N,N'-naphthalene-1,5-diylbis[4-[(2,3-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two Ames tests one with and one without azo-dye modification (OECD TG 471), a HPRT assay in CHO cells (OECD TG 476) and a chromosome aberration assay in human lymphocytes (OECD TG 473) revealed no mutagenicity or genotoxicity of the test substance, Pigment Brown 41.  

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:

10 December 2021 to 05 February 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

s9 Liver homogenate

Test concentrations with justification for top dose:

At a concentration of 0.125 mg/mL, the Relative Survival was greater than 10%. Therefore 0.125 mg/mL was selected as the highest concentration for testing in gene mutation test.

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

CHO AA8 Cells, a derivative of the CHO-K1, were used, CHO AA8 cells, Batch No.5000062 procured from American Type Culture Collection (ATCC) was used for the test.

Evaluation criteria:

A test chemical 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.
 The increase is concentration-related when evaluated with an appropriate trend test.
 Any of the results are outside the distribution of the historical negative/vehicle control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
A test chemical is considered clearly negative if, in all experimental conditions examined:
 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/vehicle control data.

Statistics:

Data of mutant frequencies were analyzed for differences among vehicle control, treatment and positive control groups by performing power transformation procedure by Snee and Irr (1981) with which, the observed mutant frequency was transformed using the formula:
Y=(X+A)B
Where,
Y = transformed mutant frequency, X = observed mutant frequency
BIO-GNT 1743 Study Report Page 19 of 36
[Where X=No. of mutant colonies per replicateACE value×100
and A, B = constants (viz. A = 1 and B = 0.15)]
Statistical analysis of the experimental data was carried out using SPSS Statistical package version 22.0. If the analysis of variance is significant at p < 0.05, Dunnett’s test was conducted, comparing each treatment group and the positive control to the vehicle control.
The statistical significances are designated by the superscripts as given below:
* Statistically significant (p<0.05) change than the vehicle control group.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility, Precipitation and pH Test
The test item was found soluble in DMSO at 200 mg/mL. Precipitation test was conducted at 0.0625, 0.125, 0.25, 0.50, 1 and 2 mg/mL concentrations. Post 3 hours and 40 minutes of incubation, no precipitation observed at 0.125 and 0.0625 mg/mL, mild precipitation was observed at 0.25 mg/mL, moderate precipitation observed at 0.5 mg/mL and heavy precipitation observed at 1 and 2 mg/mL. No change in pH was observed at the tested concentrations up to 2 mg/mL.
Initial Cytotoxicity Test
In the initial cytotoxicity test, there was no evidence of excessive cytotoxicity (˂10% RS) at and up to 0.5 mg/mL in both presence of metabolic activation and absence of metabolic activation when compared to vehicle control. In the presence of metabolic activation, the RS values ranged from 1.23 % to 45.68 % and in the absence of metabolic activation, the RS values ranged from 1.27 % to 46.84 % at the concentrations of 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/mL.
Gene Mutation Test
In the gene mutation test, the cells were treated with test item at the concentrations of 0.015625, 0.03125, 0.0625 and 0.125 mg/mL using DMSO as the vehicle in four plate cultures both in the presence of metabolic activation and absence of metabolic activation.
The test item resulted in mutant frequencies of 23.46 to 24.71 per 2×106 cells in the presence of metabolic activation with 24.18 per 2×106 cells in the vehicle control. In the absence of metabolic activation, mutant frequencies of 24.10 to 24.68 per 2×106 cells were observed with 23.86 per 2×106 cells in the vehicle control. There was no statistically significant increase in the mutant frequencies observed when compared with vehicle control at any of the tested concentrations.
There was no evidence of excessive cytotoxicity (˂10% RS) at any of the concentrations both in presence and absence of metabolic activation. In the presence of metabolic activation, the RS values ranged from 17.07 to 85.37 % and in the absence of metabolic activation the RS values ranged from 18.99 to 78.48 % respectively.
Application of the positive control, 3 μg/mL of Benzo (a) pyrene, resulted in a RS value of 89.02 % in the presence of metabolic activation, and a mutant frequency of 259.55 per 2×106 cells which was statistically significant when compared with the vehicle control.
The treatment with the positive control, 1 μg/mL of 4-Nitroquinoline N-oxide, resulted in a RS value 96.20 % in the absence of metabolic activation and a mutant frequency of 262.92 per 2×106 cells and was statistically significant when compared with that of vehicle control.

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

Based on the results obtained, the test item is considered as non-mutagenic at and up to the concentration of 0.125 mg/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.

Executive summary:

The test item was evaluated for gene mutation test in CHO AA8 cells.
The test item was found soluble in DMSO at 200 mg/mL. Precipitation test was conducted at 0.0625, 0.125, 0.25, 0.50, 1 and 2 mg/mL concentrations. After 3 hours and 40 minutes of incubation, no precipitation was observed at 0.125 and 0.0625 mg/mL, mild precipitation was observed at 0.25 mg/mL, moderate precipitation was observed at 0.5 mg/mL and heavy precipitation was observed at 1 and 2 mg/mL. No change in pH was observed at the tested concentrations up to 2 mg/mL.
On the basis of precipitation results, 0.5 mg/mL was selected as the highest concentration for the initial cytotoxicity test. Initial cytotoxicity test was conducted at the concentrations of 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/mL using DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation (3 hours and 33 minutes). Cytotoxicity was assessed by determining the Adjusted Cloning Efficiency and Relative Survival in the test.
The results of the initial cytotoxicity test indicated that the Relative Survival was greater than 10% at 0.125 mg/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Based on these results, 0.125 mg/mL was selected as highest concentration for gene mutation test.
The gene mutation test was conducted at the concentrations of 0.015625, 0.03125, 0.0625 and 0.125 mg/mL using DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation (4 hours).
Benzo(a) pyrene and 4 Nitroquinoline N-oxide were used as Positive controls for the gene mutation test.
Cytotoxicity as Relative Survival was 17.07 to 18.99 % in presence of metabolic activation and absence of metabolic activation at the highest tested concentration of 0.125 mg/mL.
There was no statistically significant increase in mutant frequencies at any of the concentrations tested when compared with the vehicle control. Moreover, treatment with the test item resulted in mutant frequencies which were within acceptable ranges with regard to historical controls.
There was statistically significant increase in mutant frequencies for positive controls when compared with the vehicle control in both metabolic activation and absence of 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:

03 December 2021 to 11 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

29th July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes:

Metabolic activation:

with and without

Metabolic activation system:

S9 Liver Homogenate

Test concentrations with justification for top dose:

Initial cytotoxicity was conducted at concentrations of 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/mL. The percentage reduction in Mitotic Index was in the range of 34.20 to 39.92 at 0.25 mg/mL. As the percentage reduction in MI 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 0.625 and 0.125 mg/mL.

Vehicle / solvent:

Dimethyl Sulphoxide

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Human Peripheral Blood Lymphocytes

Evaluation criteria:

All slides including vehicle control, treatment and positive controls of chromosomal aberration test were coded before evaluation.
 Cytotoxicity was determined by calculating percentage reduction in mitotic index (%).
 Concurrent measures of mitotic index for all treated and vehicle control cultures were determined.
 The cells were evaluated for structural aberrations in 150 metaphase plates for each replicate and the metaphases with aberrations were recorded in raw data.
 Gaps were recorded separately and reported but not included in the total aberration frequency.
 Individual culture data for chromosomal aberrations is provided

Statistics:

Data (Percentage of cells with aberrations) was analyzed using SPSS Software version 22 for differences among solvent/vehicle control, positive control and test item groups using ANOVA following Dunnett’s test at a 95% level of confidence (p<0.05) and the statistical significance was designated by the superscripts through out the report as stated below:
* Statistically significant (p<0.05) change than the vehicle control group.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The reduction in MI observed at 0.25 mg/mL was 40.15% in the presence of metabolic activation and 41.85% in the absence of metabolic activation for short term treatments. Similarly, 42.24% 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 1.33 in the absence of metabolic activation (short term treatment 3 to 6 hours) and 1.33, 1.33 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 10.00% of aberrated cells which was statistically significant compared to the vehicle control (1.00%). The reduction in mitotic index was 9.97% 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 (1.00%). The reduction in mitotic index observed was 9.90% 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 10.43% when compared with the vehicle control for long term treatment.

Conclusions:

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

Executive summary:

 

The test item, was evaluated for chromosomal aberrations in human lymphocytes.

The test item formed homogeneous brown solution in DMSO at 200 mg/mL. Precipitation and pH test was conducted at 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL. Post 24 hours and 15 minutes of incubation, heavy precipitation was observed at 1 and 2 mg/mL, moderate precipitation was observed at 0.5, mg/mL, mild precipitation was observed at 0.25 mg/mL. No precipitation was observed at 0.125 and 0.625 mg/mL. No change in pH was observed in any of the concentrations tested upto 2 mg/mL. Hence, 0.5 mg/mL was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were 0.03125, 0.0625, 0.125 and 0.25 mg/mL of test item.

In initial cytotoxicity test, the percentage reduction in Mitotic Index was in the range of 57.90 to 60.08% at 0.5mg/mL in presence of metabolic activation (short term), in absence of metabolic activation (short term) in absence of metabolic activation (long term), respectively. The percentage reduction in mitotic index was in the range of 7.90 to 39.92 at 0.03125 to 0.25 mg/mL. As the percentage reduction inmitotic 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.

In the chromosomal aberration test, the cells were treated with test item at the concentrations of 0.0625, 0.125 and 0.25mg/mLusing DMSO as the vehicle. The treatment was carried out in duplicates for the short term period (3 to 6 hours) both in the presence and absence of metabolic activation and for the long term period (20 to 24 hours) in the absence of metabolic activation. Cyclophosphamide Monohydrate (+S9 for short term) at the concentration of 10 µg/mL and Mitomycin-C at the concentration of 0.05 µg/mL (-S9 both for short term and long term) were used as positive controls.

The treated cells were harvested at about 1.5 normal cell cycle length after treatment. During harvesting of cultures, thecells were treated with a metaphase-arresting substance (colchicine), stained and metaphase cells were analysed microscopically for the structural chromosomal aberrations.

There was no statistically significant increase in the number of aberrated cells when compared with vehicle control at any of the test item concentration levels tested.Positive controls induced statistically significant aberrant cells when compared to vehicle control.

The concurrent vehicle control values were within the 95% control limits of the distribution of the laboratory’s historical vehicle control database.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant study, similar to OECD 471, Very limited information on test item

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no independent repeat assay

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

S. typhimurium, other: TA 1538

Metabolic activation:

with and without

Metabolic activation system:

induced rat liver S9

Test concentrations with justification for top dose:

10, 50, 100, 500, 1000 and 5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-AA with S9; AF2 and sodium azide without S9

Evaluation criteria:

"Significant increases"

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 September 2022 to 09 February 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted on 26th June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

At the histidine locus of Salmonella typhimurium and at the tryptophan locus of Escherichia coli tester strains.

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 homogenate of hamster liver

Test concentrations with justification for top dose:

Based on cytotoxicity results 0.1 mg/plate was considered as the highest test concentration for mutation assay along with 0.001, 0.003, 0.01, 0.03 mg/plate.

Vehicle / solvent:

Dimethyl sulphoxide

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene

Details on test system and experimental conditions:

Preincubation method was carried out with concentrations of 0.001, 0.003, 0.01, 0.03 and 0.1 mg/plate of test item, vehicle and positive control. The tester strains along with S9/PBS were transferred into sterile tubes and kept in an incubator shaker for 30 minutes at 180±5 rpm at temperature 37±1°C. Post incubation, the test constituents were transferred into sterile tubes and mixed with 2 mL molten soft agar containing histidine-biotin for Salmonella typhimurium and L-tryptophan for E.coli WP2 uvrA (pKM101) and poured on to MGA plates. Five concentrations of the test item were plated with each of the following tester strains Salmonella typhimurium TA98, TA100, TA1535, TA1537 and E.coli WP2 uvrA (pKM101) with and without metabolic activation. Plates were incubated at 37±1°C for 63 hours and 32 minutes.
The condition of the bacterial background lawn was evaluated for evidence of the test item cytotoxicity using the code system and revertant colonies for each strain within the test item dilution series were counted manually.

Rationale for test conditions:

The Bacterial reverse mutation test was considered acceptable as it meets the following criteria:
• The positive control substances produced a significant increase in mutant colony frequencies.
• The spontaneous reversion rates in the solvent and negative control (if used) are in the range of in house historical control data and preferably within the range reported in the literature or if it’s a new batch as per certificate of analysis received by the vendor.
• The tester strains must meet all required genetic characterization.
• Tester Strains used should be in the approximate range of 1×109 to 2×109 cells/mL.

• Minimum of five analyzable test item dose concentrations should be used to evaluate the mutation assay and none of these must show any signs of contamination.
• Cytotoxicity must be defined as greater than 50% reduction in mean revertants per concentration relative to the mean solvent/vehicle control value or by a thinning of the bacterial background lawn.
Study met all criteria mentioned above.

Evaluation criteria:

The conditions necessary for determining a positive result are: There should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test item either in the presence or absence of the metabolic activation system.
The test will be judged positive if the increase in mean revertants at the limit dose tested is equal to or greater than 2 times the mean vehicle control value in Salmonella typhimurium strains TA98, TA100 and Escherichia coli WP2 uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value in tester strains TA1535 and TA1537.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.
Based on the above interpretation, the test item is evaluated as negative.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Post incubation test item color interference was observed from 5 mg/plate to 0.2 mg/plate which was interfering with lawn evaluation and for counting of revertant colonies. Hence 5 mg/plate to 0.2 mg/plate was not considered further.

• SUMMARY OF INITIAL CYTOTOXICITY TEST - SALMONELLA TYPHIMURIUM TA100 TESTER STRAIN

Test Item Concentration (mg/plate)	No. of Revertants/plate
	With S9						Without S9
	R1	R2	R3	Average	±SD	Bacterial Lawn Intensity	R1	R2	R3	Average	±SD	Bacterial Lawn Intensity
Vehicle Control	110	105	108	108	2.5	*4+,4+,4+	96	104	102	101	4.2	*4+,4+,4+
0.00625	103	109	106	106	3.0	*4+,4+,4+	105	102	101	103	2.1	*4+,4+,4+
0.0125	109	104	111	108	3.6	*4+,4+,4+	106	101	103	103	2.5	*4+,4+,4+
0.025	107	105	110	107	2.5	*4+,4+,4+	104	100	102	102	2.0	*4+,4+,4+
0.05	103	105	107	105	2.0	*4+,4+,4+	99	104	103	102	2.6	*4+,4+,4+
0.1	106	108	104	106	2.0	*4+,4+,4+	99	101	104	101	2.5	*4+,4+,4+
0.2**	-	-	-	-	-	-	-	-	-	-	-	-
0.4**	-	-	-	-	-	-	-	-	-	-	-	-
0.8**	-	-	-	-	-	-	-	-	-	-	-	-
1.6**	-	-	-	-	-	-	-	-	-	-	-	-
3.2**	-	-	-	-	-	-	-	-	-	-	-	-
5**	-	-	-	-	-	-	-	-	-	-	-	-

Values of Revertants are in Mean±SD

Lawn intensity: 4+= Thick lawn: Distinguished by a healthy (Normal) background lawn compared to vehicle control plates.

  *Lawn intensity of replicates 1, 2, 3 respectively.

Note: **Color interference occurred at 0.2 to 5.0 mg/plate. Evaluation of lawn and revertant counts was not possible. These concentrations were excluded from the mutation test.

• SUMMARY OF COLONY COUNTS OF REVERTANTS - TRIAL-II

Preincubation Method

                                                                                                                                                               Refer: Appendix - 2

Treatment	Test Concentration  (mg/plate)		No. of Revertants (Mean of 3 Plates)
			With S9						Without S9
			Salmonella typhimurium				E. coli WP2 uvrA (pKM 101)		Salmonella typhimurium				E. coli WP2 uvrA (pKM 101)
			TA             98	TA 100	TA 1535	TA 1537			TA              98	TA 100	TA 1535	TA 1537
Vehicle Control	100 µL of  DMSO	Mean	32.0	108.7	22.0	9.7	78.7		28.7	105.3	17.3	7.3	72.7
		±SD	3.0	3.1	2.0	2.1	3.5		1.5	1.5	1.5	1.5	3.5
	Lawn Intensity		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,   4+		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+
Test item	0.001	Mean	31.7	109.3	22.3	11.0	78.0		28.3	101.7	18.0	8.7	71.3
		±SD	3.1	3.1	1.5	1.0	4.6		1.5	3.1	2.0	1.5	3.5
	Fold Increase		1.0	1.0	1.0	1.1	1.0		1.0	1.0	1.0	1.2	1.0
	Lawn Intensity		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,  4+		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+
	0.003	Mean	32.3	109.0	23.3	10.0	77.7		28.0	102.7	18.3	9.0	72.0
		±SD	3.1	2.0	1.5	2.0	4.2		2.0	3.1	1.5	1.0	4.6
	Fold Increase		1.0	1.0	1.1	1.0	1.0		1.0	1.0	1.1	1.2	1.0
	Lawn Intensity		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,  4+		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+
	0.01	Mean	29.0	108.0	22.7	9.3	76.7		27.7	105.7	18.0	8.3	72.3
		±SD	3.6	3.0	2.1	2.1	3.5		1.5	4.0	2.0	1.5	2.5
	Fold Increase		0.9	1.0	1.0	1.0	1.0		1.0	1.0	1.0	1.1	1.0
	Lawn Intensity		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+
	0.03	Mean	32.3	109.3	22.3	9.0	76.3		28.7	101.7	18.0	9.3	71.3
		±SD	3.5	3.1	2.1	1.0	2.5		1.5	2.5	1.0	1.2	4.5
	Fold Increase		1.0	1.0	1.0	0.9	1.0		1.0	1.0	1.0	1.3	1.0
	Lawn Intensity		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+
	0.1	Mean	32.3	107.0	23.3	10.0	76.0		27.3	102.0	18.0	7.7	71.7
		±SD	3.2	2.0	1.5	2.0	4.4		1.5	3.6	2.0	1.5	3.1
	Fold Increase		1.0	1.0	1.1	1.0	1.0		1.0	1.0	1.0	1.0	1.0
	Lawn Intensity		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+
Positive Control	100 µL of respective Positive Control	Mean	384.0	405.7	140.3	115.3	376.3		378.3	391.0	138.3	114.7	372.0
		±SD	6.6	9.3	3.8	9.5	8.7		6.5	9.0	3.1	6.0	9.5
	Fold Increase		12.0	3.7	6.4	11.9	4.8		13.2	3.7	8.0	15.6	5.1
	Lawn Intensity		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+		4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+,4+	4+,4+, 4+

Lawn intensity: 4+= Thick lawn: Distinguished by a healthy (Normal) background lawn compared to vehicle control plates.

Values of Revertants are in Mean±SD

Positive controls:

With S9:

For Salmonella typhimurium TA98, TA100, TA1535 and TA1537 = 4 µg/plate of 2-Aminoanthracene

For E.coli uvrA pKM 101 = 30 µg/plate of 2-Aminoanthracene

Without S9:

For TA98: 2 µg/plate of 2-Nitrofluorene

For TA100 and TA1535: 1 µg/plate of Sodium azide

For TA1537: 50 µg/plate of 9-Aminoacridine

For E.coli uvrA pKM 101: 5 µg/plate of 4 Nitroquinoline N-oxide

• INDIVIDUAL PLATE COLONY COUNT OF REVERTANTS - PREINCUBATION METHOD

 

Treatment	Test Concentration  (mg/plate)	Plate No.	No. of Revertants/Plate in triplicates
			With S9						Without S9
			Salmonella typhimurium				E. coli WP2 uvrA (pKM 101)		Salmonella typhimurium				E. coli WP2 uvrA (pKM            101)
			TA 98	TA 100	TA 1535	TA 1537			TA 98	TA 100	TA 1535	TA 1537
Vehicle Control	100 µL of  DMSO	1	32	108	24	12	82		27	104	17	7	69
		2	29	112	20	9	75		30	107	19	9	73
		3	35	106	22	8	79		29	105	16	6	76
		Lawn Intensity	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+		*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+
Test item	0.001	1	31	110	21	12	83		27	99	16	7	71
		2	35	106	24	10	74		30	105	18	10	75
		3	29	112	22	11	77		28	101	20	9	68
		Lawn Intensity	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+		*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+
	0.003	1	33	111	23	10	79		28	102	20	8	67
		2	29	107	25	8	81		30	106	17	10	76
		3	35	109	22	12	73		26	100	18	9	73
		Lawn Intensity	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+		*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+
	0.01	1	26	111	25	10	77		28	110	20	8	75
		2	28	105	22	7	73		29	102	18	10	70
		3	33	108	21	11	80		26	105	16	7	72
		Lawn Intensity	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+		*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+
	0.03	1	36	110	24	9	74		29	99	18	10	71
		2	32	106	23	8	79		27	102	17	8	76
		3	29	112	20	10	76		30	104	19	10	67
		Lawn Intensity	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+		*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+
	0.1	1	36	109	22	12	73		26	98	20	6	69
		2	30	105	23	10	81		29	105	16	9	75
		3	31	107	25	8	74		27	103	18	8	71
		Lawn Intensity	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+		*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+
Positive Control	100 µL of respective Positive Control	1	391	410	142	125	386		385	400	141	109	363
		2	378	412	136	106	374		372	382	135	114	371
		3	383	395	143	115	369		378	391	139	121	382
		Lawn Intensity	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+		*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+	*4+, 4+,4+

Lawn intensity: 4+= Thick lawn: Distinguished by a healthy (Normal) background lawn compared to vehicle control plates.

*Lawn intensity of replicates 1, 2, 3 respectively.

 

• INDIVIDUAL VIABLE COUNT OF TESTER STRAINS

Name of the Strain	Required CFU/mL	No. of colonies of 10^-7dilutions in triplicates			Average	Obtained CFU/mL (1 to 2 × 10^9CFU /mL)
		R1	R2	R3
Salmonella typhimurium TA98	1-2×10^9	175	171	168	171	1.71×10^9
Salmonella typhimurium TA100	1-2×10^9	174	165	180	173	1.73×10^9
Salmonella typhimurium TA1535	1-2×10^9	161	179	185	175	1.75×10^9
Salmonella typhimurium TA1537	1-2×10^9	182	170	165	172	1.72×10^9
E.coli Wp2 uvrA pKM 101	1-2×10^9	172	181	168	174	1.74×10^9

R: Replicate

 

 

 

Conclusions:

Based on the results of the study, it is concluded that the test item is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the concentration of 0.1 mg/plate under the test conditions, when tested on Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA (pKM101) tester strains.

Executive summary:

The test item was evaluated for mutagenicity in Bacterial Reverse Mutation Test.

The test item formed suspension in dimethyl sulphoxide at a concentration of 50 mg/mL and resulted in no precipitation from 0.00625 to 5 mg/plate at tested concentrations.

On the basis of precipitation test, 5 mg/plate was selected as the highest test concentration for initial cytotoxicity test.

Salmonella typhimurium TA100 was exposed to vehicle control 0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 and 5 mg/plate of test item concentrations. Post incubation test item color interference was observed from 5 mg/plate to 0.2 mg/plate which was interfering with lawn evaluation and for counting of revertant colonies. Hence 5 mg/plate to 0.2 mg/plate was not considered for further evaluation

The test item caused no cytotoxicity from 0.00625 to 0.1 mg/plate in the tester strain Salmonella typhimurium TA100 with lawn intensity 4+ (Thick lawn) in the presence and absence of metabolic activation system when compared to vehicle control 4+ (Thick lawn).

Based on the results of initial cytotoxicity test, concentrations of 0.001, 0.003, 0.01, 0.03 and 0.1 mg/plate were selected for testing in the mutation test.

The test item was assessed for its mutagenic effects using Salmonella typhimurium tester strains: TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA (pKM101). The test item was tested according to the preincubation method in presence and absence of metabolic activation system using DMSO the vehicle and appropriate positive controls  were tested simultaneously. Two trials were carried out for this study in triplicate.

Based on the experimental results obtained, the mean numbers of revertant colonies at the tested concentrations were comparable to those of the vehicle control, in both the trials in the presence and absence of metabolic activation. There was no appreciable increase in number of revertant colonies at any of the tested concentrations in both the trials. The number of revertant colonies in the positive controls resulted in 3.7 to 15.6 fold increase under comparable conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the third time in Directive EC 618/2012.