[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

other: read across from analogue substance

Adequacy of study:

weight of evidence

Study period:

from 28 Jan. 2019 to 08 Mar. 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

no

Principles of method if other than guideline:

Since the test item gave positive results in a mouse lymphoma mutation assay without any remarkable difference with and without S9 metabolic activation, a direct genotoxic mechanism was suspected. This study was intended as a further investigation to evaluate cytogenetic effects and treatments were performed only in the absence of S9 metabolism.

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of diazotised Sodium Picramate coupled with Resorcinol, subsequently coupled with diazotised 4'-Amino-4-Nitrodiphenylamin-2-Sulfonic Acid, at the end metallized with Iron (II) sulphate heptahydrate, sodium and ammonium salts

EC Number:

946-788-8

Molecular formula:

not available being an UVCB substance

IUPAC Name:

Reaction products of diazotised Sodium Picramate coupled with Resorcinol, subsequently coupled with diazotised 4'-Amino-4-Nitrodiphenylamin-2-Sulfonic Acid, at the end metallized with Iron (II) sulphate heptahydrate, sodium and ammonium salts

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

lymphocytes: human whole body

Details on mammalian cell type (if applicable):

-Source: Biopredic International (France)
- Batches used:
(i)
Sex Female
Age 33 years old
Donor information healthy, no smoker without any recent exposure to drugs or radiation
Anticoagulant Sodium heparin, 556 IU/mL of whole blood
Collection date 24 January 2019
Code Number 2019/05
(ii)
Sex Female
Age 30 years old
Donor information healthy, no smoker without any recent exposure to drugs or radiation
Anticoagulant Sodium heparin, 556 IU/mL of whole blood
Collection date 24 January 2019
Code Number 2019/06

- Culture medium composition:
RPMI 1640 1x (Dutch modification) 500mL
Foetal Calf Serum 100mL
L-Glutamine (200mM) 6.25mL
Antibiotic solution 1.25mL
The foetal calf serum was heat-inactivated at 56 °C for 20 minutes before use. For the initiation of the cultures, medium with the addition of phytohaemagglutin (PHA) was used in the following proportion: 10 mL of PHA was added to 500 mL of medium.

Metabolic activation:

without

Metabolic activation system:

Positive control: S9

Test concentrations with justification for top dose:

Dose levels were selected on the basis of the cytotoxicity results obtained in MLA Study No. A3170.
- short term treatment series:
1250, 826, 556, 371, 247, 165, 110, 73.2 and 48.8 µg/mL (expressed as organic conten)
1730, 1140, 769, 513, 342, 228, 152, 101 and 67.5 µg/mL (expressed as test item as received)
- continuous treatment:
1250, 826, 556, 371, 247, 165, 110, 73.2, 48.8 and 32.5 µg/mL (expressed as organic conten)
1730, 1140, 769, 513, 342, 228, 152, 101, 67.5 and 45.0 µg/mL (expressed as test item as received)

Vehicle / solvent:

- Test item: Minimal Medium A (RTC batch nos.: 008/222 and 008/225).
- Benzo(a)pyrene (B(a)P): DMSO (batch no.: H012S, obtained from Honeywell)

Preparation of the test item
Suspensions/solutions of the test item were prepared immediately before use in culture medium on a weight/volume basis, without correction for the displacement due to the volume occupied by the test item. Concentrations were expressed in terms of organic content. Suspensions/solutions at 12.5, 8.26 and 5.56 mg/mL (as organic content), were prepared by separate formulations (corresponding to 17.3, 11.4 and 7.69 mg/mL, as test item as received). All test item suspensions/solutions were used within 45 minutes from the initial formulation.
Treatment with the positive control Colchicine was performed under safe light conditions.

Controls

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

other: colchicine (migrated information)

Details on test system and experimental conditions:

- Preparation of the test cultures and treatment
Two treatment series were performed including negative and positive controls. Two replicate cultures were prepared at each test point. Lymphocyte cultures were treated approximately forty-eight hours after they were initiated. Before treatment, cultures were centrifuged at 1000 rpm for 10 minutes and the culture medium was decanted and replaced with treatment medium
The composition of the treatment media was as follows:
Test item solution 0.50mL
Culture medium (without PHA) 4.50mL
For the short treatment series, the treatment mediawere added to the tubes and the cultures were incubated for 3 hours at 37 °C. At the end of treatment time, the cell cultures were centrifuged and washed twice with Phosphate Buffered Saline Solution. Fresh medium was added and the cultures were incubated for a further 28 hours (Recovery Period) before harvesting. At the same time, Cytochalasin-B was added to achieve a final concentration of 6 µg/mL.
For the continuous treatment series, 3 hours after beginning of treatment, Cytochalasin-B was also added and the cultures were incubated for a further 28 hours before harvesting.

- Harvesting and slide preparation
The lymphocyte cultures were centrifuged for 10 minutes at 1000 rpm and the supernatant was removed.
The cells were resuspended in hypotonic solution. Fresh methanol/acetic acid fixative was then added. After centrifugation and removal of this solution, the fixative was changed several times by centrifugation and resuspension.
A few drops of the cell suspension obtained in this waywere dropped onto clean,wet, greasefree glass slides. Three slides were prepared for each test point and each was labelled with the identity of the culture.
The slides were allowed to air dry and kept at room temperature prior to staining with a solution of Acridine Orange in PBS.

- Slide evaluation
The cytokinesis-block proliferation index CBPI was calculated as follows:
CBPI = (mononucleated + 2×binucleated + 3×multinucleated) / total number of cells counted
where mononucleated, binucleated and multinucleated are respectively the number of mononucleated cells, binucleated cells and multinucleated cells. CBPI was used to measure the cytotoxic effect. Five hundred cells per cell culture were analysed and when negligible cytotoxicity was observed, scoring was interrupted. The highest dose level for genotoxicity assessment was selected on the basis of the cytotoxicity as calculated by the CBPI.
The percentage cytotoxicity was evaluated according to the following formula:
%Cytotoxicity = 100−100*[(CBPI T −1)/(CBPI C −1)]
where:
T = test item treated culture
C = solvent control culture
The highest dose level for genotoxicity assessment was selected as a dose which produces a substantial cytotoxicity compared with the solvent control. Ideally the cytotoxicity should be between 50 % and 60 %. In the absence of cytotoxicity, the highest treatment level is selected as the highest dose level for scoring.
Two lower dose levels were also selected for the scoring of micronuclei.
For the three selected doses, for the solvent and the positive control Mitomycin-C, 1000 binucleated cells per cell culture were scored to assess the frequency of micronucleated cells.
Concerning cultures treated with Colchicine, since it is a known mitotic spindle poison which induces mitotic slippage and cytokinesis block, a greater magnitude of response was observed in mononucleated cells. For this reason, 1000 mononucleated cells per cell culture were scored.
The criteria for identifying micronuclei were as follows:
1. The micronucleus diameter was less than 1/3 of the nucleus diameter
2. The micronucleus diameter was greater than 1/16 of the nucleus diameter
3. No overlapping with the nucleus was observed
4. The aspect was the same as the chromatin

- Solubility test
Solubility of the test item was evaluated in a preliminary trial using complete culture medium.

- Osmolality and pH results
Following treatment with the test item, variations of pH or osmolality were monitored.

- Selection of dose levels for scoring
The CBPI was calculated for each of the treatment series.

Evaluation criteria:

In this assay, the test item is considered as clearly positive if the following criteria are met:
– Significant increases in the proportion of micronucleated cells over the concurrent controls occur at one or more concentrations.
– The proportion of micronucleated cells at such data points exceeds the normal range based on historical control values (95 % control limits).
– There is a significant dose effect relationship.
The test item is considered clearly negative if the following criteria are met:
– None of the dose levels shows a statistically significant increase in the incidence of micronucleated cells.
– There is no concentration related increasewhenevaluated with theCochran-Armitage trend test.
– All the results are inside the distribution of the historical control data (95% control limits).

Statistics:

For the statistical analysis, a modified χ2 test was used to compare the number of cells with micronuclei in control and treated cultures.
Cochran-Armitage Trend Test (one-sided) was performed to aid determination of concentration response relationship.

Results and discussion

Test results

Species / strain:

lymphocytes: human whole body

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Solubility test
The test item gave an opaque formulation with heavy and moderate precipitation in culture medium at the concentrations of 50.0 and 25.0 mg/mL respectively, expressed as organic content. An opaque formulate without visible precipitation, feasible for treatment was obtained at 12.5 mg/mL. An opaque solution feasible for dilution was obtained at the concentration of 6.29 mg/mL.
On the basis of these results and the cytotoxicity results obtained in the MLA Study No. A3170, the maximum dose level of 1250 µg/mL was selected for the cytogenetic test by using a suspension at 12.5 mg/mL added to the treatment medium in the ratio 1:10.
Following treatment with the test item, no precipitate visible by eye was noticed at the beginning or by the end of treatment at any concentration tested.

- Osmolality and pH results
Following treatment with the test item, no remarkable variation of pH or osmolality was observed at any dose level, in the absence or presence of S9 metabolism.

- Selection of dose levels for scoring
Following the 3 hour treatment, no remarkable cytotoxicity was observed at any dose level.
Following the continuous treatment, a cytotoxicity of 65 % and 23 % respectively was calculated for the two highest dose levels of 1250 and 826 µg/mL, where precipitation of the test item, which interfered with the scoring of micronuclei, was seen onto slides.
Moderate cytotoxicity was seen at the lower dose level of 556 µg/mL (47 %), while no remarkable cytotoxicity was seen over the remaining dose range.
On the basis of the above results, the dose levels selected for scoring of micronuclei were as follows:
Short Treatment 1250, 826 and 556 with the respective cytotoxicity 18, 7 and 11 %
Long Treatment 556, 371 and 247 with the respective cytotoxicity 47, 18 and 15 %
For the positive control, the following dose levels were selected for scoring:
Short treament Mitomycin-C 0.500
Long treatment Colchicine 0.0800
Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controlsMitomycin-C and Colchicine, indicating the correct functioning of the test system. ForMitomycin-C the response was slightly out of the historical range (observed incidence 6% and range of incidence 1.60 - 5.35%). However, for both positive controls the results were compatible with those generated in our historical control database. The study was accepted as valid.
- Analysis of results
Adequate cell proliferation was observed in negative control cultures and the appropriate number of doses and cells was analysed.

Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the control value was observed at any dose level, in any treatment series.
All incidenceswere within the normal distribution of historical control data (95 % confidence limits) with the exception of the result obtained at the intermediate (short treatment) and high dose level (continuous treatment). These values slightly exceeded the upper confidence limit, but fell within the historical control range (0.0-1.05 and 0.1-1.20) and were consistent with those reported in literature for female donors (Fenech et al., 1994, Bonassi et al., 1995, Bonassi et al., 2001). No concentration related increase was seen, hence these results were considered without any biological relevance.

Applicant's summary and conclusion

Conclusions:

The substance does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Executive summary:

The test item was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment, according to the OECD guideline 487 (2016).

Since the test item gave positive results in a mouse lymphoma mutation assay without any difference in the absence and presence of S9 metabolic activation, a direct genotoxic mechanism was suspected.

This study was intended as a further investigation to evaluate cytogenetic effects and treatments were carried out only in the absence of S9 metabolism.

Two treatment series were performed. A short termtreatment, where the cells were treated for 3 hours and the harvest time of approximately 32 hours, corresponding to approximately two cell cycle lengths was used and a long term (continuous), treatment where the cells were treated until harvest at 31 hours.

Solutions of the test item were prepared in complete culture medium.

On the basis of the solubility of the test item and cytotoxicity results obtained in the MLA test, the maximum dose level of 1250 µg/mL expressed as organic content or 1730 µg/mL expressed as test item as received, was selected for treatment. Lower dose levels of 826, 556, 371, 247, 165, 110, 73.2 and 48.8 µg/mL expressed as organic content or 1140, 769, 513, 342, 228, 152, 101 and 67.5 µg/mL expressed as test item as received, were chosen for

the short termtreatment. For the continuous treatment, the additional dose level of 32.5 µg/mL expressed as organic content or 45.0 µg/mL expressed as test item as received was included.

The experiment included appropriate negative and positive controls. Two replicate cell cultures were prepared at each test point.

The actin polymerisation inhibitor Cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. The cytokinesisblock

proliferation index CBPI was calculated in order to evaluate cytotoxicity.

Dose levels for the scoring of micronuclei were selected with the aim to evaluate the test item concentrations at adequate levels of cytotoxicity, covering a range from the maximum

(55 ± 5%) to slight or no toxicity. In the absence of cytotoxicity, the maximum dose level was selected as the highest dose level for scoring.

Based on the results obtained, the following concentrations were selected for the scoring of micronuclei:

- short treatment: 1250, 826 and 556 µg/mL (organic content) with Cytotoxicity 18, 7 and 11 % respectively;

- continuous treatment: 556, 371 and 247 µg/mL (organic content) with Cytotoxicity 47, 18 and 15 % respectively.

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells. Adequate cell proliferation was observed in negative control cultures and the appropriate number of doses and cells was analysed. Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controlsMitomycin-C and Colchicine, indicating the correct functioning of the test system. The study was accepted as valid.

Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the concurrent solvent control value was observed at any dose level, in any treatment series. All incidences were within the distribution of historical negative control (95% control limits) with the exception of the result obtained at the intermediate dose level (short treatment) and high dose level (continuous treatment). These values slightly exceeded the upper confidence limit, but fell within the historical control range and no concentration related increase was seen. Hence, this result was considered without any biological relevance.

It is concluded that the test item does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.