Fatty acids, C16-18 (even numbered), esters with 1,2,3-propanetriol and oligomers, ethoxylated

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In order to evaluate the mutagenic potential of the test item in bacteria, a Bacterial Reverse Mutation test is available. Based on the results of the study, it can be concluded that the Hostacerin DGSB, is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 5 mg/plate under the test condition.

In order to evaluate the mutagenic potential of Hostacerin DGSB in mammalian cells, an HGPRT Test is available. Based on the results obtained in this In vitro mammalian cell gene mutation test, Hostacerin DGSB is considered non-mutagenic at and up to the concentration of 0.50 mg/mL both in the presence and absence of metabolic activation under the laboratory conditions tested.

In order to evaluate the clastogenic potential of Hostacerin DGSB an in vitro chromosome aberration test in mammalian lymphocytes is available. Based on the results obtained, Hostacerin DGSB is considered as non-clastogenic up to the concentration of 250 μg/mL both in the presence and absence of metabolic activation under the presented test conditions.

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:

2017-03-20 to 2017-10-30

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:

In accordance with the 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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Clariant / DEG4338635
- Expiration date of the batch: 2018-10-04
- Purity test date: 2016-10-04


STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Solubility of the test substance in the vehicle: Acetone
- Reactivity of the test substance with the vehicle of the cell culture medium: Soluble

Target gene:

Histidine & Tryptophan Locus

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Sodium phenobarbitone and β-naphthoflavone induced rat liver S9 homogenate was used as the metabolic activation system

Test concentrations with justification for top dose:

In mutation assay the test item was tested at the concentrations of 0.05, 0.16, 0.50, 1.58 and 5 mg/plate. The test concentrations for mutation assay were selected based on the results of solubility, precipitation and initial cytotoxicity test.

Vehicle / solvent:

Vehicle used:acetone
Justification for choice of vehicle: The test item was soluble in acetone at a concentration of 50 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

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-amioantracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar -plate incorporation & preincubation; with and without metabolic activation

DURATION
Plate incorporation: 65 hrs
- Preincubation period:30 minutes
- Postincubation period: 63 hrs 45 minutes

NUMBER OF REPLICATIONS: triplicates

- OTHER: cytotoxicity by lawn evaluation & mutation assay by counting 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 TA1537.There was no appreciable increase in number of revertant colonies at any of the tested concentrations in both the trials.

Statistics:

not applicable

Key result

Species / strain:

other: Salmonella typhimurium TA98, TA100, TA1535, and TA1537 and Escherichia coli WP2 uvrA (pKM101).

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: No, soluble in acetone
- Precipitation: The test item resulted in precipitation at 3, 4 and 5 mg/plate and minimal precipitation was observed at 2 mg/plate and no precipitation was observed at 1, 0.9, 0.8,0.7 mg/plate.


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

Plate incorporation method
Tester Strain TA 98 TA 100 TA 1535 TA 1537 E.COLI PKM 101 (With S9)
Mean 401.7 383. 140.1 119.2 383.5
SD ±23.4 ±16.9 ±8.0 ±6.6 ±9.8
Min 256 246 118 100 320
Max 440 419 1 82 149 421

Tester Strain TA 98 TA 100 TA 1535 TA 1537 E.COLI PKM 101 (Without S9)
Mean 396.6 379.7 141.1 120.1 383.1
SD ±27.4 ±18.3 ±9.9 ±7.3 ±10.4
Min 290 270 110 98 371
Max 430 446 190 158 416

Pre incubation method
Tester Strain TA 98 TA 100 TA 1535 TA 1537 E.COLI PKM 101 (With S9)
Mean 402.5 383.9 140.6 118.6 382.9
SD ±18.5 ±16.1 ±9.3 ±6.3 ±10.4
Min 290 293 103 99 330
Max 429 425 187 151 412

Tester Strain TA 98 TA 100 TA 1535 TA 1537 E.COLI PKM 101 (Without S9)
Mean 3398.3 381.3 141.3 119.5 382.8
SD ±24.5 ±16.2 ±11.0 ±6.4 ±10.3
Min 281 312 110 97 321
Max 428 450 200 173 418

TABLE 1.           SUMMARY OF INITIAL CYTOTOXICITY TEST-SALMONELLA TYPHIMURIUMTA100                                        

Initial Cytotoxicity Test                                                                                                 

Test Item Concentration (mg/plate)	No. of Revertants/plate
	With S9						Without S9
	R1	R2	R3	Average	±SD	Bacterial Lawn Intensity	R1	R2	R3	Average	±SD	Bacterial Lawn Intensity
Vehicle Control	106	98	105	103	4.4	4+	103	93	110	102	8.5	4+
2	91	88	101	93	6.8	4+	85	96	102	94	8.6	4+
1	102	108	104	105	3.1	4+	93	101	104	99	5.7	4+
0.9	93	102	102	99	5.2	4+	105	98	91	98	7.0	4+
0.8	84	103	89	92	9.8	4 +	107	101	94	101	6.5	4+
0.7	108	92	111	104	10.2	4+	107	109	90	102	10.4	4+

 

Follow-up Initial Cytotoxicity Test                                                                                                 

Test Item Concentration (mg/plate)	No. of Revertants/plate
	With S9						Without S9
	R1	R2	R3	Average	±SD	Bacterial Lawn Intensity	R1	R2	R3	Average	±SD	Bacterial Lawn Intensity
Vehicle Control	107	96	101	101	5.5	4+	98	102	92	97	5.0	4+
5	90	87	92	90	2.5	4+	85	94	91	90	4.6	4+
4	99	84	93	92	7.5	4+	92	79	88	86	6.7	4+
3	80	92	89	87	6.2	4+	88	100	93	94	6.0	4+
2.75	86	94	90	90	4.0	4+	78	101	90	90	11.5	4+
2.5	87	94	105	95	9.1	4+	81	98	94	91	8.9	4+
2.25	104	92	96	97	6.1	4+	102	95	89	95	6.5	4+
2	91	88	85	88	3.0	4+	81	87	98	89	8.6	4+
1	90	95	80	88	7.6	4+	96	84	81	87	7.9	4+

Values of Revertants are in Mean±SD

Lawn intensity: 4+= Thick lawns: Distinguished by a healthy (Normal) background lawn comparable tovehiclecontrol plates.    

TABLE 2.           SUMMARY OF COLONY COUNTS OF REVERTANTS OF TRIAL-I

Plate Incorporation Method

Treatment	Test Concentration   (mg/plate)		No. of Revertants (Mean of 3 Plates)
			With S9						Without S9
			Salmonella typhimurium				E.coliWP2 uvrA (pKM 101)		Salmonella typhimurium				E.coliWP2 uvrA (pKM 101)
			TA 98	TA 100	TA 1535	TA 1537			TA 98	TA 100	TA 1535	TA 1537
Vehicle Control	100 µL of Acetone	Mean	25.0	104.0	19.3	11.0	175.0		23.3	99.7	20.3	9.7	173.7
		±SD	3.6	10.4	2.5	2.0	9.2		4.0	4.7	3.1	2.5	5.5
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
Hostacerin DGSB	5	Mean	17.3	99.0	20.3	7.3	164.0		18.7	101.7	18.7	7.3	166.3
		±SD	3.1	14.5	3.8	2.5	5.6		2.5	10.5	2.5	2.1	5.5
	Fold Increase		0.7	1.0	1.1	0.7	0.9		0.8	1.0	0.9	0.8	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	1.58	Mean	18.0	94.7	19.0	6.0	167.3		18.7	87.7	18.3	7.0	173.7
		±SD	3.6	12.5	4.6	2.0	6.4		4.0	5.1	3.8	3.6	7.1
	Fold Increase		0.7	0.9	1.0	0.5	1.0		0.8	0.9	0.9	0.7	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.5	Mean	19.3	102.3	18.0	7.3	163.3		18.0	92.0	16.0	8.3	165.3
		±SD	4.2	7.1	1.7	4.2	8.0		4.4	11.0	2.0	2.9	6.7
	Fold Increase		0.8	1.0	0.9	0.7	0.9		0.8	0.9	0.8	0.9	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.16	Mean	18.0	91.7	15.7	8.7	165.3		18.7	97.7	16.7	9.0	170.7
		±SD	1.0	9.1	1.5	1.2	14.0		4.7	9.7	3.2	2.0	9.5
	Fold Increase		0.7	0.9	0.8	0.8	0.9		0.8	1.0	0.8	0.9	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.05	Mean	19.7	102.3	16.3	6.7	167.0		19.0	99.7	18.0	9.0	169.3
		±SD	2.5	8.6	3.5	2.5	11.4		2.6	5.9	2.0	3.6	16.0
	Fold Increase		0.8	1.0	0.8	0.6	1.0		0.8	1.0	0.9	0.9	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
Positive Control	100 µL of respective Positive Control	Mean	372.3	390.7	142.3	115.7	397.0		319.7	380.3	137.3	113.7	385.0
		±SD	20.4	19.4	12.3	8.5	15.1		22.1	22.5	7.0	5.7	28.4
	Fold Increase		14.9	3.8	7.4	10.5	2.3		13.7	3.8	6.8	11.8	2.2
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+

Values of Revertants are in Mean±SD

Positive controls:

For with S9:

ForSalmonella typhimuriumTA98, TA100, TA1535 and TA1537 = 4 µg/plate of 2-Aminoanthracene

ForEscherichia coliWP2 uvrA (pKM101)strain +S9 = 30µg/plate of 2-Aminoanthracene

For without S9:

For TA98: 2 µg/plate of 2-Nitrofluorene

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

For TA1537: 50 µg/plate of 9-Aminoacridine

ForEscherichia coliWP2 uvrA (pKM101): 5 µg/plate of4-Nitroquinoline N-oxide

TABLE 3.           SUMMARY OF COLONY COUNTS OF REVERTANTS OF TRIAL-II

Preincubation Method

Treatment	Test Concentration  (mg/plate)		No. of Revertants (Mean of 3 Plates)
			With S9						Without S9
			Salmonella typhimurium				E.coliWP2 uvrA (pKM 101)		Salmonella typhimurium				E.coliWP2 uvrA (pKM 101)
			TA 98	TA 100	TA 1535	TA 1537			TA 98	TA 100	TA 1535	TA 1537
Vehicle Control	100 µL of Acetone	Mean	25.3	106.3	22.3	10.7	177.7		24.0	102.0	21.3	12.0	178.7
		±SD	3.1	12.1	4.2	1.2	7.8		3.6	4.0	4.0	2.0	3.5
	Lawn Intensity		4+	4+	23	4+	4+		4+	4+	4+	4+	4+
Hostacerin DGSB	5	Mean	20.7	106.7	18.7	9.3	175.0		20.7	100.7	20.7	7.0	168.0
		±SD	4.2	6.8	6.1	2.5	9.2		4.7	4.5	2.1	3.0	10.5
	Fold Increase		0.8	1.0	0.8	0.9	1.0		0.9	1.0	1.0	0.6	0.9
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	1.58	Mean	20.3	102.7	18.3	8.0	172.3		17.7	92.0	18.3	6.7	174.3
		±SD	2.5	3.5	3.5	2.6	8.3		3.5	4.6	4.0	1.5	5.5
	Fold Increase		0.8	1.0	0.8	0.8	1.0		0.7	0.9	0.9	0.6	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.5	Mean	21.0	106.0	17.3	8.0	172.0		17.0	94.0	16.7	8.7	172.0
		±SD	1.7	7.5	3.2	3.6	7.2		3.6	9.0	3.1	2.5	9.5
	Fold Increase		0.8	1.0	0.8	0.8	1.0		0.7	0.9	0.8	0.7	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.16	Mean	19.3	102.0	18.0	8.7	174.0		18.0	90.3	19.7	7.7	169.7
		±SD	2.1	6.0	3.6	4.7	4.6		4.6	6.5	3.1	2.1	13.2
	Fold Increase		0.8	1.0	0.8	0.8	1.0		0.8	0.9	0.9	0.6	0.9
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.05	Mean	23.0	100.7	17.7	7.7	167.0		18.3	91.3	20.3	7.3	165.3
		±SD	2.0	12.4	2.5	2.9	13.5		2.1	10.5	4.0	3.1	9.1
	Fold Increase		0.9	0.9	0.8	0.7	0.9		0.8	0.9	1.0	0.6	0.9
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
Positive Control	100 µL of respective Positive Control	Mean	380.7	398.7	147.0	115.7	394.0		340.0	382.3	133.3	121.7	393.0
		±SD	18.9	9.3	9.6	9.7	18.7		21.7	10.0	4.5	10.4	11.5
	Fold Increase		15.0	3.7	6.6	10.8	2.2		14.2	3.7	6.3	10.1	2.2
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+

Values of Revertants are in Mean±SD

Positive controls:

For with S9:

ForSalmonella typhimuriumTA98, TA100, TA1535 and TA1537 = 4 µg/plate of 2-Aminoanthracene

ForEscherichia coliWP2 uvrA (pKM101)strain +S9 = 30µg/plate of 2-Aminoanthracene

For without S9:

For TA98: 2 µg/plate of 2-Nitrofluorene

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

For TA1537: 50 µg/plate of 9-Aminoacridine

ForEscherichia coliWP2 uvrA (pKM101): 5 µg/plate of4-Nitroquinoline N-oxide

 

Conclusions:

Based on the results of the study, it can be concluded that the test item Hostacerin DGSB, is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 5 mg/plate under the test condition.

Executive summary:

The test item, Hostacerin DGSB obtained from Clariant India Limited,was evaluated for mutagenicity in Bacterial Reverse Mutation Test as per the OECD guidelinefor testing of chemicals No. 471, “Bacterial Reverse Mutation Test”, adopted on21^stJuly 1997.

The tester strains used in the mutation assay were Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coliWP2 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 I and II) were conducted by plate incorporation method and pre incubation method in the presence and absence of metabolic activation system. In mutation assay the test item was tested at the concentrations of0.05, 0.16, 0.50, 1.58 and 5mg/plate. Vehicle control (acetone) 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 0.7, 0.8, 0.9, 1, and 2 mg/plate. The follow-up initial cytotoxicity test was performed at the 1, 2, 2.25, 2.50, 2.75, 3, 4 and 5 mg/plate concentrations. Initial cytotoxicity test was performed with TA100 both in the presence and absence of metabolic activation system. For the tester strain TA100 treated with Hostacerin DGSB at the concentration of 5 mg/plate both in the presence and absence of metabolic activation no cytotoxicity and lawn reduction was observed. Hence on the basis of cytotoxicity results 5 mg/plate was considered as the highest test concentration for mutation assay.

From the experimental results obtained, the mean numbers of revertant colonies at the tested concentrations were comparable to those of the vehicle control, in both the trials, in the presence and absence of metabolic activation. There was no appreciable increase in number of revertant colonies at any of the tested concentrations in both the trials.The number of revertant colonies in the positive controls resulted in 2.2 to 15.0 fold increase under identical conditions.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-03-23 to 2017-09-28

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 accordance with the OECD guidelines for testing of chemicals, No. 473, “In vitro Mammalian Chromosomal Aberration Test” adopted on 29th July 2016.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro Mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Clariant / DEG4338635
- Expiration date of the batch: 2018-10-04
- Purity test date: 2016-10-04


STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Solubility of the test substance in the vehicle: Acetone
- Reactivity of the test substance with the vehicle of the cell culture medium: Soluble

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 (31 and 35 years of age) with no known recent exposure to genotoxic chemicals or radiation.
- Suitability of cells:The primary cell cultures of human whole blood were selected on the basis of growth
ability in culture and stability of the karyotype. 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 requ
irements human peripheral blood lymphocytes are among the recommended test systems.
- Cell cycle length: 18 to 24 hrs
- Sex, age and number of blood donors : Two male donors of 31 and 35 years of age
- Whether whole blood or separated lymphocytes were used : Whole blood were used
- 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:

Precipitation and pH test:
0.03125, 0.0625, 0.125, 0.25, 0.50, 1 and 2 mg/mL along with vehicle control.

Initial cytotoxicity test:
31.25, 62.5, 125, 250 and 500 µg/mL

Three analyzable concentrations were used for chromosomal aberration test:
62.5, 125 and 250 µg/mL

Vehicle / solvent:

- Vehicle used: acetone
- Justification for choice of solvent/vehicle: Test item was found to be soluble in acetone at 200 mg/mL

Untreated negative controls:

yes

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:
Culture Media: RPMI Media supplemented with 10% FBS and antibiotics (1% Penicillin-Streptomycin) was used. The pH of the culture medium used was 7.33 to 7.41.
Preincubation:
Whole blood of volume 0.5 mL was added to each tube containing culture media of volume 4.5 mL supplemented with 2% phytohaemagglutinin (PHA) and incubated for 44 to 48 hours at 37±1ºC and 5±1% CO2.
Post 44 to 48 hours of incubation with PHA, cells from tubes were centrifuged at 1500 rpm for 10 minutes, supernatant was discarded.
Cell pellet was resuspended with fresh media.
Treatment details for the initial cytotoxicity test:
•For tubes with metabolic activation (+S9) - set 1, cell suspension was mixed with 50 µL each of the respective test concentrations/vehicle/negative control and 0.5 mL of S9 mix was added, volume was made up to 5 mL with culture media.
•For tubes without metabolic activation (-S9) - set 2 and 3, cell suspension was mixed with 50 µL each of the respective test concentrations/vehicle/negative control. The volume was made up to 5 mL with culture media.
•All the test item concentrations and controls were maintained in duplicates.
•Cells were incubated both with metabolic activation (+S9) without metabolic activation (-S9) for 3 to 6 hours (Set 1 and 2) and without metabolic activation (-S9) for 20 to 24 hours (Set 3) at 37±1ºC and 5±1% CO2 as mentioned in the table.
•The treatment for Set 1 and 2 tubes were terminated post 3 to 6 hours of incubation, by centrifugation at 1500 rpm for 10 minutes.
•Supernatant was discarded and cell pellet was mixed with 5 mL of culture medium and incubated further to complete 20 to 24 hours starting from the start of treatment.
•The treatment for Set 3 tubes was terminated post 20 to 24 hours of incubation, by centrifugation at 1500 rpm for 10 minutes. Supernatant were discarded and cell pellet was mixed and made up to 5 mL with culture medium.
•Before 1 to 3 hours of harvesting, Colchicine of concentration 0.3 µg/mL was added to all the tubes of set 1, 2 and 3. Post incubation of 2 hours with colchicine, cell suspension was collected to pre-labeled tubes and centrifuged for 10 minutes at 1500 rpm.

The treatment details for the initial cytotoxicity test are as mentioned below:
Set No. Metabolic
Activation Treatment Duration
1 +S9 Negative control /Vehicle control/Test item 4 Hours
2 - S9 Negative control /Vehicle control/Test item 4 Hours
3 - S9 Negative control /Vehicle control/Test item 21 Hours 15 mins

The treatment details for Chromosomal aberration test were maintained as indicated in the below table:
Set No. Metabolic
Activation Treatment Duration
1. +S9 NC/VC/test item/PC 4 Hours 10 min
2. -S9 NC/VC/test item/PC 4 Hours 10 min
3. -S9 NC/VC/test item/PC 22 Hours 5 min

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Pellets were mixed with 2 to 5 mL of freshly prepared 0.56% warm potassium chloride. Cell suspension was incubated for 10 minutes at room temperature and later centrifuged at 1800 to 2000 rpm for 10 minutes. Supernatant was discarded and cell pellet was mixed with 2 to 5 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 2200 rpm for 10 minutes. The procedure was repeated twice by adding 2 to 5 mL of cold acetic acid: methanol fixative (1:3). Finally the supernatant was discarded and cell pellet was mixed with 0.2 mL of freshly prepared cold acetic acid: methanol fixative (1:3). Clean slides were stored in a beaker with distilled water and kept in the refrigerator 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, activation, treatment and slide number. The slides were air dried.
Three slides were prepared for each treatment. Slides were stained using 5% Giemsa stain for 15 to 20 minutes.

NUMBER OF CELLS EVALUATED:
For each replicate minimum of 500 cells were scored.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
The cells were evaluated for structural aberrations in at least 300 well spread metaphases per concentration (150 well-spread metaphases for each replicate).

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.

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.

Statistics:

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

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No change in pH was observed at any of the concentrations tested at and up to 2 mg/mL
- Effects of osmolality:NA
- Evaporation from medium:NA
- Water solubility:No
- Precipitation:Slight precipitation was observed at 0.50 mg/mL and the test item precipitated at and ab
ove 1 mg/mL.

Remarks on result:

other: no mutagenic potential

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

Set No.	Treatment	Concentrations (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)	Negative control	-	0.0551	0.0651	0.0601	6.01	NA
	Vehicle control	-	0.0580	0.0620	0.0600	6.00	0.17
	Test item     (Hostacerin DGSB)	31.25	0.0569	0.0488	0.0529	5.29	11.83
		62.5	0.0483	0.0431	0.0457	4.57	23.83
		125	0.0439	0.0390	0.0415	4.15	30.83
		250	0.0362	0.0335	0.0348	3.48	42.00
		500	0.0229	0.0198	0.0213	2.13	64.50
Set 2 (-S9)  (3-6 hours)	Negative control	-	0.0486	0.0650	0.0568	5.68	NA
	Vehicle control	-	0.0516	0.0606	0.0561	5.61	1.23
	Test item     (Hostacerin DGSB)	31.25	0.0486	0.0486	0.0486	4.86	13.37
		62.5	0.0498	0.0377	0.0437	4.37	22.10
		125	0.0447	0.0377	0.0412	4.12	26.56
		250	0.0296	0.0331	0.0314	3.14	44.03
		500	0.0222	0.0210	0.0216	2.16	61.50
Set 3 (-S9) (20-24 hours)	Negative control	-	0.0484	0.0618	0.0551	5.51	NA
	Vehicle control	-	0.0524	0.0564	0.0544	5.44	1.27
	Test item     (Hostacerin DGSB)	31.25	0.0455	0.0407	0.0431	4.31	20.77
		62.5	0.0416	0.0359	0.0388	3.88	28.68
		125	0.0378	0.0325	0.0352	3.52	35.29
		250	0.0321	0.0255	0.0288	2.88	47.06
		500	0.0155	0.0169	0.0162	1.62	70.22

      

 

TABLE 2.     SUMMARY OFCHROMOSOMAL ABERRATIONSAND MITOTIC INDEX

 

                                                                                                                                                                              

Set No.	Treatment	Concentrations (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)	Negative control	-	6.26	0.95	1	0.5	0.5	0.35
	Vehicle control	-	6.32	NA	1.5	1	1	0.7
	Positive Control (Cyclophosphamide monohydrate)	10	5.64	10.76	17	16	16	10.65*
	Test item     (Hostacerin DGSB)	62.5	4.60	27.22	1.5	1	1	0.7
		125	4.45	29.59	1	0.5	0.5	0.35
		250	3.44	45.57	1.5	1	1	0.7

                            

TABLE 2 (Contd..,) SUMMARY OFCHROMOSOMAL ABERRATIONSAND MITOTIC INDEX

Set No.	Treatment	Concentrations (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 2 (-S9) (3-6 hours)	Negative control	-	5.24	1.32	1	0.5	0.5	0.35
	Vehicle control	-	5.31	NA	1	0.5	0.5	0.35
	Positive Control (Mitomycin-C)	0.05	4.70	11.49	21.5	19.5	17.5	11.65*
	Test item (Hostacerin DGSB)	62.5	4.32	18.64	1.5	1	1	0.7
		125	3.61	32.02	1	1	1	0.7
		250	2.97	44.07	1.5	1.5	1.5	1

                                                                                                                                              

MI: Mitotic Index; *: Statistically significant; +S9: With metabolic activation.

 

 

TABLE 2 (Contd..,). SUMMARY OFCHROMOSOMAL ABERRATIONSAND MITOTIC INDEX

Set No.	Treatment	Concentrations (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 3 (-S9) (20-24 hours)	Negative control	-	6.20	2.21	1	1	1	0.7
	Vehicle control	-	6.34	NA	1	0.5	0.5	0.35
	Positive Control (Mitomycin-C)	0.05	5.44	14.20	17.5	17.5	16.5	11.0*
	Test item (Hostacerin DGSB)	62.5	4.43	30.13	1	0.5	0.5	0.35
		125	3.88	38.80	1.5	1	1	0.7
		250	3.26	48.58	1.5	1	1	0.7

Conclusions:

Based on the results obtained, the test item, Hostacerin DGSB is considered as non-clastogenic up to the concentration of 250 μg/mL both in the presence and absence of metabolic activation under the presented test conditions.

Executive summary:

The test item, Hostacerin DGSB, was evaluated for chromosome aberrations in human lymphocytes, as per the OECD guideline for the testing of chemicals, No. 473. Test item was found to be soluble in Acetone at 200 mg/mL. A precipitation test was conducted at 0.03125, 0.0625, 0.125, 0.25, 0.50, 1 and 2 mg/mL. Post 25 hours of incubation, no change in pH was observed at any of the concentrations tested upto 2 mg/mL. No precipitation was observed at the tested concentrations at and up to 0.25 mg/mL. Slight precipitation was observed at 0.50 mg/mL and the test item precipitated at and above 1 mg/mL. Hence, 0.50 mg/mL was selected as highest concentration for initial cytotoxicity test.

The treatment of cultures with Hostacerin DGSB in the presence and absence of metabolic activation (short term treatment 3 to 6 hours) at all concentrations tested resulted in the decrease of Mitotic Index (MI) and the obtained reduction in MI was in the range of 11.83% to 64.50%. The reduction in MI at 250µg/mLwas in the range of 42 to 44.03%. Hence 250µg/mL was selected as the highest concentration for the chromosomal aberration test for short term treatment (3 to 6 hrs). Similarly, treatment of cultures with Hostacerin DGSB in the absence of metabolic activation (long term treatment 20 to 24 hrs) at all concentrations tested resulted in the decrease of Mitotic Index (MI) and the obtained reduction in MI was in the range of 20.77% to 70.22%. The reduction in MI at 250 µg/mL was 47.06%. Hence, 250µg/mL was selected as the highest concentration for the chromosomal aberration test for long term treatment 20 to 24 hours.

In the chromosome aberration test, the cells were treated with Hostacerin DGSB at the concentrations of 62.50, 125 and 250 µg/mL using Acetone as the vehicle. The treatment was carried out in duplicates for the short-term period (3 to 6 hours) both in the presence and absence of metabolic activation, and for the long-term period (20 to 24 hours) in the absence of metabolic activation. Negative control was also maintained in the presence and absence of metabolic activation (short term treatment 3 to 6 hours) and also in the absence of metabolic activation (long term treatment 20 to 24 hrs). Cyclophosphamide Monohydrate (+S9 for short term) at the concentration of 10 µg/mL and Mitomycin-C at the concentration of 0.05 µg/mL (-S9 both for short term and long term) were used as positive controls.

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

The results with Hostacerin DGSB indicated no statistically significant increase in the number of aberrated cells when compared with vehicle control at any of the concentrations tested, whether with or without metabolic activation.The reduction of MI observed at 62.50, 125 and 250 µg/mL for the short-term period (3 to 6 hours) in the presence of metabolic activation were 27.22%, 29.59% and 45.57% respectively. Similarly, the reduction of MI observed at the same test concentrations for the short-term period (3 to 6 hours) in the absence of metabolic activation were 18.64%, 32.02% and 44.07% respectively. The reduction of MI observed at 62.50, 125 and 250 µg/mL in the absence of metabolic activation for long term treatment (20 to 24 hours) were 30.13%, 38.80% and 48.58%, respectively.

The cultures treated with positive controls for the short-term period (3 to 6 hours) both in the presence and absence of metabolic activation, and for the long-term period (20 to 24 hours) in the absence of metabolic activation, induced 10.65%, 11.65% and 11.00% of aberrated cells respectively, which was statistically significant compared with the respective vehicle controls. This demonstrated sensitivity of the test system towards positive controls and confirmed that the test conditions were adequate.

 

 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-04-07 to 2017-09-19

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:

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Clariant / DEG4338635
- Expiration date of the batch: 2018-10-04
- Purity test date: 2016-10-04


STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Solubility of the test substance in the vehicle: Acetone
- 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±1°C and 5±1% CO2.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: alpha MEM culture medium containing 10% FBS with antibiotics (1% Penicillin and Streptomycin) and incubated at 37±1°C and 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:

Based on the results of solubility, pH and precipitation test, an initial cytotoxicity test was conducted for the selection of test concentrations for the gene mutation test. Five concentrations of 0.0312, 0.0625, 0.125, 0.25 and 0.50 mg/mL of the test item were tested in the initial cytotoxicity test.
The test item showed slight precipitation at 0.50 mg/mL, same was considered as the highest concentration in the initial cytotoxicity test.
Up to the concentration of 0.50 mg/mL the Relative Survival was more than 20%. At higher concentration, the RS value of < 10 % was obtained. Therefore 0.50 mg/mL was selected as the highest concentration for testing in the Gene mutation test.
our concentrations i.e. 0.0625, 0.125, 0.25 and 0.50 mg/mL were selected for gene mutation test, based on initial cytotoxicity test

Vehicle / solvent:

- Vehicle used: acetone
- Justification for choice of vehicle: The test item Hostacerin DGSB was found to be soluble in acetone 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

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:

METHOD OF APPLICATION: in medium;
- Cell density at seeding (if applicable):Approximately 2×106 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.
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): 6 Thioguanine

NUMBER OF REPLICATIONS: Tetraplates
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative survival;

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.
A test item, for which the results do not meet the above criteria, is considered non-mutagenic in this system.
There is no requirement for verification of a clearly positive or negative response.
In cases when the response is neither clearly negative nor clearly positive as described above, a repeat experiment possibly using modified experimental conditions is to perform.

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. The statistical significance was designated by the superscripts in the report as stated below:
* Statistically significant (p<0.05) change than the vehicle control group.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no 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

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)	Average colony count Mean± SD				*Cloning Efficiency (CE)	Adjusted Cloning Efficiency (ACE)	Relative Survival (RS) (%)
Set 1 +S9	Vehicle Control (Acetone)	-	154.67	±		10.97	0.77	0.98	-
	Hostacerin DGSB	0.0312	141.00	±		9.54	0.71	0.89	90.82
		0.0625	139.67	±		10.60	0.70	0.87	88.78
		0.125	137.00	±		10.54	0.69	0.85	86.73
		0.25	135.00	±		9.17	0.68	0.84	85.71
		0.50	139.00	±		10.54	0.70	0.80	81.63
Set 2 -S9	Vehicle Control (Acetone)	-	156.00		±	14.53	0.78	0.99	-
	Hostacerin DGSB	0.0312	142.67		±	7.37	0.71	0.90	90.91
		0.0625	139.33		±	13.05	0.70	0.87	87.88
		0.125	137.00		±	6.56	0.69	0.85	85.86
		0.25	136.00		±	12.00	0.68	0.83	83.84
		0.50	136.00		±	6.24	0.68	0.77	77.78

+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)	Average colony count Mean ± SD				Cloning Efficiency (CE)	Adjusted Cloning Efficiency (ACE)	Relative Survival (RS) (%)
Set 1 +S9	Vehicle Control (Acetone)	-	154.00	±		15.10	0.77	0.99	-
	Hostacerin DGSB	0.0625	138.33	±		11.68	0.69	0.88	88.89
		0.125	135.67	±		4.51	0.68	0.86	86.87
		0.25	131.67	±		12.01	0.66	0.83	83.84
		0.50	127.33	±		7.51	0.64	0.79	79.80
	Benzo(a)pyrene              (Positive Control)	0.003	132.00	±		13.53	0.66	0.80	80.81
Set 2 -S9	Vehicle Control (Acetone)	-	151.00	±	8.00		0.76	0.99	-
	Hostacerin DGSB	0.0625	144.67	±	13.05		0.72	0.89	89.90
		0.125	136.00	±	6.56		0.68	0.85	85.86
		0.25	133.67	±	15.04		0.67	0.82	82.83
		0.50	127.67	±	13.32		0.64	0.77	77.78
	4 Nitroquinoline 1-oxide (Positive Control)	0.001	131.00	±	8.19		0.66	0.78	78.79

  +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)	*Average colony count Mean ± SD				Cloning Efficiency in selective media	Cloning Efficiency in non-selective media	Average Mutant Colonies/ 2x10^6cells	Mutant Frequency/ 2x10^6cells
Set 1 +S9	Vehicle Control (Acetone)	-	145.67	±		21.01	0.0000100	0.73	20	27.40
	Hostacerin DGSB	0.0625	142.00	±		8.00	0.0000095	0.71	19	26.76
		0.125	146.00	±		6.56	0.0000095	0.73	19	26.03
		0.25	136.33	±		9.71	0.0000090	0.68	18	26.47
		0.50	132.33	±		8.74	0.0000085	0.66	17	25.76
	Benzo(a)pyrene                  (Positive control)	0.003	143.33	±		14.05	0.0001070	0.72	214	**297.22
Set 2 -S9	Vehicle Control (Acetone)	-	145.33	±	11.37		0.0000095	0.73	19	26.03
	Hostacerin DGSB	0.0625	145.67	±	15.57		0.0000090	0.73	18	24.66
		0.125	141.67	±	7.02		0.0000090	0.71	18	25.35
		0.25	133.00	±	8.19		0.0000085	0.67	17	25.37
		0.50	123.67	±	10.97		0.0000075	0.62	15	24.19
	4 Nitroquinoline 1-oxide  (Positive control)	0.001	142.33	±	9.07		0.0001050	0.71	210	**295.77

+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 in this In vitro mammalian cell gene mutation test, Hostacerin DGSB is considered non-mutagenic at and up to the concentration of 0.50 mg/mL both in the presence and absence of metabolic activation under the laboratory conditions tested.

Executive summary:

The test item Hostacerin DGSB, was evaluated for gene mutation test in CHO AA8 cells, as per the OECD guideline for the testing of chemicals, No. 476 “In vitro Mammalian Cell Gene Mutation Tests using the hprt and xprt genes” adopted on 29th July 2016. The test item was found to be soluble in acetone at 200 mg/mL. The test item did not precipitated at and up to 0.25 mg/mL. Slight precipitation was observed at the concentration of 0.5 mg/mL and moderate to heavy precipitation was observed at the concentration at 1 and 2 mg/mL under the inverted microscope. The pH tested at concentrations up to 2 mg/mL was comparable to the vehicle control. Based on these results, 0.5 mg/mL was chosen as the highest concentration for the initial cytotoxicity test.

Initial cytotoxicity test was conducted by the test item Hostacerin DGSB at the concentrations of 0.0312, 0.0625, 0.125, 0.25 and 0.50 mg/mL with 10 mL of culture media with CHO AA8 cells in the respective flasks. Percentage of Relative Survival (RS) for test item treated at 0.0312, 0.0625, 0.125, 0.25 and 0.50 mg/mL were 90.82, 88.78, 86.73, 85.71 and 81.63 in the presence of metabolic activation, respectively and 90.91, 87.88, 85.86, 83.84 and 77.78 in the absence of metabolic activation, respectively. The results of the initial cytotoxicity test indicated that the test item was not cytotoxic to CHO AA8 cells as the Relative Survival of the treated CHO AA8 cells at the tested concentrations up to 0.5 mg/mL was more than 20%, when compared with the respective vehicle control, both in the presence and absence of metabolic activation.

The Gene mutation test was conducted by the test item Hostacerin DGSB at the concentrations of 0.0625, 0.125, 0.25 and 0.50 mg/mL using acetone as a vehicle in tetra plates in the presence and absence of metabolic activation (3 to 6 hours). Positive controls, 3µg/mL of Benzo (a) pyrene [with metabolic activation (+S9)] and 1 µg/mL of 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.

Hostacerin DGSB resulted in mutant frequencies of 25.76 to 26.76 per 2×106 cells in the presence of metabolic activation at the different concentrations tested compared to 27.40 per 2×106 cells in the vehicle control. In the absence of metabolic activation, test item resulted in mutant frequencies of 24.19 to 25.37 per 2×106 cells compared to 26.03 per 2×106 cells 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.

Positive controls resulted in mutant frequencies of 297.22 (with metabolic activation) and 295.77 (without metabolic activation) per 2×106 cells, which was statistically significant when compared with the vehicle control.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

In order to evaluate the genetic toxicity potential of the registered substance three in vitro tests according to OECD guidelines were performed: i.e. Ames test (OECD 471), Chromosome aberration test (OECD473) and HGPRT (OECD 476) test. All of these three in vitro tests were negative in result. According to Regulation (EC) No 1272/2008 of the European Parliament and of the council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006, the test substance Hostacerin DGSB has not to be classified.