Sodium N-(2-carboxyethyl)-N-dodecyl-β-alaninate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the available information, the test item did not cause genotoxic effects in mammalian and bacterial cells under the experimental conditions selected, therefore it is considered not to be mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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:

- Name of test material (as cited in study report): DERIPHAT 160 C
- Physical state:Solid (lyophilized), white
- Analytical purity: 97% The test substance was characterized analytically (for details see the analytical report; project no.: AU 122958-1)
- Lot/batch No.:4986V1
- Expiration date of the lot/batch: until 03 Jun 2014
Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
- Storage condition of test material:Room temperature (avoid temperatures > 40°C)

Target gene:

X-linked hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from phenobarbital- and ß-naphthoflavone induced rats

Test concentrations with justification for top dose:

1st Experiment, without S9 mix: 0; 12.5; 25.0; 50.0; 100.0; 200.0; 400.0 µg/mL
1st Experiment, with S9 mix: 0; 18.8; 37.5; 75.0; 150.0; 300.0; 600.0 µg/mL

2nd Experiment, without S9 mix: 0; 18.8; 37.5; 75.0; 150.0; 300.0; 600.0 µg/mL
2nd Experiment, with S9 mix: 0; 25.0; 50.0; 100.0; 200.0; 400.0; 600.0 µg/mL

Vehicle / solvent:

Due to the good solubility of the test substance in water, culture medium (Ham's F12) was used as most suitable vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Negative controls, with and without S9 mix, were treated with culture medium without test substance in parallel to the other treatment groups.

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Attachment period:20 - 24 hours
- Exposure duration:4 hour, removal of test substance by intense washing (4 hour exposure)
- Expression time (cells in growth medium):3 days (4-hour treatment)
- Selection time (if incubation with a selection agent):6 - 7 days
- Fixation time (start of exposure up to fixation or harvest of cells):10-11 days

SELECTION AGENT (mutation assays): Hypoxanthine-free Ham's F12 medium with 6-thioguanine (10 μg/mL), stable glutamine (200 mM), fetal calf serum (FCS)
STAIN (for cytogenetic assays):colonies were fixed with methanol, stained with Giemsa

NUMBER OF REPLICATIONS:Duplicate cultures were used for all experimental
groups.

NUMBER OF CELLS EVALUATED: Cloning efficiency 200 cells per dose group were seeded in 25 cm² flasks in duplicate using 5 mL. For selection of the mutants, six 75 cm2 flasks with 3x105 cells each from every treatment group, if possible, were seeded in 10 mL selection medium.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The cloning efficiency (CE, %) was calculated for each test group as follows:
CEabsolute = total number of colonies in the test group/total number of seeded cells in the test group x 100

The uncorrected mutant frequency (MFuncorr.) per 106 cells was calculated for each test group
as follows:
MFuncorr. = total number of mutant colonies / number of seeded cells x 106
MFcorr. = MFuncorr. / CE2 absolut x 100

A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range (see Appendix 6).
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.

The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantly increased above the concurrent negative control and is within our historical negative control data range.

Statistics:

An appropriate statistical trend test was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective negative control groups. A trend is judged as statistically significant whenever the p-value (probability value) is below 0.10 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with S9 in highest tested concentration

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In both experiments, in the presence and absence of S9 mix, after 4 hours treatment the morphology and attachment of the cells were adversely influenced in at least the highest applied concentration

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Oct 2007 - 26 Nov 2007

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:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): C-SAT 070070
- Physical state: liquid, light yellow
- Analytical purity: 30% (a.i.)
- Lot/batch No.: S761700005
- Storage condition of test material: room temperature

Target gene:

his operon for S. typhimurium strains

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other: TA 98: rfa-, uvrB-, R-factor; TA 100: rfa-, uvrB-, R-factor; TA 1535: rfa-, uvrB-; TA 1537: rfa-, uvrB-; TA 102: rfa-; uvrB+. R-factor

Metabolic activation:

with and without

Metabolic activation system:

S9 liver mix prepared from the liver fraction of Wistar rats treated with Phenobarbital/ß-Naphthoflavone

Test concentrations with justification for top dose:

First experiment (initially pre-experiment, with and without metabolic activation): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Second experiment (without metabolic activation): 1, 3, 10, 33, 100, 333, 1000 and 2500 µg/plate
Second experiment (with metabolic activation): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

yes

Remarks:

(sterility control)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with S9-mix

Positive control substance:

other: 2-aminoanthracene (2-AA)

Untreated negative controls:

yes

Remarks:

(sterility control)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

methylmethanesulfonate

Untreated negative controls:

yes

Remarks:

(sterility control)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

sodium azide

Untreated negative controls:

yes

Remarks:

(sterility control)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

other: 4-nitro-o-phenylendiamine (NOPD)

Details on test system and experimental conditions:

EXPERIMENT I (PRETEST), PLATE INCORPORATION ASSAY
In the standard plate test, tubes were filled with 2 mL portions of overlay agar, 0.1 mL test solution or vehicle or positive control substance, 0.1 mL fresh bacterial culture and 0.5 mL S9 -mix or phosphate buffer. After mixing samples were poured onto selective agar plates and incubated for at least 48 hours in the dark at 37°C.
Triplicate testing is done.

EXPERIMENT II, PREINCUBATION ASSAY
For the preincubation test 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL of the S9 mix were mixed and incubated at 37°C for 60 minutes. After addition of 2 mL overlay agar (45°C) samples were poured onto minimal agar plates and incubated for at least 48 hours in the dark at 37°C.
Triplicate testing is done.

DETERMINATION OF CYTOTOXICITY
1.) decrease in the number of revertants
2.) titer reduction
3.) clearing or diminution of the background lawn

TEST CONFOUNDING EFFECTS
As long as no interference between precipitation and colony counting occurs is 5 mg/plate set as maximum dose even for relatively insoluble compounds.

Evaluation criteria:

An assay is accepted when the following criteria are met:
1.) number of colonies in the negative control is in the historical control range
2.) no indication of bacterial contamination (checked by sterility control)
3.) number of colonies in the positive controls are in the range of historical control data
4.) titer of viable bacteria is ≥ 10 E+9/mL

A test chemical is to be considered as mutagenic when:
An increase of number of revertant colonies (by a factor of 2 for TA 98, TA 100 and TA 102; by a factor of 3 for TA 1535 and TA 1537) is seen, is reproducible and dose-related.

A test chemical is to be considered as non-mutagenic when:
The number of revertants is inside the range of historical negative control data in 2 experiments performed independently from each other.

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, TA 102

Remarks:

Experiment I and Experiment II

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in none of the experiments, with and without S9 mix, an increase in number of revertants was observed

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

reduced background growth and reduction in number of revertants as cytotoxicity indicators were observed at different concentration levels, in absence and presence of S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose Level, neither in the presence or absence of metabolic activation (S9 mix).
No tendency of higher mutation rates with increasing concentration in the range below the generally acknowledged border of biological relevance

EXPERIMENT I, with S9 mix: mean number of revertant colonies per dose and strain [3 replicates]
Concentration (µg/plate)		Salmonella typhimurium strain
		TA 1535	TA 1537	TA 98	TA 100	TA 102
untreated		13	20	36	159	495
Vehicle (water) control		16	21	37	151	575
TS	3	18	18	40	145	531
	10	18	21	41	152	549
	33	19	24	43	171	568
	100	18	23	38	157	587
	333	20	29	38	165	514
	1000	15	29	48	148	477
	2500	12(RB)	17(RB)	18(RB)	90(RB)	230(RB)
	5000	1(RB, M)	1(RB, M)	0(RB, M)	15(RB, M)	0(RB, M)
Positive control (2-AA)		282	322	1645	2745	1842
EXPERIMENT I, without S9 mix: mean number of revertant colonies per dose and strain [3 replicates]
Concentration (µg/plate)		Salmonella typhimurium strain
		TA 1535	TA 1537	TA 98	TA 100	TA 102
untreated		19	18	34	142	447
Vehicle (water) control		15	14	36	150	432
TS	3	16	18	30	130	423
	10	14	18	30	141	429
	33	11	14	37	129	468
	100	13	17	34	135	429
	333	9	8(RB)	18	81(RB)	264(RB)
	1000	6(RB, M)	5(RB, M)	7(RB, M)	56(RB, M)	52(RB, M)
	2500	0(RB, M)	0(RB, M)	0(RB, M)	0(RB, M)	0(RB, M)
	5000	0(RB, M)	0(RB, M)	0(RB, M)	0(RB, M)	0(RB, M)
Respective Positive controls		2030	98	421	2170	3651
EXPERIMENTII, with S9 mix: mean number of revertant colonies per dose and strain [3 replicates]
Concentration (µg/plate)		Salmonella typhimurium strain
		TA 1535	TA 1537	TA 98	TA 100	TA 102
untreated		24	18	39	187	615
Vehicle (water) control		20	14	41	176	646
TS	3	20	16	41	166	608
	10	20	13	44	195	658
	33	20	14	41	182	673
	100	21	21	45	171	636
	333	17	20	34	167	624
	1000	12(RB)	23(RB)	30(RB)	148	536
	2500	9(RB)	14(RB, M)	8(RB, M)	106(RB)	334(RB)
	5000	2(RB, M)	0(RB, M)	0(RB, M)	0(RB, M)	3(RB, M)
Positive control (2-AA)		204	161	649	1733	2664
EXPERIMENT II, without S9 mix: mean number of revertant colonies per dose and strain [3 replicates]
Concentration (µg/plate)		Salmonella typhimurium strain
		TA 1535	TA 1537	TA 98	TA 100	TA 102
untreated		16	14	25	150	481
Vehicle (water) control		14	12	27	153	501
TS	1	21	13	30	140	439
	3	17	12	29	147	497
	10	15	10	29	131	486
	33	16	13	32	151	503
	100	19	13	34	125	446
	333	15	10	15	106	380
	1000	8(RB, M)	3(RB, M)	2(RB, M)	14(RB, M)	277(RB)
	2500	0(RB, M)	0(RB, M)	0(RB, M)	0(RB, M)	0(RB, M)
Respective Positive controls		2006	132	613	2110	3510
TS: test substance; RB: reduced background growth; M: manual count

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Dec 1989 - 06 Aug 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

lack of study details, purity of test substance not specified

GLP compliance:

not specified

Type of assay:

other: in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Name of test material (as cited in study report): Sodium Laurimindiproprionate (Deriphat 160C)
- Test article ID: (TSIN) MV#2716-040
- Physical state: light yellow liquid
- Analytical purity: not specified
- Storage condition of test material: room temperature protected from light
- Expiration date of the lot/batch: 1990-09-30

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

with S9:
8 hours harvest - 0.21, 0.28, 2.38 µL/mL
12 hour harvest - 0.28, 0.38, 0.50 µL/mL

without S9:
8 hour harvest: 0.48, 0.63, 0.84 µL/mL
12 hour harvest: 0.63, 0.84, 1.13 µL/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Triethylenemelamine (TEM)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 8 and 12 hours

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Statistics:

The cytotoxic effects of treatment are expressed relative to the solvent control (relative cloning efficiency). The number and types of aberrations found are presented for each treatment flask. Duplicate treatment flasks were compared using the Fisher's exact test and, if not statistically significant, combined for treatment comparison.
The percentage of damaged cells (numerical and structural) in the total population of cells examined was calculated for each group. A statistical analysis of the percent aberrant cells per dose was made using the Fisher's exact test.
The average number of aberrations per cell was reported but no statistical analysis was applied. The Cochran-Armitage trend test was performed between the solvent and treatment groups for each treatment condition and harvest time to test for evidence of a dose response.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Relative cloning efficiency at the highest dose level tested of 34% and 1% in the 8 hour and 12 hour non-activated studies, respectively, and 70% and 38% in the 8 hour and 12 hour S9 activated studies, respectively.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: stock concentrations were adjusted to pH7 in order to ensure neutrality of the treatment medium
- Effects of osmolality: osmolality was measured before the main experiments and concentrations chosen where no difference in osmolality was observed

RANGE-FINDING/SCREENING STUDIES:
Dose levels were chosen according to a range-finding toxicity study which was based on cloning efficiency relative to the solvent control. Based upon the findings of the toxicity study dose levels 0.5 and 1.5 µl/mL were the high doses selected for further study in the non-activated and S9 activated studies, respectively.
The average cell generation time (AGT) was calculated in the non-activated and S9 activated studies for the solvent and the two highest test concentrations yielding metaphase cells. At the absence of cell cycle delay harvest times were set at 8 and 12 hours after initiation of treatment for non-activated and S9 activated studies.

Table1: summary of cytogenetic assay results

Treatment^1	Harvest time (hour)	Mean mitotic index^2	Cells scored	Cels with structural aberrations (%)^3,4	Structural aberrations per cell (mean ± SD)^5
Non-activated study
untreated cells	8	3.4	100	0	0 .000 ± 0.000
water	8	4.1	100	1	0.010 ± 0.100
0.21 µL/mL	8	3.9	100	0	0.000 ± 0.000
0.28 µL/mL	8	2.2	100	1	0.010 ± 0.100
0.38 µL/mL	8	0.2	67	0	0.000 ± 0.000
untreated cells	12	4.2	100	0	0.000 ± 0.000
water	12	3.5	100	0	0.000 ± 0.000
0.28 µL/mL	12	2.5	100	1	0.010 ± 0.100
0.38 µL/mL	12	0.8	100	2	0.020 ± 0.141
0.50 µL/mL	12	0.2	64	1	0.016 ± 0.125
TEM, 0,25 µg/mL	12	0.9	100	11	0.130 ± 0.393
S9 activated study
untreated cells	8	2.5	100	1	0.010 ± 0.100
water	8	2.3	100	3	0.030 ± 0.171
0.48 µL/mL	8	1.9	100	1	0.010 ± 0.100
0.63 µL/mL	8	2.1	100	1	0.010 ± 0.100
0.84 µL/mL	8	0.8	100	1	0.010 ± 0.100
untreated cells	12	3.7	100	1	0.010 ± 0.100
water	12	3.3	100	1	0.010 ± 0.100
0.63 µL/mL	12	3.7	100	3	0.030 ± 0.171
0.84 µL/mL	12	4.1	100	2	0.020 ± 0.141
1.13 µL/mL	12	0.9	55	0	0.000 ± 0.000
CP, 20µg/mL	12	1.8	100	12	0.130 ± 0.367

¹ CHO cells were treated at 37°C for 4 hours

² Mitotic Index = cells in mitosis per 500 cells counted, expressed as a percentage

³ Structural aberrations include unanalyzable cells and cells with one ore more aberrations, excluding gaps

⁴ significantly increased above the control using Fisher's exact test; *p<0.025

⁵ Gaps and unanalyzable cells are not included; SD = standard deviation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro gene mutation study in bacteria:

A study, according to OECD 471 (1997) was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98. TA 100, and TA 102 (RCC, 2008). The assay was performed in two independent experiments, with and without liver micmosomal activatiion(S9 mix). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations of the active ingredient: Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000: 2500: and 5000 µg/plate, Experiment II without S9 mix: 1; 3; 10; 3: 100: 333; 1000; and 2500 µg/plate, Experiment II with S9 mix: 3; 10; 33; 100; 333; 100: 2500, and 5000 µg/plate. The plates incubated with the test item showed reduced background growth with and without S9 mix in all strains used in both experiments. Toxic effect, evident as a reduction in the number of revertents, occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose Level, neither in the presene or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentration in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Summarizing, the test item is considered not to be mutagenic in this bacterial mutagenicity test in vitro.

 

In vitro gene mutation study in mammalian cells:

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro according to OECD 476 (BASF SE, 2013). Two independent experiments were carried out, with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiments, the following doses were tested. 1st and 2nd Experiment without S9 mix 0; 12.5; 25.0; 50.0; 100.0; 200.0; 400.0 μg/mL and with S9 mix 0; 18.8; 37.5; 75.0; 150.0; 300.0; 600.0 μg/mL. Following attachment of the cells for 20-24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6-8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The negative controls gave mutant frequencies within the range expected for the CHO cellline. Both positive control substances, EMS and DMBA, led to the expected increase in the frequencies of forward mutations. In this study, in the 1st and 2nd Experiment, the highest concentrations evaluated for gene mutations were clearly cytotoxic in the absence and the presence of metabolic activation. Based on the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation. Summarizing, the test item is not considered to be mutagenic in a gene mutation test in vitro with mammalian cells.

 

In vitro cytogenicity / chromosome aberration study in mammalian cells:

The test item was tested in the chromosome aberration assay using Chinese hamster ovary cells similar to OECD 473 (CIR, 1990). The assay was conducted both in the absence and presence of an Aroclor induced rat liver S9 activation system. Dose levels of 0.21, 0.28 and 0.38 µl/mL in the 8 hour non-activated study, 0.28, 0.38 and 0.5 µl/mL in the 12 hour non-activated study, 0.48, 0.63 and 0.84 µl/mL in the 8 hour S9 activated study and dose levels of 0.63, 0.84 and 1.13 µl/mL in the 12 hour S9 activated study were selected at the highest, non-precipitating concentrations in the non-activated and S9 activated studies which could be evaluated for chromosome aberrations. Survival (relative cloning efficiency) at the highest dose level scored was 34% and 1% in the 8 and 12 hour non-activated studies, respectively, and 70% and 38% in the 8 and 12 hour S9 activated studies, respectively.Summarizing the test item did not induce a significant increase in chromosome aberrations at either harvest time, 8 or 12 hours, in the absence or presence of S9 activation. Therefore the test substance is considered negative in the CHO cytogenicity study.

Assessment of genetic toxicity:

According to the test strategy for mutagenicity mentioned in Annex VII-IX of REGULATION (EC) No 1907/2006 first in vitro test to investigate mutagenicity has to be performed. Only in cases were evidence from in vitro test (or other information) exist testing in mammals is suggested.

All in vitro tests conducted showed negative results. Therefore the test substance was considered as not mutagenic.

As no indication of mutagenicity can be observed in the available test results and also no other further information gave hints for mutagenicity no additional testing is suggested.

 

 

 

 

Justification for classification or non-classification

Based on the available information, classification for genetic toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.