Amines, N-C10-16-alkyltrimethylenedi-, reaction products with chloroacetic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative in all tests conducted:
- Ames test with S. typhimurium TA 98, TA 100, TA 1535, TA 1537, E coli WP2 (met. act.: with and without) (OECD TG 471, GLP); tested up to cytotoxic concentrations
- Mammalian cell gene mutation assay with CHO cells (HPRT test) (met. act.: with and without) (OECD Guideline 476, pre-GLP); tested up to cytotoxic concentrations
- In vitro mammalian chromosome aberration test with cultured human lymphocytes (met. act.: with and without) (OECD Guideline 473, pre-GLP); tested up to cytotoxic concentrations

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (Salmonella strains), trp operon (E. coli strain)

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : rat liver
- method of preparation of S9 mix: according to Ames et al. (1975) and Maron and Ames (1983): Male Wistar rats were injected intraperitoeally with a single dose of Aroclor 1254 in soya bean oil. Five days after injection the livers were removed S9 was prepared.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix
- quality controls of S9: protein content, cytochrome P-450 content and sterility were checked

Test concentrations with justification for top dose:

First assay: 0, 62, 185, 556, 1667 and 5000 µg/plate (in the presence and in the absence of rat liver S9 fraction)
Second assay: 0, 12.5, 25, 50, 100 and 200 µg/plate (in the presence and in the absence of rat liver S9 fraction)

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (Milli-Q-water)

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

ethylnitrosurea

other: 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 - 72 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, background growth inhibition

Evaluation criteria:

The mutagenicity study is considered valid if the mean colony counts of the control values of the strain are within acceptable ranges, if the results of the positive controls meet the criteria of positive response, and if no more than 5% of the plates are lost through contamination of other unforeseen events.

A test substance is considered to be positive in the bacterial reverse mutation test if the mean number of revertant colonies on the test plates is concentration-related increased or if a reproducible two-fold or more increase is observed compared to that on the negative control plates.

A test substance is considered to be negative in the bacterial reverse mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Genotoxicity
Without metabolic activation: test item dissolved in Milli-Q-water did not cause a two-fold or more increase in the mean number of revertant colonies in all tested strains compared to the background spontaneous reversion rate in the negative control.
The mean number of revertant colonies in the negative controls was within the range of the historical controls.
Positive controls significantly increased the reverse mutation rate.
The results are presented in Table A6.6.1- 1and Table A6.6.1- 2.

With metabolic activation: test item dissolved in Milli-Q-water did not cause a two-fold or more increase in the mean number of revertant colonies in all tested strains compared to the background spontaneous reversion rate in the negative control.
The mean number of revertant colonies in the negative controls was within the range of the historical controls
Positive controls significantly increased the reverse mutation rate.
The results are presented in Table A6.6.1- 1 and Table A6.6.1- 2.

Cytotoxicity
Yes
In the first assay cytotoxicity was observed at and above 185 µg/plate with and without metabolic activation in all tested strains.
In the second assay, cytotoxicity was observed at 200 µg in the absence and presence of S9-mix, except for TA 1535 and TA 100 where normal growth was observed with S9 mix. At 100 µg per plate pin points were observed in TA 1537 and TA 98, in the absence of S9-mix.
The results are presented in Table A6.6.1- 1 and Table A6.6.1- 2.

Table A6.6.1 -1:First assay: average numbers of revertants per plate in E.coli or S.typhimurium after treatment with the test item (plate-incorporation assay, triplicate plates per concentration).

Concentration [µg/plate]	TA 1535		TA 1537		TA 98		TA 100		E.coli		Comments
	–S9	+S9	–S9	+S9	–S9	+S9	–S9	+S9	–S9	+S9
Control (mean number of revertant colonies)	26±3	28±5	23±1	17±7	47±6	69±4	191±15	167±12	33±6	37±6
62	27±3	21±6	16±8	23±8	27±4	67±15	162±19	173±21	34±3	40±6
185	–	17±2	–	8±5	–	11±1	–	107±4	30±6	37±6	Cytotoxicity
556	–	–	–	–	–	–	–	–	–	–	Cytotoxicity
1667	0	0	0	0	0	0	0	0	0	0	Cytotoxicity
5000	0	0	0	0	0	0	0	0	0	0	Cytotoxicity
Positive controls	606±42	814±114	4087±1344	288±10	2219±82	1953±83	775±58	2879±186	222±10	1527±167
–         plate not counted due to contamination or pin points

 

Table A6.6.1-2:Second assay:average numbers of revertants per plate in E.coli or S.typhimurium after treatment with the test item (plate-incorporation assay, triplicate plates per concentration).

Concentration [µg/plate]	TA 1535		TA 1537		TA 98		TA 100		E.coli		Comments
	–S9	+S9	–S9	+S9	–S9	+S9	–S9	+S9	–S9	+S9
Control (mean number of revertant colonies)	24 ±9	29 ±7	18 ±2	26 ±12	42 ±15	68 ±4	184 ±34	173 ±17	47 ±10	48 ±8
12.5	30 ±7	23 ±3	19 ±3	27 ±4	41 ±8	69 ±6	168 ±7	162 ±14	49 ±6	64 ±2
25	24 ±5	18 ±3	16 ±3	21 ±3	32 ±6	52 ±7	172 ±13	144 ±22	37 ±9	44 ±7
50	25 ±3	18 ±6	18 ±7	29 ±5	41 ±2	55 ±8	187 ±6	179 ±13	40 ±4	44 ±5
100	25 ±6	23 ±5	–	30 ±4	-	51 ±14	164 ±37	179 ±20	51 ±8	47 ±9	Cytotoxicity (pin points observed in TA 1537; TA 98 without S9 mix)
200	–	121 ±4	–	–	–	–	–	159 ±20	–	–	Cytotoxicity, (except TA 1535 and TA 100 with S9 mix)
Positive controls	721 ± 36	500 ± 53	2396 ± 440	316 ± 20	1314 ± 141	845 ± 107	856 ± 84	2205 ± 81	174 ± 32	1695 ± 152
–         plate not counted due to pin points

Conclusions:

Interpretation of results: negative

The registration substance was not mutagenic in this bacterial reverse mutation assay in the absence and presence of metabolic activation (S9 mix).

Executive summary:

The mutagenic potential of the registration substance (99.4% a.i.) was tested in the bacterial reverse mutation test using the plate incorporation assay. The study was conducted according to OECD guideline 471 (1997).

Two independent plate incorporation assays were performed at concentrations of up to 5000 µg per plate (first assay) or up to 200 µg/plate (second assay). In the first assay cytotoxicity was observed at ≥ 185 µg/plate ± S9 mix in all tested strains. In the second assay, cytotoxicity was observed at 200 µg ± S9 mix in all strains except for TA 1535 and TA 100.

No increase in the number of revertant colonies was found in any of the tested strains with or without metabolic activation while the positive controls gave the expected increase in the mean number of revertant colonies.

Under the conditions of this study, the test item dissolved in water was not mutagenic.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration 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 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Sex, age and number of blood donors: male, 26 years old, one donor
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: no

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: RPMI 1640 medium + 20% FCS, L-glutamine, penicillin, streptomycin, phytohaemagglutinin, 5%CO2, humidified air, 37°C

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : male Wistar rats
- method of preparation of S9 mix: derived from livers of male rats which were treated by intraperitoneal injection with a single dose of Aroclor 1254. Preparation of S9 five days after treatment.
- concentration or volume of S9 mix and S9 in the final culture medium : 0.5 mL of 10% S9 mix
- quality controls of S9: checked for sterility, protein, cytochrom P-450 content

Test concentrations with justification for top dose:

0, 1.11, 3.33, 10.0 and 30.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 1640 medium

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:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 24 hours without S9 mix, 2 hours with S9 mix
- Harvest time after the end of treatment (sampling/recovery times): without S9 mix harvested directly, with S9 mix harvested after 22 hours

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used: colcemide (0.1 µg/mL final concentration) for 2 hours
- Methods of slide preparation and staining technique used including the stain used: cells were harvested by low speed centrifugation, treated for 8 minuts with hypotonic solution (0.075 M KCl), fixed three times with a 3:1 mixture of methanol and glacial acetic acid, and transferred to clean microscopic slides; slides were stained with 2% Giemsa solution
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 4 slides per culture, 100 well-spread metaphases (each containing 46 chromosomes) were analysed

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)

Evaluation criteria:

The test is considered positive if a dose-related, statistically significant increase in the number of cells with structural chromosome aberrations. However, a clear dose-response relationship can be absent because the yield of chromosome aberrations can vary markedly with post-treatment sampling time of an asynchronous population and because increasing doses of clastogens can induce increasing degrees of mitotic delay. A test substance producing neither a dose-related, statistically significant increase in the number of cells with structural chromosome aberrations, nor a statistically significant and reproducible positive response at any of the doses is considered non-clastogenic in this system.

Statistics:

Data were analysed, when apprpriate, by Fisher's exact probability test using the number of cells with aberrations to determine significant differences between test and control cultures.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Genotoxicity
Without metabolic activation: Negative
The test substance did not induce a statistically significant increase in the number of cells with structural chromosome aberrations at any of the concentrations used when compared with the vehicle control values.
The positive control compound, Mitomycin C, showed the expected statistically significant increase in the number of cells with structural chromosome aberrations.

With metabolic activation: Negative
The test substance did not induce a statistically significant increase in the number of cells with structural chromosome aberrations at any of the concentrations used when compared with the vehicle control values.
The positive control compound, Cyclophosphamide, showed the expected statistically significant increase in the number of cells with structural chromosome aberrations.
The results are summarised in Table A6.6.2- 2.

Cytotoxicity
Yes
In a first preliminary cytotoxicity test, the test substance was clearly cytotoxic at concentrations in a range of 82.3–20000 µg/mL with and without metabolic activation. In a second preliminary cytotoxicity test the mitotic index was reduced to about 50% at 27.4 µg/mL both with and without S9 mix.
In the chromosome aberration test a reduction of the mitotic index to about 60 % or 54 % of the vehicle control without or with S9 mix, respectively, was observed at the highest test concentration (30 µg/mL).

Table A6.6.2-1:Metaphase chromosome analysis of human lymphocytes after exposure to the test item or mitomycin C in the absence of S9-mix.

	Control	1.11 µg/mL	3.33 µg/mL	10 µg/mL	30 µg/mL	Mitomycin C
Cytotoxicity	An inhibition of growth of 50 % was judged to lie at a concentration of 27.4 µg/mL of the test item
No. of cells analysed	200	200	200	200	200	200
ABERRATIONS
No. of cells with aberrations
Gaps	0	1	2	1	1	11**
Breaks	0	0	1	1	0	28***
Exchanges	0	0	0	0	0	22***
Multiple	0	0	0	0	0	0
% cells with aberrations
Incl. gaps	0	0.5	1.5	1.0	0.5	26.0***
Excl. gaps	0	0	0.5	0.5	0	22.5***
Mitotic index^1	9.9	8.9	8.1	8.9	5.9	4.2
**) p<0.01 Fisher’s exact probability test (two sided) ***) p<0.001 Fisher’s exact probability test (two-sided) 1) percentage of metaphases determined in 1000 nuclei (mean value of duplicate cultures)

 

Table A6.6.2-2:Metaphase chromosome analysis of human lymphocytes after exposure to the test item or cyclophosphamide in the presence of S9-mix.

	Control	1.11 µg/mL	3.33 µg/mL	10 µg/mL	30 µg/mL	Cyclophosphamide
Cytotoxicity	An inhibition of growth of 50 % was judged to lie at a concentration of 27.4 µg/mL of the test item
No. of cells analysed	200	200	200	200	200	200
ABERRATIONS
No. of cells with aberrations
Gaps	1	0	0	0	0	30***
Breaks	2	0	1	2	1	71***
Exchanges	0	0	0	0	0	44***
Multiple	0	0	0	0	0	0
% cells with aberrations
Incl. gaps	1.5	0	0.5	1.0	0.5	51.0***
Excl. gaps	1.0	0	0.5	1.0	0.5	46.5***
Mitotic index^1	9.9	7.1	8.3	8.2	5.3	1.7
***) p<0.001 Fisher’s exact probability test (two-sided) 1) percentage of metaphases determined in 1000 nuclei (mean value of duplicate cultures)

 

Conclusions:

Interpretation of results: negative

The registration substance (20% a.i.) did not induce structural chromosome damage in cultured human lymphocytes either in the presence or in the absence of metabolic activation (S9 mix) when tested up to cytotoxic concentrations.

Executive summary:

The in vitro genotoxicity of the registration substance (20% a.i.) was tested in human lymphocytes. The study was carried out according to OECD guideline 473 (1983). No relevant deviations from the prescribed test guideline were reported.The test was performed at concentrations of 0, 1.11, 3.33, 10.0 and 30.0 µg/mL in the presence and absence of metabolic activation (S9 mix). A reduction of the mitotic index to about 60 % or 54 % of the vehicle control without or with S9 mix, respectively, was observed at the highest test concentration (30 µg/mL) indicating cytotoxicity. The positive controls resulted in the appropriate responses.

The test item did not induce structural chromosome damage in cultured human lymphocytes either in the presence or in the absence of S9-mix.

Reference 3

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)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HGPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: CHO K-1 line from Prof. Dr. A.T. Natarajan, University of Leiden, Netherlands

For cell lines:
- Absence of Mycoplasma contamination: yes
- Modal number of chromosomes: 21-22 chromosomes, stable aneuploid karyotype, 2n=22

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Ham's F-12 culture medium with 10% FCS, gentamycin, L-glutamine; humidified incubator at 37°C in air containing 5% CO2.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: male Wistar rats
- method of preparation of S9 mix: derived from livers of male rats which were treated by intraperitoneal injection with a single dose of Aroclor 1254. Preparation of S9 five days after treatment.
- concentration or volume of S9 mix and S9 in the final culture medium : 0.5 mL of 10% S9 mix
- quality controls of S9: checked for sterility, protein, cytochrom P-450 content

Test concentrations with justification for top dose:

With S9-mix
First assay: 20.0, 40.0, 60.0, 80.0 and 100.0 mg/L
Second assay: 40.0, 60.0, 80.0, 100.0 and 120.0 mg/L

Without S9-mix
First assay: 2.0, 4.0, 6.0, 8.0 and 10.0 mg/L
Second assay: 4.0, 6.0, 8.0, 10.0 and 12.0 mg/L

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Tissue culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: single
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hours
- Harvest time after the end of treatment: 18 - 20 hours

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 18 - 20 hours
- If a selective agent is used: 6-thioguanine (final concentration 10 µM), incubated for 8 days
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: mutant frequency expressed as number of mutants per 1E06 clonable cells

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: absolute clonining efficiency

Evaluation criteria:

The following criteria were used to evaluate the data obtained in this assay:
a) the survival (absolute cloning efficiency) of the negative controls should not be less than 50%
b) the mean mutant frequency of the negative controls should fall within the range of 0 to 20 6-TG resistant mutants per 1000000 clonable cells,
c) the positive controls must induce a response of a magnitude appropriate for the mutagen under the experimental conditions applied,
d) the highest test substance concentration should, if possible, result in a clear cytotoxic repsonse (e.g. 10-30% of the relative initial survival). Any apparent increase in mutant frequency at concentrations of the test substance causing more than 90% toxicity is considered to be an artifact and not indicative of genotoxicity.

The test substance is considered genotoxic if
a) a concentration-related increase in mutant frequency and
b) a reproducible positive repsonse for at least one of the test substance concentrations (e.g. the mean mutant frequency should be more than 20 mutants per 1000000 clonable cells is observed.

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Genotoxicity
Without metabolic activation: Negative
In the absence of S9 mix the test substance did not induce significant increases in the mutant frequency in both independent assays. Mutant frequency induced by EMS increased by 14 to 22-fold.
The results are summarised in Table A6.6.3- 1.

With metabolic activation: Negative
In the presence of S9 mix the test substance did not increase the mutant frequency in both independent assays. Mutant frequency induced by 2.0 mL/L DMN increased by 33 to 39-fold and by 4.0 mL/L DMN increased by 43 to 83-fold.
The results are summarised in Table A6.6.3- 2.

Cytotoxicity
Yes
In a preliminary toxicity test no cells were recovered at concentrations ranging from 13.7–10000 mg/L without S9 mix and at concentrations ranging from 124–10000 mg/L with S9 mix.
In the first HGPRT assay, the absolute initial cloning efficiency was reduced to about 88% or 68% at 10 mg/L without S9 mix and 100 mg/L with S9 mix, respectively.
In the second HGPRT assay, the relative cloning efficiency was reduced to about 50% at concentrations of 12.0 mg/L (without S9-mix) and 100.0 mg/L (with S9-mix).
Results are presented in Table A6.6.3- 1 and Table A6.6.3- 2.

Table A6.6.3-1:The effect of the test item or Ethylmethanesulphonate (EMS) on forward mutation rates to 6‑thioguanine resistance in CHO cells in absence of metabolic activation on day 8.

Concentration [mg/mL]	–S9
	First point mutation assay		Cytotoxicity	Second point mutation assay		Cytotoxicity
	Mutant frequency per 1E06 cells	Mean absolute cloning efficiency %	Mean relative cloning efficiency %^1)	Mutant frequency per 1E06 cells	Mean absolute cloning efficiency %	Mean relative cloning efficiency %^1)
0	8.3	96.1	100.0	5.9	85.2	100.0
2.0	8.1	92.4	113.7	n.e.	n.e.	n.e.
4.0	1.6	94.8	111.7	2.3	87.2	92.7
6.0	9.6	104.7	99.8	3.6	97.3	86.5
8.0	6.7	90.2	97.3	0.9	106.2	87.5
10.0	10.1	93.9	93.2	3.7	94.7	73.8
12.0	n.e.	n.e.	n.e.	3.9	88.7	55.7
EMS (0.2 mL/L)	187.3	81.7	90.2	87.5	84.0	79.8
^1)survival rate of cells about 24 hours after addition of the test substance n.e.: not examined

Table A6.6.3-2:The effect of the test item or Dimethylnitrosamine (DMN) on forward mutation rates to 6‑thioguanine resistance in CHO cells in presence of metabolic activation on day 8.

Concentration [mg/mL]	+S9
	First point mutation assay		Cytotoxicity	Second point mutation assay		Cytotoxicity
	Mutant frequency per 1E06 cells	Mean absolute cloning efficiency %	Mean relative cloning efficiency %^1)	Mutant frequency per 1E06 cells	Mean absolute cloning efficiency %	Mean relative cloning efficiency %^1)
0	4.2	107.8	100.0	2.8	88.4	100.0
20	2.0	102.3	85.0	n.e.	n.e.	n.e.
40	5.1	97.1	98.9	2.7	93.1	101.4
60	8.2	97.0	97.2	2.8	88.8	101.2
80	3.7	95.8	84.9	2.2	92.3	98.0
100	8.1	99.2	59.7	1.8	108.9	53.7
120	n.e.	n.e.	n.e.	n.d.	n.d.	n.d.
DMN (2.0 mL/L)	163.6	80.7	21.8	91.8	76.8	60.2
DMN (4.0 mL/L)	347.7	68.6	5.1	120.4	67.7	31.0
^1)survival rate of cells about 24 hours after addition of the test substance n.e.: not examined n.d.: not determined, because no cells were recovered for cloning efficiency

Conclusions:

Interpretation of results: negative

The registration substance did not induce a significant increase in the mutant frequency in cultured CHO cells either in the presence or in the absence of metabolic activation (S9 mix) when tested up to cytotoxic concentrations.

Executive summary:

The in-vitro genotoxicity of  the registration substance was tested in Chinese hamster ovary cells. The study was carried out according to OECD guideline 476 (1984). No relevant deviations from the prescribed test guideline were reported. The test was performed in two independent assays in the presence and absence of metabolic activation (S9 mix) using the following concentrations.

With S9-mix

First assay: 20.0, 40.0, 60.0, 80.0 and 100.0 mg/L

Second assay: 40.0, 60.0, 80.0, 100.0 and 120.0 mg/L

Without S9-mix

First assay: 2.0, 4.0, 6.0, 8.0 and 10.0 mg/L

Second assay: 4.0, 6.0, 8.0, 10.0 and 12.0 mg/L

In the first HGPRT assay, the absolute initial cloning efficiency was reduced to about 88% or 68% at 10 mg/L without S9 mix and 100 mg/L with S9 mix, respectively. In the second HGPRT assay, the relative cloning efficiency was reduced to about 50% at concentrations of 12.0 mg/L (without S9-mix) and 100.0 mg/L (with S9-mix). The positive controls resulted in the appropriate responses.

In the absence and in the presence of a metabolic activation system, the test item did not induce a significant increase in the mutant frequency in both independent assays.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The mutagenic potential of the registration substance (99% a.i.) was tested in the bacterial reverse mutation test using the plate incorporation assay. The study was conducted according to OECD guideline 471 (1997). The substance was tested up to cytotoxic concentrations. No increase in the number of revertant colonies was found in any of the tested strains with or without metabolic activation while the positive controls gave the expected increase in the mean number of revertant colonies. Under the conditions of this study, the test item dissolved in water was not mutagenic.

This result is supported by a second bacterial reverse mutation test using the plate incorporation assay. The study was conducted according to OECD guideline 471 (1983) and thus with some limitations to the most recent guideline (no E. coli strain, only one positive control substance for S9 mix). The substance (20% solution) was tested up to cytotoxic concentrations. No increase in the number of revertant colonies was found in any of the tested strains with or without metabolic activation while the positive controls gave the expected increase in the mean number of revertant colonies. Under the conditions of this study, the test item was not mutagenic.

The clastogenic potential of the registration substance (20% solution) was tested in human lymphocytes. The study was carried out according to OECD guideline 473 (1983). The substance was tested up to cytotoxic concentrations. The test item did not induce structural chromosome damage in cultured human lymphocytes either in the presence or in the absence of S9-mix.

The in vitro genotoxicity of registration substance (20% solution) was tested in Chinese hamster ovary cells (HPRT assay). The study was carried out according to OECD guideline 476 (1984). The substance was tested up to cytotoxic concentrations. In the absence and in the presence of a metabolic activation system, the test item did not induce a significant increase in the mutant frequency in both independent assays.

Based on the overall negative results of in vitro genotoxicity testing, the registration substance may be regarded as void of any genotoxic potential. In conclusion, there is no need to carry out in vivo tests for genetic toxicity. There are no data gaps for this endpoint.

No human data are available for genetic toxicity. However, there is no reason to believe that the negative results would not be relevant to humans.

Justification for classification or non-classification

The registration substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and the mammalian cell gene mutation assay using CHO cells. In an in vitro chromosomal aberration test, the test item did not induce structural chromosomal aberrations. Therefore, the registration substance is considered to be non-clastogenic.

In conclusion the full set of genotoxicity tests required by the REACH regulation is negative. According to GHS Regulation EC No 1272/2008 and GHS-UN no classification and labelling for mutagenic toxicity is necessary.