium: all strains possess rfa- and uvrB-; TA98 and TA100 also possess the R-factor plasmid pKM101.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: Stock cultures were prepared in Oxoid nutrient broth.
- Properly maintained: yes. Stored at approximately -196 °C in a liquid nitrogen freezer. Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Additional strain / cell type characteristics:

other: E. coli strain possesses the uvrA- mutation.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate (10 % liver S9 in standard co-factors)

Test concentrations with justification for top dose:

Preliminary Toxicity Test
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Mutation Test
Experiments 1 and 2: 0, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: The test material was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL. The test material formed the best doseable suspension in distilled water, therefore, this solvent was selected as the vehicle.
The test material was accurately weighed and approximate half-log dilutions prepared in distilled water by mixing on a vortex mixer and sonication for 10 minutes at 40 °C on the day of each experiment. All formulations were used within four hours of preparation and were assumed to be stable for this period.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

- EXPERIMENT 1
METHOD OF APPLICATION: in agar (direct plate incorporation)
0.1 mL aliquots of the bacterial cultures were dispensed into sets of test tubes, followed by 2 mL molten top agar (0.6 % agar, 0.5 % NaCl with 5 mL of 1.0 mM histidine and 1.0mM biotin for S. typhimurium or 1.0 mM tryptophan solution for E. coli added to each 100 mL of top agar), 0.1 mL of the appropriate test material solution, vehicle or positive control and 0.5 mL S9-mix (for the plates with metabolic activation) or 0.5 mL phosphate buffer (for the plates without metabolic activation). The contents were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates.

DURATION
- Exposure duration: 48 hours at 37 °C.

NUMBER OF REPLICATIONS: The tests were performed in triplicate.


- EXPERIMENT 2
METHOD OF APPLICATION: pre-incubation
0.1 mL of the appropriate bacterial culture was dispensed into a test tube followed by 0.5 mL of S9-mix or phosphate buffer and 0.1 mL of the vehicle or test material formulation and incubated for 20 minutes at 37 °C with shaking at approximately 130 rpm prior to the addition of 2 mL of molten, trace histidine or tryptophan supplemented top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates.
The positive and untreated controls were dosed using the standard plate incorporation method described above.

DURATION
- Exposure duration: 48 hours at 37 °C.

NUMBER OF REPLICATIONS: The tests were performed in triplicate.


DETERMINATION OF CYTOTOXICITY
- Method: Examined for effects on the background lawn of bacterial growth.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al, 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

The reverse mutation assay may be considered valid if the following criteria are met:
- All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks.
- All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- All tester strain cultures should be in the range of 0.9 to 9 x 10⁹ bacteria per mL.
- Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
- There should be a minimum of four non-toxic test material dose levels.
- There should be no evidence of excessive contamination.

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity test was performed with TA100 and WP2uvrA in both the absence and presence of S9-mix with ten different concentrations of the test material, ranging from 0.15 to 5000 µg/plate. The test material was not toxic at any concentration both in the absence and presence of S9-mix. Precipitation was observed at concentrations of ≥ 500 µg/plate.


DEFINITIVE STUDY
Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The mean number of revertant colonies for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiments 1 and 2, respectively.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A test material film (opaque in appearance) was noted at and above 1500 μg/plate with an associated precipitate observed at 5000 μg/plate. Neither of these observations prevented the scoring of revertant colonies.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

All of the positive control chemicals induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table 1: Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment	Mean number of colonies/plate
	Base-pair Substitution Type			Frameshift Type
	TA100	TA1535	WP2uvrA	TA98	TA1537
1	79	16	27	19	11
2	93	31	22	16	14

 

Table 2: Experiment 1

+/- S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate ± SD
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
- - - - - -	Solvent 50 150 500 1500 5000	86 ± 6.5 97 ± 18.2 91 ± 7.2 73 ± 6.1 81 ± 14.5* 60 ± 18.3**	19 ± 8.6 16 ± 7.5 16 ± 6.2 14 ± 5.1 15 ± 2.6* 12 ± 2.6**	27 ± 6.7 28 ± 1.5 24 ± 4.2 26 ± 5.3 19 ± 5.6* 27 ± 6.0**	21 ± 4.0 20 ± 4.2 25 ± 6.4 17 ± 1.7 19 ± 3.1* 17 ± 6.7**	9 ± 1.5 9 ± 2.1 8 ± 2.1 7 ± 3.5 8 ± 1.5* 8 ± 3.1**
+ + + + + +	Solvent 50 150 500 1500 5000	91 ± 9.9 96 ± 11.9 86 ± 15.0 102 ± 9.5 80 ± 12.3* 64 ± 3.6**	16 ± 4.0 15 ± 3.5 13 ± 2.1 19 ± 4.9 14 ± 5.5* 9 ± 0.6**	32 ± 6.4 26 ± 1.5 24 ± 2.5 32 ± 0.6 22 ± 4.7* 28 ± 6.5**	25 ± 6.0 25 ± 8.7 32 ± 5.9 25 ± 5.0 29 ± 5.0* 20 ± 9.0**	11 ± 4.9 7 ± 4.0 9 ± 1.5 12 ± 3.8 10 ± 5.5* 10 ± 2.0**
Positive Controls
-	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentration (µg/plate)	3	5	2	0.2	80
	Mean no. colonies/plate ± SD	834 ± 31.1	766 ± 100.4	743 ± 3.8	162 ± 11.7	650 ± 214.5
+	Name	2AA	2AA	2AA	BP	2AA
	Concentration (µg/plate)	1	2	10	5	2
	Mean no. colonies/plate ± SD	810 ± 12.7	218 ± 31.1	192 ± 23.6	165 ± 21.0	282 ± 7.8

* Test material film

** Test material film and precipitate

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = benzo(a)pyrene

SD = standard deviation

 

Table 3: Experiment 2

+/- S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate ± SD
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
- - - - - -	Solvent 50 150 500 1500 5000	91 ± 6.4 98 ± 9.6 100 ± 9.2 105 ± 10.6 83 ± 19.2* 88 ± 14.4**	32 ± 6.6 24 ± 6.1 34 ± 0.0 33 ± 1.5 28 ± 6.1* 34 ± 1.2**	28 ± 11.2 23 ± 3.1 29 ± 8.4 31 ± 4.7 33 ± 5.0* 25 ± 2.9**	14 ± 4.0 18 ± 4.0 11 ± 2.1 14 ± 6.0 17 ± 7.4* 18 ± 2.5**	15 ± 4.6 14 ± 2.6 14 ± 6.2 14 ± 2.5 12 ± 2.6* 16 ± 2.6**
+ + + + + +	Solvent 50 150 500 1500 5000	83 ± 8.9 92 ± 9.0 88 ± 6.7 86 ± 8.5 79 ± 8.7* 89 ± 17.3**	11 ± 2.3 10 ± 2.9 11 ± 3.8 15 ± 1.2 10 ± 2.1* 12 ± 2.1**	32 ± 5.1 37 ± 5.3 30 ± 2.5 39 ± 2.0 36 ± 3.8* 26 ± 3.2**	22 ± 4.7 18 ± 1..5 23 ± 3.2 19 ± 2.5 18 ± 2.9* 20 ± 7.4**	18 ± 1.5 18 ± 2.3 13 ± 2.1 11 ± 5.0 12 ± 5.7* 13 ± 4.4**
Positive Controls
-	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentration (µg/plate)	3	5	2	0.2	80
	Mean no. colonies/plate ± SD	330 ± 12.5	236 ± 57.8	702 ± 54.1	138 ± 20.3	416 ± 80.6
+	Name	2AA	2AA	2AA	BP	2AA
	Concentration (µg/plate)	1	2	10	5	2
	Mean no. colonies/plate ± SD	628 ± 200.0	140 ± 18.0	248 ± 20.0	248 ± 9.9	170 ± 16.5

* Test material film

** Test material film and precipitate

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = benzo(a)pyrene

SD = standard deviation

Conclusions:

Under the conditions of this study, the test material was considered to be non-mutagenic.

Executive summary:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14. Furthermore, the test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test material, using the plate incorporation and pre-incubation methods, at five dose levels, both with and without metabolic activation. The dose levels assessed were 50, 150, 500, 1500 and 5000 µg/plate.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A test material film (opaque in appearance) was noted at and above 1500 μg/plate with an associated precipitate observed at 5000 μg/plate. Neither of these observations prevented the scoring of revertant colonies.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

The vehicle controls gave revertant colony counts within the normal range. The positive controls gave the expected increases in revertants, validating the sensitivity of the assay and the efficacy of the S9-mix.

The test material was considered to be non-mutagenic under the conditions of this test.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

Read-across to structurally similar substance.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

Chromosome Aberration Test

The potential of the test material to cause chromosomal aberrations in human lymphocytes was determined in accordance with the standardised guidelines OECD 473 andThe Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environmental (MOE), under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by precipitate. The dose levels selected for the Main Test were:0.125, 0.25, 0.5, 1, 2, 4, 8 µg/mL.

All vehicle (Minimal Essential Medium) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive controls induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material did not demonstrate any marked toxicity and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level. 

Under the conditions of this study, the test material was considered to be non-clastogenic to human lymphocytes in vitro.

V79 HPRT Gene Mutation Assay

The mutagenicity of the test material was investigated on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line, in accordance with the standardised guideline OECD 476 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Chinese hamster (V79) cells were treated with the test material at six concentrations, in duplicate, together with vehicle (MEM medium) and positive controls in both the absence and presence of metabolic activation. 

The concentrations used in the main test were selected using data from the preliminary toxicity study. The maximum dose levels were limited by a combination of test material induced toxicity and precipitate. The concentrations of test material plated for cloning efficiency and expression of mutant colonies were as follows: 4-hour without S9: 0.13, 0.25, 0.5, 1, 2 and 3 µg/mL and 4-hour with S9 (2 %): 1, 2, 4, 8 and 16 µg/mL.

The vehicle (MEM medium) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any of the concentration levels in the main test, in either the absence or presence of metabolic activation. Optimum levels of toxicity were achieved at 3 µg/mL in the 4-hour –S9 exposure. In the 4-hour exposure +S9 the dose level of 16 µg/mL exhibited precipitate of the test material. Both exposure groups met the requirements of the OECD 476 guideline.

Under the conditions of this study, the test material was considered to be non-mutagenic to V79 cells at the HPRT locus.

Ames Test: Read-across performed with structurally similar substance (Praseodymium (III,IV) Oxide)

The objective of this study was to evaluate the potential of the test material to induce reverse gene mutations inSalmonella typhimuriumin accordance with the standardised guidelines OECD 471 and EU Method B13/14. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was tested with and without a metabolic activation system (S9 mix, prepared from a liver post mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254).

S. typhimuriumTA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test material (three plates/dose-level) ranging from 312.5 to 5000 µg/plate. After 48 to 72 hours of incubation at 37 °C, the revertant colonies were scored. Solvent control (DMSO) and positive controls were used.

The numbers of revertants for the vehicle and positive controls were within the ranges specified in the acceptance criteria. The study was therefore considered valid.

A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels ≥ 312.5 µg/plate. No noteworthy toxicity was induced in any of the five tester strains.

The test item did not induce any noteworthy increase in the number of revertants which could be considered as relevant, either with or without S9 mix, in any of the five tester strains.

Under these experimental conditions, the test material did not show any mutagenic activity in the bacterial reverse mutation test withSalmonella typhimurium.

Ames Test: Read-across performed with structurally similar substance (Praseodymium Tricarbonate)

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14. Furthermore, the test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimuriumstrains TA1535, TA1537, TA98, TA100 andEscherichia colistrain WP2uvrA were treated with the test material, using the plate incorporation and pre-incubation methods, at five dose levels, both with and without metabolic activation. The dose levels assessed were 50, 150, 500, 1500 and 5000 µg/plate.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000μg/plate. A test material film (opaque in appearance) was noted at and above 1500μg/plate with an associated precipitate observed at 5000μg/plate. Neither of these observations prevented the scoring of revertant colonies.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

The vehicle controls gave revertant colony counts within the normal range. The positive controls gave the expected increases in revertants, validating the sensitivity of the assay and the efficacy of the S9-mix.

The test material was considered to be non-mutagenic under the conditions of this test.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.