Ferrate(4-), hexakis(cyano-C)-, Et 2-[6-(ethylamino)-3-(ethylimino)-2,7-dimethyl-3H-xanthen-9-yl]benzoate copper(2+) salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium /Escherichia coli reverse mutation assay with and without metabolic activation.

 

In vitro mammalian chromosome aberration study:

The test chemical did not induce chromosomal aberrations in the cultured in the mammalian cell line with and without metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

In vitro mammalian cell gene mutation:

Due to COVID-19, the study is delayed and the related information is expected to be provided by 15 February 2023. Please also refer to the attachment.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report.

Qualifier:

according to guideline

Guideline:

other: * OECD No. 471 (July 21, 1997) * EEC Directive 92/69, B14 and B13 (December 1992)

Principles of method if other than guideline:

Standard plate test and Preincubation test was performed to determine the mutagenic nature of the test chemical

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella strains and Tryptophan for E. coli strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

other: histidine auxotrophs (his-)

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

other: tryptophan auxotrophy (trp-)

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S-9 mix

Test concentrations with justification for top dose:

0, 20, 100, 500, 2,500 or 5,000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was selected as the vehicle, which had been
demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Untreated negative controls:

yes

Remarks:

Additional plates are treated with soft agar, S-9 mix, buffer, vehiele or the test substance but without the addition of tester strains

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

with S9

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

strains: TA 1535, TA 100, TA 1537, TA 98: 2.5 µg/plate, dissolved in DMSO; Strain: Escherichia coli WP2 uvrA: 60 µg/plate, dissolved in DMSO

Untreated negative controls:

yes

Remarks:

Additional plates are treated with soft agar, S-9 mix, buffer, vehicle or the test substance but without the addition of tester strains

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

without S9

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosuguanidine (MNNG)

Remarks:

Strains: TA 1535, TA 100: 5 µg/plate, dissolved in DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine (NOPD)

Remarks:

Strain: TA 98: 10 µg/plate, dissolved in DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Strain: TA 1537: 100 µg/plate, dissolved in DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Strain: E. coli WP2 uvrA: 10 µg/plate, dissolved in DMSO

Details on test system and experimental conditions:

Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S-9 mix)

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: For PIT: 20 mins
- Exposure duration: 48 - 72 hours
- Expression time (cells in growth medium): 48 - 72 hours
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):

SPINDLE INHIBITOR (cytogenetic assays):

STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: Triplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

NUMBER OF CELLS EVALUATED:

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

- OTHER:

Rationale for test conditions:

No data

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met:

A dose-related and reproducible increase in the nuinber of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adcling a metabolizing system.

A test substance is generally considered nonmutagenic in this test if:

The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Statistics:

No data

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:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: Precipitation of the test substance was found from about 500 µg/plate onward.
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: No data

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data

Remarks on result:

other: No mutagenic potential

Standard plate assay

Table: Mutagenic potential

Strain TA1535 Without S9

 

Dose	Rev	M	SD	FAC
DMSO	16	20	3	1.0
	21
	22
20 µg	20	20	1	1.0
	19
	20
100 µg	21	20	1	1.0
	19
	19
500 µg	18P	19	1	1.0
	20P
	19P
2500 µg	5P	5	1	0.3
	4P
	6P
5000 µg	5P	3	2	0.2
	3P
	1P
MNNG 5.0µg	1004	1002	24	50.9
	977
	1024
2-AA 2.5 µg

Strain TA1535 With S9

Dose	Rev	M	SD	FAC	TITRE
DMSO	18	19	2	1.0	29
	18				22
	21				27
20 µg	19	18	1	1.0
	18
	18
100 µg	15	14	1	0.7
	13
	14
500 µg	17P	17	2	0.9
	19P
	19P
2500 µg	7P	6	2	0.3	15
	8P				14
	4P				20
5000 µg	0P				0
	0P				0
	0P				0
2-AA	176	171	14	9.0
	155
	181

 

 

Strain TA100 Without S9

 

Dose	Rev	M	SD	FAC
DMSO	124	115	8	1.0
	108
	113
20 µg	104	101	8	0.9
	107
	94
100 µg	98	102	7	0.9
	110
	99
500 µg	55P	60	5	0.5
	61P
	65P
2500 µg	11P	8	3	0.1
	5P
	8P
5000 µg	0P
	0P
	0P
MNNG 5.0µg	1312	1319	49	11.5
	1273
	1371
2-AA 2.5 µg

 

Strain TA100 With S9

Dose	Rev	M	SD	FAC	TITRE
DMSO	112	111	9	1.0	22
	102				21
	120				29
20 µg	110	110	2	1.0
	108
	111
100 µg	104	114	8	1.0
	118
	119
500 µg	57P	52	17	0.5
	66P
	33P
2500 µg	5P	5	3	0.0	1
	8P				2
	2P				5
5000 µg	0P				0
	0P				0
	0P				0
2-AA 2.5 µg	1135	1274	180	11.4
	1477
	1210

 

Strain TA1537 Without S9

 

Dose	Rev	M	SD	FAC
DMSO	9	10	1	1.0
	10
	10
20	9	9	1	1.0
	9
	10
100	9	10	1	1.0
	10
	11
500	5P	5	2	0.6
	7P
	4P
2500	1P	1	0	0.1
	1P
	1P
5000	0P
	0P
	0P
AAC 100 µg	621	649	27	67.2
	674
	653
2-AA 2.5 µg

 

Strain TA1537 With S9

Dose	Rev	M	SD	FAC	TITRE
DMSO	11	11	1	1.0	38
	11				29
	10				28
20 µg	9	9	2	0.9
	8
	11
100 µg	7	8	1	0.8
	9
	9
500 µg	8P	8	0	0.8
	8P
	8P
2500 µg	1P	1	1	0.1	2
	1P				5
	2P				1
5000 µg	0P				0
	0P				0
	0P				0
AAC 100 µg
2-AA 2.5 µg	173	185	13	17.5
	198
	185

 

Strain TA 98 Without S9

 

Dose	Rev	M	SD	FAC
DMSO	20	27	7	1.0
	29
	33
20 µg	24	21	3	0.8
	19
	21
100 µg	21	23	2	0.9
	24
	25
500 µg	21P	20	1	0.7
	19P
	19P
2500 µg	2P	3	1	0.1
	3P
	4P
5000 µg	0P
	0P
	0P
NOPD 10 µg	937	945	25	34.6
	925
	973
2-AA 2.5 µg

 

Strain TA 98 With S9

Dose	Rev	M	SD	FAC	TITRE
DMSO	42	38	3	1.0	17
	36				24
	36				24
20 µg	35	33	2	0.9
	31
	32
100 µg	34	33	2	0.9
	31
	34
500 µg	20P	22	2	0.6
	24P
	23P
2500 µg	11P	8	3	0.2	2
	8P				2
	6P				2
5000 µg	0P				0
	0P				0
	0P				0
NOPD 10 µg
2-AA 2.5 µg	936	943	13	24.8
	925
	958

 

 

StrainE.coli WP2uvrA Without S9

 

Dose	Rev	M	SD	FAC
DMSO	36	36	1	1.0
	37
	35
20 µg	31	32	4	0.9
	37
	29
100 µg	34	32	3	0.9
	33
	29
500 µg	29P	32	3	0.9
	31P
	35P
2500 µg	25P	28	3	0.8
	30P
	30P
5000 µg	15P	18	3	0.5
	17P
	21P
ENNG 10 µg	999	1009	18	28.0
	998
	1029
2-AA 60 µg

 

Strain E.coli WP2 uvrAWith S9

Dose	Rev	M	SD	FAC	TITRE
DMSO	39	40	2	1.0	40
	42				45
	39				39
20 µg	35	32	3	0.8
	31
	29
100 µg	37	35	3	0.9
	37
	31
500 µg	31P	31	1	0.8
	31P
	30P
2500 µg	20P	24	4	0.6	8
	26P				6
	27P				7
5000 µg	12P	12	1	0.3	0
	12P				0
	13P				0
ENNG 10 µg
2-AA 60 µg	326	314	40	7.9
	347
	269

Conclusions:

According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay with and without metabolic activation.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. The study was performed using Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA. The test chemical was dissolved in DMSO and used at dose level of 0, 20, 100, 500, 2,500 or 5,000 µg/plate. Concurrent solvent and positive control chemicals were also included in the study. According to the results of the present study, the test substance pigment red 169 is not mutagenic in the Salmonella typhimurium /Escherichia coli reverse mutation assay with and without metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 26 May 2022 to 03 August 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Human Peripheral Blood Lymphocytes

Details on mammalian cell type (if applicable):

Blood Procured from Sanjeevini blood bank (Vidya Nagar, Tumkur) from male healthy adult young 22 years and 26 years (male) of age with no known illness or recent exposure to genotoxic chemicals or radiation was used.

Metabolic activation:

with and without

Metabolic activation system:

Sodium phenobarbitone and β-Naphthoflavone induced rat liver S9 homogenate was used as the metabolic activation system. The S9 homogenate was prepared from liver of male Wistar Rats induced with intraperitoneal injection of sodium phenobarbtone and β-naphthoflavone at 16 mg/mL and 20 mg/mL respectively for 3 days prior to sacrifice. The S9 homogenate was prepared and stored in the test facility at -80±10ºC until use. Batch of S9 homogenate was assessed for sterility, protein content and for its ability to metabolize the promutagens 2-Aminoanthracene and Benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 tester strain.
1 mL of S9 homogenate was thawed immediately before use and mixed with the 9 mL of co-factor solution containing 4 mM NADP, 5 mM Glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in Phosphate Buffer Saline (PBS) of pH 7.35 and 7.34 for initial cytotoxicity test and chromosomal aberration test.

Test concentrations with justification for top dose:

The test item formed suspension in DMSO at 50 mg/mL. Precipitation and pH test was conducted at 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/mL. Post 23 hours and 20 minutes of incubation, heavy precipitation was observed at 0.5 mg/mL, moderate precipitation was observed at 0.25 mg/mL. No precipitation was observed at 0.125, 0.0625 and 0.03125 mg/mL. No change in pH was observed in any of the concentrations tested upto 0.5 mg/mL. Hence, 0.25 mg/mL was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were 0.015625, 0.03125, 0.0625 and 0.125 mg/mL of test item.
In initial cytotoxicity test, the percentage reduction in Mitotic Index was in the range of 45.62 to 47.71% at 0.25 mg/mL in presence of metabolic activation (short term), in absence of metabolic activation (short term) in absence of metabolic activation (long term), respectively. As the percentage reduction in mitotic index was not more than 45±5% at 0.25 mg/mL, same was selected as the highest concentration for the chromosomal aberration test. Other concentrations tested were 0.125 and 0.0625 mg/mL.

Vehicle / solvent:

A solubility test was conducted to determine the maximum concentration or workable solution of the test item in the vehicle compatible with the test system at 200 mg/mL. Solubility was conducted using distilled water, dimethyl sulphoxide, ethanol, acetone, acetonitrile and n-hexane. The test item formed suspension in dimethyl sulphoxide at 50 mg/mL. Based on the results of solubility test and compatibility dimethyl sulphoxide was selected as vehicle for test item formulation.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Not specified

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 item concentrations exhibits a statistically significant increase compared with the concurrent negative/vehicle control.
The increase is dose-related when evaluated with an appropriate trend test.
any of the results are outside the distribution of the historical negative/vehicle control data (e.g. Poisson-based 95% control limits).
When all of these criteria are met, the test item is then considered to be able to induce chromosomal aberrations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in all experimental conditions examined:
None of the test item concentrations exhibits a statistically significant increase compared with the concurrent negative/vehicle control.
There is no concentration-related increase when evaluated with an appropriate trend test.
All results are inside the distribution of the historical vehicle control data.
Based on the above, the test item is then considered unable to induce chromosomal aberrations in cultured mammalian cells in this test system.

Key result

Species / strain:

lymphocytes: Human Peripheral Blood Lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Initial Cytotoxicity Test
The percentage reduction in Mitotic Index at 0.015625, 0.03125, 0.0625, 0.125 and 0.25 mg/mL was 7.17, 13.28, 23.37, 34.13 and 46.48% in presence of metabolic activation (short term), 7.73, 12.63, 23.58, 33.51 and 45.62% in absence of metabolic activation (short term) and 8.36, 14.29, 24.80, 34.10 and 47.71% in absence of metabolic activation (long term) respectively.

Chromosomal Aberration Test
The reduction in MI observed at 0.25 mg/mL was 46.21% in the presence of metabolic activation and 46.51% in the absence of metabolic activation for short term treatments. Similarly, 48.55% in the absence of metabolic activation system for long term treatment.
The observed mean percent aberrated cells at 0.0625, 0.125 and 0.25 mg/mL were 1.00, 1.00 and 1.33 in the presence of metabolic activation (short term treatment 3 to 6 hours), 1.00, 1.00 and 1.00 in the absence of metabolic activation (short term treatment 3 to 6 hours) and 1.00, 1.00 and 1.33 in the absence of metabolic activation (long term 20 to 24 hours) respectively.
There was no statistically significant increase in the number of aberrated cells when compared with vehicle control at any of the concentration levels tested.
Positive control, 10 µg/mL of Cyclophosphamide Monohydrate, in the presence of metabolic activation (3 to 6 hours), induced 9.67% of aberrated cells which was statistically significant compared to the vehicle control (1.00%). The reduction in mitotic index was 10.35% when compared with the vehicle control for short term treatment.
Positive control, 0.05 µg/mL of Mitomycin-C, in the absence of metabolic activation (3 to 6 hours), induced 10.00% of aberrated cells which was statistically significant to the vehicle control (1.33%). The reduction in mitotic index observed was 9.82% when compared with the vehicle control for short term treatment.
Positive control, 0.05 µg/mL of Mitomycin-C, in the absence of metabolic activation (20 to 24 hours), induced 10.34% of aberrated cells which was statistically significant to the vehicle control (1.33%). The reduction in mitotic index observed was 11.13% when compared with the vehicle control for long term treatment.

Remarks on result:

other: No mutagenic potential was observed.

Conclusions:

Based on the results obtained, the test item, Pigment Red 169 is considered non-clastogenic up to the concentration of 0.25 mg/mL both in the presence and absence of metabolic activation under the presented test conditions.

Executive summary:

The cytogenetic effect of the test chemical Pigment Red 169 (CAS no.: 12237-63-7) on cultured cells, chromosomal aberration test was performed using Human Peripheral Blood Lymphocytes. The study was performed according to Guidelines for OECD Guideline No. 473. Human Peripheral Blood Lymphocytes was used as the test cell line. Sodium phenobarbitone and β-Naphthoflavone induced rat liver S9 homogenate was used as the metabolic activation system. The S9 homogenate was prepared from liver of male Wistar Rats induced with intraperitoneal injection of sodium phenobarbtone and β-naphthoflavone at 16 mg/mL and 20 mg/mL respectively for 3 days prior to sacrifice. The S9 homogenate was prepared and stored in the test facility at -80±10ºC until use. 1 mL of S9 homogenate was thawed immediately before use and mixed with the 9 mL of co-factor solution containing 4 mM NADP, 5 mM Glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in Phosphate Buffer Saline (PBS) of pH 7.35 and 7.34 for initial cytotoxicity test and chromosomal aberration test. The test item formed suspension in DMSO at 50 mg/mL. Precipitation and pH test was conducted at 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/mL. Post 23 hours and 20 minutes of incubation, heavy precipitation was observed at 0.5 mg/mL, moderate precipitation was observed at 0.25 mg/mL. No precipitation was observed at 0.125, 0.0625 and 0.03125 mg/mL. No change in pH was observed in any of the concentrations tested upto 0.5 mg/mL. Hence, 0.25 mg/mL was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were 0.015625, 0.03125, 0.0625 and 0.125 mg/mL of test item. In initial cytotoxicity test, the percentage reduction in Mitotic Index was in the range of 45.62 to 47.71% at 0.25 mg/mL in presence of metabolic activation (short term), in absence of metabolic activation (short term) in absence of metabolic activation (long term), respectively. As the percentage reduction in mitotic index was not more than 45±5% at 0.25 mg/mL, same was selected as the highest concentration for the chromosomal aberration test. Other concentrations tested were 0.125 and 0.0625 mg/mL. Based on the results of solubility test and compatibility dimethyl sulphoxide was selected as vehicle for test item formulation. The reduction in MI observed at 0.25 mg/mL was 46.21% in the presence of metabolic activation and 46.51% in the absence of metabolic activation for short term treatments. Similarly, 48.55% in the absence of metabolic activation system for long term treatment. The observed mean percent aberrated cells at 0.0625, 0.125 and 0.25 mg/mL were 1.00, 1.00 and 1.33 in the presence of metabolic activation (short term treatment 3 to 6 hours), 1.00, 1.00 and 1.00 in the absence of metabolic activation (short term treatment 3 to 6 hours) and 1.00, 1.00 and 1.33 in the absence of metabolic activation (long term 20 to 24 hours) respectively. There was no statistically significant increase in the number of aberrated cells when compared with vehicle control at any of the concentration levels tested. Based on the results obtained, the test item, Pigment Red 169 is considered non-clastogenic up to the concentration of 0.25 mg/mL both in the presence and absence of metabolic activation under the presented test conditions. Hence, the test chemical can be considered to be non mutagenic to Human Peripheral Blood Lymphocytes.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

other justification

Justification for data waiving:

other:

Justification for type of information:

The study is ongoing and this information will be submitted later based on ECHA communication/decision number CCH-D-2114557769-27-01/F.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on the data from various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE derived based on the experimental data from various test chemicals

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

6 and 7

Details on mammalian cell type (if applicable):

- Type and identity of media: Fischer's medium
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

6. S9 metabolic activation system was prepared from the livers of Aroclor 1254 induced male Fischer 344/N rats
7. Freshly prepared 89 metabolic activation factors were obtained from the liver of either Aroclor 1254-induced or noninduced male F344 rats

Test concentrations with justification for top dose:

6. Without S9: Trial 1: 0, 5, 10, 20, 30 , 40 or 60 nL/mL
Trial 2: 0, 20, 30, 40, 50, 60 or 80 nL/mL
Trial 3: 0, 10, 20, 30, 40, 60 or 80 nL/mL
Trial 4: 0, 10, 20, 30 or 40 nL/mL
With S9: Trial 1: 0, 30, 40, 50, 60, 80 or 100 nL/mL
Trial 2: 0, 30, 40, 50, 60, 80 or 100 nL/mL
7. Without S9: Trial 1: 0, 1.25, 2.5, 3.75, 5.0 or 7.5 µg/mL
Trial 2: 0, 2, 3, 4, 5, 6 or 8 µg/mL
Trial 3: 0, 2, 3, 4, 5, 6, 8 or 10 µg/mL
With S9: Trial 1: 0, 2.5, 5.0, 7.5, 10 or 15 µg/mL

Vehicle / solvent:

6 and 7. - Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The test chemical was soluble in ethanol

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

6 and 7

Details on test system and experimental conditions:

6. METHOD OF APPLICATION: in medium
Cells at start of experiment: 6000000 cells

DURATION
- Preincubation period: No data
- Exposure duration: 4 hrs
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): 10-12 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: 4 trial were performed without S9 and 2 trials were performed with S9.

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data
7. METHOD OF APPLICATION: in medium
Cells at start of experiment: 6000000 cells

DURATION
- Preincubation period: No data
- Exposure duration: 4 hrs
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): 10-12 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

6 and 7. The responses had to be significant (P<0.05) for the test chemical to be considered positive, i.e., capable of inducing TFT resistance. A single significant response led to a "questionable" conclusion, and the absence of both a trendand peak response resulted in a "negative" call

Statistics:

6 and 7. All data were evaluated statistically for trend and peak responses.

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

6

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

7

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

No data

Remarks on result:

other: No mutagenic potential

Conclusions:

Based on the presented data, the test chemical is regarded to be non-mutagenic in mouse lymphoma cells both in the presence and absence of S9 metabolic activation system.

Executive summary:

Data available for the test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Mouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in ethanol and used at dose level of 0, 5, 10, 20, 30 , 40 or 60 nL/mL (trial 1), 0, 20, 30, 40, 50, 60 or 80 nL/mL (trial 2), 0, 10, 20, 30, 40, 60 or 80 nL/mL (trial 3) or 0, 10, 20, 30 or 40 nL/mL (trial 4) without S9 and 0, 30, 40, 50, 60, 80 or 100 nL/mL (trial 1) and 0, 30, 40, 50, 60, 80 or 100 nL/mL (trial 2) with S9. Mouse lymphoma L5178Y cells were maintained at 37˚C as suspension cultures in supplemented Fischer's medium; normal cycling time was approximately 10 hours. To reduce the number of spontaneously occurring trifluorothymidine-resistant cells, subcultures were exposed to medium containing THMG (thymidine, hypoxanthine, methotrexate, and glycine) for 1 day, to medium containing THG (thymidine, hypoxanthine, and glycine) for 1 day, and to normal medium for 3 to 5 days. For cloning, the horse serum content was increased and Noble agar was added. All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated. Treated cultures contained 6 x 10⁶cells in 10 mL medium. This volume included the S9 fraction in those experiments performed with metabolic activation. Incubation with butyl benzyl phthalate continued for 4 hours, at which time the medium plus butyl benzyl phthalate was removed and the cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48-hour expression period, 3 x 10⁶cells were plated in medium and soft agar supplemented with trifluorothymidine (TFT) for selection of TFT-resistant (TK-1) cells; 600 cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37" C in 5% CO2 for 10 to 12 days. The test was initially performed without 59.' If a clearly positive response was not obtained, the test was repeated using freshly prepared S9 from the livers of Aroclor 1254 induced maleFischer344/N rats. Based on the observations made, thetest chemical did not induce gene mutation inMouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

Above study is supported with another In vitro mammalian cell gene mutation assay performed to determine the mutagenic nature of the test chemical. The study was performed using Mouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in ethanol and used at dose level of 0, 1.25, 2.5, 3.75, 5.0 or 7.5µg/mL (trial 1), 0, 2, 3, 4, 5, 6 or 8µg/mL (trial 2), 0, 2, 3, 4, 5, 6, 8 or 10µg/mL (trial 3) without S9 and 0, 2.5, 5.0, 7.5, 10 or 15µg/mL (trial 1) with S9. Mouse lymphoma L5178Y cells were maintained at 37˚C as suspension cultures in supplemented Fischer's medium; normal cycling time was approximately 10 hours. To reduce the number of spontaneously occurring trifluorothymidine-resistant cells, subcultures were exposed to medium containing THMG (thymidine, hypoxanthine, methotrexate, and glycine) for 1 day, to medium containing THG (thymidine, hypoxanthine, and glycine) for 1 day, and to normal medium for 3 to 5 days. For cloning, the horse serum content was increased and Noble agar was added. All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated. Treated cultures contained 6 x 10⁶cells in 10 mL medium. This volume included the S9 fraction in those experiments performed with metabolic activation. Incubation with butyl benzyl phthalate continued for 4 hours, at which time the medium plus butyl benzyl phthalate was removed and the cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48-hour expression period, 3 x 10⁶cells were plated in medium and soft agar supplemented with trifluorothymidine (TFT) for selection of TFT-resistant (TK-1) cells; 600 cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37˚C in 5% CO2 for 10 to 12 days. Based on the observations made, thetest chemical did not induce gene mutation in Mouse lymphoma L5178Y cells in the presence S9 metabolic activation system. It however induced gene mutation I the cell line in the absence of S9 metabolic activation system.

Based on the presented data, the test chemical is regarded to be non-mutagenic in mouse lymphoma cells both in the presence and absence of S9 metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Genetic Toxicity (In-Vivo):

Based on all the available observations and results, it was concluded that the test chemical was non-mutagenic (negative) in sex-linked recessive lethal (SLRL) mutation induction test in Drosophila melanogaster.

Link to relevant study records

Reference

Endpoint:

in vivo insect germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from a publication

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.20 (Sex-linked Recessive Lethal Test in Drosophila melanogaster)

Principles of method if other than guideline:

Equivalent or similar to EU Method B.20 (Sex-linked Recessive Lethal Test in Drosophila melanogaster)

GLP compliance:

not specified

Type of assay:

Drosophila SLRL assay

Species:

Drosophila melanogaster

Strain:

not specified

Sex:

male

Details on test animals or test system and environmental conditions:

The males to be treated were collected from a Canton-S wild-type stock within 8 hr of emergence and kept on regular culture medium until exposure.

Route of administration:

other: The chemical was first tested by feed. If non-mutagenic by this route, the chemical was injected.

Vehicle:

Dietary exposure: 5% aqueous sucrose
Injection: 0.7% aqueous NaCl

Details on exposure:

Males to be fed were shaken into vials containing glass fiber filter material that was soaked with the feeding solution or mixture. At 24 hr and again at 48 hr, the flies were transferred to vials with freshly prepared feeding mixture and were mated at 72 hr. Males to be injected were held on regular food for 1-3 days. They were then injected with 0.7% NaCl solution containing the test chemical. They were held for 24 hr to recover, then mated.

Duration of treatment / exposure:

1-2 days

Frequency of treatment:

Feeding: 1-2 days of exposure
Injection: one injection

Post exposure period:

Injection: 24 hr recovery period

Remarks:

0 and 50000 ppm (feed)
0 and 10000 ppm (injection)

No. of animals per sex per dose:

no data

Control animals:

yes, concurrent vehicle

yes, plain diet

Positive control(s):

AF-2

Tissues and cell types examined:

Germ cells

Evaluation criteria:

Generally, a test was considered positive if the frequency of lethals in the treated series exceeded 0.2% over the control frequency.

Statistics:

Data were then tested for significance using the normal test as described by Margolin et al (Environ Mutagen 1983; 5: 705-16). A SLRL result was called negative if P > 06, equivocal if P = 0.04-0.06, either questionable or positive if P = 0.01-0.04 (depending on the control frequency), and positive if P < 0.01.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

19% mortality bu injection, 17% mortality by feeding

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks:

AF-2

Remarks on result:

other: Negative

Additional information on results:

The sex-linked recessive lethal frequency was similar between insects fed at 0 and 50000 ppm (0.09% vs 0.06%, respectively) and similar between insects injected at 0 and 10000 ppm (0.05% vs 0.08%, respectively). Mortality was in the range of 17-19% for insects treated with the test chemical. No cases of sterility were observed in the study.

Conclusions:

The test chemical was non-mutagenic (negative) in sex-linked recessive lethal (SLRL) mutation induction test in Drosophila melanogaster.

Executive summary:

The test chemical was tested for sex-linkedrecessive lethal (SLRL) mutation inductionin Drosophila melanogaster. Initially, the test chemical was tested by feeding, whenno significant mutagenicitywas observed,it was re-tested by injection.Doses used in the mutagenicity test was selected based on the results of a preliminary dose-finding study. The test substance was dissolved in water anddiluted with aqueous 5%sucrose for feedingand with aqueous 0.7% NaCl for injection. Toxicity tests were performed both forfeeding and injection treatments using a series of concentrations.Doses were chosen to induce mortalityof about 30% after 72 hoursof feeding or 24 hoursafter injection. The males to be treated werekept on regular culture medium until exposure. Males to befed were shaken into vials containing glass fiber filter material that was soaked withthe feeding solution ortest chemicalmixture. At 24 hoursand again at 48hours, the flies were transferredto vials with freshly prepared feeding mixture and were mated at 72 hours.Males to be injected were held on regular food for 1-3 days. They were theninjected with0.7% NaCl solution containing the test chemical. They were held for24 hoursto recover, then mated. The flies for the SLRL assay were exposed for 4hours. Treated and control males were matedindividuallyto threeharems ofBascvirgin females to produce three broods of 3,2, and 2 days.Thus,primarily post-meiotic germ cells were tested. To reduce the chances of recoveringseveral lethals from the same male, no more than 40 F1 females were mated individuallyfrom each brood of each male. Thus, no more than 120 chromosomes weretested from each P1male. F2 cultures were scored as presumptive lethals if thenumber of wild-type males recovered was 0, 1, or fewer than5% of the number ofBascmales (or Basc/+females). Presumptive lethals were confirmed by repeating the matings and scoring the F3.The test chemical was used at doses of 0 (solvent control) and 50000 ppm for feeding experiments and 10000 ppm for injection. No mutagenic response was observed in broods examined when the test chemical solution was fed or injected to male flies. The percents of lethals (total lethals) were statistically comparable in treated and control groups using both treatment methods. The mortality was 19 and 17% in experiments with injection and feeding treatment, respectively In conclusion, the test chemical was non-mutagenic (negative) insex-linkedrecessive lethal (SLRL) inductiontest in Drosophila melanogaster.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames assay:

 

1. The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. The study was performed using Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA. The test chemical was dissolved in DMSO and used at dose level of 0, 20, 100, 500, 2,500 or 5,000 µg/plate. Concurrent solvent and positive control chemicals were also included in the study. According to the results of the present study, the test substance pigment red 169 is not mutagenic in the Salmonella typhimurium /Escherichia coli reverse mutation assay with and without metabolic activation.

 

In vitro mammalian chromosome aberration study:

 

The cytogenetic effect of the test chemical Pigment Red 169 (CAS no.: 12237-63-7) on cultured cells, chromosomal aberration test was performed using Human Peripheral Blood Lymphocytes. The study was performed according to Guidelines for OECD Guideline No. 473. Human Peripheral Blood Lymphocytes was used as the test cell line. Sodium phenobarbitone and β-Naphthoflavone induced rat liver S9 homogenate was used as the metabolic activation system. The S9 homogenate was prepared from liver of male Wistar Rats induced with intraperitoneal injection of sodium phenobarbtone and β-naphthoflavone at 16 mg/mL and 20 mg/mL respectively for 3 days prior to sacrifice. The S9 homogenate was prepared and stored in the test facility at -80±10ºC until use. 1 mL of S9 homogenate was thawed immediately before use and mixed with the 9 mL of co-factor solution containing 4 mM NADP, 5 mM Glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in Phosphate Buffer Saline (PBS) of pH 7.35 and 7.34 for initial cytotoxicity test and chromosomal aberration test. The test item formed suspension in DMSO at 50 mg/mL. Precipitation and pH test was conducted at 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/mL. Post 23 hours and 20 minutes of incubation, heavy precipitation was observed at 0.5 mg/mL, moderate precipitation was observed at 0.25 mg/mL. No precipitation was observed at 0.125, 0.0625 and 0.03125 mg/mL. No change in pH was observed in any of the concentrations tested upto 0.5 mg/mL. Hence, 0.25 mg/mL was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were 0.015625, 0.03125, 0.0625 and 0.125 mg/mL of test item. In initial cytotoxicity test, the percentage reduction in Mitotic Index was in the range of 45.62 to 47.71% at 0.25 mg/mL in presence of metabolic activation (short term), in absence of metabolic activation (short term) in absence of metabolic activation (long term), respectively. As the percentage reduction in mitotic index was not more than 45±5% at 0.25 mg/mL, same was selected as the highest concentration for the chromosomal aberration test. Other concentrations tested were 0.125 and 0.0625 mg/mL. Based on the results of solubility test and compatibility dimethyl sulphoxide was selected as vehicle for test item formulation. The reduction in MI observed at 0.25 mg/mL was 46.21% in the presence of metabolic activation and 46.51% in the absence of metabolic activation for short term treatments. Similarly, 48.55% in the absence of metabolic activation system for long term treatment. The observed mean percent aberrated cells at 0.0625, 0.125 and 0.25 mg/mL were 1.00, 1.00 and 1.33 in the presence of metabolic activation (short term treatment 3 to 6 hours), 1.00, 1.00 and 1.00 in the absence of metabolic activation (short term treatment 3 to 6 hours) and 1.00, 1.00 and 1.33 in the absence of metabolic activation (long term 20 to 24 hours) respectively. There was no statistically significant increase in the number of aberrated cells when compared with vehicle control at any of the concentration levels tested. Based on the results obtained, the test item, Pigment Red 169 is considered non-clastogenic up to the concentration of 0.25 mg/mL both in the presence and absence of metabolic activation under the presented test conditions. Hence, the test chemical can be considered to be non mutagenic to Human Peripheral Blood Lymphocytes.

 

In vitro mammalian cell gene mutation assay:

 

1. Due to COVID-19, the study is delayed and the related information is expected to be provided by 15 February 2023. Please also refer to the attachment.

 

2. Data available for the test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

 

In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Mouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in ethanol and used at dose level of 0, 5, 10, 20, 30 , 40 or 60 nL/mL (trial 1), 0, 20, 30, 40, 50, 60 or 80 nL/mL (trial 2), 0, 10, 20, 30, 40, 60 or 80 nL/mL (trial 3) or 0, 10, 20, 30 or 40 nL/mL (trial 4) without S9 and 0, 30, 40, 50, 60, 80 or 100 nL/mL (trial 1) and 0, 30, 40, 50, 60, 80 or 100 nL/mL (trial 2) with S9. Mouse lymphoma L5178Y cells were maintained at 37˚C as suspension cultures in supplemented Fischer's medium; normal cycling time was approximately 10 hours. To reduce the number of spontaneously occurring trifluorothymidine-resistant cells, subcultures were exposed to medium containing THMG (thymidine, hypoxanthine, methotrexate, and glycine) for 1 day, to medium containing THG (thymidine, hypoxanthine, and glycine) for 1 day, and to normal medium for 3 to 5 days. For cloning, the horse serum content was increased and Noble agar was added. All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated. Treated cultures contained 6 x 10⁶cells in 10 mL medium. This volume included the S9 fraction in those experiments performed with metabolic activation. Incubation with butyl benzyl phthalate continued for 4 hours, at which time the medium plus butyl benzyl phthalate was removed and the cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48-hour expression period, 3 x 10⁶cells were plated in medium and soft agar supplemented with trifluorothymidine (TFT) for selection of TFT-resistant (TK-1) cells; 600 cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37" C in 5% CO2 for 10 to 12 days. The test was initially performed without 59.' If a clearly positive response was not obtained, the test was repeated using freshly prepared S9 from the livers of Aroclor 1254 induced maleFischer344/N rats. Based on the observations made, thetest chemical did not induce gene mutation inMouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

 

Above study is supported with another In vitro mammalian cell gene mutation assay performed to determine the mutagenic nature of the test chemical. The study was performed using Mouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in ethanol and used at dose level of 0, 1.25, 2.5, 3.75, 5.0 or 7.5µg/mL (trial 1), 0, 2, 3, 4, 5, 6 or 8µg/mL (trial 2), 0, 2, 3, 4, 5, 6, 8 or 10µg/mL (trial 3) without S9 and 0, 2.5, 5.0, 7.5, 10 or 15µg/mL (trial 1) with S9. Mouse lymphoma L5178Y cells were maintained at 37˚C as suspension cultures in supplemented Fischer's medium; normal cycling time was approximately 10 hours. To reduce the number of spontaneously occurring trifluorothymidine-resistant cells, subcultures were exposed to medium containing THMG (thymidine, hypoxanthine, methotrexate, and glycine) for 1 day, to medium containing THG (thymidine, hypoxanthine, and glycine) for 1 day, and to normal medium for 3 to 5 days. For cloning, the horse serum content was increased and Noble agar was added. All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated. Treated cultures contained 6 x 10⁶cells in 10 mL medium. This volume included the S9 fraction in those experiments performed with metabolic activation. Incubation with butyl benzyl phthalate continued for 4 hours, at which time the medium plus butyl benzyl phthalate was removed and the cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48-hour expression period, 3 x 10⁶cells were plated in medium and soft agar supplemented with trifluorothymidine (TFT) for selection of TFT-resistant (TK-1) cells; 600 cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37˚C in 5% CO2 for 10 to 12 days. Based on the observations made, thetest chemical did not induce gene mutation in Mouse lymphoma L5178Y cells in the presence S9 metabolic activation system. It however induced gene mutation I the cell line in the absence of S9 metabolic activation system.

 

Based on the presented data, the test chemical is regarded to be non-mutagenic in mouse lymphoma cells both in the presence and absence of S9 metabolic activation system.

 

Genetic Toxicity (In-Vivo):

 

The test chemical was tested for sex-linkedrecessive lethal (SLRL) mutation inductionin Drosophila melanogaster. Initially, the test chemical was tested by feeding, whenno significant mutagenicitywas observed,it was re-tested by injection.Doses used in the mutagenicity test was selected based on the results of a preliminary dose-finding study. The test substance was dissolved in water anddiluted with aqueous 5%sucrose for feedingand with aqueous 0.7% NaCl for injection. Toxicity tests were performed both forfeeding and injection treatments using a series of concentrations.Doses were chosen to induce mortalityof about 30% after 72 hoursof feeding or 24 hoursafter injection. The males to be treated werekept on regular culture medium until exposure. Males to befed were shaken into vials containing glass fiber filter material that was soaked withthe feeding solution ortest chemicalmixture. At 24 hoursand again at 48hours, the flies were transferredto vials with freshly prepared feeding mixture and were mated at 72 hours.Males to be injected were held on regular food for 1-3 days. They were theninjected with0.7% NaCl solution containing the test chemical. They were held for24 hoursto recover, then mated. The flies for the SLRL assay were exposed for 4hours. Treated and control males were matedindividuallyto threeharems ofBascvirgin females to produce three broods of 3,2, and 2 days.Thus,primarily post-meiotic germ cells were tested. To reduce the chances of recoveringseveral lethals from the same male, no more than 40 F1 females were mated individuallyfrom each brood of each male. Thus, no more than 120 chromosomes weretested from each P1male. F2 cultures were scored as presumptive lethals if thenumber of wild-type males recovered was 0, 1, or fewer than5% of the number ofBascmales (orBasc/+females). Presumptive lethals were confirmed by repeating the matings and scoring the F3.The test chemical was used at doses of 0 (solvent control) and 50000 ppm for feeding experiments and 10000 ppm for injection. No mutagenic response was observed in broods examined when the test chemical solution was fed or injected to male flies. The percents of lethals (total lethals) were statistically comparable in treated and control groups using both treatment methods.The mortality was 19 and 17% in experiments with injection and feeding treatment, respectivelyIn conclusion, the test chemical was non-mutagenic (negative) insex-linkedrecessive lethal (SLRL) inductiontest in Drosophila melanogaster.

 

Based on the data available, the test chemical does not exhibit gene mutation in vitro and in-vivo. Hence the test chemical is not likely to be mutagenic in vitro as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available, the test chemical does not exhibit gene mutation in vitro and in-vivo. Hence the test chemical is not likely to be mutagenic in vitro as per the criteria mentioned in CLP regulation.