Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
Ames test: negative (Zeiger, 1987; OECD471, NTP, 2011) HPRT locus assay: negative ; (BASF, 2012; OECD476) In vitro micronucleus assay: positive (BASF, 2012; OECD487) In vivo micronucleus assay: positive (BASF, 2014; OECD474)
Link to relevant study records
Reference
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:
2013-09-09 - 2014-07-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE Experimental Toxicology and Ecology
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: 28.73 g (mean body weight)(Exp.1); 27.66 g (mean body weight)(Exp.2)
- Assigned to test groups randomly: yes, randomization plan prepared with an appropriate computer program
- Housing: single housing in Makrolon cages, type M II cages
- Diet: Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland); ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Fully air-conditioned rooms with central air conditioning
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: due to the good solubility of the test substance
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The substance was dissolved in deionized water
- To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly.
- All test substance formulations were prepared immediately before administration.
Duration of treatment / exposure:
single administration with a volume of 10 mL/kg body weight of the test substance preparation, positive control or vehicle
Frequency of treatment:
once orally
Post exposure period:
Depending on the test group in the 1st Experiment 24 and 48 hours and in the 2nd Experiment 48 and 72 hours after test substance administration, the animals were sacrificed and both femora each were excised for the preparation of the bone marrow smears.
Remarks:
Doses / Concentrations:
150, 300 and 600 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5 male animals per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive control No. 1: 20 mg/kg bw cyclophosphamide (CPP)
Positive control No. 2: 0.15 mg/kg bw vincristine sulfate (VCR)

The positive controls, both dissolved in deionized water, were administered to animals once orally (cyclophosphamide, CPP) or intraperitoneally (vincristine sulfate, VCR) each in a volume of 10 mL/kg body weight.
The stability of CPP and VCR is well-defined under the selected conditions, since both substances are well-established reference clastogens and aneugens, respectively.
Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a pretest to determine the acute oral toxicity in males and females, deaths were observed at 1 000 mg/kg body weight. At 500 mg/kg, all animals
survived but weak signs of toxicity were observed. However, there were no distinct differences in clinical observations between male and female animals. Thus, only male animals were used for the cytogenetic investigations as requested by the current OECD Guideline 474.
Based on the data of the pretest a dose of 600 mg/kg body weight was defined as MTD (maximum tolerated dose) and was selected as the highest
dose in the present cytogenetic study. 300 mg/kg and 150 mg/kg body weight were administered as further doses.
TREATMENT AND SAMPLING TIMES:
In the 1st Experiment, the animals were treated once orally (gavage) with a volume of 10 mL/kg body weight of the test substance and the vehicle.
The positive controls, both dissolved in deionized water, were administered to male animals once orally (CPP) or intraperitoneally (VCR) each in a
volume of 10 mL/kg body weight. The animals were sacrificed 24 hours (all test substance concentrations, vehicle, both positive controls) and 48hours (highest test substance concentration, vehicle) after the treatment, respectively.
In the 2nd Experiment, the animals were treated once orally (gavage) with a volume of 10 mL/kg body weight of the test substance and the vehicle.he animals were sacrificed 48 hours (all test substance concentrations, vehicle) and 72 hours (highest test substance concentration, vehicle)
after the treatment, respectively. With regard to animal welfare in the 2nd Experiment only one positive control, dissolved in deionized water, was
administered to male animals once orally (CPP) with a volume of 10 mL/kg body weight. The animals of this dose group were sacrificed 24 hours
after administration because experiences on the genotoxic potency of CPP after prolonged 48 hours exposure were missing in our laboratory.
The positive control animals were sacrificed in parallel to all dose groups prepared at 48 hours sacrifice interval.
DETAILS OF SLIDE PREPARATION:
- The slides were stained with eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes
- After briefly rinsing in deionized water, the preparations were soaked in deionized water for about 2 - 3 minutes
- Subsequently, the slides were stained with Giemsa solution (15 mL Giemsa plus 185 mL deionized water) for about 15 minutes
- After rinsing twice in deionized water and clarifying in xylene, the preparations were mounted in Corbit-Balsam

Evaluation criteria:
The test is considered valid if:
• The quality of the slides must allow the evaluation of a sufficient number of analyzable cells; i. e. ≥ 2 000 PCEs per animal and a clear differentiation between PCEs and NCEs.
• The ratio of PCEs/NCEs in the concurrent vehicle control animals has to be within the normal range for the animal strain selected.
• The number of cells containing micronuclei in vehicle control animals has to be within the range of the historical vehicle control data for PCEs.
• The administered positive control substances have to induce a distinct increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.

A finding is considered positive if:
• Statistically significant and dose-related increase in the number of PCEs containing micronuclei.
• The number of PCEs containing micronuclei has to exceed both the concurrent vehicle control value and the range of the historical vehicle control data.

A test substance is considered negative if:
• The number of cells containing micronuclei in the dose groups is not statistically significant increased above the concurrent vehicle control value and is within the range of the historical vehicle control data.
Statistics:
Used program system: MUKERN (BASF SE)
The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether
there are statistically significant differences between the untreated control group and the treated dose groups with regard to the micronucleus rate
in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test. Statistical significances were identified as follows:
* p ≤ 0.05
** p ≤ 0.01
However, both biological relevance and statistical significance were considered together.
Sex:
male
Genotoxicity:
positive
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Exp.1 Summary Table: Induction of micronuclei in bone marrow cells

 

Sacrifice

Interval

[hrs]

Animal

No.

PCE with Micronuclei

PCEs per

2 000

erythrocytesc

totala

[‰]

largeb

[‰]

Vehicle control

deionized water

24

5

1.0

0.0

1365

Test substance

150 mg/kg bw.

24

5

2.2**

0.1

1370

Test substance

300 mg/kg bw.

24

5

2.4

0.0

1373

Test substance

600 mg/kg bw.

24

5+

2.2**

0.1

1407

Positive control cyclophosphamide

20 mg/kg bw.

24

5

15.1**

0.1

1417

Positive control

vincristine sulfate

0.15 mg/kg bw.

24

5

44.2**

10.2**

1306

Vehicle control

deionized water

48

5

1.4

0.0

1398

Test substance

600 mg/kg bw.

48

5+

4.3**

0.0

1350

Exp. 2 Summary Table: Induction of micronuclei in bone marrow cells

 

Sacrifice

Interval

[hrs]

Animal

No.

Micronuclei in PCE

PCEs per

2 000

erythrocytesc

totala

[‰]

large MNb

[‰]

Vehicle control

deionized water

24

5

1.3

0.0

1315

Test substance

150 mg/kg bw.

24

5

1.1

0.0

1270

Test substance

300 mg/kg bw.

24

5

4.2**

0.0

1295

Test substance

600 mg/kg bw.

24

5

6.0**

0.1

1199

Positive control cyclophosphamide

20 mg/kg bw.

24

5

18.4**

0.1

1279

Vehicle control

deionized water

48

5

3.0

0.2

1322

Test substance

600 mg/kg bw.

48

5

3.5

0.5

908

PCE = polychromatic erythrocytes

NCE = normochromatic erythrocytes

bw. = body weight

a = sum of small and large micronuclei

b = large micronuclei (indication for spindle poison effect)

c = calculated number of PCEs per 2 000 erythrocytes (PCE + NCE) when scoring a sample of up to 10 000 PCE per test group

+ = sample of evalutation was increased up to 4 000 erythrocytes (PCE + NCE) per animal to confirm the data

* = p ≤ 0.05

** = p ≤ 0.01

Clinical observations1st and 2nd Experiment:

- Dose: 150 mg/kg bw: no clinical signs

- Dose: 300 mg/kg bw: no clinical signs

- Dose: 600 mg/kg bw (24 hrs): all animals showed piloerection 1h, 2h ,4h and 1d after test substance application; hunched posture was observed 1h, 2h, and 4h after test substanc application in all animals

- Dose: 600 mg/kg bw (48 hrs): all animals showed piloerection 1h, 2h, 4h and 1d after test substance application; hunched posture was observed 1h, 2h and 4h after test substance application in all animals. No clinical signs 2d after substance application .

-Dose: 600 mg/kg (72 hrs- only 2nd Experiment): all animals showed piloerection 1h, 2h, 4h and 1d after test substance application; hunched posture was observed 1h, 2h, 4h after test substance application in all animals. No clinicals signs 2d and 3d after substance application.

Conclusions:
Interpretation of results (migrated information): positive
Executive summary:

Thus, under the experimental conditions chosen, the test substance Dipropylene triamine showed the potency to induce chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in bone marrow cells of NMRI mice in vivo at delayed 48-hour and 72-hour sacrifice interval.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vivo:

In-vitro study: Bacterial systems

Dipropylenetriamine was tested in the reverse mutation assay according to OECD TG 471 (adopted 1983) using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 at 33 to 10000 µg/plate (preincubation test) with and without metabolic activation. Dipropylenetriamine was only slightly cytotoxic at the highest concentrations in all 4 strains of S. typhimurium. The number of revertants was not inreased in any of the test strains. In conclusion, Dipropylenetriamine was not mutagenic under the conditions of the study.

In-vitro study: Mammalian cell gene mutation test

The study was performed according to OECD guideline 476 in compliance with GLP. The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Four independent experiments were carried out, all with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation).

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations. In this study, in all experimental parts in the absence and presence of metabolic activation evaluated for gene mutations at least the highest concentrations applied were clearly cytotoxic. On the basis from the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in four experiments performed independently of each other.

Thus, under the experimental conditions of this study, the test substance Dipropylene triamine is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

In-vitro study: in vitro micronucleus assay

The study was performed according to OECD TG 487 in compliance with GLP. The test substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity) both in the absence and the presence of a metabolizing system.

The highest test concentration corresponded to appr. 10 mM, which is the max. conc. acc. to OECD guideline. A sample of at least 1 000 cells for each culture were analyzed for micronuclei, i.e. 2000 cells for each test group. The vehicle controls gave frequencies of micronucleated cells within historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei. In the main experiments, no cytotoxicity indicated by clearly reduced relative increase in cell count (RICC) or proliferation index (PI) was observed up to the highest applied test substance concentration.

On the basis of the results of the present study, the test substance caused a statistically significant increase in the number of cells containing micronuclei without S9 mix at intermediate concentrations showing no cytotoxicity in two experiments carried out independently of each other.

Thus, under the experimental conditions described, the test substance is considered to be genotoxic under in vitro conditions in V79 cells in the absence of metabolic activation.

In-vivo study: in vivo micronucleus assay

The study was performed according to OECD TG 474 in compliance with GLP. The test substance was assessed for its potential to induce

chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in NMRI mice after administration once orally to male animals at dose levels of 150 mg/kg, 300 mg/kg and 600 mg/kg body weight. Both positive control substances, cyclophosphamide and vincristine sulfate, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. The MTD (maximum tolerated dose) of 600 mg/kg body weight was defined due to severe signs of toxicity and several deaths at 1 000 mg/kg body weight in the pretest. In the 1st Experiment, the single oral administration of Dipropylene triamine led to statistically significant increased numbers of polychromatic erythrocytes containing either small or large micronuclei in all dose groups. To corroborate the data an increased sample of 4 000 PCEs per animal was scored for the top dose group at both sacrifice intervals. The values at 24-hour sacrifice interval were clearly within the historical negative control data range (0.3 – 3.2‰ micronucleated polychromatic erythrocytes), but at 48-hour sacrifice interval the micronucleus rate clearly exceeded the borderline of 3.2‰ micronucleated polychromatic erythrocytes. For corroboration of this finding, a 2nd Experiment with delayed sacrifice intervals of 48 and 72 hours was performed. In the 2 nd Experiment a clear inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected at 72 hours sampling time at 600 mg/kg body weight compared with the concurrent vehicle control group as clear indication that the test substance reached the target organ bone marrow.

A dose-related increase in the number of micronucleated polychromatic erythrocytes was observed at 48-hour sacrifice interval in the 2 nd experiment. The values at both higher dose groups were statistically significant increased. They clearly exceeded both the concurrent negative control values and the laboratory’s historical negative control data range. Additionally, the single high dose administered at 72-hour sacrifice interval induced a slightly increased micronucleus rate which exceeded either the concurrent negative control values or the historical negative control data range. Thus, under the experimental conditions of this study, Dipropylene triamine had the potency to increase the number of micronucleated PCEs from bone marrow of NMRI mice in vivo 48 and 72 hours after administration.

Justification for classification or non-classification

Based on the results of in vitro and in vivo micronucleus tests, Dipropylene triamine was considered to be clastogenic. Thus, classification is warranted according to EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 (R68; GHS cat. 2).