Glycine, N-[5-(acetylamino)-4-[2-(2-bromo-4,6-dinitrophenyl)diazenyl]-2-methoxyphenyl]-N-(2-methoxy-2-oxoethyl)-, methyl ester

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 December 2014 to 05 March 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Disperse Blue ANT

IUPAC Name:

Disperse Blue ANT

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Test item name : Disperse Blue ANT

Test animals

Species:

rat

Strain:

Sprague-Dawley

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 206-237 g for males and 180-187 g for females, were received from Charles River Italia S.p.A., Calco (Lecco), Italy. After arrival the weight range for each sex was determined 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 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.
In-life phase: 31 december 2015 - 05 March 2015 (last necropsy)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

The vehicle for the test item was Polyethylene glycol (PEG) 400 in softened water (by reverse osmosis) - 1:1 v/v.

Positive Control item
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.

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 required amount of Disperse Blue ANT (Br) was suspended in the vehicle, Polyethylene glycol (PEG) 400 in softened water (by reverse osmosis) - 1:1 v/v. The formulations were prepared daily (concentrations of 12.5, 50 and 200 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.


Positive Control group
The 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:

Males
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 37 and 38 of study). Males were treated for a total of 36 or 37 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.


Positive Control group (Group 7)
Animals received a single dose approximately 24 hours before sacrifice.

Frequency of treatment:

once a day

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
62.5, 250, 1000 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
12.5, 50 and 200 mg/mL
Basis:
nominal conc.

No. of animals per sex per dose:

Each main group comprised 10 male ( and 10 female rats not included in the test) (Groups 1 to 4).
Two groups (control and high dose levels) of 5 animals per sex were sacrificed after 3 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 (Mitomycin) 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. Determination of the stability and concentration of solutions of the positive control item will not be undertaken.

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 hours following the final treatment and approximately 48 hours following the second last
treatment 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 smears

The 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 overnight and then fixed with
methanol for 10 minutes. Subsequently slides were stained with haematoxylin and eosin solutions. Finally slides were rinsed in distilled water and allowed to dry.


Scoring of the slides and data analysis

For 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 of cells was 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 is considered valid if the following criteria are met:

1 -The incidence of micronucleated PCEs in the vehicle control group fell within the historical vehicle control range.
2 -The positive control item induced a significant increase in the frequency of micronucleated PCEs.
3 -At least 5 males per group are available for slide analysis.


Evaluation of results

The test item is considered to induce micronuclei if a statistically significant (p<0.05) and biologically meaningful increase in micronucleus incidence
is observed in any treatment group. A dose-effect relationship should be observed.
Where increases in the incidence of micronucleated PCEs was observed which were statistically significant, but fall within the range of vehicle control values
within this laboratory, then concurrent and historical control data was used to demonstrate that these increases did not had biological significance.

Statistics:

Statistical analysis of data

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

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
No signs of genotoxicity were detected by measuring the micronuclei in polychromatic erytrocytes.

Executive summary:

The ability of the test item to induce cytogenetic damage and/or disruption of the mitotic apparatus in rat bone marrow was investigated measuring the induction of micronuclei in polychromatic erythrocytes. The test was only performed in males, as no toxicological relevant difference in target organ toxicity was noted.

Samples of bone marrow were collected approximately 24 hours following the final treatment and approximately 48 hours following the second last treatment from 5 males of the main groups randomly selected and from all animals of the positive control group consisting of 5 males. One femur of each animal was removed and bone marrow cells obtained by flushing with foetal calf serum. The cells were centrifuged and a concentrated suspension prepared to make smears on slides. These slides were air-dried, fixed with methanol and then stained with haematoxylin and eosin solutions and mounted with Eukitt. Three slides were made from each animal.

The slides were randomly coded by a person not involved in the subsequent microscope scoring and examined under low power to select one or more slides from each animal according to staining and quality of smears. Four thousand polichromatic erythrocytes (PCEs) per animal were examined for the presence of micronuclei at high power (x 100 objective, oil immersion). At the same time, the numbers of normal and micronucleated normochromatic erythrocytes (NCEs) were also recorded

Findings noted during treatment (discoloured urine/bedding) were considered a proof of absorption.

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.

Following treatment with the test item, no relevant increase in the number of micronucleated PCEs was observed at any dose level.

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

A summary of the results obtained is presented in the following table:

Dose level (mg/kg/day)	Incidence in micronucleated PCEs			PCE/s(PCEs+NCEs) % over the mean control value
	Mean	SE	Range
0.00	0.4	0.2	0.0-1.0	100
62.5	1.2	0.3	0.5-2.3	93
250	1.0	0.4	0.0-2.3	92
1000	1.2	0.4	0.5-2.5	100
Mitomycin-C 2.00 mg/kg	11.3	1.7	7.0 - 17.3	89

The incidence of micronucleated PCEs of the negative control group fell within the historical control range (95% confidence limit). Statistically significant increases in the incidence of micronucleated PCEs over the negative control values were seen in the positive control group. The induced response was compatible with the historical control range, demonstrating the laboratory proficiency in the conduct of the test. Five animals per groups were available for micronucleus slide analysis. Based on the stated criteria, the assay was therefore accepted as valid.

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