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Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-11-24 to 2021-07-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 29th July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
3-[(3-{[(3-acetoxy-2,2-dimethylpropylidene)amino]methyl}-3,5,5-trimethylcyclohexyl)imino]-2,2-dimethylpropyl acetate
EC Number:
805-722-7
Cas Number:
1064082-81-0
Molecular formula:
C24H42N2O4
IUPAC Name:
3-[(3-{[(3-acetoxy-2,2-dimethylpropylidene)amino]methyl}-3,5,5-trimethylcyclohexyl)imino]-2,2-dimethylpropyl acetate
Test material form:
liquid

Test animals

Species:
mouse
Strain:
NMRI
Details on species / strain selection:
The NMRI mouse is a recommended test system for the Mammalian Erythrocyte Micronucleus Test
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc.
- Age at study initiation: 6 – 10 weeks
- Weight at study initiation: 30.4 - 36.7 g
- Assigned to test groups randomly: Yes
- Housing: Singly in Makrolon Type II / III cages with wire mesh top and granulated soft wood bedding
- Diet: Ad libitum (certified 2018C Teklad Global 18% protein rodent diet)
- Water: Ad libitum (tap water)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2
- Humidity (%): 45-65
- Air changes (per hr): At least 8
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From day 1 to day 2 (positive control animals) or from day 1 to day 3 (negative control and treated animals)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: Corn oil
- Justification for choice of solvent/vehicle: The vehicle was chosen due to its relative non-toxicity for the animals and its ability to formulate a suitable dosing preparation since the test item is not stable in water.
- Amount of vehicle: 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The preparations were made freshly before each dosing occasion.
Duration of treatment / exposure:
Animals used for micronuclei evaluation: 48 hours (2 applications at an interval of 24 hours + 24 hours post treatment period)

Animals used for plasma sampling: 25 hours and 28 hours (2 applications at an interval of 24 hours + 1 hour or 4 hours post treatment period)
Frequency of treatment:
Animals were treated twice (test item treatment + negative control) at an interval of 24 hours or once (positive control)
Post exposure period:
24 hours
Doses / concentrationsopen allclose all
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
vehicle
No. of animals per sex per dose:
6 animals per sex per dose for micronucleus evaluation and 6 high dose animals (3 animals 1 hour after last application and 3 animals 4 hours after last substance application) and 3 vehicle animals for plasma sampling
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: Oral (gavage)
- Doses / concentrations: 40 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The maximum tolerated dose was selected on the basis of a pre-experiment with two animals per sex at a dose level of 2000 mg/kg bw under identical conditions as in the mutagenicity study concerning animal strain, vehicle, route, frequency, and volume of administration. As no deaths or severe suffering occured, 2000 mg/kg bw was selected as the highest concentration recommended in the guideline.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed using CO2 followed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald/Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 4000 polychromatic erythrocytes (PCE) per animal were analysed for micronuclei. To describe a cytotoxic effect, the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per total erythrocytes. The analysis was performed with coded slides. All animals per test group were evaluated as described.
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test substance is classified as positive in the assay if
a) at least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control;
b) this increase is dose-related at least at one sampling time when evaluated with an appropriate trend test and
c) any of these results are outside the distribution of the historical negative control data (e.g., Poisson-based 95 % control limits).

Providing that all acceptability criteria are fulfilled, a test substance is considered clearly negative in the assay if:
a) none of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control;
b) there is no dose-related increase at any sampling time when evaluated by an appropriate trend test;
c) all results are inside the distribution of the historical negative control data (e.g., Poisson-based 95 % control limits), and
d) bone marrow exposure to the test substance(s) occurred.

There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment and/or further investigations.
Statistics:
Statistical methods (as appropriate, Mann-Whitney Test, linear regression analysis) were used as an aid in evaluating the results.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500 - 2000 mg/kg bw per application
- Solubility: Soluble in corn oil at all tested concentrations
- Clinical signs of toxicity in test animals: One animal showed partially closed eyes 2-4 hours after the first treatment. Two animals showed piloerection 2-4 hours after the first treatment. The animals did not express any clinical symptoms after the second treatment.
- Evidence of cytotoxicity in tissue analysed: The mean number of polychromatic erythrocytes was not substantially decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that the test item did not have any cytotoxic properties in the bone marrow.
- Rationale for exposure: The maximum recommended dose outlined in OECD guideline 474 was used in the pre-experiment as no severe toxicity was expected based on information from the acute oral toxicity and the repeated dose toxicity studies.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: See "Any other information on results incl. tables"
- Ratio of PCE/NCE: See "Any other information on results incl. tables"
- Appropriateness of dose levels and route: The dose levels were chosen according to the maximum recommended dose outlined in OECD guideline 474. This dose induced no severe suffering or deaths in the pre-experiment and was thus considered well tolerated in the main experiment. The substance was considered to have the highest bioavailability via the oral route as the substance rapidly hydrolyses in contact with aqueous solutions (e.g. stomach fluid) and its hydrolysis products are thus considered to become bioavailable via the oral route.
- Statistical evaluation: See "Any other information on results incl. tables"

Any other information on results incl. tables

Table 1: Summary of micronucleus test results











































































Test
Group



Dose
mg/kg
b.w.



Sampling
time
(after 2nd application)



Mean MN/4000 PCE



SD  MN/4000 PCE



Range



Ratio
 PCE /total Ery



% ratio
Vehicle



min



max



Vehicle



0



24



7.0



3.1



2



11



0.732



100.00



Dose 1



500



24



4.5



2.3



1



8



0.750



102.46



Dose 2



1000



24



5.7



1.8



3



8



0.714



97.54



Dose 3



2000



24



5.7



2.4



3



10



0.716



97.81



Positive



40



24



115.7



32.9



61



144



0.729



99.59



 


 Table 2: Biometry. Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test using the validated statistical program RScript Wilcoxon_2.Rnw. The Holm-Bonferroni Adjustment method was used to correct for the Familiywise error rate of multiple comparisons.




































Negative control versus test group



Significance



p



p adjusted



500 mg test item/kg b.w.; 24 h



-



0.139



0.417



1000 mg test item/kg b.w.; 24 h



-



0.325



0.650



2000 mg test item/kg b.w.; 24 h



-



0.418



0.650



Positive Control - 40 mg CPA/kg b.w.; 24 h



+



0.005



0.020



-      =     not significant
+     =     significant


Furthermore, a linear regression analysis was performed (least squares, calculated using the validated statistical program RScript LM_v02.Rnw) in order to assess a possible dose dependent increase of mean micronuclei values. The mean number of micronuclei obtained for the groups treated with the test item was compared to the vehicle control group.


A trend was judged as significant whenever the p-value (probability value) is below 0.05. A p-value of 0.7372 was obtained, demonstrating that there was no dose dependent increase of mean micronuclei values.


 


Table 3: Content of the test item in dose formulation samples (corn oil)































































Sample ID


 



Nominal test concentration


[mg/mL]



Timing



Test item


analysed
[mg/mL]



% of nominal test concentration


[%]



1 a



Control



After the
first application



n.d.



n.a.



2 a



50



45.2



90



3 a



100



94.8



95



4 a



200



185



93



5 a



Control



After the second application



n.d.



n.a.



6 a



50



45.8



92



7 a



100



94.2



94



8 a



200



188



94



n.d.: not detectable (< 7.5 mg/mL); n.a.: not applicable


 


Table 4: Contents of the hydrolysis products 2,2-dimethyl-3-oxopropyl acetate and 3-aminomethyl-3,5,5-trimethylcyclohexylamine in blood plasma































































Sample ID *


 



Timing



Treatment

[mg/kg]



2,2-dimethyl-3-oxopropyl acetate analysed
[mg/L]



3-aminomethyl-3,5,5-trimethylcyclohexylamine analysed
[mg/L]



P-1 a/b



1 h after second treatment



Control (Vehicle)



n.d.



n.d.



P-2 a/b



n.d.



n.d.



P-3 a/b



n.d.



n.d.



P-4 a/b



2000 mg/kg



n.d.



< LOQ



P-5 a/b



n.d.



< LOQ



P-6 a/b



n.d.



< LOQ



P-7 a/b



4 h after second treatment



2000 mg/kg



n.d.



14.5



P-8 a/b



n.d.



9.02



P-9 a/b



n.d.



< LOQ



* two aliquots of each sample were provided by the test facility: aliquot a was used for analysis of 2,2-dimethyl-3-oxopropyl acetate analysed, aliquot b was used for analysis of 3-aminomethyl-3,5,5-trimethylcyclohexylamine; n.d.: not detectable (< 2.4 mg/L); < LOQ: below limit of quantification (i.e. < 8 mg/L)

Applicant's summary and conclusion

Conclusions:
In conclusion, in can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test item is considered to be non-genotoxic in this in vivo micronucleus assay.
Executive summary:

A study according to OECD guideline 474 and GLP was peformed to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was dissolved in corn oil, which was also used as vehicle control. The dose volume administered orally (twice) was 10 mL/kg b.w. The administered volume of the positive control was 10 mL/kg. 24 h after the second administration of the test item the bone marrow cells were collected for micronuclei analysis. Six males per test group were evaluated for the occurrence of micronuclei. Per animal 4000 polychromatic erythrocytes were scored for micronuclei. To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per total erythrocytes. The following dose levels of the test item were investigated: 500, 1000, and 2000 mg/kg bw.


The highest dose (maximum guideline-recommended dose) was estimated by a pre experiment to be suitable. The animals treated with the test item and the vehicle control as well as with the test item did not exhibit any clinical symptoms. Accuracy of dose formulations was confirmed within phase number 20I13137-01-RATX. Recoveries were between 90 and 95% of nominal concentrations. Furthermore, within the same phase, bioavailability was confirmed by analytical detection of the test item in plasma (namely, the analytes 2,2-dimethyl-3-oxopropyl acetate (CAS 16184-79-5) and 3-aminomethyl-3,5,5-trimethylcyclohexylamine (CAS 2855-13-2)). 2,2-dimethyl-3-oxopropyl acetate could not be detected in plasma at any time point, whereas 3-aminomethyl-3,5,5-trimethylcyclohexylamine was present in plasma in test item treated animals and could be quantified in two of three animals at the 4 h after the second application time point (9.02 and 14.5 mg/L, respectively). In the third animal with blood withdrawal 4 h after the second application, 3-aminomethyl-3,5,5-trimethylcyclohexylamine could also be detected, but at a level which was below the Limit of Quantification (LOQ, <8 mg/L) but above the Limit of Detection (LOD, >2.4 mg/L). In conclusion, exposure of the blood and consequently also the bone marrow could be demonstrated. At 1 h after the second application, 3-aminomethyl-3,5,5-trimethylcyclohexylamine concentration in plasma was below LOQ in the three animals sacrificed at that time point.


After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test item not exert any cytotoxic effects in the bone marrow.


In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronulclei after administration of the test item with any dose level used. 40 mg/kg b.w. cyclosphosphamide administered orally was used as positive control which induced a substantial increase in cells with micronuclei.


In conclusion, in can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test item is considered to be non-clastogenic in this in vivo micronucleus assay.