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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Macrolex Rot E2G (14H-benz[4,5]isoquino[2,1-a]perimidin-14-one, CAS 6829-22-7) was investigated in 2 Salmonella/microsome tests (Ames test) with 4 and 5 strains. Result: positive with mutagenic activation in S. typhimurium strains TA98, TA100, TA102, and TA1537.
Macrolex Rot E2G was evaluated as negative in a CHO/HGPRT test.

Link to relevant study records

Referenceopen allclose all

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
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
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 -/+
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)
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

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.

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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HGPRT locus on CHO cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Additional strain / cell type characteristics:
other: CHO cells
Metabolic activation:
with and without
Metabolic activation system:
S9-fraction of Sprague-Dawley rat liver induced with Aroclor 1254
Test concentrations with justification for top dose:
Cytotoxicity test:
non-activated: 0.38 - 150 µg/ml
activated: 23 - 200 µg/ml
CHO/HGPRT Mutagenesis Assay
non-activated: 50, 75, 90, 100, 125 µg/ml
activated: 25, 50, 75, 100, 125 µg/ml
Vehicle / solvent:
DMF
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
Preliminary cytotoxicity test, then dose selection for the mutagenicity test according to guideline.
Evaluation criteria:
An assay will be considered negative if none of the doses tested induced a reproducible mutants frequency which is considered significant.
Statistics:
POUSSON heterogenicity test.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: precipitation occurred when solution of 125 µg/ml test compound in DMF was added to Medium
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Precipitation occurred when solution of 125 µg/ml test compound in DMF was added to medium. Therefore, this concentration was chosen as the highest concentration in the mutagenicity test.
From the lack of any increases in mutant frequency, the test article is considered non-mutagenic in the CHO-HGPRT Forward Mutation Assay both with and without metabolic activation, according to the applied evaluation criteria.
Conclusions:
Interpretation of results: negative

Macrolex Red E2G is considered non-mutagenic in the CHO-HGPRT Forward Mutation Assay both with and without metabolic activation.
Executive summary:

Macrolex Red E2G dissolved in DMF was tested for point mutations in mammalian cell system according to OECD TG 476 by using CHO cells. As positive controls EMS and DMB for the non-activated and the activated assay, respectively, was chosen.

Precipitation occurred when solution of 125 µg/ml test compound in DMF was added to Medium. Therefore, this concentration was chosen as the highest concentration in the mutagenicity test with and without metabolic activation system. From the lack of any increases in mutant frequency, the Macrolex Red E2G is considered nonmutagenic in the CHO-HGPRT Forward Mutation Assay both with and without metabolic activation. The positive controls were functional.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In an in-vivo MNT, NMRI mice were used to examine the in vivo clastogenic activity of Macrolex Red E2G according to OECD TG 474. Based on a pilot study, a dose of 10000 mg Macrolex Rot E2G/kg bw, dissolved in aqueous cremophor emulsion, was chosen for intraperitoneal injection. Animals were sacrificed 16, 24, and 48 hours after the administration and the femoral marrow was prepared for evaluation.
The treated animals showed symptoms of toxicity including apathy, rough fur, staggering gait, spasm, eyelids stuck together and one animal died before the end of the test.
There was an altered ratio between polychromatic and normochromatic erythrocytes. No indications of a clastogenic effect of Macrolex Red E2G were found after a single intraperitoneal treatment with 10000 mg/kg bw Macrolex Red E2G. Therefore the test is negative.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 29 - 43 g
- Housing: females in groups of 3; males singly
- Diet ad libitum
- Water ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 43-50
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
intraperitoneal
Vehicle:
Aequeous cremophor emulsion
Details on exposure:
Each group comprised tne mice , 5 males and 5 females. each respective substance was administered once. Animals were sacrificed 16, 24, and 48 hours after the administration and the femoral marrow was prepared for evaluation.
Duration of treatment / exposure:
Once
Frequency of treatment:
Once
Post exposure period:
Up to 48 hours
Remarks:
Doses / Concentrations:
10000 mg/kg bw
Basis:

No. of animals per sex per dose:
10 (5 males and 5 females)
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
Tissues and cell types examined:
bone marrow from femur (erythroblasts)
Details of tissue and slide preparation:
bone marrow from femur (erythroblasts)
Evaluation criteria:
Coded slides were evaluated using a light microscope at a magnification about 1000. Micronucei appear as stainde chromatinparticles in the anucleated erythrocytes: 1000 polychromatic erythrocytes were counted per animal
A test is considered positive if, at any of the intervals, there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control.
Statistics:
Wilcoxon's non-parametric rank sum test, one-sided chi-square test
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
After single intraperitoneal administration of 10000 mg/kg bw Macrolex Red E2G treated animals showed the following compound-related symptoms until sacrifice: apathy, rough fur, staggering gait, spasm, eyelids stuck together. Their feeding behaviour was normal. One of the Macrolex Red E2G treated animal died during the test period. All intestinal organs of this animal were covered red and sticked together. The stomach was filled with a light-red liquid.
The ratio of polychromatic to normochromatic erythrocytes was altered by the treatment with Macrolex Red E2G in the 24 hours and in the 48 hours group.
There was no biologically important or statistically significant variations between the negative control and the groups treated intraperitoneally with 10000 mg/kg bw Macrolex E2G with respect to the incidence of micronucleated polychromatic erythrocytes.
Conclusions:
Interpretation of results: negative
There was no indication of a clastogenic effect of an intraperitoneal dose of 10000 mg/kg bw Macrolex Red E2G in the micronucleus test on the mouse.
Executive summary:

NMRI mice were used to examine the in vivo clastogenic activity of Macrolex Red E2G in a MNT according to OECD TG 474 and GLP. Based on a pilot study a dose of 10000 mg/kg bw dissolved in aequeous cremophor emulsion was chosen for intraperitoneal injection. Animals were sacrificed 16, 24, and 48 hours after the administration and the femoral marrow was prepared for evaluation.

The treated animals showed symptoms of toxicity including apathy, rough fur, staggering gait, spasm, eyelids stuck together and one animal died before the end of the test.

There was an altered ratio between polychromatic and normochromatic erythrocytes. No indications of a clastogenic effect of Macrolex Red E2G were found after a single intraperitoneal treatment with 10000 mg/kg bw Macrolex Red E2G.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In the Ames tests Macrolex Rot E2G (14H-benz[4,5]isoquino[2,1-a]perimidin-14-one, CAS 6829-22-7) was positive with metabolic activation. Macrolex Rot E2G was evaluated as negative in a CHO/HGPRT test.

No indications of a clastogenic effect of Macrolex Red E2G were found in erythrocytes after a single intraperitoneal treatment with 10000 mg/kg bw Macrolex Red E2G.

Due to the positive results of the Ames tests, the conduction of an in-vivo Comet assay is proposed to generate an overall result. At this point in time the data are inconclusive. Although the compound is not bioavailable (see chapter toxicokinetics) and consequently it is unlikely that the compound is systemically available the compound will be allocated to the “medium” hazard band until the in vivo Comet data become available.

Justification for classification or non-classification

Positive results on the available Ames tests and negative results were found in a CHO/HGPRT test as well as in an in-vivo MNT on red blood cells.

A justification for classification or non-classification will be done after the availability of the results on the proposed in-vivo Comet assay. At this point in time the data are inconclusive.