[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

DHX2 was evaluated for its mutagenic potential in the strains of Salmonella typhimurium, TA100, TA1535, TA98 and TA1537, and Escherichia coli WP2uvrA with or without metabolic activation according to OECD Guideline 471 and GLP principles. A chromosome aberration study with DHX2 was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. An in vivo micronucleus study with DHX2 in the mouse (5 animals per sex/dose, intraperitoneal injection) was conducted according to OECD 474 and GLP guidelines.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 17, 2005 - March 08, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(1997)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(2000)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: NMRI BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: Young adult animals (6-8 weeks old)
- Weight at study initiation: males: 34.2-38.6 g; females: 25.6-30.8 g
- Assigned to test groups: randomly
- Housing: Group housing of 5 animals per sex per cage in labelled polycarbonate cages.
- Diet (e.g. ad libitum): Free access to standard pelleted laboratory animal diet (Altromin (code VRF 1), Lage, Germany)
- Water (e.g. ad libitum): Free access to tap-water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30-70
- Air changes (per hr): Approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: physiological saline

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test substance was formulated in physiological saline. DHX concentrations were dosed within 2.5 hours after preparation.

The mice received an intraperitoneal injection of a maximum tolerated (high), an intermediate and a low dose of DHX2. The route of administration was chosen to maximize the chance of the test article reaching the target tissue. The route and frequency of administration and the volume administered of the negative control was the same as those of the test substance.

Dosing volume administered: 10 mL/kg body weight.

Duration of treatment / exposure:

Negative control: 24 hours.
Test substance: 24 and 48 hours.
Positive control: 48 hours.

Frequency of treatment:

Single.

Remarks:

Doses / Concentrations:
290 mg/kg bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
575 mg/kg bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
750 mg/kg bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
1150 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CP) in physiological saline
- Route of administration: Single intraperitoneal injection
- Doses / concentrations: 50 mg/kg body weight (10 mL/kg body weight)

Tissues and cell types examined:

Measuring the increase of the number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in mouse bone marrow.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Selection of an adequate dose for the Micronucleus test was based on a preliminary study (see additional information on results).

SAMPLING TIMES ( in addition to information in specific fields):
Sampling of the bone marrow was done 24 and 48 72 hours after treatment.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed by cervical dislocation. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approx. 2 mL of fetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm for 5 minutes and the supernatant was removed. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide. The drop was spread by moving a clean slide with roundwhetted sides. The preparations were air-dried, fixed for 5 min in methanol and air-dried overnight. The slides were automatically stained using the Wright-stain-procedure in an Ames HEMA-tek slide stainer. The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.

METHOD OF ANALYSIS:
Slides were scored at a magnification of 1000x. The number of micronucleated polychromatic erythrocytes (PCE) was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

Evaluation criteria:

The test substance was considered positive if:
It induced a biologically as well as a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.
The test substance was considered negative if:
None of the tested concentrations or sampling times showed a statistically significant increase in the incidence of micronucleated polychromatic erythrocytes neither in the combined data for both sexes nor in data for male or female groups separately.

Statistics:

Wilcoxon Rank Sum Test, two-sided test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

in the groups treated with 750 and 1150 mg DHX/kg body weight.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range study 1: 2000, 200, 500, 1000, 1500, 1250 and 1150 mg/kg body weight
- Dose range study 2: 1000 and 750 mg/kg body weight (performed in response to the death of 12 animals at 1150 mg/kg body weight in main study 1)
- Solubility: the test substance was formulated in physiological saline
- Clinical signs of toxicity in test animals: see "Tables dose range finding in the attached background material

RESULTS OF DEFINITIVE STUDY, see "Any other information on results incl. tables"

- Mortality and systemic toxic signs: The animals of the groups treated with 575 and 290 mg DHX2/kg body weight and the animals of the negative and positive control groups showed no abnormalities. The following clinical observations were made in the groups treated with 1150 mg DHX2/kg body weight: During the first hour after dosing nine male and three female animals were lethargic and showed ventral recumbency. One female animal also had their eyes closed. Six male and twelve female animals died. Within 19 hours after dosing the surviving animals recovered from the treatment.
750 mg DHX2/kg body weight: During the first hour after dosing all animals were lethargic and five animals also showed ataxia. Within 18 hours after dosing all animals recovered from the treatment.
- Induction of micronuclei (for Micronucleus assay): No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of DHX2 treated animals compared to the vehicle treated animals.
- Ratio of PCE/NCE (for Micronucleus assay): The animals of the groups, which were treated with DHX2, showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this compound on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.
- As animals died it is clear the test substance as systemically available, which means it was present in the blood. Bone marrow is a well perfused tissue and the test substance is considered to have reached the bone marrow.

Summary of results

Test group males	Dose mg/kg bw	Sampling time (h)	PCEs with micronuclei	Ratio PCE/NCE
Vehicle	0	24	0.6 ± 0.9	0.87 ±0.17
Test substance	1150	24	1.5 ± 1.9	0.94 ±0.07
Test substance	1150	48	0.8 ± 1.8	1.27 ±0.22
Test substance	575	24	1.0 ± 1.0	0.99 ±0.12
Test substance	290	24	1.2 ± 1.1	1.22 ±0.19
Cyclophosphamide	50	48	68.8±24.2	0.33 ±0.15

 

Test group females	Dose mg/kg bw	Sampling time (h)	PCEs with micronuclei	Ratio PCE/NCE
Vehicle	0	24	1.4 ± 1.5	0.96±0.04
Test substance	575	24	0.8 ± 1.1	1.01±0.10
Test substance	290	24	0.8 ± 1.3	1.01±0.21
Cyclophosphamide	50	48	29.0 ±11.3	0.42±0.08

 

Test group females	Dose mg/kg bw	Sampling time (h)	PCEs with micronuclei	Ratio PCE/NCE
Vehicle	0	24	0.2 ± 0.4	1.11±0.05
Test substance	750	24	0.8 ± 0.8	1.17±0.26
Test substance	750	48	1.2 ± 1.1	1.05±0.06
Cyclophosphamide	50	48	23.2±3.8	0.52±0.24

 

Cyclophosphamide showed a distinct increase of induced micronucleus frequency.

Conclusions:

An in vivo micronucleus study with DHX2 in the mouse (5 animals per sex/dose, intraperitoneal injection) was conducted according to OECD 474 and GLP guidelines. It is concluded that DHX2 is not clastogenic in the micronucleus test.

Executive summary:

An in vivo micronucleus study with DHX2 in the mouse (5 animals per sex/dose, single intraperitoneal injection) was conducted according to OECD 474 and GLP guidelines. As deaths occurred, the test substance was systemically available and as bone marrow is a well-perfused tissue, the test substance is considered to have reached the bone marrow. The animals treated with DHX2, showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this compound on the erythropoiesis.

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of DHX2 treated animals compared to the vehicle treated animals. It is concluded that DHX2 is not clastogenic in the in vivo micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Ames test:

DHX2 was tested in theSalmonella typhimuriumreverse mutation assay with TA1535, TA1537, TA100 and TA98 and in theEscherichia colireverse mutation assay with WP₂uvrA,in accordance with OECD 471, EU Method B.14 and according to GLP principles. Toxicity was observed in all tester strains in the absence and presence of S9-mix,except in tester strain WP₂uvrA in the presence of S9-mix in experiment 1. DHX2 did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains and in the number of revertant (Trp+) colonies in the tester strain both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded that DHX2 is not mutagenic in theSalmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay.

Chromosoom aberration test:

In a chromosome aberration study, cultured peripheral human lymphocytes were exposed to different concentrations of DHX2, in the presence and absence of S9 -mix according to OECD 473 and GLP principles.The substance was tested up to cytotoxic concentrations in two experiments.At the 24 h continuous treatment time without S9 -mixat the cytotoxic concentrations of 15 and 20 µg/ml, a statistically significant and biologically relevant increase in the number of cells with chromosome aberrations was observed in the presence of a dose-response relationship, both when gaps were included and excluded. At the 48 h continuous treatment time without S9 -mix and in the presence of S9 -mix (3 h treatment time), DHX2 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.No effects of DHX2 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that DHX2 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations.

Since DHX2 induced a statistically significant and dose dependent increase in the number of cells with chromosome aberrations in the absence of S9-mix at the 24 hour continuous treatment time, it is concluded that DHX2 is clastogenic in human lymphocytes. The clastogenicity was observed in the absence of metabolic activation, and only confined to cytotoxic concentrations.

Mouse lymphoma assay:

In accordance with Column 1 of Annex VIII Section 8.4.3. of the REACH regulation, the in vitro gene mutation study in mammalian cells needs to be conducted if a negative result in Annex VII, section 8.4.1. and Annex VIII, section 8.4.2. is obtained. The in vitro gene mutation study in mammalian cells does not need to be conducted as the substance showed a negative result in the Ames test and a positive result in the Chromosome aberration study.

In vivo micronucleus:

An in vivo micronucleus study with DHX2 in the mouse (5 animals per sex/dose, single intraperitoneal injection) was conducted according to OECD 474 and GLP guidelines. As deaths occurred, the test substance was systemically available and as bone marrow is a well-perfused tissue, the test substance is considered to have reached the bone marrow. The animals treated with DHX2, showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this compound on the erythropoiesis.

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of DHX2 treated animals compared to the vehicle treated animals. It is concluded that DHX2 is not clastogenic in the in vivo micronucleus test.

Justification for selection of genetic toxicity endpoint
One Ames test, one chromosome aberration study and one in vivo micronucleus test are available.

Justification for classification or non-classification

DHX2 does not have to be classified and has no obligatory labelling requirement for genetic toxicity in accordance with Regulation EC No. 1272/2008.