Propanamide, N-[4-(2,4-dihydroxyphenyl)-2-thiazolyl]-2-methyl-

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Basic data given: This micronucleus assay is part of a 90-day repeated dose toxicity study performed according to OECD 408. The test item was assessed for its cumulative toxicity when administered daily to rats by gavage for a period of 90 days and for its potential to induce the formation of micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of Wistar rats. No positive control was used.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

In this study, the animals were treated orally by gavage for period of at least 90 days; no positive control

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

N-[4-(2,4-dihydroxyphenyl)-1,3-thiazol-2-yl]-2-methylpropanamide

EC Number:

807-621-3

Cas Number:

1428450-95-6

Molecular formula:

C13H14N2O3S

IUPAC Name:

N-[4-(2,4-dihydroxyphenyl)-1,3-thiazol-2-yl]-2-methylpropanamide

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Horst, The Netherlands
- Age at study initiation: 7 weeks
- Weight at study initiation: 212 - 256 g (males), 140 - 160 g (females)
- Housing: Animals were housed in groups of 3 or 4 in Makrolon type-4 cages with wire mesh tops and standard softwood bedding including paper enrichment.
- Diet: Pelleted standard HarlanTeklad 2914C rat / mouse maintenance diet, ad libitum
- Water: Community tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 29 Nov 2012
To: 28 Feb 2013

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: PEG 300
- Batch no.: BCBJ2013V

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared weekly. The test substance was weighed into a glass beaker on a tared balance and the vehicle added. The mixtures were stirred using a magnetic stirrer. Homogeneity of the test substance in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
50, 200, 800 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

7

Control animals:

yes, concurrent vehicle

Positive control(s):

none

Examinations

Tissues and cell types examined:

Tissue: bone marrow
Cell type: bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: This micronucleus assay is part of 90 day repeated dose toxicity study. The dose levels were selected based on a previous dose range finding toxicity study.

TREATMENT AND SAMPLING TIMES: 24 hours after the last treatment of the animals, animals were sacrificed and the right femurs were removed.

DETAILS OF SLIDE PREPARATION: The epiphyses were cut off and the marrow was flushed out with warmed fetal calf serum (FCS), using a syringe. The nucleated cells were separated from the erythrocytes using the method of Romagna. Briefly, the cell suspensions were passed through a column consisting of α-Cellulose (Sigma) and Cellulose (Sigmacell type 50). The columns were then washed with Hank´s buffered saline. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. The pellet was resuspended in a small drop of FCS and spread on slides. The smears were air-dried, fixed in methanol and 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. At least 4000 immature erythrocytes per animal should be scored for the incidence of micronucleated immature erythrocytes. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal 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 2000 erythrocytes. The analysis was performed with coded slides.

Evaluation criteria:

A test item can be classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
A test item producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points, is considered non-mutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test, if necessary. However, both biological and statistical significance should be considered together.

Acceptance Criteria
The study is considered valid if the following criteria show that:
- at least 5 animals per group and sex are evaluable
- PCE to erythrocyte ratio should not be less than 20% of the negative control.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

RESULTS
- Induction of micronuclei: In comparison to the corresponding vehicle controls, there was no statistically significant or biologically relevant enhancement of the frequency of the detected micronuclei in males and females at any dose level. The mean values of micronuclei observed after treatment with the test item were below or near the value of the respective vehicle control group (see Table 1+2) and within the historical control data (see Table 3).
- Ratio of PCE/NCE: The ratio between polychromatic and normochromatic erythrocytes was determined in the same sample to evaluate any cytotoxic effect due to treatment with the test substance and reported as the number of PCEs per 2000 erythrocytes. After treatment with the test substance, the mean number of PCEs per 2000 erythrocytes was not decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control, thus indicating that the test substance did not induce cytotoxic effects in the bone marrow of exposed rats.

Any other information on results incl. tables

Table 1: Males

Test group	Dose [mg/kg bw/day]	PCEs with micronuclei [%]	Range	PCE per 2000 erythrocytes
Vehicle (PEG 300)	0	0.314	1-11	948
Test item	50	0.350	1-17	965
Test item	200	0.357	0-11	954
Test item	800	0.257	3-9	1008

 

 Table 2: Females

Test group	Dose [mg/kg bw/day]	PCEs with micronuclei [%]	Range	PCE per 2000 erythrocytes
Vehicle (PEG 300)	0	0.414	0-21	1039
Test item	50	0.364	4-11	1144
Test item	200	0.250	1-7	1095
Test item	800	0.393	2-23	1135

Table 3: Historical controls 2006-2011

Micronuclated cells	Males	Females	Total	Males	Females	Total
Mean ± SD [%]	0.184± 0.054	0.160± 0.053	0.175± 0.055	2.154± 0.820	1.034± 0.432	1.723± 0.884
Range of mean group [%]	0.085- 0.320	0.058- 0.283	0.058- 0.320	0.670- 4.058	0.470– 2.875	0.470– 4.058
Range (individual animal data)	0-12	0-12	0-12	8-136	5-74	5-136
No. of Experiments	51	35	51	49	32	49

The values included in the historical control data base were obtained from single time application using differentroutes(e.g. orally, intravenously, intraperitoneally).

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
Under the experimental conditions reported, the test substance did not induce micronuclei in the bone marrow of the rats exposed to the test substance for 90 days as part of a 90-day repeated dose toxicity study. Therefore, test substance is considered to be non-mutagenic in this micronucleus assay.