[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 October 2019 TO 09 April 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted on 29th July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Test material

Test material information

1

Reference substance name:

Reaction products of DL-methionine and C18 unsaturated fatty acid chloride and isopropanol

Molecular formula:

not applicable (UVCB)

IUPAC Name:

Reaction products of DL-methionine and C18 unsaturated fatty acid chloride and isopropanol

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Clariant Ibérica Producción,S.A.
- Batch: ESD0033040
- Expiration date of the batch: August 2021
- Purity : UVCB substance; purity ca 97%

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Solubility of the test substance in the solvent/vehicle: miscible in DMSO

Method

Target gene:

HPRT gene

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The derivative of the CHO-K1, CHO AA8 Cells

Metabolic activation:

with and without

Metabolic activation system:

induced rat liver S9 homogenate

Test concentrations with justification for top dose:

moderate precipitation was observed at 0.25 µL/mL and the Relative Survival was greater than 10%. Therefore 0.125 µL/mL was selected as the highest concentration for testing in gene mutation test.
(four concentrations i.e. 0.015625, 0.03125, 0.0625 and 0.125 µL/mL were selected for gene mutation test)

Vehicle / solvent:

dimethyl sulphoxide

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Details on test system and experimental conditions:

CHO AA8 cells, Batch No.5000062 procured from American Type Culture Collection (ATCC).

Evaluation criteria:

• The concurrent vehicle control is considered acceptable for addition to the laboratory historical vehicle control database
• Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative/vehicle control
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
[Where X=(No. of mutant colonies per replicate)/(ACE 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.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No change in pH was observed in any of the test concentrations.
- Water solubility: No
- Precipitation: No change in precipitation was observed at the tested concentrations 0.03125 and 0.0625 µL/mL, slight precipitation was observed at 0.125 µL/mL, moderate precipitation was observed at 0.25 µL/mL and heavy precipitation was observed at 0.50, 1 and 2 µL/mL.


RANGE-FINDING STUDIES: Pre study conducted to select highest test concentration.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Positive Control- Benzo(a)pyrene and 4- Nitroquinoline N-oxide
With Metabolic Activation
(3 to 6 hours)
[Benzo(a)pyrene] Without Metabolic Activation
(3 to 6 hours)
[4 Nitroquinoline N-oxide]
Mean Data of Mutant Frequency/2x106 Cells 261.94 264.60
Standard
Deviation 27.28 18.52
Margin of Error 17.82 12.10
Upper bound 279.76 276.70
Lower bound 244.12 252.50

- Negative (solvent/vehicle) historical control data:
Vehicle-DMSO
With Metabolic Activation
(3 to 6 hours) Without Metabolic Activation
(3 to 6 hours)
Mean Data of Mutant Frequency/2x106 Cells 24.51 25.43
Standard
Deviation 2.81 1.89
Margin of Error 1.95 1.31
Upper bound 26.46 26.74
Lower bound 22.56 24.12

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Yes

Remarks on result:

other:

Remarks:

Non mutagenic

Any other information on results incl. tables

 TABLE 1.           SUMMARY OF INITIAL CYTOTOXICITY TEST

Refer: Appendix 1

Set No.		Treatment		Concentration (µL/mL)		Average Colony Count± SD				Cloning           Efficiency (CE)		Adjusted Cloning Efficiency (ACE)		Relative Survival (RS) (%)
Set 1 +S9		Vehicle Control  (DMSO)		-		183.33		±		7.57		0.92		1.17		-
		Dodicor V 5654		0.0078125		172.00		±		3.00		0.86		1.07		91.45
			0.015625		163.00		±		8.19		0.82		0.99		84.62
			0.03125		143.00		±		4.36		0.72		0.85		72.65
			0.0625		103.00		±		4.58		0.52		0.50		42.74
			0.125		76.67		±		5.13		0.38		0.31		26.50
Set 2 -S9		Vehicle Control   (DMSO)		-		179.00		±		6.56		0.90		1.10		-
		Dodicor V 5654		0.0078125		162.67		±		4.04		0.81		0.97		88.18
			0.015625		152.33		±		7.51		0.76		0.90		81.82
			0.03125		136.00		±		7.21		0.68		0.73		66.36
			0.0625		101.33		±		3.51		0.51		0.48		43.64
			0.125		62.33		±		4.93		0.31		0.27		24.55

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

 Adjusted CE = CE × 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 × 100.

 CE = Number of colonies/Number of cells plated.

 

 

 

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

Refer: Appendix 2

Set No.	Treatment	Concentration (µL/mL)	Average Colony Count ± SD			Cloning Efficiency (CE)	Adjusted Cloning Efficiency (ACE)	Relative Survival (RS) (%)
Set 1 +S9	Vehicle Control (DMSO)	-	182.00	±	3.61	0.91	1.14	-
	Dodicor V 5654	0.015625	174.00	±	4.36	0.87	1.08	94.74
		0.03125	173.00	±	6.56	0.87	1.03	90.35
		0.0625	95.67	±	8.33	0.48	0.47	41.23
		0.125	72.00	±	4.36	0.36	0.31	27.19
	Benzo(a)pyrene              (Positive Control)	3 µg/mL	145.33	±	10.02	0.73	0.77	67.54
Set 2 -S9	Vehicle Control (DMSO)	-	178.67	±	10.02	0.89	1.09	-
	Dodicor V 5654	0.015625	172.00	±	4.00	0.86	1.04	95.41
		0.03125	166.33	±	4.04	0.83	0.98	89.91
		0.0625	102.67	±	8.62	0.51	0.50	45.87
		0.125	68.33	±	14.05	0.34	0.31	28.44
	4 Nitroquinoline N-oxide (Positive Control)	1 µg/mL	140.33	±	8.50	0.70	0.71	65.14

 +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 × 100.

 CE = Number of colonies/Number of cells plated.

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

 

 

 

Set No.	Treatment	Concentration (µL/mL)	*Average Colony Count ± SD			Cloning Efficiency in selective media	Cloning Efficiency in non-selective media	Total number of Mutant Colonies/ 2×10^6cells	Mutant Frequency/ 2×10^6cells
Set 1 +S9	Vehicle Control (DMSO)	-	188.00	±	6.24	0.0000115	0.94	23	24.47
	Dodicor V 5654	0.015625	175.00	±	5.29	0.0000115	0.88	23	26.14
		0.03125	181.33	±	9.02	0.0000120	0.91	24	26.37
		0.0625	168.33	±	3.06	0.0000110	0.84	22	26.19
		0.125	159.00	±	4.58	0.0000100	0.80	20	25.00
	Benzo(a)pyrene              (Positive Control)	3 µg/mL	163.67	±	8.50	0.0001060	0.82	212	258.54**
Set 2 -S9	Vehicle Control (DMSO)	-	183.00	±	7.21	0.0000120	0.92	24	26.09
	Dodicor V 5654	0.015625	180.33	±	1.53	0.0000115	0.90	23	25.56
		0.03125	174.00	±	7.00	0.0000105	0.87	21	24.14
		0.0625	176.00	±	8.54	0.0000115	0.88	23	26.14
		0.125	164.33	±	5.51	0.0000105	0.82	21	25.61
	4 Nitroquinoline N-oxide (Positive Control)	1 µg/mL	152.67	±	10.07	0.0001030	0.76	206	271.05**

TABLE 3.           SUMMARY OF GENE MUTATION TEST

Refer: Appendix 3

+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.

Applicant's summary and conclusion

Conclusions:

Based on the results obtained, the test item, Dodicor V 5654 is considered as non-mutagenic at and upto the concentration of 0.125 µL/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.

Executive summary:

The test itemDodicor V 5654was evaluated for gene mutation test in CHO AA8 cells.

The test item was found miscible in DMSO at 200 µL/mL. Precipitation test was conducted at 0.03125, 0.0625, 0.125, 0.25, 0.50, 1 and 2 µL/mL. Post 3 hours and
3 minutes of incubation, no change in precipitation was observed at the tested concentrations of 0.03125 and 0.0625 µL/mL, slight precipitation was observed at
0.125 µL/mL, moderate precipitation was observed at 0.25 µL/mL and heavy precipitation was observed at 0.50, 1 and 2 µL/mL. No change in pH was observed in any of the test concentrations.

On the basis of precipitation results, 0.125 µL/mL was selected as the highest concentration for the initial cytotoxicity test. Initial cytotoxicity test was conducted at the concentrations of 0.0078125, 0.015625, 0.03125, 0.0625 and 0.125 µL/mL using DMSO as a vehicle in tetra plates/group in the presence and absence of metabolic activation (3 to 6 hours). 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% (26.50% in presence of metabolic activation and24.55% in absence of metabolic activation) at 0.125 µL/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Based on these results, 0.125 µL/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 µL/mLusing DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation (3 to 6 hours).

Benzo(a) pyrene and4 Nitroquinoline N-oxidewere used asPositive controlsfor the gene mutation test.

Cytotoxicity as Relative Survival was 27.19% in presence of metabolic activation and28.44% in absence of metabolic activation) at the highest tested concentration of 0.125 µL/mL.

There was no statistically significant increase in mutant frequencies at any of the concentrations tested when compared with the vehicle control. Moreover, treatment withDodicor V 5654resulted in mutant frequencies which fell within acceptable ranges with regard to historical controls.

There was statistically significant increase in mutant frequenciesfor positive controlswhen compared with the vehicle controlin both the phases of the experiment.