Nonanamide, N,N-dimethyl-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genotoxicity of Genagen PA/ N,N-Dimethylnonanamide was investigated in three in-vitro tests. Genagen PA/ N,N-Dimethylnonanamide is not mutagenic in Ames test, not clastogenic in chromosome aberration test and not mutagenic in HPRT Assay.

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:

21 November 2016 to 08 May 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD guideline for testing of chemicals No. 471, “Bacterial Reverse Mutation Test”, adopted on 21st July 1997.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan locus

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: Genetic Characterization of Tester Strains, histidine & tryotophan requirement

Metabolic activation:

with and without

Metabolic activation system:

1 mL of S9 homogenate was mixed with 9 mL of co-factor solution.

Test concentrations with justification for top dose:

0.01, 0.03, 0.10, 0.31 and 1 µL /plate , based on the results of solubility, precipitation and initial cytotoxicity test

Vehicle / solvent:

Vehicle(s)used: DMSO
Justification for choice of vehicle: The test item at the given concentration of 50 µL/mL was miscible in dimethyl sulphoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoantracene

Details on test system and experimental conditions:

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


DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: triplicates


- OTHER: cytotoxicity by lawn evaluation and mutagenicity by evauatimg revertant colonies

Rationale for test conditions:

not applicable

Evaluation criteria:

The test will be judged positive if the increase in mean revertants at the peak of the dose response 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 TA1537In the two independent trials conducted, the test item concentrations tested resulted in no appreciable increase in the number of revertant colonies over the vehicle control.

Statistics:

not applicable

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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:

cytotoxicity

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:

cytotoxicity

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:

cytotoxicity

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:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: insoluble
- Precipitation:no precipitation upto tested concentration


HISTORICAL CONTROL DATA (with ranges, means and standard deviation) : Attached as Annexure 1 in study report (Page no. 32 of 35)

Conclusions:

The mutagenicity of Genagen PA/ N, N-Dimethylnonanamide was investigated according to the Guideline OECD 471 (Ames Test). No mutagenic acitivity was found.

Executive summary:

The test item, Genagen PA/ N, N-Dimethylnonanamide was evaluated for mutagenicity in Bacterial Reverse Mutation Test as per the OECD guideline for testing of chemicals No. 471, “Bacterial Reverse Mutation Test”, adopted on 21^stJuly 1997.

The tester strains used in the mutation assay were Salmonella typhimurium TA98, TA100, TA1535, and TA1537 and Escherichia coli WP2 uvrA (pKM101).

The test concentrations tested in the mutation assay were selected based on the results of solubility, precipitation and initial cytotoxicity test. The two independent trials (trial 1 and 2) were conducted by plate incorporation method in the presence and absence of metabolic activation system. In mutation assay the test item was tested at the concentrations of0.01, 0.03, 0.10, 0.31 and 1 µL /plate. Vehicle control (dimethyl sulphoxide) and appropriate positive controls (2-nitrofluorene, sodium azide and 9-Aminoacridine, 4-nitroquinoline 1-oxide for trials “without metabolic activation” and 2-Aminoanthracene for trials “with metabolic activation”) were tested simultaneously.

On the basis of test item solubility and precipitation tests, the initial cytotoxicity test was performed at 1, 2, 3, 4, and 5 µL/plate. Initial cytotoxicity test was performed with TA100 both in the presence and absence of metabolic activation system.

The tester strain, TA100 treated with test item, Genagen PA/ N, N-Dimethylnonanamide at the concentration of 3, 4 and 5 µL /plate both in the presence and absence of metabolic activation revealed extremely reduced lawn (grade1+) when compared to vehicle control. Similarly, TA100 treated with test item at 2 and 1 µL/plate showed moderately reduced lawn (grade 3+) and slightly reduced lawn (grade 2+) respectively when compared to vehicle control. On the basis of cytotoxicity results 1 µL /plate was considered as the highest test concentration for mutation assay.

The results of mutation assay performed in two independent experiment (Trial 1 and 2) clearly revealed that there was no appreciable increase in the mean revertant colonies compared to those of the vehicle control, in both the trials, in the presence and absence of metabolic activation, at any of the tested concentrations.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 December 2016 to 15 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Not Applicable

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

In vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes adopted on 29th July 2016

Deviations:

no

Principles of method if other than guideline:

Not Applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material:112-2016-11 D
- Expiration date of the batch:May 2018
- Purity test date:98.3 % (w/w)


STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:Cool and dry (+2 to 8°C)

- Solubility of the test substance in the vehicle:DMSO
- Reactivity of the test substance with the vehicle of the cell culture medium: Soluble

Target gene:

Hprt and xprt genes

Species / strain / cell type:

other: CHO-AA8

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:ATCC
- Suitability of cells:The CHO AA8 cells are one of the recommended test systems by regulatory agencies for conducting In vitro mammalian gene mutation test.
- Methods for maintenance in cell culture if applicable:Cells were maintained in alpha MEM culture medium containing 10% FBS with antibiotics (1% Penicillin and Streptomycin) and incubated at 37±1ºC with 5±1% CO2

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:alpha MEM, incubated at 37±1ºC with 5±1% CO2

- Properly maintained: [yes]
- Periodically checked for Mycoplasma contamination: [yes]
- Periodically checked for karyotype stability: [yes)
- Periodically 'cleansed' against high spontaneous background: [yes]

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Not Applicable

Metabolic activation:

with and without

Metabolic activation system:

One mL of S9 homogenate was thawed immediately before use and mixed with 9 mL of co-factor solution

Test concentrations with justification for top dose:

The test item did not precipitated at 1 mg/mL, 1 mg/mL was considered as the highest concentration in the initial cytotoxicity test.

Up to the concentration of 0.125 mg/mL the Relative Survival was more than 20%. At higher concentrations the RS < 10% was obtained. Therefore 0.125 mg/mL was selected as the highest concentration for testing in the Gene mutation test.

Four concentrations i.e. 0.0156, 0.0312, 0.0625, and 0.125 mg/mL were selected for gene mutation test, based on the results of initial cytotoxicity test.

Vehicle / solvent:

- Vehicle: DMSO
- Justification for choice of vehicle:The test item, Genagen PA/ N,N-Dimethylnonanamide was soluble in DMSO at 200 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

- Cell density at seeding (if applicable):Approximately 20 Lakh cells per culture flask were seeded using culture medium with 10% FBS with antibiotics (1% Penicillin and Streptomycin).

DURATION
- Exposure duration:3 to 6 hours
- Expression time (cells in growth medium):9 days of expression period
- Selection time (if incubation with a selection agent):7 to 9 days

SELECTION AGENT (mutation assays):6 Thioguanine

NUMBER OF REPLICATIONS:Tetraplates

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth:

Rationale for test conditions:

Not Applicable

Evaluation criteria:

There are several criteria for determining a positive result, such as a concentration-related increase or a reproducible increase in mutant frequency. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determining factor for a positive response.
Since the results did not meet the above criteria, the test item is considered non-mutagenic in this system.

Statistics:

Data of mutant frequencies were analyzed for differences among vehicle control, treatment and positive control groups using SPSS Software version 22 at a 95% level (p<0.05) of significance

Key result

Species / strain:

other: The derivative of the CHO-K1, CHO AA8 Cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No Change
- Water solubility:No
- Precipitation:Yes

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:Yes
- Negative (vehicle) historical control data:Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RICC, Cloning efficiency, Relative survival.

TABLE 1.            SUMMARY OF INITIAL CYTOTOXICITY TEST

Set No.	Treatment	Concentration (mg/mL)	*Colony count		Cloning Efficiency (CE)	Adjusted Cloning Efficiency (ACE)	Relative Survival (RS) (%)
			Mean	±SD
Set 1 +S9	Vehicle Control (DMSO)	-	152.67	13.58	0.76	1.00	-
	Genagen PA/N,N-Dimethylnonanamide	0.0156	146.67	13.61	0.73	0.92	92.00
		0.0312	150.00	5.57	0.75	0.88	88.00
		0.0625	136.67	9.61	0.68	0.50	50.00
		0.125	94.67	3.06	0.47	0.22	22.00
		0.25	59.67	9.50	0.30	0.07	7.00
		0.5	32.67	10.69	0.16	0.02	2.00
		1	19.67	8.50	0.10	0.01	1.00
Set 2 -S9	Vehicle Control (DMSO)	-	148.33	8.50	0.74	0.96	-
	Genagen PA/N,N-Dimethylnonanamide	0.0156	145.00	7.55	0.73	0.92	95.83
		0.0312	136.33	7.77	0.68	0.79	82.29
		0.0625	127.67	11.24	0.64	0.46	47.92
		0.125	98.00	5.57	0.49	0.21	21.88
		0.25	65.00	9.54	0.33	0.09	9.38
		0.5	26.00	11.53	0.13	0.02	2.08
		1	14.00	4.00	0.07	0.01	1.04

 +S9: with metabolic activation; -S9: without metabolic activation;  

*Note: Cloning Efficiency = 200 cells plated for each replicate.

 Adjusted CE = CE x Number of cells at the end of treatment/number of cells at the beginning of treatment.

RS = Adjusted CE in treated culture/Adjusted CE in the vehicle control x 100.

CE = Number of colonies/Number of cells plated.

TABLE 2.           SUMMARY OF PARALLEL CYTOTOXICITY TEST- GENE MUTATION TEST

Set No.	Treatment	Concentration (mg/mL)	*Colony count		Cloning Efficiency (CE)	Adjusted Cloning Efficiency (ACE)	Relative Survival (RS) (%)
			Mean	±SD
Set 1 +S9	Vehicle Control (DMSO)	-	146.33	6.03	0.73	0.99	-
	Genagen PA/N,N-Dimethylnonanamide	0.0156	134.67	11.06	0.67	0.90	90.91
		0.0312	128.00	9.54	0.64	0.84	84.85
		0.0625	116.33	4.73	0.58	0.53	53.54
		0.125	89.00	7.94	0.45	0.24	24.24
	Benzo(a)pyrene (Positive Control)	0.003	132.33	7.09	0.66	0.86	86.87
Set 2 -S9	Vehicle Control (DMSO)	-	143.67	7.02	0.72	0.97	-
	Genagen PA/N,N-Dimethylnonanamide	0.0156	139.00	9.54	0.70	0.91	93.81
		0.0312	126.00	12.49	0.63	0.81	83.51
		0.0625	127.00	9.17	0.64	0.55	56.70
		0.125	99.00	7.55	0.50	0.25	25.77
	4- Nitroquinoline 1-oxide (Positive Control)	0.001	133.67	14.29	0.67	0.84	86.60

+S9: with metabolic activation;  -S9: without metabolic activation;   

*Note: Cloning Efficiency = 200 cells plated for each replicate.  

 RS = Adjusted CE in treated culture/Adjusted CE in the vehicle control x 100.

 CE = Number of colonies/Number of cells plated.

  Adjusted CE = CE x Number of cells at the end of treatment/number of cells at the beginning of treatment.

TABLE 3.           SUMMARY OF GENE MUTATION TEST

Set No.	Treatment	Concentration (mg/mL)	*Colony count		Cloning Efficiency in selective media	Cloning Efficiency in non-selective media		Average Mutant Colonies/ 2x10^6cells	Mutant Frequency/ 2x10^6cells
			Mean	±SD
Set 1 +S9	Vehicle Control (DMSO)	-	179.33	7.02	0.0000070	0.90		14	15.56
	Genagen PA/N,N-Dimethylnonanamide	0.0156	174.33	6.03	0.0000065	0.87		13	14.94
		0.0312	157.33	9.71	0.0000060	0.79		12	15.19
		0.0625	129.33	8.50	0.0000035	0.65		7	10.77
		0.125	99.00	8.19	0.0000015	0.50		3	6.00
	Benzo(a)pyrene (Positive Control)	0.003	174.33	12.90	0.0000645	0.87		129	148.28**
Set 2 -S9	Vehicle Control (DMSO)	-	177.33	7.51	0.0000070	0.89	14		15.73
	Genagen PA/N,N-Dimethylnonanamide	0.0156	172.00	9.85	0.0000055	0.86	11		12.79
		0.0312	157.67	10.50	0.0000060	0.79	12		15.19
		0.0625	135.00	6.56	0.0000040	0.68	8		11.76
		0.125	107.33	5.03	0.0000015	0.54	3		5.56
	4-Nitroquinoline 1-oxide (Positive Control)	0.001	175.67	11.59	0.0000655	0.88	131		148.86**

+S9: with metabolic activation; -S9: without metabolic activation;                                                                                                                   

 *Note: Cloning efficiency = 200 cells plated for each replicate.  

   **: Statistically significant (p˂0.05). 

Mutant Frequency = Cloning efficiency of mutant colonies in selective medium/Cloning efficiency in non-selective  medium

Conclusions:

The mutagenicity of Genagen PA/ N, N-Dimethylnonanamide was investigated according to the Guideline OECD 476 (HPRT). No mutagenic acitivity was found.

Executive summary:

The test item, Genagen PA/ N,N-Dimethylnonanamide was evaluated for gene mutation test in CHO AA8 cells, as per the OECD guideline for the testing of chemicals,No. 476 “In vitroMammalian Cell Gene Mutation Tests using theHprtandxprtgenes” adopted on 29^thJuly 2016.

The test item was soluble in DMSO at 200 mg/mL. The test item showed moderate to heavy precipitation at 2 mg/mL. The pH tested at concentrations up to 2 mg/mL was comparable to the vehicle control. Based on these results, 1 mg/mL was chosen as the highest concentration for the initial cytotoxicity test.

Initial cytotoxicity test was conducted at the concentrations of 0.0156, 0.0312, 0.0625, 0.125, 0.25, 0.5 and 1 mg/mL. Slight precipitation was observed under the inverted microscope at 1 mg/mL at the end of the treatment. Percentage of relative survival for test item treated at 0.0156, 0.0312, 0.0625, 0.125, 0.25, 0.5 and 1 mg/mL were 92.00, 88.00, 50.00, 22.00, 7.00, 2.00 and 1.00 in the presence of metabolic activation and 95.83, 82.29, 47.92, 21.88, 9.38, 2.08 and 1.04 in the absence of metabolic activation respectively. The results of the initial cytotoxicity test indicated that the test item was not excessive cytotoxic to CHO AA8 cells at the concentrations up to 0.125 mg/mL, the Relative Survival of the treated CHO AA8 cells being > 10%, both in the presence and absence of metabolic activation.  

The Gene mutation test was conducted at the concentrations of 0.0156, 0.0312, 0.0625, and 0.125 mg/mL using DMSO as a vehicle in duplicates in the presence and absence of metabolic activation (3 to 6 hours).

Positive controls, 3µg/mL ofBenzo (a) pyrene [with metabolic activation (+S9)] and   1µg/mLof 4-Nitroquinoline 1- oxide [without metabolic activation (-S9)] were used for the gene mutation test.

Cytotoxicity was assessed by determining the Adjusted Cloning Efficiency and Relative Survival in the test. 

The test item, Genagen PA/ N,N-Dimethylnonanamid eresulted inmutant frequencies of 6.00to 15.19 per 2×10⁶cellsin the presence of metabolic activation at the different concentrations tested with 15.56 per 2×10⁶cells in the vehicle control. In the absence of metabolic activation,test itemresulted inmutant frequencies of 5.56 to 15.19per 2×10⁶cells with 15.73 per 2×10⁶cells in the vehicle control. There was no statistically significant increase in the number of mutant colonies at any of the concentrations tested when compared with vehicle control.

No mutagenic acitivity was found for Genagen PA/ N,N-Dimethylnonanamid.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 November 2016 to 23 May 2017

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:

In vitro Mammalian Chromosomal Aberration Test” adopted on 29 July 2016.

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro Mammalian Chromosomal Aberration Test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material:112-2016-11 D
- Expiration date of the batch:May 2018
- Purity test date:98.3 %


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:Cool and dry(+2 to +8°C)
- Solubility of the test substance in the vehicle: soluble in DMSO at 200 mg/mL.


FORM AS APPLIED IN THE TEST - liquid

Target gene:

not applicable

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Human peripheral lymphocytes from the blood of healthy, young, non-smoking male donors (28 and 24 years of age) with no known recent exposure to genotoxic chemicals or radiation
- Suitability of cells: This provides the opportunity to test using the same test system which the in vitro test is predictive of in vivo genotoxic events. Further as per the regulatory requirements the human peripheral blood lymphocytes is one of the recommended test system.
- Cell cycle length : 18 to 24
- Sex, age and number of blood donors : two male donors, 28 and 24 years of age
- Whether whole blood or separated lymphocytes were used: whole blood
- Modal number of chromosomes: 46

MEDIA USED
- Type and identity of media including CO2 concentration :RPMI Media supplemented with 10% FBS and antibiotics (1% Penicillin-Streptomycin), 5±1% CO2
- Properly maintained: [yes]

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

One mL of S9 homogenate was thawed immediately before use and mixed with the 9 mL of co-factor solution

Test concentrations with justification for top dose:

In the initial cytotoxicity test the test item, Genagen PA/ N,N Dimethylnonanamide was found to be cytotoxic and the reduction in mitotic index was in the range of 18.40 % to 100 % at all the tested concentrations. Therefore on the basis of this results the test concentrations selected for chromosomal aberration test and were found to cover a range from the maximum to little or no toxicity.

Based on the results of initial cytotoxicity test, the concentrations selected for the chromosome aberration test were 31.25, 62.5 and 125 µg/mL of Genagen PA/ N, N-Dimethylnonanamide as low, mid and high concentrations respectively

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of vehicle:Test item was found to be soluble in DMSO at 200 mg/mL.

Details on test system and experimental conditions:

METHOD OF APPLICATION:

DURATION
-- Exposure duration:3 to 4 hrs for short term and 20 to 24 hrs for long term treatment

- Fixation time (start of exposure up to fixation or harvest of cells): 10 minutes per stage


SPINDLE INHIBITOR (cytogenetic assays):colchicine



NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:Slides were stained using 5% Giemsa stain for 15 to 20 minutes.

NUMBER OF CELLS EVALUATED:500 cells were scored for mitotic indix

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 150


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Rationale for test conditions:

not applicable

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 vehicle control data.

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.
The test item is then considered unable to induce chromosomal aberrations in cultured mammalian cells in this test system.

Statistics:

ANOVA following Dunnett’s test at a 95% level of confidence (p < 0.05) (SPSS Software version 22)

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH : No

- Water solubility: no
- Precipitation: Slight precipitation at 2000 µg/mL.

ADDITIONAL INFORMATION ON CYTOTOXICITY:mitotic index

Remarks on result:

other: non-clastogenic

TABLE 1.           SUMMARY OF PERCENTAGE MITOTIC INDEX FOR INITIAL CYTOTOXICITY TEST

Set No.	Treatment	Dose (µg/mL)	Mitotic Index		Mean Mitotic Index	Mean Percentage Mitotic Index	Percentage Reduction in Mitotic Index
			Replicate 1	Replicate 2
Set 1 (+S9)  (3-6 hours)	Vehicle control	-	0.0531	0.0647	0.0589	5.89	-
	Test item [Genagen PA/ N, N-Dimethylnonanamide]	62.5	0.0453	0.0508	0.0481	4.81	18.40
		125	0.0341	0.0353	0.0347	3.47	41.10
		250	0.0187	0.0160	0.0174	1.74	70.48
		500	0.0000	0.0000	0.0000	0.00	100.00
		1000	0.0000	0.0000	0.0000	0.00	100.00
		2000	0.0000	0.0000	0.0000	0.00	100.00
Set 2 (-S9) (3-6 hours)	Vehicle control	-	0.0518	0.0625	0.0571	5.71	-
	Test item [Genagen PA/ N, N-Dimethylnonanamide]	62.5	0.0487	0.0424	0.0455	4.55	20.27
		125	0.0275	0.0358	0.0316	3.16	44.64
		250	0.0172	0.0189	0.0180	1.80	68.42
		500	0.0000	0.0000	0.0000	0.00	100.00
		1000	0.0000	0.0000	0.0000	0.00	100.00
		2000	0.0000	0.0000	0.0000	0.00	100.00
Set 3 (-S9) (20-24 hours)	Vehicle control	-	0.0617	0.0698	0.0657	6.57	-
	Test item [Genagen PA/ N, N-Dimethylnonanamide]	62.5	0.0519	0.0494	0.0506	5.06	23.00
		125	0.0359	0.0356	0.0357	3.57	45.61
		250	0.0188	0.0176	0.0182	1.82	72.33
		500	0.0000	0.0000	0.0000	0.00	100.00
		1000	0.0000	0.0000	0.0000	0.00	100.00
		2000	0.0000	0.0000	0.0000	0.00	100.00

+S9: With metabolic activation; -S9: Without metabolic activation  

TABLE 2: SUMMARY OF CHROMOSOMAL ABERRATIONS AND MITOTIC INDEX

Set No.	Treatment	Dose (µg/mL)	Mean % MI	Mean % Reduction in MI	Mean of Total Aberrations with Gaps	Mean of Total Aberrations without Gaps	Mean of Total Aberrant cells without Gaps	Mean of Percentage Aberrated Cells
Set 1 (+S9) (3-6 hours)	Vehicle control	-	4.45	NA	0.5	0.5	0.5	0.35
	Positive Control (Cyclophosphamide)	10	3.83	13.93	33	32	16	10.65*
	Test item	31.25	3.95	11.24	1.5	1.5	1	0.7
		62.5	3.48	21.8	1	0.5	0.5	0.35
		125	2.61	41.35	1.5	1	1	0.7
Set 2 (-S9) (3-6 hours)	Vehicle control	-	5.09	NA	2.5	1.5	1.5	1
	Positive Control (Mitomycin-C)	0.05	4.40	13.56	39	34.5	17.5	11.65*
	Test item	31.25	4.34	14.73	1.5	1	0.5	0.35
		62.5	3.89	23.58	2	1	1	0.35
		125	2.94	42.24	1	0.5	0.5	0.35
Set 3 (-S9) (20-24 hours)	Vehicle control	-	5.66	NA	1	0.5	0.5	0.35
	Positive Control (Mitomycin-C)	0.05	4.83	14.66	33.5	31.5	18	12*
	Test item	31.25	4.53	19.96	1.5	1.5	1	0.7
		62.5	4.24	25.09	1	0.5	0.5	0.35
		125	3.08	45.58	1	1	1	0.7

Note :MI: Mitotic Index; *: Statistically significant; -S9: Without metabolic activation, Test Item =Genagen PA/ N, N Dimethylnonanamide]

TABLE 3 -1: INDIVIDUAL DATA OFCHROMOSOMAL ABERRATIONAND MITOTIC INDEX

Set No.	Treatment	Dose (µg/mL)	Replicate	Mitotic Index				Aberrations										Total No. of Aberrations	Total No. of Aberrations without Gaps	Total no. of Aberrant cells	Total % of Aberrated cells
								Gap		Break		Exchange		Fragments	Ring	Deletion	Dicentric
				Total No. of MP	Total No. of Blast cells	Mitotic Index	Percentage of MI	Chromatid	Chromosome	Chromatid	Chromosome	Chromatid	Chromosome
Set 1 (+S9) (3-6 hours)	Vehicle control	-	R1	28	572	0.0467	4.67	0	0	0	0	0	0	1	0	0	0	1	1	1	0.7
			R2	26	590	0.0422	4.22	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Positive control (Cyclophosphamide )	10	R1	22	583	0.0364	3.64	0	3	0	5	0	6	22	0	0	0	36	33	17	11.3
			R2	25	598	0.0401	4.01	0	1	0	3	1	7	18	0	0	0	30	31	15	10.0
	Test item	31.25	R1	25	558	0.0429	4.29	0	0	0	0	0	0	1	0	0	0	1	1	1	0.7
			R2	21	562	0.0360	3.60	0	0	0	0	0	0	2	0	0	0	2	2	1	0.7
		62.5	R1	21	564	0.0359	3.59	0	1	0	0	0	0	0	0	0	0	1	0	0	0
			R2	19	546	0.0336	3.36	0	0	0	1	0	0	0	0	0	0	1	1	1	0.7
		125	R1	14	538	0.0254	2.54	0	1	1	0	0	0	0	0	0	0	2	1	1	0.7
			R2	16	580	0.0268	2.68	0	0	0	0	0	0	1	0	0	0	1	1	1	0.7

MI: Mitotic Index, MP: Metaphase Plates,R1: Replicate 1, R2: Replicate 2, +S9: With metabolic activation,Test Item =Genagen PA/ N, N Dimethylnonanamide]

TABLE 3 -2:INDIVIDUAL DATA OF CHROMOSOMAL ABERRATIONAND MITOTIC INDEX

Set No.	Treatment	Dose (µg/mL)	Replicate	Mitotic Index				Aberrations										Total No. of Aberrations	Total No. of Aberrations without Gaps	Total no. of Aberrant cells	Total % of Aberrated cells
								Gap		Break		Exchange		Fragments	Ring	Deletion	Dicentric
				Total No. of MP	Total No. of Blast cells	Mitotic Index	Percentage of MI	Chromatid	Chromosome	Chromatid	Chromosome	Chromatid	Chromosome
Set 2 (-S9) (3-6 hours)	Vehicle control	-	R1	28	512	0.0519	5.19	0	0	0	0	0	0	1	0	0	0	1	1	1	0.7
			R2	29	551	0.05	5.00	0	2	0	0	0	0	2	0	0	0	4	2	2	1.3
	Positive control (Mitomycin C)	0.05	R1	23	506	0.0435	4.35	0	4	0	8	0	6	21	0	0	0	39	35	18	12
			R2	26	559	0.0444	4.44	0	5	0	5	0	3	26	0	0	0	39	34	17	11.3
	Test item	31.25	R1	26	540	0.0459	4.59	0	0	0	0	0	0	2	0	0	0	2	2	1	0.7
			R2	23	539	0.0409	4.09	0	1	0	0	0	0	0	0	0	0	1	0	0	0
		62.5	R1	20	540	0.0357	3.57	0	0	0	0	0	0	0	0	0	0	0	0	0	0
			R2	24	545	0.0422	4.22	0	1	0	0	0	0	1	0	0	0	2	1	1	0.7
		125	R1	15	560	0.0261	2.61	0	1	0	0	0	0	1	0	0	0	2	1	1	0.7
			R2	18	531	0.0328	3.28	0	0	0	0	0	0	0	0	0	0	0	0	0	0

MI: Mitotic Index, MP: Metaphase Plates,R1: Replicate 1, R2: Replicate 2, +S9: With metabolic activation,Test Item =Genagen PA/ N, N Dimethylnonanamide]

TABLE 3 -3:INDIVIDUAL DATA OF CHROMOSOMAL ABERRATIONAND MITOTIC INDEX

Set No.	Treatment	Dose (µg/mL)	Replicate	Mitotic Index				Aberrations										Total No. of Aberrations	Total No. of Aberrations without Gaps	Total no. of Aberrant cells	Total % of Aberrated cells
								Gap		Break		Exchange		Fragments	Ring	Deletion	Dicentric
				Total No. of MP	Total No. of Blast cells	Mitotic Index	Percentage of MI	Chromatid	Chromosome	Chromatid	Chromosome	Chromatid	Chromosome
Set 3 (-S9) (20-24 hours)	Vehicle control	-	R1	34	514	0.062	6.20	0	1	0	0	0	0	0	0	0	0	1	0	0	0
			R2	29	538	0.0511	5.11	0	0	0	0	0	0	1	0	0	0	1	1	1	0.7
	Positive control       (Mitomycin C)	0.05	R1	30	556	0.0512	5.12	0	2	0	4	0	3	22	0	0	0	31	29	17	11.3
			R2	25	526	0.0454	4.54	0	2	7	0	0	3	24	0	0	0	36	34	19	12.7
	Test item	31.25	R1	26	525	0.0472	4.72	0	0	0	1	0	0	0	0	0	0	1	1	1	0.7
			R2	23	507	0.0434	4.34	0	0	0	0	0	0	2	0	0	0	2	2	1	0.7
		62.5	R1	25	526	0.0454	4.54	0	1	0	0	0	0	0	0	0	0	1	0	0	0
			R2	22	536	0.0394	3.94	0	0	0	0	0	0	1	0	0	0	1	1	1	0.7
		125	R1	16	531	0.0293	2.93	0	0	0	0	0	0	1	0	0	0	1	1	1	0.7
			R2	17	508	0.0324	3.24	0	0	0	0	0	0	1	0	0	0	1	1	1	0.7

MI: Mitotic Index, MP: Metaphase Plates, R1: Replicate 1, R2: Replicate 2, -S9: Without metabolic activation,Test Item =Genagen PA/ N, N Dimethylnonanamide]

Conclusions:

The genotoxicity (clastogenicity) of Genagen PA/ N, N-Dimethylnonanamide was investigated according to the Guideline OECD 473. No clastogenic acitivity was found.

Executive summary:

The test item,Genagen PA/ N, N-Dimethylnonanamide, was evaluated for chromosome aberrations in human lymphocytes, as per the OECD guideline for the testing of chemicals, No. 473 “In vitroMammalian Chromosomal AberrationTest” adopted on 29 July 2016.

Test item was found to be soluble in DMSO at 200 mg/mL. Precipitation test was conducted at 62.5, 125, 250, 500, 1000 and 2000 µg/mL. Post 24 hours of incubation, no change in pH was observed at any of the concentrations tested upto 2000 µg/mL. Slight precipitation was observed at the tested concentrations at 2000 µg/mL. Hence, 2000 µg/mL was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were 62.5, 125, 250, 500 and 1000 µg/mL.

The treatment of cultures withGenagen PA/ N, N-Dimethylnonanamide at all concentrations testedresulted in the decrease of Mitotic Index (MI) and the obtained reduction in MI was in the range of 18.40% to 100%. The reduction in MI at 125 µg/mL was in the range of 41.10 to 45.61%. Hence 125 µg/mL was selected as the highest concentration for the chromosomal aberration test for both short term (3 to 6 hrs) and long term (20 to 24 hrs) treatment. The other concentrations tested were 62.5 and 31.25 µg/mL.   

In the chromosomal aberration test, the cells were treated withGenagen PA/ N, N-Dimethylnonanamideat the concentrations of 31.25, 62.5 and 125 µg/mLrespectivelyusing DMSO as the vehicle. Cyclophosphamide Monohydrate (+S9) at10 µg/mL andMitomycin-C (-S9 both for short term and long term) at 0.05µg/mLwere used as positive controls. The treatment was carried out in duplicates for short term period
(3 to 6 hours) both in the presence and absence of metabolic activation and long term (20 to 24 hours) in the absence of metabolic activation.

Later, 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), harvested, stained and metaphase cells were analysed microscopically for the structural chromosomal aberrations.

The results with Genagen PA/ N, N-Dimethylnonanamide indicated no statistically significant increase in the number of aberrated cells when compared with vehicle control at any of the concentrations tested.The reduction in MI observed at
125 µg/mL was 41.35% in the presence of metabolic activation and 42.24% and 45.58% in the absence of metabolic activation for short and long term treatments, respectively.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The genotoxicity of Genagen PA/ N,N-Dimethylnonanamide was investigated in three in-vitro tests. Genagen PA/ N,N-Dimethylnonanamide is not mutagenic in Ames test, not clastogenic in chromosome aberration test and not mutagenic in HPRT Assay. No classification is justified.