7-acetamido-4-hydroxy-3-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2-sulphonic acid, sodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reactive Orange 72/78 is not mutagenic in the absence and presence of rat S-9 Mix using the standard Ames Test procedure. In the presence of hamster liver S-9 Mix and preincubation the test compound did induce a minimal increase in the number of revertant colonies (MF 2.1) with the strain TA 98 at 2500 µg/plate. As this is a marginal effect that was not dose dependent and was not seen in the highest concentration, this effect was considered to be incidental. Reactive Orange 72/78 and its structural analogue were negative in the HPRT assay in mammalian cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-09-12 until 2013-11-13

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

Principles of method if other than guideline:

first experiment 4 hours treatment with and without metabolic activation
second experiment 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Experiment I:
without metabolic activation: 568.8; 1137.5; 2275.0; 4550.0; 6825.0 µg/mL
with metabolic activation: 568.8; 1137.5; 2275.0; 4550.0; 9100.0 µg/mL
Experiment II:
without metabolic activation: 71.1; 142.2; 284.4; 568.8; 716.2 µg/mL
with metabolic activation: 568.8; 1137.5; 2275.0; 4550.0; 9100.0 µg/mL

Vehicle / solvent:

Deionised water. The final concentration of deionised water in the culture medium was 10% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: >1,5x10exp. 6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT(R)11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.42 in the solvent control versus pH 7.43 at 9100 µg/mL) measured in the range finding pre-experiment
- Effects of osmolality: No relevant increase (281 mOsm in the solvent control versus 340 mOsm at 9100 µg/mL, and 320 mOsm at 4550 µg/mL) measured in the pre-experiment
- Evaporation from medium: Not examined
- Water solubility: Soluble
- Precipitation: No precipitation of the test item was observed up to the maximum concentration in all experiments.
- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
The range finding pre-experiment was performed using a concentration range of 71.1 to 9100 µg/mL to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24-hours treatment) of metabolic activation. The highest applied concentration in the pre-test on toxicity (9100 µg/ml) was equal to 5 mg/mL of the pure substance.

Relevant toxic effect occurred after the 4-hour treatment at 4550 µg/mL and above without metabolic activation. Following the 24-hour treatment without metabolic activation toxic effects were noted at 568.8 µg/mL and above. Another low value of the cloning efficiency was noted at 568.8 µg/mL following 4-hour treatment without metabolic activation. This effect was judged as irrelevant fluctuation rather than a true cytotoxic effect however, as the relative cloning efficiency remained above 50% at the next two higher concentrations.

The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. No precipitation or phase separation was observed up to the maximum concentration with and without metabolic activation following 4 and 24 hours treamtment.

There was no relevant shift of the pH of the medium even at the maximum concentration of the test item. The osmolarity was increased from 283 to 340 mOsm at 9100 µg/mL and 320 mOsm at 4550 µg/mL. Both levels of osmolarity are sufficiently close to the physiological level of approximately 300 mOsm.
The dose range of the first experiment was set according to the cytotoxicity data generated in the pre-experiment. The dose range of the second experiment was adjusted to data produced in the pre-experiment (without metabolic activation) and in the first experiment (with metabolic activation). The individual concentrations were generally spaced by a factor of 2.0. A narrower spacing was used at higher concentrations to cover the cytotoxic range more closely.
To overcome problems with possible deviations in toxicity the main experiments were started with more than four concentrations.

Table 1 Doses applied in the gene mutation assay with Reactive Orange 72-78
Experiment I
4 hours treatment without S9 mix: 142.2; 284.4; 568.8; 1137.5; 2275.0; 4550.0; 6825.0 µg/mL
4 hours treatment with S9 mix: 284.4; 568.8; 1137.5; 2275.0; 4550.0; 9100.0 µg/mL
Experiment II
24 hours treatment without S9 mix: 17.8; 35.6; 71.1; 142.2; 284.4; 568.8; 716.2 µg/mL
4 hours treatment with S9 mix: 284.4; 568.8; 1137.5; 2275.0; 4550.0; 9100.0 µg/mL

In experiment I and II the cultures at the two lowest concentrations without metabolic activation and the culture at the lowest concentration with metabolic activation were not continued since a minimum of only four concentrations is required by the guidelines.

COMPARISON WITH HISTORICAL CONTROL DATA: Complies

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects, indicated by a relative cloning efficiency I or a relative cell density at first subcultivation of less than 50% in both parallel cultures, occurred in the first experiment at 4550 µg/mL and above without metabolic activation. In the second experiment relevant cytotoxic effects as described above were noted at 568.8 µg/mL and above without metabolic activation.

Summary Table

			relative	relative	relative	mutant		relative	relative	relative	mutant
	conc.	S9	cloning	cell	cloning	colonies/	induction	cloning	cell	cloning	colonies/	induction
	µg/mL	mix	efficiency I	density	efficiency II	10^6cells	factor	efficiency I	density	efficiency II	10^6cells	factor
			%	%	%			%	%	%
Column	1	2	3	4	5	6	7	8	9	10	11	12
Experiment I / 4 h treatment			culture I					culture II
Solvent contro with water		-	100.0	100.0	100.0	17.2	1.0	100.0	100.0	100.0	15.4	1.0
Positive control (EMS)	150.0	-	96.4	58.2	108.2	167.6	9.8	97.5	108.8	98.1	168.3	10.9
Test item	142.2	-	99.3	culture was not continued^#				85.4	culture was not continued^#
Test item	284.4	-	82.0	culture was not continued^#				83.1	culture was not continued^#
Test item	568.8	-	91.2	69.4	111.4	14.5	0.8	95.2	135.9	102.2	11.5	0.7
Test item	1137.5	-	100.7	53.2	100.5	19.8	1.2	91.1	117.4	101.3	21.3	1.4
Test item	2275.0	-	80.4	57.1	99.0	13.2	0.8	79.9	112.1	100.6	13.1	0.8
Test item	4550.0	-	32.0	56.3	98.2	20.2	1.2	28.3	108.2	95.6	20.9	1.4
Test item	6825.0	-	4.9	30.4	97.6	21.7	1.3	4.1	73.2	103.6	19.1	1.2
Solvent contro with water		+	100.0	100.0	100.0	6.3	1.0	100.0	100.0	100.0	7.5	1.0
Positive control (DMBA)	1.1	+	99.2	80.3	103.2	159.0	25.4	93.6	108.4	75.9	214.1	28.6
Test item	284.4	+	100.0	culture was not continued^#				98.1	culture was not continued^#
Test item	568.8	+	103.9	90.9	76.8	14.9	2.4	94.4	109.5	66.3	11.8	1.6
Test item	1137.5	+	96.4	82.5	93.8	12.2	1.9	91.7	133.5	90.2	9.6	1.3
Test item	2275.0	+	97.5	80.9	77.0	6.6	1.1	103.1	110.7	81.4	11.9	1.6
Test item	4550.0	+	83.2	73.4	80.2	10.4	1.7	83.9	114.1	81.9	19.5	2.6
Test item	9100.0	+	52.9	79.8	80.6	15.3	2.4	48.9	86.9	87.7	9.2	1.2
Experiment II / 24 h treatment			culture I					culture II
Solvent contro with water		-	100.0	100.0	100.0	15.5	1.0	100.0	100.0	100.0	19.8	1.0
Positive control (EMS)	150.0	-	94.5	89.8	89.8	269.9	17.4	89.0	85.3	97.5	318.3	16.1
Test item	17.8	-	101.8	culture was not continued^#				99.9	culture was not continued^#
Test item	35.6	-	100.3	culture was not continued^#				99.1	culture was not continued^#
Test item	71.1	-	103.6	117.2	89.3	21.2	1.4	97.9	73.6	101.7	11.7	0.6
Test item	142.2	-	97.3	74.7	92.3	18.9	1.2	98.7	89.4	102.8	15.0	0.8
Test item	284.4	-	102.6	89.5	113.9	13.1	0.9	96.1	110.4	104.7	18.4	0.9
Test item	568.8	-	21.7	92.0	98.3	12.0	0.8	19.0	131.0	103.9	15.8	0.8
Test item	716.2	-	0.8	102.2	77.0	10.9	0.7	3.3	107.0	104.6	31.7	1.6
Experiment II / 4 h treatment			culture I					culture II
Solvent contro with water		+	100.0	100.0	100.0	11.8	1.0	100.0	100.0	100.0	14.2	1.0
Positive control (DMBA)	1.1	+	88.1	91.0	95.8	77.7	6.6	92.7	147.4	89.7	92.5	6.5
Test item	284.4	+	83.7	culture was not continued^#				112.5	culture was not continued^#
Test item	568.8	+	78.5	132.2	122.4	14.0	1.2	91.9	129.3	92.5	14.5	1.0
Test item	1137.5	+	76.2	167.0	110.2	12.2	1.0	101.5	142.1	93.8	16.2	1.1
Test item	2275.0	+	80.9	166.2	117.0	13.9	1.2	94.1	127.8	92.8	15.5	1.1
Test item	4550.0	+	95.5	182.7	102.7	28.8	2.4	103.4	159.2	82.4	17.8	1.3
Test item	9100.0	+	72.2	78.8	121.8	2.9	0.2	94.9	133.2	91.8	6.9	0.5

#       culture was not continued since a minimum of only four analysable concentrations is required

Conclusions:

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, Reactive Orange 72/78 is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

 

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

 

The main experiments were evaluated at the following concentrations:

 

exposure period	S9 mix	concentrations in µg/mL
		Experiment I
4 hours	-	568.8	1137.5	2275.0	4550.0	6825.0
4 hours	+	568.8	1137.5	2275.0	4550.0	9100.0
		Experiment II
24 hours	-	71.1	142.2	284.4	568.8	716.2
4 hours	+	568.8	1137.5	2275.0	4550.0	9100.0

 

No precipitation of the test item was observed up to the maximum concentration in all experiments.

 

Relevant cytotoxic effects, indicated by a relative cloning efficiency I or a relative cell density at first subcultivation of less than 50% in both parallel cultures, occurred in the first experiment at 4550 µg/mL and above without metabolic activation. In the second experiment relevant cytotoxic effects as described above were noted at 568.8 µg/mL and above without metabolic activation.

 

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls.

 

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

 

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 6.3 up to 19.9 mutants per 10⁶cells; the range of the groups treated with the test item was from 2.9 up to 31.7 mutants per 10⁶cells.

 

EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. The positive control with DMBA in the first culture of the second experiment with metabolic activation did not quite reach the historical range of positive controls (77.7 versus 91.4 - 2666.3 colonies per 10⁶cells). The positive control was valid however, as the induction factor was 6.6 and the positive control of the parallel culture remained within the historical range of positive controls.

 

 In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 09,1989 to September 28, 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Version / remarks:

The Salmonella typhimurium reverse mutation assay HG-Gene Muta-S. typhimurium, August 1982 0ffice of Toxic Substances

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

rat and hamster S9

Test concentrations with justification for top dose:

Toxicity test: 0, 4, 20, 100, 500, 2500, 10000 µg/plate
Mutagenicity test: up to 5000 µg/plate

Vehicle / solvent:

Aqua bidest

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

with 10% or 30% rat liver S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

other: benzidine; 2-aminoanthracene

Remarks:

with 30% hamster liver S9

Details on test system and experimental conditions:

PREPARATION AND STORAGE OF A LIVER HOMOGENATE FRACTION (S9)
Liver preparations were performed from liver of Aroclor induced Sprague Dawley rats and from non pretreated Syrien hamsters. Male Sprague Dawley rats (200 - 300 g) receive a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bodyweight) 5 days before sacrifice. Preparation is performed at O to 4 °C using cold sterile solution and glassware. The livers from at least 5 - 6 Sprague Daw¬ley rats or from 5 - 6 male Syrien golden hamsters (7 - 8 weeks old) are removed and pooled, washed in 150 mM KCl (approximately 1 ml/g wet livers). The washed livers are cut into small pieces and homogenized in three volumes of KCl. The homogenate is centrifuged at 9000 g for 10 minutes. The supernatant is the S-9 fraction. It is divided into small portions, rapidly frozen and stored at -80 °C for not longer than three months.

Preparation of S-9 Mix:
Sufficient S-9 fraction is thawed immediately before each test at room temperature. One or three volumes of S-9 fraction is mixed with 9 or 7 volumes of the S-9 cofactor solution and kept on ice until used. This preparation is termed S-9 Mix. The concentrations of the different compounds in the S-9 Mix of the rat liver are: 8 mM MgCl2; 33 mM KCl; 5 mM glucose-6-phosphate; 4 mM NADP+; 100 mM phosphate buffer pH 7.4

According to the modification proposed by Prival using 30 minutes preincubation in the presence of 30 % hamster S-9 (Syrien golden hamster), the S-9 Mix consisted of: 8 mM MgCl2; 33 mM KCl; 20 mM glucose-6-phosphate; 2.8 units/ml glucose-6-phosphate dehydrogenase; 4 mM NADP+; 2 mM NADH; 2 mM FMN (Riboflavin-5'-phosphate-Na-salz); 100 mM phosphate buffer pH 7.4

BACTERIA
Bacteria are grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No 2 /liter) at 37 °C. The suitable amount of bacteria in the cell suspension is checked by nephelometry. For inoculation, stock cultures which are stored at -80 °C, are used. The compound is tested with the strains Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538.

RANGE-FINDING/SCREENING STUDIES:
Preliminary toxicity tests were performed with five or four tester strains using three plates per dose to get information on mutagenicity and toxicity for calculation of an appropriate dose range. A reduced rate of spontaneously occuring colonies as well as visible thinning of the bacterial lawn were used as indicator for toxicity. Thinning of the bacterial lawn was controlled microscopically. In combination with the main experiments, toxicity testing was performed as follows: 0.1 ml of the different dilutions of the test compound were thoroughly mixed with 0.1 ml of 10^-6 dilution of the overnight culture of TA 100 and plated with histidine and biotin rich top agar (3 plates per dose). The solvent control is compared with the number of colonies per plate in the presence of the test compound. Results are given as a ratio of these values (= surviving fraction).

PROCEDURE
AMES TEST
- Method of application: in agar
Top agar is prepared for the Salmonella strains by mixing 100 ml agar (0.6 % agar, 0.6 % NaCl) with 10 ml of a 0.5 mM histidine-biotin solution. The following ingredients are added (in order) to 2 ml of molten top agar at 45 °C:
0.1 ml test compound solution
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.5 ml S-9 Mix or buffer

- Duration
Preincubation period: 48 to 72 hours at 37 °C in the dark

- Number of replications:2

PRIVAL TEST
- Method of application: in suspension
0.1 ml test solution, 0.1 ml bacterial suspension and 0.5 ml S-9 Mix are incubated at 30 °C for the duration of 30 minutes. Subsequently, 2 ml of soft agar which consists of 100 ml agar (0.6 % agar + 0.6 % NaCl) and 10 ml aminoacid solution (minimal aminoacid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) is added. After mixing, the samples are poured on to the Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.

- Duration
Preincubation period:30 min

- Number of replications:2

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

rat S9

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

rat S9

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

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

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

hamster S9

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

hamster S9; MF: 2.1

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

The test compound did not cause a significant increase in the number of revertant colonies with any of the tester strains either in the absence or in the presence of rat liver S-9 Mix. No dose dependent effect was obtained.

In the presence of hamster liver S-9 using the preincubation method according to Prival the test compound did show relevant increases in the number of revertant colonies under the experimental conditions described with the tester strain TA 98. Therefore the test was repeated with this strain and the same results were obtained.

It is concluded that Remazol-Goldorange 3G, FW is not mutagenic in the absence and presence of rat S-9 Mix using the standard Ames Test procedure.

In the presence of hamster liver S-9 Mix and preincubation the test compound did induce a minimal increase (MF 2.1) in the number of revertant colonies with the strain TA 98

Conclusions:

Reactive Orange 72/78 is not mutagenic in the absence and presence of rat S-9 Mix using the standard Ames Test procedure. In the presence of hamster liver S-9 Mix and preincubation the test compound did induce a minimal increase in the number of revertant colonies (MF 2.1) with the strain TA 98 at 2500 µg/plate. As this is a marginal effect that was not dose dependent and was not seen in the highest concentration, this effect was considered to be incidental.

Executive summary:

Reactive Orange 72/78 was tested for mutagenicity with the tester strains TA 100, TA 1535, TA 1537, TA1538 and TA 98 of Salmonella typhimurium.

The mutagenicity studies were conducted in the standard plate test (Ames Test) and in a modified preincubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolizing system derived from rat or hamster liver homogenate. A dose range of 7 different doses from 4 µg/ plate to 5000 µg/plate was used. Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

Toxicity:

The test compound proved to be not toxic to the bacterial strains. 5000 µg/plate was chosen as top dose level for the mutagenicity study.

a) Ames Test:

Mutagenicity: In the absence of the metabolic activation system the test compound did not show a relevant increase in the number of revertants in any of the five bacterial strains. In the presence of a metabolic activation system, treatment of the cells with Reactive Orange 72/78 did not result in relevant increases in the number of revertant colonies.

b) Prival Test:

In the presence of hamster liver S-9Mix (30 %) using the preincubation method according to Prival Reactive Orange 72/78 did induce a slight increase (2.1 -fold) in the number of revertant colonies with the tester strain TA 98 at 2500 µg/plate only. This effect was only marginal, was not seen in the highest concentration and not dose dependant and despite it could also be seen in the repeat experiment was hence considered incidental. In the presence of rat liver S-9Mix (30 %) Reactive Orange 72/78 did not induce a significant increase in the number of revertant colonies in any other strains.

Summarizing, it can be stated that Reactive Orange 72/78 is not mutagenic in the standard plate test (Ames Test) and is also considered not mutagenic in the preincubation method according to Prival.

Reference 3

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:

From June 12, 1985 to August 1, 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

0, 4, 20, 100, 500, 2500, 10000 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 on strain TA 100 and TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 on strain TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9 on strain TA 1538 TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 on WP2uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9, all strains

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoantracene

Remarks:

with S9, all strains

Details on test system and experimental conditions:

PREPARATION AND STORAGE OF A LIVER HOMOGENEATE FRACTION ("S-9")
Male Sprague Dawley rats (200 - 300 g) receive a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bodyweight) 5 days before sacrifice. Preparation is performed at 0° to 4°C using cold sterile solution and glassware. The livers from at least 5 - 6 animals are removed and pooled, washed in 150 mM KCl (approximately 1 ml/g wet livers). The washed livers are cut into small pieces and homogenized in three volumes of KCl. The homogenate is centrifuged at 9000 g for 10 minutes. The supernatant is the. S-9 fraction.It is divided into small portions, rapidly frozen and stored at -80°c for not longer than three months.

Preparation of S-9 Mix:
Sufficient S-9 fraction is thawed immediately before each test at room temperature. One volume of S-9 fraction is mixed with 9 volumes of the S-9 cofactor solution and kept on ice until used. This preparation is termed S-9 Mix. The concentrations of the different compounds in the S-9 Mix are:
8 mM MgC12
33 mM KC1
5 mM glucose-6-phosphate
4 mM NADP+
100 mM phosphate buffer pH 7.4

BACTERIA
Bacteria are grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No 2 /liter) at 37 °C. The suitable amount of bacteria in the cell suspension is checked by nephelometry. For inoculation, stock cultures which are stored at -80 °C, are used. The compound is tested with the strains Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 and E. coliWP2uvrA.

RANGE-FINDING/SCREENING STUDIES:
Preliminary toxicity tests were performed with all tester strains using two plates per dose to get information on mutagenicity and toxicity for calculation of an appropriate dose range. A reduced rate of spontaneously occurring colonies as well as visible thinning of the bacterial lawn were used as indicator for toxicity. Thinning of the bacterial lawn was controlled microscopically.In combination with the second experiment, -toxicity testing was performed as follows: 0.1 ml of the different dilutions of the test compound were thoroughly mixed with 0.1 mi of 10-6 dilution ,of the overnight culture of TA 100 and plated with histidine and biotin rich top agar (3 plates per dose).The solvent control is compared with the number of colonies per plate in the presence of the test compound. Results are given as a ratio of these values (= surviving fraction).

AMES TEST
METHOD OF APPLICATION: in agar
Top agar is prepared for the Salmonella strains by mixing 100 ml agar (0.6 % agar, 0.6 % NaCl) with 10 ml of a 0.5 mM histidine-biotin solution. The following ingredients are added (in order) to 2 ml of molten top agar at 45 °C:
0.1 ml test compound solution
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.5 ml S-9 Mix or buffer

DURATION
- Preincubation period: 48 to 72 hours at 37 °C in the dark

NUMBER OF REPLICATIONS:2

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The test compound did not cause a significant increase in the number of revertant colonies with any of the tester strains neither in the absence nor presence of S-9 Mix. No dose dependent effect was obtained.

It is concluded that the test substance is not mutagenic in these bacterial test systems neither in the absence nor in the presence of an exogenous metabolizing system.

Conclusions:

It is concluded that the test substance is not mutagenic in these bacterial test systems neither in the absence nor in the presence of an exogenous metabolizing system.

Executive summary:

Structural analogue 01 as lithium salt, was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA. The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. A dose range of 6 different doses from 4 µg/plate to 5000 µg/plate was used. Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies. Toxicity: The test compound proved to be not toxic to the bacterial strains. 5000 µg/plate was chosen as top dose level for the mutagenicity study. Mutagenicity: In the absence of the metabolic activation system the test com¬pound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with the test substance did not result in relevant increases in the number of revertant colonies.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test substance was not clastogenic in the in vivo micronucleus test in mice.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September, 1985 to September 19, 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: HOECHST AG, Kastengrund, SPF breeding colony
- Age at study initiation: 8 weeks
- Weight at study initiation: male=32.63 g (27-37 g), female=25.74 g (22-30 g)
- Fasting period before study:
- Housing: in fully air conditioned rooms in Macrolon cages, on softwood granulate in group of 5 animals
- Diet: Altromin 1324, ad libitum
- Water: tap water in plastic bottles, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2°C
- Humidity (%): 55±10%
- Lighting time: 12 hours daily

Route of administration:

oral: gavage

Vehicle:

- Vehicle: water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

-The test substance dilution were freshly prepared each day. 7500 mg was weighed into a beaker, mixed with deionized water, washed out into a 25 mL flask and topped up to the calibration mark with water, a solution was formed.

- Endoxan stock solution: 25mL distilled water were added to 100mg Endoxan in an injection phial and shaken to form a clear solution. The daily solution for administration were prepared from this stock solution. For this purpose, 2 mL of the 2% stock solution were mixed with 6 mL distilled water.

Duration of treatment / exposure:

- Tratment: single administration
- Duration of exposure: 72 hours

Frequency of treatment:

- once

Post exposure period:

The animals were killed after 24, 48 and 72 hours

Dose / conc.:

3 000 mg/kg bw (total dose)

No. of animals per sex per dose:

5 animals per sex per dose per time point

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: cyclophosphamide
- Route of administration: gavage
- Doses / concentrations: 50 mg/Kg bw.

Tissues and cell types examined:

erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based on a preliminary study for determinati on of the acute toxicity the dose level for the micronucleus test was selected. Oral application at a dose of 4000 or 5000 mg per kg body weight caused diarrhoe and/or heavy vomiting. The highest dose which could be administered was 3000 mg per kg body weight. This dose level was used for the main study.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed according to the test procedure 24, 48 or 72 hours after application by carbon dioxide asphyxiation. For each animal about 3 mL foetal bovine serum was poured into a silicone-coated centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at 1200 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared on a cleaned slide, identified by project code and animal number, air-dried for about 24 hours.

Staining
- 5 minutes in methanol
- 3 minutes in May-Grünwalds solution
- 2 minutes in May-Grünwalds solution diluted 1:1 with distilled water
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts distilled water
- rinsing in distilled water
- drying
- providing with Entellan

Statistics:

The number of polychromatic erythrocytes with micronuclei occurring in 1000 counted polychromatic erythrocytes, and the number of normocytes with micronuclei occurring in 1000 counted normocytes, were evaluated statistically; comparison of dose groups with the simultaneous control group was performed according to wilcoxon (paired, one—sided, increase).
The results of the test substance at each killing time and dose were compared with the corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two sided).

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The incidence of micronucleated polychromatic erythrocytes in each dose group was not significantly different from the data in the negative control groups. The number of erythrocytes with micronuclei did not differ significantly from the values of the simultaneous control animals for each of the three killing times investigated. This is true for the polychromatic erythrocytes as well as for the normocytes. A small increase in the number of polychromatic erythrocytes with micronuclei in female mice 72 hours after dosing was not considered as biologically significant. It was due to an increase of micronuclei in only 1 out of 5 animals and was within the normal range of erythrocytes with micronuclei (mean,value). The ratio of polychromatic erythrocytes to normocytes remained uneffected by the test compound.
Cyclophosphamid induced a marked and statistically significant increase of the number of polychromatic erythrocytes with micronuclei in both males and females. The ratio of polychromatic erythrocytes to normocytes was not shifted (within the normal range).

All animal survived after the application of 0 or 3000 mg per Kg body weight.

Clinical signs of toxicity in test animals: diarrhoe (3/30 red coloured faeces), piloerection, back-arched position, chenge in gait, narrowed eye opening).

Conclusions:

Under the conditions described the application of the test substance did not lead to an elevated occurence of micronuclei in the polychromatic or normochromatic erythrocytes. It is concluded that the test substance is not mutagenic in the micronucleus test.

Executive summary:

The test substance was tested in the micronucleus test. The test compound was administered orally by gavage to male and female mice. The following doses were tested: 0 and 3000 mg per kg bodyweight. The dose level 3000 mg per kg bodyweight was chosen because a preliminary study had shown this dose level to be the maximum tolerated dose.

The animals were treated once with the test substance and sacrificed according to the test procedure 24, 48 or 72 hours after administration of the test compound.

The incidence of micronucleated polychromatic erythrocytes was not significantly effected by the test substance in comparison with the negative control. The number of normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained uneffected by the treatment with the test substance .

The results indicate that the test substance is not mutagenic in the micronucleus test under the conditions of the present study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Reactive Orange 72/78 was tested for mutagenicity with the tester strains TA 100, TA 1535, TA 1537, TA1538 and TA 98 of Salmonella typhimurium.

The mutagenicity studies were conducted in the standard plate test (Ames Test) and in a modified preincubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolizing system derived from rat or hamster liver homogenate. A dose range of 7 different doses from 4 µg/ plate to 5000 µg/plate was used. Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

Toxicity: The test compound proved to be not toxic to the bacterial strains. 5000 µg/plate was chosen as top dose level for the mutagenicity study.

a) Ames Test:

Mutagenicity: In the absence of the metabolic activation system the test compound did not show a relevant increase in the number of revertants in any of the five bacterial strains. In the presence of a metabolic activation system, treatment of the cells with Reactive Orange 72/78 did not result in relevant increases in the number of revertant colonies.

b) Prival Test:

In the presence of hamster liver S-9Mix (30 %) using the preincubation method according to Prival Reactive Orange 72/78 did induce a slight increase (2.1 -fold) in the number of revertant colonies with the tester strain TA 98 at 2500 µg/plate only. This effect was only marginal, was not seen in the highest concentration and not dose dependant and despite it could also be seen in the repeat experiment was hence considered incidental. In the presence of rat liver S-9Mix (30 %) Reactive Orange 72/78 did not induce a significant increase in the number of revertant colonies in any other strains.

Summarizing, it can be stated that Reactive Orange 72/78 is not mutagenic in the standard plate test (Ames Test) and is also considered not mutagenic in the preincubation method according to Prival.

The structural analogue was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA. The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. A dose range of 6 different doses from 4 µg/plate to 5000 µg/plate was used. Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies. Toxicity: The test compound proved to be not toxic to the bacterial strains. 5000 µg/plate was chosen as top dose level for the mutagenicity study. Mutagenicity: In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with the test substance did not result in relevant increases in the number of revertant colonies.

The test item Reactive Orange 72/78 was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The main experiments were evaluated at the following concentrations: 

exposure period	S9 mix	concentrations in µg/mL
		Experiment I
4 hours	-	568.8	1137.5	2275.0	4550.0	6825.0
4 hours	+	568.8	1137.5	2275.0	4550.0	9100.0
		Experiment II
24 hours	-	71.1	142.2	284.4	568.8	716.2
4 hours	+	568.8	1137.5	2275.0	4550.0	9100.0

 

No precipitation of the test item was observed up to the maximum concentration in all experiments.

Relevant cytotoxic effects, indicated by a relative cloning efficiency I or a relative cell density at first subcultivation of less than 50% in both parallel cultures, occurred in the first experiment at 4550 µg/mL and above without metabolic activation. In the second experiment relevant cytotoxic effects as described above were noted at 568.8 µg/mL and above without metabolic activation.

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 6.3 up to 19.9 mutants per 10⁶cells; the range of the groups treated with the test item was from 2.9 up to 31.7 mutants per 10⁶cells.

EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. The positive control with DMBA in the first culture of the second experiment with metabolic activation did not quite reach the historical range of positive controls (77.7 versus 91.4 - 2666.3 colonies per 10⁶cells). The positive control was valid however, as the induction factor was 6.6 and the positive control of the parallel culture remained within the historical range of positive controls.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Reactive Orange 72/78 is considered to be non-mutagenic in this HPRT assay.

The test substance (Structural Analogue 01) was tested in the micronucleus test. The test compound was administered orally by gavage to male and female mice. The following doses were tested: 0 and 3000 mg per kg bodyweight. The dose level 3000 mg per kg bodyweight was chosen because a preliminary study had shown this dose level to be the maximum tolerated dose.

The animals were treated once with the test substance and sacrificed according to the test procedure 24, 48 or 72 hours after administration of the test compound.

The incidence of micronucleated polychromatic erythrocytes was not significantly effected by the test substance in comparison with the negative control. The number of normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained uneffected by the treatment with the test substance .

The results indicate that the test substance is not mutagenic in the micronucleus test under the conditions of the present study.

Justification for classification or non-classification

not genotoxic