3-(5-chlorobenzoxazol-2-yl)-7-(diethylamino)-2-benzopyrone

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

April 14th, 1997 to June 20th, 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guideline For Testing Of Chemicals, 476; "Genetic Toxicology: In vitro Mammalian Cell Gene Mutation Test". Adopted: April 4, 1984

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: In vitro Mammalian Cell Gene Mutation Test EEC Directive 87/302

Version / remarks:

In vitro Mammalian Cell Gene Mutation Test EEC Directive 87/302, L133, pp. 61-63, March 1987

Deviations:

no

GLP compliance:

yes

Type of assay:

other: In vitro Mammalian Cell Gene Mutation Test

Test material

Test material information

Constituent 1

Reference substance name:

3-(5-chlorobenzoxazol-2-yl)-7-(diethylamino)-2-benzopyrone

EC Number:

252-722-2

EC Name:

3-(5-chlorobenzoxazol-2-yl)-7-(diethylamino)-2-benzopyrone

Cas Number:

35773-43-4

Molecular formula:

C20H17ClN2O3

IUPAC Name:

3-(5-chloro-1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one

Test material form:

solid: particulate/powder

Details on test material:

Disperse Yellow 184:1

Specific details on test material used for the study:

No further details specified in the study report.

Method

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase) locus.

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Source of biological material: cell bank of "Genetic Toxicology", Hoechst AG
Test organism: cell line V79 of Chinese hamster lung fibroplasts
Cell culture medium: MEM (minimal essential medium) with Hanks-salts and 25 mM Hepes-buffer

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

The compound was dissolved in DMSO and tested at the following concentrations:
1. Experiment
without S9-mix: 0.05, 0.1, 0.5, 1, 2.5 and 4 μg/ml
with S9-mix: 0.1, 0.5, 1, 2.5, 5 and 7.5 μg/ml

2. Experiment
without S9-mix: 0.5, 1, 2.5, 5, 7.5, 10* and 15* μg/ml
with S9-mix: 1, 2. 5, 5, 7. 5, 10, 12.5 and 15 μg/ml
* = because of high cytotoxicity no mutant selection was performed

The concentration ranges were based on the results of preliminary tests for solubility and toxicity.

Following treatment in the absence of S9 metabolic activation, high toxicity was observed at 10 ug/ml and above. Survival declined in a dose-related manner reaching 3.3 % of the solvent control value at the dose level 25 ug/ml in the first preliminary experiment and 2.4% at a concentration of 5 ug/ml in the second. On the basis of these results, a concentration of 4 ug/ml was used as maximum concentration for the first main assay and five lower dose levels were included in the treatment series. In the presence of S9 metabolic activation survival declined also in a dose-dependent manner. At a concentration of 25 ug/ml survival was reduced to 2.2 % of the solvent control value in the first preliminary experiment, while in the second preliminary experiment at the highest dose level of 10 ug/ml survival was reduced to 1.3 %. Based on these results 7.5 ug/ml was selected as the maximum dose level for the first main mutation experiment in the presence of S9-mix. Five lower concentrations down to 0.1 ug/ml were also included.

Vehicle / solvent:

Formulation of test compounds: dissolved in DMSO at appropriate concentrations immediately before use.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Test groups 1. main experiment:
a: without metabolic activation:
0.05, 0.1, 0.5, 1, 2.5 and 4 μg/ml
b: with metabolic activation:
0.1, 0.5, 1, 2.5, 5 and 7.5 μg/ml

Test groups 2. main experiment:
a: without metabolic activation:
0.5, 1, 2.5, 5, 7 .5, 10* and 15* μg/ml
b: with metabolic activation:
1, 2.5, 5, 7.5, 10, 12.5 and 15 μg/ml
*= because of high toxicity in the main experiment no mutant selection was performed

Control groups
negative controls:
a: untreated control
b: cultures treated with the solvent

positive controls:
a: without metabolic activation:
EMS (Ethyl methane sulfonate)
b: with metabolic activation:
DMBA (9, 10-dimethyl-1 ,2-benzanthracene)

Formulation of test compounds: dissolved in DMSO at appropriate concentrations immediately before use.

Formulation of reference compounds:
EMS dissolved in cell culture medium on the day of treatment, final concentration: 1.0 mg/ml = 8 mM.
DMBA dissolved in DMSO and frozen in small portions. Aliquot thawed on the day of treatment, final concentration in cell culture medium: 7.7 μg/ml = 30 μM

Experimental conditions in vitro: approx. 37 °C and approx. 4 % CO2 in plastic flasks

Toxicity experiments and dose range finding
A preliminary toxicity test was undertaken in order to select appropriate dose levels for the mutation assay. In this test a wide range of dose levels of test compound was used. Cell cultures were subjected to the same treatment conditions as in mutation assays, and the survival of the cells was subsequently determined.
The test included the following treatments:
Solvent control: the maximum final concentration of organic solvents was approx. 1 % (v/v).
Test compound: the highest dose level for the preliminary toxicity test was determined by the solubility of the test compound up to the maximum of 10 mM or 5000 μg/ml.
Treatments were performed both in the presence and absence of S9 metabolic activation system using a single cell culture at each test point

Test procedure
In preliminary toxicity experiments approximately 4500 cells were seeded in each well of a microliter plate, allowed to attach overnight and then exposed to the test and control compound for four hours.
For each concentration at least 6 wells were used. Approx. 24 hours after treatment, the cells were fixed and stained with crystal violet.
Survival was determined by measurement of the crystal violet extinction.
In the main mutation experiments the cultures for assessing toxicity were prepared and treated with the test compound in the same way as for the preliminary experiment. 24 hours after seeding of approx. 4500 cells per well in a microliter plate, the medium was replaced with serum-reduced (5 % v/v) medium containing the test compound to which either buffer or S9-mix was added as appropriate. After 4 hours the treatment medium was replaced with normal medium after rinsing twice with this. The cultures were stained with crystal violet and survival was determined after an incubation period of approx. 24 hours.

Mutagenicity test
Two-day old, exponentially growing cultures which were more than 50% confluent were trypsinated and a single cell suspension was prepared. The trypsin concentration was approx. 0.25 % (v/v) in Ca-Mg-free salt solution. The Ca-Mg-free salt solution was prepared as follows (per liter): NaCI6.8 g; Ka 0.4 g; glucose 1 g; NaHC03 2.2 g; phenol red 5 mg; trypsin 2.5 g.
Subsequently the cells were replated for mutagenicity testing and for determination of plating efficiency.
The treatment schedule of the mutagenicity test is described below:
Day 1: Subculturing of an exponentially growing culture
a) Approx. 4500 cells in each well of a microtiterplate for determination of the plating efficiency.
b) 6E+5 – 1E+6 cells in 175 cm2 flasks with 30 mil medium for the mutagenicity test, one flask per experimental point.

Day 2: Treatment of a) and b) with the test compound in the presence and absence of S9-mix (final protein concentration: approx. 0.3 mg/ml) for 4 hours.

Day 3: Fixation and staining of the cells in a) for the determination of the plating efficiency.

Day 5: Subculturing of b) in 175 cm2 flasks

Day 9: Subculturing of b) in five 75 cm2 flasks with culture medium containing 6-thioguanine:
Mutant selection (about 300 000 cells/flask); subculturing of b) in two 25 cm2 flasks for plating efficiency (about 400 cells per flask)

Day 16: Fixation and staining of colonies of b) - from subcultures seeded on day 9.

All incubations were carried out at approx. 37 °C and 4 % CO2
Staining was performed with approx. 10 % (v/v) methylene blue in approx. 0.01 % (w/v) KOH solution.
Only colonies with more than 50 cells were counted

Rationale for test conditions:

In accordance with test guidelines.

Evaluation criteria:

Criteria for a valid assay
The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory's normal range
- the plating efficacy for the solvent control was greater than 50 %

Criteria for a positive response
The test compound is classified as mutagenic if:
- it reproducibly induces with one of the test compound concentrations a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment
- there is a reproducible dose-related increase in the mutation frequency. Such an evaluation may be considered independently from the number induced mutants
- survival of the responding dose group is at least 30 %
However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data.

Statistics:

The biometry of the results for the test compound is performed off-line with the MANN-WHITNEY-U-TEST

Results and discussion

Test results

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:

valid

Positive controls validity:

valid

Additional information on results:

Solubility and toxicity
Evaluation of the solubility of Macrolex Fluoreszensgelb 10 GN trocken in cell culture medium showed that a concentration of 3685 μg/ml (this concentration corresponds to 10 mM, which is the highest dose level tolerated for the test system) produced a precipitation in the cell culture medium.

In spite of the precipitation a preliminary toxicity study was carried out using dose levels of 3685 μg/ml down to 100 μg/ml. In this preliminary experiment an evaluation could not be performed because of precipitation and toxicity of the test compound at all concentrations tested. Therefore a second toxicity experiment with dose levels from 1 to 100 μg/ml and a third toxicity experiment with a concentration range of 0.1 to 10 μg/ml were performed. Precipitation of the test compound was observed at concentrations of 50 μg/ml and above with and without metabolic activation.

Following treatment in the absence of S9 metabolic activation, high toxicity was observed at 10 μg/ml and above. Survival declined in a dose-related manner reaching 3.3% of the solvent control value at the dose level 25 μg/ml in the first preliminary experiment and 2.4% at a concentration of 5 μg/ml in the second. On the basis of these results, a concentration of 4 μg/ml was used as maximum concentration for the first main assay and five lower dose levels were included in the treatment series.

In the presence of S9 metabolic activation survival declined also in a dose-dependent manner. At a concentration of 25 μg/ml survival was reduced to 2.2 % of the solvent control value in the first preliminary experiment, while in the second preliminary experiment at the highest dose level of 10 μg/ml survival was reduced to 1.3 %.
Based on these results 7.5 μg/ml was selected as the maximum dose level for the first main mutation experiment in the presence of S9-mix. Five lower concentrations down to 0.1 μg/ml were also included.

Mutagenicity
Experimental design
Two independent mutation assays to examine the resistance to 6-thioguanine were performed.
In the absence of S9 metabolic activation using dose levels of 0.05, 0.1, 0.5, 1, 2.5 and 4 μg/ml in the first main experiment. The second experiment was performed with the dose levels of 0.5, 1, 2.5, 5, 7.5, 10 and 15 μg/ml. Because of high cytotoxicity at the concentrations of 10 and 15 μg/ml no mutant selection was performed.
In the presence of S9 metabolic activation concentrations of 0.1, 0.5. 1, 2.5. 5 and 7.5 μg/ml were chosen in the first main experiment and concentrations of 1, 2.5, 5, 7.5, 10, 12.5 and 15 μg/ml were selected for the second mutation assay.
Before treatment, the pH values and osmolality of the treatment media were determined. The addition of test compound solutions did not have any effect on these parameters.

Survival after treatment
In the absence of 59 metabolic activation in both mutation experiments a dose-related decrease in survival was observed starting at a concentration of 0.5 μg/ml and reaching 57.5% of the solvent control value in the microtiter plates at the dose level of 4 μg/ml in the first main experiment. For testing the mutagenic potential of the test substance at concentrations with higher cytotoxic effects, the dose levels in the second main experiment were increased. In this experiment the survival rate was decreased to 29.4% at a concentration of 15 μg/ml.
In the presence of 59 metabolic activation survival decreased in a dose-related manner starting at a concentration of 5 μg/ml reaching 22.8 % of the solvent control value in the microtiter plates after treatment at the highest dose level, 7.5 μg/ml. In the second main experiment also higher concentrations of the test substance were used.
At a dose level of 15 μg/ml only 3.8 % of the cells survived in relation to the solvent control group.

Mutation results
The test compound Macrolex Fluoreszensgelb 10 GN trocken was assessed for its mutagenic potential in vitro in the HPRT-test in two independent experiments without metabolic activation and two independent experiments with metabolic activation.
Only in the first experiment a statistically significant enhancement of the mutation rate over the range of the solvent controls was induced, without metabolic activation only at the concentration of 0.1 μg/ml of the test compound and with S9-mix at the dose levels of 5 and 7.5 μg/ml. In all cases the mutation rates were not three fold higher than the solvent control level and in the range of the historical controls. No increases in the mutant colonies or mutant frequency over the range of the solvent control were found with any of the concentrations used, either with or without metabolic activation by S9-mix in the repeat main experiment.
Obviously all observed effects were not three fold increased, not reproducible, in the absence of S9-mix also not dose dependent and therefore considered as not relevant.
The sensitivity of the test system and efficacy of the S9-mix was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.

Any other information on results incl. tables

Toxicity Data (Preliminary experiment)

	Dose μg/ml	S9-mix	Extinction in microwell plates mean less blank values	Standard deviation	Relative survival*
Solvent control Macrolex Fluoreszensgelb 10 GN	0 1 2.5 5 10 25 50 75 100	- - - - - - - - -	0.476 0.318 0.313 0.066 0.010 0.016 0.034 0.029 0.043	0.10 0.04 0.06 0.03 0.02 0.02 0.04 0.01 0.04	100.0 66.9 65.9 13.9 2.0 3.3 7.2# 6.2# 9.1#

 

	Dose μg/ml	S9-mix	Extinction in microwell plates mean less blank values	Standard deviation	Relative survival*
Solvent control Macrolex Fluoreszensgelb 10 GN	0 1 2.5 5 10 25 50 75 100	+ + + + + + + + +	0.488 0.503 0.401 0.172 0.024 0.011 0.054 0.099 0.064	0.08 0.15 0.13 0.12 0.03 0.02 0.04 0.06 0.02	100.0 103.1 82.3 35.3 5.0 2.2 11.0# 20.2# 13.2#

# precipitate of the test substance was dyed

* = relative survival (mean value / mean value corresponding control x 100)

Solvent = DMSO

 

Toxicity Data (Preliminary experiment)

	Dose μg/ml	S9-mix	Extinction in microwell plates mean less blank values	Standard deviation	Relative survival*
Solvent control Macrolex Fluoreszensgelb 10 GN	0 0.1 0.25 0.5 0.75 1 2.5 5 10	- - - - - - - - -	0.331 0.320 0.261 0.236 0.229 0.230 0.234 0.008 -0.001	0.03 0.01 0.04 0.02 0.01 0.01 0.01 0.00 0.00	100.0 96.7 78.9 71.2 69.1 69.6 70.6 2.4 -0.4

 

	Dose μg/ml	S9-mix	Extinction in microwell plates mean less blank values	Standard deviation	Relative survival*
Solvent control Macrolex Fluoreszensgelb 10 GN	0 0.1 0.25 0.5 0.75 1 2.5 5 10	+ + + + + + + + +	0.312 0.309 0.408 0.335 0.331 0.335 0.278 0.157 0.004	0.02 0.03 0.13 0.02 0.02 0.01 0.02 0.02 0.00	100.0 99.1 130.7 107.3 106.1 107.4 89.2 50.1 1.3

* = relative survival (mean value / mean value corresponding control x 100)

Solvent = DMSO

 

Toxicity Data (Main mutation experiment)

	Dose μg/ml	S9-mix	Extinction in microwell plates mean less blank values	Standard deviation	Relative survival*
Negative control Solvent control Positive control Macrolex Fluoreszensgelb 10 GN	0.0 0.0 1000.0 0.05 0.1 0.5 1.0 2.5 4.0	- - - - - - - - -	0.484 0.499 0.404 0.442 0.467 0.337 0.336 0.309 0.287	0.05 0.09 0.04 0.03 0.05 0.08 0.01 0.04 0.04	96.9 100.0 80.8 88.4 93.5 67.4 67.3 62.0 57.5

 

	Dose μg/ml	S9-mix	Extinction in microwell plates mean less blank values	Standard deviation	Relative survival*
Negative control Solvent control Positive control Macrolex Fluoreszensgelb 10 GN	0.0 0.0 7.5 0.1 0.5 1.0 2.5 5.0 7.5	+ + + + + + + + +	0.620 0.568 0.368 0.583 0.554 0.585 0.456 0.380 0.129	0.05 0.10 0.05 0.08 0.03 0.09 0.06 0.08 0.02	109.2 100.0 64.8 102.6 97.6 103.1 80.2 66.9 22.8

* = relative survival (mean value / mean value corresponding control x 100)

Solvent = DMSO

Positive control without S9-mix = EMS

Positive control with S9-mix = DMBA

 

Toxicity Data (Repeat mutation experiment)

	Dose μg/ml	S9-mix	Extinction in microwell plates mean less blank values	Standard deviation	Relative survival*
Negative control Solvent control Positive control Macrolex Fluoreszensgelb 10 GN	0.0 0.0 1000.0 1.0 2.5 5.0 7.5 10.0 15.0	- - - - - - - - -	0.482 0.466 0.416 0.324 0.398 0.256 0.212 0.174 0.138	0.01 0.06 0.02 0.02 0.23 0.03 0.02 0.01 0.01	103.1 100.0 88.9 69.2 85.1 54.8 45.3 37.1 29.4

 

	Dose μg/ml	S9-mix	Extinction in microwell plates mean less blank values	Standard deviation	Relative survival*
Negative control Solvent control Positive control Macrolex Fluoreszensgelb 10 GN	0.0 0.0 7.7 2.5 5.0 7.5 10.0 12.5 15.0	+ + + + + + + + +	0.412 0.425 0.282 0.370 0.276 0.072 0.048 0.062 0.016	0.03 0.17 0.01 0.11 0.02 0.01 0.01 0.07 0.00	97.0 100.0 66.2 86.9 65.0 16.9 11.2 14.5 3.8

* = relative survival (mean value / mean value corresponding control x 100)

Solvent = DMSO

Positive control without S9-mix = EMS

Positive control with S9-mix = DMBA

 

Mutagenicity Data – Part 1 (Main mutation experiment)

	Dose μg/ml	S9-mix	Number of cells per flask				Factor* calculated	Cells** seeded	Cells*** survived
			Seeded	Found		Mean
			I / II	I	II
Negative control	0.0	-	401	383.0	369.5	376.3	0.94	289800	271913
Solvent control (DMSO)	0.0	-	402	390.5	363.5	377.0	0.94	355500	333392
Positive control (EMS)	1000.0	-	401	344.0	322.5	333.3	0.83	287700	239092
Macrolex Fluoreszensgelb 10 GN	0.05	-	405	327.0	346.0	336.5	0.83	249600	207384
	0.1	-	403	331.0	293.5	312.3	0.77	325800	252434
	0.5	-	402	431.0	453.5	422.3	1.10	265500	292083
	1.0	-	399	367.0	347.5	357.3	0.90	255300	228586
	2.5	-	399	284.0	304.0	294.0	0.74	297600	219284
	4.0	-	400	345.0	339.0	342.0	0.86	311100	265991
Negative control	0.0	+	395	331.0	311.0	321.0	0.81	296700	241116
Solvent control (DMSO)	0.0	+	398	323.5	339.5	331.5	0.83	324300	270114
Positive control (DMBA)	7.7	+	400	255.0	286.0	270.5	0.68	267600	180965
Macrolex Fluoreszensgelb 10 GN	0.1	+	400	286.0	297.0	291.5	0.73	324600	236552
	0.5	+	402	381.5	416.5	399.0	0.99	254700	252799
	1.0	+	401	389.0	443.5	416.3	1.04	248700	258158
	2.5	+	398	280.5	285.0	282.8	0.71	255600	181585
	5.0	+	399	281.0	287.0	284.0	0.71	300300	213747
	7.5	+	398	350.5	350.5	350.5	0.88	309600	272650

 

Mutagenicity Data – Part 1 (Repeat mutation experiment)

	Dose μg/ml	S9-mix	Number of cells per flask				Factor* calculated	Cells** seeded	Cells*** survived
			Seeded	Found		Mean
			I / II	I	II
Negative control	0.0	-	402	334.0	344.5	339.3	0.84	276900	233677
Solvent control (Lömi eintr.)	0.0	-	398	301.5	316.5	309.0	0.78	304200	236175
Positive control (EMS)	1000.0	-	390	304.5	313.3	308.8	0.79	296700	234888
Macrolex Fluoreszensgelb 10 GN	0.5	-	401	366.5	370.0	368.3	0.92	350700	322058
	1.0	-	402	341.5	337.0	339.3	0.84	328800	277476
	2.5	-	401	319.5	3223.5	321.5	0.80	283200	227054
	5.0	-	397	353.0	3580.5	351.8	0.89	396700	262882
	7.5	-	392	325.5	324.0	324.8	0.83	300900	249279
Negative control	0.0	+	413	296.0	304.5	300.3	0.73	291900	212211
Solvent control (Lömi eintr.)	0.0	+	399	280.5	282.5	281.5	0.71	257700	181811
Positive control (DMBA)	7.7	+	399	317.5	300.0	308.8	0.77	295500	228661
Macrolex Fluoreszensgelb 10 GN	1.0	+	399	318.0	339.0	328.5	0.82	331800	273174
	2.5	+	397	312.0	297.0	304.5	0.77	277200	212613
	5.0	+	401	321.0	300.0	310.5	0.77	323700	250646
	7.5	+	385	327.5	329.0	328.3	0.85	256200	218435
	10.0	+	399	362.0	370.5	366.3	0.92	330000	302914
	12.5	+	398	317.0	316.0	316.5	0.80	312900	248826
	15.0	+	401	339.5	311.5	325.5	0.81	285900	232071

*             factor calculated mean value / number of cells per flask seeded

**            cells seeded in 6-thioguanine (TG) containing medium

***          cells survived after plating in (TG) containing medium (cells seeded x factor calculated)

 

Mutagenicity Data – Part 2 (Main mutation experiment)

	Dose μg/ml	S9-mix	Number of mutant colonies						Standard deviation	Mutation frequency	Stat. sig.
			I	II	III	IV	V	Mean
Negative control	0.0	-	1	2	3	2	0	1.6	1.14	5.9
Solvent control (DMSO)	0.0	-	4	2	4	5	3	3.6	1.14	10.8
Positive control (EMS)	1000.0	-	118	163	123	139	129	134.4	17.80	562.1	*
Macrolex Fluoreszensgelb 10 GM	0.05	-	0	2	1	1	0	0.8	0.84	3.9
	0.1	-	3	4	4	5	5	4.2	0.84	16.6	*
	0.5	-	4	4	3	6	4	4.2	1.10	14.4
	1.0	-	0	1	1	2	1	1.0	0.71	4.4
	2.5	-	1	5	4	4	3	3.4	1.52	15.5
	4.0	-	2	3	2	3	4	2.8	0.84	10.5
Negative control	0.0	+	1	0	1	4	2	1.6	1.52	6.6
Solvent control (DMSO)	0.0	+	1	2	1	1	4	1.8	1.30	6.7
Positive control (DMBA)	7.7	+	23	30	21	20	30	24.8	4.87	137.0	*
Macrolex Fluoreszensgelb 10 GN	0.1	+	5	6	6	0	3	4.0	2.55	16.9
	0.5	+	0	1	1	1	0	0.6	0.55	2.4
	1.0	+	4	3	2	1	2	2.4	1.14	9.3
	2.5	+	5	2	2	2	0	2.2	1.79	12.1
	5.0	+	4	6	2	6	2	4.0	2.00	18.7	*
	7.5	+	9	4	3	3	8	5.4	2.88	19.8	*

 

Mutagenicity Data – Part 2 (Repeat mutation experiment)

	Dose μg/ml	S9-mix	Number of mutant colonies						Standard deviation	Mutation frequency	Stat. sig.
			I	II	III	IV	V	Mean
Negative control	0.0	-	4	7	6	5	4	5.2	1.30	22.3
Solvent control (Lömi eintr.)	0.0	-	4	2	8	2	1	3.4	2.79	14.4
Positive control (EMS)	1000.0	-	100	112	107	128	110	111.4	10.33	474.3	*
Macrolex Fluoreszensgelb 10 GM	0.5	-	5	4	7	1	4	4.2	2.17	13.0
	1.0	-	4	10	4	10	12	8.0	3.74	28.8
	2.5	-	2	2	5	3	3	3.0	1.22	13.2
	5.0	-	3	6	11	5	7	6.4	2.97	24.3
	7.5	-	0	0	0	0	0	0.0	0.00	0.0
Negative control	0.0	+	2	2	4	0	3	2.2	1.48	10.4
Solvent control (Lömi eintr.)	0.0	+	5	4	9	3	7	5.6	2.41	30.8
Positive control (DMBA)	7.7	+	38	34	35	39	32	35.6	2.88	155.7	*
Macrolex Fluoreszensgelb 10 GN	1.0	+	7	2	5	7	8	5.8	2.39	21.2
	2.5	+	8	7	2	5	8	6.0	2.55	28.2
	5.0	+	2	2	1	2	4	2.2	1.10	8.8
	7.5	+	5	2	1	3	2	2.6	1.52	11.9
	10.0	+	2	2	1	7	7	3.8	2.95	12.54
	12.5	+	6	1	3	7	2	3.8	2.59	15.3
	15.0	+	3	4	2	3	5	3.4	1.14	14.7

Mutation frequency (mutant colonies per 1 million cells): mean value / cells surviving

*Statistical significant (p ≤0.05) Mann-Whitney-U-Test

Applicant's summary and conclusion

Conclusions:

Macrolex Fluoreszensgelb 10 GN trocken was not mutagenic in this HPRT-test with cells of the V79 Chinese hamster cell line.
The substance is not classified in accordance with CLP criteria.

Executive summary:

The study was performed to investigate the potential of Macrolex Fluoreszensgelb 10 GN trocken to induce gene mutations at the HPRT locus in V 79 cells of the Chinese hamster in vitro.

 

Two independent experiments were conducted both with and without an exogenous rat liver microsomal activation system (S9-mix)

.

The compound was dissolved in DMSO and tested at the following concentrations:

1. Experiment

without S9-mix: 0.05, 0.1, 0.5, 1, 2.5 and 4 μg/ml

with S9-mix: 0.1, 0.5, 1, 2.5, 5 and 7.5 μg/ml

 

2. Experiment

without S9-mix: 0.5, 1, 2.5, 5, 7.5, 10* and 15* μg/ml

with S9-mix: 1, 2. 5, 5, 7. 5, 10, 12.5 and 15 μg/ml

* = because of high cytotoxicity no mutant selection was performed

 

The concentration ranges were based on the results of preliminary tests for solubility and toxicity. The test substance produced dose-dependent cytotoxic effects with and without metabolic activation.

 

Up to the highest investigated dose no reproducible increase in mutant colony numbers was obtained in two independent experiments.

 

Appropriate reference mutagens used as positive controls showed a distinct increase in induced mutant colonies, thus indicating the sensitivity of the assay, and the efficacy of the S9-mix.

 

In conclusion, Macrolex Fluoreszensgelb 10 GN trocken does not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system, under the experimental conditions described.

 

Macrolex Fluoreszensgelb 10 GN trocken is therefore considered to be non-mutagenic in this HPRT assay.