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

Genetic toxicity: in vitro

Administrative data

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:

other: Guideline study, GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report Date:

2003

Materials and methods

Test guideline

Qualifier:

according to

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. certificate)

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material: DOPA-Glycinate

Method

Target gene:

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

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: Amino-acid requiring strains, histidine for S. typhimurium and tryptophane for E. coli

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

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

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (Milli-Q-water)

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

ethylnitrosurea

other: (Absence of S9-mix)

Controls 2

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene; (Presence of S9-mix)

Details on test system and experimental conditions:

Way of application:
Test substance was dissolved in Milli-Q-water at 50 mg/mL. The stock solution was sterilized by passage through a micropore filter (0.45 µM).
For the plate incorporation tests, 0.1 mL of a fully grown culture of the appropriate strain, 0.1 mL of the appropriate test substance solution and 0.5 mL S9-mix for the tests with metabolic activation or 0.5 mL sodium phosphate for the tests without metabolic activation were added to top agar mix and poured onto minimal glucose agar plates. All determinations were made in triplicate. The cultures were incubated at 37°C for 48–72 hours.

Pre-incubation time:
None

Other modifications:
None

Examinations:
Number of revertant colonies

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

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 (migrated information):
negative

DOPA-Glycinate 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 DOPA-Glycinate (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.