2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Nourycryl MA128 was not mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the TK mutation test with L5178Y mouse lymphoma cells. The in vitro MN resulted to positive responses, albeit at levels close to high cytotoxicity and showing precipitation. Subsequent evaluation in an in vivo MN study confirmed lack of cytogenic activity in vivo.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 1 December 1986 and 12 December 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Chemical name: Methacrylic acid , 2-(1 oxa-4-aza spiro [4 , 5] dec-4 -yl) ethyl ester
Commercial name/code: Nourycryl MA 128
Batch no . 860930 - DT 4
Storage: At ambient temperature in the dark in the presence of silica gel
Appearance: clear slightly yellow liquid

Target gene:

histidine locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 from homogenised liver of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Preliminary toxicity test:
1.0, 3.3, 10.0, 33.3, 100, 333, 1000, 3330, 5000 µg/plate
5000 μg/plate is the maximum test substance concentration that should be used, according to the OECD guidelines

Mutagenticity test (experiment 1 and 2)
100, 333, 1000, 3330 and 5000 µg/plate
In the preliminary test, the highest dose tested caused no increase in toxicity

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

1 µg/plate for strain TA1535

Positive control substance:

sodium azide

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

60 µg/plate for strain TA1537

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/plate for strain TA1538

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

2 µg/plate for strain TA98

Positive control substance:

other: daunomycine

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

650 µg/plate for strain TA100

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

0.5 µg/plate for all strains

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Bacterial cultures
Samples of frozen stock cultures of bacteria are transferred into enriched nutrient broth (Oxoid No.2) and incubated in a shaking water bath (37 °C, 150spm) until the cultures reach an O.D. of 0.4 at 700 nm (10^9 cells /ml) . Freshly grown cultures of each strain are used for a test.

Test procedure
Standard plate test
Top agar in top agar tubes is melted and heated to 45ºC. The following solutions are succesively added to 3 ml of top agar: 0.1ml of a fresh bacterial culture (10^9 cells/ml) of one of the tester strains, 0.1ml of a dilution of the test substance in DMSO, and in the case of activation of assays 0.5ml of S9-mix. The ingredients are mixed in a vortex and the contents of the top agar tube are poured onto a selective agar plate. After solidifcation of the top agar, the plates are turned and incubated in the dark at 37ºC for 48h. After this period revertant colonies (histidine independent) are counted automatically with an Artek model 880 colony counter or manually.
Each concentration was tested in triplicate.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the Ames test if:
a) The total number of revertants in any tester strain ay any concentration is not greater than two times the solvent control value with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) is the Ames test if:
a) it induced at least a 2-fold increase in the number of revertants with respect to the number induced by the solvent control in any of the test strains, either with or without metabolic activation. Moreover, the positive response shouls be dose related. If the test substance shows in the first test only a positive response at one or two concentrations, the assay is repeated with doses just below and exceeding those showing positive effects in the first test.
b) The positive response should be reproducible in at least one independently repeated experiment

The preceding criteria are not absolute and other extenuating factors may enter into the final evaluation decision.

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:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

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:

not applicable

Positive controls validity:

valid

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:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary test
Nine serial dilutions of the test substance, in approximately half-log steps, were plated with an appropriately diluted TA100 culture (equal numbers of bacterial cells/plate) into non-selective agar (viability determination). The percentage survival of the TA100 culture is determined by comparing the number of colonies on the solvent control plate with those on the plates containing the test substance. However, even at the highest test substance concentration used the survival of strain TA100 is not reduced. Based on these data, the test substance was tested up to a concentration of 5000 µg/plate.


Main test
All bacterial strains showed negative responses over the entire dose range of the test substance, i.e. no dose-related increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values fell within laboratory background historical ranges, indicating that the test conditions were optimal and that the metabolic activation system functioned properly. Based on these results, the test substance can be considred as not mutagenic in the Ames Salmonella/microsome assay.

Conclusions:

All bacterial strains showed negative responses over the entire dose range of the test substance, i.e. no dose-related increase in the number of revertants in two independently repeated experiments. Therefore, the test substance can be considered as not mutagenic in the Ames Salmonella/microsome assay.

Executive summary:

Nourycryl MA128 was tested in the Ames Salmonella/microsome test up to 5000 µg/plate. The test substance induced no dose-related increase in the numbers of revertant (His+) colonies in each of the five tester strains (TA1535, TA1537, TA1538, TA98 and TA100). These results were confirmed in an independently repeated experiment. The test substance can, therefore, be considered as non mutagenic in this test system.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 23 November 2016 Experimental completion date 24 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This report describes the results of an in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Identification: 2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate
Sponsors Identification: Nourycryl MA128
Supplier: Akzo Nobel Functional Chemicals b.v.
Product Number: 436661
EC Name: 2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate
CAS Number: 4203-89-8
Chemical Name: 2-(2,2-pentamethylene-1,3-oxazolidyl-3)ethyl methacrylate
Description: Extremely pale yellow slightly viscous liquid
Purity/concentration: 94.5% 2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl 2-methylprop-2-enoate and 1.4% water.
Batch Number: 1609501020
Date of Expiry: 10 May 2017
Storage conditions: Approximately 4 °C in the dark

Target gene:

Nuclei of normal human lymphocytes

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:
Preliminary Toxicity Test: male, aged 27 years
Main Experiment: male, aged 27 years
Confirmatory Experiment: male, aged 27 years

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM).

Test concentrations with justification for top dose:

Three exposure groups were used for Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest. The dose range of test item used was 0, 250, 375, 500, 750, 1000, 1500, and 2000 µg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest. The dose range of test item used was 0, 250, 375, 500, 750, 1000, 1500 and 2000 µg/mL.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest. The dose range of test item used was 0, 62.5, 125, 250, 375, 500, 750 and 1000 µg/mL.

Vehicle / solvent:

Dimethyl sulphoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.2 µg/mL for 4-hour exposure

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Demecolcine (DEME-C)

Remarks:

Absence of S9 mix: 0.075 µg/mL for 24-hour continuous exposure

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

5 g/mL for 4-hour exposurePresence of S9 mix:

Details on test system and experimental conditions:

The molecular weight of the test item was given as greater than 200, therefore, the maximum dose level was 2000 µg/mL, which was the maximum recommended dose level. The purity of the test item was 94.5% and was not accounted for in the test item formulations at the request of the Sponsor.
The test item was immiscible in aqueous media at 20 mg/mL but was fully miscible in DMSO at 200 mg/mL in solubility checks performed in house. Prior to each experiment, the test item was accurately weighed, formulated in DMSO and serial dilutions prepared.
The solubility of the test item was investigated in the Envigo Research Limited Mouse Lymphoma Assay, Study number RX39DH.
There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991). The osmolality and pH of the test item was investigated in the Envigo Research Limited Mouse Lymphoma Assay, Study number RX39DH.

Rationale for test conditions:

The test item was formulated within two hours of it being applied to the test system; it is assumed that the test item formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Evaluation criteria:

Please see section below "Any other information on materials and methods"

Statistics:

The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible.

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in the absence of metabolic activation. A small increase was noted in the 4-hour exposure group at 1500 µg/mL but this increase was just within the upper range of the in-house historical control range. Any increase in the frequency of binucleate cells within the in-house control data the test item is considered to be negative, particularly if all of the criteria for a valid assay are met.

However, in the presence of S9, the test item induced statistically significant increases in the frequency of binucleate cells with micronuclei over two separate experiments. In the Main Test, an increase was observed at 1500 µg/mL in the A culture but not in the B culture due to poor quality slides. Therefore, new slides from both cultures were prepared and analyzed.

In deviation to the General Study Plan 2000 cells were assessed for micronuclei in the cultures detailed above after a discrepancy in the results obtained between both cultures. This change to the number of cells assessed was undertaken due to the poor quality slides initially scored, where new slides of improved quality were prepared for analysis. The second analysis evened the response between the cultures and the resulting mean micronuclei value was in excess of the in-house control range, indicating a positive response. Therefore a Confirmatory Experiment was performed using a tightened dose range in an effort to confirm the response. The result of this experiment returned statistically significant dose related increases in all the dose levels analyzed confirming the reproducibility of the response in the initial experiment. It should be noted that the response in both experiments is occurring on or around the optimum level of toxicity and may be driven by a cytotoxic mechanism rather than a genotoxic mechanism.

It should be noted that the test item was also investigated in a Mouse Lymphoma Assay (MLA) Envigo project number RX39DH where similar dose levels were employed using similar exposure conditions. During the performance of the MLA test at the end of the exposure periods the cultures are maintained for 48-hours during an expression period where cells exposed to toxic dose concentrations are lost. The result of the MLA study was negative with no mutagenic response being observed at dose levels with acceptable levels of toxicity. There was a marked increase in mutant frequency at 1500 µg/mL in the presence of metabolic activation. However, this was excluded from the analysis because the toxicity observed exceeded the maximum acceptable level for the study. The response observed in the MLA study in the presence of metabolic activation is also observed at around the optimum toxicity levels and may also be driven by a cytotoxic mechanism rather than a genotoxic mechanism.

Remarks on result:

other: Negative when in the absence of S9

 PLEASE NOTE, ALL TABLES ARE PROVIDED AS AN ATTACHMENT

ppreliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was 7.81 to 2000 µg/mL. The maximum dose was the maximum recommended dose level. 

A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 1000 µg/mL in the 4-hour exposure group in the presence of metabolic activation (S9) only. No precipitate in the blood-free cultures were observed in the absence of S9, however, precipitate was observed in the blood cultures at and above 1000 µg/mL and at 2000 µg/mL in the 24-hour continuous exposure and 4-hour exposure groups, respectively. The culture observations are presented inthe table below where precipitate from either culture type has been designated with the same symbol (P).

                       

Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 2000 µg/mL in the 4-hour exposure, both in the presence and absence of S9. The maximum dose with binucleate cells present in the 24-hour continuous exposure was 500 µg/mL.  The test item induced some evidence of toxicity in human lymphocytes.

The selection of the maximum dose level for the Main Experiment was based on the maximum recommended dose level for the 4-hour exposure groups and was limited by toxicity for the 24-hour exposure group; however, the onset of precipitate also coincided with these observations.

Micronucleus Test – Main Experiment

The dose levels of the controls and the test item are given in the table below:

Group	Final concentration of test item2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate (CAS 4203-89-8)(µg/mL)
4-hour without S9	0*, 250, 375, 500, 750*, 1000*, 1500*, 2000, MMC0.2*
4-hour with S9 (2%)	0*, 250, 375, 500, 750*, 1000*, 1500*,2000, CP5*
24-hour without S9	0*, 62.5, 125, 250*, 375*, 500*, 750, 1000,DC0.075*

The qualitative assessment of the slides determined that the toxicity was slightly more severe to that observed in the Preliminary Toxicity Test and that the onset of toxicity coincided with the onset of precipitate in the 4-hour exposure groups. Despite this, there were binucleate cells suitable for scoring at the maximum dose level of test item, 2000 µg/mL. In the 24-hour continuous exposure group, there were binucleates suitable for scoring up to 500 µg/mL. 

Precipitate of test item was noted at and above 1500 µg/mL for the 4-hour exposure groups and at 1000 µg/mL in the 24-hour continuous exposure group.

The CBPI data for the short exposure groups and for the 24-hour exposure group are given inTable 2andTable 3, respectively. They confirm the qualitative observations in that a dose‑related inhibition of CBPI was observed in all three exposure groups.

In the absence of S9, the 4-hour exposure group observed 12%, 22% and 58% cytostasis at 750, 1000 and 1500 µg/mL, respectively. Above this dose level, there were not enough binucleates available for analysis. Therefore the maximum dose level selected for analysis of binucleate cells was 1500 µg/mL, which was optimum toxicity as defined by the OECD 487 Guideline (55±5%). 

In the 24-hour exposure group, 14%, 38% and 49% cytostasis was observed at 250, 375 and 500 µg/mL, respectively. Above this dose level, there were no binucleates available for analysis. Therefore the maximum dose level selected for analysis of binucleate cells was 500 µg/mL, because this dose was only slightly below the lower range for optimum toxicity as defined by the OECD 487 Guideline (55±5%).

 

In the presence of S9, a steeper dose-related inhibition of CBPI was observed with 8%, 9% and 41% cytostasis observed at 750, 1000 and 1500 µg/mL, respectively. Above this dose level, there were not enough binucleates available for analysis. Therefore the maximum dose level selected for analysis of binucleate cells was 1500 µg/mL, because this dose was the lowest precipitating dose level, it was below the lower range for optimum toxicity as defined by the OECD 487 Guideline (55±5%) and because there were not enough binucleates available for analysis at the dose level above (2000 µg/mL).

The micronucleus data are given inTable 5,Table 6andTable 7. The vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in the absence of metabolic activation. However, there was a statistically significant increase in the frequency of binucleate cells with micronuclei in the presence of metabolic activation at 1500 µg/mL, even after an additional 1000 cells had been analyzed due to poor quality slides.

 Micronucleus Test – Confirmatory Experiment

Due to the results of the Main Experiment, a Confirmatory Experiment was performed in the presence of S9-mix only to see if the initial response is reproducible.

The dose levels of the controls and the test item are given in the table below:

Group	Final concentration of test item2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate (CAS 4203-89-8)(µg/mL)
4-hour with S9 (2%)	0*, 250, 500, 1000, 1500*, 1600*, 1700*, CP5*

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Main Experiment and that the onset of toxicity coincided with the onset of precipitate with binucleate cells suitable for scoring at the maximum dose level of test item, 1700 µg/mL.

A precipitate of test item was noted at the end of exposure at 1700 µg/mL, withhemolysis observed at and above 250µg/mL. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.

The CBPI data for the 4-hour exposure group in the presence of S9 in provided inTable 4and confirm the qualitative observations in that a moderate dose‑related inhibition of CBPI was observed where 32%, 45% and 65% cytostasis observed at 1500, 1600 and 1700 µg/mL, respectively. These dose levels were selected for binucleate analysis because they were the maximum dose levels and they exhibited a moderate increase in cytostasis. Therefore the two maximum dose levels selected for analysis of binucleate cells were 1600 and 1700 µg/mL, because these doses were below and above the range for optimum toxicity as defined by the OECD 487 Guideline (55±5%).

The micronucleus data are given inTable 8. The vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive control item induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Again, there was a statistically significant and dose related increase in the frequency of binucleate cells with micronuclei in the presence of metabolic activation at all three dose levels analyzed.

*              = Dose levels selected for analysis of micronucleus frequency in binucleate cells

MMC = Mitomycin C

CP            = Cyclophosphamide

DC          = Demecolcine

CP            = Cyclophosphamide

                 

Conclusions:

The test item, 2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate (CAS 4203-89-8), did not induce a statistically significant increase in the frequency of binucleate cells with micronuclei in the absence of a metabolizing system. However in the presence of the metabolizing system reproducible, dose related and statistically significant increases were observed. The test item was therefore considered to be mutagenic to human lymphocytes in vitro.

Executive summary:

 Summary

 

Introduction

This report describes the results of anin vitrostudy for the detection of the clastogenic and aneugenic potential of the test item,2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate (CAS 4203-89-8)on the nuclei of normal human lymphocytes. 

 

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls. Up to three exposure conditions over two experiments were used for the study. The Main Experiment was performed using a 4‑hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. A confirmatory Study was performed usinga 4‑hour exposure in the presence of S9 (2%) using a tighter dose range. At the end of the exposure period in both experiments, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

 

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited on both precipitate and/or toxicity, depending on the exposure group. The dose levels selected for the Main Test were as follows:

Group	Final concentration of test item2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate (CAS 4203-89-8)(µg/mL)
4-hour without S9	0, 250, 375, 500, 750, 1000, 1500, 2000
4-hour with S9 (2%)
24-hour without S9	0, 62.5, 125, 250, 375, 500, 750, 1000

 

A Confirmatory Experiment was conducted after the results of the Main Experiment. The maximum dose level was limited by both precipitate and toxicity. The dose levels selected for the Confirmatory Experiment were as follows:

 

Group		Final concentration of test item2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate (CAS 4203-89-8)(µg/mL)
4-hour with S9 (2%)		0, 250, 500, 1000, 1500, 1600, 1700

Results

All vehicle (dimethyl sulphoxide (DMSO)) controls from both experiments had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.

 

The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validatedover both experiments.

 

In the Main Experiment, the test item was moderately toxic to human lymphocytes in all three exposure groups. In the absence of metabolic activation, the test item did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that induced approximately 50% reduction in CBPI. However, in the presence of metabolic activation, the test item did induce a statistically significant increase in the frequency of cells with micronuclei, using a dose range that included a dose level that approximately 50% reduction in CBPI.

 

Therefore, a Confirmatory experiment was performed in an effort to confirm and/or reproduce the result of the Main Experiment. Again,statistically significant increases were observed in the frequency of cells with micronuclei using a dose range that included dose levels that were slightly below and above optimum toxicity.

 

Conclusion

The test item,2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate (CAS 4203-89-8)was considered to be mutagenic to human lymphocytesin vitro.

However, some comments can be made to these. Precipitation has been observed from concentrations of 1000 μg/mL and too high cytotoxicity (more than recommended reduction in CBPI to 45%) was observed above 1500 μg/mL. All positive increases have been observed at 1500 μg/mL and one at 1600 μg/mL (only in confirmatory experiment with S9) the highest possible concentration in view of toxicity. And combining all data in a graph (see attached) shows that there are actually not very significant differences between all experiments.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 10 November 2016 Experimental Completion Date: 06 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian gene mutation assay

Specific details on test material used for the study:

Identification: 2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate
Sponsors Identification: Nourycryl MA128
Supplier: Akzo Nobel Functional Chemicals b.v.
Product Number: 436661
Description: Extremely pale yellow slightly viscous liquid
Purity/concentration: 94.5% 2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl 2-methylprop-2-enoate and 1.4% water. No correction made for purity
Batch Number: 1609501020
Date of Expiry: 10 May 2017
Storage conditions: Approximately 4 °C in the dark

Target gene:

thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Cell line
The L5178Y TK +/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit in Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time. The L5178Y cell line has successfully been used in in vitro experiments for many years. L5178Y cells are characterized by a high proliferation rate (doubling time 10 - 12 h in stock cultures) and cloning efficiency of untreated cells of usually more than 50 %, both characteristics are necessary for the appropriate performance of the study. The stock of cells is stored in liquid nitrogen at approximately -196 °C. Master stocks of cells were tested and found to be free of mycoplasma.

Cell culture
The culture medium used will be RPMI 1640 with Glutamax-l and 20mM HEPES buffer supplemented with Penicillin (100 units/ml), Streptomycin (100 µg/ml), Sodium Pyruvate (1mM) and Amphotericin B (2.5 µg/mL) giving R0 media.
For use, a sample of cells will be removed from cryogenic storage before the start of the study and grown in R0 media supplemented with 10% horse serum (known as R10 media) at approximately 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and are sub-cultured accordingly. R10 media is used for general maintenance of cells and cell washing at the end of the test item exposure period. R20 media, (R0 with 20% horse serum) is used for the seeding of viability and mutant frequency plates and for maintenance of cultures during the expression period. The serum concentration during the exposure period will be 5% for the 4-hour groups and 10% for the 24 hour group.

Cell cleansing
TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before a stock of cells is frozen these homozygous mutants (TK -/-) must be removed. The cells are cleansed of mutants by culturing in THMG medium for 24 hours. This medium contains Thymidine (9 µg/mL), Hypoxanthine (15 µg/ml), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24 hours the cells are cultured in THG medium (THMG medium without Methotrexate) before being returned to R10 media. The "cleansed" cells are frozen in 1 ml aliquots at between 1 to 3 x 106 cells/mL and stored in liquid nitrogen freezers at approximately –196 °C. A fresh ampoule is removed from the frozen stock and cultured to provide adequate numbers of cells for testing.

Metabolic activation:

with and without

Metabolic activation system:

S9 microsomal fraction from rats ineduced with Phenobarbital/B-Naphthoflavone

Test concentrations with justification for top dose:

The test item was a molecular weight of greater than 200 and therefore the maximum dose level in the solubility test was set at 2000 µg/mL, with no correction for purity (94.5%).

Preliminary test: 0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/ml

Main experiment (4-hour exposure, with and without S9-mix): 0, 31.25, 62.5, 125, 250, 500, 1000, 1250, 1500, 1750 and 2000 µg/ml
Main experiment (24-hour exposure, without S9-mix): 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 187.5, 250, 375 and 500 µg/ml

Vehicle / solvent:

DMSO was chosen as the vehicle following solubility checks

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

400 µg/ml and 150 µg/ml for 4-hour and 24-hour exposures respectively

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activtion

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

1.5 µg/ml

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 7.81 to 2000 µg/mL for all three of the exposure groups. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted using a Coulter counter and then serially diluted to 2 x 10^5 cells/mL.

The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 10^5 cells/mL. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.

Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:

i) Maximum recommended dose level, 2000 µg/mL or 10 mM.

ii) The presence of excessive precipitate where no test item-induced toxicity was observed.

iii) Test item-induced toxicity, where the maximum dose level used should produce approximately 10 to 20% survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al 2002).


Mutagenicity Test
Main Experiment
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. For the 4-hour exposure groups both with and without metabolic activation, the cells were counted and processed to give 1 x 10^6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals. In the 24-hour exposure in absence of metabolic activation the cells were processed to give 0.3 x 10^6 cells/mL in 10 mL cultures established in 25 cm2 tissue culture flasks. The treatments were performed in duplicate (A + B), at up to 10 dose levels of the test item (31.25 to 2000 µg/mL in the 4-hour exposure groups and 3.91 to 500 µg/mL in the 24-hour exposure), vehicle and positive controls. To each universal was added 2 mL of S9-mix (2%) if required, 0.2 mL of the treatment dilutions, (0.15 or 0.2 mL for the positive control) and sufficient R0 medium to bring the total volume to 20 mL (R10 is used for the 24-hour exposure group). The treatment vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

Measurement of Survival, Viability and Mutant Frequency
At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures were incubated at 37 °C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained.

On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5 trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.

The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Plate Scoring
Microtitre plates were scored using a magnifying mirror box after ten to fourteen days’ incubation at 37 °C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded (Cole et al., 1990). Colonies are scored manually by eye using qualitative judgement. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for approximately two to three hours. MTT is a vital stain that is taken up by viable cells and metabolized to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.

Evaluation criteria:

Interpretation of Results
The normal range for mutant frequency per survivor is 50-170 x 10-6 for the TK+/- locus in L5178Y cells at this laboratory. Vehicle controls results should ideally be within this range, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 200 x 10^6 mutant frequency per survivor are not normally acceptable and will be repeated.

Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.

Dose levels that have RTG survival values markedly less than 10% are excluded from any statistical analysis, as any response they give would be considered to have no biological or toxicological relevance.

For a test item to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. A Global Evaluation Factor (GEF) value of 126 x 10^6 was set for the microwell method. Therefore, any test item dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10^6 and demonstrates a positive linear trend will be considered positive. However, if a test item produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test item induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Cytotoxicity Test
In the 4-hour exposures, both in the absence and presence of metabolic activation (S9), there was evidence of marked reductions in the relative suspension growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. In the 24-hour exposure in the absence of S9 there was evidence of marked reductions of %RSG values of cells treated with test item. In both 4-hour exposures a precipitate of the test item was observed at 2000 µg/mL at the end of exposure. In the 24-hour exposure in the absence of metabolic activation a precipitate was not observed at the end of exposure. In the subsequent mutagenicity experiments the maximum dose was set at the maximum recommended dose level of 2000 µg/mL, with the knowledge of possible limitations due to precipitate in the 4-hour exposure groups. In the 24-hour exposure group the maximum dose level was set at 500 µg/mL.

Mutagenicity Test
4-hour exposure in the absence and presence of metabolic activation
There was evidence of toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. There was evidence of marked reductions in viability (%V); therefore indicating that residual toxicity had occurred. Optimum levels of toxicity were considered to have been achieved in both 4-hour exposure groups. The excessive toxicity observed at and above 1500 µg/mL in the absence of metabolic activation and at and above 1750 µg/mL in the presence of metabolic activation resulted in these dose levels not being plated for viability or 5-TFT resistance. The dose level at 1250 µg/mL in the absence of metabolic activation and at 1500 µg/mL in the presence of metabolic activation were plated out, but later excluded form analysis due to excessive toxicity. Acceptable levels of toxicity were seen with both positive control substances.

Neither of the vehicle control mutant frequency values were outside the normal in-house range of 50 to 170 x 10^6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The test item induced a very small statistically significant dose related (linear-trend) increase in the mutant frequency in both exposure groups. However, the GEF value was not exceeded in any of the analyzable dose levels, and of these dose levels, all were within the acceptable range for a vehicle control. Therefore the response is considered to be artefactual and of no toxicological significance

24-hour exposure in the absence of metabolic activation

There was evidence of toxicity following exposure to the test item as indicated by the %RSG and RTG values. There was no evidence of reductions in viability (%V), therefore indicating that residual toxicity had not occurred in this exposure group. Optimum levels of toxicity were achieved based on the %RSG value. The excessive toxicity observed at and above 375 µg/mL resulted in these dose levels not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with the positive control substance.

The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had a marked effect on the toxicity of the test item. The vehicle control mutant frequency was within the normal range of 50 to 170 x 10^6 viable cells. The positive control produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily.

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency in the 24-hour exposure in the absence of metabolic activation. The GEF value was not exceeded at any test item dose level, and all of the mutant frequency values were within the acceptable range for a vehicle control.

Summary of Results

Main Experiment

Treatment (µg/ml)		4-hours-S-9				Treatment (µg/ml)		4-hours+S-9
		%RSG	RTG	MF§				%RSG	RTG	MF§
0		100	1.00	104.66		0		100	1.00	96.56
31.25	Ø	95				31.25	Ø	88
62.5		94	1.00	99.65		62.5	Ø	91
125		92	1.12	86.65		125		93	1.18	78.35
250		89	1.02	78.15		250		82	0.86	108.16
500		63	0.68	109.87		500		69	0.78	108.18
1000		27	0.19	157.92	*	1000		40	0.46	101.40
1250	X	17	0.09	219.59		1250		23	0.18	178.44	*
1500	Ø	6				1500	X	15	0.07	339.35
1750	Ø	3				1750	Ø	5
2000	Ø	1				2000	Ø	2
Linear trend				**		Linear trend				**
EMS						CP
400		64	0.63	1113.05		1.5		64	0.54	764.84
Treatment (µg/ml)		24-hours-S-9
		%RSG	RTG	MF§
0		100	1.00	129.67
3.91	Ø	94
7.81	Ø	90
15.63		94	1.08	112.43
31.25		81	1.04	117.52
62.5		70	0.88	115.32
125		51	0.73	111.86
187.5		37	0.61	107.85
250		16	0.32	142.58
375	Ø	1
500	Ø	0
Linear trend				NS
EMS
150		49	0.43	1306.35

RSG = Relative suspension growth rate

RTG =  Relative Total Growth

MF = Mutation Frequency

Conclusions:

The test item, 2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate (CAS 4203-89-8) did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10^6, consequently it is considered to be non-mutagenic in this assay.

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In VitroMammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

 

 

Methods…….

One main experiment was performed. In the main experiment, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at up to 10 dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4‑hour exposure groups both in the absence and presence of metabolic activation (2% S9) and a 24-hour exposure in the absence of metabolic activation.

 

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated out for expression of mutant colonies were as follows:

 

Main Experiment

Group	Concentration of 2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate (CAS 4203-89-8) (µg/mL)  plated for mutant frequency
4-hour without S9	62.5, 125, 250, 500, 1000, 1250
4-hour with S9 (2%)	125, 250, 500, 1000, 1250, 1500
24-hour without S9	15.63, 31.25, 62.5, 125, 187.5, 250

 

 

Results……..

The maximum dose level analyzed was limited by a combination of test item induced toxicity, and precipitate in the 4 hour exposure groups. In the 24-hour exposure group the maximum dose level analyzed was limited by test item induced toxicity. At the end of exposure a precipitate of the test item was observed at 1750 and 2000 µg/mL in the 4-hour exposures. No precipitate was observed at the end of exposure in the 24-hour exposure group. The vehicle (solvent) controls had acceptable mutant frequency values that were within the acceptable range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

 

The test item did not induce any toxicologically significant or dose-related (linear-trend) increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

 

Conclusion

The test item 2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate (CAS 4203-89-8), did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded theGlobal Evaluation Factor (GEF) of 126 x 10^-6, consequently it is considered to be non-mutagenic in this assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Evaluation in an in vivo MN study (OECD 474) confirmed lack of cytogenic activity in vivo.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 21 JAN 2022 to 1 MAR 2022

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Han Wistar
Charles River (UK) Ltd., Margate, UK / Envigo, Blackthorn, UK (as available).

Sex:

male

Details on test animals or test system and environmental conditions:

Age range: 7-9 weeks on the first day of dosing
Approximate supplier weight range for 7-9 week animals:
Males: 175-375 g
Females: 125-250 g
RF in 3 male and 3 female showed no substantial differences in toxicity between the sexes, and male animals were selected for the main study.

Environment:
Housing: Wire topped, solid bottom cages.
Cages conforming to the 'Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes’ (Home Office, London, 2014).
Housing Density Up to three animals per cage, with sexes separated.
Target Temperature Range: 19 to 25°C
Target Humidity Range: 40 to 70%
Air Changes: minimum of 15 air changes/hour.
Photo-Period 12 hours nominal.

Diet: Ad libitum access to 5LF2 EU Rodent Diet.
Water: Ad libitum access to mains water via water bottles.
Bedding: Suitable wood bedding (Aspen) changed weekly.
Environmental Enrichment: Wooden Aspen chew blocks and rodent retreats.
Analysis and Certification:
- Diet and bedding - per batch (reviewed prior to use).
- Water – periodic analysis.
- Environmental enrichment – as available.
No contaminants are expected to be present in diet, water or bedding at levels which might interfere with achieving the objective of the study. Results of analyses performed on diet, water, bedding and environmental enrichment are held centrally at Labcorp Laboratories.

Route of administration:

oral: gavage

Vehicle:

Arachis Oil (The vehicle was used with this compound in previous in vivo studies. )

Details on exposure:

Method of Preparation: Developed at Labcorp.
Frequency of Preparation: Once.
Stability: The test article formulation in vehicle at 12.5 and 250 mg/mL is stable for 20 days when stored at 2 to 8°C, protected from light (Envigo Study number RM34VX).
Homogeneity: The test article formulation in vehicle is homogeneous at 3.75 and 250 mg/mL (Envigo Study number RM34VX).
Storage Conditions in Dispensary: 2 to 8°C, protected from light.

Dose levels:
RF: (3 male & 3 female): 2000 mg/kg /day, in 10 mL/kg
Main Experiment in 6 males/group: 0, 500, 1000 and 2000 mg/kg/day; in 10 mL/kg

Oral by gavage (unfasted animals). Two administrations, at 0 and 24 hours.

Duration of treatment / exposure:

Two administrations on day 1 and day 2 (at 0 and 24 hours); necropsy day 3 (48 hours)

Frequency of treatment:

Two administrations, at 0 and 24 hours.

Post exposure period:

Animals sampled at approximately 48 hours.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle control

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

6 males/group

Control animals:

yes, concurrent vehicle

Positive control(s):

Three positive control slides from animals dosed with cyclophosphamide (prepared under Labcorp study 8449759) were stained and analysed alongside the study slides.
These slides were used to verify adequate slide staining of the micronuclei and confirm the ability of the slide analyst to detect a positive response within a coded system.

Tissues and cell types examined:

One femur were removed and bone marrow isolated from all animals at scheduled kill.
Slides were prepared from cell suspension from bone marrow.

Details of tissue and slide preparation:

Bone Marrow Isolation: Flushed from femur with foetal bovine serum.
Sample Filtration: Additional foetal bovine serum was added to sample prior to filtration through cellulose column (Sun et al., 1999).
Cell Suspension: Cells pelleted by centrifugation. Supernatant decanted and pellet resuspended in serum. Centrifugation repeated and majority of supernatant decanted.
Number of Slides: At least 3.
Slide Preparation: Pellet gently resuspended in remaining serum. Smears made on microscope slides and air-dried.
Fixation: Absolute methanol.
Staining: At least 1 slide per animal stained, with remaining slides held as reserves.
Fixed slides are rinsed in distilled water, then stained in 12.5 µg/mL acridine orange.
Stained slides were rinsed in phosphate buffer, air-dried and stored in the dark at room temperature prior to analysis.

Statistics:

MN PCE in the test article treated groups (Groups 2 to 4) and from the positive control slides are compared against the vehicle control group (Group 1) using ranks of the data via the Wilcoxon Rank Sum Test (Lehmann, 1975).
The tests are interpreted with one-sided risk for increased frequency with increasing dose.
A Terpstra-Jonckheere test will be conducted to evaluate dose response (Jonckheere, 1954).
Probability values of p≤0.05 is accepted as significant.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

Tested up to maximum dose of 2000 mg/kg bw

Negative controls validity:

valid

Positive controls validity:

valid

Assay validity: 

The vehicle control data (%PCE and MN PCE) were within the laboratory’s historical vehicle control observed data ranges.

The positive control slides demonstrated a statistically significant increase in MN PCE (over the concurrent vehicle control group) that was above the laboratory’s positive control data, but confirmed that micronuclei could be detected under coded scoring conditions and was therefore accepted as valid.

 

• : NPT 2042: Summary of Bone Marrow 48hr Micronucleus Data – Male

 

Group/ Dose Level	% PCE		% MN PCE		WRS P-value	Significance
(mg/kg/day)	Mean	SD	Mean	SD
1/ Vehicle (0)	51.03	1.48	0.06	0.05	N/A	N/A
2/ Nourycryl MA 128 (500)	54.73	2.94	0.08	0.05	0.3333	NS
3/ Nourycryl MA 128 (1000)	51.27	5.45	0.10	0.04	0.1613	NS
4/ Nourycryl MA 128 (2000)	46.37	4.81	0.10	0.08	0.2835	NS
5/ CPA (10)	51.00	4.69	4.94	0.28	0.0119	*
Terpstra-Jonckheere dose response p-value (Groups 1,2,3,4 ): 0.2161 NS

MN    Micronucleated
PCE   Polychromatic erythrocyte
SD    Standard deviation
WRS Wilcoxon Rank Sum Test
NS    Not significant (p>0.05)
*      p≤0.05

Conclusions:

Nourycryl MA 128 did not induce micronuclei in the polychromatic erythrocytes of the bone marrow when tested up to 2000 mg/kg/day (the maximum recommended dose).

Executive summary:

Nourycryl MA 128: was tested for its potential to induce micronuclei (MN) in the polychromatic erythrocytes (PCE) of the bone marrow of treated rats.
Strain / Species: Han Wistar Rats
The dose range finding study indicated that 2000 mg/kg/day (the regulatory recommended maximum dose) was tolerated. The main study therefore was performed using 3 groups of 6 male rats that dosed with the test substance at 500, 1000 and 2000 mg/kg bw in 10 mL arachis oil/kg bw in two administrations 24 hours apart. An additional groupd of 6 males received vehicle alone (negative control).
Positive control slides, prepared under Labcorp Study Number 8449759 were stained and coded alongside the study slides.
Evaluations consisted of clinical signs (prior to dosing, immediate, 0.5, 1, 2 and 4-6 hours post dosing on day 1 and 2, and prior to necropsy on day 3) and BW.
Slides prepared from the bone marrow of the femurs of the animals and evaluated for ration polychromatic erythorcytes (PCE) and normochromatic erythrocytes (NCE) and for the presence of micronuclei (MN) in at least 4000 PCE/animal.

In the Range-Finder Experiment of this study a group of 3 male and 3 female rats were dosed with Nourycryl MA 128 at a dose level of 2000 mg/kg/day.
On day 1 all animals exhibited decreased activity 0.5 hr and 1 hour post dose. All three female animals had returned to a normal state 2 hours post dose but the three male animals still exhibited decreased activity, which was also noted in 2/3 male animals approximately 4 hours post dose. On day 2 all animals were marked normal prior to dosing. Immediately after dosing salivation was noted in all animals, which persisted up to 1 hour post dose. Decreased activity was noted in all animals from 0.5 hour post dose which lasted throughout the rest of the day. Transient raised fur was noted in 1 female animal 1 hour post dose and 1 male animal 4 hours post dose. A single male was also noted to have staining around the mouth 4 hours post dose. On the morning of Day 3 all animals had returned to a normal state and no clinical signs were noted for any animals throughout the day. The mean % bodyweight changes from Day 1 to Day 3 were -9.9% for the male animals and -6.4% for the female animals.
there are no data available to suggest differences in metabolism or bioavailability, or gender specific exposure. As there were no substantial differences in toxicity between the sexes male animals only were used for the main study.

Assay validity was confirmed. The vehicle control data (%PCE and MN PCE) were within the laboratory’s historical vehicle control observed data ranges.
The positive control slides demonstrated a statistically significant increase in MN PCE (over the concurrent vehicle control group) that was above the laboratory’s positive control data, but confirmed that micronuclei could be detected under coded scoring conditions and was therefore accepted as valid.

Animals treated with Nourycryl MA 128:
All doses exhibited group mean %PCE that were similar to the concurrent vehicle control group and which fell within the laboratory’s historical vehicle control data, thus confirming there was no evidence of test article related bone marrow toxicity. However, results from earlier OECD 422 study clearly shows that systemic exposures occur. In this study dose levels of 0, 50, 150 and 375 mg/kg bw/day resulted to changes  hematology and blood chemistry parameters suggestive of mild anemia, and to liver and kidney effects, resulting to a NOAEL of 50 mg/kg bw/day.

All doses exhibited MN PCE frequencies that were similar to the concurrent vehicle control group and that fell within laboratory's historical vehicle control data. There were no statistically significant increases in MN frequency for any of the groups receiving the test article, compared to the concurrent vehicle controls.

It is concluded that Nourycryl MA 128 did not induce micronuclei in the polychromatic erythrocytes of the bone marrow when tested up to 2000 mg/kg/day (the maximum recommended dose), under the experimental conditions employed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Nourycryl MA128 was tested in the Ames Salmonella/microsome test up to 5000 µg/plate. The test substance induced no dose-related increase in the numbers of revertant (His+) colonies in each of the five tester strains (TA1535, TA1537, TA1538, TA98 and TA100). These results were confirmed in an independently repeated experiment. The test substance can, therefore, be considered as non mutagenic in this test system.

 

Nourycryl MA128 was evaluated for its possible induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The study was performed under GLP and according to the most recent OECD (TG 490, Jul 2016) and EU guidelines (B.17, May 2008).
The maximum dose level analysed was limited by a combination of test item induced toxicity, and precipitate in the 4 hour exposure groups. In the 24-hour exposure group the maximum dose level analysed was limited by test item induced toxicity. At the end of exposure a precipitate of the test item was observed at 1750 and 2000 µg/mL in the 4-hour exposures.  No precipitate was observed at the end of exposure in the 24-hour exposure group.  The vehicle (solvent) controls had acceptable mutant frequency values that were within the acceptable range for the L5178Y frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.The test item did not induce any toxicologically significant or dose-related (linear-trend) increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.Therefore, Nourycryl MA128 is not mutagenic in the TK mutation test.

 

Nourycryl MA128 did not induce a statistically significant increase in the frequency of binucleate cells with micronuclei in the absence of a metabolizing system. However, in the presence S9, reproducible, dose related, and statistically significant increases were observed. The test item was therefore considered to be mutagenic to human lymphocytes in vitro.
However, some comments can be made to these. Precipitation has been observed from concentrations of 1000 μg/mL and too high cytotoxicity (more than recommended reduction in CBPI to 45%) was observed above 1500 μg/mL. All positive increases have been observed at 1500 μg/mL and one at 1600 μg/mL (only in confirmatory experiment with S9) the highest possible concentration in view of toxicity. And combining all data in a graph shows that there are actually not very significant differences between all experiments.

 

In order to address the raised concerns from the in vitro MN study, further evaluation of the possibility of Nourycryl MA128 to cause cytogenetic damage in vivo was proposed. In this study performed according to OECD 474, Nourycryl MA128 did not induce micronuclei in the polychromatic erythrocytes of the bone marrow when tested up to 2000 mg/kg/day in rats (the maximum recommended dose). Although biomonitoring is not feasible for this substance in view of its almost instantaneous hydrolysation, results from earlier OECD 422 study clearly demonstrate that systemic exposures occur: In this study dose levels of 0, 50, 150 and 375 mg/kg bw/day resulted to changes haematology and blood chemistry parameters suggestive of mild anaemia, and to liver and kidney effects, resulting to a NOAEL of 50 mg/kg bw/day.

Justification for classification or non-classification

Nourycryl MA128 was not mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the TK mutation test with L5178Y mouse lymphoma cells. The in vitro MN resulted to positive responses, albeit at levels close to high cytotoxicity and showing precipitation. Subsequent evaluation in an in vivo MN study confirmed lack of cytogenic activity in vivo.