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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Amines, N-C10–C16-alkyltrimethylenedi-, reaction products with chloroacetic acid

Cas Number:

139734-65-9

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

Amines, N-C10–C16-alkyltrimethylenedi-, reaction products with chloroacetic acid

Details on test material:

- Name of test material: DOPA-Glycinate

Method

Target gene:

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

Species / 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

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.