3-methyl-6-(p-toluidino)-3H-dibenz[f,ij]isoquinoline-2,7-dione

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are two bacterial reverse mutation test wirth different results available:

1. Test:

MACROLEX Rot 5B was screened with one plate per dose using the Salmonella/microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 µg per plate on five Salmonella typhimurium LT2 mutants. These com­ prised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102. Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. Substance precipitation occurred at the dose 1581 µg per plate and above. Evidence of mutagenic activity of MACROLEX Rot 5B was seen. On Salmonella typhimurium TA 1537, TA 98 and TA 102, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Except for TA 1537 positive response was found only with S9 mix. The lowest effective dose was 16 µg per plate for Salmonella ty­ phimurium TA 1537 and 50 µg per plate for TA 98 and TA 102. The Salmonella/microsome test thus showed MACROLEX Rot 5B to have a mutagenic effect.

2. Test:

No bacterial toxicity was observed up to 5000 µg/plate and no mutagenic activity was observed in the absence and presence of S9 in the two Salmonella Typhimurium strains investigated (TA98 and TA100)

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only one plate per dose

Principles of method if other than guideline:

MACROLEX Rot 5B was screened with one plate per dose using the Salmonella/microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 µg per plate on five Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

name of test substance: MACROLEX Rot SB
manufacturer: Bayer AG
batch number: 976-012
content: not indicated by the sponsor
approved: not indicated by the sponsor
appearance: black powder
synonyms: C.I. Solvent Red 52; C.I. 68210
chemical name: 3H-Dibenz[f,i,j]isoquionoline-2,7-dione, 3-methyl-6- [(4-methylphenyl) amino]-
molecular weight: 366
CAS No.: 81-39-0

Target gene:

Histidine-deficient mutants

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

There was no indication of a bacteriotoxic effect of MACROLEX Rot SB at doses of up to and including 5000 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well. At 1581 µg per plate, the substance started to precipitate.

Vehicle / solvent:

MACROLEX Rot SB was dissolved in DMSO and formed a dark violet solution. The positive controls were dissolved in DMSO.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: The positive controls sodium azide, nitrofurantoin, 4-nitro- 1,2-phenylene diamine and cumene hydroperoxide were used without S9 mix; the positive control 2-aminoanthracene was used with S9 mix.

Details on test system and experimental conditions:

Comparable to OECD TG 471

Rationale for test conditions:

Comparable to OECD TG 471

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 150 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guide­ lines may be overruled by good scientific judgement.

In case of questionable results, investigations should con­ tinue, possibly with modifications, until a final evaluation is possible.

Statistics:

not applied

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Evidence of mutagenic activity of MACROLEX Rot 5B was seen. On Salmonella typhimurium TA 1537, TA 98 and TA 102, a bio­ logically relevant increase was found in the mutant count compared to the corresponding negative control. Except for TA 1537 positive response was found only with S9 mix. The lowest effective dose was 16 µg per plate for Salmonella ty­ phimurium TA 1537 and 50 µg per plate for TA 98 and TA 102. The Salmonella/microsome test thus showed MACROLEX Rot 5B to have a mutagenic effect.

The positive controls sodium azide, nitrofurantoin, 4-nitro- 1,2-phenylene diamine, cumene hydroperoxide and 2-amino­ anthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Executive summary:

MACROLEX Rot 5B was screened with one plate per dose using the Salmonella/microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 µg per plate on five Salmonella typhimurium LT2 mutants. These com­ prised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102.

Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. Substance precipitation occurred at the dose 1581 µg per plate and above.

Evidence of mutagenic activity of MACROLEX Rot 5B was seen. On Salmonella typhimurium TA 1537, TA 98 and TA 102, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Except for TA 1537 positive response was found only with S9 mix. The lowest effective dose was 16 µg per plate for Salmonella ty­ phimurium TA 1537 and 50 µg per plate for TA 98 and TA 102. The Salmonella/microsome test thus showed MACROLEX Rot 5B to have a mutagenic effect.

The positive controls sodium azide, nitrofurantoin, 4-nitro- 1,2-phenylene diamine, cumene hydroperoxide and 2-amino­ anthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo Comet test in liver, glandular stomach and jejunum:

A study is available according to OECD TG 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" and OECD TG 489 “In vivo Mammalian Alkaline Comet Assay” is available.

The test item was administered by gavage to three groups (Groups 2, 3 and 4), each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days (females) and twenty-nine consecutive days (males) at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group (Group 1) of five males and five females was dosed with vehicle alone (Polyethylene glycol 400). Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all Group 1 to 4 animals at the end of the study. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed.

In conclusion, a dosage of 1000 mg/kg bw/day (the highest dosage tested) is considered to be the No Observed Adverse Effect Level (NOAEL) for this twenty-eight day toxicity study since no toxicologicalyy significant effects were reported in this guideline study.

The protential genotoxicity was investigated in this study sub-acute with a Comet test according to OECD TG 489 “In vivo Mammalian Alkaline Comet Assay”. Samples of the liver, glandular stomach and jejunum were taken from male animals and processed to provide single cell suspensions with sufficient numbers of cells for the Comet Assay to detect DNA strand breaks in cells or nuclei.

The test item did not demonstrate any significant increases in the percentage tail intensity or median percentage tail intensity of the jejunum, glandular stomach and liver, and was considered not to induce DNA damage in these tissues.

In vivo Micronucleus Test in bone marrow:

SOLVENT RED 52 was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow. Four groups each comprising 5 males, received an intraperitoneal injection. Two groups were dosed with 2000 mg/kg body weight, one group was dosed with 1000 mg/kg body weight and one group was dosed with 500 mg/kg body weight. After dosing, the animals of the dose levels of 1000 and 2000 mg/kg body weight showed the following toxic signs: lethargy, rough coat, a hunched posture and closed eyes. The animals of the dose level of 500 mg/kg body weight showed no abnormalities after dosing, except one animal, which showed a rough coat.

No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with SOLVENT RED 52. The groups that were treated for 24 hours with SOLVENT RED 52 showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The group that was treated for 48 hours with SOLVENT RED 52 and the group that was treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control. lt is concluded that SOLVENT RED 52 is not mutagenic in the micronucleus test under the experimental conditions described in this report.

Link to relevant study records

Reference

Reference 1

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 24 May 2017. Experimental Completion Date: 2 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

Identification: Macrolex Rot 5B
Physical State/Appearance: Red Solid
IUPAC Name: 3-methyl-6-[(4-methylphenyl)amino]-3H-naphtho[1,2,3-de]quinoline-2,7-dione
Storage Conditions: Room temperature in the dark, used/formulated in the light
Expiry Date: 19 August 2021

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Information
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals initially allocated to the study were acclimatized for eight days during which time their health status was assessed. A total of forty-six animals (twenty-five males and twenty-one females) were accepted into the study. At the start of treatment the males weighed 190 to 215g, the females weighed 141 to 169g, and were approximately six to eight weeks old. An additional female was added to the study on Day 5 to replace a female that was found dead on Day 3. Procedures were the same for this animal as the others on the study but study timings were based on its own start of treatment date (Day 1) rather than the start date for the study.

Animal Care and Husbandry
The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited., Oxon, UK) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.
The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Male rats were used in the comet assay.

Route of administration:

oral: gavage

Vehicle:

For the purpose of this study the test item was prepared at the appropriate concentrations as a suspension in Polyethylene glycol 400.

Details on exposure:

A purity adjustment (for 90% purity) was made when preparing the dosing formulations.
The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least 18 days when stored refrigerated in the dark. Formulations were therefore prepared three times during the treatment period and stored at approximately 4 ºC in the dark.
Samples of each test item formulations for Groups 1 to 4 were taken on two occasions and analyzed for concentration of Macrolex Rot 5B at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within
94-104% of the nominal concentration.

The test item was administered to Group 1 to 4 (see table in section any other information on materials and methods) animals daily, for twenty-eight consecutive days (females) and twenty-nine consecutive days (males), by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 6 mL/kg of Polyethylene glycol 400. Group 5 males, used as positive controls for comet assay assessment remained untreated until Day 28 when they were dosed with N-Nitroso-N-methylurea on Days 28 and 29 alone.
All males (including positive controls for the comet assay) were dosed on Day 28 approximately 27 hours before scheduled euthanasia and Day 29 approximately 3 hours before scheduled euthanasia.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals. The volume of reference item administered to positive control animals was based on the scheduled body weight on Day 28.

Duration of treatment / exposure:

29 days for animals used in the comet assay

Frequency of treatment:

daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

Positive control (comet assay)

No. of animals per sex per dose:

5 males and 5 females per dose, except for the positive control where 5 males only were treated.

Control animals:

yes, concurrent vehicle

other: concurrent positive control

Positive control(s):

N-Nitroso-N-methylurea was dissolved in distilled water at the appropriate concentration. A purity adjustment (for 90% purity) was made when preparing dosing formulations. Fresh formulations were prepared each day and dosed within two hours of preparation.
N-Nitroso-N-methylurea is recommended for its use by the OECD TG 489, regulatory authorities and it is widely cited in literature for its use in Comet assay, and historical control data are available in the performing laboratory. The N-Nitroso-N-methylurea formulation was not analyzed for stability, homogeneity or concentration. This does not affect the purpose or integrity of the study and an exception is included in the GLP Compliance Statement.

Tissues and cell types examined:

For examinations made in the repeated dose aspect of this study, see dossier section 7.5.1

For the males from Groups 1 to 4 as well as all positive control males, samples of the liver (after recording of liver weight), glandular stomach and jejunum were processed appropriately by Envigo Research Ltd., Shardlow, UK Cell and Molecular Sciences Department to provide single cell suspensions with sufficient numbers of cells for the Comet Assay. The procedure was performed under subdued lighting and the Comet Assay tissues/cells were processed as quickly as possible, using ice-cold buffers to maintain the tissues and cell preparations at low temperature.

Details of tissue and slide preparation:

Following removal and weighing (where applicable), tissue samples taken for the comet assay were placed in a small volume of the appropriate ice-cold buffer and quickly transferred to the Department of Cell and Molecular Sciences where they were processed for the comet assay.

The tissue samples were processed under subdued lighting and over ice to provide single cell suspensions, providing sufficient cells for scoring for the comet assay as follows:

Glandular Stomach – A small section of the glandular stomach was immersed in stomach buffer (Hanks balanced salt solution supplemented with EDTA and EGTA) and incubated for approximately 15 minutes on ice. The mucosal layer of the glandular stomach was removed by gentle scraping and then a single cell suspension was obtained by scraping the remaining tissue into a small volume of stomach buffer.

Jejunum - Approximately a 2 cm piece of Jejunum was processed. This was immersed briefly in stomach buffer and then scraped gently to remove any contents, incubated in stomach buffer (approximately 10 mL), on ice for 5 to 10 minutes. A single cell suspension was obtained by gentle scraping into approximately 1 mL of fresh liver buffer.
Liver - A small piece of liver (approximately 1 cm3) was washed in liver buffer, (Hanks balanced salt solution supplemented with EDTA), before being minced and filtered to provide a single cell suspension.


Slide Preparation
Adequate numbers of slides were pre-coated with 0.5% normal melting point agarose and stored at room temperature. The slides were labelled for animal number, study number and tissue type prior to use for the comet assay.
Once the cell suspensions were obtained, approximately 30 µL of the cell suspension was added to 270 µL of 0.5% low melting point (LMP) agarose, mixed thoroughly and 50 µL of this agarose/cell suspension mix was placed onto a pre-coated slide. Two gels were placed on each slide, and 4 gels were prepared for each tissue. Two of the gels were scored for Comets (A and B replicates) and two (C and D replicates) were kept in reserve in case further scoring was required or the gels were damaged during processing. The agarose/cell suspension mix was immediately covered with a glass coverslip, transferred to a cold room at approximately 4 °C in the dark for approximately 20 minutes to allow it to solidify.
Once the LMP agarose had set the coverslips were removed and the slides gently lowered into freshly prepared lysing solution (pH 10) and refrigerated in the dark overnight. All slides went through the subsequent processing.
After the lysis phase had been completed the slides were removed from the lysing buffer, rinsed with neutralization buffer (0.4M Tris pH 7.5) to remove residual detergents and salts and then placed randomly into an electrophoresis unit. The electrophoresis unit was filled with chilled electrophoresis buffer (pH >13) until the slide surface was just covered. The slides were left for approximately 20 minutes to allow the DNA to unwind, after which electrophoresis proceeded at approximately 0.7 V/cm (calculated between the electrodes), 300 mA for 20 minutes. The buffer in the bath was chilled during the electrophoresis period and the temperature of the electrophoresis buffer was monitored at the start of unwinding, the start of electrophoresis and the end of electrophoresis to ensure the electrophoresis solution was maintained at low temperature (2-10 °C).
At the end of the electrophoresis period the bath was switched off, the slides gently removed and rinsed three times with neutralization buffer for approximately 5 minutes each time. The slides were then carefully drained and fixed in cold 100% methanol for 5 minutes and allowed to air dry, after which they were stored prior to staining and scoring. For each tissue, two of the four processed slide gels were scored and the remaining two slides were stored as backup slides.

Scoring
The processed Comet slides were coded to allow “blind” scoring using a computer generated code and stained just prior to analysis for comets. To each dry slide gel, 75 µL of propidium iodide (20 µg/mL) was placed on top of the slide and overlaid with a clean cover slip. After a short period to allow hydration and staining of the DNA, the slide was placed onto the stage of a fluorescence microscope and scored for comets using a CCD camera attached to a PC-based image analysis program (Comet IV version 4.3.1).
Two slide gels for each tissue for each animal were scored with a maximum of 100 cells per slide gel giving an accumulative total of 200 cells per tissue per animal. The slide score data for each tissue was processed using the Excel macro program provided in Comet IV version 4.3.1. Comparison between the vehicle control group response and that of the test item dose groups was made. The primary end-point was percentage DNA in the tail (percentage Tail intensity), although other endpoints such as tail moment and length may also be utilized.
Each slide was also assessed for the incidence of ‘hedgehog’ cells to give an indication of cell integrity. Hedgehogs are cells that exhibit a microscopic image consisting of a small or non-existent head, and large diffuse tails and are considered to be heavily damaged cells, although the etiology of the hedgehogs is uncertain.

Evaluation criteria:

Evaluation and Interpretation of Results
Providing that all the acceptability criteria are fulfilled, a test item is considered to be clearly Negative if:
·        None of the test concentrations exhibits a significant increase compared with the concurrent negative control.
·        There is no evidence of a dose-related response.
·        The results are within the laboratory historical vehicle control range.
·        There is evidence, direct or indirect, to demonstrate exposure or toxicity to the target tissue has been achieved.
The test item is then considered unable to induce DNA strand breakage in the tissues studied in the test system.
Providing that all the acceptability criteria are fulfilled, a test item is considered to be clearly Positive if:
·        At least one of the test doses exhibits a statistically significant increase compared to the concurrent negative control.
·        The response is considered to be dose-related.
·        The results are substantially outside the laboratory historical vehicle control range.
The test item can be considered to induce DNA strand breakage in a particular tissue if all three conditions are met. 
There is no requirement for verification of a clearly positive or negative response.

Statistics:

The mean percentage tail intensity and the median percentage tail intensity for each slide was determined followed by calculation of the mean and median percentage tail intensities for each animal. The mean of the individual animal means was calculated to give a group mean.
When there was no indication of any increase in % Tail Intensity at any dose level, statistical analysis was not performed. In all other circumstances, comparisons were made between the appropriate vehicle control value and each individual dose level, using an appropriate statistical method.
Where considered appropriate, statistical analysis is performed on the mean % Tail Intensity and median % Tail Intensity data using a Students t-test on transformed data using a √(x+1) transformation. However, there were no marked increases in the % Tail Intensity and median % Tail Intensity over the vehicle control group which required statistical analysis in this study.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Evaluation of Comet Assay Slides

Vehicle and Positive Controls
The vehicle control group induced percentage tail intensities which were consistent with the current laboratory historical control data ranges. The positive control item (MNU) produced a marked increase in the percentage tail intensity and median percentage tail intensity in the liver, glandular stomach and jejunum indicating that the test method itself was operating as expected and was considered to be valid under the conditions of the test.

Glandular Stomach
The glandular stomach did not demonstrate any marked increases in the percentage tail intensity or the median percentage tail intensity over the vehicle control. The group tail intensity and median tail intensity values were all within the current laboratory historical control data range for a vehicle. Therefore it is considered that the test item did not induce DNA damage in the glandular stomach tissue investigated under the conditions of the test. There were no dose-related increases in the percentage hedgehog frequencies in the glandular stomach.

Jejunum
There were no significant increases in the percentage mean tail intensity or median tail intensity in the jejunum for any of the test item dose groups when compared to the vehicle control. The group mean and median percentage tail intensities for the vehicle group and test item dose groups generally fell within the current historical control data ranges. The percentage tail intensity for the high dose group marginally exceeded the upper end of the historical control range for a vehicle but this small increase could be attributed mainly to two animals in this group (animals 34 and 35) which may have been more sensitive to the effects of the test item. The incidence of hedgehog cells in the jejunum also exceeded the upper end of the historical control range for a vehicle but this was seen in both the vehicle and the test item dose groups and was therefore considered to be of no consequence.

Liver
There were no significant increases in the percentage mean tail intensity or median tail intensity in the test item dose groups of the liver when compared to the vehicle control group. The percentage tail intensities and median percentage tail intensities for the vehicle and test item dose groups all fell within the historical control range for a vehicle.
There were no hedgehogs seen in the vehicle or test item dose groups of the liver.

For results of the repeated dose aspect of this study, see dossier section 7.5.1

Comet Assay – Summary ofGroupData

GlandularStomach

Dose Level	Group Mean % Hedgehogs	Group Mean % TailIntensity	Group Mean of Median % Tail Intensity perAnimal	Group Mean Standard Deviation of % Tail Intensity
Vehicle	8.96	6.02	3.95	6.32
Low	8.33	4.56	2.28	5.89
Intermediate	6.95	4.67	2.40	6.04
High	8.18	6.14	4.19	6.12
Positive (MNU)	12.20	30.91	29.96	12.40

Jejunum

Dose Level	Group Mean % Hedgehogs	Group Mean % TailIntensity	Group Mean of Median % Tail Intensity perAnimal	Group Mean Standard Deviation of % Tail Intensity
Vehicle	12.25	4.51	2.18	6.42
Low	13.79	4.67	2.09	6.55
Intermediate	11.41	4.17	1.67	6.01
High	14.08	5.46	3.20	7.08
Positive (MNU)	16.96	40.03	39.96	16.06

Liver

Dose Level	Group Mean % Hedgehogs	Group Mean % TailIntensity	Group Mean of Median % Tail Intensity perAnimal	Group Mean Standard Deviation of % Tail Intensity
Vehicle	0	0.54	0.09	1.04
Low	0	0.51	0.05	1.15
Intermediate	0	0.41	0.04	0.98
High	0	0.51	0.06	1.14
Positive (MNU)	3.81	31.62	31.93	8.48

Conclusions:

The test item did not demonstrate any significant increases in the percentage tail intensity or median percentage tail intensity of the jejunum, glandular stomach and liver and was considered to not induce DNA damage in these tissues under the conditions of the test.

Executive summary:

Introduction

The study was designed to investigate the systemic toxicity of the testitem and to detect DNA strand breaks in cells or nuclei and is compatible with the following regulatory guidelines:

 

·        Commission Directive 96/54/EC (Method B7).

·        The OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 03 October 2008).

·        Commission Regulation (EC) No 440/2008 of 30 May 2008, test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

·        OECD Guidelines for Testing of Chemicals No. 489 “In vivo Mammalian Alkaline Comet Assay” (Adopted 29 July 2016)

Methods

The test item was administered by gavage to three groups (Groups 2, 3 and 4), each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days (females) and twenty-nine consecutive days (males) at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group (Group 1) of five males and five females was dosed with vehicle alone (Polyethylene glycol 400). A group of five males (Group 5) used as a positive control for comet assay assessment, remained untreated until Day 28 when they were dosed with 25 mg/kg bw/day N-Nitroso-N-methylurea on Days 28 and 29.

All males were dosed on Day 28 approximately 27 hours before scheduled euthanasia, and dosed on Day 29 approximately 3 hours before scheduled euthanasia, and selected tissues from these animals were used for comet assay assessment.

Clinical signs,functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all Group 1 to 4 animals at the end of the study.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed. 

Additionally, samples of the liver, glandular stomach and jejunum were taken from male animals and processed to provide single cell suspensions with sufficient numbers of cells for the Comet Assay to detect DNA strand breaks in cells or nuclei.

 

For comet assay assessment, the five males from each dose group (Groups 1 to 4) were dosed once daily with the test item for twenty nine consecutive days and killed approximately 3 hours after the last dose. Another set of five males were dosed with N-nitroso-N-methylurea (NMU) on Days 28 and 29 only (approximately 27 and 3 hours before euthanasia) and used as positive controls. The dosing regimen details are given below:

 

Group	Animal Numbers	Dose level (mg/kg bw/day)	Treatment volume (ml/kg)	Concentration (mg/ml)	Treatment period
1. Control	1-5	0$	6	0	29 days
2. Low	11-15	100	6	16.7	29 days
3. Intermediate	21-25	300	6	50	29 days
4. High	31-35	1000	6	166.7	29 days
5. Positive Control	41-45	25@	10	2.5	2 days#

$Animals treated with Vehicle Control.

@Animals dosed with N-nitroso-N-methylurea

# Dosed on days 28 and 29 only

 

Samples of liver, glandular stomach and jejunum were taken at necropsy from these males and transferred to the Cell and Molecular Sciences Department followed by processing to provide slides for the comet assay.

Results

The test item did not demonstrate any significant increases in the percentage tail intensity or median percentage tail intensity of the jejunum, glandular stomach and liver, and was considered not to induce DNA damage in these tissues.

Conclusion

The test item did not demonstrate any significant increases in the percentage tail intensity or median percentage tail intensity of the jejunum, glandular stomach and liver and was considered to not induce DNA damage in these tissues under the conditions of the test.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Study conducted 2001; guideline not mentioned in the report.

Deviations:

not applicable

Remarks:

Study conducted 2001; guideline not mentioned in the report. Animals are exposed via I.P. injection

Principles of method if other than guideline:

Five male mice were used per sampling time in each treatment group. The animals were dosed once and sampeld after 24 and/or 48 hours.

GLP compliance:

not specified

Type of assay:

other: Micronucleus Test

Specific details on test material used for the study:

Solvent Red 52
brown solid
batch SJW6099
Purity: not indicated

Stabilty in corn oil: not indicated

Species:

mouse

Strain:

other: NMRI BR mice (SPF)

Details on species / strain selection:

Recommended test system in internatfonal guidelines (e.g. EPA, FDA, OECD, EEC).

Sex:

male

Details on test animals or test system and environmental conditions:

Conditions
A controlled environment was maintained in the room with optimal conditions of approximately 15 air changes per hour, a temperature of 21± 3°C, a relative humidity of 30-70% and 12 hours artificial fluorescent light and 12 hours dark per day. Fluctuations from these optimal conditions were noted, but were considered not to have affected the integrity of the study.

Accommodation
Group housing of 5 animals per sex per cage in labelled polycarbonate cages containing purified sawdust as bedding material (Sawi, Jelu Werk, Rosenberg, Germany). Certificates of analysis were examined and then retained in the NOTOX archives. Acclimatisation period was at least 5 days before start of treatment under laboratory conditions.

Diet
Free access to standard pelleted laboratory animal diet (Altromin (code VRF 1), Lage, Germany). Certificates of analysis were examined and then retained in the NOTOX archives.

Water
Free access to tap-water. Certificates of analysis (performed quarterly) were examined and then retained in the NOTOX archives.

Route of administration:

intraperitoneal

Vehicle:

SOLVENT RED 52 was suspended in corn oil (OPG, Utrecht, The Netherlands). SOLVENT RED 52 concentrations were treated with ultra-sonic waves to obtain a homogeneous suspension. SOLVENT RED 52 concentrations were dosed within 4 hours after preparation.

Details on exposure:

The mice received an intraperitoneal injection of a maximum tolerated (high), an intermediate and a low dose of SOLVENT RED 52. The route of administration was selected taking into account the possible route of human exposure during manufacture, handling and use.

The dosing volume was 10 ml/kg body weight.

The route and frequency of administration and the volume administered of the negative and the positive control was the same as those of the test article.

Duration of treatment / exposure:

24 and 48 h

Frequency of treatment:

single injection

Post exposure period:

24 and 48 h

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

5 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 male mice per sampling time in each treatment group.

Control animals:

yes, concurrent vehicle

Positive control(s):

50 mg/kg cyclophosphamide

Tissues and cell types examined:

The animals were sacrificed by cervical dislocation 24 or 48 h after dosing SOLVENT RED 52, 24 h after dosing of the vehicle and 48 h after dosing the positive control. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml offoetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min.

Details of tissue and slide preparation:

The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide which was previously cleaned (24 h immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue) and marked (with the NOTOX study identification number and the animal number). The drop was spread by moving a clean slide with round­ whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol and air-dried overnight. Two slides were prepared per animal.

Evaluation criteria:

All slides were randomly coded before examination. An adhesive label with NOTOX study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were weil spread, undamaged and weil stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

A micronucleus test is considered acceptable if it meets the following criteria:
a) The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
b) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range (mean ± three times the standard deviation): Males: 0.70%0 ± 2.67%0 indicated are means for n=80.

Statistics:

Wilcoxon Rank Sum Test

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

In a dose range finding study four animals (two mafes and two females per group) were dosed intraperitoneally with 2000 mg/kg body weight (group A). The results of this dose range finding study are lethargy; rough coat and hunched posture.

Since there were no substantial differences between the sexes in toxicity after dosing the animals with 2000 mg/kg body weight, the micronuclues test was performed with male animals only. Based on the results of the dose range finding studY, dose levels of 2000, 1000 and 500 mg/kg body weight were selected as appropriate doses for the micronucleus test. Five male animals were used in each treatment group.

The clinical observations made in the groups treated with SOLVENT RED 52 are lethargy; rough coat and hunched posture.

Micronucleated polychromatic erythrocytes

The mean number of micronucleated polychromatic erythrocytes per group and the mean ratio of polychromatic to normochromatic erythrocytes are presented in Table 4. The individual data are described in Appendix 1. The mean number of micronucleated polychromatic erythrocytes scored in SOLVENT RED 52 treated groups was compared with the corresponding solvent control group.

No biologically significant increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of SOLVENT RED 52 treated animals compared to the vehicle treated animals.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes (Appendix 2). Hence, the acceptability criteria of the test were met.

The groups that were treated for 24 hours with SOLVENT RED 52 showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The dose group of 2000 mg SOLVENT RED 52 /kg body weight (48 hours treatment) and the group that was treated with,cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control.

Executive summary:

SOLVENT RED 52 was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow.

Four groups each comprising 5 males, received an intraperitoneal injection. Two groups were dosed with 2000 mg/kg body weight, one group was dosed with 1000 mg/kg body weight and one group was dosed with 500 mg/kg body weight. After dosing, the animals of the dose levels of 1000 and 2000 mg/kg body weight showed the following toxic signs: lethargy, rough coat, a hunched posture and closed eyes. The animals of the dose level of 500 mg/kg body weight showed no abnormalities after dosing, except one animal, which showed a rough coat.

A vehicle treated group served as negative control, a group treated with an intraperitoneal injection of cyclophosphamide (CP) at 50 mg/kg body weight served as positive control.

Bone marrow of the groups treated with SOLVENT RED 52 was sampled 24 or 48 hours after dosing. Bone marrow from the negative control group was harvested at 24 hours after dosing only and bone marrow from the positive control group was harvested at 48 hours after dosing only.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes.

No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with SOLVENT RED 52.

The groups that were treated for 24 hours with SOLVENT RED 52 showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The group that was treated for 48 hours with SOLVENT RED 52 and the group that was treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control.

lt is concluded that SOLVENT RED 52 is not mutagenic in the micronucleus test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

In vitro bacterial reverse mutation tests gave positive and negative results. In vivo

In vivo tests ( 1- Comet test investigationg liver, glandular stomach and jejunum after 28 days of exposure, 2 - in vivo Micronucleus Test in bone marrow after I.P. injection) were negative. It can be concluded that Macrolex Rot 5B is not genotoxic in vivo.