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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Macrolex Rot E2G (CAS no 6829-22-7) was investigated for point mutagenic effects in the Salmonella/microsome test (plate incorporation test). The test item, dissolved in DMSO, was administered in doses of up to and including 5000 µg per plate without and with S9 mix on the five Salmonella typhimurium LT2 mutant strains TA1535, TA100, TA1537, TA98 and TA102, according to the OECD guideline 471.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Test Item: Macrolex Rot E2G
CAS No.: 6829-22-7
Molecular Formula: C22H12N20
Chemical Name: 14H-Benz[4,5]isoquino[2, 1-a]perimidin-14-one
CAS Name: 14H-Benz[4,5]isoquino[2, 1-a]perimidin-14-one
Molecular Mass (g/mol): 320.3
Purity: 98.9%
Appearance: red powder
Storage: room temperature

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
The original strains were obtained from Prof. Bruce Ames (Berkeley, CA, USA). TA1535 and TA100 bear the base-pair substitution, his G 46, and TA100 additionally contains the plasmid pKM 101. This R factor also contained in TA98 and TA102, codes for an ampicillin resistance and should raise the sensitivity of the strains. TA102 carries the ochre mutation his G 428 on the multicopy plasmid pAQl, which codes in addition for tetracycline resistance. TA1537 and TA98 bear frameshift markers. TA1537 exhibits the +1 mutant, his C 3076, while TA98 bears the +2 type, his D 3052.
Furthermore, the strains have other properties, which should increase their sensitivity. They are all deep rough, i.e. partly deficient in lipopolysaccharide side chains in their cell walls, enabling larger molecules to penetrate the bacterial cell wall and produce mutations. With the exception ofTA102, all strains have reduced capability to repair DNA-damage which increases the likelihood that such damage results in mutations.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Test Substances [µg per plate] S9 mix
Solvent Control 0 -/+
Test Item 50 -/+
160 -/+
500 -/+
1600 -/+
5000 -/+
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
mitomycin C
other: 4-N PDA ( 4-Nitro-o-phenylenediamine), 2-AA (Anthracene-2-amine)

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
other: Substance precipitation occurred at the dose of 5000 µg per plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Macrolex Rot E2G, further described as test item in this report, was investigated for point mutagenic effects in the Salmonella/ microsome test (plate incorporation test). The test item, dissolved in DMSO, was administered in doses of up to and including 5000 µg per plate without and with S9 mix on the five Salmonella typhimurium LT2 mutant strains TA1535, TAl00, TA1537, TA98 and TA102, according to the OECD guideline 471. No bacteriotoxic effects were observed. Substance precipitation occurred at the dose of 5000 µg per plate.

The employed positive controls had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding solvent controls.

No evidence of mutagenic effects were seen in Salmonella typhimurium TA1535 in the absence or presence of S9 mix. Evidence of mutagenic activity of the test item was seen for Salmonella typhimurium TAl00, TA1537 TA98 and TA102. A biologically relevant increase was found in the mutant count compared to the corresponding solvent control in the presence of S9 mix. Positive response started strain specifically at 50 µg/plate.

Therefore, the test item was considered to be mutagenic to Salmonella typhimurium TA100, TA1537 TA98 and TA102 in the presence of S9 mix in the plate incorporation of the Salmonella/microsome test.

Due to these clear positive effects in the plate incorporation test, an independent repeat using the preincubation modification was relinquished.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: positive with metabolic activation
Executive summary:

Macrolex Rot E2G (CAS no 6829-22-7) was investigated for point mutagenic effects in the Salmonella/microsome test (plate incorporation test). The test item, dissolved in DMSO, was administered in doses of up to and including 5000 µg per plate without and with S9 mix on the five Salmonella typhimurium LT2 mutant strains TA1535, TA100, TA1537, TA98 and TA102, according to the OECD guideline 471.

No bacteriotoxic effects were observed. Substance precipitation occurred at the dose of 5000 µg per plate.

The employed positive controls had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding solvent controls.

No evidence of mutagenic effects were seen in Salmonella typhimurium TA1535 in the absence or presence of S9 mix. Evidence of mutagenic activity of the test item was seen for Salmonella typhimurium TAl00, TA1537 TA98 and TA102. A biologically relevant increase was found in the mutant count compared to the corresponding solvent control in the presence of S9 mix. Positive response started strain specifically at 50 µg/plate.

Therefore, the test item was considered to be mutagenic to Salmonella typhimurium TA100, TA1537 TA98 and TA102 in the presence of S9 mix in the plate incorporation of the Salmonella/microsome test.