Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
genetic toxicity in vivo
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2006

Materials and methods

Type of assay:
other: The EU has carried out a full hazard review as part of the EU Risk assessment of 2,4-TDA. The text in the results section below is the In vivo hazard assessment from the risk assessment.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
2,4-TDA

Results and discussion

Any other information on results incl. tables

George and Westmoreland (1991) investigated the effects of single oral administrations of 150 mg/kg bw (2-h and 16-h treatment) and 300 mg/kg bw (16-h treatment) on hepatocytes of male rats (Fischer-344). A weak positive result was obtained at 16-h sampling with 150 mg/kg bw, no effect was found with 300 mg/kg bw (for 150 mg/kg the mean net grain value was 1.86 as compared to -3.19 in the negative control). Dosing of 150 mg/kg bw was negative at 2-h sampling time. No data about toxicity were given.

Table 1. In vivo tests: Tests for induction of unscheduled DNA synthesis (UDS) in rats.

Test system

Doses

Expos. regimen

Sampl.

times

Result

Local cytotox.

General toxicity

Remarks

Reference

Fischer-344 rat liver

150 mg/kg bw

1 x p.o.

2 h,

12 h

positive

no data

no data

autoradio-graphy

Mirsalis et al., 1982

Fischer-344 rat liver

150 - 300 mg/kg bw

1 x p.o.

2 h,

16 h

positive

no data

pilot study: 1/4 animals dosed with 150 mg/kg died

autoradio-graphy

George and Westmore-land, 1991

Rodent germ cell tests

2,4-TDA was negative in dominant lethal and sperm abnormality tests with mice.

According to Soares and Lock (1980) 2,4-TDA was negative in dominant lethal tests with male mice (strain: DBA/2J) both after oral or intraperitoneal administration of 40 mg/kg bw on two consecutive days. At 48 h after treatment, each treated male was paired with three CD-1 female mice. After seven days the females were replaced by new females. This mating scheme was repeated for a total of seven weeks post-treatment. There was no induction of abnormalities in sperm morphology after both types of administration. Sperms were investigated eight weeks post-treatment.

The i.p. application of 2,4-TDA doses from 111 mg/kg to 375 mg/kg bw to C57Bl/6xC3H mice (10 animals per dose group) resulted in a dose-dependent reduction of murine testicular DNA synthesis (Greene et al. 1981). The test substance was applied 3h prior to i.p. injection of 10 uCi[125]iododeoxyuridine and 3.5 h prior to removal of testes; physiological saline was used as negative control and an oral dose of 100 mg/kg bw dimethylnitrosamine served as positive control. There are no data on toxicity in the main experiment. In a parallel experiment 2,4-TDA induced a dose-dependent reduction of body temperature. The authors conclude that the inhibition of murine testicular DNA synthesis induced by 2,4-TDA could not be entirely explained by drop in body temperature (which in itself may lead to reduced testicular DNA synthesis). This study is regarded as additional information as inhibition of DNA synthesis is not a genotoxicity endpoint but a relatively unspecific parameter of cytotoxicity.

Table 2. In vivo tests: Rodent germ cell tests with mice.

Test system

Doses

Exposure

regimen

Result

General toxicity

Reference

Testicular DNA synthesis

111, 167, 250, 375 mg/kg bw

1 x i.p.

positive

hypo-thermia

Greene et al., 1981

Dominant lethal test, DBA/2J mice

40 mg/kg bw

2 x p.o., treated on two consecutive days

negative

no data

Soares and Lock, 1980

2 x i.p., treated on two consecutive days

negative

no data

Sperm morphology test, DBA/2J mice

40 mg/kg bw

2 x p.o., treated on two consecutive days

negative

no data

Soares and Lock, 1980

2 x i.p., treated on two consecutive days

negative

no data

Drosophila melanogaster

2,4-TDA induced positive effects in sex-linked recessive lethal tests with Drosophila (SLRL-test).

In sex-linked recessive lethal tests with Drosophila (strain: Berlin K) positive results were reported after feeding of males with doses of 5.9 mmol/l (721 µg/ml) 2,4-TDA and 15.2 mmol/l (1857 µg/ml) 2,4-TDA for 3 days (Blijleven, 1977).

Fahmy and Fahmy (1977) described that 2,4-TDA was positive in a test for sex linked recessive lethals in Drosophila (strain: Oregon-K) after single microinjection of doses ranging from 5.0 to 20 mmol/l (611 - 2443 µg/ml) into the haemocoel of males.

Table 3. In vivo tests: tests with Drosophila melanogaster.

Test system

Doses

Exposure

regimen

Result

General toxicity

Reference

Sex-linked recessive lethal test

5.9 - 15.2 mmol/l

(721 -

1857 µg/ml)

3 days;

feeding of males

positive

no data

Blijleven, 1977

Sex-linked recessive lethal test

5.0 - 20 mmol/l;

(611 - 2443 µg/ml)

1 x injection into the haemocoel of males

positive

no data

Fahmy and Fahmy, 1977

DNA strand breaks

2,4-TDA was positive for induction of DNA strand breaks in several rodent tissues.

Induction of DNA strand breaks in cells of different mouse organs was analyzed by Sasaki et al. (1999; 1997) using the alkaline single-cell gel electrophoresis assay (Comet-assay).

Male mice (strain: ddY) were treated with the maximum tolerated doses of 60 mg/kg bw. After single oral administration a statistically significant increase in DNA damage was reported for cells of stomach, liver and kidney. The strongest effect was shown in liver. Negative results were observed in cells of colon, bladder, lung, brain and bone marrow. The sampling times were 3 h, 8 h and 24 h; data on toxic effects were not given (Sasaki et al., 1999).

After single intraperitoneal administration of 240 mg/kg bw to male mice (strain CD-1) and exposure times of 3 h and 24 h positive effects were found in cells of liver, kidney and lung. In liver cells the strongest positive response was determined. No effects were observed in cells of spleen and bone marrow. No data on toxic effects were given (Sasaki et al., 1997).

Multiple organs of male Wistar rats were analysed for DNA strand breaks in the comet assay (Sekihashi et al., 2002). After a single oral gavage of 130 mg/kg bodyweight a statistically significant increase in migration of nuclear DNA was reported for stomach, colon, kidney and brain. The strongest effect was shown in stomach. No effects were observed in liver, bladder, lung and bone marrow. The dose was reported to be equivalent to the 0.5 x LD50 value.

Induction of DNA strand breaks in male rat liver (strain: Sprague-Dawley) was analyzed by Brambilla et al. (1985) using the viscometric technique, a measure of viscosity profiles of DNA. Animals were treated with single intraperitoneal injection of 37 and 147 mg/kg bw, sampling was after 2 h, 4 h, 12 h and 24 h. Both doses induced a positive effect; the highest tested dose was equivalent to the LD-50 value. There were no data on toxicity.

Table 4. In vivo tests: DNA strand breaks.

Test system

Doses

Expos. regimen

Sampl.

times

Result

General toxicity

Positive organs

Negative organs

Reference

Comet-assay on ddY mice

60 mg/kg bw

1 x p.o.

3 h,

8 h,

24 h

positive

no data

liver

kidney

stomach

lung

bone marrow

colon

bladder

brain

Sasaki et al., 1999

Comet-assay on CD-1 mice

240 mg/kg bw

1 x i.p.

3 h,

24 h

positive

no data

liver

kidney

lung

bone marrow spleen

Sasaki et al., 1997

Comet assay on Wistar rats

130 mg/kg bw

1 x p.o.

3h,

8 h,

24 h

positive

0.5 x LD50

stomach, colon, kidney, brain

liver, bladder, lung, bone marrow

Sekihashi et al., 2002

Viscometric technique, Sprague-Dawley rats

37 - 147 mg/kg bw

1 x i.p.

2 h,

4 h, 

12 h,

24 h

positive

147 mg/kg = LD50

hepato-cytes

.

Brambilla et al., 1985

DNA adducts

2,4-TDA formed adducts with DNA of various organs of rats.

32P-post-labeling technique (with nuclease P1 enrichment):

Delcos et al. (1996) observed a time- and dose-dependent formation of adducts in DNA from livers and mammary glands of female rats (strain: Fischer-344) fed 10 up to 180 ppm (1.2 - 22.1 mg/kg bw) 2,4-TDA. Sampling times were 0.5, 1, 3 and 6 weeks after beginning of the test. The maximum yield of DNA adducts, about 200x107 relative adduct level (RAL), was found at the highest tested dose after exposure for 6 weeks. A single major DNA adduct and two minor adducts were identified. No data on toxicity were given.

Wilson et al. (1996) reported on DNA-binding in hepatic cells of male rats (strain: Fischer-344) after single intraperitoneal injection of doses ranging from 0.5 up to 250 mg/kg bw; sampling time was 24 h. The effect was dose-dependent: the highest concentration of adducts (about 120 nmol/g DNA) was determined at the highest tested dose of 250 mg/kg bw. After a single intraperitoneal injection of 150 mg/kg bw a time course was investigated: adduct concentration was measured at different time points (0.5 - 30 days); maximum adduct level (about 60 nmol/g DNA) occurred 24 h post-exposure. No toxicity data were given.

Taningher et al. (1995) investigated 2,4-TDA for the ability to induce DNA-adducts in liver cells of male rats (strain: Fischer-344). After single intraperitoneal injection of 125 and 250 mg/kg bw a dose-dependent positive effect was observed after an exposure for 18 h. The quantitative evaluation of autoradiograms displayed one major and two faint spots. Data on toxicity were not given.

La and Froines (1994) examined DNA adduct formation in male and female rats (strain: Fischer-344) and compared the adduct formation between target organs for carcinogenicity (liver and mammary gland) and non-target organs (kidney and lung). The results demonstrated organ-specific and dose-dependent effects; there were no differences in adduct persistence. Relative adduct levels were given as ratios of adducted nucleotids to total nucleotids. With single intraperitoneal doses of 5.0 and 50 mg/kg bw the authors could show that the quantitative formation of the DNA adducts in rat liver cells is dose-dependent; maximum yields were obtained after 18 - 24 h at both doses and lasted over a 2-week period. Animals treated with a single intraperitoneal injection of 50 mg/kg bw showed a formation of three distinct DNA adducts in the liver and mammary gland at 18-h sampling; in kidney and lung only one adduct was observed. Among the organs examined, DNA binding was highest in liver (adduct 1: 17.5x107 RAL; adduct 2: 2.12x107 RAL; adduct 3: 1.05 x 107 RAL) followed by mammary gland (adduct 1: 1.8x 107 RAL; adduct 2: 0,3x 107 RAL; adduct3: 0.15x107 RAL) and the two non-target organs lung (adduct 1: 0.57 x 107 RAL) and kidney (adduct 1: 0.37x 107RAL).

Also in an earlier publication La and Froins (1992) described DNA adduct formation by 2,4-TDA in liver, lung and kidney of male rats and in mammary gland of female rats (strain: Fischer-344) after single intraperitoneal injection; sampling time was 18 h. DNA binding in liver cells was detected in a dose-range beginning at the lowest tested dose of 4.1 up to 2046 umol/kg bw (0.5 - 250 mg/kg bw); the effect was dose-dependent. At a dose of 2046 umol/kg bw (250 mg/kg) DNA binding of was described in all four organs, with each producing one major and two minor adduct spots. The adducts induced were qualitatively identical among the organs, but quantitative differences were observed. The liver (RAL, 29.2x107) and mammary gland (RAL, 4.2x107) showed major adduct yields, which were up to 30 times higher than those for kidney (RAL, 1.1x107) and lung (RAL, 1.4x107). The yields of the minor adducts were approximately 1/10 that for the major adduct.

No adducts were detected by Delcos et al. (1996) in DNA of T-lymphocytes isolated from spleen of male rats (strain: Fischer-344) fed 40 or 180 ppm (4.9 or 22.1 mg/kg bw) 2,4-TDA daily for 32 weeks.

Detection for tritiated DNA-adducts

A single intraperitoneal injection of 100 mg/kg bw of tritated 2,4-TDA (2,4-(3H)-TDA) to male rats (strain: Wistar) did not lead to interaction with DNA of liver cells but to binding to liver protein and ribosomal RNA in liver cells (Aune et al.; 1979). The detection limit for tritated DNA-adducts is far less than that of the postlabelling method.

Hemoglobin adducts

Using the gas chromatography/mass spectrometry Wilson et al. (1996) showed a 2,4-TDA induced hemoglobin adduct formation in male rats (strain: Fischer-344) after single intraperitoneal injection. The hemoglobin adduct formation increased with dose and time: After administration of 150 mg/kg bw a maximum adduct level was detected after exposure of 24 h (ca. 0.32 nmol/g hemoglobin). To examine a relationship between administered doses and hemoglobin adduct levels, animals were exposed to doses in a range from 0.5 up to 250 mg/kg bw for 24 h. The maximum adduct concentration of about 0.36 nmol/g hemoglobin was determined at the highest tested dose of 250 mg/kg bw. Data on toxicity were not given.

Table 5. In vivo tests: DNA adducts in rats

Test system

Doses

Expos. regimen

Sampl.

times

Result

Positive tested organs

General toxicity

Reference

32P-post-labeling, Fischer-344 rats

10 - 180 ppm (1.2 - 22.1 mg/kg day)

feeding daily for 6 weeks

week:

0.5, 1, 3, 6

positive

liver

mammary gland

no data

Delcos et al., 1996

32P-post-labeling, Fischer-344 rats

0.5 - 250 mg/kg bw

1 x i.p.

0.5 - 30 days

positive

liver

no data

Wilson et al., 1996

32P-post-labeling, Fischer-344 rats

125 - 250 mg/kg bw

1 x i.p.

18 h

positive

liver

no data

Taningher et al., 1995

32P-post-labeling, Fischer-344 rats

5.0 - 50

mg/kg bw

1 x i.p.

0.5 - 14 days

positive

liver

no data

La and

Froines, 1994

50 mg/kg bw

1 x i.p.

18 h

positive

liver

mammary gland

kidney

lung

no data

32P-post-labeling, Fischer-344 rats

4.1 - 2046 µmol/kg bw (0.5 - 250 mg/kg)

1 x i.p.

18 h

positive

liver

mammary gland

kidney

lung

no data

La and Froines, 1992

32P-post-labeling, Fischer-344 rats

40 - 180 ppm (4.9 - 22.1 mg/kg/ day)

feeding daily for 32 weeks

week: 1, 4, 8, 20, 32

negative (spleen T lymph.)

.

no data

Delcos et al., 1996

Tritiated DNA-adducts; Wistar rats

100 mg/kg bw

1 x i.p.

4 h

negative

liver

no data

Aune et al., 1979

Hb adducts, Fischer-344 rats

0.5 - 250 mg/kg bw

1 x i.p.

0.5 - 30 days

positive

.

no data

Wilson et al., 1996

<p></p>

<p>

Transgenic mouse

2,4 -TDA was positive in transgenic mouse assays.</p>

<p>Suter et al. (1996) described a weak genotoxic potential of 2,4-TDA on liver DNA of transgenic C57BL/6 Big Blue(trademark) mice. Male and female mice were used to examine the induction of mutations in the lacI gene in liver. In consequence of determination of the maximal tolerated dose in non-transgenic C57BL/6 mice the test animals were given ten daily oral doses of 80 mg/kg bw per day with a treatment free interval of 2 days between treatment five and six. Sampling times were 10 and 28 days after the last treatment. After an expression time of 28 days, 2,4-TDA treatments induced doublings of spontaneous mutation frequencies in both sexes (males, 8.46x10-5 as compared to 4.32x10-5 in the negative control; females, 9.67x10-5 as compared to 4.32x10-5). After 10 days expression a marginal response was obtained in females only (7.48x10-5; negative control, 5.15x10-5). Ten days after treatment hepatic cell proliferation was induced.</p>

<p>In a study with Big Blue (trademark) transgenic B6C3F1 mice Hayward et al. (1995) reported on an increase of mutant frequency at lacI gene in liver. Male mice were exposed to 2,4-TDA at 1000 ppm (123 mg/kg bw) in the diet for 30 and 90 days. No effect was observed after a feeding of 30 days, while after 90 days the mutant frequency was higher (mean mutant frequency, 12.1x10-5 as compared to 5.7x10-5 in the control group). The tested dose corresponded to the highest non-toxic dose in a 90-day subchronic study.</p>

Table 6. In vivo tests: Transgenic mouse

Test system

Doses

Expos. regimen

Sampl.

times

Result

Tested

organ

General

toxicity

Reference

Big Blue mouse

80 mg/kg bw/d

oral gavage (ten daily doses)

10 and 28 days after the last treatment

positive

liver

80 mg/kg = MTD

Suter et al., 1996

Big Blue mouse

1000 ppm (123 mg/kg/d)

feeding for 30 and 90 days

on the day after feeding period

positive

liver

1000 ppm =  highest non-toxic dose in a 90 day study

Hayward et al., 1995

<p></p>

Table 7. OVERVIEW ON IN VIVO FINDINGS

Negative effects

Questionable effects

Positive effects

Mutation tests in vivo

Micronuclei in mice and rats (bone marrow; peripheral blood)

Micronuclei in rats at a highly toxic dose (bone marrow)

Gene mutations in transgenic mice (liver)

Dominant lethals in mice

.

Drosophila (SLRL test)

Indicator tests in vivo

Sperm morphology in mice

.

SCE in mice (bone marrow)

.

.

UDS in rats (liver)

.

.

DNA strand-breaks in mice and rats (liver, kidney, lung, stomach)

.

.

DNA adducts in rats (liver, mammary gland, kidney, lung)

.

.

Reduction of testicular DNA-synthesis in mice

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): positive
The Rapporteur in the EU RISK Assessment concluded "2,4-Toluenediamine induces genotoxic effects in bacteria (gene mutations) and cultivated mammalian cells (chromosomal aberrations, SCE, UDS, DNA strand breaks, DNA adducts).

In general, rodent in vivo micronucleus tests were negative in bone marrow or peripheral blood; a weak positive effect in a rat strain was limited to a dose with high acute toxicity. However, in other tissues clear genotoxic effects were obtained which were not limited to highly toxic doses, e.g. gene mutations, UDS, DNA strand breaks and DNA adducts were observed in rodent livers.

Negative results were found in dominant lethal and sperm morphology tests; however, due to low sensitivity these findings are not adequate for exclusion of germ cell mutagenesis.

Although there is some indication for effects on the testes from a non-standard assay measuring a reduction of murine testicular DNA synthesis after 2,4-TDA we do not regard the available mutagenicity data as sufficient to classify 2,4-TDA as a category-2 mutagen.

On the basis of the positive findings on somatic cells in vitro and in vivo we rather propose to classify the substance as a category-3 mutagen."