EPTC

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacteria reverse mutation (Ames) test is negative.

In the mouse lymphoma cell mutagenesis assay performed in 1986 the EPTC proved to be negative without metabolic activation and positive with metabolic activiation.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

other: Clives et.al 1979

Version / remarks:

Clives et.al 1979, Mutat.Res.59, 61-108

Deviations:

not specified

Principles of method if other than guideline:

The method-Clives et.al 1979, Mutat.Res.59, 61-108 - is comparable to OECD TG 490 guideline.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L517BY mouse lym~homa cells heterozygous for thFmldlne klnase (TK+/-),
clone 3.7.2C initially obtained from D. Clive, Burroughs Wellcome, NC
(Clive et al., 1979), were used as target cells.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

na

Metabolic activation:

with and without

Metabolic activation system:

An Aroclor 1254-1nduced rat-liver homogenate preparation ($9) was used as the metabolic activation system. Liver enzymes were induced by in~ecting adult m~le rats with Aroclor 1254 (500 mg/kg) 5 days before sacrifice.

Test concentrations with justification for top dose:

The results of the preliminary cytotoxlcity experiments (Table I),
suggested that the maximum concentrations for the mutagenlcity assays,
based on the toxicity of the test article, should be between 140 and 233
~g/ml without activation, and between 233 and 389 ~g/ml with activation.
Concentrations of EPTC from 389 to 5000 ~g/ml were immiscible in the
culture medium at the time of exposure; the medium containing 389 tp 1800
~g/ml was cloudy and oily drbplets appeared at 3000 and 5000 Bg/ml. After
the 4-hour exposure period, the cloudiness had dissipated but the oily
droplets appeared~s opaque, white droplets that floated on the surface of
the culture medium.

Vehicle / solvent:

ethanol (final concentration less than 1 % of the final volume)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

ethylmethanesulphonate

Details on test system and experimental conditions:

Procedural Detail
The experimental procedure was adapted from one described by Clive
and associates (1975, 1979). For each culture, 6 x 106 cells In i0 ml of
medium, with or without the metabolic activation mixture, ’were prepared in
a sterile 50-ml centrifuge tube. After addition of the test or control
articles, the centrl6uge tubes were rotated for 4 hr in a roller drum at
37°C. Following the exposure, the treatment solutions were removed and
the cells were washed by a series of low-speed centrlfugations (i000 rpm,
8 mln), each followed by removal of the supernatant and resuspenslon of
the cells in 20 ml of fresh Fl0p. The tubes were then rotated in a roller
drum for 2 days at 37°C for expression of any mutations. Cell growth was
monitored daily by.countlng a tenfold dilution of the cells in 0.1%
trypsin with a Coulter counter. The cells were diluted to 3 x 105
cells/ml each day during the expression period.
After the expression period, approximately 3 x 106 cells from each
culture were seeded in cloning medium supplemented with TFT for selection
of TFTr cells, and approximately 600 cells were seeded in cloning medium
without TFT to determine the percentage of viable TK+/- cells. ~fter
cultivation of the cells for ii to 12 days at 37°C in an atmosphere containing 5% C02-1n-alr, the colonies of cells in each petrl dish were
counted using an automatic colony counter with a standard 50-mm lens.

Evaluation criteria:

Analysis of Results. ~
The following criteria are based on SRI’s experience and on values
published in the literature. Only experiments meeting the criteria for
acceptability are evaluated for mutagenic response~ and experiments that
do not meet the criteria are repeated. Because the mouse lymphoma mutegenesis assay is a complex biological system~ exceptions to these criteriamay be Justlfled. In these cases, the reasons are clearly stated in the report. The final interpretation of the results is the responsibility of the Study Director.

Statistics:

The mutation frequency of each culture (the ratio of the number of
mutant cells to the number of viable cells) was calculated by dividing the
number of TFTr colonies by 5,000 times the number of unselectad (viable)
colonies obs.erved in the plates without TFT. The average mutation frequencies of the solvent and positive control cultures and each set of cell
cultures treated with a single concentration of the test article were calculated. Toxicity was evaluated based on the growth of treated cells in
suspension and in the cloning medium relative to the solvent control cultures (relative total growth).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Sizing measurements were made on TFT-reststant ~olonies from the
mutagenesis experiment with activation (App~dlx B). The dfstrihutlons
colony sizes are shown in Figure I. The spproximate numbers of small and
large colonies in each culture are listed in Table 4. These data demonstt~ate that the test article induces an increase primarily in the frequency of sm~ll TFTr colonies, by a mechanism which is not yet known.

Conclusions:

In the mouse lymphoma cell mutagenesis assay performed in 1986 the EPTC proved to be negative without metabolic activation and positive with metabolic activiation.

Executive summary:

The study to investigate the mutagenlc potential of EPTC in the mouse
lymphoma cell assay was initiated on 23 May 1985, and the laboratory work
was comple.ted on 18 June 1985. The study was conducted according to the
specific protocol "Mouse lymphoma cell mutagenesis a~say (TK +/- ÷ TK -/-)
of EPTC~’° Results of the experiments conducted urlng this study are
shown in Tables I-4 and Figure i. All experiments fullfiled the criteria
for acceptable experiments and were used to evaluate the potential
mutagenicity of the test article.
The results of the preliminary cytotoxlcity experiments (Table I),
suggested that the maximum concentrations for the mutagenlcity assays,
based on the toxicity of the test article, should be between 140 and 233
~g/ml without activation, and between 233 and 389 ~g/ml with activation.
Concentrations of EPTC from 389 to 5000 ~g/ml were immiscible in the
culture medium at the time of exposure; the medium containing 389 tp 1800
~g/ml was cloudy and oily drbplets appeared at 3000 and 5000 Bg/ml. After
the 4-hour exposure period, the cloudiness had dissipated but the oily
droplets appeared~s opaque, white droplets that floated on the surface of
the culture medium.
Six concentrations of EPTC ranging from i18 to 200 ~g/ml.were tested
without activation. Neither a concentration-related increase in the
number Of mutant colonies nor a two-fold increase in the average mutation
frequency relative to the solvent controls was observed in the experiment
without activation (Table 2). Although the treatment with the highest
concentration resulted in relatively low toxicity (an average relative
total growth of 69%), th~’s concentration was within a one-tenth log
interval of a higher-concentration (233 ~g/ml) that reduced the relative
growth of cells to less than i% in the prior study. Therefore, based on
the criteria stated in the protocol, the test results for EPTC were
considered negative without activation.
In cultures treated with six concentrations of EPTC (from 42 to 250
~g/ml) with activation, the numbers of mutant colonies were 3 to 4 times
the number in the solvent control cultures. The average mutation frequency at each concentration increased from 3.7 to 6.1 times the solvent control levels (Table 3). These results fulfilled the criteria for a positive
response.
Sizing measurements were made on TFT-reststant ~olonies from the
mutagenesis experiment with activation (App~dlx B). The dfstrihutlons
colony sizes are shown in Figure I. The spproximate numbers of small and
large colonies in each culture are listed in Table 4. These data demonstt~ate that the test article induces an increase primarily in the frequency of sm~ll TFTr colonies, by a mechanism which is not yet known.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

EPTC did not reported to be germ cell mutagen based on in vivo test (mouse).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This animal study is not performed according to GLP, but the test parameters are well documented and scientifically acceptable.

Qualifier:

no guideline followed

Principles of method if other than guideline:

1. A felhasznált állatok adatai
2. A kezelés leírása
3. A csontvelő preparálása
4. Festés
5. A lemezek értékelése
6. Statisztika
7. Eredmények

GLP compliance:

no

Type of assay:

sister chromatid exchange assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

A kezelt és kontroll csoportok 5 hím és 5 nőstény állatból álltak.

Route of administration:

oral: unspecified

Vehicle:

víz, Tween 80

Details on exposure:

a, negatív kontroll: csapvíz + 0,5% Tween 80, 10 ml/kg
b, EPTC hatóanyag: 2x 500mg/kg, csapvíz + 0,5% Tween 80 vivőszer
c, EPTC hatóanyag : 2x 1000mg/kg, csapvíz + 0,5% Tween 80 vivőszer
d, pozitív kontroll: cyclophosphamid 30 mg/kg, csapvíz vivőszer

A tesztanyagokkal történt második kezeléssel egyidőben az egerek 333 mg/kg , hat órával később 666 mg/kg EUdR-t kaptak, 5%-os lágy agarban szuszpendálva, szubkután.

Duration of treatment / exposure:

2 nap gyomorszondán át

Frequency of treatment:

7 days

Remarks:

Doses / Concentrations:
2x 500 mg/kg
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
2x1000 mg/kg
Basis:
nominal in diet

No. of animals per sex per dose:

5 male and 5 female

Control animals:

yes, plain diet

Positive control(s):

d, pozitív kontroll: cyclophosphamid 30 mg/kg, csapvíz vivőszer

Tissues and cell types examined:

csontvelő ( bone marrow )

Details of tissue and slide preparation:

Bone marrow cells were separated, concentrated and stained with fluorochrom and Giemsa.

A csontvelőt mindkét femurból izotóniás sóoldattal mostuk ki. Felszuszpendálás után 200 x g-n centrifugáltuk 10 percig. A felülúszót eldobtuk, az üledékben lévő sejteket 37 c-on, 20 percig 0,56%-os KCl oldattal hipotonizáltuk. + 4 C-os metanol-jégecet ( 3:1) eleggyel négyszer fixáltuk. A sejtszuzspenziót Pasteur pipettával cseppentettük tárgylemezre, melyet levegőn szárítottunk.

Evaluation criteria:

Minden állatból 25 diferenciálódottan festődött második sejtciklusú metafázist értékeltünk.

Statistics:

A kezelt és a negatív csoportok összehasonlítása Student "t" próbával történt.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Az EPTC két különböző dózisban alkalmazva /maximálisan tolerálható dózis és a fele/ a kezelt állatokban nem növelte az Sister chromated exchange ( SCE) előfordulás gyakoriságát - a negatív kontroll csoporthoz viszonyítva.
A Cyclophosphamid "pozitív kontroll" csoporttal végzett kísérletben /per os 30 mg/kg / 22,0 o - 23,3 o SCE per sejt átlagértéket kaptunk, mely jelentősen /p<0,001/ nagyobb volt a "negatív kontroll" értéknél.
A továbbiakban megadott kísérleti körülmények között az EPTC hatóanyag nem mutatkozott mutagénnek.

A negatív kontroll csoport / csapvíz + 0,5% Tween 80 / állatainak csontvelősejtjeiben a hímek esetében 3,4 +-1,7 a nőstényeknél 4,5 +-2,5 SCE/sejt értékeket kaptunk.

2x 500 mg/kg EPTC technikai hatóanyag hatására az SCE frekvencia gyakorlatilag egyik nemnél sem változott /hím: 3,7 +- 1,8, nőstény: 3,9 +-1,6 SCEpers sejt/.

2x 1000 mg/kg dózisú EPTC a nőstényeknél nem /4,2 +-2/ a hímeknél kis mértékben / 4,7 +-2,5 növelte az SCE gyakoriságot. /p 0,001/. A növekedés mértéke biológiailag nem jelentős /5/.

A pozitív kontroll Cyclophosphamid 22,0 +- 6,1 /hím/ és 23,3 +- 8,2 /nőstény/ sejtenkénti átlag SCE értékeket produkált.

Conclusions:

Interpretation of results (migrated information): negative
A megadott kísérleti körülmények között az EPTC hatóanyag nem mutatkozott mutagénnek.

Executive summary:

EPTC is reported to be not mutagen.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

The potential of EPTC to cause genetic toxicity (chromosome abberrations: mamalian germ cell cytogenetic assay and DNA damage and /or repair: sister chromated exchange assay) was investigated in the bone marrow of NMRI mice.

EPTC did not reported to be germ cell mutagen based on in vivo tests.

Justification for selection of genetic toxicity endpoint

In vivo test

Justification for classification or non-classification

EPTC is not classified as germ cell mutagenicity based on in vivo genetic toxicity tests.