[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For the test substance registered no Ames test is available. Read across to a structural analogue substance is made. Due to structural similarities it is assumed that both substances reveal almost the same metabolism and thus are of similar toxicity. Read across justification for this approach is attached to section 13 of this IUCLID. In conclusion, it can be stated that during the described mutagenicity test (AMES test) and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used, both in the presence and absence of metabolic activation.

Further, two in vitro genetic toxicity studies with the test item registered are available, i.e.  a mammalian chromosomal aberration test in human lymphocytes and an HPRT test using CHO AA8 cells.

Based on the results obtained with a mammalian chromosomal aberration test in human lymphocytes, Dodicor V 5654 is considered as non-clastogenic both in the presence and absence of metabolic activation under the presented test conditions.

Dodicor V 5654 is considered as non-mutagenic at and up to the concentration of 0.125 µL/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions in the HPRT Test.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 October 2019 TO 09 April 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted on 29th July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Specific details on test material used for the study:

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

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

Target gene:

HPRT gene

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The derivative of the CHO-K1, CHO AA8 Cells

Metabolic activation:

with and without

Metabolic activation system:

induced rat liver S9 homogenate

Test concentrations with justification for top dose:

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

Vehicle / solvent:

dimethyl sulphoxide

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Details on test system and experimental conditions:

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

Evaluation criteria:

• The concurrent vehicle control is considered acceptable for addition to the laboratory historical vehicle control database
• Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative/vehicle control
A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
• At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
• The increase is concentration-related when evaluated with an appropriate trend test.
• Any of the results are outside the distribution of the historical negative/vehicle control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
• A test chemical is considered clearly negative if, in all experimental conditions examined:
• None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
• There is no concentration-related increase when evaluated with an appropriate trend test.
• All results are inside the distribution of the historical negative/vehicle control data.

Statistics:

Data of mutant frequencies were analyzed for differences among vehicle control, treatment and positive control groups by performing power transformation procedure by Snee and Irr (1981) with which, the observed mutant frequency was transformed using the formula:
Y=〖(X+A)〗^B
Where,
Y = transformed mutant frequency, X = observed mutant frequency
[Where X=(No. of mutant colonies per replicate)/(ACE value)×100
and A, B = constants (viz. A = 1 and B = 0.15)]
Statistical analysis of the experimental data was carried out using SPSS Statistical package version 22.0.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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


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


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

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

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

Remarks on result:

other:

Remarks:

Non mutagenic

 TABLE 1.           SUMMARY OF INITIAL CYTOTOXICITY TEST

Refer: Appendix 1

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

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

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

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

 CE = Number of colonies/Number of cells plated.

 

 

 

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

Refer: Appendix 2

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

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

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

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

 CE = Number of colonies/Number of cells plated.

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

 

 

 

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

TABLE 3.           SUMMARY OF GENE MUTATION TEST

Refer: Appendix 3

+S9: with metabolic activation; -S9: without metabolic activation;                                                                                                                      *Note: Cloning efficiency = 200 cells plated for each replicate.  

 **: Statistically significant (p˂0.05). 

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

Conclusions:

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

Executive summary:

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

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

On the basis of precipitation results, 0.125 µL/mL was selected as the highest concentration for the initial cytotoxicity test. Initial cytotoxicity test was conducted at the concentrations of 0.0078125, 0.015625, 0.03125, 0.0625 and 0.125 µL/mL using DMSO as a vehicle in tetra plates/group in the presence and absence of metabolic activation (3 to 6 hours). Cytotoxicity was assessed by determining the Adjusted Cloning Efficiency and Relative Survival in the test.

 The results of the initial cytotoxicity test indicated that the Relative Survival was greater  than 10% (26.50% in presence of metabolic activation and24.55% in absence of metabolic activation) at 0.125 µL/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Based on these results, 0.125 µL/mL was selected as highest concentration for gene mutation test.

The gene mutation test was conducted at the concentrations of 0.015625, 0.03125, 0.0625 and 0.125 µL/mLusing DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation (3 to 6 hours).

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

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

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

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

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 November 2019 to 09 July 2020

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:

adopted on 29th July 2016.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro Mammalian Chromosomal Aberration Test

Specific details on test material used for the study:

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

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Solubility of the test substance in the solvent/vehicle:
Test item was soluble in DMSO at 500 mg/mL.

Target gene:

Human Peripheral Blood Lymphocytes

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Human peripheral lymphocytes from the blood of healthy, young, non-smoking donors [22 (Female), 28 (Male) and 29 (Male) years of age] with no known recent exposure to genotoxic chemicals or radiation were used

Test concentrations with justification for top dose:

Based on the results of initial cytotoxicity test 0.01953 mg/mL was selected has highest testing concentration.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test item was 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

DURATION
- Exposure duration: 3 to 6 hours and 20 to 24 hours
- Expression time (cells in growth medium): 44 to 48 hours
- Selection time (if incubation with a selection agent): 44 to 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 27 hours

STAIN (for cytogenetic assays): 5% Giemsa stain

NUMBER OF REPLICATIONS: 02

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Pellets were mixed with 4 mL of freshly prepared warm 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 3 to 4 mL of freshly prepared cold acetic acid:methanol fixative (1:3). Cell suspension was 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 4 mL of cold acetic acid: methanol fixative (1:3). Clean slides were stored in a beaker 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 were 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 was air dried. Minimum of 3 slides were prepared for each treatment replicate. Slides were stained using 5% Giemsa stain for 15 minutes.

NUMBER OF CELLS EVALUATED: 500 cells were scored for initial cytotoxicity and 150 metaphases for chromosomal abbrevation test.

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

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy:Yes
- Determination of endoreplication: Yes

Rationale for test conditions:

The following set of criteria was followed for the selection of concentrations for chromosomal aberration test.
• Three analyzable concentrations were used for chromosomal aberration test.
• The percentage reduction in Mitotic Index was in the range of 7.62% to 45.42% at 0.00488, 0.00977 and 0.01953 mg/mL. As the percentage reduction in MI was not more than 45±5% at 0.01953 mg/mL, same was selected as the highest concentration for the chromosomal aberration test. Other concentrations tested were 0.00488 and 0.00977 mg/mL.

Evaluation criteria:

• All slides including vehicle control, Negative control, treatment and positive controls of chromosomal aberration test were coded before evaluation.
• Cytotoxicity was determined by calculating percentage reduction in mitotic index (%).
• Concurrent measures of mitotic index for all treated and vehicle control cultures were determined.
• The cells were evaluated for structural aberrations in 150 metaphase plates for each replicate and 300 (per concentration), the metaphases with aberrations were recorded in raw data.
•Gaps were recorded separately and reported but not included in the total aberration frequency.

Statistics:

Yes

Key result

Species / strain:

lymphocytes: Human blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:No change in pH was observed in any of the concentrations tested upto 5 mg/mL
- Water solubility: Insoluble
- Precipitation: Milld and moderate precipitation was observed at 1.25 and 2.5 mg/mL respectively

RANGE-FINDING/SCREENING STUDIES: Yes, Initial cytotoxicity

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: 95% Confidence level
With S9 - cyclophosphamide monohydrate
(3 to 6 hours) Without S9 - Mitomycin C (3 to 6 hours) Without S9 - Mitomycin C (20 to 24 hours)
Average (% of Aberrated cells) 9.88 9.90 10.42
Standard Deviation 1.12 1.26 1.89
Sample size 16.00 16.00 16.00
Margin of error 0.55 0.62 0.93
Upper bound 10.43 10.52 11.34
Lower bound 9.33 9.28 9.49
95% Confidence level 1.96 1.96 1.96
Max 12.00 13.35 16.00
Min 8.67 8.67 8.67


- Negative (solvent/vehicle) historical control data:
95% Confidence level
With S9
(3-6 hours) Without S9 (3-6 hours) Without S9 (20-24 hours)
Average (% of Aberrated cells) 0.76 0.90 0.81
Standard Deviation 0.24 0.24 0.29
Sample size 12.00 12.00 12.00
Margin of error 0.14 0.14 0.16
Upper bound 0.90 1.04 0.97
Lower bound 0.62 0.76 0.65
95% Confidence level 1.96 1.96 1.96
Max 1.00 1.33 1.30
Min 0.35 0.67 0.34



ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Mitotic index
- Other observations when applicable: metaphase counting

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

Set No.	Treatment	Concentrations (mg/mL)	Average % Mitotic Index	% Reduction of Mitotic Index
Set 1 (+S9)                       (3 to 6 hours)	Vehicle Control	-	6.98	-
	Dodicor V 5654	0.3125	0	100
		0.625	0	100
		1.25	0	100
		2.5	0	100
		5	0	100
Set 2  (-S9)  (3 to 6 hours)	Vehicle Control	-	6.67	-
	Dodicor V 5654	0.3125	0	100
		0.625	0	100
		1.25	0	100
		2.5	0	100
		5	0	100
Set 3      (-S9)                      (20 to 24 hours)	Vehicle Control	-	6.46	-
	Dodicor V 5654	0.3125	0	100
		0.625	0	100
		1.25	0	100
		2.5	0	100
		5	0	100

TABLE 1 (Contd…). SUMMARY OF PERCENTAGE MITOTIC INDEX FOR INITIAL CYTOTOXICITY TEST

Follow up Initial cytotoxicity test

Set No.	Treatment	Concentrations (mg/mL)	Average % Mitotic Index	% Reduction of Mitotic Index
Set 1 (+S9)                       (3 to 6 hours)	Vehicle Control	-	7.61	-
	Dodicor V 5654	0.00488	7.03	7.62
		0.00977	5.77	24.18
		0.01953	4.43	41.79
		0.03906	2.05	73.06
		0.07812	0.67	91.20
Set 2  (-S9)  (3 to 6 hours)	Vehicle Control	-	7.83	-
	Dodicor V 5654	0.00488	6.85	12.52
		0.00977	6.10	22.09
		0.01953	4.51	42.40
		0.03906	2.56	67.31
		0.07812	0.29	96.30
Set 3      (-S9)                      (20 to 24 hours)	Vehicle Control	-	8.08	-
	Dodicor V 5654	0.00488	7.44	7.92
		0.00977	6.34	21.53
		0.01953	4.41	45.42
		0.03906	2.47	69.43
		0.07812	0.68	91.58

TABLE 2.           SUMMARY OFCHROMOSOMAL ABERRATIONS AND MITOTIC INDEX

Set No.	Treatment	Concentrations (mg/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 to 6 hours)	Vehicle Control	-	8.18	NA	1.5	1.5	1.5	1.00
	Positive Control (Cyclophosphamide monohydrate)	10 µg/mL	7.68	6.11	19.5	18.5	17.0	11.33*
	Dodicor V 5654	0.00488	7.40	9.54	1.5	1.5	1.5	1.00
		0.00977	6.34	22.49	3.0	2.0	2.0	1.34
		0.01953	4.76	41.81	2.5	2.5	2.5	1.68

Set No.	Treatment	Concentrations (mg/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 to 6 hours)	Vehicle Control	-	7.89	NA	1.5	1.5	1	0.67
	Positive Control (Mitomycin-C)	0.05 µg/mL	7.39	6.34	21.5	18.5	15.5	10.34*
	Dodicor V 5654	0.00488	7.27	7.86	1.5	1.5	1	0.67
		0.00977	6.25	20.79	2	2	1.5	1.00
		0.01953	4.51	42.84	2.5	2.5	2.5	1.67

Set No.	Treatment	Concentrations (mg/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 to 24 hours)	Vehicle Control	-	7.89	NA	2	2	2	1.33
	Positive Control (Mitomycin-C)	0.05 µg/mL	7.40	6.21	17	16	15	10.00*
	Dodicor V 5654	0.00488	7.08	10.27	1.5	1.5	1.5	1.00
		0.00977	6.29	20.28	2.5	2.5	2	1.33
		0.01953	4.49	43.09	2.5	2	2	1.34

Conclusions:

Based on the results obtained, the test item, Dodicor V 5654 is considered as non-clastogenic both in the presence and absence of metabolic activation under the presented test conditions.

Executive summary:

The test item,Dodicor V 5654was evaluated for chromosomal aberrations test in human lymphocytes.Test item was soluble in DMSO at 500 mg/mL. Precipitation test was conducted at 0.3125, 0.625, 1.25, 2.5 and 5 mg/mL. Post 23 hours and 37 minutes of incubation, mild and moderate precipitation was observed at 2.5 and 5 mg/mL respectively. No precipitation was observed at any of the other concentrations tested. No change in pH was observed in any of the concentrations tested upto 5 mg/mL. Hence, 5 mg/mL was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were0.3125, 0.625, 1.25 and 2.5 mg/mL of test item. In initial cytotoxicity test, complete cytotoxicity was observed down to 0.3125 mg/mL both in short and long term treatments.Hence a follow up initial cytotoxicity test was performed to select concentrations for the chromosomal aberration test.   

Follow up initial cytotoxicity testwas conducted with the cocentrations of 0.00488, 0.00977, 0.01953, 0.03906 and 0.07812 mg/mL.The percentage reduction in Mitotic Index was in the range of 7.62% to 45.42% at 0.00480, 0.00977 and 0.01953 mg/mL. As the percentage reduction in MI was not more than 45±5% at 0.01953 mg/mL, same was selected as the highest concentration for the chromosomal aberration test. Other concentrations tested were 0.00488 and 0.00977 mg/mL. 

In the chromosomal aberration test, the cells were treated with test item at the concentrations of 0.00488, 0.00977 and 0.01953 mg/mL using DMSO 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. 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, thecells were treated with a metaphase-arresting substance (colchicine), harvested, stained and metaphase cells were analysed microscopically for the structural chromosomal aberrations.There was no statistically significant increase in the number of aberrated cells when compared with vehicle control at any of the concentration levels tested.The cultures treated with positive controlsfor 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 aberrated cells which was statistically significant compared with the respective vehicle control. This demonstrated sensitivity of the test system towards positive controls and confirmed that the test conditions were adequate. The concurrent vehicle control values were within the 95% control limits of the distribution of the laboratory’s historical vehicle control database.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: According to the OECD Guideline, under GLP

Justification for type of information:

Justification for Read across to analogue substance is attached (refer to Read across statement Section 13 of this IUCLID)

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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
- Batch No. of test material: ESD0003028:
- Purity: 25 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stable in water for days at room temperature (not quantified)
- Solubility: Solvent water was chosen because of its solubility properties

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added:
No precipitation of the test item occurred up to the highest investigated dose of 5000 µg/plate.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

induced rat liver S9-mix

Test concentrations with justification for top dose:

3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I and experiment II

Vehicle / solvent:

Deionised water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in agar plate incorporation; preincubation

DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3 plates

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed .
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration .
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

stains affected: see additional information on results.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Summary Tables:

    Summary of Results Pre-Experiment and Experiment I

Study Name: 1211000	Study Code: Harlan CCR 1211000
Experiment: 1211000 VV Plate	Date Plated: 17/09/2008
Assay Conditions:	Date Counted: 24/09/2008

Metabolic Activation	Test Group	Dose Level (µg/plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	Deionised water			20 ± 3	11 ± 3	35 ± 2	137 ± 5	55 ± 4
	Untreated			18 ± 3	13 ± 3	37 ± 10	126 ± 4	47 ± 3
	Hostapon SG	3 µg		20 ± 3	11 ± 4	29 ± 1	134 ± 12	50 ± 4
		10 µg		19 ± 4	14 ± 2	33 ± 5	124 ± 8	52 ± 6
		33 µg		18 ± 3	11 ± 4	35 ± 6	132 ± 10	55 ± 1
		100 µg		19 ± 4	8 ± 0	37 ± 6	139 ± 16	52 ± 4
		333 µg		13 ± 2	10 ± 4	32 ± 8	122 ± 15	63 ± 3
		1000 µg		17 ± 2	7 ± 1	25 ± 5	84 ± 11^R	62 ± 2
		2500 µg		8 ± 2^M R	2 ± 2^M R	19 ± 2^M R	36 ± 5^M R	59 ± 2
		5000 µg		2 ± 2^M R	0 ± 1^M R	5 ± 2^M R	27 ± 3^M R	58 ± 2
	NaN3	10 µg		2009 ± 31			2369 ± 114
	4-NOPD	10 µg				424 ± 9
	4-NOPD	50 µg			83 ± 3
	MMS	3.0 µL						1017 ± 33
With Activation	Deionised water			23 ± 3	20 ± 5	37 ± 4	148 ± 5	68 ± 4
	Untreated			24 ± 6	19 ± 7	42 ± 8	148 ± 14	64 ± 9
	Hostapon SG	3 µg		24 ± 3	19 ± 5	39 ± 5	129 ± 14	64 ± 4
		10 µg		21 ± 5	17 ± 5	44 ± 8	135 ± 3	71 ± 2
		33 µg		25 ± 3	19 ± 3	43 ± 6	132 ± 9	60 ± 11
		100 µg		23 ± 1	15 ± 2	38 ± 4	134 ± 5	60 ± 4
		333 µg		22 ± 5	13 ± 2	47 ± 6	136 ± 13	73 ± 11
		1000 µg		15 ± 4	16 ± 3	35 ± 3	116 ± 13	69 ± 3
		2500 µg		9 ± 3^M R	8 ± 1^M R	28 ± 2^M R	48 ± 7^M R	66 ± 1
		5000 µg		5 ± 1^M R	1 ± 1^M R	13 ± 2^M R	34 ± 5^M R	59 ± 4
	2-AA	2.5 µg		245 ± 16	191 ± 30	1267 ± 70	1506 ± 43
	2-AA	10.0 µg						232 ± 24

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	R M	Reduced background growth Manual count

     Summary of Results Experiment II

Study Name: 1211000	Study Code: Harlan CCR 1211000
Experiment: 1211000 HV2 Pre	Date Plated: 01/10/2008 / 14/10/2008*
Assay Conditions:	Date Counted: 08/10/2008 / 17/10/2008*

Metabolic Activation	Test Group	Dose Level (µg/plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98*	TA 100	WP2 uvrA
Without Activation	Deionised water			19 ± 6	12 ± 4	22 ± 2	131 ± 5	51 ± 8
	Untreated			16 ± 6	10 ± 2	24 ± 4	137 ± 12	46 ± 8
	Hostapon SG	3 µg		17 ± 4	14 ± 5	23 ± 2	131 ± 8	55 ± 1
		10 µg		18 ± 4	15 ± 2	24 ± 2	127 ± 9	52 ± 8
		33 µg		18 ± 3	11 ± 6	24 ± 4	122 ± 1	55 ± 6
		100 µg		22 ± 6	14 ± 3	23 ± 1	101 ± 15^R	56 ± 2
		333 µg		12 ± 5	10 ± 4	15 ± 2	75 ± 7^R	47 ± 4
		1000 µg		5 ± 2^R M	2 ± 3^M R	10 ± 1^M R	39 ± 5^M R	49 ± 5
		2500 µg		2 ± 2^M R	0 ± 0^M R	3 ± 2^M R	31 ± 10^M R	46 ± 5
		5000 µg		1 ± 1^M R	0 ± 0^M R	0 ± 0^M R	5 ± 2^M R	37 ± 7
	NaN3	10 µg		1799 ± 67			1998 ± 31
	4-NOPD	10 µg				1637 ± 116
	4-NOPD	50 µg			105 ± 5
	MMS	3.0 µL						418 ± 20
With Activation	Deionised water			17 ± 4	16 ± 5	26 ± 3	138 ± 9	58 ± 5
	Untreated			18 ± 4	19 ± 7	29 ± 3	142 ± 37	64 ± 7
	Hostapon SG	3 µg		19 ± 4	17 ± 4	27 ± 0	131 ± 11	59 ± 3
		10 µg		22 ± 4	17 ± 4	25 ± 2	132 ± 8	58 ± 3
		33 µg		20 ± 2	19 ± 3	24 ± 2	141 ± 15	59 ± 6
		100 µg		21 ± 1	15 ± 3	22 ± 5	140 ± 7	57 ± 5
		333 µg		18 ± 4	14 ± 4	19 ± 3	110 ± 5^R	53 ± 2
		1000 µg		11 ± 3^M R	12 ± 2^M R	10 ± 2^M R	74 ± 7^R	54 ± 1
		2500 µg		9 ± 3^M R	8 ± 2^M R	4 ± 3^M R	31 ± 2^M R	59 ± 4
		5000 µg		0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	31 ± 4^M R	61 ± 3
	2-AA	2.5 µg		256 ± 8	105 ± 10	1616 ± 151	1346 ± 157
	2-AA	10.0 µg						334 ± 24

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	R M	Reduced background growth Manual count

* Repeated experiment

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

The test item Hostapon SG was assessed for its potential to induce gene mutations in the plate incorporation test (experi­ment I) and the pre-incubation test (experiment II) using Salmo­nella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escheri­chia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Due to irregular background growth in strain TA 98 in experiment II no data were evaluated. This part was repeated under identical conditions and is reported as part of experiment II. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I and II:         3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed reduced background growth at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	2500 - 5000	2500 - 5000	1000 - 5000	1000 - 5000
TA 1537	2500 - 5000	2500 - 5000	1000 - 5000	1000 - 5000
TA 98	2500 - 5000	2500 - 5000	1000 - 5000	1000 - 5000
TA 100	2500 - 5000	2500 - 5000	100 - 5000	333 - 5000
WP2 uvrA	/	/	/	/

/ = no reduced background growth

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	2500 - 5000	2500 - 5000	1000 - 5000	5000
TA 1537	2500 - 5000	2500 - 5000	1000 - 5000	5000
TA 98	5000	5000	1000 - 5000	1000 - 5000
TA 100	2500 - 5000	2500 - 5000	1000 - 5000	2500 - 5000
WP2 uvrA	/	/	/	/

/ = no toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5)

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Hostapon SG at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher muta­tion rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease of induced revertant colonies.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on all available data (i.e mammalian chromosomal aberration test in human lymphocytes and an HPRT test using CHO AA8 cells) with the registered substance as well as available data (Ames test) with the analogue substance, registered substance is not considered to be mutagenic. Accordingly, classification & labelling does not apply.