bis(diethylamino)-N,N-diethylmethaniminium chloride

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: OECD 471 (1998); GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Method

Test concentrations with justification for top dose:

6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 microg per plate (Preliminary toxicity assay). 100, 333, 1000, 3333 and 5000 microg per plate (Initial and independent repeat mutagenicity assays)

Vehicle / solvent:

Sterile distilled water (CAS No. 7732-18-5); from Invitrogen Corporation

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Remarks:

All strains with S9 activation

Positive control substance:

other: 2-aminoanthracene (CAS No. 613-13-8)

Remarks:

1.0 microg/plate all Salmonella strains; 10 microg/plate WP2 uvrA

Controls 2

Positive controls:

yes

Remarks:

TA98 without S9 activation

Positive control substance:

2-nitrofluorene

Remarks:

1.0 microg/plate

Migrated to IUCLID6: (CAS No. 607-57-8)

Controls 3

Positive controls:

yes

Remarks:

TA100, TA1535 without S9 activation

Positive control substance:

sodium azide

Remarks:

1.0 microg/plate

Migrated to IUCLID6: (CAS No. 26628-22-8)

Controls 4

Positive controls:

yes

Remarks:

TA1537 Without S9 activation

Positive control substance:

9-aminoacridine

Remarks:

75 microg/plate

Migrated to IUCLID6: (CAS No. 90-45-9)

Controls 5

Positive controls:

yes

Remarks:

WP2 uvrA without S9 activation

Positive control substance:

methylmethanesulfonate

Remarks:

1,000 microg/plate

Migrated to IUCLID6: (CAS No. 66-27-3)

Details on test system and experimental conditions:

Preliminary Toxicity Assay: The preliminary toxicity assay was used to establish the dose range over which the test article would be assayed. Vehicle control and ten dose levels of the test article were plated, one plate per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor induced rat liver S9.
Mutagenicity Assay: The mutagenicity assay (initial and independent repeat assays), was used to evaluate the mutagenic potential of the test article. Five dose levels of test article along with vehicle control and appropriate positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA in the presence and absence of Aroclor induced rat liver S9. All dose levels of test article, vehicle control and positive controls were plated in triplicate.
Plating and Scoring Procedures: The test system was exposed to the test article via the plate incorporation method. On the day of its use, minimal top agar was melted and supplemented. Top agar, not used with S9 or Sham mix, was supplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, all references to water imply sterile, deionized water produced by the Milli Q Reagent Water System. Bottom agar was supplemented Vogel Bonner minimal medium E.
Each plate was labeled with a code system that identified the test article, test phase, dose level, tester strain and activation.
One half (0.5) milliliter of S9 or Sham mix, 100 microL of tester strain and 100 microL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45±2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 microL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2 8°C until colony counting could be conducted.
The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification.
Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the assay was the preliminary toxicity assay or the plate exhibited toxicity.

Evaluation criteria:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2.0-times the mean vehicle control value.

Results and discussion

Test results

Species / strain:

other: S.typhimurium TA98, TA100, TA1535 and TA1537 & E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the preliminary toxicity assay, the doses tested were 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 microg/plate. Neither precipitate nor appreciable toxicity was observed. Based on the preliminary toxicity findings the doses tested for the mutagenicity assay were 100, 333, 1000, 3333 and 5000 microg/plate.

In the Initial Mutagenicity Assay (Experiment B1), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

For the Independent Repeat Mutagenicity Assay, in the initial assay (Experiment B2), no positive mutagenic responses were observed with tester strains TA98 and TA1535 in the absence of S9 activation. Due to unacceptable positive control values, all tested strains in the presence of S9 activation and test strain TA100 in the absence of S9 activation were not evaluated but were retested in Experiment B3. Due to a dosing error in which the top two test article doses did not receive an aliquot of tester strain, tester strains TA1537 and WP2 uvrA in the absence of S9 activation were not evaluated but were also retested in Experiment B3.

In Experiment B3, no positive mutagenic responses were observed with any of the tester strains in the presence of S9 activation and with tester strains TA100, TA1537 and WP2 uvrA in the absence of S9 activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Initial Mutagenicity Assay

Any other information on results incl. tables

Initial Mutagenicity Assay Mean Number of Revertants Per Plate, Activation:  None

Dose (microg/plate)	TA98	TA100	TA1535	TA1537	WP2 uvrA
Vehicle (Water)	19 ± 3	163 ± 53	21 ± 6	9 ± 4	25 ± 1
100	14 ± 2	177 ± 71	18 ± 4	5 ± 2	25 ± 5
333	16 ± 1	137 ± 32	17 ± 5	6 ± 4	17 ± 1
1000	16 ± 4	192 ± 93	16 ± 1	4 ± 3	19 ± 5
3333	16 ± 3	112 ± 17	15 ± 5	4 ± 2	19 ± 5
5000	12 ± 1	136 ± 6	16 ± 5	5 ± 1	19 ± 2
Positive Control	90 ± 7	616 ± 8	267 ± 17	290 ± 33	96 ± 11

Initial Mutagenicity Assay Mean Number of Revertants Per Plate, Activation:  S9

Dose (microg/plate)	TA98	TA100	TA1535	TA1537	WP2 uvrA
Vehicle (Water)	19 ± 4	187 ± 45	14 ± 3	4 ± 1	18 ± 2
100	18 ± 3	156 ± 19	12 ± 3	6 ± 3	17 ± 1
333	14 ± 2	160 ± 4	11 ± 3	4 ± 2	21 ± 2
1000	17 ± 2	151 ± 27	12 ± 2	3 ± 2	17 ± 2
3333	14 ± 2	147 ± 16	10 ± 2	4 ± 0	18 ± 3
5000	22 ± 3	155 ± 12	10 ± 3	4 ± 1	16 ± 4
Positive Control	375 ± 62	654 ± 24	70 ± 8	78 ± 3	241 ± 6

Independent Repeat Mutagenicity Assay Mean Number of Revertants Per Plate, Activation:  None

Dose (microg/plate)	TA98*	TA100	TA1535*	TA1537	WP2 uvrA
Vehicle (Water)	22 ± 3	226 ± 10	15 ± 6	8 ± 3	23 ± 5
100	16 ± 1	197 ± 11	13 ± 2	5 ± 4	20 ± 5
333	14 ± 5	209 ± 26	14 ± 1	9 ± 3	21 ± 2
1000	17 ± 7	214 ± 16	12 ± 5	7 ± 1	20 ± 2
3333	16 ± 4	249 ± 26	17 ± 2	6 ± 3	20 ± 2
5000	13 ± 1	238 ± 10	18 ± 3	6 ± 3	20 ± 3
Positive Control	99 ± 8	734 ± 47	196 ± 34	459 ± 157	134 ± 12
*Data from Experiment B3

Independent Repeat Mutagenicity Assay Mean Number of Revertants Per Plate, Activation:  S9

Dose (microg/plate)	TA98	TA100	TA1535	TA1537	WP2 uvrA
Vehicle (Water)	25 ± 7	233 ± 4	17 ± 1	8 ± 2	21 ± 2
100	29 ± 10	242 ± 38	16 ± 5	10 ± 5	21 ± 5
333	29 ± 4	251 ± 14	17 ± 2	6 ± 2	18 ± 4
1000	29 ± 11	215 ± 17	16 ± 2	8 ± 1	19 ± 3
3333	34 ± 3	265 ± 13	16 ± 5	9 ± 3	16 ± 7
5000	28 ± 4	250 ± 28	16 ± 5	6 ± 0	19 ± 1
Positive Control	1378 ± 104	1283 ± 83	185 ± 25	215 ± 54	721 ± 129

Applicant's summary and conclusion

Conclusions:

The results of the Bacterial Reverse Mutation Assay with an Independent Repeat Assay indicate that, under the conditions of this study, the test material did not cause a positive response in either the presence or absence of Aroclor induced rat liver S9.