Phenothiazine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed GLP study according to standard NTP protocols, which are similar to recent OECD TG.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: standard NTP protocol

Deviations:

no

Remarks:

However, no report date is indicated. Therefore, 1999-01-01 was chosen as report date for the reference

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:

bacterial reverse mutation 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

Method

Target gene:

- Histidine

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 97, TA 98, TA 100, TA 102, TA 104, TA 1535

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Separately tested with Rat S9 and Hamster S9

Test concentrations with justification for top dose:

0 µg/plate, 33 µg/plate, 100 µg/plate, 333 µg/plate, 1000 µg/plate, 3333 µg/plate, 10000 µg/plate

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

w/o S9

Migrated to IUCLID6: TA 98

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

w/o S9

Migrated to IUCLID6: TA 100 and TA 1535

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

w/o S9

Migrated to IUCLID6: TA 97

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

w/o S9

Migrated to IUCLID6: TA 102

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

w/o S9

Migrated to IUCLID6: TA 104

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

used for all strains with S9

Details on test system and experimental conditions:

PREINCUBATION PROTOCOL
- Test tube containing a suspension of one strain of Salmonella typhimurium plus S9 mix or plain buffer without S9
- Incubation for 20 minutes at 37º C with the test chemical
- Control cultures, with all the same ingredients except the test chemical, are also incubated
- In addition, positive control cultures are prepared (containing the particular bacterial tester strain under investigation, the various culture ingredients, and a known potent mutagen)
- After 20 minutes, agar is added to the cultures and the contents of the tubes are thoroughly mixed
- Then the mixture is poured onto the surface of Petri dishes containing standard bacterial culture medium
- The plates are incubated, and bacterial colonies that do not require an excess of supplemental histidine appear and grow
- These colonies are comprised of bacteria that have undergone reverse mutation to restore function of the histidine-manufacturing gene
- The number of colonies is usually counted after 2 days.

Evaluation criteria:

- Spontaneous mutations (those that occur by chance, not by chemical treatment) will appear as colonies on the control petri dishes.
- If the test chemical was mutagenic to any particular strain of bacterium, the number of histidine-independent colonies arising on those plates will be significantly greater than the corresponding control plates for that strain of bacteria.
- The positive control plates are also counted, and the number of mutant colonies appearing on them must be significantly increased over the spontaneous control number for the test to be considered valid.
- Failure of the positive control chemical to induce mutation is reason to discard the experiment.

Statistics:

- No data given, according to OECD TG statistics are not obligate for conduction of the Ames-Test.
- However, mean values and standard errors are given in the results table

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 97, TA 98, TA 100, TA 102, TA 104, TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- Precipates observed onwards from 1000 µg/plate
- One replicate of TA 100 w/o activation resulted in equivocal results and was repeated twice with negative results
- One replicate of TA 98 w/ Hamster S9@30% was equivocal (weakly positive). However, a follow-up experiment applying identical conditions was negative, as well as all other TA98 experiments w/ an w/o S9.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

1) Summary Data

Chemical Name:	Phenothiazine
CASRN:	92-84-2
Study Type:	Salmonella
Study ID:	A72526
Study Result:	Negative
Year Completed:	1999
Vehicle Control:	Dimethyl Sulfoxide
Protocol:	Preincubation

2) Results for this Salmonella study's detailed data

Individual strain data is presented as mean ± standard error.
Abbreviations are noted at bottom of page.
Trial summary calls are shown in parentheses.

Strain: TA1535

Dose	No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		30% HLI (Negative)		10% RLI (Negative)		30% RLI (Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	11	1.7	13	2.8	16	2.6	14	2.3	13	2.7	16	1.5
33	10	0.3					11	2			16	1.9
100	14	0.9	11	1.2	11	0.6	14	2.1	14	2.7	11	1.7
333	9	4.6	15	0	14	2.5	11	1.5	11	2	12	0.9
1000	10p	0.6	12p	0	13p	1.5	13p	1.5	14p	0.6	15p	1.5
3333	16p	1.8	12p	2.6	10p	1.7	13p	2.4	16p	2.4	16p	1.8
10000	9p	0.7	15p	2	12p	2.7	16p	0.9	10p	1.7	11p	1.3
Positive Control	939	17.8	960	10.4	169	10.5	103	4.6	135	12.6	107	7.2

Strain: TA97

Dose	No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		30% HLI (Negative)		10% RLI (Negative)		30% RLI (Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	130	3.3	124	3.7	150	7	138	8.4	154	11.8	138	2
33	134	2.9					133	5.2			134	3.4
100	121	3.2	139	6.7	150	13.3	147	2.2	145	9.5	149	2
333	144	7.2	132	7.8	147	5.8	133	15.3	146	9.9	135	2.3
1000	135p	3	124p	8.4	158p	2.3	135p	4.9	149p	8.1	134p	10.7
3333	117p	6.1	132p	11.5	151p	3.8	144p	4.3	137p	6.4	132p	10
10000	117p	1.9	124p	3.2	141p	5.8	149p	12.2	121p	2.5	117p	6.7
Positive Control	448	29.6	558	19.5	619	10.7	593	13.8	551	24.5	552	12.6

Strain: TA100

Dose	No Activation (Equivocal)		No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		30% HLI (Negative)		10% RLI (Negative)		30% RLI (Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	115	4	137	3.9	112	3.3	122	8.4	110	8.7	116	2.4	122	3
33	90	0.3	133	2.7					131	9.2			141	3.2
100	142	1.9	121	3.2	106	8.2	118	6.7	129	5.2	110	2.5	124	2.6
333	136	4.3	134	5.5	101	3.5	119	2.4	117	8.4	110	7.4	110	5.3
1000	135p	7.8	125p	1.7	118p	3.8	121p	4	114p	6.7	114p	8.7	122p	4
3333	134p	3.2	123p	4.1	111p	1.5	119p	11	93p	3.2	109p	6.4	109p	5.5
10000			124p	15	114p	3	115p	2			107p	0.7
Positive Control	878	19.5	855	24.8	931	9	767	15.4	620	12.8	721	16.8	570	11.9

Strain: TA104

Dose	No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		30% HLI (Negative)		10% RLI (Negative)		30% RLI (Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	317	12.1	316	6	413	21.4	370	30.6	417	3.8	372	28.5
100	317	9.4	319	1.8	423	24.9	385	13.9	421	2	420	6.8
333	288	5.5	304	9.2	420	11.2	347	18	363	27.9	403	8.4
1000	262p	25.3	346p	27.4	374p	29.5	366p	11.4	379p	9.7	387p	4.9
3333	285p	19.1	277p	4.9	425p	30.7	392p	16.4	367p	28.8	392p	9.4
10000	259p	37.6	311p	8.7	391p	8.1	361p	22.7	392p	6.5	383p	26.8
Positive Control	835	20.2	933	21.2	1216	19.5	1182	9.8	1107	52	1076	32.3

Strain: TA98

Dose	No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		30% HLI (Equivocal)		30% HLI (Negative)		10% RLI (Negative)		30% RLI (Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	19	1.8	21	4.8	29	2.2	20	2.5	29	1.8	33	2.3	23	1.8
33	24	2.2					17	1.5	34	2.2			20	0.9
100	24	1.2	25	3.3	27	2.4	35	10.2	24	1.5	27	4.2	19	4.2
333	19	3.5	23	1.2	27	1.5	41	1.2	30	6.6	28	3.1	22	1.3
1000	20p	1.5	23p	0.9	21p	3.7	37p	1	26p	1	17p	2.3	20p	1.2
3333	19p	1.2	26p	3.6	21p	2.7	22p	2	30p	1.5	26p	1	15p	1.2
10000			25p	2	24p	2.6			22p	2.3	22p	3.2
Positive Control	500	14.2	407	29.1	604	4.9	561	10.4	446	20.2	541	19.1	449	16.3

Strain: TA102

Dose	No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		30% HLI (Negative)		10% RLI (Negative)		30% RLI (Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	183	13.5	192	12.5	257	13.7	261	15.3	235	12	252	15.7
100	203	9.5	192	10	228	10	248	15.6	229	26.3	278	11.6
333	186	20.1	171	7.6	248	3.2	250	3.2	251	8.4	260	11.8
1000	173p	14.1	180p	8.1	273p	2.8	273p	16.4	217p	0.7	239p	19.4
3333	209p	20.5	176p	14.6	267p	25.2	281p	6.5	264p	8.7	243p	18
10000	175p	14	178p	11.3	241p	20.3	242p	18.8	214p	7.4	257p	22.9
Positive Control	700	18.9	788	31	961	19.1	968	21.7	946	21.3	913	10.4

Abbreviations:
RLI = induced male Sprague Dawley rat liver S9
HLI = induced male Syrian hamster liver S9
p = Precipitate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Based on the results of this NTP-study, phenothiazine is non-mutagenic in the Salmonella mutagenicity assay (Ames test) when tested with and without metabolic activation using several Salmonella strains. Tests were conducted up to precipitating concentrations.

Executive summary:

Phenothiazine was tested in 6 different concentrations up to 10000 µg/plate. Precipitation was observed onward 1000 µg/plate. Six different Salmonella strains (TA 97, TA 98, TA 100, TA 102, TA 104 and TA 1535) were used for testing. Based on the results of this NTP-study, phenothiazine is non-mutagenic in the Salmonella mutagenicity assay (Ames test) when tested with and without metabolic activation.