Sodium 4-[4-[[2-methyl-4-[[(p-tolyl)sulphonyl]oxy]phenyl]azo]anilino]-3-nitrobenzenesulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non genotoxic

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:

From January 17th to February 02nd, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 29 July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In Vitro Mammalian Cell Gene Mutation Test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: the CHO cell line was originally derived from the ovary of a female Chinese hamster. The CHO K1 is a sub-line of CHO cell line. The CHO K1 cell line was purchased.
- Supplier: ECACC (European Collection of Cell Cultures)
- Storage: the cell stocks are kept in liquid nitrogen.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
- Periodically checked for Mycoplasma contamination: each batch of cells was free for mycoplasma infections, tested by Central Agricultural Office, National Animal Health Institute, Budapest, Hungary; results were fully documented within the raw data file.
- Preparation of cells: for each experiment the cells were thawed rapidly, the cells diluted in Ham's F12 medium containing 10 % foetal bovine serum and incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air. Growing cells were subcultured in an appropriate number of flasks.

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix

Test concentrations with justification for top dose:

Treatment concentrations for the mutation assay were selected on the basis of the result of a Pre-test on Toxicity.
Without S9 Mix: 125, 250, 500, 650 and 750 µg/ml
With S9 Mix: 250, 500, 750, 1000 and 1250 µg/ml

Vehicle / solvent:

- Solvent used: the test item was prepared in DMSO and diluted prior to treatment.
- Justification for choice of solvent/vehicle: solvent was chosen based on the results of the preliminary solubility test and its suitability is confirmed with the available laboratory’s historical database.

Untreated negative controls:

yes

Remarks:

solvent

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

CULTURE MEDIUM: on the day of treatment the culture medium of exponentially growing cell cultures were replaced with medium (F12-5) containing the test item.

DURATION
- Preincubation period: 5 x10^6 cells were each placed in sterile dishes and incubated for approximately 24 hours before treatment at 37 °C in a humidified atmosphere of 5 % CO2.
- Exposure duration: 5-hour treatment Following the exposure period the cells were washed with F12-5 medium and incubated in fresh F12-10 medium for 19 hours.
- Expression time: during the phenotypic expression period the cultures were subcultured. Aliquots of approximately 2x10^6 cells were taken on days 1, 3, 6 and evaluated on day 8.
- Selection time: at the end of the expression period, cultures from each dose level were adjusted to 2 x 10^5 cells / dish ( 4 x five dishes) in selection medium (hypoxanthine Ham's F12-SEL medium) containing 3.4 µg/ml of thioguanine (6-TG).
- Plating viability: at the end of the expression period cell number in the samples was adjusted to 2 × 10^5 cells/ml. Cells were plated in 3 parallel dishes (diameter is approx. 60 mm) for a viability test as described in “Plating for Survival“ section for the survival test.
- Fixation time: after the selection period, the colonies were fixed with methanol for five minutes, stained with Giemsa and counted for either mutant selection or cloning efficiency determination

NUMBER OF REPLICATIONS: duplicate cultures were used at each test item concentration, for negative (solvent) controls and the positive controls for treatment without and with S9-mix.

CELLS COUNT
After the 19-hour incubation period, cells were washed twice with F12-10 medium and suspended by treatment with trypsin-EDTA solution and counted using a Bürker chamber.

DETERMINATION OF CYTOTOXICITY
A non GLP Pre-test on Toxicity was performed.
During the Pre-test on Toxicity (cytotoxicity assay), the cultures (more than 50 % confluent) was trypsinised and cell suspensions were prepared in Ham's F12-10 medium. Cells was seeded into petri dishes (tissue cultures quality: TC sterile) at 5x10^6 cells each and incubated with culture medium. After 24 hours the cells were treated with the suitable concentrations of the test item in absence or in presence of S9 mix (50 µl/ml) and incubated at 37 °C for 5 hours. After the treatment cells were washed and incubated in fresh Ham's F12-10 medium for 19 hours. 24 hours after the beginning of treatment, the cultures were washed with Ham's F12-5 medium and the cells were covered with trypsin-EDTA solution, counted and the cell concentration was adjusted to 40 cells/ml with Ham's F12-10 medium. For each concentration of test solution or control solution, 5 ml was plated in parallel into 3 sterile dishes (diameter is approx. 60 mm). The dishes were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 6 days for colony growing. Colonies were then fixed with methanol and was stained with Giemsa and the colonies were counted. In order to determine cytotoxicity, survivals were assessed by comparing the colony forming ability of the treated groups to the negative (solvent) control. Precipitation of the test item in the final culture medium was visually examined at beginning and end of the treatments. In addition, pH and osmolality was considered for dose level selection.

GROWING CONDITIONS
The CHO K1 cells for the study were grown in Ham's F12 medium supplemented with 1 % Antibiotic-antimycotic solution (containing 10000 U/ml penicillin, 10 mg/ml streptomycin and 25 µg/ml amphotericin-B) and heat-inactivated bovine serum (final concentration 10 %).
During the 5 treatments with the test item, solvent (negative control) and positive controls, the serum content was reduced to 5 %. The selection medium for TG resistant mutants contained 3.4 µg/ml 6-thioguanine (6-TG).

MAMMALIAN MICROSOMAL FRACTION S9
- Supplier: the S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH.
- Composition: N-2-Hydroxyethylpiperazine-N-2-Ethane Sulphonic Acid 0.2 ml/ml, KCl 0.1 ml/ml, MgCl2 0.1 ml/ml, β-Nicotinamide Adenine Dinucleotide Phosphate 0.1 ml/ml, D-Glucose 6 phosphate (Monosodium salt) 0.1 ml/ml, Ham’s F12 medium 0.1 ml/ml, S9 0.3 ml/ml.
- Storage: before adding to the culture medium the S9 Mix was kept in an ice bath.

ASSAY ACCEPTANCE CRITERIA
The assay was considered valid as all the following criteria were met:
- the mutant frequency of concurrent negative controls is within the 95 % control limits of the distribution of the laboratory’s historical negative control database.
- the positive control chemicals induced a statistically significant and biologically relevant increase in mutant frequency compared to the concurrent negative control. The increases are compatible with the laboratory historical positive control data base.
- adequate number of cells and concentrations were analysable.
- two experimental conditions with and without metabolic activation were tested.
- the highest concentration is adequate.
- the cloning efficiency of the negative controls is between the range of 60 % to 140 % on Day 1 and 70 % to 130 % on Day 8.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- any of the results are outside the distribution of the laboratory historical negative control data (based 95 % control limit),
- the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.

Providing that all acceptability criteria were fulfilled, a test chemical is considered clearly negative because:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data (based 95 % control limit).

Statistics:

Statistical Analysis was performed with SPSS PC+ software for the following data:
- mutant frequency between the negative (solvent) control group and the test item or positive control item treated groups.
- mutant frequency between the laboratory historical negative (solvent) control group and concurrent negative (solvent) control, the test item or positive control item treated groups

Species / strain:

Chinese hamster Ovary (CHO)

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

Additional information on results:

MAIN MUTATION ASSAY
On Day 1, there was very clear evidence of toxicity with the test item in presence and absence of metabolic activation (S9 mix) when compared to the negative (solvent) controls, confirming the response seen in the dose selection cytotoxicity assays. The Day 8 cloning efficiency data indicate that in general the cells had recovered during the expression period.
There were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no significant differences between treatment and control groups and no dose-response relationships were noted. All values were within the range of the laboratory historical solvent control data and no dose-related increase was observed in any of the cultures.

The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures with Ethyl methanesulfonate (1.0 µL/ml) and 7,12-Dimethyl benz[a]anthracene (20 µg/ml). The mutation frequencies of the positive and negative control cultures were consistent with the historical control data from the previous studies performed at this laboratory. Thus, the study is considered valid.

The osmolality and pH values of test item solutions did not show any significant alterations compared to the concurrent control groups in the Pre-test on Toxicity and Main Mutation Assay.

SOLUBILITY AND CONCENTRATION SELECTION
The test item was dissolved in DMSO and the concentrations were selected on the basis of preliminary cytotoxicity investigations (without and with metabolic activation using S9-mix).
A clear solution was obtained up to a concentration of 100 mg/ml. For examined test item concentrations no precipitation in the medium was noted. A Pre-test on Toxicity was performed to establish an appropriate concentration range for the main Mutation Assay, both in the absence and in the presence of metabolic activation (rodent S9-mix).

Summarized results of the pre-test on toxicity (concentration selection) (5h treatment with and without S9 -mix)

Test group	Dose µg/ml	S9-mix	Treatment/ time/ hour	Number of colonies/200cells/dish			Mean	Relative^a survival (%)
				dish 1	dish 2	dish 3
Untreated Control	-	-	5	202	199	200	200.3	101
Solvent Control (DMSO)	-	-	5	196	200	197	197.7	100
Test item	31.3	-	5	199	197	202	199.3	101
	62.5	-	5	196	195	194	195	99
	125	-	5	189	190	188	189	96
	250	-	5	177	176	175	176	89
	500	-	5	148	146	149	147.7	75
	750	-	5	35	36	34	35	18
	1000	-	5	0	0	0	0	0
Untreated Control	–	+	5	187	189	189	188.3	101
Solvent Control (DMSO)	–	+	5	185	187	185	185.7	100
Test item	31.3	+	5	183	182	185	183.3	99
	62.5	+	5	179	179	180	179.3	97
	125	+	5	178	179	176	177.7	96
	250	+	5	176	177	175	176	95
	500	+	5	146	145	144	145	78
	750	+	5	122	118	120	120	65
	1000	+	5	68	66	69	67.7	36

^a: Relative to Solvent Control

Conclusions:

The substance tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency over the background (negative solvent control) in this in vitro test in Chinese hamster ovary cells.

Executive summary:

The test item was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver.

Mutation Assay was performed at the following concentrations and treatment intervals: mutation assay 5-hour treatment period without S9-mix at 125, 250, 500, 650 and 750 µg/ml; mutation assay 5-hour treatment period with S9-mix at 250, 500, 750, 1000 and 1250 µg/ml. In the performed Mutation Assay the concentration levels were chosen mainly based on the cytotoxicity concentration. 

Phenotypic expression was evaluated up to 8 days following exposure.

In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups when was compared to the concurrent and historical control groups and no dose-response relationships were noted.

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

The mutation frequency found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

Conclusion 

The substance tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency over the background (negative solvent control) in this in vitro test in Chinese hamster ovary cells.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September from 10th to 29th, 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

Only four out of five strains reccomanded into the updated OECD guideline were tested

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983

Qualifier:

according to guideline

Guideline:

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

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: the bacterial strains TA 1535, TA 98, and TA 100 were obtained from Ames (University of California, Berkeley, U.S.A.). The bacterial strain TA 1537 was obtained from BASF (D-67063 Ludwigshafen).
- Storage: the strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.
- Preculture: from the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred into 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 μl ampicillin (25 μg/ml) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre: 8 g Merck Nutrient Broth, 5 g NaCl.

Metabolic activation:

with and without

Test concentrations with justification for top dose:

MAIN TEST (exp. I and II): 33, 100, 333, 1000, 2500 and 5000 μg/plate
PRE-EXPERIMENT FOR TOXICITY: 3, 10.0, 133, 100, 333, 1000, 2500 and 5000 μg/plate

Vehicle / solvent:

- Solvent: on the day of the experiment, the test article was dissolved in DMSO.
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria. No precipitation of the test article occurred up to the highest investigated dose.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine // 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION
- Method: experiment I plate incorporation test; experiment II pre-incubation test.
- Incubation: the bacterial culture was incubated in a shaking water bath for 8 hours at 37 °C.
- Overlay Agar content per litre: 6.0 g MERCK Agar Agar, 6.0 g NaCl, 10.5 mg L-Histidine x HCl x H20, 12.2 mg Biotin.
- Sterilisations: performed at 121 °C in an autoclave.

REPLICATES: for each strain and dose level, including the controls three plates were used.

EXPERIMENTAL PERFORMANCE
- The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μl test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control);
500 μl S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation);
100 μl Bacteria suspension (cf. test system, pre-culture of the strains);
2000 μl Overlay agar
- Pre-incubation assay: 100 μl test solution, 500 μl S9 mix / S9 mix substitution buffer and 100 μl bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes.
- Post pre-incubation: after pre-incubation 2.0 ml overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates.
- Incubation: after solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

PRE-EXPERIMENT FOR TOXICITY
- Strains: pre-experiment was performed with strains TA 98 and TA 100.
- Concentrations: 8 concentrations were tested.
- Replicate: mutatation induction with each 3 plates.
- Test conditions: the experimental conditions in this pre-experiment were the same as described for the experiment I (plate incorporation test).
- Toxicity definition: toxicity of the test article can be evidenced by a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
- Anima source: the S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats, strain Wistar Hanlbm (weight approx. 220 - 320 g) which received daily applications of 80 mg/kg b.w. Phénobarbital i.p. dissolved in deionised water and ß-Naphthoflavone orally dissolved in corn oil on three subsequent days.
- Liver collection: after cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The livers were prepared 24 hours after the last treatment.
- Homogenate: it was diluted 1+3 in KCl and centrifuged at 9000 g for 10 minutes at 4° C.
- Storage of stock solution: a stock of the supernatant containing the microsomes was frozen in ampoules and stored at -80 °C. Small numbers of the ampoules are kept at -20 °C for up to one week before use.
- Protein content: the protein content was determined using an analysis kit of Bio-Rad Laboratories. The protein concentration in the S9 preparation was 24.7 mg/ml.
- S9 mix preparation: before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15 % v/v. The composition of the co-factor solution was chosen to yield the following concentrations in the S9 mix: 8mM MgCl2, 33 mM KCl, 5mM Glucose-6-phosphate, 5mM NADP, in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. The S9 mix preparation was performed according to Ames et al.
- Storage: during the experiment the S9 mix was stored in an ice bath.

ACCEPTED CONDITIONS
The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates.
Range of spontaneous reversion frequencies: TA 1535 10-29; TA 1537 5 - 28; TA 98 15 - 57; TA 100 77 - 189.

Evaluation criteria:

A test article is considered positive if either a biologically relevant and reproducible dose related increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.
A test article producing neither a biologically relevant and reproducible dose related increase in the number of revertants nor a biologically relevant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A biologically relevant response is described as follows:
a test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98 and TA 100 or thrice on TA 1535 and TA 1537.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the criteria described above or not.

Species / strain:

S. typhimurium, other: 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

Additional information on results:

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.
The plates incubated with the test article showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.
No relevant increase in revertant colony numbers of any of the four tester strains was observed following treatment at any concentration level, neither in the
presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.
In conclusion, it can be stated that during the described test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Summary of revertants/plate, mean from three plates - without S9

Concentration μg/plate	TA 1535		TA 1537		TA 98		TA 100
	I	II	I	II	I	II	I	II
Negative control	12	28	15	9	17	25	95	101
Solvent control	15	25	12	9	17	20	96	97
Positive control*	927	997	135	114	522	668	700	924
33	16	25	12	15	12	19	109	105
100	17	21	10	12	11	22	101	106
333	18	24	12	14	12	25	97	116
1000	16	28	9	12	12	22	96	121
2500	14	21	8	10	13	21	89	102
5000	18	16	8	11	15	30	86	107

*Sodium azide (10.0 μg/plate) strains TA 1535 and TA 100; 4-nitro-o-phenylene-diamine strains TA 1537 (50 μg/plate) and TA 98 (10.0 μg/plate)

Summary of revertants/plate, mean from three plates - with S9

Concentration μg/plate	TA 1535		TA 1537		TA 98		TA 100
	I	II	I	II	I	II	I	II
Negative control	17	20	13	26	18	30	116	131
Solvent control*	19	23	19	21	18	28	118	110
Positive control	194	191	83	86	426	445	594	453
33	22	19	17	22	19	33	114	132
100	16	20	14	18	17	30	132	123
333	22	22	17	18	15	35	114	131
1000	15	21	14	25	19	28	118	138
2500	16	19	20	18	17	39	119	125
5000	14	21	18	20	15	33	108	133

*2-aminoanthracene (2.5 μg/plate) strains TA 1535, TA 1537, TA 98, and TA 100

Pre-Experiment for Toxicity

Substance	Concentration μg/plate	TA 98		TA 100
		-	+	-	+
Negative control	-	17	18	95	116
Solvent control	-	17	18	96	118
4-NOPD	10.0	522	/	/	/
NaN3	10.0	/	/	700	/
2-AA	2.5	/	426	/	594
Test item	3	17	21	90	112
	10.0	16	17	99	116
	133	12	19	109	114
	100	11	17	101	132
	333	12	15	97	114
	1000	12	19	96	118
	2500	13	17	89	119
	5000	15	15	86	108

Conclusions:

The test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

The study was performed to investigate the potential of test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33, 100, 333, 1000, 2500 and 5000 μg/plate.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. The plates incubated with the test article showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used. No relevant increase in revertant colony numbers of any of the four tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Conclusion

In conclusion, under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No clastogenic activity in the mouse micronucleus test.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From May 19th to July 02nd, 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

The read across approach is detailed into the document attached below.

Principles of method if other than guideline:

The substance was tested for clastogenic effects using the mouse micronucleus test, following the method of Salamone et al (1981). Cyclophosphamide was used as a positive control to assess the sensitivity of the test system.

Ref: Salamone M.F., Heddle J.A. and Katz M., 1981. Mutagenic activity of 41 compounds in the in vivo micronucleus assay. Prog Mlutat Res 1 686-697.

GLP compliance:

no

Type of assay:

mammalian germ cell cytogenetic assay

Species:

mouse

Strain:

C57BL

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Breeding Unit, Alderley Park, Macclesfield, Cheshire.
- Age at study initiation: 6 - 8 weeks old.
- Housing: on arrival the mice were housed ten per cage on mobile mouse racks. After acclimatisation the animals were housed five per cage.
- Diet: food (Porton Combined Diet [PCD] supplied by BP Nutrition Ltd), ad libitum.
- Water: ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C (range 21 - 23 °C).
- Humidity: 58 % (range 39 - 76 %).
- Air changes: 22 air changes per hr.
- Photoperiod: 12 hrs dark / 12 hrs light.

Route of administration:

intraperitoneal

Details on exposure:

DOSING FOR MAIN TEST
Animals were dosed with two consecutive intraperitoneal injections, adminstered 24 hours apart, using 1000 and 625 mg/kg as there were no deaths on the LD50 study.

LD50 DETERMINATION
The LD50 was calculated on the deaths observed over a seven day period and was determined on a single daily intraperitoneal dose.
The compound was administered as single intraperitoneal dose at the dose levles of 7000, 750, 500, 250 and 150 mg/kg. 5 animals per dose lavel. The animals were observed daily for deaths.

Frequency of treatment:

Animals were dosed with two consecutive intraperitoneal injections.

Post exposure period:

5 males and 5 females were sacrified after 24 hours from the start of exposure; further 5 males and 5 females after 48 hours; further 5 males and 5 females after 72 hours.

Dose / conc.:

1 000 other: mg/kg

Dose / conc.:

625 other: mg/kg

Remarks:

The lower dose level was used to ensure a test result in the event of deaths in the top dose group and to allow the observation of a dose response, should the higher dose give a positive result.

No. of animals per sex per dose:

15 males and 15 females

Control animals:

yes

Positive control(s):

Cylophosphamide. Solvent used was physiological saline.
Dose: 60 mg/kg

Tissues and cell types examined:

Polychromatic erythrocytes

Details of tissue and slide preparation:

The animals were killed by cervical dislocation at 24, 48 and 72 hours after the last dose of compound.
Femurs were removed and stripped clean of muscle. The iliac end of the femur was removed and a fine paint brush wetted with a solution of albumen (6 % v/v in saline) was dipped into the marrow canal.
3 to 4 streaks of marrow suspension were then applied to appropriately is labelled clean, dry microscope slides. The slides were allowed to air dry.
e The slides were then stained in Wright's stain using an Ames Hema-Tek staining machine (Hema-Tek, Miles Laboratory Limited, Stoke Court, Stoke Poges, Slough, UK).
Slides were coded and scored blind.
500 polychromatic erythrocytes were examined and the number containing micronuclei scored. The samples were also examined for any evidence of cytotoxicity.

Statistics:

The results were analysed for significant differences from the control group using a one sided Student's 't' test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

deaths at the higher dose level

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Test item gave negative results at all three sampling times, even though there were a number of deaths which occurred in the higher dose level animals. These deaths were unexpected based on the LD50 data and were probably due to administration of the compound as split dose rather than a single treatment. However, the 625 mg/kg dose group would qualify as a maximum tolerated dose at the 48 and 72hr sampling times.

The positive control cyclophoshamide gave an elevated and statistically significant increase in the incidence of micronuclei at both the 24 and 48 hr sampling times.

Incidence of micronuclei/1000 polychromatic erythrocytes at three sampling times

Compound	Dose	Incidence of micronuclei/1000 cells
		24 hrs	48 hrs	72 hrs
Control (corn oil)	0.1 ml/10 g	1.0	2.0	1.9
Cyclophosphamide	60 mg/kg	52.1*	7.6**	2.4
Test item	1000 mg/kg	1.5	1.8	2.0
Test item	625 mg/kg	1.0	2.0	1.3

* Statistically significantly different p < 0.001

** Statistically significantly different p<0.01 (one sided).

Individual animal data

Compound	Dose	Sex	Incidence of micronuclei/1000 cells
			24 hrs	48 hrs	72 hrs
Control (corn oil)	0.1 ml/10 g	Male	0, 2, 2, 0, 1	41, 0, 2, ND, 4	2, 2, 61, 3, 2
		Female	2, 1, 0, 0, 2	3, 4, 0, 2, 2	0, 4, 2, 3, 0
Cyclophosphamide	60 mg/kg	Male	48, ND, 37, 60, 47	10, ND, 8, 1, 6	0, 1, 2, 3, 1
		Female	ND, 65, 47, 63, 50	10, 7, 12, 6, 10	1, 3, 5, 2, 6
Test item	1000 mg/kg	Male	3, 1, 2, 0, ND, 4	3, 1, 2, 0, 4	D, D, D, D, 2
		Female	0, 2, 1, 2, 2	1, 2, D, D, D	D, 2, D, D, D
Test item	625 mg/kg	Male	1, 3, 0, 2, 0	4, 6, 1, 1, 0	0, 0, 3, 2, 0
		Female	1, 0, 2, 0, 1	4, 1, 2, 0, 1	3, 0, 2, 1, 2

ND: sample not counted, insufficient cells or poor sample

D: animal found dead

LD50 DETERMINATION

Compound	Dose (mg/kg)	N. animal	N. deaths
Test item	1000	5	0
Test item	750	5	0
Test item	500	5	0
Test item	250	5	0
Test item	150	5	0

Conclusions:

The results indicate thatthe substance has no clastogenic activity in the mouse micronucleus test.

Executive summary:

The substance was tested for clastogenic effects using the mouse micronucleus test, following the method of Salamone et al (1981). Cyclophosphamide was used as a positive control to assess the sensitivity of the test system.

The substance was tested for clastogenic activity in the mouse micronucleus test using C57BL/6J mice.

Test item did not induce any statistically significant increase in the frequency of micronuclei when compared with control animals.

Throughout the study the positive control substance, cyclophosphamide, gave the expected responses.

Conclusion

These results indicate that substance is not clastogenic in the mouse micronucleus test.

Ref: Salamone M.F., Heddle J.A. and Katz M., 1981. Mutagenic activity of 41 compounds in the in vivo micronucleus assay. Prog Mlutat Res 1 686-697.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Investigation on the genetic toxicity has been performed with the integrated evaluation of the following studies: in vitro Ames tests, in vitro gene mutation on mammalian cells and in vivo chromosomal aberration assay, performed on a structural analogous. The read across approach is detailed into the document attached to the related IUCLID endpoint record (section 7.6.2).

IN VITRO BACTERIA GENE MUTATION ASSAY

The potential of test item to induce gene mutations in bacteria was evaluated by AMES assay, according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100. The assay was performed in two independent experiments both with and without liver microsomal activation, at concentrations up to 5000 μg/plate. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. The plates incubated with the test article showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used. No relevant increase in revertant colony numbers of any of the four tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Thus, the test article resulted not inducing gene mutations by base pair changes or frameshifts in the genome of the strains used.

IN VITRO MAMMALIAN CELL GENE MUTATION ASSAY

The test item was tested in CHO-K1 cells. In the performed Mutation Assay the concentration levels were chosen mainly based on the cytotoxicity concentration. In all the experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups when was compared to the concurrent and historical control groups and no dose-response relationships were noted.

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

The mutation frequency found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study was considered valid and the substance tested resulted not able to induce increases in mutant frequency, both without and with metabolic activation (S9 mix).

IN VIVO CHROMOSOMAL ABERRATION

The chromosomal aberration potential of the substance under investigation has been investigated taken into consideration the available data on a structural analogous.

The analogous was tested for clastogenic effects using the mouse micronucleus test. The substance was tested in the mouse micronucleus test using C57BL/6J mice. Test item did not induce any statistically significant increase in the frequency of micronuclei when compared with control animals.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 1272/2008, for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The available information suggest that test substance did not show any reasons of concern from the genotoxicity point of view.

 

In conclusion, the substance does not meet the criteria to be classified for genetic toxicity according to the CLP Regulation (EC) No 1272/2008.