1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-C18(unsaturated) acyl derivs., hydroxides, inner salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genotoxicity of the registration substance was investigated in three in vitro test systems, namely bacterial reverse mutation test (Ames test), mammalian cytogenetic assay (Chromosome aberration test), and mammalian gene muatation test (HPRT). In all three tests, negative results were obtained.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

The test material corresponded to the approximately 45% of the registration substance. The given dose refers to the amount of the registration substance.

Species / strain / cell type:

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

Additional strain / cell type characteristics:

DNA polymerase A deficient

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 homogenate

Test concentrations with justification for top dose:

Preliminary Toxicity Test - (a) 50, (b) 100, (c) 200, (d) 400, (e) 800, (f) 1600, (g) 3200 and (h) 5000 µg/plate
Initial mutation assay - (a) 8, (b) 25, (c) 80, (d) 253 and (e) 800 µg/plate
Confirmatory mutation assay - (a) 41, (b) 86, (c) 181, (d) 380 and (e) 800 µg/plate.

Vehicle / solvent:

One hundred microliters (100 µL) of DMSO was used as the vehicle control.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene

Details on test system and experimental conditions:

- Source of the Test System:
-Salmonella typhimurium: Health Protection Agency, National Collection of Type, Cultures (NCTC), 61, Colindale Avenue, London NW9 5EQ, Great Britain
- Escherichia coli: The National Collection of Industrial and Marine Bacteria Ltd. (NCIMB), Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland, U.K.

- Storage of Test System
Stock cultures of tester strains were stored in Oxoid nutrient broth No. 2 in the test facility as frozen permanents in liquid nitrogen. Laboratory stocks were maintained on respective minimal glucose agar plates as master plates of each strain, for a maximum period of 2 months and refrigerated at 2 to 8ºC. The master plates prepared on 14 March 2016 were used in the study.

- Genotypic Characterization of Test System
The growth requirements and the genetic identity of strains like histidine or tryptophan requirement, sensitivity to UV radiation, resistance of strains
TA98, TA100 and WP2uvrA (pKM101) to ampicillin and rfa mutation of Salmonella typhimurium strains were checked along with the range of spontaneous revertants after preparation of the master plates

Evaluation criteria:

- Evaluation and Interpretation : To determine a positive result, there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations 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 peak of the dose response is equal to or greater than 2 times the mean vehicle control value for strains TA98, TA100 and WP2uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value for 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 the 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.

Key result

Species / strain:

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

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

Table 1: Results of Preliminary Toxicity Test
Treatment (mg/plate)	TA 100 revertant colonies/plate^*
	Presence of S9			Absence of S9
	Mean	Background lawn	Precipitation	Mean	Background lawn	Precipitation
DMSO (100mL)	114	4+	Nil	112	4+	Nil
50	108	4+	Nil	107	4+	Nil
100	103	4+	Nil	101	4+	Nil
200	72	4+	Nil	70	4+	Nil
400	56	3+	Nil	51	3+	Nil
800	46	3+	Nil	43	3+	Nil
1600	37	3+	Nil	33	3+	Nil
3200	9	1+	Nil	7	1+	Nil
5000	4	0	Nil	3	0	Nil

*: Mean of two replicates

4+ - Normal

3+ - Slightly reduced   

1+ - Severely reduced

0  - Absent

Table 2: Viable Counts of the Overnight Culture of the Tester Strains
Tester Strains	Viable Counts ( x 10^9CFU/mL*)
	Initial Mutation Assay	Confirmatory Mutation Assay
TA98	1.55	1.55
TA100	1.64	1.54
TA1535	1.53	1.52
TA1537	1.55	1.52
WP2uvrA (pKM101)	1.62	1.63

* Required Cell count: 1-2x10⁹Colony Forming Units (CFU)/mL

Table 3: Summary Results of Initial Mutation Assay in the Presence of Metabolic Activation
Treatment (µg/plate)	No. of revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA(pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
Vehicle control DMSO	26	2	NA	116	1	NA	14	2	NA	11	2	NA	134	3	NA
8	26	2	1.00	114	1	0.98	13	2	0.93	10	2	0.91	130	3	0.97
25	28	1	1.08	112	1	0.97	11	2	0.79	11	2	1.00	127	3	0.95
80	26	2	1.00	112	2	0.97	14	1	1.00	12	1	1.09	129	2	0.96
253	21	2	0.81	71	2	0.61	9	2	0.64	7	1	0.64	105	3	0.78
800	14	1	0.54	44	4	0.38	2	2	0.14	3	2	0.27	93	2	0.69
Positive controls	545^c	6^c	20.96^c	882^c	11^c	7.60^c	139^c	9^c	9.93^c	126^c	1^c	11.45^c	572^d	4^d	4.27^d
^aValues are means of three replicates calculated from Appendix 2 and are rounded off to the nearest whole number ^bRatio of treated/vehicle control (mean revertants per plate) ^cTA98, TA100, TA1535, TA1537: 2-Aminoanthracene (4 µg/plate)                          ^dWP2uvrA(pKM101): 2-Aminoanthracene (30 µg/plate) NA: Not applicable                             SD: Standard deviation

Table 4: Summary Results of Initial Mutation Assay in the Absence of Metabolic Activation
Treatment (µg/plate)	No. of revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA(pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
Vehicle control DMSO	27	1	NA	115	2	NA	13	3	NA	12	2	NA	132	3	NA
8	27	2	1.00	112	2	0.97	13	2	1.00	12	1	1.00	129	4	0.98
25	27	2	1.00	112	3	0.97	14	3	1.08	11	2	0.92	129	3	0.98
80	27	1	1.00	112	1	0.97	14	2	1.08	10	2	0.83	128	2	0.97
253	21	1	0.78	75	3	0.65	9	1	0.69	8	1	0.67	109	2	0.83
800	15	1	0.56	40	1	0.35	2	1	0.15	3	2	0.25	91	2	0.69
Positive controls	241^c	5^c	8.93^c	530^d	6^d	4.61^d	143^d	5^d	11.00^d	128^e	2^e	10.67^e	572^f	7^f	4.33^f
^aValues are means of three replicates calculated from Appendix 3 and are rounded off to the nearest whole number  ^bRatio of treated/vehicle control (mean revertants per plate) ^cTA98: 2-Nitrofluorene (2 µg/plate),             ^dTA100, TA1535: Sodium azide (1 µg/plate),            ^eTA1537: 9-Aminoacridine (50 µg/plate),      ^fWP2uvrA(pKM101): 4-Nitroquinoline-1-oxide (4 µg/plate)             NA: Not applicable                                         SD: Standard deviation

Table 5: Summary Results of Confirmatory Mutation Assayin the Presence of Metabolic Activation
Treatment [µg/plate]	No. of revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA (pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
Vehicle control DMSO	25	2	NA	114	2	NA	14	2	NA	11	2	NA	133	4	NA
41	25	2	1.00	112	1	0.98	14	2	1.00	11	2	1.00	135	5	1.02
86	26	1	1.04	111	1	0.97	15	1	1.07	12	1	1.09	131	5	0.98
181	27	1	1.08	96	2	0.84	14	2	1.00	10	1	0.91	130	3	0.98
380	23	3	0.92	63	2	0.55	8	1	0.57	8	2	0.73	102	4	0.77
800	15	2	0.60	44	3	0.39	3	1	0.21	3	1	0.27	91	2	0.68
Positive control	557^c	17^c	22.28^c	876^c	10^c	7.68^c	134^c	6^c	9.57^c	129^c	4^c	11.73^c	580^d	6^d	4.36^d
^aValues are means of three replicates calculated from Appendix 4 and are rounded off to the nearest whole number          ^bRatio of treated/vehicle control (mean revertants per plate) ^cTA98, TA100, TA1535, TA1537: 2-Aminoanthracene (4 µg/plate) ^dWP2uvrA(pKM101): 2-Aminoanthracene (30 µg/plate) NA: Not applicable                                                                             SD: Standard deviation

Table 6: Summary Results of Confirmatory Mutation Assay in the Absence of Metabolic Activation
Treatment [µg/plate]	No. of revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA (pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
Vehicle control DMSO	25	1	NA	114	2	NA	15	1	NA	12	1	NA	130	4	NA
41	26	1	1.04	111	2	0.97	15	2	1.00	11	2	0.92	131	5	1.01
86	25	1	1.00	111	1	0.97	14	2	0.93	12	1	1.00	128	3	0.98
181	26	1	1.04	96	3	0.84	15	1	1.00	10	1	0.83	127	2	0.98
380	21	1	0.84	64	3	0.56	9	2	0.60	6	1	0.50	99	3	0.76
800	16	3	0.64	43	2	0.38	2	2	0.13	3	1	0.25	90	2	0.69
Positive control	246^c	6^c	9.84^c	537^d	3^d	4.71^d	133^d	6^d	8.87^d	133^e	4^e	11.08^e	584^f	5^f	4.49^f
^aValues are means of three replicates calculated from Appendix 5 and are rounded off to the nearest whole number ^bRatio of treated/vehicle control (mean revertants per plate) ^cTA98: 2-Nitrofluorene (2 µg/plate),             ^dTA100, TA1535: Sodium azide (1 µg/plate)             ^eTA1537: 9-Aminoacridine (50 µg/plate),      ^fWP2uvrA(pKM101): 4-Nitroquinoline-1-oxide (4 µg/plate)             NA: Not applicable                                         SD: Standard deviation

Conclusions:

The genotoxicity of the registration substance was investigated in Bacterial Reverse Muation Test (Ames test) according to the Guideline OECD 471. No mutagenicity was found.

Executive summary:

The genotoxicity of the registration substance was investigated in Bacterial Reverse Muation Test (Ames test) according to the Guideline OECD 471. In the preliminary cytotoxicity test, the S.typhimurium TA 100 cultures were treated up to 5000 µg/plate with and without metabolic activation. Significantly reduced background lawn was observed at 400 µg/plate or higher. Two independent experiments for mutagenicity were performend, each with and without metabolic activation up to the concentration of 800µg/plate. No increase of number of reverstants were found in any of performed experiments.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

The test material corresponded to the approximately 45% of the registration substance. The given dose refers to the amount of the registration substance.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1, (ATCC CCL-61, Lot 4765275)

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 homogenate

Test concentrations with justification for top dose:

Experiments 1 & 2:a) 3 b) 10 and c) 30 µg/mL (factor of 3)
Experiment 3:b) 2 b) 5 and c) 15 µg/mL (factor of 3)

Vehicle / solvent:

DMSO was used as the vehicle control.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

- Source of the Test System:
American Type Culture Collection, P. O. Box 1549, Manassas, VA 20108, USA

- Storage of Test System
Stock cultures of the CHO-K1 cell line were stored in the test facility as frozen permanents in liquid nitrogen.

Evaluation criteria:

- Evaluation and Interpretation : When all the validity criteria are fulfilled:

a.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 in number of aberrations compared with the concurrent vehicle control
•The increase is dose-dependent when evaluated with an appropriate trend test
•Any of the results are outside the distribution of the historical vehicle control data

b.A test chemical is considered to be clearly negative if, in all experimental conditions examined:

•None of the test concentrations exhibits a statistically significant increase in number of aberrations compared with the concurrent 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

c.The results will be considered equivocal if they do not meet the criteria specified for a positive or negative response. Additional experimental work may be required to clarify such results.

Statistics:

Statistical analysis of the experimental data was carried out using validated SYSTAT Statistical package ver.12.0. Data were analyzed for proportions of aberrant metaphases in each sample excluding gaps as aberrations. Pooled data from each test concentration and the positive control were compared with the vehicle control using Fischer exact test. All analysis and comparisons were evaluated at 5 % (p < 0.05) level.

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

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

 

TABLE 1. Results of Preliminary Cytotoxicity Test

Treatment (mg/mL)	Presence of metabolic activation (3-hour exposure)			Absence of metabolic activation (3-hour exposure)			Absence of metabolic activation (21-hour exposure)
	Final – initial cell count (1x10^6/flask)	Cell growth index RICC (%)	Cell growth inhibition (%)	Final – initial cell count (1x10^6/flask)	Cell growth index RICC (%)	Cell growth inhibition (%)	Final – initial cell count (1x10^6/flask)	Cell growth index RICC (%)	Cell growth inhibition (%)
DMSO	2.715	100	0	2.590	100	0	2.240	100	0
10	2.465	91	9	1.965	76	24	1.490	67	33
20	2.315	85	15	1.615	62	38	0.915	41	59
40	0.69	25	75	0.44	17	83	Negligible cell counts
80	Negligible cell counts			Negligible cell counts
160	No cell monolayer			No cell monolayer			No cell monolayer
320
640
1280
2000
Note: Baseline cell count (No. of cells at the beginning of treatment) 2.31x 10^5cells/mL Relative Increase in Cell Count (RICC) = [Increase in number of cells in treated cultures (final – initial)] x 100 [Increase in number of cells in control cultures (final – initial)]

TABLE 2.    Summary Results of Chromosomal Aberration Assay - Experiment 1

eatment (µg/mL)	No. of metaphases scored	No. (%) of metaphases with aberrations^@							Total No.(%) of aberrant metaphases^*		Cell Growth Inhibition (%)
		Gaps		Breaks		Exchanges			Including Gaps	Excluding Gaps
		Cs	Ct	Cs	Ct	Cs	Ct	RC
DMSO (150 µL)	300	0	1 (0.3)	0	0	0	0	0	1 (0.3)	0	0
3	300	0	2 (0.7)	0	0	0	0	0	2 (0.7)	0	10
10	300	0	0	0	0	0	0	0	0	0	17
30	300	0	1 (0.3)	0	0	0	0	0	1 (0.3)	0	46
CPA 55	300	7 (2.3)	7 (2.3)	16 (5.3)	91 (30.3)	35 (11.7)	8 (2.7)	0	137 (45.7)	+ 137 (45.7)	39

*: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations

@: values are the sum of two replicates and values in parenthesis represent %   

Cs: Chromosome type             Ct: Chromatid type      RC: Ring chromosome    

CPA: Cyclophosphamide monohydrate            +: Significantly higher than control (p <0.05) by Fischer exact test

Note: There were no incidences of polyploidy and endoreduplicated cells

 

 

 TABLE 3.    Summary Results of Chromosomal Aberration Assay - Experiment 2

Treatment (µg/mL)	No. of metaphases scored	No. (%) of metaphases with aberrations^@							Total No.(%) of aberrant metaphases^*		Cell Growth Inhibition (%)
		Gaps		Breaks		Exchanges			Including Gaps	Excluding Gaps
		Cs	Ct	Cs	Ct	Cs	Ct	RC
DMSO (150 µL)	300	0	2 (0.7)	0	0	0	0	0	2 (0.7)	0	0
3	300	1 (0.3)	1 (0.3)	0	0	0	0	0	2 (0.7)	0	15
10	300	3 (1.0)	0	0	0	0	0	0	3 (1.0)	0	24
30	300	0	5 (1.7)	0	0	0	0	0	5 (1.7)	0	52

*: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations

@: values are the sum of two replicates and values in parenthesis represent %   

Cs: Chromosome type             Ct: Chromatid type                  RC: Ring chromosome 

Note: There were no incidences of polyploidy and endoreduplicated cells

 

 

 

 TABLE 4.    Summary Results of Chromosomal Aberration Assay - Experiment 3

Treatment (µg/mL)	No. of metaphases scored	No. (%) of metaphases with aberrations^@							Total No.(%) of aberrant metaphases^*		Cell Growth Inhibition (%)
		Gaps		Breaks		Exchanges			Including Gaps	Excluding Gaps
		Cs	Ct	Cs	Ct	Cs	Ct	RC
DMSO (150 µL)	300	0	1 (0.3)	0	0	0	0	0	1 (0.3)	0	0
2	300	1 (0.3)	2 (0.7)	0	0	0	0	0	3 (1.0)	0	18
5	300	0	3 (1.0)	0	0	0	0	0	3 (1.0)	0	35
15	300	1 (0.3)	1 (0.3)	0	0	0	0	0	2 (0.7)	0	47
EMS600	300	12 (4.0)	23 (7.7)	10 (3.3)	93 (31.0)	49 (16.3)	47 (15.7)	0	145 (48.3)	+ 145 (48.3)	40

*: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations

@: values are the sum of two replicates and values in parenthesis represent %   

Cs: Chromosome type             Ct: Chromatid type      RC: Ring chromosome    

EMS: Ethyl methanesulfonate   +: Significantly higher than control (p <0.05) by Fischer exact test

Note: There were no incidences of polyploidy and endoreduplicated cells

 

Conclusions:

The genotoxicity of the registration substance was investigated using in-vitro chromosome aberration test according to the Guideline OECD 473. No significant effect was found.

Executive summary:

The genotoxicity of the registration substance was investigated using in-vitro chromosome aberration test according to the Guideline OECD 473.

The CHO cells were treated at 3, 10 and 30 µg/mL for 3 hours with (+S9) or without metabolic activation (-S9), which resulted in the cell growth inhibition of up to 46% (+S9) and 52% (-S9) when compared to the solvent control.

The CHP cells were treated at 2, 5, 15 µg/mL for 21 hours without metabolic activation, which results in the cell growth inhibition of up to 47% when compared to the solvent control.

No significant clastogenic effect was found in all performed experiments and no polyploidy and no endoreduplicated cells were found.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

The test material corresponded to the approximately 45% of the registration substance. The given dose refers to the amount of the registration substance.

Target gene:

This mammalian cell mutation assay system detects point mutations involving base substitutions, deletions, frameshifts and rearrangements within the locus. The enzyme hypoxanthine guanine phosphoribosyl transferase (hprt) catalyses phosphorylation of purines in one of the purine salvage pathways. The selective agent used in this assay, 6-Thioguanine (6-TG), is also a substrate for this enzyme and cells that retain the functional hprt enzyme are susceptible to the cytotoxic effects of 6-TG. Forward mutations that result in the loss of the functional hprt gene render cells resistant to 6-TG. These mutant cells can be quantitated after an expression period by cloning in culture medium supplemented with 6-TG, the selective agent.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster (Cricetulus grieus) ovary cell line CHO-K1 (ATCCCCL-61, Lot 4765275)

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 homogenate

Test concentrations with justification for top dose:

A) 10 B) 20 C) 40 and D) 80 µg/mL (factor of 2)

Vehicle / solvent:

DMSO was used as the vehicle control.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Details on test system and experimental conditions:

- Source of the Test System:
American Type Culture Collection, P. O. Box 1549, Manassas, VA 20108, USA

- Storage of Test System
Stock cultures of the CHO-K1 cell line were stored in the test facility as frozen permanents in liquid nitrogen.

Evaluation criteria:

- Evaluation and Interpretation : When all the validity criteria are fulfilled:

a.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 in number of aberrations compared with the concurrent vehicle control
•The increase is dose-dependent when evaluated with an appropriate trend test
•Any of the results are outside the distribution of the historical vehicle control data

b.A test chemical is considered to be clearly negative if, in all experimental conditions examined:

•None of the test concentrations exhibits a statistically significant increase in number of aberrations compared with the concurrent 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

c.The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Statistics:

A power transformation procedure (Snee and Irr, 1981) was used with which, the observed mutant frequency was transformed.
Statistical analysis of the experimental data was carried out using validated copies of SYSTAT Statistical package version 12.0. In cases where analysis of variance was significant at p < 0.05, a Dunnett’s test was conducted, comparing each treatment group and the positive control to the vehicle control (p < 0.05).

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

TABLE 1.         Determination of pH of Test Medium

Treatment (mg/mL)	pH at the beginning of exposure to treatment		pH at the end of exposure to treatment
	With S9	Without S9	With S9	Without S9
DMSO	7.05	7.13	7.00	7.10
10	7.02	7.18	6.98	7.24
20	7.02	7.18	6.99	7.21
40	7.01	7.21	7.04	7.20
80	7.05	7.21	7.03	7.21
160	7.05	7.23	7.06	7.26
320	7.01	7.22	7.09	7.29
640	7.02	7.18	7.10	7.32
1280	7.00	7.25	7.12	7.31
2000	7.00	7.25	7.06	7.24

TABLE 2.         Determination of Osmolality of Test Medium

Treatment (mg/mL)	Osmolality at the beginning of exposure to treatment (OSMOL/kg)		Osmolality at the end of exposure to treatment (OSMOL/kg)
	With S9	Without S9	With S9	Without S9
DMSO	0.493	0.477	0.479	0.460
2000	0.454	0.449	0.449	0.433

TABLE 3.         Results of Preliminary Cytotoxicity Test

Treatment (mg/mL)	3-hour exposure with metabolic activation				3-hour exposure without metabolic activation
	Cloning Efficiency (CE)	Cells at end of treatment (1x10^5/mL)	Adjusted Cloning Efficiency	Relative Survival (%RS)*	Cloning Efficiency (CE)	Cells at end of treatment (1x10^5/mL)	Adjusted Cloning Efficiency	Relative Survival (%RS)*
DMSO	0.96	11.90	1.31	100	0.97	11.50	1.27	100
10	0.90	11.50	1.18	90	0.92	10.30	1.08	85
20	0.77	10.05	0.88	67	0.87	9.30	0.92	72
40	0.58	8.05	0.53	40	0.63	8.50	0.61	48
80	0.32	5.00	0.18	14	0.44	4.00	0.20	16
160	0.16	1.50	0.03	2	0.20	1.00	0.02	2
320	-	Absence of cells	-	-	-	Absence of cells	-	-
640	-		-	-	-		-	-
1280	-		-	-	-		-	-
2000	-		-	-	-		-	-

TABLE 4.         Parallel Cytotoxicity Test Results from Experiment 1

Treatment µg/mL	No. of Colonies /Flask			CE*	ACE	RS %
	1	2	3
DMSO	191	190	192	0.96	1.39	100
	192	194	195
10	179	182	188	0.91	1.28	92
	180	184	181
20	152	163	158	0.78	0.97	70
	150	156	154
40	124	130	123	0.63	0.65	47
	126	122	127
80	69	63	62	0.32	0.21	15
	62	62	65
3-MCA	104	110	113	0.54	0.41	29
	108	117	101

TABLE 5.         Parallel Cytotoxicity Test Results from Experiment 2

Treatment µg/mL	No. of Colonies /Flask			CE*	ACE	RS %
	1	2	3
DMSO	198	192	193	0.97	1.37	100
	196	190	191
10	180	187	183	0.92	1.17	85
	186	183	185
20	173	179	164	0.86	1.00	73
	170	176	173
40	131	133	136	0.67	0.69	50
	135	139	127
80	88	93	96	0.44	0.24	18
	81	89	84

TABLE 6.         Summary Results of the Gene Mutation Assay in the Presence of Metabolic Activation (Experiment 1)

Treatment µg/mL	Mutation Assay Flasks						Cloning Efficiency of Mutant Colonies	Cloning Efficiency Flasks				6-TG Mutants per 10^6Clonable Cells (MF)
	TG Colonies/Flask							No. of Colonies/Flask
	1	2	3	4	5	Total		1	2	3	CE*
DMSO	2	2	3	3	3	25	0.0000125	192	189	194	0.97	12.89
	2	2	2	3	3			193	195	197
10	2	1	3	1	2	17	0.0000085	184	188	179	0.90	9.44
	3	0	2	1	2			176	181	175
20	2	2	2	1	0	16	0.000008	168	157	159	0.81	9.88
	2	1	2	3	1			154	170	163
40	1	2	2	2	2	19	0.0000095	152	152	155	0.76	12.50
	3	2	1	2	2			153	149	148
80	1	1	1	1	2	15	0.0000075	109	108	117	0.58	12.93
	2	1	3	1	2			129	111	116
3-MCA	36	32	33	34	32	337	0.0001685	144	158	149	0.75	224.67
	38	34	30	35	33			157	143	148

TABLE 7.         Summary Results of the Gene Mutation Assay in the Absence of Metabolic Activation (Experiment 2)

Treatment µg/mL	Mutation Assay Flasks						Cloning Efficiency of Mutant Colonies	Cloning Efficiency Flasks				6-TG Mutants per 10^6Clonable Cells (MF)
	TG Colonies/Flask							No. of Colonies/Flask
	1	2	3	4	5	Total		1	2	3	CE*
DMSO	2	3	2	3	3	25	0.0000125	190	187	192	0.95	13.16
	2	2	2	4	2			194	192	190
10	2	2	2	1	2	20	0.00001	178	177	168	0.87	11.49
	3	2	3	1	2			174	176	170
20	1	1	2	2	1	18	0.000009	158	164	149	0.77	11.69
	2	2	2	3	2			148	149	154
40	1	2	2	2	2	16	0.000008	135	139	136	0.67	11.94
	1	0	0	4	2			133	135	129
80	2	2	1	2	2	16	0.000008	120	128	130	0.64	12.50
	0	2	1	2	2			127	130	128

Conclusions:

The genotoxicity of the registration substance was investigated using in-vitro mammalian cell gene mutation tes (HPRT) according to the Guideline OECD 476. No significant effect was found.

Executive summary:

The genotoxicity of the registration substance was investigated in in-vitro mammalian gene mutation test (HPRT) according to the Guideline OECD 476.

The CHO cells were treated at 10, 20, 40 and 80 µg/mL for 3 hours with (+S9) or without metabolic activation (-S9), which resulted in reduction of the relative survival down to 15% (+S9) and 18% (-S9) when compared to the solvent control.

No significant mutagenic effect was found.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The genotoxicity of the registration substance was investigated in three in vitro test systems, namely bacterial reverse mutation test (Ames test), mammalian cytogenetic assay (Chromosome aberration test), and mammalian gene muatation test (HPRT). In all three tests, negative results were obtained. No classification is justified.