cyclic C12/14-Glucamide;cyclic N-methyl-N-dodecanoyl/tetradecanoyl-glucamine;anhydro-N-dodecanoyl/tetradecanoyl-N-methylglucamine;Amides, C12-C14, N-(1-deoxy-D-glucitol-1-yl)-N-methyl, dehydrated

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 December 2020 to 04 May 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

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: The purpose of this study is to evaluate the mutagenic potential of the test item, Cyclic Glucamide C12-C14 based on quantitation of forward mutations at the Hprt locus of CHO AA8 cells.

Test material

Test material information

Constituent 1

Reference substance name:

D-Glucitol, anhydro-1-deoxy-1-(methylamino)-, N-[C12-14(even numbered) acyl] derivs.

Molecular formula:

C19H37NO5 C21H41NO5

IUPAC Name:

D-Glucitol, anhydro-1-deoxy-1-(methylamino)-, N-[C12-14(even numbered) acyl] derivs.

Test material form:

semi-solid (amorphous): gel

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Clariant Produkte (Deutschland) GmbH
- batch No.of test material: S148866
- Expiration date of the batch: 30.09.2022
- Purity : 100 % due to “ UVCB”

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

Method

Target gene:

Hprt locus of CHO AA8 cells

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO AA8 cell line

Metabolic activation:

with and without

Metabolic activation system:

Sodium phenobarbitone and β-Naphthoflavone induced rat liver S9 homogenate

Test concentrations with justification for top dose:

At concentration of 0.078125 mg/mL, the Relative Survival was greater than 10%. Therefore 0.078125 mg/mL was selected as the highest concentration for testing in gene mutation test.

Vehicle / solvent:

Dimethyl sulphoxide (DMSO)

Controls

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, Batch No.5000062 procured from American Type Culture Collection (ATCC) was used for the test.
Frozen stock of cryovial was thawed immediately at 37±1°C in the water bath. Cells were transferred in to a sterile flask with culture medium containing 10% FBS with antibiotics (1% Penicillin and Streptomycin) and incubated at 37±1°C and 5±1% CO2 for 3 days. The cell lines were trypsinized using trypsin-EDTA and the trypsinized cultures were subcultured 3 times for (Initial cytotoxicity test) and 3 times for (Gene mutation test) before using for the experiment.

Rationale for test conditions:

Approximately 2×106 (Initial cytotoxicity test and Gene mutation test) cells per culture flask were seeded using culture medium with 10% FBS with antibiotics (1% Penicillin and Streptomycin). Four additional flasks were seeded and kept for incubation along with flasks for treatment to determine cell count at the beginning of the treatment to determine the Adjusted Cloning Efficiency. The flasks were incubated at 37±1oC with 5±1% CO2 for 23 hours and 30 minutes (Initial cytotoxicity test) and 25 hours (Gene mutation test). Cells free of mycoplasma were used for the experiment.

Evaluation criteria:

Colony counts in selective and non-selective media

Statistics:

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:

cytotoxicity

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: Insoluble
- Precipitation: Post 4 hours of incubation, no precipitation was observed at the tested concentrations of 0.078125, 0.15625, 0.3125, 0.625 mg/mL, moderate precipitation was observed at 1.25 mg/mL, heavy precipitation was observed at 2.5 and 5 mg/mL
-
RANGE-FINDING/SCREENING STUDIES: yes

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: Relative survival

Remarks on result:

other: Cyclic Glucamide C12-C14 is considered as non-mutagenic at and upto the concentration of 0.078125 mg/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.

Any other information on results incl. tables

TABLE 1.           SUMMARY OF INITIAL CYTOTOXICITY TEST

Set No.	Treatment	Concentration (mg/mL)	Average Colony Count± SD			Cloning  Efficiency (CE)	Adjusted Cloning Efficiency (ACE)	Relative Survival (RS) (%)
Set 1 +S9	Vehicle Control  (DMSO)	-	185.67	±	3.79	0.93	1.16	-
	Cyclic Glucamide C12-C14	0.078125	40.67	±	1.53	0.20	0.12	10.34
		0.15625	12.00	±	2.00	0.06	0.02	1.72
		0.3125	0.00	±	0.00	0.00	0.00	0.00
		0.625	0.00	±	0.00	0.00	0.00	0.00
		1.25	0.00	±	0.00	0.00	0.00	0.00
Set 2 -S9	Vehicle Control  (DMSO)	-	183.33	±	6.66	0.92	1.14	-
	Cyclic Glucamide C12-C14	0.078125	43.67	±	5.69	0.22	0.13	11.40
		0.15625	7.67	±	4.93	0.04	0.02	1.75
		0.3125	0.00	±	0.00	0.00	0.00	0.00
		0.625	0.00	±	0.00	0.00	0.00	0.00
		1.25	0.00	±	0.00	0.00	0.00	0.00

 +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

Set No.	Treatment	Concentration (mg/mL)	Average Colony count ± SD			Cloning Efficiency (CE)	Adjusted Cloning Efficiency (ACE)	Relative Survival (RS) (%)
Set 1 +S9	Vehicle Control (DMSO)	-	185.67	±	4.04	0.93	1.16	-
	Cyclic Glucamide C12-C14	0.009765625	132.67	±	11.24	0.66	0.65	56.03
		0.01953125	104.00	±	16.37	0.52	0.48	41.38
		0.0390625	85.33	±	5.03	0.43	0.29	25.00
		0.078125	48.67	±	6.03	0.24	0.14	12.07
	Benzo(a)pyrene (Positive Control)	3 µg/mL	179.33	±	4.51	0.90	1.07	92.24
Set 2 -S9	Vehicle Control (DMSO)	-	185.67	±	5.86	0.93	1.17	-
	Cyclic Glucamide C12-C14	0.009765625	139.00	±	6.56	0.70	0.69	58.97
		0.01953125	102.67	±	8.33	0.51	0.46	39.32
		0.0390625	74.33	±	9.5	0.37	0.26	22.22
		0.078125	51.33	±	6.03	0.26	0.15	12.82
	4 Nitroquinoline N-oxide (Positive Control)	1 µg/mL	182.67	±	6.43	0.91	1.07	91.45

 +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.

TABLE 3.           SUMMARY OF GENE MUTATION TEST

Set No.	Treatment	Concentration (mg/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)	-	184.00	±	6.24	0.0000105	0.92	21	22.83
	Cyclic Glucamide C12-C14	0.009765625	176.67	±	8.08	0.0000105	0.88	21	23.86
		0.01953125	167.67	±	6.03	0.0000100	0.84	20	23.81
		0.0390625	162.67	±	5.51	0.0000095	0.81	19	23.46
		0.078125	159.00	±	2.65	0.0000095	0.80	19	23.75
	Benzo(a) pyrene               (Positive Control)	3 µg/mL	181.67	±	3.51	0.0001115	0.91	223	245.05**
Set 2 -S9	Vehicle Control (DMSO)	-	185.00	±	3.61	0.0000115	0.93	23	24.73
	Cyclic Glucamide C12-C14	0.009765625	174.33	±	4.51	0.0000110	0.87	22	25.29
		0.01953125	172.00	±	7.94	0.0000105	0.86	21	24.42
		0.0390625	164.33	±	3.51	0.0000105	0.82	21	25.61
		0.078125	158.33	±	7.51	0.0000095	0.79	19	24.05
	4 Nitroquinoline N-oxide (Positive Control)	1 µg/mL	180.67	±	2.08	0.0001150	0.90	230	255.56**

+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, Cyclic Glucamide C12-C14 is considered as non-mutagenic at and upto the concentration of 0.078125 mg/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.

Executive summary:

The test itemCyclic Glucamide C12-C14was evaluated for gene mutation test in CHO AA8 cells.

The test item was found soluble in DMSO at 500 mg/mL. Precipitation test was conducted at 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 mg/mL concentrations. Post 4 hours of incubation, no precipitation was observed at the tested concentrations of 0.078125, 0.15625, 0.3125 and 0.625 mg/mL, moderate precipitation was observed at 1.25 mg/mL, and heavy precipitation were observed at 2.5 and 5 mg/mL. No change in pH was observed in any of the test concentrations.

On the basis of precipitation results, 1.25 mg/mL was selected as the highest concentration for the initial cytotoxicity test. Initial cytotoxicity test was conducted at the concentrations of 0.078125, 0.15625, 0.3125, 0.625 and 1.25 mg/mL using DMSO as a vehicle in tetra plates/group in the presence and absence of metabolic activation (3 hours and 28 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% (10.34 % in presence of metabolic activation and11.40 % in absence of metabolic activation) at 0.078125 mg/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Based on these results, 0.078125 mg/mL was selected as highest concentration for gene mutation test.

The gene mutation test was conducted at the concentrations of 0.009765625, 0.01953125, 0.0390625 and 0.078125 mg/mLusing DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation (3 hours and 14 minutes).Benzo(a) pyrene and4 Nitroquinoline N-oxidewere used aspositive controlsfor the gene mutation test.

Cytotoxicity as Relative Survival was 12.07 % in presence of metabolic activation and12.82 % in absence of metabolic activationat the highest tested concentration of 0.078125 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 withCyclic Glucamide C12-C14 resulted 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 bothmetabolic activation andabsence of metabolic activation.