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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in-vitro gene mutation study (Ames, OECD 471): negative

in-vitro mammalian cell gene mutation study (HRPT, OECD 476): negative

in-vitro mammalian cell micronucleus study (MN, OECD 487): negative

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:

02 December 2020 to 04 May 2021

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:

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.

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

Target gene:

Hprt locus of CHO AA8 cells

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)

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.

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.

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 December 2020 TO 02 June 2021

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 26thJune 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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 %

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

Target gene:

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

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: Genetic characterization

Metabolic activation:

with and without

Metabolic activation system:

S9 homogenate

Test concentrations with justification for top dose:

Based on the results of the initial cytotoxicity test, the test concentrations of 0.001, 0.003, 0.01, 0.032 and 0.1 mg/plate were selected.

Vehicle / solvent:

The test item found soluble in DMSO at the concentration of 50 mg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other:

Remarks:

2-Aminoanthracene and 4-Nitroquinoline N-Oxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; i
- Tester strain density: 18×108 cells/mL.

DURATION
- Preincubation period: 20 to 22 minutes

SELECTION AGENT (mutation assays): Minimal glucose agar plates.

NUMBER OF REPLICATIONS: 03

DETERMINATION OF CYTOTOXICITY
- Method: Lawn intewnsity and number of revertant colonies

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 control are in the range of in house historical control data and preferably within the range reported in the literature and as per new batch of certificate of analysis received by the vendor.
• The tester strains must met all required genetic characterization.
• Tester Strains used were in the approximate range of 1×109 to 2×109 cells/mL.
• Minimum of five analyzable test item dose concentrations were used to evaluate the mutation assay and none of these must show any signs of contamination.
• Cytotoxicity was 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.

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 that 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

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

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

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

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

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

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

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

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

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

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Insoluble
- Precipitation: The test item resulted slight precipitation at 3.2 and 5 mg/plate and no precipitation from 0.1 to 1.6 mg/plate at tested concentrations

RANGE-FINDING/SCREENING STUDIES: Initial cytotoxixity test

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Plate Incorporation Method:
Positive Control Mean 383.1 402.9 141.0 120.0 394.7 363.9 393.5 132.1 112.4 387.8
±SD 11.8 10.3 8.5 8.7 11.7 13.2 10.0 11.2 8.2 11.7
Min 320 370 112 96 356 310 348 94 91 352
Max 416 425 165 142 421 398 416 159 143 412

Preincubation Method :
Positive Control Mean 381.9 402.7 140.1 119.2 396.1 365.7 394.6 131.6 111.4 389.1
±SD 11.1 10.2 9.5 7.9 11.8 12.0 9.8 11.9 8.2 12.2
Min 351 370 107 93 348 329 365 100 90 352
Max 415 430 163 140 421 402 426 158 134 415

- Negative (solvent/vehicle) historical control data:
Plate Incorporation Method:
Vehicle Control (Dimethyl Sulphoxide) Mean 29.0 108.0 19.8 9.8 73.2 26.0 102.3 18.2 8.4 69.4
±SD 4.9 6.7 2.6 1.8 6.8 5.1 5.9 2.4 1.6 5.8
Min 16 95 15 5 57 16 90 13 5 56
Max 42 123 26 14 86 41 120 26 12 81

Preincubation Method :
Vehicle Control (Dimethyl Sulphoxide) Mean 28.6 108.0 19.8 9.7 73.1 26.2 103.1 18.3 8.4 69.6
±SD 4.9 6.5 2.7 1.8 6.8 4.9 5.8 2.4 1.6 5.5
Min 18 93 14 6 58 16 90 13 5 56
Max 41 123 26 14 88 42 120 24 13 80

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: number of revertant colonies and lawn intensity.

Conclusions:

Based on the results of the study it is concluded that the test item Cyclic Glucamide C12-C14 is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 0.1 mg/plate under the test conditions when tested on Salmonella typhimurium TA98, TA100, TA1535, TA1537 and E.coli WP2 uvrA (pKM101) tester strains.

Executive summary:

The test item,Cyclic Glucamide C12-C14was evaluated for mutagenicity in Bacterial Reverse Mutation Test.

The test itemfound solublein DMSO at a concentration of 50mg/mL and slight precipitation was observed at 3.2 and 5 mg/plate, based on which 5 mg/platewas selected as the highest test concentration for initial cytotoxicity test.

In the initial cytotoxicity test, Salmonella typhimuriumTA100 tester strain was exposed to vehicle control and range of concentrations from 0.00625 to5 mg/plate.

The test item resulted cytotoxicity from 0.8 mg/plate to 5 mg/plate with lawn intensity    0 (No lawn), 0.4 mg/plate with lawn intensity 1+ (Very thin lawn), 0.2 mg/plate with lawn intensity 2+ (Thin lawn) and slight cytotoxicity at0.1 mg/platewith lawn intensity3+ (Slightly thin lawn) andno cytotoxicity at 0.00625, 0.0125, 0.025, and 0.05 mg/platewith 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, 0.1mg/plate was selected as highest test concentration for mutation assay with the other test concentrations 0.001, 0.003, 0.01 and 0.032 mg/plate.

The test item was assessed for its mutagenic effects usingSalmonella typhimuriumtester strains: TA98, TA100, TA1535, TA1537 andEscherichiacoliWP2 uvrA (pKM101).  The test item was tested for plate incorporation method (trail I) and for preincubation method (trail II) in the presence and absence of metabolic activation system using DMSOasvehicle and appropriate positive controls (2-nitrofluorene, sodium azide,  9-Aminoacridine and4-nitroquinoline N-oxidefor trials “without metabolic activation” and 2-Aminoanthracene for trials “with metabolic activation”) were tested simultaneously. The trials were carried out 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 (Except 0.1 mg/plate) in both the trials, (0.032 mg/plate in trail II inSalmonella typhimuriumTA100 ) 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 3.8 to 16.1 fold increase underidenticalconditions.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 December 2020 to 16 April 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

data waiving: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted on 29th July 2016.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

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

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

Target gene:

Human peripheral blood lymphocytes were used

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Human peripheral blood lymphocytes were used

Cytokinesis block (if used):

cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

S9 homogenate

Test concentrations with justification for top dose:

0.0048828125, 0.009765625 and 0.01953125 mg/mL based on the results of initial cytotoxixity test.

Vehicle / solvent:

found soluble in DMSO at 500 mg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium RPMI

DURATION
- Exposure duration:
Set No. Metabolic
Activation Treatment Duration
1 +S9 Vehicle control/Test item/Positive control (10 µg/mL Cyclophosphamide Monohydrate) 3 hours and 2 minutes
2 - S9 Vehicle control/Test item/Positive control (0.03 µg/mL Colchicine) 3 hours and 2 minutes
3 - S9 Vehicle control/Test item/Positive control
(0.075 µg/mL Mitomycin C) 23 hours and
40 minutes .

STAIN : Acridine orange

NUMBER OF REPLICATIONS:02

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Pellet was mixed with 4 mL of freshly prepared 0.56% Potassium chloride. Cell suspension was incubated for 10 minutes at room temperature and later it was centrifuged at 1800 rpm for 10 minutes. Supernatant was discarded and cell pellet was mixed with 4 mL of freshly prepared cold acetic acid: methanol fixative (1:3). Cells were incubated for 10 minutes at room temperature and later suspension was centrifuged at 1500 to 2000 rpm for 10 minutes. The procedure was repeated twice by adding 4 mL of cold acetic acid: methanol fixative (1:3) and slides were prepared.
Clean slides were stored in a container with distilled water and kept in the refrigerator for 1 hour before use. The cell suspension was mixed using a pipette and few drops of the suspension was aspirated and dropped onto the chilled slide pre labeled with study number, with (+S9) or without metabolic activation(-S9), treatment/group and slide number. The slides were air dried. Smear was stained using Acridine Orange stain by allowing the stain to retain for 5 minutes. Slides were evaluated for cytotoxicity by observing under 40X

NUMBER OF CELLS EVALUATED: at least 2000 binucleates per concentration (at least 1000 per culture).

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
•The cytoplasm has remained essentially intact, and
•The daughter nuclei are of approximately equal size
A micronucleus was only recorded as it meets the following criteria:
•The micronucleus should have the same staining characteristics and a similar morphology to the main nuclei and
•Any micronucleus present is separate in the cytoplasm or only just touching a main nucleus
•Micronuclei should be smooth edged and smaller than approximately one third the diameter of the main nuclei

DETERMINATION OF CYTOTOXICITY
- Cytokinesis-Block Proliferation Index (CBPI). CBPI was determined from 500 cells per culture

Rationale for test conditions:

•The concurrent negative control is considered acceptable for addition to the historical negative control database as described OECD 487.
•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 control.
•Cell proliferation criteria in the solvent control should be fulfilled.
•All experimental conditions were tested unless one resulted in positive results.
•Adequate number of cells and concentrations are analysable (according to OECD 487 (2016)).
•The criteria for the selection of top concentration are consistent with those described in OECD 487 (2016).

Evaluation criteria:

•All slides including vehicle control, treatment and positive controls of main study were coded before evaluation.
•Concurrent measures of cytotoxicity for all treated and vehicle control cultures were determined using a single slide from each treatment.
•The CBPI will be calculated for all treated and control cultures as measurement of cell cycle delay.
•Slides were evaluated for the presence of micronuclei in at least 2000 binucleates per concentration (at least 1000 per culture).
•Any of the results are outside the distribution of the historical negative control data.
•A test item is considered to be clearly positive if:
At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
The increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test.
•A test item is considered to be clearly negative if:
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.

Statistics:

% Micronucleus data was analyzed using SPSS software version 22 for differences among 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 throughout the study report as stated below.
*: Statistically significant (p≤0.05).

Key result

Species / strain:

lymphocytes: Human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Water solubility: Insoluble
- Precipitation: heavy precipitation was observed at 2.5 and 5 mg/mL, moderate and mild precipitation was observed at 1.25 and 0.625 mg/mL and no precipitation was observed at 0.15625 and 0.3125 mg/mL

RANGE-FINDING/SCREENING STUDIES:

CYTOKINESIS BLOCK (if used) : Cytochalasin B
- Distribution of mono-, bi- and multi-nucleated cells: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI
- Other observations when applicable: frequency of binucleated cells

 

TABLE 1.     SUMMARY OFCYTOKINESIS BLOCK PROLIFERATION INDEXAND CYTOTOXICITY

 

Treatment	Metabolic Activation	Concentration (mg/mL)	Duration (hours)	Replicates No.	Total Number of Cells	CBPI	Mean CBPI	% of Cytotoxicity
Vehicle Control	With S9	0	3 to 6	1	505	1.63	1.63	NA
				2	523	1.63
CyclicGlucamide C12-C14		0.078125		1	524	1.18	1.18	71.43
				2	501	1.18
		0.15625		1	515	1.07	1.07	88.89
				2	524	1.06
		0.3125		1	511	1.02	1.02	96.83
				2	526	1.01
		0.625		1	514	1.00	1.00	100.00
				2	506	1.00
		1.25		1	514	1.00	1.00	100.00
				2	523	1.00
Vehicle Control	Without S9	0	3 to 6	1	524	1.63	1.64	NA
				2	513	1.65
CyclicGlucamide C12-C14		0.078125		1	516	1.16	1.17	73.44
				2	520	1.17
		0.15625		1	506	1.06	1.06	90.63
				2	523	1.06
		0.3125		1	518	1.02	1.01	98.44
				2	517	1.01
		0.625		1	523	1.00	1.00	100.00
				2	524	1.00
		1.25		1	518	1.00	1.00	100.00
				2	520	1.00
Vehicle Control	Without S9	0	20 to 24	1	534	1.61	1.62	NA
				2	525	1.63
CyclicGlucamide C12-C14		0.078125		1	530	1.17	1.16	74.19
				2	511	1.16
		0.15625		1	547	1.07	1.06	90.32
				2	527	1.05
		0.3125		1	520	1.01	1.01	98.39
				2	514	1.02
		0.625		1	524	1.00	1.00	100.00
				2	522	1.00
		1.25		1	504	1.00	1.00	100.00
				2	508	1.00

CBPI:Cytokinesis Block Proliferation Index; R: Replicate

 

Follow up initial cytotoxicity test :

 

 

Treatment	Metabolic Activation	Concentration (mg/mL)	Duration (hours)	Replicates No.	Total Number of Cells	CBPI	Mean CBPI	% of Cytotoxicity
Vehicle Control	With S9	0	3 to 6	1	501	1.60	1.60	NA
				2	525	1.60
CyclicGlucamide C12-C14		0.00244140625		1	512	1.52	1.53	11.67
				2	521	1.54
		0.0048828125		1	501	1.45	1.44	26.67
				2	505	1.43
		0.009765625		1	510	1.36	1.35	41.67
				2	504	1.33
		0.01953125		1	504	1.28	1.28	53.33
				2	517	1.28
		0.0390625		1	505	1.22	1.21	65.00
				2	557	1.20
Vehicle Control	Without S9	0	3 to 6	1	507	1.57	1.59	NA
				2	505	1.60
CyclicGlucamide C12-C14		0.00244140625		1	517	1.52	1.52	11.86
				2	504	1.53
		0.0048828125		1	511	1.46	1.44	25.42
				2	508	1.42
		0.009765625		1	542	1.35	1.35	40.68
				2	562	1.34
		0.01953125		1	510	1.27	1.26	55.93
				2	512	1.25
		0.0390625		1	506	1.21	1.20	66.10
				2	517	1.19
Vehicle Control	Without S9	0	20 to 24	1	523	1.62	1.62	NA
				2	540	1.61
CyclicGlucamide C12-C14		0.00244140625		1	525	1.53	1.52	16.13
				2	537	1.52
		0.0048828125		1	547	1.45	1.45	27.42
				2	532	1.45
		0.009765625		1	555	1.35	1.35	43.55
				2	566	1.35
		0.01953125		1	507	1.27	1.26	58.06
				2	525	1.26
		0.0390625		1	512	1.19	1.20	67.74
				2	510	1.20

CBPI:Cytokinesis Block Proliferation Index; R: Replicate

 

 

TABLE 2.     SUMMARY OF MICRONUCLEI INCIDENCE WITH METABOLIC ACTIVATION FOR 3 TO 6 HOURS

Treatment	Concentration (mg/mL)	Replicate No.	Average CBPI	Average % of Cytotoxicity	Total No. of Binucleates	Total No. of Micronucleus	Average Percentage of Micronucleus
Vehicle Control	0	1	1.60	0.0	2020	3	0.15
		2
CyclicGlucamide C12-C14	0.0048828125	1	1.45	25.00	2034	4	0.20
		2
	0.009765625	1	1.35	41.67	2007	3	0.15
		2
	0.01953125	1	1.29	51.67	2077	4	0.20
		2
Cyclophosphamide Monohydrate	10 µg/mL	1	1.56	6.67	2057	23	1.12*
		2

 

SUMMARY OF MICRONUCLEI INCIDENCE WITHOUT METABOLIC ACTIVATION FOR 3 TO 6 HOURS

 

 

Treatment	Concentration      (mg/mL)	Replicate No.	Average CBPI	Average % of Cytotoxicity	Total No. of Binucleates	Total No. of Micronucleus	Average Percentage of Micronucleus
Vehicle Control	0	1	1.62	0.0	2084	4	0.19
		2
CyclicGlucamide C12-C14	0.0048828125	1	1.48	22.58	2062	3	0.15
		2
	0.009765625	1	1.37	40.32	2021	4	0.20
		2
	0.01953125	1	1.29	53.23	2099	4	0.19
		2
Colchicine	0.03 µg/mL	1	1.58	6.45	2036	24	1.18*
		2

 

TABLE 2. SUMMARY OF MICRONUCLEI INCIDENCE WITHOUT METABOLIC ACTIVATION FOR                     20 TO 24 HOURS

 

 

Treatment	Concentration      (mg/mL)	Replicate No.	Average CBPI	Average % of Cytotoxicity	Total No. of Binucleates	Total No. of Micronucleus	Average Percentage of Micronucleus
Vehicle Control	0	1	1.63	0.0	2065	4	0.19
		2
CyclicGlucamide C12-C14	0.0048828125	1	1.46	26.98	2098	4	0.19
		2
	0.009765625	1	1.36	42.86	2030	4	0.20
		2
	0.01953125	1	1.27	57.14	2062	4	0.20
		2
Mitomycin C	0.075 µg/mL	1	1.57	9.52	2044	23	1.13*
		2

CBPI:Cytokinesis Block Proliferation Index,*: Statistically Significant

 

 

Conclusions:

Based on the results obtained, it is concluded that the test item Cyclic Glucamide C12-C14 is non clastogenic and/or non aneugenic in cultured human lymphocytes at and up to 0.01953125 mg/mL both in short term and long term treatments (presence and absence of metabolic activation) as it showed no evidence of increase in the induction of micronuclei under the test conditions

Executive summary:

The test item, CyclicGlucamide C12-C14was evaluated for formation of micronuclei in the cytoplasm of interphase cells using human lymphocytes.

Test item was found soluble in dimethyl sulphoxide at 500 mg/mL. Post incubation, heavy precipitation was observed at 2.5 and 5 mg/mL, moderate and mild precipitation was observed at 1.25 and 0.625 mg/mL and no precipitation was observed at 0.3125 and 0.15625 mg/mL and no pH change was observed at any of the other concentrations tested. Hence 1.25 mg/mL was selected as highest concentration for initial cytotoxicity test and other concentrations tested were 0.078125, 0.15625, 0.3125 and 0.625 mg/mL.The experiment was conducted with and without metabolic activation. The test item was assessed in proliferated lymphocytes in duplicates by exposing for a short term  (3 to 6 hours, with and without metabolic activation) and a long term (20 to 24 hours, without metabolic activation).Cytokinesis was blocked using Cytochalasin B, the cells were harvested and slides were prepared. In order to assess the cytotoxicity of the test item,the Cytokinesis-Block Proliferation Index (CBPI) was calculated for cultures treated with the test item and vehicle control.  

In initial cytotoxicity test, the % cytotoxicity ranged from 71.43% to 100% at 0.078125, 0.15625, 0.3125, 0.625 and 1.25 mg/mL. As the % cytotoxicity of test item was more than 55±5% at and up to 1.25 mg/mL hence follow up initial cytotoxicity test was performed.In follow up initial cytotoxicity test,the% cytotoxicitywas in the range of 65.00 to 67.74 at 0.0390625 mg/mL. The% cytotoxicitywas in the range of 11.67 to 58.06 at 0.00244140625, 0.0048828125, 0.009765625 and 0.01953125mg/mL. As the% cytotoxicitywas not more than 55±5% at 0.01953125 mg/mL, same was selected as the highest concentration for the micronucleus test. Other concentrations tested were 0.0048828125 and 0.009765625 mg/mL. 

In micronucleus test, the test item was tested at the concentrationsof0.0048828125, 0.009765625 and 0.01953125mg/mL for short term in presence and absence ofmetabolic activation and long term treatment in the absence of metabolic activation system. The test item induced cytotoxicity at0.01953125mg/mL (51.67 to 57.14%) when compared to vehicle control. There was no statistical significant increase in the percentage of micronuclei in binucleated cells were observed in any of the tested concentrations when compared to the vehicle control.Further, the micronucleus frequencies observed for test item treatments fell within acceptable ranges with regard to in-house historical control data.The positive controls resulted increase of the micronuclei frequencies with statistical significance at 95% level of confidence (p<0.05) under identical conditions, when compared with the vehicle control. This demonstrated the sensitivity of test system towards positive control and confirmed that the test conditions were adequate and within the range of historical control

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

The registration substance is “non-mutagenic” in a Bacterial Reverse Mutation Test (OECD 471) and an in-vitro mammalian cell gene mutation test (OECD 476). It is also negative in an in-vitro mammalian cell micronucleus test (OECD 487). Based on available information, the registration substance is not classfied for genetic toxicity.