N1,N2-dimethyl-N1-{2-[methyl(propan-2-yl)amino]ethyl}-N2-(propan-2-yl)ethane-1,2-diamine

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:

February 21, 2021 - March 3, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

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:

(Adopted July 2016)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

methylbis({2-[methyl(propan-2-yl)amino]ethyl})amine

EC Number:

950-627-7

Cas Number:

1042950-30-0

Molecular formula:

C13H31N3

IUPAC Name:

methylbis({2-[methyl(propan-2-yl)amino]ethyl})amine

Test material form:

liquid

Specific details on test material used for the study:

Batch No.: UW20226411
Purity: 98%
Expiry Date: 14 February 2022
Storage conditions: Room Temperature

Test animals

Species:

rat

Strain:

CD-1

Remarks:

[Hsd: ICR(CD-1)]

Details on species / strain selection:

The specieswere those stated in the regulations, giving a valid model for the assessment of in vivo genotoxicity.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy - Age at study initiation:
- Weight at study initiation: 181.0 -199.3 g.
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Housing: Animals were housed up to 5 animals/cage in polisulphone H-temp solid bottomed cages with nesting material provided into suitable bedding bags
- Diet (e.g. ad libitum): commercially available laboratory rodent diet ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ±15
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

The vehicle used in this study was sterile water of injectable grade (batch nos. 20B0703 and
20C3004 obtained from Baxter, Italy).

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Fresh suspensions of the test item were prepared for each day’s work in the vehicle. Concentrations of solutions were calculated and expressed in terms of test item as received. All dose levels in this Report are expressed to three significant figures.
For the Main Assay, dose levels were selected on the basis of a preliminary toxicity experiment,
where two groups of two male and two female animals each were dosed on two consecutive days at 600 and 300mg/kg body weight/day. The maximum dose level of 600mg/kg body weight/day was selected on the basis of information received from the Sponsor (Safety Data Sheet).
Animals were inspected approximately 1 hour after dosing, 3 hours after dosing and at the end of working day daily throughout the study for signs of reaction to treatment. Besides, they were weighed daily starting from Day 1 of treatment.
Animals were sacrificed approximately twenty-four hours after dosing and bone marrow smear slides were prepared . Scoring was performed on slides prepared from the femurs of animals from the high dose group.

Duration of treatment / exposure:

around 23-24 hours for administration with test item; 24 hours for administration with positive control. An additional group of 3 satellite rats was dosed once with test item at 600mg/kg to performthe blood samplings for proof of exposure, pre-dose, 1 and 2 hours after dosing.

Frequency of treatment:

24 h interval between 2 administrations

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

five animals per sex and dose

Control animals:

yes, concurrent vehicle

Positive control(s):

The vehicle used in this study was sterile water of injectable grade (batch nos. 20B0703 and
20C3004 obtained from Baxter, Italy).

Examinations

Tissues and cell types examined:

bone marrow erythrocytes

Details of tissue and slide preparation:

Animals were sacrificed at the appropriate sampling time as indicated in the experimental scheme. One femur of each animal was rapidly dissected out and cleaned of surrounding tissue. In order to extract the bone marrow, the bone was cut at the proximal end and irrigated with foetal calf serum using a syringe. The suspension of cells was aspirated and this procedure wasrepeated several times. The suspension thus obtained was centrifuged at 1000 rpm for 5 minutes and the supernatant completely removed. The cells of the sediment were then resuspended and transferred onto clean microscope slides as smear preparations.These slides were air-dried and then fixed with methanol for 10 minutes. Subsequently slides were stained with haematoxylin and eosin solutions. Three slides were made from each animal.The slides were randomly coded by a person not involved in the subsequent microscope scoring. The slides were examined under low power and one or two slides from each animal were selected according to staining and quality of smears. Immature polychromatic erythrocytes (PCE) stain a pink-purple colour (since they retain basic ribosomal material for approximately 24 h after enucleation) and can be distinguished from the pink normochromatic
erythrocytes (NCE). The polychromatic cells are also slightly larger and have more diffuse boundaries. Erythrocytes lack nuclei, making micronuclei obvious when present. The criteria of Schmid (1976) will be used to score micronuclei.At least four thousand PCEs per animal were examined for the presence of micronuclei at high power (x 100 objective, oil immersion). At the same time, the numbers of normal and micronucleated normochromatic erythrocytes (NCEs) were also recorded.

Evaluation criteria:

Acceptance criteria: The incidence of micronucleated PCEs of the vehicle control group falls within the historical negative control range. The positive control item induces a significant increase in the frequency of micronucleated PCEs and the response falls within the historical positive control range.The appropriate number of doses and cells has been analysed.

Criteria for outcome of assay: The test item is considered to induce micronuclei if: At least one of the treatment groups exhibits a statistically significant increase (p<0.05) in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control. The increase is dose-related when evaluated with an appropriate trend test. Any of these results are outside the distribution of the historical negative control data (95% control limits).
Where increases in the incidence of micronucleated PCEs are observed, which are statistically
significant, but fall within the range of negative control values of this laboratory, then these data are used to demonstrate that these increases do not have biological significance

Statistics:

After completion of microscopic analysis, the data for each slide were decoded and the results analysed as described below. Only counts obtained from polychromatic cells were subjected to statistical analysis. Using the original observations (and not the micronucleus frequencies per 1000 cells), a modified χ2 calculation was employed to compare treated and control groups. The degree of heterogeneity within each group was first calculated and where this was significant, it was considered in the comparison between groups. In this case the variance ratios (F) were calculated from the between-groups and within group χ2 values. In addition, a test for a lineartrend (Snedecor and Cochran) was performed in order to evaluate dose effect relationship.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: two male and two female animals each were dosed on two consecutive days at 600 and 300mg/kg body weight/day
- Clinical signs of toxicity in test animals: body weight loss (300 mg/kg bw); body weight loss, semiclosed yes, hypoactivity
- Evidence of cytotoxicity in tissue analysed: no

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Following treatment with the test item, no increase in the incidence of micronucleated PCEs over the concurrent vehicle control was observed in any group. Increases in the frequency of micronucleated PCEs were observed in the positive control group, indicating the correct functioning of the test system.
- Appropriateness of dose levels and route: Preparations of test item were analysed with respect to concentration and homogeneity (high and low concentrations) by the Analytical Chemistry Department of the test institute-
- Bone Marrow cell Toxicity: The ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analysed to evaluate the bone marrow cell toxicity. Based on these results, no inhibitory effect on erythropoietic cell division was observed at any dose level. The results confirmed the identity of the test item and concentrations and homogeneity of all preparations were within the acceptance limits (85-115%; precision CV < 10%)
- Observations: Following treatment with the test item, hypoactivity, semiclosed eyes and body weight loss were observed in the animals from the high dose group. Body weight loss was also seen in the intermediate dose group. No signs were observed in any other treatment group.
- Statistical evaluation: The incidence of micronucleated PCEs in the vehicle control fell within the distribution of the historical negative control range. A statistically significant increase in the incidence of micronucleated PCEs over the control values was seen in the positive control group and the response was compatible with those generated in the historical control database. The appropriate number of doses and cells was analysed. The study was accepted as valid since the above mentioned criteria were met. Following treatment with test item, no statistically significant increase in the incidence of micronucleated PCEs over the control value was observed in any treatment group and no dose effect relationship was noted. Bone marrow exposure was indicated by bioanalytical results. On the basis of the above mentioned results and in accordance with the criteria for outcome of the study, the test item was not considered to induce micronuclei in the polychromatic erythrocytes of treated rats

Applicant's summary and conclusion

Conclusions:

On the basis of the results obtained, it is concluded that test item, administered by oral gavage, does not induce micronuclei in the polychromatic erythrocytes of treated rats, under the reported experimental conditions.