Hexane, 1,6-diisocyanato-, homopolymer, 2-hydroxyethyl acrylate- and propylene glycol monoacrylate-blocked

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 21, 2016 to August 02, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial forward mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Hexane, 1,6-diisocyanato-, homopolymer, 2-hydroxyethyl acrylate- and propylene glycol monoacrylate-blocked

EC Number:

806-984-5

Cas Number:

1392411-89-0

IUPAC Name:

Hexane, 1,6-diisocyanato-, homopolymer, 2-hydroxyethyl acrylate- and propylene glycol monoacrylate-blocked

Test material form:

liquid: viscous

Details on test material:

Name: Ebecryl 4738, Batch no.: DR0004393

Specific details on test material used for the study:

Batch no.: DR0004393; Purity: 100% (UVCB); Appearance: viscous liquid

Method

Species / strain

Species / strain / cell type:

other: S. typhimurium TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

The following nominal concentrations were prepared for experiment 1a:
50, 150, 500, 1500 and 5000 μg/plate
The following nominal concentrations were prepared for experiment 1b:
15, 50, 150, 500 and 5000 μg/plate

The following nominal concentrations were prepared for experiment 2 (from stock solutions 50 g/L and 15 g/L) for the strains TA98, TA100, TA102 and TA1535:
156, 313, 625, 1250, 2500 and 5000 μg/plate
The following nominal concentrations were prepared for experiment 2 for the strain TA97a:
47, 94, 188, 375, 750 and 1500 μg/plate

Vehicle / solvent:

DMSO was chosen as vehicle, because the test substance was sufficiently soluble in it, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations. The stock solution of 50 g/L was used to prepare the geometric series of the concentrations to be tested.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 2-Amino-Anthracene, 4-Nitro-1,2-phenylene Diamine

Details on test system and experimental conditions:

- Salmonella typhimurium (all strains used) were obtained from TRINOVA BioChem GmbH (batch of the bacteria strains: TA97a: 4997D, TA98: 5011D, TA100: 4996D, TA102: 4982D, TA1535: 5012D) and were stored as lyophilisates in the fridge at 2-8°C. The lyophilisates were used to prepare permanent cultures which were filled into vials and stored at < - 75°C. Eight hours before the start of each experiment, an aliquot of a permanent culture per strain to be used was taken from the deep freezer to inoculate a culture vessel containing nutrient broth. After incubation overnight for eight hours at 37 ± 1°C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.

- General preparation:
Per strain and dose, three plates with S9 and three plates without S9 mix were used. Top agar basis was melted in a microwave oven, after melting, 10 mL of histidine-biotinsolution 0.5 mM per 100 mL basis was added and the bottle was placed in the water bath at 43 ±1°C.

Plate incorporation method: the following materials were gently vortexed in a test tube and poured onto the selective agar plates:
- 100 μL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control);
- 500 μL S9 mix (see chapter 6.4.18, for test with metabolic activation) or phosphate
buffer (for test without metabolic activation);
- 100 μL bacteria suspension (see chapter 6.3.2, test system, culture of the strains)
- 2000 μL overlay agar (top agar);
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ±1°C.

Rationale for test conditions:

In the experiment 1a, the test substance caused cytotoxicity towards the strain TA97a and TA1535 in the highest concentration (5000 μg/plate). In experiment 1b, cytotoxicity was observed towards the strain TA97a in the highest concentration (5000 μg/plate) only.

Evaluation criteria:

The colonies were counted visually and the numbers were recorded. The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f (l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test substance solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given. A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Statistics:

A spreadsheet software (Microsoft Excel) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: S. typhimurium TA97a

Remarks:

number of revertant colonies was reduced in the absence and presence of metabolic activation in the highest concentration (5000 μg/plate)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Results for experiments 1a, 1b and 2.
- Confirmation of the Criteria and Validity:
All strains met the criterion of at least billion bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (historical data of the laboratory). All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
- Solubility and Toxicity:
In this experiment, the test substance showed no precipitates on the plates in none of the tested concentrations. The test substance showed signs of toxicity towards the bacteria strains TA97a and TA1535 in both the absence and presence of metabolic activation in the highest concentration (5000 μg/plate), only. The number of revertant colonies was reduced. In contrast to experiment 1a, TA1535 did not show toxicity in the highest concentration in experiment 1b, therefore the toxicity towards the strain TA1535 in experiment 1a was considered being due to an experimental error.
- Mutagenicity:
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found. Therefore, the test substance is stated as not mutagenic under the test conditions.
Due to the toxicity result, a further experiment was performed under the same conditions for the strains TA97a and TA1535.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance was not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. The study was performed using five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535). Three experiments (1a, 1b and 2) were performed in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254). Bacteria were exposed to test substance concentrations ranging from 15 μg/plate to 5000 μg/plate. In none of the experiments precipitation of the test substance was observed at any of the tested concentrations up to 5000 μg/plate. In the experiment 1a, the test substance caused cytotoxicity towards the strain TA97a and TA1535 at the highest concentration (5000 μg/plate). In experiment 1b, cytotoxicity was observed for the strain TA97a at the highest concentration (5000 μg/plate) only. No significant increase in the number of revertant colonies at any of the concentrations tested with and without metabolic activation could be observed. Under the study conditions the test substance was not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation (Andres, 2017).