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

Genetic toxicity: in vivo

Administrative data

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)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

224-116-8

EC Name:

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

Cas Number:

4203-89-8

Molecular formula:

C14H23NO3

IUPAC Name:

2-{1-oxa-4-azaspiro[4.5]decan-4-yl}ethyl 2-methylprop-2-enoate

Test material form:

liquid

Remarks:

Pale yellow slightly viscous liquid

Details on test material:

Chem. Name: 2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate;
CAS 4203-89-8
Batch number: 2110501010
Date of CoA: 08.12.2021
appearance: pale yellow slightly viscous liquid
Mw: 253.34
Purity info: 95%
Production date 5 Oct 2021
expiry date: 5 Oct 2024
Storage: dry; refrigerator between 0 and 5°C

Test animals

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.

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

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.

Examinations

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.

Results and discussion

Test results

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.