N-methoxy-1-(2,4,6-trichlorophenyl)propan-2-amine

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration The in vivo micronucleus study was conducted solely to comply with non-EU national registration requirement, and has been provided here in accordance with REACH, Article 22(1)e

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 October 2014 to 09 December 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

methoxy[1-(2,4,6-trichlorophenyl)propan-2-yl]amine

EC Number:

941-634-6

Cas Number:

1228284-78-3

Molecular formula:

C10H12Cl3NO

IUPAC Name:

methoxy[1-(2,4,6-trichlorophenyl)propan-2-yl]amine

Test material form:

other: liquid

Details on test material:

- Physical state: Brown liquid
- Purity test date: 18 October 2013
- Expiration date of the lot/batch: End September 2015
- Stability under test conditions: Recertification date End September 2015
- Storage condition of test material: <30°C

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6-7 Weeks
- Weight at study initiation: 155-193 g
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: In groups of up to three, by sex, in grid-floor cages suspended over paper-lined trays.
- Diet: LabDiet 5L0S EURodent Diet (pelleted) ad libitum
- Water: Mains tap water ad libitum
- Acclimation period: 5 days (main study)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-20
- Humidity (%): 42-70
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12/12

EXPERIMENTAL DATES: From: 02 October 2014 (1st animal arrival) To: 09 December 2014 (last day of slide scoring)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 1.0 % w/v carboxymethylcellulose with 1.0 % v/v Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was formulated for dosing as a suspension in the vehicle. Separate formulations were prepared for each dose level, with the weighed quantity of test item (adjusted for purity) being suspended in the appropriate quantity of vehicle. Formulations were prepared on the day of use. A weighed quantity of test item was added to a suitable container calibrated to the final preparation volume. Approximately 90 % of the final volume of vehicle was added gradually, whilst stirring. The formulation was then stirred and heated to a maximum of 51°C, until a homogenous mixture was formed. The formulation was then allowed to cool before adding vehicle up to the final volume and stirring until mixed. The formulation was then transferred to an amber glass bottle.

Cyclophosphamide monohydrate (CPA), the positive control item, was a 3 mg/mL solution of CPA in UHP water.

Duration of treatment / exposure:

2 days

Frequency of treatment:

once per day

Post exposure period:

24 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 45, 90, 175 mg/kg/day
Basis:
other: nominal in vehicle

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide monohydrate (CPA)
- Route of administration: Oral gavage
- Doses / concentrations: 15 mg/kg (single dose at a dose volume of 5 mL/kg)

Examinations

Tissues and cell types examined:

Bone marrow cells (from femur)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A dose-sighting phase was carried out (2 males/group, dose levels 175, 200, 320 mg/kg/day, 2 doses) in order to select the highest dose of the test item that did not produce mortality or severe signs of clinical toxicity up to the MTD level. Then a dose range-finding phase was carried out (175 mg/kg/day - 3 males and 3 females, 275 and 450 mg/kg/day - 3 females/group only, 2 doses) to confirm that the MTD level identified in the dose-sighting phase was tolerated in males and females.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Three groups, each of six male rats were dosed with 45, 90 or 175 mg/kg/day test item on two successive days, separated by approximately 24 hours (Groups 2 to 4). A group of six male rats (negative control - Group 1) was dosed with the vehicle alone on two successive days, separated by approximately 24 hours and a positive control group (Group 5), also of six male rats, was given a single 15 mg/kg oral (gavage) dose of Cyclophosphamide monohydrate (CPA).

Animals were killed approximately 24 hours after their second dose (negative control and test item-treated groups) or first dose (positive control group). Bone marrow was harvested from each animal and smears prepared. The stained slides were coded, 2000 polychromatic erythrocytes (PCE) per animal were scored for the presence of micronuclei and the group frequencies were statistically analysed.

DETAILS OF SLIDE PREPARATION: One femur from each animal was exposed by dissection of the surrounding muscle and connective tissues and the shank of the bone removed. The bone marrow cells from each femur were aspirated into labelled centrifuge tubes using a syringe containing foetal bovine serum. The bone marrow cells were centrifuged, the supernatant withdrawn, and the cells re-suspended in a minimal volume of foetal bovine serum. One drop of cell suspension was placed on each of two slides and spread by drawing an edge of a clean glass microscope slide along from the drop to the end of the slide. All slides were left to air dry and age overnight before fixing for 5 minutes in methanol. Fixed slides were stained for 20 to 30 minutes in 11.5 % (v/v) Giemsa in Sorensen’s buffer pH 6.0, based on the method of Gollapudi and Kamra (Gollapudi, B. and Kamra, O.P. Application of a Simple Giemsa-Staining Method in the Micronucleus Test. Mutation Research. (1979) 64 45-46.).

METHOD OF ANALYSIS: 2000 polychromatic erythrocytes (PCE), including micronucleated PCE (MN-PCE), were counted for each animal. The numbers of normochromatic (NCE) and micronucleated NCE (MN-NCE) erythrocytes were also recorded for the first 1000 cells scored. Only areas of slides of good technical quality and appropriate staining characteristics were scored. As NCE were only recorded for the first 1000 erythrocytes scored, the PCE/NCE ratio was calculated as follows: PCE/NCE = (1000-NCE count)/NCE count)

OTHER: A proof of exposure phase was conducted to demonstrate that the bone marrow was exposed to the test item. This was demonstrated by analysis of test item in the whole blood of treated animals. The presence of the test item was confirmed by analysis of the study samples alongside samples of blank matrix and matrix spiked with the test item.

Evaluation criteria:

The test was considered to be positive, i.e. test item was considered to induce clastogenic/aneugenic damage, if all of the following were observed:
• A statistically significant increase in the frequency of MN-PCE occurred at one or more dose levels.
• The incidence and distribution of MN-PCE in individual animals at the dose level(s) showing statistical significance exceeded the laboratory’s historical negative control data.
• A dose-related increase in the frequency of MN-PCE (where more than two dose levels are analysed) was observed.
Results which only partially satisfy the above criteria would be dealt with on a case-by-case basis. Evidence of a dose-related effect was considered useful but not essential in the evaluation of a positive result. Biological relevance was taken into account, for example consistency of response within and between dose levels.
A test was considered to be negative, i.e. non-clastogenic/aneugenic, if there was neither a dose-response curve nor any group showed statistically significant increases in the frequency of micronucleated PCEs compared to the negative controls.

Statistics:

The data analysed were the proportion of MN-PCEs and the ratio of polychromatic to normochromatic cells (PCE/NCE). The preferred approach for the MN-PCE data is to combine the data within each group and construct a 2x2 contingency table for each treated group with the negative control. The groups are then compared using a one tailed Fisher Exact test. However, this approach must first be validated by carrying out a test for between animal heterogeneity using a chi
square test. If the heterogeneity test is significant at the 1% level then an exact Wilcoxon Rank Sum test is used instead of the Fisher Exact test. The same method is used to compare the positive and negative controls. For the PCE/NCE data, the treated groups were compared with the negative control using one tailed exact Wilcoxon Rank Sum tests.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

in males at concentrations of 175 mg/kg/day

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF DOSE-SIGHTING STUDY:
- Doses: 175, 200, 320 mg/kg/day (males only)
- Clinical signs of toxicity in test animals: Yes at 200 and 320 mg/kg/day (piloerection, tremors, hunched posture, vocalisation on handling, incoordination, eyes partially closed, unsteady gait). Animals killed approximately 6 hours post second dose of 320 mg/kg/day due to clinical signs.

RESULTS OF RANGE-FINDING STUDY
- Doses: 175 (males and females), 275 (females), 450 (females) mg/kg/day
- Clinical signs of toxicity in test animals: Yes in females only (abnormal sensitivity to touch or disturbance, intermittent tremors @ 175 and 275 mg/kg/day; unsteady and abnormal gait @ 275 and 450 mg/kg/day; decreased activity, tremors, intermittent twitching, piloerection, vocalisation on handling, incoordination @450 mg/kg/day). Females killed approximately 0.75 hours post second dose of 450 mg/kg/day due to clinical signs.

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: Yes at 175 mg/kg/day (abnormal sensitivity to touch or disturbance)
- Induction of micronuclei (for Micronucleus assay): No
- Ratio of PCE/NCE (for Micronucleus assay): 0.74, 0.98, 0.85, 0.93, 0.89 for 0, 45, 90, 175 mg/kg/day test item and 15 mg/kg CPA, respectively.
- Appropriateness of dose levels and route: Satisfactory
- Statistical evaluation: There were no statistically significant differences in any parameter between test item-treated groups and the vehicle control group. The mean MN-PCE for the positive control group was statistically significantly different (p<0.001, Fischer's Exact test) from the vehicle controls.

Any other information on results incl. tables

Table 1: Micronucleus results

	Dose level (mg/kg/day)
	0 mg/kg/day (control)	45 mg/kg/day	90 mg/kg/day	175 mg/kg/day	15 mg/kg CPA (positive control)
Mean MN-PCE	1.00±1.26	1.33±0.82	0.83±0.98	1.83±1.33	15.17±2.99***
Mean PCE/NCE ratio	0.74±0.17	0.98±0.17	0.85±0.19	0.93±0.28	0.89±0.11
*** Statistically significant p<0.001 (Fisher’s Exact test)

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
There was no evidence of clastogenicity or aneugenicity following oral (gavage) administration of the test item up to the MTD of 175 mg/kg/day in male rats. Therefore, the test item is considered to be neither clastogenic nor aneugenic in this bone marrow micronucleus assay.

Executive summary:

The MTD was confirmed (in dose-sighting and range-finding phases) as 175 mg/kg/day in male rats and 275 mg/kg/day in female rats. As there were no substantial inter-sex differences in toxicity, the main study was conducted in males only, with the high dose selected as 175 mg/kg/day.

 

Three groups, each of six male rats were dosed, by oral gavage, with 45, 90 or 175 mg/kg/day test item on two successive days, separated by approximately 24 hours. A group of six male rats (negative control) was dosed with the vehicle alone on two successive days, separated by approximately 24 hours and a positive control group, also of six male rats, was given a single 15 mg/kg oral (gavage) dose of Cyclophosphamide monohydrate (CPA). Animals were killed approximately 24 hours after their second dose (negative control and test item-treated groups) or first dose (positive control group). Bone marrow was harvested from each animal and smears prepared. The stained slides were coded, 2000 polychromatic erythrocytes (PCE) per animal were scored for the presence of micronuclei and the group frequencies were statistically analysed.

 

There were no statistically significant increases in micronucleus frequency in male rats treated at any dose level with the test item, compared with the negative control group. There was no evidence of a statistically significant reduction in the PCE/NCE ratio in male rats treated with the test item, indicating a lack of toxicity to the bone marrow. Proof of exposure was demonstrated in the range-finding and main study phases. The animals dosed with CPA, the positive control item, had statistically significant increases in the number of micronucleated cells compared to the concurrent control group, which demonstrated that the test system was capable of detecting a known clastogen and that the scorers were capable of detecting micronuclei. There was no statistically significant decrease in the PCE/NCE ratio in the positive control group, indicating a lack of toxicity to the bone marrow.

 

In conclusion, there was no evidence of clastogenicity or aneugenicity following oral (gavage) administration of the test item up to the MTD of 175 mg/kg/day in male rats. Therefore, the test item is considered to be neither clastogenic nor aneugenic in this bone marrow micronucleus assay.