alkaline distillation residues from epsilon-caprolactam

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: Study conducted in accordance with OECD guideline 471 and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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

Test material information

Constituent 1

Reference substance name:

alkaline distillation residues from epsilon-caprolactam

EC Number:

938-820-4

Molecular formula:

Unspecified (UVCB substance)

IUPAC Name:

alkaline distillation residues from epsilon-caprolactam

Constituent 2

Reference substance name:

Reaction mass of caprolactam, amino caproic acid and oligomers

IUPAC Name:

Reaction mass of caprolactam, amino caproic acid and oligomers

Constituent 3

Reference substance name:

Reaction mass of caprolactam, amino acproic acid and oligomers

IUPAC Name:

Reaction mass of caprolactam, amino acproic acid and oligomers

Constituent 4

Reference substance name:

Lactamoil 70%

IUPAC Name:

Lactamoil 70%

Details on test material:

Composition:
ca. 43.82 % w/w) ε-caprolactam / azepan-2-one /2H-Azepin-2-one, hexahydro- /105-60-2
ca. 13.18 % (w/w) sodium hydroxide / sodium hydroxide /Sodium hydroxide (Na(OH)) / 1310-73-2
ca. 1.1 % (w/w) 1,8-Diazacyclotetradecane-2,9-dione / 1,8-Diazacyclotetradecane-2,9-dione / 56403-09-9
ca. 0.33 % (w/w) 1,8,15-Triazacycloheneicosane-2,9,16-trione / 1,8,15-Triazacycloheneicosane-2,9,16-trione / 56403-08-8
ca. 0.12 % (w/w) 1,8,15,22-Tetraazacyclooctacosane-2,9,16,23-tetrone / 1,8,15,22-Tetraazacyclooctacosane-2,9,16,23-tetrone / 5834-63-9
ca. 9.36 % (w/w) aminocaproic acid / 6-aminohexanoid acid / 60-32-2
ca. 9.57 % (w/w) Hexanoic acid, 6-[(6-amino-1-oxohexyl)amino]- / Hexanoic acid, 6-[(6-amino-1-oxohexyl)amino]- / 2014-58-6
ca. 7.12 % (w/w) Hexanoic acid, 6-[[6-[(6-amino-1-oxohexyl)amino]-1-oxohexyl]amino]- / Hexanoic acid, 6-[[6-[(6-amino-1-oxohexyl)amino]-1-oxohexyl]amino]- / 5776-78-3
ca. 4.38 % (w/w) Hexanoic acid, 6-[[6-[[6-[(6-amino-1-oxohexyl)amino]-1-oxohexyl]amino]-1-oxohexyl]amino]- / Hexanoic acid, 6-[[6-[[6-[(6-amino-1-oxohexyl)amino]-1-oxohexyl]amino]-1-oxohexyl]amino]- / 5834-64-0
ca. 2.53 % (w/w) Hexanoic acid, 6-[[6-[[6-[[6-[(6-amino-1-oxohexyl)amino]-1-oxohexyl]amino]-1-oxohexyl]amino]-1-oxohexyl]amino]- / Hexanoic acid, 6-[[6-[[6-[[6-[(6-amino-1-oxohexyl)amino]-1-oxohexyl]amino]-1-oxohexyl]amino]-1-oxohexyl]amino]- / 16605-19-9
ca. 1.41 % (w/w) Hexanoic acid, 6-[(34-amino-1,8,15,22,29-pentaoxo-7,14,21,28-tetraazatetratriacont-1-yl)amino]- / Hexanoic acid, 6-[(34-amino-1,8,15,22,29-pentaoxo-7,14,21,28-tetraazatetratriacont-1-yl)amino]- / 10259-02-6
ca. 0.82 % (w/w) Hexanoic acid, 6-[(41-amino-1,8,15,22,29,36-hexaoxo-7,14,21,28,35-pentaazahentetracont-1-yl)amino]- / Hexanoic acid, 6-[(41-amino-1,8,15,22,29,36-hexaoxo-7,14,21,28,35-pentaazahentetracont-1-yl)amino]- / 16580-03-3
ca. 626 % (w/w) non-specified constituents / non-specified constituents

Method

Target gene:

His-, Trp-

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 liver fraction from male Wistar rats, induced by 80 mg/kg b.w. phenobarbital (i.p.) and beta-naphthoflavone (oral) on three consecutive days. Cofactors in S9 mix: MgCl2 (8 mM), KCl (33 mM), G6P (5mM), NADP (4 mM), phosphate buffer (15 mM, pH 7.4).

Test concentrations with justification for top dose:

0, 33, 100, 333, 1000, 3333 and 10000 ug/plate. Due to missing information concerning purity of the test substance, 10 mg/plate was chosen as the top dose, instead of 5 mg/plate as recommended by the guideline.

Vehicle / solvent:

Ethanol (suitable for bacterial reverse mutation test, historical control data available)

Controls

Untreated negative controls:

other: Sterility control: Soft agar plates with S9, buffer, vehicle/test substance but without tester strains.

Negative solvent / vehicle controls:

yes

Remarks:

With and without S9 mix, contained vehicle at same concentration and volume for all tester strains.

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene, N-methyl-N'-nitro-N-nitrosoguianidine), 4-nitro-o-phenylenediamine

Remarks:

2-aminoanthracene: E.coli WP2 uvrA + S9, TA 1535/100/1537/98 + S9; N-methyl-N'-nitro-N-nitrosoguianidine: TA 1535/TA100 - S9; 4-nitro-o-phenylenediamine: TA98 - S9; 9-aminoacridine: TA 1537 - S9; 4-nitroquinoline-N-oxide: E.coli Wp2 uvrA - S9.

Details on test system and experimental conditions:

STANDARD PLATE TEST
Test substance, bacterial culture (Salmonella typhimurium or E. coli) and, if applicable, S9 mix, were added to soft agar heated up to 42-45 °C. Samples were mixed and immediately poured onto minimal glucose (Salmonella typhimurium) or minimal (E. coli) agar plates. After incubation at 37 °C for 48-72 hours in the dark, his+ (Salmonella typhimurium) or trp+ (E. coli) revertants were counted.

PREINCUBATION TEST
Test substance, bacterial suspension and, if applicable, S9 mix were incubated on a shaker at 37 °C for 20 minutes. Subsequently, soft agar was added. Samples were mixed and immediately poured onto agar plates. After incubation at 37 °C for 48-72 hours in the dark, bacterial colonies were counted.

REPLICATES
3 test plates per dose or per control

Evaluation criteria:

Positive: Dose-related and reproducible increase in number of revertant colonies, i.e. about
doubling of spontaneous mutation rate in at least one tester strain either without
S9 mix or after adding a metabolizing system.
Negative: Number of revertants for all tester strains within historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Statistics:

3 plates were tested per dose or per control.

Results and discussion

Test results

Test results 1

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

Remarks:

Weak bacteriotoxic effect at 10 000 ug/plate (standard plate test), bacteriotoxicity from about 3 333 ug/plate onward (preincubation assay).

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Bacteriotoxicity from about 3 333 ug/plate onward (preincubation assay). Weak bacteriotoxic effect at 10000 ug/plate (standard plate test).

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Lactamoil 70 % was found to be non-mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Executive summary:

Lactamoil 70% was tested for mutagenicity in the bacterial reverse mutation test according to OECD guideline 471 using the following strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. The study was conducted as two independent experiments: standard plate test and preincubation assay, both in the presence and absence of S9 mix. In both experiments, dose ranges of 33 -10 000 ug/plate were used. The test substance did not lead to a relevant increase in the number of revertant colonies in any of the experiments, whereas the positive control substances caused a significant increase in the number of revertant colonies within the range of historical data or above. Thus, Lactamoil 70% is considered to be non-mutagenic in the bacterial reverse mutation assay.