Trichloro-12,24-dihydro-5H-naphtho[2,3-h]naphth[2'',3'':6',7']indolo[2',3':6,7]anthra[2,1,9-mna]acridine-5,10,13,18,25-pentone

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

27 May 2015 to 10 March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of 3,9-dibromobenzanthrone condensed with 2 equivalents of 1-aminoanthraquinone, subsequently further condensed under oxidative conditions

EC Number:

944-232-9

Molecular formula:

Not available - UVCB substance

IUPAC Name:

Reaction product of 3,9-dibromobenzanthrone condensed with 2 equivalents of 1-aminoanthraquinone, subsequently further condensed under oxidative conditions

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and because there is ample experience
and background data on this species and strain.

Sex:

male

Details on test animals or test system and environmental conditions:

A total of 125 Sprague Dawley [Crl:CD(SD)BR] rats (65 males and 60 virgin females), 7 to 8 weeks old and weighing 200 to 225 g for males and 175 to 200 g for females, were ordered from and supplied by Charles River Italia S.p.A., Calco (Lecco), Italy.After arrival, on 21 May 2015, the weight range for each sex was determined (189-216 g for females, 226-250 g for males, slightly outside the range at order) and the animals were temporarily identified within the cage by means of a coloured mark on the tail.A health check was then performed by a veterinarian. An acclimatisation period of at least 14 days was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C +/- 2°C and 55% +/- 15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

The vehicle for the test item was sesame oil.
The vehicle for the positive control item was sterile water for injection.
Test item
The required amount of Vat Black 25 was dissolved/suspended in the vehicle. The formulations were prepared daily (concentrations of 12,5, 50 and 200mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.
Positive Control item
The required amount of positive control item was dissolved in the vehicle. The formulation was prepared on the day of dosing at a concentration of0.2 mg/mL.

Details on exposure:

The test item was administered orally by gavage at a dose volume of 5 mL/kg body weight. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
Analysis was not performed to confirm that the proposed formulation procedure was acceptable and that the stability of the formulation was satisfactory as the test item is neither extractable in hydrophilic nor in lipophilic solvents. The correct concentration preparation was monitored in the weighing record of the test item in each formulation process.
Positive Control
Animals received a single dose approximately 24 hours before sacrifice. Mitomycin-C (positive control) was administered once by intraperitoneal injection at the dose volume of 10 mL/kg body weight. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

Duration of treatment / exposure:

Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing, through the mating period and thereafter through the day before necropsy (Days 43 and 44 of study). They were treated for a total of 42 or 43 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Frequency of treatment:

Once a day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

62.5 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

Each main group comprised 10 male and 10 female rats (Groups 1 to 4). Two groups (control and high dose levels) included 5 animals per sex which were sacrificed after 2 weeks of recovery (Groups 5 and 6). For genotoxicity endpoint, a satellite control group (Positive Control group) comprised 5 male rats (Group 7).

Control animals:

yes, concurrent vehicle

Positive control(s):

For genotoxicity endpoint, a satellite control group (Positive Control group) comprised 5 male rats (Group 7).The required amount of positive control item was dissolved in the vehicle (sterile water for injection). The formulation was prepared on the day of dosing at a concentration of 0.2 mg/mL. No documents on Mitomycin-C are included in the report. Determination of the stability and concentration of solutions of the positive control item was not undertaken.Mitomycin-C (positive control) was administered once by intraperitoneal injection at the dose volume of 10 mL/kg body weight. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.Positive Control group animals were killed under carbon dioxide asphyxiation.

Examinations

Tissues and cell types examined:

Bone marrow from one femur of males only.

Details of tissue and slide preparation:

Extraction of bone marrow
Samples of bone marrow were collected approximately 24 and 48 hours after the 2 final treatments, from the same 5 males of the main groups randomly selected for clinical pathology investigation (see section 4.4). Samples of bone marrow were also collected approximately 24 hours after the single treatment from all males of Group 7 (Positive Control group). One femur of each animal was rapidly dissected out and cleaned of surrounding tissue. In order to extract the bone marrow, the bone was cut at the proximal end and irrigated with foetal calf serum using a syringe. The suspension of cells was aspirated, and this procedure was repeated several times.Preparation of the smearsThe suspension thus obtained was centrifuged at 1000 rpm for at least 5 minutes and the supernatant was completely removed. The cells of the sediment were resuspended and transferred onto clean microscope slides as smear preparations. They were air-dried and then fixed with methanol for 10 minutes. Subsequently, slides were stained with haematoxylin and eosin solutions. Finally, the slides were rinsed in distilled water and allowed to dry.Scoring of the slides and data analysisFor each animal, at least three slides were prepared. These slides were randomised and coded by staff not subsequently involved in the scoring.The adequate quality and the sufficient number of cells were evaluated before scoring. Scoring was performed using a microscope and high-power objective. Immature polychromatic erythrocytes (PCEs) stain a pink-purple colour (since they retain basic ribosomal material for approximately 24 hours after enucleation), and can be distinguished from the pink normochromatic erythrocytes (NCE).Erythrocytes lack nuclei, making micronuclei obvious when present; the criteria of Schmid (1976) were used to score micronuclei. At least four thousand polychromatic erythrocytes per animal were scored for the presence of micronuclei. At the same time the number of normochromatic erythrocytes was recorded, as well as the number of micronucleated NCE. The proportion of immature erythrocytes among total erythrocytes gives an indication of the toxicity of the treatment; a reduction in the proportion indicates inhibition of cell division. Finally, the incidence of micronucleated PCE provides an index of induced genetic damage.

Evaluation criteria:

Acceptance criteria
The assay was considered valid if the following criteria were met:
– The incidence of micronucleated PCEs of the vehicle control group fell within the historical negative control range.
– The positive control item results fell within the historical control range and were significantly increased, at statistical analysis, when comparedwith the concurrent negative control.
– 5 males per group were available for slide analysisEvaluation of results
The test item was considered to induce micronuclei if a statistically significant increase in the micronucleus incidence of polychromatic erythrocytes(at p< 0.05) was observed in any treatment group and a dose-effect relationship was demonstrated. Where statistically significant increases in the incidence of micronucleated PCEs were observed, but all results were inside the distribution of negative control values within this laboratory, then historical control data were used to demonstrate that these increases did not have any biological significance.

Statistics:

Only counts from polychromatic cells were subjected to statistical analysis. Using the original observations (and not the micronucleus frequencies per 1000 cells), a modified chi-squared calculation was employed to compare treated and control groups. The degree of heterogeneity within each group was first calculated and where significant it was taken into account in the comparison between groups. If there was no significant within-group heterogeneity, the chi-squared test was used to compare treated groups with the controls. If there was significant within-groups heterogeneity, then that group was compared with the controls using a variance ratio (F) value calculated from the between-group and within-group chi-squared values.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Incidence of micronucleated cells
Following treatment with the test item, a slight increase in the number of micronucleated PCEs over the concurrent negative control was observed for animals from the low and high dose groups; however, the incidences of micronucleated PCEs were comparable to RTC historical control data for negative control animals. A marked increase in the frequency of micronucleated PCEs was observed in the positive control group.Bone marrow cell toxicityThe ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analysed to evaluate the bone marrow cell toxicity. Based on these results, no relevant inhibitory effect on erythropoietic cell division was observed at any dose level.
Analysis of resultsFollowing treatment with the test item, statistically significant increases in the incidence of micronucleated PCEs over the negative control value were observed for animals from the low and high dose levels (p<0.01). A significant dose effect relationship was found after a trend test evaluation (p<0.05). The dose-related and statistically significant increases of micronucleated PCEs could be attributable to the low incidence of micronucleated cells in the vehicle control group, which fell in the lower part of the distribution range of historical control data. In addition, the frequency of micronucleated immature erythrocytes observed at all dose levels was inside the distribution of historical negative control data (95% upper confidence limit = 2.0), therefore the observed increases were not considered biologically meaningful.

Any other information on results incl. tables

 

Dose level (mg/kg/day)	Incidence in micronucleated PCEs			PCE/s(PCEs+NCEs) % Over the mean control value
	Mean	SE	Range
0.00 62.5 250 1000 Mitomycin-C 2.00 mg/kg	0.2 0.9 0.3 0.9 8.2	0.1 0.3 0.1 0.2 1.2	0.0 – 0.5 0.3 – 1.8 0.0 – 0.5 0.5 – 1.5 5.5 – 12.5	100 101 97 96 97

 

SUMMARY OF INCIDENCE OF MICRONUCLEATED CELLS

INCIDENCE OF MICRONUCLEATED CELLS/1000 CELLS
Dose level mg/kg/day	Scored cells		NCE/PCE Ratio	PCE/(PCE+NCE) Ratio	% over the Mean control value	Polychromatic				Normochromatic
	PCE	NCE				Mean	SE	Min	Max	Mean	SE	Min	Max
0.00 62.5 250 1000	20000 20000 20000 20000	22374 21840 23814 24165	1.12 1.09 1.19 1.21	0.47 0.48 0.46 0.45	100 101 97 96	0.2 0.9 0.3 0.9	0.1 0.3 0.1 0.2	0.0 0.2 0.0 0.5	0.5 1.8 0.5 1.5	0.0 0.0 0.0 0.0	0.0 0.0 0.0 0.0	0.0 0.0 0.0 0.0	0.0 0.2 0.0 0.2
Mitomycin C 2.00	20000	23531	1.18	0.46	97	8.2	1.2	5.5	12.5	0.0	0.0	0.0	0.0

NCE/PCE ratio: The ratio of NCE/PCE calculated as the mean of the ratio values for the individual animals.

PCE/(PCE+NCE) ratio: The ratio of PCE/(PCE+NCE) calculated as the ratio of the total PCEs over the total erythrocytes scored

% over the mean control: Percentage of the PCE/(PCE+NCE) ratio of each treated group value over the negative control value

MEAN:    The group mean incidence of micronucleated PCEs and NCEs

SE:          The standard error of the mean incidence

MIN:       Minimum value observed in an individual animal

MAX:      Maximum value observed in an individual animal

 

HISTORICAL CONTROL DATA

INCIDENCES OF MICRONUCLEATED PCEs (%) (1991 – 2015)
Male animals
	Vehicle controls	Positive controls
Mean	0.7	16.4
SD (σn-1)	0.70	8.41
n	165	164
Upper confidence limit (95%)	2.0	NC
Maximum	3.5	47.5
Minimum	0.0	0.5

SD = standard deviation

n = number of animals

NC = not calculated

Applicant's summary and conclusion

Conclusions:

On the basis of the results obtained, it is concluded that Vat Black 25 does not induce micronuclei in the polychromatic erythrocytes of treated rats, under the reported experimental conditions.

Executive summary:

The purpose of this study was to provide information on toxic effects on male and female rats after repeated dosing with the test item, the micronucleus test was included in order to assess the ability of the test item to induce cytogenetic damage in rat bone marrow, as measured by the induction of micronuclei in polychromatic erythrocytes.

 

Experimental procedures were based on the following guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

 

Incidence of micro nucleated cells

Following treatment with the test item, a slight increase in the number of micro nucleated PCEs over the concurrent negative control was observed for animals from the low and high dose groups; however, the incidences of micro nucleated PCEs were comparable to RTC historical control data for negative control animals.

A marked increase in the frequency of micro nucleated PCEs was observed in the positive control group.

 

Bone marrow cell toxicity

The ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analysed to evaluate the bone marrow cell toxicity. Based on these results, no relevant inhibitory effect on erythropoietic cell division was observed at any dose level.

 

Analysis of results

Following treatment with the test item, statistically significant increases in the incidence of micro nucleated PCEs over the negative control value were observed for animals from the low and high dose levels (p<0.01). A significant dose effect relationship was found after a trend test evaluation (p<0.05). The dose-related and statistically significant increases of micro nucleated PCEs could be attributable to the low incidence of micro nucleated cells in the vehicle control group, which fell in the lower part of the distribution range of historical control data. In addition, the frequency of micro nucleated immature erythrocytes observed at all dose levels was inside the distribution of historical negative control data (95% upper confidence limit = 2.0), therefore the observed increases were not considered biologically meaningful.

 

Conclusions

On the basis of the results obtained, it is concluded that Vat Black 25 does not induce micronuclei in the polychromatic erythrocytes of treated rats, under the reported experimental conditions.