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Genetic toxicity: in vitro

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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
Study period:
July 9, 1993 - August 2, 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The test has been conducted in compliance with GLP and according to Hazleton protocol No. 401 (edition 17) comparable to OECD Guideline 471 with acceptable restrictions (no strain to detect cross mutagen was included). The test is well documented and scientifically acceptable.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(bacterial strain E. coli or TA102 not included in the study)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-methylenebis(N-sec-butylcyclohexamine)
EC Number:
679-514-8
Cas Number:
154279-60-4
Molecular formula:
C21H42N2
IUPAC Name:
4,4'-methylenebis(N-sec-butylcyclohexamine)
Constituent 2
Reference substance name:
4,4-METHYLENBIS(N-SEC BUTYLCYCLOHEXANAMINE)
IUPAC Name:
4,4-METHYLENBIS(N-SEC BUTYLCYCLOHEXANAMINE)
Details on test material:
- Name of test material (as cited in study report): XPA-143-93/ Unilink 4200H
- Physical state: Cloudy white semi-solid
- Storage condition of test material: Room temperature under nitrogen
- Date received: 07/08/93

Method

Species / strain
Species / strain / cell type:
S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538
Additional strain / cell type characteristics:
other: rfa wall mutation and deletion of uvrB gene; presence of R factor plasmid pKM101 in strains TA98 and TA100 to further increase the sensitivity of these strains to some mutagens
Metabolic activation:
with and without
Metabolic activation system:
- S9 homogenate (Liver microsomal enzymes) purchased from Molecular Toxicology, Inc., Annapolis (42.8 mg of protein/mL); homogenate prepared from male SD rats by injecting 500 mg/kg bw , i.p. of Aroclor 1254 (200 mg/mL in corn oil) (Ames et al, 1975)
Test concentrations with justification for top dose:
- Dose range finding study: 0 (vehicle 50 µL), 6.67, 10, 33.3, 66.7, 100, 333, 667, 1000, 3330 and 5000 µg/plate using tester strain TA100 with and without metabolic activation
- Main study: 100, 333, 667, 1000, 3330 and 5000 µg/plate
Vehicle / solvent:
- Solvent used: ethanol
- Justification for choice of solvent: The substance´s solubility is less than 32.3mg/ml to water and less than 50 mg/ml to DMSO. When compared the suspended conditions, distilled water was better.The 50 mg/ml suspension prepared with the distilled water showed no change in colour tone and exothermic reaction up to 2 hours after the preparation and was determined to be stable. Therefore the distilled water was selected as solvent.

The above explanation was given in the test report. However it is not entirely consistent. Due the poor solubility in water, and the great differences in molecular weight between water and the test substance, it is likely , that the suspension will not be stable, but two phases fill be formed within time. This would result uneven distribution of the test substance in the plate, if not used shortly after preparation.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
For strain TA98 and TA1538; without S9 mix

Migrated to IUCLID6: Concentration: 1 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
For strain TA100 and TA1535; without S9 mix

Migrated to IUCLID6: Concentration: 2 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (Concentration 2.5 µg/plate)
Remarks:
For strain TA98, TA100, TA1535, TA1537 and TA1538; with S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR-191 (Concentration: 2 µg/plate)
Remarks:
For strain TA1537; without S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation)

INCUBATION
- Temperature: 37 ± 2°C
- Duration: 48 ± 8 hrs

NUMBER OF REPLICATIONS: Three

DETERMINATION OF CYTOTOXICITY
- Method: Decrease in number of revertant colonies per plate and/or thinning or disappearance of the bacterial background lawn was considered as an indication of cytotoxicity.

Evaluation criteria:
CRITERIA FOR A VALID ASSAY:
-Tester strain integrity:
a. rfa wall mutation- to demonstrate the rfa wall mutation, tester strain cultures exhibited sensitivity to crystal violet
b. pKM101 Plasmid- to demonstrate the presence of R factor plasmid, pKM101, tester strain cultures of TA98 and TA100 exhibited resistance to ampicillin
c. Characteristic number of spontaneous revertants- to demonstrate the requirement for histidine, the tester strain cultures exhibited a characteristic number of spontaneous revertants per plate when plated along with the vehicle under selective conditions. The acceptable range for the vehicle control were 8-60 for TA98; 60-240 for TA100; 4-45 for TA1535; 2-25 for TA1537 and 3-35 for TA1538.
d. Tester strain culture density: to demonstrate that appropriate number of bacteria are plated, the density of tester strain cultures were greater than or equal to 0.5*10-9 bacteria/ mL and/or had reached a target level of turbidity demonstrated to produce cultures with a density greater than or equal to 0.5*10-9 bacteria/mL
e. Positive control values: To demonstrate the tester strains were capable of identifying a mutagen (with and without metabolic activation), the mean value of a positive control for a respective tester strain exhibited at least a 3-fold increase over the mean value of the vehicle control for that strain.

- Cytotoxicity:
A minimum of three non-toxic dose levels were required to evaluate assay data.

CRITERIA FOR A POSITIVE RESPONSE:
For a test article to be considered positive it had to produce at least a 2-fold (for tester strain TA98 and TA100) and 3-fold (for tester strain TA1535, TA1537, and TA1538) increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.
Statistics:
For all replicate platings, the mean revertants per plate and the standard deviations were calculated. The results of these calculations are presented in tabular form in attached background material named, "Data tables"

Results and discussion

Test results
Species / strain:
other: TA98, TA100, TA1535, TA1537 and TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING STUDY:
No cytotoxicity was observed in the dose range finding study up to test substance concentration of 5 mg/plate with and without metabolic activation.


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

XPA-143 -93/Unilink 4200H (trade name Clearlink 1000) was tested for its ability to induce point mutations in strains S. typhimurium TA1535, TA 1537, TA 1538, TA 98 and TA 100. All the strains were tested with and without metabolic activation with six doses from 100 up to 5000 µg/plate. The test method is comparable to OECD 471 with acceptable restrictions (no strain to detect cross linking properties was included) and the test was conducted in compliance with GLP.

Under the test conditions, test substance did not cause a positive increase in the number of histidine revertants per plate of any of the tester strain with or without metabolic activation and the result of the study was determined to be negative.

The study is classified as acceptable and satisfies the guideline requirements for bacterial reverse mutation test. However, this study does not detect the cross-linking mutagens.