REAKTIV-ORANGE DYPR 934

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999-02-17-1999-03-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to GLP and guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

-Name of test material (as cited in study report): Reaktiv-Orange DYPR934

Method

Target gene:

Test organism:
Salmonella typhinium strains:
TA98 hisD3052 rfa uvrB + R
TA 100 hisG46 rfa uvrB +R
TA 1535 hisG46 rfa uvrB,
TA 1537 hisC3076 rfa uvrB
and
E. coli WP2 uvrA pkm101

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Bacteria:
The strains of Salmonella typhimurium were obtained from professor B.N. Ames, University of California, U.S.A. The strain of E. coli was obtained from the national Collection of Industrial Bacteria, Aberdeen, Scotland.
Bacteria were grown overnight in nutrient broth (25 g Oxid Nutrient Broth No. 2 /liter) at approx. 37 °C. The amount of bacteria in the cell suspension was checked by nephelometry. Inoculation was performed with stock cultures which had been stored at approx. -80 °C. The different bacterial strains are checked half-yearly with regard to their respective biotin, histidine requirements, membrane permeability, ampicillin resistance, crystal violet sensitivity, UV resistance and response to diagnostic mutagens. All criteria for a valid assay were fulfilled.

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix aus Rattenleber und Hamsterleber Mix

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 50 ... 5000 µg/plate
Concentration range in the main test (without metabolic activation): 50 ... 5000 µg/plate

Vehicle / solvent:

Solvent: d. Wasser

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

See under " details on test system and conditions"

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

sodium azide

Remarks:

Used with tester strains TA100 and TA1535

Controls 2

Untreated negative controls:

yes

Remarks:

See under " details on test system and conditions"

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Used with tester Strain TA 1537

Migrated to IUCLID6: Without metabolic activation

Controls 3

Untreated negative controls:

yes

Remarks:

See under " details on test system and conditions"

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

2-nitrofluorene

Remarks:

Used with tester strain TA 98

Controls 4

Untreated negative controls:

yes

Remarks:

See under " details on test system and conditions"

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

other: MNNG( N-methyl-N-nitro-N-nitrosoguanidine

Remarks:

Used with tester strain WP2uvrA

Controls 5

Untreated negative controls:

yes

Remarks:

See under " details on test system and conditions"

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Used with tester strain WP2uvrA

Controls 6

Untreated negative controls:

yes

Remarks:

See under " details on test system and conditions"

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

with metabolic activatiobn (10% rat liver)

Positive control substance:

other: 2-aminoanthracene

Remarks:

Used with all tester strains

Controls 7

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

with metabolic activation (30% Syrian golden hamster liver and preincubation)

Positive control substance:

other: 2-aminoanthracene

Remarks:

Used with tester Strains TA 100, TA 1535 and TA 1537

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation; in suspension;

Test System:
Test Groups: PLATE INCORPORATION TEST
a) Without metabolic activation: 50; 160, 500, 1600 and 5000 µg/plate
b) With metabolic activation (10 % rat liver):
50, 160, 500, 1600 and 5000 µg/plate
PREINCUBATION TEST
a) Without metabolic activation:
50; 160, 500, 1600 and 5000 µg/plate
b) With metabolic activation (30 % rat liver)
50, 160, 500, 1600 and 5000 µg/plate


CONTROL GROUPS:
Negative controls:
a) Untreated control
b: solvent control
Positive controls:
a) without metabolic activation (Sodium-azide for Strain TA 100 and TA 1535, 9 aminoacridine for strain TA 1537, 2-Nitrofluorene for strain 98, MNNG for strain WP2uvrA and 4-NQO for strain WP2uvrA
b): with metabolic activation (10 % rat liver)
2-aminoanthracene for all tester strains
c)-with metabolic activation (30 % Syrian golden hamster liver and preincubation)
2-aminoanthracene for strain TA 100, TA 1535 and TA 1537
Congo red for Strain TA98

Formulation of test compound: dissolved in deionized water at appropriate concentrations immediately before use.

DURATION
- Preincubation period: 30 min preincubation in the presence of 30 % (v/v) Syrian golden hamster S9-mix.
Three volumes of S9 fraction was mixed with 7 volumes of the S9 cofactor solution.

Evaluation criteria:

Assay considered valid if:
-Solvent control data are within the laboratory´s normal control range for the spontaneous mutant frequency
-Positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory´s normal range
Test compound is classified as mutagenic if has either following effects:
-it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
-it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn If test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system Sterility of S9-mix and the test compound were indicated by the absence of contamination on the test material and S9-mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies, i.e. values were within the laboratories control range. Test compound proved to be not toxic to the bacterial strains. In all independent mutation tests Reaktiv-Orange DYPR 934 was tested for mutagenicity with concentrations as described in table above. Number of colonies per plate with each strain as well as mean values of 3 plates is given.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

Test compound proved to be not toxic to the bacterial strains

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

Test compound proved to be not toxic to the bacterial strains

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: NA

- Water solubility:
The test compound did not precipitate on the plates until the highest investigated dose of 5000 µg/plate
The test substance proved to be not toxic to the bacterial strains.
HISTORICAL CONTROL DATA (see table under "overall remarks”)

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

RESULTS AND COMMENTS:

RESULTS:

STERILITY CHECKS AND CONTROL PLATES

Sterility of S9-mix and the test material and S9-mix sterility check plates. Control plates gave the expected number of colonies, i.e. values were within the laboratory's historical range.

SOLUBILITY AND TOXICITY

The test compound was dissolved in deionized water and a stock solution of 50 mg/mL was prepared for the highest concentration, which provided a final concentration of 5000 µg/plate. Further dilutions of 1600, 500, 160 and 50 µg/plate were used in all experiments.

The test compound proves to be not toxic to the bacterial strains.

MUTAGENICITY:

In all independent mutation tests Reaktiv-Orange DYPR 934 was tested for mutagenicity with the same concentration.

AMES TEST:

Reaktiv-Orange DYPR 934 did not cause a significant increase in the number of revertant colonies at any dose level with any of the tester strains either in the absence or in the presence of rat liver S9-mix in either mutation test. No dose dependant effect was obtained.

PRIVAL TEST:

In the presence of hamster liver S9-mix (30 % (v/v)) using the preincubation method according to prival the test compound did not cause a significant increase in the number of revertant colonies under the experimental conditions described.

All positive controls produced significant increases in the number of revertant colonies. Thus the sensitivity of the assay and the efficacy of the exogenous metabolic activation system were demonstrated.

Table I: AMES TEST RESULTS Controls Study ZR0611 Date tested: 1999-02-17 Date counted: 1999-02-19 Positive controls: QN0073/87    Solvent Controls: QN0076/40      Negative Controls: QN0078/84 Solvent Control comments: none Negative control comments: none Positive control comments: none
STRAIN	Dose levels (µg/plate)	Mean	Standard deviation	Ratio: BL Test/ Control		No Revertants/plate   Plate 1	No Revertants/plate   Plate 2	No Revertants/plate   Plate 3
Ta 100 (+S9)	Solvent controls	116.0	13.7			131	104	113
	Negative controls	104.3	14.2	0.9		88	113	112
	Positive controls 0.5	P00001/001/001				2-Aminoanthracene
		1464.0	21.9	12.0		1448	1489	1455
Ta 100 (-S9)	Solvent controls	109.3	6.5			103	116	109
	Negative controls	113.0	27.9	1.0		105	144	90
	Positive controls 1	P00002/001/001				Sodium-Azide
		453.7	38.4	4.2		498	432	431
Ta 1535 (+S9)	Solvent controls	10.0	3.0			7	10	13
	Negative controls	6.3	2.5	0.6		9	6	4
	Positive controls 1	P00001/001/001				2-Aminoanthracene
		165.0	13.1	16.5		151	177	167
Ta 1535 (-S9)	Solvent controls	8.7	4.0			5	8	13
	Negative controls	7.7	3.5	0.9		8	11	4
	Positive controls 1	P00002/001/001				Sodium-Azide
		321.7	45.2	37.0		341	354	270
Ta 1537 (+S9)	Solvent controls	7.7	2.1			6	7	10
	Negative controls	7.3	1.2	0.9		8	8	6
	Positive controls 1	P00001/001/001				2-Aminoanthracene
		200.0	16.5	26.0		191	190	219
Ta 1537 (-S9)	Solvent controls	10.3	3.8			13	12	6
	Negative controls					4	7	8
	Positive controls 50	P00003/001/001				9-Aminoacridine
		119.0	25.0	11.6		91	127	139
TA 98 (+S)	Solvent controls	24.3	2.5			22	24	27
	Negative controls					25	20	28
	Positive controls 0.5	P00001/001/001				2-Aminoanthracene
		1042.7	118.5	42.9		1161	924	1043
TA 98 (-S)	Solvent controls	18.7	4.5			14	19	23
	Negative controls	17.0	2.0	0.9		15	19	17
	Positive controls 2.5	P00004/001/001				2-Nitrofluorene
		420	56.7	22.5		479	366	415
Wp2uvrA (+S9)	Solvent controls	24.0	7.8			28	29	15
	Negative controls	22.3	4.0	0.9		26	23	18
	Positive controls 30	P00001/001/001				2-Aminoanthracene
		98.3	9.0	4.1		103	88	104
Wp2uvrA (-S9)	Solvent controls	21.7	5.0			27	21	17
	Negative controls	17.3	5.1	0.8		13	23	16
	Positive controls 4	P00005/001/001				MNNG (N-Methyl-N-nitro-N-nitrosoguanine)
		109.0	16.7	5.0		91	112	124

Table II: AMES TEST RESULTS Study ZR0611 Date tested: 1999-02-17 Date counted: 1999-02-19 Compound D00897/001/001 Reaktiv Orange DYPR 934 Batch: DYPR 934 Comments: Votox 99.0014 All sterility control plates were steriled
STRAIN	Dose levels (µg/plate)	Mean	Standard deviation	Ratio: BL Test/ Control		No Revertants/plate   Plate 1	No Revertants/plate   Plate 2	No Revertants/plate   Plate 3
Ta 100 (+S9)	0.	116.0	13.7			131	104	113
	50.	104.3	14.2	1.1		140	120	122
	160.
		113.7	26.4	1.0		106	92	143
	500.	119.3	5.9	1.0		126	115	117
	1600	116.0	14.5	1.0		102	131	115
	5000.
		105.0	20.1	0.9		126	86	103
Ta 100 (-S9)	0.	109.3	6.5			103	116	109
	50.	109.7	13.3	1.0		121	113	95
	160.
		92.7	17.1	0.8		104	101	73
	500.	92.7	28.1	0.8		66	122	90
	1600	94.7	18.6	0.9		75	112	97
	5000.
		79.3	4.0	0.7		84	77	77
Ta 1535 (+S9)	0.	10.0	3.0			7	10	13
	50.	9.3	0.6	0.9		10	9	9
	160.
		10.0	0.0	1.0		10	10	10
	500.	9.3	2.3	0.9		8	12	8
	1600	12.0	2.0	1.2		12	14	10
	5000.
		11.3	1.2	1.1		10	12	12
Ta 1535 (-S9)	0	8.7	4.0			5	8	13
	50.	7.7	2.5	0.9		8	10	5
	160.
		8.7	2.5	1.0		11	6	9
	500.	11.0	2.0	1.3		9	11	13
	1600	7.0	1.0	0.8		6	7	8
	5000.
		8.3	5.1	1.0		7	4	14
Ta 1537 (+S9)	0.	7.7	2.1			6	7	10
	50.	6.0	1.7	0.8		7	7	4
	160.
		6.7	2.1	0.9		9	6	5
	500.	6.0	1.0	0.8		5	6	7
	1600	5.7	2.1	0.7		5	4	8
	5000.
		9.3	2.9	1.2		6	11	11

Table II: AMES TEST RESULTS TEST Study ZR0611 Date tested: 1999-02-17 Date counted: 1999-02-19 Compound D00897/001/001 Reaktiv Orange DYPR 934 Batch: DYPR 934 Comments: Votox 99.0014 All sterility control plates were sterile
STRAIN	Dose levels (µg/plate)	Mean	Standard deviation	Ratio: BL Test/ Control		No Revertants/plate   Plate 1	No Revertants/plate   Plate 2	No Revertants/plate   Plate 3
Ta 1537 (-S9)	0.	10.3	3.8			13	12	6
	50.	7.3	1.5	0.7		9	7	6
	160.
		5.3	2.1	0.5		6	3	7
	500.	8.3	4.9	0.8		14	5	6
	1600	7.7	1.2	0.7		7	7	9
	5000.
		8.3	0.6	0.8		8	9	8
Ta 98 (+S9)	0.	24.3	2.5			22	24	27
	50.	26.0	1.7	1.1		27	24	27
	160.
		18.3	2.3	0.8		17	21	17
	500.	19.0	4.0	0.8		15	19	23
	1600	18.3	3.6	0.7		22	15	17
	5000.
		15.3	7.2	0.6		20	7	19
Ta 98 (-S9)	0.	18.7	4.5			14	19	23
	50.	22.7	8.1	1.2		32	25	17
	160.
		22.3	2.3	1.2		21	25	21
	500.	24.3	2.9	1.3		21	26	26
	1600	21.7	0.6	1.2		22	22	21
	5000.
		17.7	2.1	0.9		17	20	16
WP2uvrA (+S9)	0	24.0	7.8			28	29	15
	50.	24.0	7.2	1.0		16	30	26
	160.
		22.3	3.2	0.9		26	21	20
	500.	25.0	2.0	1.0		27	23	25
	1600	22.0	5.3	0.9		28	20	18
	5000.
		26.7	0.6	1.1		27	27	26
WP2uvrA (-S9)	0.	21.7	5.0			27	21	17
	50.	20.0	1.0	0.9		24	16	20
	160.
		20.0	1.0	0.9		24	16	20
	500.	18.3	2.9	0.8		20	20	15
	1600	19.3	1.5	0.9		19	18	21
	5000.
		22.7	4.2	1.0		16	24	26

(to be continued)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Summarizing, it can be assumed that Reaktiv-Orange DYPR 934 is not mutagenic in the standard plate test; Ames Test as well as in the preincubation method according to Prival in the investigated dose levels.

Executive summary:

Reaktiv-Orange DYPR 934 was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA.

Two independent mutagenicity studies were conducted one standard plate test ( Ames Test) and a modified preincubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolizing system derived from a rat liver homogenate or a hamster liver homogenate. Additionally a repeat of the preincubation test was performed with the strain WP2uvrA in the absence of S9 -mix.

For all studies, the compound was dissolved in deionized water, and each bacterial strain was exposed to 5 dose levels. Doses for all studies ranged from 50 to 5000 µg/plate.

Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control range and similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies. Except in the first preincubation test with the strain WP2uvrA in the absence of S9 -mix, the positive control showed not the expected increase in the number of revertant colonies, this doubtful effect is caused by a decompose of N-Methyl-N-nitrosoguanidine (MNNG). In a repeat of this strain with an alternative positive compound 4 -Nitroquinoline-N-Oxide (4 -NQO) the sensitivity of the assay was demonstrated.

Toxicity: In the plate incorporation test and also in the preincubation test toxicity was not observed either with or without metabolic activation.

Ames Test:

Mutagenicity: in the absence of the metabolic activation system the test compound did not result in relevant increases in the number of revertants in any of the bacterial strains. Also in the presence of rat liver activation system (10 % (v/v)), treatment of the cells with Reaktiv-Orange DYPR 934 did not result in relevant increases in the number of revertant colonies.

 

Prival Test:

In the presence of hamster liver S9-mix (30 % (v/v)) using preincubation method according to Prival the test compound did not cause a significant increases in the number of revertant colonies with any of the tester strains under experimental conditions described.

Therefore, it can be assumed that Reaktiv-Orange DYPR 934 is not mutagenic in the standard plate test; Ames Test as well as in the preincubation method according to Prival in the investigated dose levels.