Esterification products of alkenylsuccinic anhydride and 2-dialkylaminoethanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro. Gene mutation in bacteria (Ames)

The mutagenicity of the test material was evaluated in a bacterial reverse mutation assay (Ames test) conducted to a method equivalent to OECD 471 and EU Method B13/14. The study was conducted with Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and E.coli WP2 in the presence and absence of S9 metabolic activation up to limit concentrations (5 mg/plate) or up to cytotoxic concentrations.

Under the conditions of the test, the test material was determined to be non-mutagenic in all strains tested. The assays were conducted in the presence and absence of metabolic activation up to cytotoxic concentrations (where appropriate), or up to limit concentrations.

 

Genetic toxicity in vitro. Chromosome Aberration

The clastogenicity of the test material was evaluated in an in vitro mammalian chromosome aberration test conducted to a method comparable to OECD 473. Chinese hamster ovary cells were exposed to the test material in the presence and absence of metabolic activation (S9) up to cytotoxic concentrations. Under the conditions of the test, the test material was concluded to be non-clastogenic in the presence and absence of metabolic activation.

 

Genetic toxicity in vitro. Gene mutation in mammalian cells

The mutagenicity of the test material was evaluated in a study conducted in accordance with OECD 476 and EU Method B.17. Two experiments were conducted up to precipitating cytotoxic concentrations in the presence and absence of metabolic activation for 3-hour and 24-hour exposure times. Under the conditions of the test, no toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cell were observed and is therefore considered to be non-mutagenic under the conditions of the 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:

29 June 1993 to 16 August 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro cytogenicity / chromosome aberration study in mammalian cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The Merck Institute for Therapeutic Research, West Point, PA
- Cell cycle length, doubling time or proliferation index: Doubling time 14 hours
- Number of passages if applicable: 9 and 10
- Methods for maintenance in cell culture if applicable: Master vials were stored at -80 °C. Working cultures were maintained in a 37 °C, 5 % CO2 and 95 % realtive humidity incubator in culture medium.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: 5 % CO2. Maintained in McCoy's 5A with NaHCO3 buffer, 10 % foetal bovine serum (FBS), 50 units/mL penicillin, 50 µg/mL streptomycin and 2 mL L-glutamine.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 microsome fraction

Test concentrations with justification for top dose:

0.05, 0.15, 0.5, 1.5, 5, 15 and 50 µg/mL - Nonactivated, 24 and 14 hour assays
0.015, 0.05, 0.15, 0.5, 1.5, 5, and 15 µg/mL - Activated, 24 hour assay
0.00051, 0.0015, 0.0051, 0.015, 0.051, 0.15, 0.51, 1.5, 5.1 and 15 µg/mL - Activated, 14 hour assay

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding : Seeded at a cell density of 5E+05 cells per flask

DURATION
- Exposure duration:14 or 24 hours in the nonactivated assay and 2 hours in the activated assay
- Expression time (cells in growth medium): 14 or 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): Cells were fixed at the end of the expression period

SPINDLE INHIBITOR (cytogenetic assays): Vinblastine sulfate (0.26 µg/mL added 2 hours prior to harvest)

STAIN : 5 % Giemsa

NUMBER OF REPLICATIONS: Performed in duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At the end of the incubation period, metaphase cells were collected by treatment with trypsin (0.25 % trypsin and 0.02 % EDTA) followed by concentration of cells by centrifugation. Due to the presence of the test material and positive control in the flasks in the nonactivated assay, these cells were washed with phosphate buffered saline (PBS) solution (pH 7.3) or with medium. The cells for all assays were suspending in hypotonic solution (0.03 M KCl and 0.01 M sodium citrate) for 12 minutes at 37 °C and fixed 3 times 3:1 methanol:acetic acid by centrifugation and resuspension. Drops of the concentrated cell suspension were placed on glass slides, air dried and stained in 5 % Giemsa for approximately 5 minutes at room temperature.

NUMBER OF CELLS EVALUATED: 100

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (a minimum of 500 cells were counted)

Rationale for test conditions:

Two range finder assays were performed to determine toxic concentrations of the test material in the presence of metabolic activation in the test system. The first range finding assay was performed using concentrations of 523, 1577 and 5257 µg/mL. The second assay was performed with concentrations of 1.4 and 148 µg/mL. The test concentrations of the definitive test were selected based of the results of the two range finding tests.

Evaluation criteria:

Metaphase cells were analysed for chromosomal aberrations through a microscope using a 100 x objective. The mitotic index was determined by counting a minimum of 500 total cells. A minimum of 25 cells were analysed for each positive control flask. In the 14 hour activated assay, the highest test concentration, 15 µg/mL, was analysed uncoded due to the low number of metaphase cells (0.7 %) observed coupled with the low percentage of confluency noted (less than 10 %, toxic spots). One hundred metaphase cells per flask for at least three selected test sample concentrations and the negative control were analysed for the following types of chromosomal aberrations:
Chromatid gap
Chromatid break
Chromosome gap
Chromosome break
Quadriradial
Interstitial deletions
Double minute chromosomes
Triradial
Dicentric chromosome
Cell with at least one pulverised chromosome
Complex rearrangements
Rings
Cells with greater than 10 aberrations
A positive response was determined by a statistically significant increase in aberrations.

Statistics:

Coordinates of cells with aberrations were recorded.
% Cells with aberrations = (Number of cells with aberrations/total number of cells analysed) * 100
Number of Aberrations per Cell = Total number aberrations/Total cells analysed
% Mitotic Index (MI) = (Number of metaphase cells/Total number of cells) * 100
The number of aberrations per cell and the percent of cells with aberrations were calculated by two methods. In one calculation the number of chromatid and chromosome gaps were counted as aberrations. IN the other calculation, the more traditional method was followed in which gaps were not counted as aberrations. A cell with a large number of aberrations was counted as having 10 aberrations. No cells with pulverised chromosomes were observed.
The data were analysed statistically using the method described in Margolin et al (1986) Statistical Analyses for In Vitro Cytogenetic Assays Using Assays Chinese Hamster Ovary Cells, Environ. Mutagen. 8: 183-204.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The test material was found to be cytotoxic at concentrations greater than 49.3 µg/mL in the presence of metabolic activation.

Table 1: Results of the range finding studies

Concentration (µg/mL)	1st Range Finding Study (Mitotic Index)	2nd Range Finding Study (Mitotic Index)
5257	Toxic
1577	Toxic
523	Toxic
148		Toxic
49.3		Toxic
14.8		11/543 (2.0 %)
		5/506 (0.79 %)
4.93		18/506 (3.6 %)
		16/545 (2.9 %)
1.48		13/534 (2.4 %)
		17/514 (3.3 %)
1 % DMSO	35/498 (7.0 %)	14/509 (2.8 %)
	28/514 (5.4 %)	10/502 (2.0 %)

 

Table 2: 14-hour assay without metabolic activation (individual results)

Treatment		No. Cells Analysed	% Mitotic Index	Number and type of chromosome aberration
				Simple			Complex						Other
				TB	SB	DM	ID	TR	QR	D	R	CR	PU	10+	TG	SG
Mitomycin C	0.5 µg/mL	25	2.6	12	3	0	1	4	3	0	0	0	0	0	8	3
		25	1.8	9	5	0	1	7	4	0	0	0	0	1	6	1
DMSO	1.00%	100	10.4	1	0	0	0	0	0	0	0	0	0	0	4	1
		100	6.4	0	0	0	0	0	0	1	0	0	0	0	1	0
Test material	1.5 µg/mL	100	9.4	1	2	0	0	0	0	1	0	0	0	0	2	1
		100	7.2	2	0	0	0	0	0	0	0	0	0	0	4	0
	5.0 µg/mL	100	11.8	0	0	0	0	0	0	0	0	0	0	0	3	0
		100	4.8	0	1	0	0	0	0	0	0	0	0	0	4	0
	15 µg/mL	100	3.7	0	1	0	0	0	0	0	1	0	0	0	7	0
		100	8.7	0	3	0	0	0	0	0	0	0	0	0	3	0
	50 µg/mL	Toxic
Chromatid gap – TG; Chromatid break – TB; Chromosome gap – SG; Chromosome break – SB; Quadriradial – QR; Interstitial deletions – ID; Double minute chromosomes – DM; Triradial – TR; Dicentric chromosome – D; Cell with at least one pulverised chromosome – PU; Complex rearrangements – CR; Rings – R; Cells with greater than 10 aberrations – 10+

 

Table 3: 14-hour assay without metabolic activation (summary)

Treatment		No. Cells Analysed	Gaps Excluded			Gaps Included
			Total No. Aberrations	No. Aberrations Per Cell	% Cells with Aberrations	Total No. Aberrations	No. Aberrations Per Cell	% Cells with Aberrations
Mitomycin C	0.5 µg/mL	25	23	0.92	56	34	1.36	68
		25	36*	1.44	68	43*	1.72	72
DMSO	1.00%	100	1	0.01	1	6	0.06	5
		100	1	0.01	1	2	0.02	2
Test material	1.5 µg/mL	100	4	0.04	3	7	0.07	6
		100	2	0.02	2	6	0.06	5
	5.0 µg/mL	100	0	0	0	3	0.03	3
		100	1	0.01	1	5	0.05	5
	15 µg/mL	100	4	0.04	4	11	0.11	10
		100	3	0.03	2	6	0.06	4
	50 µg/mL	Toxic
* Data indicated pulverised cells and/or damaged cells with more than 10 chromosome aberrations per cell

 

Table 4: 24-hour assay without metabolic activation (individual results)

Treatment		No. Cells Analysed	% Mitotic Index	Number and type of chromosome aberration
				Simple			Complex						Other
				TB	SB	DM	ID	TR	QR	D	R	CR	PU	10+	TG	SG
Mitomycin C	0.3 µg/mL	25	1.6	10	2	0	2	3	5	0	3	9	0	1	3	1
		25	1.2	20	4	0	2	9	6	0	1	5	0	0	7	1
DMSO	1.00%	100	6.0	1	2	0	0	0	0	1	0	0	0	0	3	1
		100	7.0	0	3	0	0	0	0	0	1	0	0	0	0	0
Test material	1.5 µg/mL	100	6.0	1	0	0	0	0	0	0	0	0	0	0	1	0
		100	5.8	0	2	0	0	0	0	0	0	0	0	0	0	0
	5.0 µg/mL	100	5.9	0	2	0	0	0	0	2	0	0	0	0	1	0
		100	3.9	0	2	0	0	0	0	0	0	0	0	0	1	0
	15 µg/mL	100	6.0	0	1	0	0	0	0	0	0	0	0	0	1	0
		100	6.3	1	2	0	0	0	0	0	0	0	0	0	1	0
	50 µg/mL	Toxic
Chromatid gap – TG; Chromatid break – TB; Chromosome gap – SG; Chromosome break – SB; Quadriradial – QR; Interstitial deletions – ID; Double minute chromosomes – DM; Triradial – TR; Dicentric chromosome – D; Cell with at least one pulverised chromosome – PU; Complex rearrangements – CR; Rings – R; Cells with greater than 10 aberrations – 10+

 

Table 5: 24-hour assay without metabolic activation (summary)

Treatment		No. Cells Analysed	Gaps Excluded			Gaps Included
			Total No. Aberrations	No. Aberrations Per Cell	% Cells with Aberrations	Total No. Aberrations	No. Aberrations Per Cell	% Cells with Aberrations
Mitomycin C	0.5 µg/mL	25	44*	1.76	84	48*	1.92	84
		25	47	1.88	72	55	2.20	76
DMSO	1.00%	100	4	0.04	3	8	0.08	6
		100	4	0.04	3	4	0.04	3
Test material	1.5 µg/mL	100	1	0.01	1	2	0.02	2
		100	2	0.02	2	2	0.02	2
	5.0 µg/mL	100	4	0.04	4	5	0.05	5
		100	2	0.02	2	3	0.03	3
	15 µg/mL	100	1	0.01	1	2	0.02	2
		100	3	0.03	2	4	0.04	3
	50 µg/mL	Toxic
* Data indicated pulverised cells and/or damaged cells with more than 10 chromosome aberrations per cell

 

Table 6: 14-hour assay with metabolic activation (individual results)

Treatment		No. Cells Analysed	% Mitotic Index	Number and type of chromosome aberration
				Simple			Complex						Other
				TB	SB	DM	ID	TR	QR	D	R	CR	PU	10+	TG	SG
Benzo(a)pyrene	15 µg/mL	50	0.6	8	1	0	0	3	1	0	0	1	0	0	4	0
		50	2.9	4	7	0	5	7	0	0	2	0	0	0	6	2
DMSO	1.00%	100	4.4	0	6	0	0	0	0	0	1	0	0	0	1	0
		100	2.4	1	8	0	0	1	0	0	1	0	0	0	5	0
Test material	0.51 µg/mL	100	2.8	4	9	0	0	0	0	0	1	0	0	1	3	1
		100	2.2	0	6	0	0	0	0	0	2	0	0	0	3	0
	1.5 µg/mL	100	4.3	0	1	0	0	0	0	0	0	0	0	0	4	0
		100	2.4	3	4	0	0	1	0	0	0	0	0	0	4	0
	5.1 µg/mL	100	3.8	0	2	0	0	0	0	0	1	0	0	1	5	1
		100	2.0	2	3	1*	0	0	0	0	0	0	0	0	6	0
	15 µg/mL	100	2.2	1	4	0	0	0	0	0	2	0	0	0	0	1
		100	0.7	2	0	0	0	0	0	0	1	0	0	0	2	1
Chromatid gap – TG; Chromatid break – TB; Chromosome gap – SG; Chromosome break – SB; Quadriradial – QR; Interstitial deletions – ID; Double minute chromosomes – DM; Triradial – TR; Dicentric chromosome – D; Cell with at least one pulverised chromosome – PU; Complex rearrangements – CR; Rings – R; Cells with greater than 10 aberrations – 10+ * 4 pairs of double minutes in one cell

 

Table 7: 14-hour assay with metabolic activation (summary of results)

Treatment		No. Cells Analysed	Gaps Excluded			Gaps Included
			Total No. Aberrations	No. Aberrations Per Cell	% Cells with Aberrations	Total No. Aberrations	No. Aberrations Per Cell	% Cells with Aberrations
Benzo(a)pyrene	15 µg/mL	50	14	0.28	24	18	0.36	30
		50	25	0.50	36	33	0.66	44
DMSO	1.00%	100	7	0.07	5	8	0.08	6
		100	11	0.11	7	16	0.16	11
Test material	0.51 µg/mL	100	25*	0.25	6	29*	0.29	7
		100	8	0.08	2	11	0.11	5
	1.5 µg/mL	100	1	0.01	1	5	0.05	5
		100	9	0.09	7	13	0.13	11
	5.1 µg/mL	100	13*	0.13	4	19*	0.19	9
		100	7	0.07	5	13	0.13	10
	15 µg/mL	100	7	0.07	6	8	0.08	7
		100	4	0.04	4	7	0.07	5
* Pulverised cells/and or damaged cells with more than ten chromosome aberrations per cell.

 

Table 8: 24-hour assay with metabolic activation (individual results)

Treatment		No. Cells Analysed	% Mitotic Index	Number and type of chromosome aberration
				Simple			Complex						Other
				TB	SB	DM	ID	TR	QR	D	R	CR	PU	10+	TG	SG
Cyclophosphamide	50 µg/mL	25	1.1	20	3	0	3	10	2	1	1	3	0	0	4	1
		25	1.9	12	1	0	1	13	4	1	2	10	0	2	0	2
Benzo(a)pyrene	15 µg/mL	25	5.0	0	1	0	0	0	0	0	0	0	0	0	0	1
		25	8.5	0	1	0	0	1	0	0	0	0	0	0	0	0
DMSO	1.00%	100	4.8	3	3	0	0	0	0	1	0	0	0	0	1	1
		100	7.7	1	5	0	0	0	0	0	1	0	0	0	1	1
Test material	1.5 µg/mL	100	4.9	1	0	0	0	0	0	0	0	0	0	0	0	0
		100	5.0	1	3	1*	0	0	0	0	0	0	0	0	1	0
	5.1 µg/mL	100	3.8	2	6	0	0	0	0	0	0	0	0	0	1	2
		100	5.9	2	2	0	0	0	0	0	0	0	0	0	0	0
	15 µg/mL	100	5.1	1	5	0	0	0	0	0	0	0	0	0	1	1
		100	3.7	3	4	0	0	0	0	0	0	0	0	0	1	0
Chromatid gap – TG; Chromatid break – TB; Chromosome gap – SG; Chromosome break – SB; Quadriradial – QR; Interstitial deletions – ID; Double minute chromosomes – DM; Triradial – TR; Dicentric chromosome – D; Cell with at least one pulverised chromosome – PU; Complex rearrangements – CR; Rings – R; Cells with greater than 10 aberrations – 10+ * 7 pairs of double minutes in one cell

 

Table 9: 24-hour assay with metabolic activation (summary of results)

Treatment		No. Cells Analysed	Gaps Excluded			Gaps Included
			Total No. Aberrations	No. Aberrations Per Cell	% Cells with Aberrations	Total No. Aberrations	No. Aberrations Per Cell	% Cells with Aberrations
Cyclophosphamide	50 µg/mL	25	43	1.72	64	48	1.92	68
		25	64*	2.56	92	66*	2.64	92
Benzo(a)pyrene	15 µg/mL	25	1	0.04	4	2	0.08	8
		25	2	0.08	8	2	0.08	8
DMSO	1.00%	100	7	0.07	6	9	0.09	8
		100	7	0.07	2	9	0.09	4
Test material	1.5 µg/mL	100	1	0.01	1	1	0.01	1
		100	5	0.05	4	6	0.06	5
	5.1 µg/mL	100	10	0.10	5	13	0.13	6
		100	4	0.04	3	4	0.04	3
	15 µg/mL	100	7	0.07	6	9	0.09	7
		100	8	0.08	6	9	0.09	7
* Pulverised cells/and or damaged cells with more than ten chromosome aberrations per cell.

 

Conclusions:

The test material was evaluated for its ability to induce chromosomal damage in vitro using CHO cells. The test material was tested with and without metabolic activation. The positive controls were found to be valid under the conditions of the test. Under the conditions of the test, there were no increases in the percentage of aberrant cells (including and excluding gaps) with or without metabolic activation.
The test material was therefore concluded to not be clastogenic under the conditions of the test.

Executive summary:

The clastogenicity of the test material was evaluated in an in vitro mammalian chromosome aberration test conducted to a method comparable to OECD 473. Chinese hamster ovary cells were exposed to the test material in the presence and absence of metabolic activation (S9) up to cytotoxic concentrations. Under the conditions of the test, the test material was concluded to be non-clastogenic in the presence and absence of metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 July 1993 to 22 October 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine biosynthetic pathway - Salmonella
Tryptophan pathway - E.coli

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Concentrations of 0.00015 mg/plate to 0.05 mg/plate were chosen for the study. At concentrations above 0.05 mg/plate, the test material was found to be cytotoxic in the range-finding study to strain TA100.
0.00015, 0.0005, 0.0015, 0.005, 0.015 and 0.05 mg/plate were used in the definitive test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

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

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)
2.5 mL aliquots of molten top agar containing either 50 µM biotin and 50 µM histidine (for Salmonella), or 50 µM biotin and 50 µM tryptophan (for E.coli) were added to 100 µL of bacterial culture (between 1E+09 and 2E+09 bacteria/mL), 100 µL of the appropriate concentration of test substance, or 100 µL of positive or negative (vehicle) control, and 500 µL of S9 mix or PBS (for the non-activated assay). The tubes were mixed and poured onto plates containing 25-30 mL of minimal glucose agar made. Test plates were allowed to harden, then were incubated at 37 °C for approximately 3 days. The plates were counted immediately after removal from incubation or stored at 4 °C before counting.
- Cell density: Between 1E+09 and 2E+09 bacteria/mL

DURATION
- Exposure duration: 3 days

SELECTION AGENT (mutation assays): biotin and histidine (for Salmonella), or biotin and tryptophan (for E.coli)

NUMBER OF REPLICATIONS: Performed in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: bacterial lawn

Rationale for test conditions:

Concentrations were determined from the range finding study. Further repititions were performed based on observed toxicity during the definitive tests.

Evaluation criteria:

The bacterial plates were scored on an electronic colony counter, or they were counted manually. The mean number of revertants per plate and the standard deviation was calculated for each concentration and strain. A test result was considered positive if for any strain a significant increase over the negative control in the number of revertant per plate was observed which was concentration-dependent. A significant increase was a two-fold increase when the background was 50 revertants/plate or greater; a three-fold increase when the background was between 10 and 49 revertants/plate; a four-fold increase when the background was less than 10 revertants/plate.

Statistics:

The mean and standard deviation were calcualted for each concentration and strain.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

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:

S. typhimurium TA 98

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:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2

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: The initial range finding study (TA100 tested in the absence of metabolic activation) indicated that the test material was toxic to bacteria at 0.05 mg/plate and above. The bacterial lawn was not visible at 1.5 and 5 mg/plate. At lower concentrations, 0.015 mg/plate and below, the bacterial lawn was normal and the number of revertant colonies were similar to the control. Based on the range finding study, experiment 1 was conducted with a concentration range between 0.00015 and 0.05 mg/plate.

HISTORICAL CONTROL DATA
- Positive historical control data: The postive control values were within established historical parameters.
- Negative (solvent/vehicle) historical control data: The negative control values were within established historical parameters.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: In the first definitive test, the test material was found to be cytotoxic at concentrations of 0.05 mg/plate for the strain TA100. Although the test material was non-mutagenic to TA100 in this experiment, these data were used as range-finding information for the other four strains.
A subsequent non-activated experiment (Experiment 2) was conducted with strains TA98, TA1535, TA1537 and WP2 utilising a maximum concentration of 0.2 mg/plate. No increase in revertant colonies was observed in this experiment. No cytotoxicity was observed, indicating that the limit of testing hadn’t been reached.
In Experiment 3, all 5 strains were tested up to a maximum concentration of 0.8 mg/plate. The test material was not mutagenic in strain TA100, and cytotoxicity was observed. The bacterial lawn was significantly reduced at 0.8 and 0.4 mg/plate. In the presence of metabolic activation, the test material was non-mutagenic. Cytotoxicity was observed at 0.2, 0.4 and 0.8 mg/plate. Cytotoxicity in strain TA1535 were similar to those found with TA100. In the absence of metabolic activation, 0.8 and 0.4 mg/plate were also toxic to TA1535. The number of revertant colonies was found to be reduced at these concentrations in the presence of metabolic activation. The test material was not mutagenic to TA1535. The test material was not mutagenic in all other strains tested in this experiment, however these strains were not tested up to limit concentrations (5 mg/plate or cytotoxic concentrations). Toxicity was evident in the bacterial lawn density of TA98, however this did not percent evaluation for revertant colonies.
In Experiment 4, conducted in the absence of metabolic activation, the test material was determined to be non-mutagenic to TA1535. 0.4 mg/plate was determined to be extremely toxic to the bacterial cultures. The test material was tested up to the maximum concentration in all other strains. Toxicity was evident in the bacterial lawn density of TA98, however this did not percent evaluation for revertant colonies.
Experiment 5 was conducted in the presence of metabolic activation. The test material was found to be non-mutagenic in all strains tested up to 5 mg/plate.
Experiment 6 was conducted to confirm the negative results observed with TA1537 and WP2. The test material was not mutagenic up to the maximum concentration 5 mg/plate.

Table 1: Range Finding Study

	S9	Number of revertants per plate (Individual Plates)			Mean Revertants/Plate Mean (SD)
		TA100
DMSO Control	-	104	153	140	132 (25)
Positive Control (ENNG) 9 µg/plate	-	1034	975	547	852 (266)
Test compound 5 mg/plate	-	x	x	x	x
Test compound 1.5 mg/plate	-	x	x	x	x
Test compound 0.5 mg/plate	-	x	x	x	x
Test compound 0.15 mg/plate	-	x	x	x	x
Test compound 0.05 mg/plate	-	x	x	x	x
Test compound 0.015 mg/plate	-	122t	86t	162t	123 (38)
Test compound 0.005 mg/plate	-	138t	144t	133t	138 (6)
Test compound 0.0015 mg/plate	-	145t	108t	152t	135 (24)
X - cytotoxic t: Chemical demonstrated some cytotoxicity, but didn't prevent scoring ENNG = N-ethyl-N'nitro-N-nitrosaguanidine

 

Table 2: Results Experiment 1

	S9	Number of revertants per plate  (Individual Plates)			Mean Revertants/Plate Mean (SD)	Number of revertants per plate  (Individual Plates)			Mean Revertants/Plate Mean (SD)	Number of revertants per plate  (Individual Plates)			Mean Revertants/Plate Mean (SD)	Number of revertants per plate  (Individual Plates)			Mean Revertants/Plate Mean (SD)	Number of revertants per plate  (Individual Plates)			Mean Revertants/Plate Mean (SD)
		TA98				TA100				TA1535				TA1535				WP2
DMSO Control	-	32	30	26	29 (3)	168	165	185	173 (11)	29	21	15	22 (7)	15	12	15	14 (2)	44	43	28	38 (9)
	+	45	30	29	35 (9)	168	197	204	190 (19)	17	21	20	19 (2)	8	11	20	14 (8)	52	60	49	54 (6)
Positive Control	-	391	389	401	394 (6)	1288	1207	1504	1333 (154)	236	233	229	233 (4)	399	388	229	418 (43)	700	722	857	760 (85)
Positive Control	+	351	240	314	302 (57)	1121	1098	956	1058 (89)	129	124	258	137 (18)	429	327	158	385 (53)	692	506	530	576 (101)
Test compound 0.05 mg/plate	-	10	20	16t	15 (5)	x	z	x	x	12	15	16t	14 (2)	5	5	16t	6 (2)	47	50	53	50 (3)
Test compound 0.015 mg/plate	-	21	25	26	24 (3)	183	157	145t	162 (19)	15	15	17	16 (1)	6	5	17	7 (2)	45	57	49	50 (6)
Test compound 0.005 mg/plate	-	22	32	21	25 (6)	181	187	173	180 (7)	10	15	14	13 (3)	10	13	14	13 (3)	43	37	45	42 (4)
Test compound 0.0015 mg/plate	-	25	24	32	27 (4)	156	172	205	178 (25)	11	16	15	14 (3)	19	12	15	16 (4)	43	50	40	44 (5)
Test compound 0.0005 mg/plate	-	26	26	30	27 (2)	182	148	157	162 (18)	12	15	10	12 (3)	13	21	10	16 (5)	49	50	40	46 (6)
Test compound 0.00015 mg/plate	-	38	21	23	27 (9)	193	182	156	177 (19)	12	15	12	13 (2)	12	19	12	15 (4)	54	39	53	49 (8)
Test compound 0.05 mg/plate	+	42	43	48t	44 (3)	187	181	164t	177 (12)	18	12	11t	14 (4)	4	9	11t	5 (4)	72	66	66	68 (3)
Test compound 0.015 mg/plate	+	54	54	52	53 (1)	195	188	171	185 (12)	12	16	16	15 (2)	14	15	16	15 (2)	70	60	45	58 (13)
Test compound 0.005 mg/plate	+	35	40	40	38 (3)	175	205	183	188 (16)	20	20	12	17 (5)	16	17	12	17 (5)	66	55	48	56 (9)
Test compound 0.0015 mg/plate	+	33	30	29	31 (2)	211	213	194	206 (10)	20	16	11	16 (5)	8	14	11	16 (5)	52	55	55	54 (2)
Test compound 0.0005 mg/plate	+	33	43	49	42 (8)	171	182	196	183 (13)	13	15	21	16 (4)	15	14	21	16 (4)	72	65	63	67 (5)
Test compound 0.00015 mg/plate	+	30	35	53	39 (12)	220	211	192	208 (14)	13	21	21	18 (5)	12	20	21	18 (5)	64	77	76	72 (7)
TA98 Positive control -S9 2-Nitrofluorene (2NF) 3 µg/plate; +S9 2-aminoanthracene (2AA) 0.5 µg/plate TA100 Positive control -S9 N-ethyl-N'nitro-N-nitrosaguanidine (ENNG) 9 µg/plate; +S9 2-aminoanthracene (2AA) 1 µg/plate TA135 Positive control -S9 Sodium Azide 0.5 µg/plate; +S9 2-aminoanthracene (2AA) 2 µg/plate TA137 Positive control -S9 9-Aminoacridine 80 µg/plate; +S9 2-aminoanthracene (2AA) 3 µg/plate WP2 Positive control -S9 N-ethyl-N'nitro-N-nitrosaguanidine (ENNG) 2 µg/plate; +S9 2-aminoanthracene (2AA) 20 µg/plate X - cytotoxic t: Chemical demonstrated some cytotoxicity, but didn't prevent scoring

 

Table 3: Results from Experiment 2

	S9	Number of revertants per plate  (Individual Plates)			Mean Revertants/Plate Mean (SD)	Number of revertants per plate  (Individual Plates)			Mean Revertants/Plate Mean (SD)	Number of revertants per plate  (Individual Plates)			Mean Revertants/Plate Mean (SD)	Number of revertants per plate  (Individual Plates)			Mean Revertants/Plate Mean (SD)
		TA98				TA1535				TA1537				WP2
DMSO Control	-	33	34	25	31 (5)	15	14	14	14 (1)	20	32	26	26 (6)	44	70	44	53 (15)
Positive Control	-	820	750	736	769 (45)	236	282	183	234 (50)	616	570	554	580 (32)	603	692	654	650 (45)
Test compound 0.20 mg/plate	-	25	31	13t	23 (9)	8	5	8t	7 (2)	24	18	19	20 (3)	60	63	50	58 (7)
Test compound 0.10 mg/plate	-	29	17	17	21 (7)	15	9	7t	10 (4)	15	18	9	17 (2)	59	53	59	57 (3)
Test compound 0.05 mg/plate	-	16	20	22	19 (3)	13	9	14	12 (3)	15	14	18	16 (2)	51	63	53	56 (6)
Test compound 0.025 mg/plate	-	22	21	24	22 (2)	8	10	12	10 (2)	27	22	17	22 (5)	63	52	46	54 (9)
Test compound 0.0125 mg/plate	-	19	18	30	22 (7)	9	6	14	10 (4)	14	22	12	16 (5)	51	58	43	51 (8)
Test compound 0.0063 mg/plate	-	31	18	29	26 (7)	11	15	13	13 (2)	17	23	16	19 (4)	71	48	53	57 (12)
TA98 Positive control -S9 2-Nitrofluorene (2NF) 3 µg/plate TA135 Positive control -S9 Sodium Azide 0.5 µg/plate TA137 Positive control -S9 9-Aminoacridine 80 µg/plate WP2 Positive control -S9 N-ethyl-N'nitro-N-nitrosaguanidine (ENNG) 2 µg/plate t: Chemical demonstrated some cytotoxicity, but didn't prevent scoring

 

Table 4: Results from Experiment 3

	S9	Number of revertants per plate  (Individual Plates)			Mean Revertants/Plate Mean (SD)	Number of revertants per plate  (Individual Plates)			Mean Revertants/Plate Mean (SD)	Number of revertants per plate  (Individual Plates)			Mean Revertants/Plate Mean (SD)	Number of revertants per plate  (Individual Plates)			Mean Revertants/Plate Mean (SD)	Number of revertants per plate  (Individual Plates)			Mean Revertants/Plate Mean (SD)
		TA98				TA100				TA1535				TA1537				WP2
DMSO Control	-	16	18	21	18 (3)	94	96	118	103 (13)	13	10	8	10 (3)	11	11	11	11 (0)	29	24	nd	27 (4)
	+	30	54	29	38 (14)	143	150	138	144 (6)	13	9	9	10 (2)	21	11	17	16 (5)	34	32	30	32 (2)
Positive Control	-	415	657	520	531 (121)	693	784	804	760 (59)	253	123	234	203 (70)	1038	973	930	980 (54)	340	493	409	414 (77)
Positive Control	+	437	489	525	484 (44)	867	860	765	831 (57)	125	179	nd	152 (38)	89	73	79	80 (8)	1038	704	960	901 (175)
Test compound 0.8 mg/plate	-	13	14	10t	12 (2)	x	x	x	x	x	x	x	x	17	8	8	11 (5)	23	16	15	18 (4)
Test compound 0.4 mg/plate	-	10	8	15t	11 (4)	x	x	x	x	x	x	x	x	16	17	15	16 (1)	25	26	22	24 (2)
Test compound 0.2 mg/plate	-	11	14	18t	14 (4)	36	26	33t	32 (5)	2	2	6t	3 (2)	16	11	17	15 (3)	19	22	27	23 (4)
Test compound 0.1 mg/plate	-	12	16	11	13 (3)	33	37	36t	35 (2)	7	4	10	7 (3)	8	15	13	12 (4)	28	26	21	25 (4)
Test compound 0.05 mg/plate	-	17	17	20	18 (2)	48	60	47	52 (7)	12	6	9	9 (3)	14	23	18	18 (5)	26	14	22	21 (6)
Test compound 0.025 mg/plate	-	16	21	20	19 (3)	60	48	85	64 (19)	9	9	11	10 (1)	7	13	16	12 (5)	21	33	26	27 (6)
Test compound 0.8 mg/plate	+	18	29	23t	23 (6)	28	32	32t	31 (2)	4	11	4t	6 (4)	18	19	29	22 (6)	26	29	28	28 (2)
Test compound 0.4 mg/plate	+	31	32	28	30 (2)	35	53	47t	45 (9)	12	7	5t	8 (4)	17	25	14	19 (6)	32	40	34	35 (4)
Test compound 0.2 mg/plate	+	33	25	45	34 (10)	50	55	57t	54 (4)	8	7	10	8 (2)	13	23	26	21 (7)	30	38	28	32 (5)
Test compound 0.1 mg/plate	+	38	36	43	39 (4)	97	123	130	117 (17)	20	13	14	16 (4)	21	19	20	20 910	35	37	34	35 (2)
Test compound 0.05 mg/plate	+	40	38	29	36 (6)	108	122	90	107 (16)	11	15	7	11 (4)	20	23	20	21 92)	28	26	39	31 (7)
Test compound 0.025 mg/plate	+	47	34	40	40 (7)	143	145	120	136 (14)	14	10	13	12 (2)	9	15	16	13 94)	32	36	26	31 (5)
TA98 Positive control -S9 2-Nitrofluorene (2NF) 3 µg/plate; +S9 2-aminoanthracene (2AA) 0.5 µg/plate TA100 Positive control -S9 N-ethyl-N'nitro-N-nitrosaguanidine (ENNG) 9 µg/plate; +S9 2-aminoanthracene (2AA) 1 µg/plate TA135 Positive control -S9 Sodium Azide 0.5 µg/plate; +S9 2-aminoanthracene (2AA) 2 µg/plate TA137 Positive control -S9 9-Aminoacridine 80 µg/plate; +S9 2-aminoanthracene (2AA) 3 µg/plate WP2 Positive control -S9 N-ethyl-N'nitro-N-nitrosaguanidine (ENNG) 2 µg/plate; +S9 2-aminoanthracene (2AA) 20 µg/plate X - cytotoxic t: Chemical demonstrated some cytotoxicity, but didn't prevent scoring nd - not done, only two plates poured

 

Table 5: Results from Experiment 4

	S9	Number of revertants per plate  (Individual Plates)			Mean Revertants/Plate Mean (SD)	Number of revertants per plate  (Individual Plates)			Mean Revertants/Plate Mean (SD)	Number of revertants per plate  (Individual Plates)			Mean Revertants/Plate Mean (SD)	Number of revertants per plate  (Individual Plates)			Mean Revertants/Plate Mean (SD)
		TA98				TA1535				TA1537				WP2
DMSO Control	-	24	21	26	24 (3)	13	13	11	12 (1)	13	8	14	12 (3)	414	35	36	37 (3)
Positive Control	-	479	455	478	471 (14)	274	250	220	248 (27)	22	246	491	253 (235)	236	184	199	206 (27)
Test compound 5 mg/plate	-	ND	ND	ND	ND	ND	ND	ND	ND	17	10	24	17 (7)	48	22	27	32 (14)
Test compound 3.2 mg/plate	-	14	15	16t	15 (1)	ND	ND	ND	ND	16	19	12	16 (4)	36	42	48	42 (6)
Test compound 1.6 mg/plate	-	21	19	18t	19 (2)	ND	ND	ND	ND	16	11	7	11 (5)	31	36	46	38 (8)
Test compound 0.8 mg/plate	-	14	11	16t	14 (3)	ND	ND	ND	ND	11	15	9	12 (3)	46	34	36	39 (6)
Test compound 0.4 mg/plate	-	21	15	18t	18 (3)	x	x	x	x	12	14	12	13 (1)	41	42	44	42 (2)
Test compound 0.2 mg/plate	-	9	9	15	11 (3)	5	5	6t	5 (1)	18	18	15	17 (2)	33	27	39	33 (6)
Test compound 0.1 mg/plate	-	19	21	22	21 (2)	2	7	10t	6 (4)	12	13	12	12 (1)	ND	ND	ND	ND
Test compound 0.05 mg/plate	-	15	21	18	18 (3)	6	8	14	9 (4)	ND	ND	ND	ND	ND	ND	ND	ND
Test compound 0.025 mg/plate	-	29	26	27	27 (2)	6	15	4	8 (6)	ND	ND	ND	ND	ND	ND	ND	ND
Test compound 0.0125 mg/plate	-	ND	ND	ND	ND	15	9	11	12 (3)	ND	ND	ND	ND	ND	ND	ND	ND
TA98 Positive control -S9 2-Nitrofluorene (2NF) 3 µg/plate
TA135 Positive control -S9 Sodium Azide 0.5 µg/plate
TA137 Positive control -S9 9-Aminoacridine 80 µg/plate
WP2 Positive control -S9 N-ethyl-N'nitro-N-nitrosaguanidine (ENNG) 2 µg/plate
t: Chemical demonstrated some cytotoxicity, but didn't prevent scoring

 

Table 6: Results from Experiment 5

	S9	Number of revertants per plate  (Individual Plates)			Mean Revertants/Plate Mean (SD)	Number of revertants per plate  (Individual Plates)			Mean Revertants/Plate Mean (SD)	Number of revertants per plate  (Individual Plates)			Mean Revertants/Plate Mean (SD)	Number of revertants per plate  (Individual Plates)			Mean Revertants/Plate Mean (SD)
		TA98				TA1535				TA1537				WP2
DMSO Control	-	ND	ND	ND	ND	ND	ND	ND	ND	6	17	12	12 (6)	44	36	40	40 (4)
	+	37	43	40	40 (3)	15	13	16	15 (2)	15	26	25	22 (6)	30	30	34	31 (2)
Positive Control	-	ND	ND	ND	ND	ND	ND	ND	ND	324	405	436	388 (58)	636	721	694	684 (43)
Positive Control	+	864	713	761	779 (77)	169	181	178	176 (6)	279	307	321	302 (210	194	196	132	174 (36)
Test compound 5.0 mg/plate	-	ND	ND	ND	ND	ND	ND	ND	ND	9	12	13	11 (2)	24	22	26	24 (2)
Test compound 3.2 mg/plate	-	ND	ND	ND	ND	ND	ND	ND	ND	15	17	18	17 (2)	33	37	32	34 (3)
Test compound 1.6 mg/plate	-	ND	ND	ND	ND	ND	ND	ND	ND	6	3	13	7 (5)	54	26	30	37 (15)
Test compound 0.8 mg/plate	-	ND	ND	ND	ND	ND	ND	ND	ND	7	11	14	11 (4)	43	23	24	30 (11)
Test compound 0.4 mg/plate	-	ND	ND	ND	ND	ND	ND	ND	ND	6	14	16	12 (5)	18	35	45	33 (14)
Test compound 0.2 mg/plate	-	ND	ND	ND	ND	ND	ND	ND	ND	6	10	13	10 (4)	32	22	23	26 (6)
Test compound 5.0 mg/plate	+	26	15	20	20 (6)	4	10	4t	6 (3)	28	24	27	26 (2)	41	38	39	39 (2)
Test compound 3.2 mg/plate	+	50	44	51	48 (4)	11	3	11t	8 (5)	18	17	29	21 (7)	21	35	35	30 98)
Test compound 1.6 mg/plate	+	27	26	25	26 (1)	8	10	15	11 (4)	22	12	15	16 (5)	16	35	35	29 (11)
Test compound 0.8 mg/plate	+	45	34	41	40 (6)	6	6	10	7 (2)	25	16	15	19 (6)	40	24	36	33 (8)
Test compound 0.4 mg/plate	+	45	41	57	48 (8)	12	9	11	11(2)	52	39	36	42 (9)	37	40	32	36 (4)
Test compound 0.2 mg/plate	+	46	27	38	34 (11)	19	11	6	12 (7)	25	31	28	28 (3)	37	43	45	42 (4)
Test compound 0.1 mg/plate	+	38	38	46	41 (5)	12	10	18	13 (4)	19	19	20	19 (1)	ND	ND	ND	ND
TA98 Positive control -S9 2-Nitrofluorene (2NF) 3 µg/plate; +S9 2-aminoanthracene (2AA) 0.5 µg/plate
TA100 Positive control -S9 N-ethyl-N'nitro-N-nitrosaguanidine (ENNG) 9 µg/plate; +S9 2-aminoanthracene (2AA) 1 µg/plate
TA135 Positive control -S9 Sodium Azide 0.5 µg/plate; +S9 2-aminoanthracene (2AA) 2 µg/plate
TA137 Positive control -S9 9-Aminoacridine 80 µg/plate; +S9 2-aminoanthracene (2AA) 3 µg/plate
WP2 Positive control -S9 N-ethyl-N'nitro-N-nitrosaguanidine (ENNG) 2 µg/plate; +S9 2-aminoanthracene (2AA) 20 µg/plate
X - cytotoxic
t: Chemical demonstrated some cytotoxicity, but didn't prevent scoring
ND = Not determined

 

Table 7: Results from Experiment 6

	S9	Number of revertants per plate  (Individual Plates)			Mean Revertants/Plate Mean (SD)	Number of revertants per plate  (Individual Plates)			Mean Revertants/Plate Mean (SD)
		TA1537				WP2
DMSO Control	+	16	15	19	17 (2)	39	28	44	37 (8)
Positive Control	+	183	276	180	213 (55)	196	217	202	205 (11)
Test compound 5.0 mg/plate	+	10	16	21	16 (6)	27	26	29	27 (2)
Test compound 3.2 mg/plate	+	12	20	7	13 (7)	30	25	20	25 (5)
Test compound 1.6 mg/plate	+	11	42	26	26 (16)	31	34	32	32 (2)
Test compound 0.8 mg/plate	+	12	17	10	13 (4)	34	33	30	32 (2)
Test compound 0.4 mg/plate	+	39	39	37	38 (1)	35	32	40	35 (4)
Test compound 0.2 mg/plate	+	37	25	37	33 (7)	32	27	29	29 (3)
Test compound 0.1 mg/plate	+	19	26	21	22 (4)	ND	ND	ND	ND
TA137 Positive control +S9 2-aminoanthracene (2AA) 3 µg/plate
WP2 Positive control +S9 2-aminoanthracene (2AA) 20 µg/plate
ND = Not determined

Conclusions:

Under the conditions of the test, the test material was determined to be non-mutagenic in all strains tested. The assays were conducted in the presence and absence of metabolic activation up to cytotoxic concentrations (where appropriate), or up to limit concentrations.

Executive summary:

The mutagenicity of the test material was evaluated in a bacterial reverse mutation assay (Ames test) conducted to a method equivalent to OECD 471 and EU Method B13/14. The study was conducted with Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and E.coli WP2 in the presence and absence of S9 metabolic activation up to limit concentrations (5 mg/plate) or up to cytotoxic concentrations.

Under the conditions of the test, the test material was determined to be non-mutagenic in all strains tested. The assays were conducted in the presence and absence of metabolic activation up to cytotoxic concentrations (where appropriate), or up to limit concentrations.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 July 1998 and 3 September 1998

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro gene mutation study in mammalian cells

Target gene:

TK +/- locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: MRC Cell Mutation Unit, University of Sussex, Brighton, UK
- Cell cycle length, doubling time or proliferation index: The cells have a generation time of approximately 12 hours

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cell stocks were stored in liquid nitrogen at -196 °C. Cells were routinely cultured in RPMI 1640 medium supplemented with 10 % donor horse serum and 20 mM Hepes buffer (R10) at 37 °C with 5 % CO2 in air. The cells were subcultured accordingly.
- Properly maintained: yes
- Periodically 'cleansed' against high spontaneous background: yes (before stocks were frozen, they were cleansed of homozygous (TK-/-) mutants by culturing in THMG medium for 24 hours).

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

The molecular weight of the test material was calculated to be 315, therefore the maximum dose level was 3150 µg/mL equivalent to 10 mM. Test concentrations for the definitive test were based on the results on the range finding tests.
The following concentrations were used in the definitive tests:
Experiment 1
With and without S9: 0, 6, 12, 24, 36 and 42 µg/mL
Experiment 2
With and without S9: 0, 6, 12, 24, 36, 48 and 60 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 5E+05 cells/mL
DURATION
- Exposure duration: 3 hours in the presence and absence of metabolic activations, and a continuous 24-hour exposure in the absence of metabolic activation.
- Expression time (cells in growth medium): 2 days
STAIN : 0.025 mL of MTT solution (2.5 mg/mL in PBS) was added to each well of the mutation plates. The plates were incubated for roughly two hours. MTT stain assisted in the scoring of viable mutant colonies.
SELECTION AGENT (mutation assays): Selective medium containing 4 µg/mL 5-trifluorothymidine (TFT)
NUMBER OF REPLICATIONS: Performed in duplicate
DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth and cloning efficiency. At the end of the treatment period for each experiment, the test material was removed by centrifugation and the cells were washed twice in R10 medium and resuspended in R20 medium at a cell density of 2E+05 cells/mL. A sample of cells were removed for dilution to 10 cells/mL and plating in non-selective medium (2 cells/well) to determine Day 0 cloning efficiency and viability (% S) immediately after treatment. The % S value was adjusted to account for immediate post-treatment toxicity as a comparison to the vehicle control using the cell count data. A comparison of each treatment % S value to the vehicle control value was performed to give a Relative Survival value (% RS). The remaining cells were incubated and subcultured daily for the required expression period by counting and dilution to 2E+05 cells/mL. The daily cell counts were used to obtain a relative total growth (RTG) value.

Evaluation criteria:

The normal range for mutant frequency per survivor is 10-125E-06 at the conducting laboratory.
Vehicle controls should be within this range. Experiments with values greater than 150E-06 should be repeated. Positive control chemicals should give a significant increase (at least 3-5 fold).
For a significant positive response, 2 or more of the following criteria should be met:
1. A statistically significant increase in mutant frequency
2. A greater than 3-fold increase in the mutant frequency per survivor over the negative control
3. A dose-related increase in the mutant frequency per survivor
4. An increase in the absolute number of mutants
A test material may be reported as equivocal if only one of the above are met.

Statistics:

Plating efficiency was calculated using the zero term of the Poisson distribution method.
P(0) = Number of negative wells/total wells plated
P.E. % = (-ln P(0) x 100 )/ number cells per well
Mutation frequency per survivor was calculated using the following:
MF (per survivor) = [(-lnP(0) selective medium)/cells per well in selective medium]]/surviving fraction in non-selective medium

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

Remarks:

Both vehicle control mutant frequencies were within the range of 10 to 125E-06 viable cells typical for the conducting laboratory.

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The preliminary toxicity test was performed with cell cultures at 5E+05 cells/mL, using a 3 hour exposure time both with and without metabolic activation and a continuous 24 hour exposure without metabolic activation. The dose range initially used was 49.22, 98.44, 196.88, 393.75, 787.5, 1575 and 3150 µg/mL.
After the 3 hour exposure period, there were no viable cells at and above 98.44 µg/mL, the toxicity test was repeated for all three exposure conditions using a revised dose range of 3.13 to 75 µg/mL. The cells were washed twice with R10, resuspended in R20 medium, counted using a coulter counter and then serially diluted to 10 cells/mL. The cells were plated for survival in selective media (2 cells/well) in 96 well microtitre plates. The plates were incubated at 37 °C for 10-14 days and the wells were scored for colony formation. The toxicity values obtained using the 96 well plate data was adjusted to account for immediate post-treatment toxicity as a comparison to the vehicle control using cell count data.
Precipitation was noted in the highest dose levels. In both the absence and presence of metabolic activation, marked toxicity was observed with the test material at and above 50 µg/mL, with a rapid onset of toxicity in the 3 hour pulse treatments.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Previous 20 studies
Without metabolic activation (S9)
Mean MF 1152.7 (SD 641.7) (Range 362.33-3108.19)
With metabolic activation (S9)
Mean MF 969.0 (SD 355.7) (Range 226.33-1559.97)
- Negative (solvent/vehicle) historical control data:
Without metabolic activation (S9)
Mean MF 100.474 (SD 44.2) (Range 31.91-194.07)
With metabolic activation (S9)
Mean MF 103.1 (SD 32.8) (Range 54.34-194.86)

Table 1: Mutant Frequencies from Experiment 1 – without metabolic activation

Treatment (µg/mL)	Viable		Small Colonies			Large Colonies			Proportion small colony mutants (µg/mL)
			Mutants		MF*	Mutants		MF*
	Yv	Nv	Ym	Nm		Ym	Nm
0	142	768	722	768	36.6	694	768	60.0	0.38
6	74	384	357	384	44.3	350	384	56.3	0.44
12	65	384	354	384	45.8	343	384	63.6	0.42
24	86	384	348	384	65.8	356	384	50.6	0.56
36	68	384	357	384	42.1	350	384	53.6	0.44
42	96	384	352	384	62.8	355	384	56.6	0.52
800 EMS	150	384	326	384	174.2	182	384	794.3	0.22
EMS = Ethylmethanesulphonate * TFT resistant mutants/1E+06 viable cells 2 days after treatment Yv = Number of wells without colonies, day 2 viability plates Nv = Number of wells scored, day 2 viability plates Ym = Number of wells without colonies, mutation plates Nm = Number of wells scored, mutation plates

 

 

Table 2: Summary of results from Experiment 1 – without metabolic activation

Treatment (µg/mL)	%S	%RS	%V	RTG	MF
0	68.28	84.40	1.00	100.65	100.65
6	67.24	82.33	1.01	105.06	105.06
12	80.87	88.81	1.07	115.10	115.10
24	71.34	74.81	0.83	121.85	121.85
36	57.33	86.56	0.90	99.93	99.93
42	17.78	69.31	0.28	124.79	124.79
Positive control (EMS) 800	56.53	47.00	0.39	1202.50	1202.50
%RS = Percent Relative Survival %S = Percent Survival Day 0 %V = Percent Viability Day 2 RTG = Relative Total Growth MF = 5-TFT resistant mutants/1E+06 viable cells 2 days after treatment EMS = Ethylmethanesulphonate Test for linear trend: Slope 2.887E-07 Variance: 2.911E-013 b²/Sb: 0.286

 

Table 3: Mutant Frequencies from Experiment 1 – with metabolic activation

Treatment (µg/mL)	Viable		Small Colonies			Large Colonies			Proportion small colony mutants (µg/mL)
			Mutants		MF*	Mutants		MF*
	Yv	Nv	Ym	Nm		Ym	Nm
0	153	768	726	768	34.9	685	768	70.9	0.34
6	67	384	359	384	38.6	340	384	69.7	0.36
12	62	384	351	384	49.3	345	384	58.7	0.46
24	72	384	352	384	52.0	343	384	67.5	0.44
36	76	384	357	384	45.0	360	384	39.8	0.53
42	62	384	352	384	47.7	355	384	43.1	0.52
7.5 CP	169	384	270	384	429.1	313	384	249.1	0.62
CP = Cyclophosphamide * TFT resistant mutants/1E+06 viable cells 2 days after treatment Yv = Number of wells without colonies, day 2 viability plates Nv = Number of wells scored, day 2 viability plates Ym = Number of wells without colonies, mutation plates Nm = Number of wells scored, mutation plates

 

 

Table 4: Summary of results from Experiment 1 – with metabolic activation

Treatment (µg/mL)	%S	%RS	%V	RTG	MF
0	73.25	100.00	80.67	1.00	110.11
6	71.88	98.13	87.30	1.11	113.45
12	69.11	94.35	91.18	1.09	113.87
24	73.49	100.32	83.70	0.93	125.94
36	68.85	93.98	81.00	0.97	87.97
42	38.05	51.94	91.18	0.77	94.87
Positive control (CP) 7.5	44.83	61.20	41.04	0.45	800.90
%RS = Percent Relative Survival %S = Percent Survival Day 0 %V = Percent Viability Day 2 RTG = Relative Total Growth MF = 5-TFT resistant mutants/1E+06 viable cells 2 days after treatment CP = Cyclophosphamide Test for linear trend: Slope -4.745E-007 Variance: 2.281E-013 b²/Sb: 0.987

 

Table 5: Mutant Frequencies from Experiment 2 – without metabolic activation

Treatment (µg/mL)	Viable		Small Colonies			Large Colonies			Proportion small colony mutants (µg/mL)
			Mutants		MF*	Mutants		MF*
	Yv	Nv	Ym	Nm		Ym	Nm
0	117	768	723	768	32.1	695	768	53.1	0.38
6	51	384	366	384	23.8	349	384	47.3	0.34
12	52	384	369	384	19.9	346	384	52.1	0.28
24	43	384	365	384	23.2	349	384	43.7	0.35
36	77	384	352	384	54.2	368	384	26.5	0.67
48	113	384	358	384	57.3	363	384	46.0	0.55
60 X	152	384	350	384	100.0	364	384	57.7	0.63
150 EMS	83	384	296	384	169.9	150	384	613.7	0.27
EMS = Ethylmethanesulphonate X = Excluded from statistical analysis due to toxicity * TFT resistant mutants/1E+06 viable cells 2 days after treatment Yv = Number of wells without colonies, day 2 viability plates Nv = Number of wells scored, day 2 viability plates Ym = Number of wells without colonies, mutation plates Nm = Number of wells scored, mutation plates

 

Table 6: Summary of results from Experiment 2 – without metabolic activation

Treatment (µg/mL)	%S	%RS	%V	RTG	MF
0	73.67	100.00	94.08	1.00	88.66
6	81.76	110.99	100.94	1.11	73.57
12	68.75	93.33	99.97	1.19	74.28
24	29.07	39.47	109.47	1.30	69.22
36	13.41	18.20	80.34	0.64	83.10
48	5.54	7.52	61.16	0.28	106.73
60 X	0.87	1.18	46.34	0.14	163.53
Positive control (EMS) 150	49.85	67.67	76.59	0.73	1190.43
%RS = Percent Relative Survival %S = Percent Survival Day 0 %V = Percent Viability Day 2 RTG = Relative Total Growth MF = 5-TFT resistant mutants/1E+06 viable cells 2 days after treatment EMS = Ethylmethanesulphonate X = Treatment excluded from statistical analysis due to toxicity Test for linear trend: Slope 1.492E-007 Variance: 2.132E-013 b²/Sb: 0.104

 

Table 7: Mutant Frequencies from Experiment 2 – with metabolic activation

Treatment (µg/mL)	Viable		Small Colonies			Large Colonies			Proportion small colony mutants (µg/mL)
			Mutants		MF*	Mutants		MF*
	Yv	Nv	Ym	Nm		Ym	Nm
0	151	768	689	768	66.7	682	768	73.0	0.48
6	62	384	358	384	38.4	333	384	78.1	0.34
12	61	384	355	384	42.7	337	384	71.0	0.38
24	59	384	350	384	49.5	341	384	63.4	0.44
36	79	384	350	384	58.6	339	384	78.8	0.43
48	126	384	360	384	57.9	344	384	98.7	0.38
60 X	353	384	380	384	124.4	380	384	124.4	0.5
7.5 (CP)	253	384	253	384	1000.0	324	384	407.2	0.69
CP = Cyclophosphamide X = Excluded from statistical analysis due to toxicity * TFT resistant mutants/1E+06 viable cells 2 days after treatment Yv = Number of wells without colonies, day 2 viability plates Nv = Number of wells scored, day 2 viability plates Ym = Number of wells without colonies, mutation plates Nm = Number of wells scored, mutation plates

 

Table 8: Summary of results from Experiment 2 – with metabolic activation

Treatment (µg/mL)	%S	%RS	%V	RTG	MF
0	93.66	100.00	81.33	1.00	148.71
6	105.22	112.35	91.18	1.06	122.73
12	89.34	95.39	91.99	1.09	119.88
24	78.44	83.75	93.66	1.04	119.48
36	60.85	64.98	79.06	0.85	145.67
48	8.67	9.25	55.72	0.12	163.61
60 X	1.44	1.54	4.21	0.00	250.12
Positive control (CP) 7.5	24.53	26.19	20.86	0.18	1648.77
%RS = Percent Relative Survival %S = Percent Survival Day 0 %V = Percent Viability Day 2 RTG = Relative Total Growth MF = 5-TFT resistant mutants/1E+06 viable cells 2 days after treatment CP = Cyclophosphamide Test for linear trend: Slope 2.529E-007 Variance: 3.683E-013 b²/Sb: 0.174

 

Conclusions:

Under the conditions of the test, no toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cell were observed and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

The mutagenicity of the test material was evaluated in a study conducted in accordance with OECD 476 and EU Method B.17. Two experiments were conducted up to precipitating cytotoxic concentrations in the presence and absence of metabolic activation for 3-hour and 24-hour exposure times. Under the conditions of the test, no toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cell were observed and is therefore considered to be non-mutagenic under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

In accordance with Annex I of Regulation EC 1272/2008, the substance does not require classification for germ cell mutagenicity.