N,N-dimethyl-alkyl-1-amines, reaction products with alkali hydroxide and chloroacetic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro:
Bacterial reverse mutation assay (OECD 471): negative with and without metabolic activation (EC 931-700-2)
Mammalian chromosome aberration test (OECD 473): negative with and without metabolic activation (EC 931-700-2)
Mammalian cell gene mutation test (OECD 476): negative with and without metabolic activation (EC 931-700-2)

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

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

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

His-operon

Species / strain / cell type:

S. typhimurium, other: TA97a, TA98, TA100, TA1535, TA102

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and β-Naphtoflavone induced rat liver S9

Test concentrations with justification for top dose:

1. Study: 0.016, 0.05, 0.16, 0.50, 1.60 mg/plate (TA97a, TA100 ± S9; TA1535 - S9); 0.05, 0.16, 0.50, 1.60, 5.00 mg/plate (TA98, TA102 ± S9; TA1535 + S9);
2. Study: 0.005, 0.016, 0.05, 0.16, 0.50 mg/plate (TA97a, TA102 ± S9; TA100, TA1535 - S9); 0.016, 0.05, 0.16, 0.50, 1.60 mg/plate (TA98, TA102 ± S9; TA100, TA1535 + S9)

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

aqua bidest

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR 191, 0.5 µg/plate

Remarks:

TA97a without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

aqua bidest

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-o-phenylene-diamine, 0.5 µg/plate

Remarks:

TA98 without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

aqua bidest

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Nitrofurantoine, 0.2 µg/plate

Remarks:

TA100 without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

aqua bidest

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535 without S9 Migrated to IUCLID6: 0.25 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

aqua bidest

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene, 2 µg/plate

Remarks:

TA97a, TA98, TA100, TA1535 with S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

aqua bidest

True negative controls:

no

Positive controls:

yes

Positive control substance:

cumene hydroperoxide

Remarks:

TA102 without S9 Migrated to IUCLID6: 100 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

aqua bidest

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Danthron, 30 µg/plate

Remarks:

TA102 with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours


SELECTION AGENT (mutation assays): histidine prototrophy


NUMBER OF REPLICATIONS: triplicates


DETERMINATION OF CYTOTOXICITY
- Method: other: reduction of background lawn

Evaluation criteria:

The test item was interpreted as mutagenic if a concentration effect relationship occurred and the induction rate was equal to or greater than 2.
Spontaneous revertants/plate had to be within historical ranges, and the induction rates of the positive controls had to be equal to or greater than 2 to consider the test valid.

Statistics:

Arithmetic mean values and standard deviations were calculated from colonies per plate of three replicates. For evaluation of the results the induction rate of the mean values was calculated by division of the number of revertant colonies of the test item by the number of revertant colonies of the corresponding control.

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Lowest cytotoxic concentration without and with S9 (mg/plate): TA97a (0.5 and 1.6, resp.)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Lowest cytotoxic concentration without and with S9 (mg/plate): TA98 (1.6 and 5.0, resp.)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Lowest cytotoxic concentration without and with S9 (mg/plate): TA 100 (1.6 without S9 only)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Lowest cytotoxic concentration without and with S9 (mg/plate): 5.0 and 5.0, resp

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Lowest cytotoxic concentration without and with S9 (mg/plate): 1.6 and 5.0, resp.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Test concentrations were selected based on the results of a preliminary test (non-GLP)

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous revertants/plate (negative controls) had to be within the following ranges:
TA97a ± S9: 150 - 450;
TA98 ± S9: 15 - 50;
TA100 ± S9: 60 - 200;
TA102 ± S9: 300 - 600;
TA1535 ± S9: 5 - 30

Study 1:

Maximum number of revertants (mean ± SD):

Strain	-S9			+S9
	Control	Test group (mg/plate)	Induction rate	Control	Test group (mg/plate)	Induction rate
TA97a	251 ± 10.5	288 ± 1.1 (0.16)	1.1	282 ± 15.3	252 ± 14.5 (0.16)	0.9
TA98	35 ± 7.1	33 ± 7.5 (0.05)	1.0	38 ± 6.1	39 ± 12.0 (0.16)	1.0
TA100	93 ± 8.2	104 ± 12.2 (0.016)	1.1	73 ± 8.2	82 ± 9.5 (0.5)	1.1
TA102	429 ± 8.3	444 ± 36.7 (0.5)	1.0	519 ± 16.3	519 ± 11.5 (0.16)	1.0
TA1535	18 ± 4.0	28 ± 3.1 (0.05)	1.5	7 ± 2.5	14 ± 3.6 (0.05)	1.9

 

Study 2:

Maximum number of revertants (mean ± SD):

Strain	-S9			+S9
	Control	Test group (mg/plate)	Induction rate	Control	Test group (mg/plate)	Induction rate
TA97a	281 ± 26.1	239 ± 12.2 (0.05)	0.8	298 ± 41.6	273 ± 21.1 (0.05)	0.9
TA98	22 ± 10.4	41 ± 5.5 (1.6)	1.4	33 ± 2.6	35 ± 1.5 (0.5)	1.1
TA100	127 ± 14.2	111 ± 13.1 (0.016)	0.9	91 ± 8.0	91 ± 7.1 (0.5)	1.0
TA102	349 ± 20.4	355 ± 41.7 (0.05)	1.0	451 ± 34.3	462 ± 19.1 (0.5)	1.0
TA1535	29 ± 2.5	32 ± 1.7 (0.005)	1.1	11 ± 2.1	10 ± 3.1 (0.016)	0.9

 

Conclusion:

The test substance was not mutagenic to bacteria under the conditions chosen.

Conclusions:

Interpretation of results (migrated information):
negative

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:

2010-02-26 to 2010-04-16

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

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary (CHO K1) cells (repository number CCL 61) were obtained from American Type Culture Collection, Manassas, VA. In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 20. The frozen lot of cells was tested using the Hoechst staining procedure and found to be free of mycoplasma contamination. This cell line has an average cell cycle time of 10-14 hours with a modal chromosome number of 20.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

4h without S9-mix: 10, 25, 50, 100, 125, 150, 175, 200 µg/mL
20h without S9-mix: 1, 5, 10, 25, 35, 50, 75, 100 µg/mL
4h with S9-mix: 5, 10, 50, 75, 100, 125, 150 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Deionized water was used as the solvent based on the information provided by the Sponsor and compatibility with the target cells.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9-mix Migrated to IUCLID6: 10 and 15 µg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9-mix Migrated to IUCLID6: 0.1 and 0.2 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 16-24h
- Exposure duration: In the non-activated study, the cells were exposed to the test article for 4 hours or continuously for 20 hours up to the cell harvest; In the S9-activated study, the cells were exposed for 4 hours
- Expression time (cells in growth medium): 16h for the 4h treatments

SPINDLE INHIBITOR (cytogenetic assays): Colcemid® at a final concentration of 0.1 µg/mL
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; relative total growth

Evaluation criteria:

All conclusions were based on sound scientific basis; however, as a guide to interpretation of the data, the test article was considered to induce a positive response when the percentage of cells with aberrations is increased in a dose responsive manner, with one or more concentrations being statistically significant (p ≤ 0.05). However, values that are statistically significant but do not exceed the range of historical solvent controls may be judged as not biologically significant. Test articles not demonstrating a statistically significant increase in aberrations will be concluded to be negative.

Statistics:

Statistical analysis of the percent aberrant cells was performed using the Fisher's Exact test. Fisher's Exact test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's Exact test at any test article dose level, the Cochran-Armitage test was used to measure dose-responsiveness.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥280 µg/mL in both the non-activated and S9-activated 4-hour exposure groups, and at concentrations ≥84 µg/mL in the non-activated 20-hour continuous exposure group in a preliminary toxicity assay

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation/water solubility: The test article was soluble in water and in the treatment medium at all dose levels tested at the beginning and conclusion of the treatment period.

RANGE-FINDING/SCREENING STUDIES: Substantial toxicity (i.e., at least 50% cell growth inhibition, relative to the solvent control) was observed at dose levels ≥280 µg/mL in both the non-activated and S9-activated 4-hour exposure groups, and at dose levels ≥84 µg/mL in the non-activated 20-hour continuous exposure group in a preliminary toxicity assay.

Table 1: Cell viability and chromosomal changes at representative concentrations with high cell viability

Test item	Concentration	Cell viability	Mean	Aberrant cells in %
	in µg/mL	in %	mitotic index	Numerical	Structural
Exposure period 4 hrs without S9 mix
Water	--	99	13.1	1.5	0.0
MMC	0.1	96	7.9	3.0	28.0
ADB	25	98	13.2	3.0	0.0
	60	97	13.4	3.0	0.5
	80	95	12.9	5.5	0.5
Exposure period 20 hrs without S9 mix
Water	--	100	14.7	1.0	1.5
MMC	0.1	97	10.2	1.0	34.0
ADB	10	98	14.9	1.5	1.5
	25	95	14.7	1.5	2.5
	50	93	14.6	2.9	0.5
Exposure period 4 hrs with S9 mix
Water	--	98	13.8	3.0	1.0
CP	10	97	2.8	4.0	42.0
ADB	10	96	13.2	3.0	1.5
	50	93	12.5	4.5	0.0
	75	93	11.3	4.0	2.5

                  MMC: Mitomycin C

                       CP: Cyclophosphamide

                       ADB: Alkyl dimethyl betaine

Alky dimethyl betaine induced no chromosomal aberrations in CHO cells after exposure times of 4 or 20h with and without metabolic activation. Because positive controls were valid, alkyl dimethyl betaine was considered to be not clastogenic in mammalian cells.

Conclusions:

Interpretation of results (migrated information):
negative

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:

2010-03-02 to 2010-03-31

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

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1 cells were obtained from American Type Culture Collection in Manassas, VA. CHO cells were cleansed in medium supplemented with hypoxanthine, aminopterin and thymidine (HAT). Cells used in the mutation assay were within four subpassages from cleansing in order to assure karyotypic stability.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Range finding study: 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 2790 µg/mL with and without S9-mix
Main study: 5, 10, 25, 50, 75 without S9-mix and 5, 10, 25, 50, 100 µg/mL with S9-mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: destilled water
- Justification for choice of solvent/vehicle: supplied by the sponsor in destilled water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9-mix Migrated to IUCLID6: 0.2 µL/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9-mix Migrated to IUCLID6: 4 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24h
- Exposure duration: 18-24h
- Expression time (cells in growth medium): 7-9 days

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 2x10e6

DETERMINATION OF CYTOTOXICITY
- cloning efficiency and relative total growth

Evaluation criteria:

The test article was considered to induce a positive response if there was a concentration-related increase in mutant frequencies with at least two consecutive concentrations showing mutant frequencies of > 40 mutants per 10e6 clonable cells. If a single point above 40 mutants per 10e6 clonable cells was observed at the highest concentration, the test article was considered suspect.
If no culture exhibited a mutant Hequency of > 40 mutants per 10e6 clonable cells, the test article was considered negative.
The cloning efficiency of the solvent control must be greater than 50%. The spontaneous mutant frequency in the solvent control must fall within the range of 0-25 mutants per 10e6 clonable cells. The positive control must induce a mutant frequency at least three times that of the solvent control
and must exceed 40 mutants per 10e6 clonable cells. There must be at least four analyzable test article concentrations with mutant frequency data.

Statistics:

not performed

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥50 µg/mL without and ≥100 µg/mL with S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test article was provided as a 30% solution of active ingredient in water; therefore, sterile distilled water used as the solvent for the test article.
- Precipitation: There was no precipitation observed at the chosen concentrations

RANGE-FINDING/SCREENING STUDIES: Cloning efficiency relative to the solvent controls (RCE) was 0% at ≥150 µg/mL with and without S9 activation. Based on the results of the toxicity test, the concentrations chosen for the mutagenesis assay ranged from 5.0 to 150 µg/mL for the non-activated cultures and 5.0 to 200 µg/mL for the S9-activated cultures.

Table 1: Cloning efficancy, mutation rate and relative total growth

Treatment	Concentration [µg/mL]
		Cloning efficiency [%]		Mutants/10e6 clonable cells		Relative total growth
		with S9	without S9	with S9	without S9	with S9	without S9
Solvent	--	70	80	0	3.8	100	100
B(a)P	4	76	--	98.9	--	44	--
EMS	0.2 µL/mL	--	56	--	512.9	--	13
Test substance	5	54	79	20.2	8.2	103	84
	10	67	80	0.7	0	94	98
	25	57	75	0.9	2.0	94	89
	50	34	70	0	0	87	14
	100	45	--	0	--	27	--

B(a)P: Benz(a)pyrene

EMS: Ethylmethanesulphonate

All criteria for a valid study were met as described in the protocol. The results of the CHO/HGPRT Mutation Assay indicate that, under the conditions of this study, alkyl dimethyl betaine did not induce gene mutations in mammalian cells.

Conclusions:

Interpretation of results (migrated information):
negative

Reference 4

Endpoint:

genetic toxicity in vitro, other

Remarks:

gene mutation, chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium, other: TA97a, TA98, TA100, TA1535, TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Lowest cytotoxic concentration without and with S9 (mg/plate): TA97a (0.5 and 1.6, resp.); TA98 (1.6 and 5.0, resp.); TA100 (1.6 without S9 only); TA102 (5.0 and 5.0, resp.); TA1535 (1.6 and 5.0, resp.)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥280 µg/mL in both the non-activated and S9-activated 4-hour exposure groups, and at concentrations ≥84 µg/mL in the non-activated 20-hour continuous exposure group in a preliminary toxicity assay

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥50 µg/mL without and ≥100 µg/mL with S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Ames test

Conclusions:

Betaines, C12-14 (even numbered)-alkyldimethyl, potassium salt; aqueous commercial product(s) is considered to be not genotoxic.

Executive summary:

Three in vitro genetic toxicity assays are available from the structural analogue EC 931-700-2: Ames test, chromosome aberration test and a HPRT test. In all three test the structural analogue EC 931-700-2 did not show any genotoxic potential and thus this is also estimated for Betaines, C12-14 (even numbered)-alkyldimethyl, potassium salt; aqueous commercial product(s). As explained in the justification for type of information, the differences in molecular structure between the target and the source are unlikely to lead to differences in genetic toxicity that are higher than the typical experimental error of the test method.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There is no data available on the genetic toxicity of N,N-dimethyl-alkyl-1-amines, reaction products with alkali hydroxide and chloroacetic acid. However, there is reliable data for the structural related substance EC 931-700-2. The target substance and the source substance show pronounced structural and physico-chemical similarities. Therefore, read-across was performed based on an analogue approach. For a detailed justification of the analogue approach, please refer to section 13 of the technical dossier.

There are three in vitro studies available addressing mutagenicity in bacteria, and clastogenicity and mutagenicity in mammalian cells, which are acceptable for assessment.

 

Mutagenicity in bacteria was investigated by the Bacterial Reverse Mutation Assay (Ames test) with 5 strains of Salmonella typhimurium (Fiebig, 2003). In this study the tested bacteria strains TA97a, TA98, TA100, TA1535 and TA102 were exposed to respective concentrations of 0.016 to 5.000 mg/plate and 0.005 to 1.600 mg/plate of the test substance in two independent experiments for 48 hours with or without metabolic activation by phenobarbital- and beta-naphtoflavone-induced rat liver S9. Depending on the strain and the application of metabolic activation the upper and lower concentrations of the applied concentration ranges varied within each experiment. Under the conditions of the study the test substance did not induce gene mutations in the tested strains. Cytotoxic effects were observed starting at 1.6 and 0.5 mg/plate with and without S9, respectively, in TA97a, 5.0 and 1.6 mg/plate with and without S9, respectively, in both TA98 and TA1535, 5.0 mg/plate with and without S9 in TA102, and 1.6 mg/plate without S9 only in TA100.

 

The ability to induce clastogenic effects in mammalian cells was investigated with the In Vitro Chromosome Aberration Test (Madraymootoo, 2010), performed in Chinese Hamster Ovary cells (CHO K1 cells) with and without metabolic activation by Aroclor 1254-induced rat liver S9 mix. Cells were exposed to the test article EC 931-700-2 for 4 or 20 hours without S9 mix, and for 4 hours only with S9 mix. Cells were exposed to maximal concentrations of 150 and 200 µg/mL in the 4-hour exposure groups with and without S9 mix, respectively, and to 100 µg/mL in the 20-hour exposure group without S9 mix. Cytotoxicity was observed at concentrations higher than 280 µg/mL in the 4-hour exposure groups with and without S9 mix, and at concentrations higher than 84 µg/mL in the non-activated 20-hour exposure group in a preliminary toxicity assay which served as range finder for the actual study. Under the conditions of the study the test substance did not induce chromosomal aberrations in the CHO cells and is therefore not considered to be clastogenic.

Gene mutation in mammalian cells was addressed by a CHO/HGPRT Mutation Assay in Chinese Hamster Ovary cells (CHO K1) with EC 931-700-2 (Clarke, 2010). In a preliminary range-finder concentrations up to 2790 µg/mL were tested with and without metabolic activation by Aroclor 1254-induced rat liver S9 mix. As no precipitation occurred up to the highest concentration, the actual concentrations for the test were chosen based on cloning efficiency. Substantial toxicity, i.e. cloning efficiency lower than 50%, was observed at concentrations equal to or greater than 50 µg/mL without activation and at concentrations equal to or greater than 150 µg/mL with activation in the preliminary toxicity assay. Based on these findings the concentrations chosen for the mutagenesis assay ranged from 5.0 to 150 µg/mL for the non-activated cultures and from 5.0 to 200 µg/mL for the activated ones. In the actual mutagenesis assay no visible precipitate was observed in the treatment medium, either. Toxicity, i.e. cloning efficiency equal to or lower than 50% of the solvent control, was observed at concentrations equal to or greater than 50 µg/mL without activation and 100 µg/mL with activation. Under the conditions of the study the test substance was negative in the CHO/HGPRT Mutation Assay, i.e. no treated cultures with mutant frequencies higher than 40 mutants per 10e6 clonable cells were observed.

According to Regulation (EC) No 1907/2006, 8.4, column 2, an in vivo mutagenicity study does not have to be considered as there were no positive results obtained from any of the in vitro genotoxicity studies in Annex VII and VIII.

Justification for classification or non-classification

The available data on genetic toxicity of the analogue substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.