14H-benz[4,5]isoquino[2,1-a]perimidin-14-one

Endpoint summary

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

Reference

Reference 1

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

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.

Reference 2

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

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.

In an in vivo Comet assay, no effects were noted up to 2000 mg/kg in the stomach, liver and duodenum.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

in vivo Comet asay

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2022-05-20 - 2022-12-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD 489

Qualifier:

according to guideline

Guideline:

other: EU method B.62

Version / remarks:

Commission Regulation (EU) 2017/735 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No. 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH): B62 In vivo Mammalian Alkaline Comet Assay

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

9th Addendum to the OECD Guideline for Testing of Chemicals, Section 4, No. 489, adopted July 29, 2016 „In vivo Mammalian Alkaline Comet Assay“.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Recommended test system

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 6 - 10 weeks (start of treatment)
- Weight at study initiation: 208.6 - 239.7 g
- Assigned to test groups randomly: yes, based on body weight differences
- Fasting period before study: not stated
- Housing: group
Cage Type: Makrolon Type IV, with wire mesh top
Bedding: granulated soft wood bedding
- Diet (e.g. ad libitum): 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): approx. 45-65%
- Air changes (per hr): approximately fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Carboxy Methyl Cellulose, 0.5% in water
- Justification for choice of solvent/vehicle: Suitability was proven in previous studies. The vehicle was chosen due to its relative non-toxicity for the animals and ability to formulate a suitable dosing preparation (suspension). All animals were treated twice orally (24 h and 4 h prior to preparation).
- Concentration of test material in vehicle: In this study the doses 500, 1000 and 2000 mg/kg b.w. were applied based upon the pre-experimental results. The doses were chosen in accordance with the Sponsor. The administered volume was 10 mL/kg b.w.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in 0.5% aqueous carboxy methyl cellulose.
The test item was weighed into an agate mortar on a tared balance. Grinding of the test substance in the agate mortar with an agate pestle, whilst gradually adding a part of the vehicle, was used to formulate the substance, in order for achieve a homogeneous formulation. When appearing homogeneous, the respective formulation was entirely transferred into a beaker by use of a pipette, and carboxy methyl cellulose was added up to the calibration mark.
The formulations were constantly stirred during application using a magnetic stirrer.

Duration of treatment / exposure:

The animals received the test item or the vehicle control twice orally at 24 and 4 hours prior to sacrifice.

Frequency of treatment:

The animals were treated twice orally (24 h and 4 h before sacrifice) with the test item.

Post exposure period:

4h

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

methylmethanesulfonate (MMS)
- Route of administration: orally
- Doses / concentrations: Purity: 99%, dissolved in 0.9% NaCl solution, 25 mg/kg b.w.

Tissues and cell types examined:

liver, small intestine and stomach

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly, or 2000 mg/kg b.w. as the upper limit for test items which induce no substantial signs of toxicity.
The maximum tolerated dose level is determined to be the dose that induced slight toxic effects relative to the duration of the study period (i.e., clear clinical signs such as abnormal behaviour or reactions, minor body weight depression or target tissue cytotoxicity), but no death or evidence of pain, suffering or distress necessitating euthanasia.
In this study the doses 500, 1000 and 2000 mg/kg b.w. were applied based upon the pre-experimental results.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): At the beginning of each treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the body weight of the animals. The animals received the test item or the vehicle control twice orally at 24 and 4 hours prior to sacrifice. The positive control substance was administered once orally at 4 h prior to sacrifice. Six males were treated per dose group. The animals of all dose groups were examined for acute toxic symptoms at intervals of 0-1 h, 2-4 h, 5-6 h and 20 h after the first administration of the test item or vehicle and 0-1 h, 2 h and 4 h after the second administration of the test item or vehicle. The positive control group received the positive control substance (MMS) once only (4 h prior to preparation).
Four hours after the second administration of the test item or vehicle control and four hours after the administration of the positive control substance (MMS), the animals were sacrificed using CO2 followed by exsanguination (decapitation).

DETAILS OF SLIDE PREPARATION:
Isolation of Cells from the Liver
A piece of the liver (about 1 cm diameter) was cut off and rinsed several times in ice-cold mincing buffer in order to remove adjacent blood and then minced in 1 mL ice-cold mincing buffer using forceps. The resulting cell suspension was filtered through a 70 µm cell strainer. An adequate volume of the cell suspension was centrifuged at about 10000 g for 1 min and the resulting pellet suspended in prewarmed 0.7 % agarose. A cell preparation would have been rejected for further processing if it appeared not suitable for further use upon visual inspection (e.g., discoloured, claggy, etc.).
 
Isolation of Cells from the Stomach
The stomach was removed and cut open. The main part of the glandular stomach was washed several times in ice-cold mincing buffer to remove residual food. The glandular stomach was stretched and fixed with needles on a pre-cooled paraffin filled dish, rinsed several times with ice-cold mincing buffer and subsequently kept on ice. The mucosa was scraped with a cell scraper and discarded. After rinsing the glandular stomach with approximately 5 mL ice-cold mincing buffer, the glandular stomach was covered with additional approximately 400 µL and was then scraped with a cell scraper. The cells on the cell scraper were discarded. The cells on the surface were removed via a pipette and filtered through a 70 µm cell strainer. In addition, the removed cells on the surface of the glandular stomach were resuspended in approximately 400 µL ice-cold mincing buffer and filtered through a 70 µm cell strainer. Approximately 45 µL of the filtered cell suspension was suspended in 405 µL prewarmed
0.7 % agarose. A cell preparation would have been rejected for further processing if it appeared not suitable for further use upon visual inspection (e.g., discoloured, claggy, etc.).
Isolation of Cells from the Small Intestine
A piece of the small intestine (duodenum/jejunum, approx. 5 cm distal from the pylorus) was cut off and rinsed several times in cold HBSS, excess fat was removed and a piece of about
1 cm length was minced in 1 mL ice-cold mincing buffer using fine scissors. The resulting cell suspension was filtered through a 70 µm cell strainer. An adequate volume of the cell suspension was centrifuged at about 10000 g for 1 min and the resulting pellet suspended in prewarmed 0.7 % agarose. A cell preparation would have been rejected for further processing if it appeared not suitable for further use upon visual inspection (e.g., discoloured, claggy, etc.).

Preparation of Microscopic Slides
For the Comet Assay, four slides per tissue and per animal were prepared with 10 % cell suspension and 90 % of a 0.7 % (w/v) agarose (low melting point agarose) solution. 100 µL of this mixture were applied per slide. The slides were cooled before being submerged in lysis buffer.
The following steps of protocol were performed with the slides:
Lysis 1h up to 2 days in Lysis buffer pH 10
Alkaline treatment 20 min in electrophoresis buffer
Electrophoresis 30 min in electrophoresis buffer
300 mA, 25 V, performed in a cool and dark environment (2 – 8°C)
(Fisherbrand Horizontal Electrophoresis Systems Large Gel;
20 x 25cm gel; Fisher Scientific)
Neutralisation about 10 min in neutralisation buffer
Dehydration approx. 2 min in 99 % ethanol
After dehydration the slides were air-dried and stored protected from dust and light until evaluation.
3.6 Analysis of Cells
The DNA of the cells was stained with the fluorescence dye ethidium bromide (20 µg/mL; 40 µL per slide), immediately before evaluation.
All animals per test group, 150 cells per animal, 50 cells per slide, were evaluated on coded slides with a fluorescence microscope using a 20 x objective. The damage of each nucleus was measured and recorded by a validated image analysis program (Comet Assay IV, Perceptive Instruments, ICCR-Roßdorf validation number: DE0084COMET2). One slide per animal was kept as a reserve for possible re-evaluation.
An increasing extent of DNA migration detected with the Comet assay results in an increase of the median of tail % intensity of one test group compared to the vehicle control. Tail % intensity is expressed as a percentage of the Comet’s total intensity. Additionally, the number of nuclei from apoptotic or necrotic cells (‘hedgehogs’) per 1500 total nuclei, 500 nuclei per slide, was determined.

METHOD OF ANALYSIS: The following criteria were used for analysis of slides:
• Only clearly defined non-overlapping cells were scored
• Nuclei from dead/ apoptotic cells were not scored
• Cells with unusual staining artefacts were not scored
• All other normal cells, 150/animal, were scored where possible

OTHER:

Evaluation criteria:

The study is considered valid as the following criteria are met:
- At least five animals per group are available for analysis
- The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database
- The concurrent positive control produces a statistically significant increase compared with the concurrent negative control and induces a response compatible with those generated in the historical control database
- Adequate numbers of cells and doses have been analysed
- The criteria for the selection of the highest dose are consistent with those described in chapter 3.5.2 (maximum tolerated dose).
3.8 Interpretation of Results
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if:
a) At least one of the test doses exhibits a statistically significant increase compared with the concurrent negative control,
b) The increase is dose-related when evaluated with an appropriate trend test
c) Any of the results are outside the distribution of the historical negative control data
When all of these criteria are met, the test chemical is then considered to be able to induce DNA strand breakage in the tissues studied in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if:
a) None of these test item concentrations exhibits a statistically significant increase compared to vehicle control values
b) there is no concentration-related increase when evaluated with an appropriate trend test
c) All results are inside the distribution of the historical negative control data
d) direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) has been demonstrated

Statistics:

According to the recommendations in the OECD 489 Test Guideline, a possible dose response should be evaluated by an appropriate trend test. No statistical method, however, has been singled out as the method of choice, although a number of reports on various statistical methods to evaluate such data have been published.
The use of R is recommended by the FDA (‘Regulatory Compliance and Validation Issues – A Guidance Document for the Use of R in Regulated Clinical Trial Environments’, The R Foundation for Statistical Computing, 2013). The in-house developed and validated R-script LM_V02.Rnw (Pearson’s r, Multiple R-squared, Adjusted R-squared) is used for assessment of possible dose-response relationships. This script provides a standard Linear Model for the calculation and prediction of dose-dependent trend in mutagenicity studies. It performs the following functions: Linear Regression and Trend Analysis.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

Clinical signs were mild and unspecific and included piloerection and increased secretion of Harderian glands.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

See attachments

Conclusions:

Discussion
This study was performed to investigate the potential of the test item to induce mutagenic / genotoxic effects in the rat by the assessment of single DNA strand breaks in cells isolated from the liver, the stomach and the small intestine. DNA strand breaks were analysed using the alkaline single cell gel electrophoresis (Comet) assay.
The test item was formulated in 0.5% aqueous carboxy methyl cellulose which was also used as negative control. The animals received the test item, the vehicle or positive control at a constant dose volume of 10 mL/kg b.w. by oral administration. The test item was administered twice to six males per test group at intervals of 24 h and 4 h prior to preparation. After the treatment period liver cells, stomach cells and cells of the small intestine were isolated for the assessment of single DNA strand breaks.
The test item was investigated at 500, 1000 and 2000 mg/kg b.w.. The highest dose (maximum tolerated dose, MTD, Guideline-recommended maximum dose level) was estimated by a pre-experiment to be suitable.
Clinical signs were very mild, unspecific and transient included piloerection and increased secretion of Harderian glands.
Liver
As a cytotoxic parameter the dead cell index (dead cells (apoptotic/necrotic cells) per 1500 nuclei per animal) was determined in the Comet assay and did not show any increased cytotoxic effects in the liver cells in comparison to the vehicle control.
For the analysis of the single DNA strand breaks 150 cells per animal (3 slides for each animal, 50 cells evaluated per slide) were evaluated. The relevant parameter for DNA damage is the percentage of DNA in the Comet tail measured as intensity relative to the intensity of the nucleus (% Median Tail intensity).
The Comet assay on cells of the liver did not reveal any statistically significant and biologically relevant increase in DNA damage at any of the tested dose levels compared to the corresponding vehicle controls on the evaluated parameter (% Median Tail Intensity). Additionally, a dose-dependent increase was not observed which was confirmed by linear regression analysis. Furthermore, the values were in the range of the historical vehicle control data.
Stomach
As a parameter for cytotoxicity the dead cell index (dead cells (apoptotic/necrotic cells) per 1500 nuclei per animal) was determined in the Comet assay. and did not show any increased cytotoxic effects in the stomach cells in comparison to the vehicle control.
For the analysis of the single DNA strand breaks 150 cells per animal (3 slides prepared for each animal, 50 cells evaluated per slide) were evaluated. The relevant parameter for DNA damage is the percentage of DNA in the Comet tail measured as intensity relative to the intensity of the nucleus (% Median Tail intensity).
The Comet assay on cells of the stomach did not reveal any statistically significant and biologically relevant increase in DNA damage at any of the tested dose levels compared to the corresponding vehicle controls on the evaluated parameter (% Median Tail Intensity). Additionally, a dose-dependent increase was not observed, which was confirmed by linear regression analysis. Furthermore, the values were in the range of the historical vehicle control data.
Small Intestine
As a cytotoxic parameter the dead cell index (dead cells (apoptotic/necrotic cells) per 1500 nuclei per animal) was determined in the Comet assay and did not show any increased cytotoxic effects in the cells of the small intestines in comparison to the vehicle control.
For the analysis of the single DNA strand breaks 150 cells per animal (3 slides for each animal, 50 cells evaluated per slide) were evaluated. The relevant parameter for DNA damage is the percentage of DNA in the Comet tail measured as intensity relative to the intensity of the nucleus (% Median Tail intensity).
The Comet assay on cells of the small intestinbes did not reveal any statistically significant and biologically relevant increase in DNA damage at any of the tested dose levels compared to the corresponding vehicle controls on the evaluated parameter (% Median Tail Intensity). Additionally, a dose-dependent increase was not observed which was confirmed by linear regression analysis. Furthermore, the values were in the range of the historical vehicle control data.
For all three tissues, the vehicle controls were in the range to ensure a valid performance of the study. The reference mutagen (MMS, 25 mg/kg b.w. oral) showed a distinct and statistically significant increase of DNA damage as detected by % Median Tail intensity analysis, corroborating validity of results.

Conclusion
In conclusion, it can be stated that under the experimental conditions reported, double oral administration of 500, 1000 and 2000 mg/kg b.w., respectively, of the test item did not induce any biologically relevant DNA damage in the in vivo Comet assay.
With regard to the results described, the test item is considered to be non-genotoxic for the stomach, small intestine and liver.

Executive summary:

This study was performed to investigate the potential of 14H-benz[4,5]isoquino[2,1-a]perimidin-14-one to induce mutagenic / genotoxic effects in the rat by the assessment of single DNA strand breaks in cells isolated from the liver, the small intestine and the stomach. DNA strand breaks were analysed using the alkaline single cell gel electrophoresis (Comet) assay according to OECD 489 under GLP.

The test item was formulated in 0.5% aqueous carboxy methyl cellulose which was also used as negative control. The animals received the test item, the vehicle or positive controls at a constant dose volume of 10 mL/kg b.w. by oral administration. The test item was administered twice to six males per test group at intervals of 24 h and 4 h prior to preparation. After the treatment period liver cells, stomach cells and cells of the small intestine were isolated for the assessment of single DNA strand breaks.

The test item was investigated at 500, 1000 and 2000 mg/kg b.w.. The highest dose (maximum tolerated dose, MTD) was estimated by a pre-experiment to be suitable.

Clinical signs were mild and unspecific and included piloerection and increased secretion of Harderian glands.

As a cytotoxic parameter the dead cell index (dead cells (apoptotic/necrotic cells) per 1500 nuclei per animal) was determined for each tissue and animal. No increased cytotoxic effects were observed in test item treated tissues in comparison to the corresponding vehicle control.

For the analysis of the single DNA strand breaks 150 cells per animal (3 slides for each animal, 50 cells evaluated per slide) were evaluated for each tissue. The relevant parameter for DNA damage is the percentage of DNA in the Comet tail measured as intensity relative to the intensity of the nucleus (% Median Tail intensity).

No statistically significant and biologically relevant increase in DNA damage was noted in any tissue at any of the tested dose levels compared to the corresponding vehicle controls on the evaluated parameter (% Median Tail Intensity). Additionally, a dose-dependent increase was not observed. Furthermore, all values were in the range of the historical vehicle control data.

For all three tissues, the vehicle controls were in the range to ensure a valid performance of the study. The reference mutagen (MMS, 25 mg/kg b.w. oral) showed a distinct and statistically significant increase of DNA damage in each tissue as detected by % Median Tail intensity analysis, corroborating validity of results.

Conclusion

In conclusion, it can be stated that under the experimental conditions reported, double oral administration of 500, 1000 and 2000 mg/kg b.w., respectively, of the test item did not induce any biologically relevant DNA damage in the in vivo Comet assay.

With regard to the results described, the test item 14H-benz[4,5]isoquino[2,1-a]perimidin-14-one is considered to be non-genotoxic for the stomach, small intestine and liver.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

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 or test system 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 is done after the availability of the results on the proposed in-vivo Comet assay. In consequence, the substance does not nee to be classified as genotoxic.