Phenothiazine

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

Study period:

1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well perfomed GLP study according to standard NTP protocols.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: standard NTP protocol

Deviations:

not specified

GLP compliance:

yes

Remarks:

According to the NTP-website only GLP-studies are accepeted for publication (http://ntp.niehs.nih.gov/files/Specifications_2006Oct1.pdf)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Phenothiazine

EC Number:

202-196-5

EC Name:

Phenothiazine

Cas Number:

92-84-2

Molecular formula:

C12H9NS

IUPAC Name:

10H-phenothiazine

Details on test material:

- Test item: phenothiazine

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

- The NTP has specific requirements for the testing laboratories to comply with the Laboratory Animal Welfare Act of 1966 and adhere to the principles enunciated in the "Guide for the Care and Use of Laboratory Animals" NRC, 1996.
- URL: http://ntp.niehs.nih.gov/go/type
- At the end of the study animals were killed by CO2 overdose

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Corn oil

Duration of treatment / exposure:

- 3 treatments
- Treatments are administered in 24hr intervals
- Sample collection time 24hrs after last treatment

Frequency of treatment:

- 3 treatments

Post exposure period:

- 24hrs after last treament (sample collection time) animals were killed by CO2 overdose

Doses / concentrations

Remarks:

Doses / Concentrations:
625 mg/kg, 1250 mg/kg, 2500 mg/kg
Basis:
actual ingested
Doses extend up to the maximum tolerated dose

No. of animals per sex per dose:

5 males per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

- Cyclophosphamide
- Dose: 25 mg/kg
- No of animals: 4 males

Examinations

Tissues and cell types examined:

- Bone marrow is flushed from the femurs and spread onto slides
- 24hrs after last exposure about 50% of the erythrocytes in the bone marrow are immature, newly formed erythrocytes, and these are the cell types that are checked for presence of micronuclei

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- Doese extend up to the maximum tolerated dose

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- 3 treatments in 24hr intervals
- sampling time 24hrs after last treatment

DETAILS OF SLIDE PREPARATION:
- Bone marrow is flushed from the femurs and spread onto slides
- Slides are air dried, fixed and stained with a fluorescent DNA-specific stain (acridine orange) that illuminates any micronuclei that may be present

METHOD OF ANALYSIS:
- 2000 polychromatic erythrocytes (PCEs, reticulocytes; immature erythrocytes) are scored per animal for frequency of micronucleated cells
- This is done for each of 5 animals per dose group
- In addition, the %PCEs among the total erythrocyte population in the bone marrow is scored for each dose group as an indicator of chemical-induced toxicity
- In non-treated healthy mice and rats, the %PCE in bone marrow is usually around 50-60%
- If a chemical interferes with the production of erythrocytes in the bone marrow, then the %PCE in the bone marrow may decline from the typical normal level
- Conversely, if erythrocyte production is stimulated by chemical exposure, then a higher percentage of immature erythrocytes may be observed

Evaluation criteria:

- See details in section "Any other information on materials and methods incl. tables" below

Statistics:

- See details in section "Any other information on materials and methods incl. tables" below

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- Please find the study results summarised (and in detail) in the section "Remarks on results including tables and figures" below

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no
- Ratio of PCE/NCE (for Micronucleus assay): dose dependent, between 0.30 to 0.90 with test item and 11.88 with positive control
- Appropriateness of dose levels and route: doses were extended up to maximum tolerated dose, route of adminstration is according to NTP protocol and considered relevant for the test item
- Statistical evaluation: is included in the results tables in the section "Remarks on results incl. tables and figures" below

Any other information on results incl. tables

Study Summary: Bone Marrow

Study ID	Result
	Male	Female
A12280	Negative	Not Tested

Start Date	Sample Collection Time	Sex	Cell	Dosing Regimen	Route	Trend Test P-Value
01/20/1998	24 Hours	Male	PCE	GAV x 3, 72 Hours	Gavage	0.305
			Dose (mg/kg)	Number of Animals Scored	Mean MN-PCE/1000 PCE ± SEM	Pairwise P
Vehicle Control		Corn Oil	0	5	0.90 ± 0.19
Test Chemical		Phenothiazine	625	5	0.30 ± 0.12	0.958
			1250	5	0.70 ± 0.34	0.692
			2500	5	0.90 ± 0.53	0.500
Positive Control		Cyclophosphamide	25	4	11.88 ± 1.56	< 0.0001

Detailed results: Bone Marrow

		Dose (mg/kg)	Animal Number	Polychromatic Erythrocytes Trend P = 0.305
				No. Examined	Total MN Cells	Percent PCE	MN Cells per 1000
Vehicle Control	CRNO	0	20	2000	1	47.5	0.5
		0	41	2000	2	38.0	1.0
		0	47	2000	1	30.0	0.5
		0	55	2000	2	54.0	1.0
		0	65	2000	3	56.0	1.5
Average ± SEM						45.10 ± 4.91	0.90 ± 0.19
Test Chemical		625	38	2000	0	49.5	0.0
		625	44	2000	1	51.5	0.5
		625	50	2000	0	34.0	0.0
		625	57	2000	1	38.0	0.5
		625	62	2000	1	49.0	0.5
Average ± SEM						44.40 ± 3.51	0.30 ± 0.12
Pairwise P							0.958
Test Chemical		1250	14	2000	1	47.0	0.5
		1250	42	2000	1	31.0	0.5
		1250	48	2000	1	41.0	0.5
		1250	59	2000	4	49.0	2.0
		1250	67	2000	0	37.0	0.0
Average ± SEM						41.00 ± 3.29	0.70 ± 0.34
Pairwise P							0.692
Test Chemical		2500	39	2000	1	57.5	0.5
		2500	46	2000	0	45.0	0.0
		2500	52	2000	6	23.5	3.0
		2500	543	2000	1	42.5	0.5
		2500	64	2000	1	55.0	0.5
Average ± SEM						44.70 ± 6.02	0.90 ± 0.53
Pairwise P							0.500
Positive Control	CPA	25	37	2000	18	20.5	9.0
		25	45	2000	32	16.5	16.0
		25	53	2000	25	1.0	12.5
		25	58	0	0	0	0.0
		25	61	2000	20	2.0	10.0
Average ± SEM						10.00 ± 4.98	11.88 ± 1.56
Pairwise P							< 0.0001
Abbreviations:
CRNO = Corn Oil
CPA = Cyclophosphamide
MN = Micronucleated Cells

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the conditions described the test item phenothiazine is not inducing any micronuclei.

Executive summary:

Following the standard NTP protocol for the assessment of in vivo micronuclei induction (which is similar to recent OECD TG), phenothiazine is not increasing the number of micronucleated cells and hence considered to be non-genotoxic.