Butyl hydrogen maleate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 October 2012 and 04 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

other: Acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances.

Deviations:

not applicable

Principles of method if other than guideline:

Please note that there was an exception to GLP Guidance, this was noted in the GLP Statement: No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration. This exception is considered not to affect the purpose or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Butyl hydrogen maleate

EC Number:

213-116-3

EC Name:

Butyl hydrogen maleate

Cas Number:

925-21-3

Molecular formula:

C8H12O4

IUPAC Name:

(2Z)-4-butoxy-4-oxobut-2-enoic acid

Test material form:

other: liquid

Details on test material:

Sponsor's identification : Butyl Hydrogen Maleate
Description : Pale yellow liquid
Purity : ≥95%
Batch number : 120224
Date received : 20 March 2012
Expiry date : 30 March 2013
Storage conditions : Room temperature in the dark

Method

Target gene:

Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (phenobarbital/ß-naphthoflavone)

Test concentrations with justification for top dose:

Dose (µg/ml).............pH.............mOsm
0...................................7.35..........430
6.73.............................7.36..........431
13.45...........................7.36..........433
26.91...........................7.34..........431
53.81...........................7.34..........429
107.63.........................7.36..........418
215.25.........................7.32..........419
430.5...........................7.24..........423
861..............................7.14..........413
1722............................6.91..........418

Vehicle / solvent:

DMSO

Controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/ml, using a 4-hour exposure time both with and without metabolic activation (20 %S9-mix, 2% final concentration), and at 1.5 x 10^5 cells/ml using a 24-hour exposure without S9. The dose range used in the preliminary toxicity test was 6.73 to 1722 μg/ml for all three of the exposure groups. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 10^5 cells/ml.
The cultures were incubated at 37 °C with 5% CO 2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 10^5 cells/ml. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a % Relative Suspension Growth (%RSG) value.
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) Maximum recommended dose level, 5000 μg/ml or 10 mM.
ii) The presence of excessive precipitate where no test item-induced toxicity was observed.
iii) Test item-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al 2002).

Experiment 1
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10^6 cells/ml in 10 ml aliquots in R10 medium in sterile plastic universals. The treatments were performed in duplicate (A + B), both with and without metabolic activation (20 %S9-mix, 2% final concentration) at eight dose levels of the test item (6.72 to 215 μg/ml in the absence of metabolic activation, and 26.88 to 860 μg/ml in the presence of metabolic activation), vehicle and positive controls. To each universal was added 2 ml of S9-mix if required, 0.2 ml of the treatment dilutions, (0.2 ml for the positive control) and sufficient R0 medium to bring the total volume to 20 ml.
The treatment vessels were incubated at 37°C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.

Experiment 2
As in Experiment 1, an exponentially growing stock culture of cells was established. The cells were counted and processed to give 1 x 10^6 cells/ml in 10 ml cultures in R10 medium for the 4-hour treatment with metabolic activation cultures. In the absence of metabolic activation the exposure period was extended to 24 hours therefore 0.3 x 10^6 cells/ml in 10 ml cultures were established in 25 cm2 tissue culture flasks. The treatments were performed in duplicate (A + B), both with and without metabolic activation (20% S9-mix, 2% final concentration) at eight dose levels of the test item (1.69 to 108 μg/ml in the absence of metabolic activation, and 13.5 to 432 μg/ml in the
presence of metabolic activation), vehicle and positive controls. To each universal was added 2 ml of S9-mix if required, 0.2 ml of the treatment dilutions, (0.2 ml for the positive control) and sufficient R0 medium to give a final volume of 20 ml (R10 is used for the 24-hour exposure group).
The treatment vessels were incubated at 37°C with c ontinuous shaking using an orbital shaker within an incubated hood for 24 hours in the absence of metabolic activation and4 hours in the presence of metabolic activation.

Measurement of Survival, Viability and Mutant Frequency
At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/ml. The cultures were incubated at 37°C with 5% CO 2 in air and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 105 cells/ml, unless the mean cell count was less than 3 x 10^5 cells/ml in which case all the cells were maintained.
On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/ml and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/ml 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/ml and plated (2 cells/well) for viability (%V) in non-selective medium. The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Evaluation criteria:

Plate Scoring
Microtitre plates were scored using a magnifying mirror box after ten to fourteen days’ incubation at 37°C with 5% CO 2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded. Colonies are scored manually by eye using qualitative judgement. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 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 approximately two hours.
MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black colour, thus aiding the visualisation of the mutant colonies, particularly the small colonies.

Statistics:

Calculation of Percentage Relative Suspension Growth (%RSG)
The cell counts obtained immediately post treatment and over the 2-day expression period were used to calculate the Percentage Relative Suspension Growth.
4-Hour Suspension Growth (SG) = (24-hour cell count/2) x (48-hour cell count/2)
24-Hour Suspension Growth (SG) = (0-hour cell count/1.5) x (24-hour cell count/2) x (48-hour cell count/2)
Day 0 Factor = dose 0-hour cell count / vehicle control 0-hour cell count
%RSG = [(dose SG x dose Day 0 Factor) / vehicle control SG] x 100

Calculation of Day 2 Viability (%V)
Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the day 2 viability (%V) was calculated using the zero term of the Poisson distribution [P(0)] method.
P(0) = number of negative wells / total wells plated
%V = (- ln P(0) x 100) / number of cells per well

Calculation of Relative Total Growth (RTG)
For each culture, the relative cloning efficiency, RCE, was calculated:
RCE =(%V/Mean Solvent Control %V)x100%
Finally, for each culture RTG is calculated:
RTG = (RCE x RSG)/100%

Calculation of Mutation Frequency (MF)
MF per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.
The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al, 1989).

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Preliminary Toxicity Test

The dose range of the test item used in the preliminary toxicity test was 6.73 to 1722 μg/ml. The results for the Relative Suspension Growth (%RSG) were as follows:

Dose Level (µg/ml)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	10	100
6.73	101	102	100
13.45	96	98	86
26.91	86	108	69
53.81	76	91	38
107.63	35	77	3
215.25	4	62	0
430.5	0	27	0
861	1	1	0
1722	2	3	0

In all three of the exposure groups there was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. The very steep nature of the toxicity curve was taken to indicate that achieving optimum toxicity may be difficult. Precipitate of the test item was not observed at any of the dose levels. Based on the %RSG values observed, the maximum dose levels in the subsequent Mutagenicity Test were limited by test item-induced toxicity for all of the exposure groups.

Mutagenicity Test

Experiment 1

Treatment (µg/ml)		4-Hour (-S9)			Treatment (µg/ml)		4-Hour (+S9)
		%RSG	RTG	MF§			%RSG	RTG	MF§
0		100	1	132.72	0		100	1	120.5
6.72	Ø	101			26.88	Ø	94
13.44		106	1	118.23	53.75		96	0.89	120.12
26.88		88	0.94	125.99	107.5		88	0.8	119.48
53.75		49	0.5	140.35	215		52	0.47	153.11
80.63		31	0.29	196.12	322.5		35	0.29	151.63
107.5		26	0.15	228.59	430		23	0.09	207.51
161.25	X	3	0.02	355.67	645	X	4	0.02	427.32
215	Ø	1			860	Ø	1
Linear Trend				...	Linear trend				...
EMS					CP
400		80	0.55	1275.75	2		67	0.29	1387.86

Ø = Not plated due to toxicity or surplus to requirements

X = Treatment excluded from test statistics due to toxicity

%RSG= Relative Suspension Growth

RTG = Relative Total Growth

MF§ = 5-TFT resistant mutants/10⁶viable cells 2 days after treatment

There was once again evidence of marked dose-related toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values. There was also evidence of dose related reductions in viability (%V); therefore indicating that residual toxicity had occurred in both the absence and presence of metabolic activation. Based on the RTG and / or %RSG values it was considered that optimum levels of toxicity had been achieved in both the absence and presence of metabolic. The excessive toxicity observed at 215 μg/ml in the absence of metabolic activation, and at 860 μg/ml in the presence of metabolic activation, resulted in these doses not being plated for viability or 5-TFT resistance. The toxicity observed at 161.25 μg/ml in the absence of metabolic activation, and at 645 μg/ml in the presence of metabolic activation, exceeded the upper acceptable limit of 90%, therefore, these doses were excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances.

Neither of the vehicle control mutant frequency values were outside the acceptable rangeof 50 to 200 x 10^-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional. The test item induced statistically significant dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell in both the absence and presence of metabolic activation. Statistically significant increases in mutant frequency were also observed at the upper two surviving dose levels in the absence of metabolic activation, and at the upper surviving dose level in the presence of metabolic activation. However, whilst the mutant frequency values observed marginally exceeded the upper limit for vehicle controls, the GEF was not exceeded at these or any of the other dose levels with acceptable levels of toxicity. There was also no evidence of any marked increases in absolute numbers of mutant colonies or a shift towards small colony formation that would have indicated clastogenic activity. Increases in mutant frequency that exceeded the GEF and that were significantly higher than the acceptable range for vehicle controls were observed at 161.25 μg/ml in the absence of metabolic activation, and at 645 μg/ml in the presence of metabolic activation. However, these dose levels had been excluded for the statistical analysis due to excessive toxicity and the increases in mutant frequency observed were considered to be due to cytotoxicity and not a true genotoxic response. Therefore, the responses observed were considered to be of no toxicological significance. Precipitate of test item was not observed at any of the dose levels.

Experiment 2

Treatment (µg/ml)		24-Hour (-S9)			Treatment (µg/ml)		4-Hour (+S9)
		%RSG	RTG	MF§			%RSG	RTG	MF§
0		100	1	155.39	0		100	1	139.73
1.69		84	0.86	132.28	13.5	Ø	97
3.38		90	0.95	123.87	27	Ø	83
6.75		67	0.8	114.5	54		85	0.92	128.55
13.5		57	0.77	100	108		77	0.76	118.37
27		32	0.56	163.36	216		50	0.52	124.91
54		6	0.11	247.69	288		39	0.37	132.9
81.108	Ø	1			360		33	0.21	184.53
108	Ø	0			432		14	0.1	216.43
Linear Trend				...	Linear Trend				...
EMS					CP
150		52	0.42	1360.26	2		65	0.38	1360.38

Ø = Not plated due to toxicity or surplus to requirements

X = Treatment excluded from test statistics due to toxicity

%RSG= Relative Suspension Growth

RTG = Relative Total Growth

MF§ = 5-TFT resistant mutants/10⁶ viable cells 2 days after treatment

As was seen previously, there was evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values. There was also once again evidence of dose related reductions in viability (%V) in both the absence and presence of metabolic activation, therefore indicating that residual toxicity had occurred. Based on the RTG values observed, it was considered that optimum levels of toxicity had been achieved in both the absence and presence of metabolic activation. The excessive toxicity observed at and above 81 μg/ml in the absence of metabolic activation

resulted in these dose levels not being plated for viability or 5-TFT resistance. Both positive controls induced acceptable levels of toxicity.

The 24-hour exposure without metabolic activation demonstrated that the extended time point had a significant effect on the toxicity of the test item.

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10^-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

As was seen in Experiment 1, the test item induced statistically significant dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell in both the absence and presence of metabolic activation. Statistically significant increases in mutant frequency were also observed at the upper surviving dose level in both the absence and presence of metabolic activation. However, whilst the mutant frequency values observed marginally exceeded the upper limit for vehicle controls, the GEF was not exceeded at these or any of the other dose levels. There was also once again no evidence of any marked increases in absolute numbers of mutant colonies or a shift towards small colony formation that would have indicated clastogenic activity. The responses observed were once again considered to be due to cytotoxicity and not a true genotoxic response and, therefore, of no toxicological significance. Precipitate of test item was not observed at any of the dose levels.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

Introduction. The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances.

Methods. Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (2% S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test item was selected following the results of a preliminary toxicity test, and in Experiment 1 was 6.72 to 215 μg/ml in the absence of metabolic activation, and 26.88 to 860 μg/ml in the presence of metabolic activation. In Experiment 2 the dose range was 1.69 to 108 μg/ml in the absence of metabolic activation, and 13.5 to 432 μg/ml in the presence of metabolic activation.

Results. The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. Precipitate of test item was not observed at any of the dose levels in the study. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose levels exhibiting acceptable levels of toxicity, either with or without metabolic activation in either the first or the second experiment.

Conclusion. The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.