Reaction mass of 2,2',2''-nitrilotriethanol and disodium succinate and sodium acetate and sodium bromide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-12-05 to 2013-05-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study comparable to OECD TG 487 (2010)

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD TG 487 (2010)

Deviations:

yes

Remarks:

see below

Principles of method if other than guideline:

A series of in-house non-GLP validation experiments was performed to get proper responses of statistical significance when using the specified positive controls. To achieve such response the test design, specifically for the treatment, the expression phase and harvest time, was slightly modified compared to the OECD Guideline 487. The optimum in responses was found with the following schedule, which was decided to be used in the main study:Experiment I: 4 hours pulse treatment (with and without metabolic activation), expression phase 16 hours, cytokinesis block 20 hours.Experiment II: 4 hours pulse treatment (with metabolic activation), expression phase 16 hours, cytokinesis block 20 hours. 20 hours continuous treatment (without metabolic activation), cytokinesis block 20 hours.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of CXN1-55

IUPAC Name:

Reaction mass of CXN1-55

Details on test material:

- Name of test material (as cited in study report): Reaction mass of CXN1-55- Substance type: multi-constituent substance- Physical state: Paste- Stability under test conditions: substance considered stable under normal ambient conditions- Storage condition of test material: at room temperature at 20 ± 5 °C, in the dark.

Method

Target gene:

The occurrence of micronuclei in interphase blood lymphocytes provides an indirect but easy and rapid measure of chromosomal damage and aneugenicity.

Species / strain

Species / strain / cell type:

lymphocytes: blood samples

Details on mammalian cell type (if applicable):

- Type and identity of media: Blood samples were obtained from healthy, non-smoking donors not receiving medication. For this study, blood was collected from a female donor (24 years old) for Experiment I and from a female donor (34 years old) for Experiment II. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes. Blood samples were drawn by venous puncture and collected in heparinized tubes by Dr. V. Theodor (64380 Rossdorf, Germany). The tubes were sent to Harlan CCR to initiate cell cultures within 24 hrs after blood collection. The blood was stored before use at 4 °C.- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 obtained from phenobarbital/beta-naphthoflavone induced rats were used as the metabolic activation system.

Test concentrations with justification for top dose:

Experiment I: 32.5, 56.8, 99.5, 174.1, 304.6, 533.1, 932.9, 1632.7, 2857.1, 5000.0 µg/mLExperiment II: 1632.7, 2857.1, 5000.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water- Justification for choice of solvent/vehicle: test item soluble in water

Details on test system and experimental conditions:

METHOD OF APPLICATION: About 48 hours after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test group.DURATION- Preincubation period: approx. 48 hours- Exposure duration: 4 hours or 20 hours- Expression time (cells in growth medium)/Fixation time (start of exposure up to fixation or harvest of cells):4 hours of exposure + 16 hours of recovery + 20 hours with Cytochalasin B (4 µg/mL)20 hours of exposure + 20 hours with Cytochalasin B (4 µg/mL)SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B (4 µg/mL)STAIN (for cytogenetic assays): Giemsa (MERCK, 64293 Darmstadt, Germany)NUMBER OF REPLICATIONS: 2 per treatment and per sampling dateNUMBER OF CELLS EVALUATED: 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.DETERMINATION OF CYTOTOXICITY- Method: Cytokinesis-block proliferation index

Evaluation criteria:

A test item can be classified as non-mutagenic if:1. the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory historical control data and/or2. no statistically significant or concentration-related increase in the number of micronucleated cells is observed.A test item can be classified as mutagenic if:1. the number of micronucleated cells is not in the range of the historical laboratory control data and2. either a concentration-related increase of micronucleated cells in three test groups or a statistically significant increase of the number of micronucleated cells is observed.

Statistics:

Statistical significance was confirmed by means of the Chi square test. However, both biological and statistical significance should be considered together. If the criteria for the test item mentioned above are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: noExp. I:Solvent control 7.65000 mg/L 7.6Exp. II:Solvent control 7.65000 mg/L 7.6- Effects of osmolality: noExp. I:Solvent control 282 mOsm5000 mg/L 355 mOsm/L2857.1 mg/L 329 mg/L1632.7 mOsm/LExp. II:Solvent control 283 mOsm5000 mg/L 355 mOsm/L2857.1 mg/L 326 mg/L- Evaporation from medium: not relevant- Water solubility: soluble up to and including 5 mg/mL- Precipitation: not observed- Other confounding effects:noRANGE-FINDING/SCREENING STUDIES:A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of reduction in the CBPI in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate. The experimental conditions in this pre-experimental phase were identical to those required and described for the mutagenicity assay. The pre-experiment was performed with 10 concentrations of the test item and the solvent and positive controls. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix) and the cells were prepared 40 hrs after start of the exposure.COMPARISON WITH HISTORICAL CONTROL DATA: yes

Applicant's summary and conclusion

Conclusions:

In a valid, reliable and conclusive study according to OECD TG 487 (2010), the test item Reaction mass of CXN1-55 did not induce micronuclei in human lymphocytes in vitro when tested up to the highest required concentration under the experimental conditions. Therefore, Reaction mass of CXN1-55 is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

Executive summary:

In a valid, reliable and conclusive study according to OECD TG 487 (2010), the test item Reaction mass of CXN1-55, dissolved in deionised water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments. The following study design was used:

	Without S9-Mix		With S9-Mix
	Exp. I	Exp. II	Exp. I and II
Exposure period	4 hrs	20 hrs	4 hrs
Recovery	16 hrs	-	16 hrs
Cytochalasin B exposure	20 hrs	20 hrs	20 hrs
Preparation interval	40 hrs	40 hrs	40 hrs
Total culture period	88 hrs	88 hrs	88 hrs

In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were evaluated for cytogenetic damage. No precipitation of the test item in the culture medium was observed.

No relevant cytotoxicity, indicated by reduced CBPI and described as cytostasis, could be observed up to the highest applied concentration either with or without metabolic activation.

The highest applied concentration in Experiment I (5000.0 µg/mL of the test item) was chosen with respect to the current OECD Guideline 487.

In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. In Experiment I with S9 mix two statistically significant increases (0.90 and 1.20 % micronucleated cells) were observed after treatment with 2857.1 and 5000.0 µg/mL. Since these values are clearly in the range of the historical solvent control data (0.20 – 1.70 % micronucleated cells) the findings are not biologically relevant.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.