Enbucrilate

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 and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

LID-1187A (n-butylcyanoacrylate)

IUPAC Name:

LID-1187A (n-butylcyanoacrylate)

Constituent 2

Reference substance name:

Enbucrilate

EC Number:

229-552-2

EC Name:

Enbucrilate

Cas Number:

6606-65-1

Molecular formula:

C8H11NO2

IUPAC Name:

butyl 2-cyanoprop-2-enoate

Test material form:

other: solution

Details on test material:

- LID-1187A (n-butylcyanoacrylate, CAS 6606-65-1, 97% a.i., ref.#1577-19), a pale yellow liquid, contained in white plastic bottles, was received on 17 September 1990. The bottles were individually packed in sterile sachets, also containing tubing and applicator nozzles. It was stored at approximately 4°C (except for a 14-day period at ambient temperature between 24 September and 8 October) and protected from light until required.

- Histoacryl (n-butylcyanoacrylate, CAS 6606-65-1, lot 2/9484), a blue liquid, contained in plastic ampoules within plastic tubes, was received on 15 March 1990. It was stored at approximately 4°C and protected from light until required.

- Solutions, which were freshly prepared immediately before use, were made in acetone. The LID-1187A was passed through the plastic tubing and nozzle provided by means of a peristaltic pump before the preparation of an acetone solution, and both materials were tested within one hour of exposure to air. All concentrations cited in this report refer to the samples as received. No determinations of stability, homogeneity or achieved concentration were undertaken on the materials solutions. Temperature and relative humidity levels were measured and recorded, both in the laboratory and within the safety cabinet in which all the tests were conducted, during all the tests.

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: Histidine auxotroph

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

2.5, 5, 25, 50, 250, 500, 2500 and 5000 µg/plate (pre-experiment)
25, 70, 250, 790, 2500 µg/plate (main experiment)

Vehicle / solvent:

aceton

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

TEST ORGANISMS:
Cultures of the histidine-dependent strains of Salmonella typhimurium were derived from cultures provided by Prof. Bruce Ames, University of California. The characteristics of the individual strains are as follows:
- TA 1535 - contains a histidine missense mutation but is also deficient in a DNA repair system (uvr B) and has a defective lipopolysaccharide coat on the cell wall. It is reverted by many agents causing base-pair substitutions, but is not sensitive to frameshift mutagens.
- TA 100 - is the same as TA 1535 but contains a resistance transfer factor conferring ampicillin resistance and increasing sensitivity to same mutagens (plasmid pKM 101). In addition to base-pair substitutions, it is also able to detect certain frameshift mutagens.
- TA 1537 - bears a histidine frameshift mutation. Like TA 1535, it is defective in a DNA repair system and lipopolysaccharide coat. It is sensitive to agents causing frameshift mutations involving insertion or deletion of a single base-pair.
- TA 98 - contains another histidine frameshift mutation. Again it has a defective DNA repair system and lipopolysaccharide coat but also contains the pKM 101 plasmid. It is reverted by agents causing deletion of two adjacent base-pairs (double frameshift mutations), but not by simple alkylating agents causing base-pair substitutions.
Cultures of all organisms were prepared by overnight incubation of nutrient broth (Oxoid No.2) freshly inoculated from a frozen culture stock.

HUMIDITY AND TEMPERATURE CONDITIONS: Temperature and humidity levels were recorded both in the laboratory (22°C / 46-64% R.H.) and within the safety cabinet (25-27°C / 32-50% R.H.).

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

RESULTS

 

PRELIMINARY TOXICITY TESTS

LID-1187A and Histoacryl were tested in concentrations of 2.5, 5, 25, 50, 250, 500, 2500 and 5000 µg/plate. For both test materials, the lowest level to cause visible thinning of the background lawn of non-revertant cells was 2500 µg per plate. This was therefore selected as the top exposure level for use in the main assays.

 

STERILITY CHECKS, SPONTANEOUS REVERSION RATE AND VIABILITY CHECKS

The absence of colonies on test material and S-9 mix sterility check plates indicates that these preparations were free of significant microbial contamination. The total colony counts on plates containing the 10^-6dilution of bacterial culture only confirmed the viability and high cell density of the cultures of the individual organisms. The counts recorded on appropriate negative control plates confirmed the characteristically low spontaneous reversion rates of the tester strains and the absence of effects on these rates of acetone inclusion.

 

MUTAGENIC ACTIVITY OF POSITIVE CONTROL CHEMICALS

Appropriate positive control chemicals (with S-9 mix where required) induced marked increases in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S-9 mix.

 

ACTION OF LID-1187A AND HISTOACRYL

No increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to either test material at levels from 25 to 2500 µgper plate.

Inhibition of growth, observed as thinning of the background lawn of non-revertant cells and reduction in revertant colony numbers, occurred in all strains following exposure to both test materials at 2500µgper plate.

 

MEAN COLONY COUNTS FOR LID-1187A

 

No.	Addition (µg)	S-9 mix	TA98 1.	TA98 2.	TA100 1.	TA100 2.	TA1535 1.	TA1535 2.	TA1537 1.	TA1537 2.
1	None (sterility check)	+	0	0	0	0	0	0	0	0
2	LID-1187A (2500), sterility check	-	0	0	0	0	0	0	0	0
3	2500	+	7	16	57	72	5	6	2	6
4	790	+	26	26	106	98	10	8	5	8
5	250	+	32	31	115	105	9	9	6	9
6	79	+	30	30	118	114	11	10	7	8
7	25	+	33	32	116	108	14	14	7	6
8	Acetone (0.1 mL)	+	31	31	117	107	15	13	7	7
9	2500	-	14	10	46	60	6	4	1	4
10	790	-	22	24	100	98	11	8	4	4
11	250	-	28	29	106	109	10	9	5	6
12	79	-	28	30	108	111	11	12	5	5
13	25	-	31	27	112	110	13	12	5	5
14	Acetone (0.1 mL)	-	32	30	109	107	16	13	5	6
18	None (dilution of bact. culture)	-	141	125	145	136	127	120	139	123

 

MEAN COLONY COUNTS FOR HISTOACRYL

 

No.	Addition (µg)	S-9 mix	TA98 1.	TA98 2.	TA100 1.	TA100 2.	TA1535 1.	TA1535 2.	TA1537 1.	TA1537 2.
1	None (sterility check)	+	0	0	0	0	0	0	0	0
2	Histoacryl (2500), sterility check	-	0	0	0	0	0	0	0	0
3	2500	+	10	12	69	55	5	5	2	5
4	790	+	18	24	95	87	12	10	4	7
5	250	+	29	24	105	94	11	8	6	8
6	79	+	29	29	114	104	13	10	6	9
7	25	+	31	30	114	109	8	11	5	7
8	Acetone (0.1 mL)	+	31	31	117	107	15	13	7	7
9	2500	-	5	6	32	62	5	7	0	0
10	790	-	22	23	95	105	10	7	5	3
11	250	-	27	25	105	105	11	10	4	5
12	79	-	29	27	105	104	11	12	5	4
13	25	-	32	25	110	107	12	13	4	6
14	Acetone (0.1 mL)	-	32	30	109	107	16	13	5	6
18	None (dilution of bact. culture)	-	141	125	145	136	127	120	139	123

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test materials, LID-1187A and Histoacryl, were devoid of mutagenic activity under the conditions of the test.

Executive summary:

SUMMARY

LID-1187A and Histoacryl were examined for mutagenic activity in four histidine-dependent auxotrophs of Salmonella typhimurium, strains TA 98, TA 100, TA 1535 and TA 1537, using pour-plate assays. The procedures used complied with OECD Guideline for Testing of Chemicals No. 471 (issued 1983) and EPA Toxic Substances Control Act Test Guideline § 798.5265 (first issued 1985, amended 1987). Each test, in each strain, was conducted on two separate occasions.

The studies, which were conducted in the absence and presence of an activating system derived from rat liver (S-9 mix), employed a range of levels of both test materials from 25 to 2500 µg per plate, selected following a preliminary toxicity test in strain TA 98. All tests included solvent (acetone) controls with and without S-9 mix.

No increases in reversion to prototrophy were obtained with any of the four bacterial strains following exposure to either LID-1187A or Histoacryl at the levels tested, either in the presence or absence of S-9 mix. Inhibition of growth, observed as thinning of the background lawn of non-revertant cells and reduction in revertant colony numbers, occurred in all strains following exposure to both test materials at 2500 µg per plate.

Marked increases in the number of revertant colonies were induced by the known mutagens benzo[a]pyrene, 2-nitrofluorene, 2-aminoanthracene, 9-aminoacridine and sodium azide when examined under similar conditions.

It was concluded that both LID-1187A and Histoacryl were devoid of mutagenic activity under the conditions of the test.