REAKTIV-ORANGE DYPR 934

Genetic toxicity: in vivo

Administrative data

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:

1999-05-03 to 1999-05-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to GLP and guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Details on test material:

-Name of test material (as cited in study report): Reaktiv-Orange DYPR934

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Species: Mouse
Strain: Hsd Win:NMRI
- Source: ORIGINE (supplier) of animals: Harlan Winkelmann GmbH, Gartenstrasse 27, 33178 Borchen
- Age at study initiation: male/female animals approx. 7 weeks
- Weight at study initiation: MALE ANIMALS: mean = 32.3 g (= 100 %)
min = 29 g (-10.2 %)
max= 35 g (+8.4 %)
n = 15
FEMALE ANIMALS: mean =26.7 g (= 100 %)
min = 24 g (-10.1 %)
max= 32g (+19.9 %)
n=15
- Assigned to test groups randomly: [yes, under following basis:] randomization schemes 1999.0149 and 1999.0150
ANIMAL MAINTENANCE:
In fully air conditioned rooms in macrolon cages type 3 (five animals per cage) on soft wood granulate.
Food: rat/mice diet ssniff ® R/M-H (V1534) ad libitum
- Fasting period before study:
- Housing:
ANIMAL IDENTIFICATION: fur marking with KMn04 and cage numbering
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum): Tap water in plastic bottles, ad libitum.
- Acclimation period: 5 days under study conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 hours daily

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Deionized water.
- Amount of vehicle (if gavage or dermal): The vehicle was administered in the negative control groups (twice at interval of 24 hours orally by gavage at a dose of 2000 mg per kg body weight.)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test compound was dissolved in deionized water and was given twice at an interval of 24 hours as an orally dose of 2000 mg/kg body weight.
In a preliminary dose range finding study, oral administration of 2000 mg Reaktiv-Orange DYPR 934 per kg body weight did not cause any toxic effects in male and female
1 st dose: 2000 mg Reaktiv-Orange DYPR 934 per kg body weight
Observation period: 1999-04-20 to 1999-04-28
Number of animals used: 3 males and 3 females
DIET PREPARATION
- FREQUENCY OF ADMINISTRATIONS: twice at interval of 24 hours

FORMULATION OF TEST COMPOUND: On the days of administration the test substance was dissolved in deionized water at appropriate concentration.
A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed.

FORMULATION OF REFERENCE COMPOUND: Cyclophosphamide (CPA) dissolved in distilled water on the second day of experiment; final concentration 0.5 % (w/v)
FORMULATION OF TEST COMPOUND: On the days of administration the test substance was dissolved in deionized water at appropriate concentration.
A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed.

FORMULATION OF REFERENCE COMPOUND: Cyclophosphamide (CPA) dissolved in distilled water on the second day of experiment; final concentration 0.5 % (w/v)

Duration of treatment / exposure:

1 day (twice at an interval of 24 hours)

Frequency of treatment:

Once

Post exposure period:

Test item group animals dosed at 2000 mg/kg body weight were sacrificed 24 hrs following dosing.
Vehicle control animals were sacrificed 24 hrs following dosing.
Positive control animals were sacrificed 24 hrs following dosing.

Doses / concentrations

Remarks:

Doses / Concentrations:
2000 mg/kg
Basis:
nominal in water

No. of animals per sex per dose:

Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Female: 2000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (=Endoxan®)
- Justification for choice of positive control(s): produce micronuclei in vivo at exposure levels expected to give a detectable increase over background.
- Route of administration: oral /gavage
- Dose(s) / concentration(s): 50 mg/kg body weight

Examinations

Tissues and cell types examined:

The animals were examined for clinical signs of toxicity after dosing. Femural bone marrow was prepared from all control and dose group animals at 24 hr post dosing in animals receiving the high 2000 mg/kg body weight dose.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Preliminary experiment: Preliminary studies were conducted to determine the highest administrable non lethal dose level up to the limit dose of 2000 mg body weight according to the OECD
PREPARATION AND STAINING
Animals were killed by carbon dioxide asphyxiation 24 hours after dosing. For each animal, about 3 mL fetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at approx. 1200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 hours.
STAINING:
Staining was performed as follows:
-5 minutes in methanol
-5 minutes in May-Grünwald’s solution
-brief rinsing in twice distilled water
-10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
-rinsing in distilled water
-drying
-coating with Entellan®

Evaluation criteria:

Evaluation:
2000 polychromatic erythrocytes were counted for each mouse. The number of cells with micronuclei was recorded, not the number of individual micronuclei. In addition, the ratio of polychromatic erythrocytes to 200 total erythrocytes was determined. Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the substance, was the proportion of polychromatic erythrocytes with micronuclei out of 2000 counted erythrocytes. All bone marrow smears for evaluation were coded to ensure that the group from which they were taken remained unknown to the investigator.
A one-sided Wilcoxon Test was evaluated to check the validity of the study. The study was considered as valid in case the proportion of polychromatic erythrocytes with micronuclei in the positive control was significantly higher in the negative control (p = 0.05).
If the validity of the study has been shown the following sequential test procedure for the examination of the mutagenicity was applied: Based on a monotone -dose-relationship one-sided Wilcoxon tests were performed starting with the highest dose group. These tests were performed with a multiple significance of 5 %.

 

Statistics:

Biological and statistical significances.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations: All animals survived after treatment. No signs of toxicity; but orange discolored feces and urine were observed up to the end of study.

Any other information on results incl. tables

  

Summary tables and Statistics: Test compound: Reaktiv-Orange DYPR 934
Sex	Dose Mg/kg b.w.	Killing time	Number of animals	Poly counted	Poly/Ery Mean	Poly/Ery SD Mean	Poly with MN Mean	Poly with MN [%] Mean	Poly with MN SD Mean
male	0-Control	24h	5	2000	0.46	0.08	1.6	0.08	0.07
male	2000	24h	5	2000	0.45	0.06	1.0	0.05	0.05
male	50-Endoxan®	24h	5	2000	0.43	0.06	87.0	4.35	1.65
female	0-Control	24h	5	2000	0.44	0.13	2.2	0.11	0.07
female	2000	24h	5	2000	0.37	0.08	3.2	0.18	0.05
female	50-Endoxan®	24h	5	2000	0.42	0.09	63.8	3.19	0.88

 

	Summary tables and Statistics: Test compound: Reaktiv-Orange DYPR 934
Sex		Dose Mg/kg b.w.	Killing time	Number of animals	Poly counted	Poly/Ery Mean	Poly/Ery SD Mean	Poly with MN Mean	Poly with MN [%] Mean	Poly with MN SD Mean	Mutagenetic Index
pooled		0-Control	24h	10	2000	0.45	0.10	1.90	0.1	0.07	0.07 Mutagenetic Index: 1.0
pooled		2000	24h	10	2000	0.41	0.08	2.10	0.1	0.1	0.08 Mutagenetic Index: 1.1
pooled		50-Endoxan®	24h	10	2000	0.42	0.07	75.40 (significant difference from control (p<0.05)	3.8	3.8	1.39 Mutagenetic Index: 39.7

 

 Control= vehicle (deionized water)

A cross comparaison of individual data and pooled data may show discrepancies since the values are rounde.

TABLE OF INDIVIDUAL DATA                                             TEST COMPOUND: REAKTIV-ORANGE DYPR 934
Group	Animal number	Sex	Dose Mg/kg body weight	Poly/200 Ery	Poly With MN	Poly/Ery	Poly With MN [%]
1	1	male	o-Control	93	1	0.47	0.05
1	2	male	o- Control	82	1	0.41	0.05
1	3	male	o- Control	87	0	0.44	0.00
1	4	male	o- Control	117	3	0.59	0.15
1	5	male	o- Control	77	3	0.39	0.15
1	6	female	o- Control	122	2	0.81	0.10
1	7	female	o- Control	58	0	0.29	0.00
1	8	female	o- Control	83	4	0.42	0.20
1	9	female	o- Control	68	2	0.34	0.10
1	10	female	o- Control	105	3	0.53	0.15
2	11	male	2000	102	1	0.51	0.05
2	12	male	2000	75	0	0.38	0.00
2	13	male	2000	78	0	0.39	0.00
2	14	male	2000	101	2	0.51	0.10
2	15	male	2000	89	2	0.49	0.10
2	16	female	2000	53	4	0.32	0.20
2	17	female	2000	73	2	0.37	0.10
2	18	female	2000	81	4	0.41	0.20
2	19	female	2000	56	4	0.28	0.20
2	20	female	2000	97	2	0.49	0.10
3	21	male	50- Endoxan®	68	130	0.44	6.50
3	22	male	50- Endoxan®	75	110	0.38	5.50
3	23	male		103	61	0.52	3.05
3	24	male	50- Endoxan®	87	83	0.44	4.15
3	25	male		73	51	0.37	2.55
3	26	female	50- Endoxan®	67	73	0.34	3.65
3	27	female		90	49	0.45	2.45
3	28	female	50- Endoxan®	90	49	0.38	3.55
3	29	female		75	71	0.38	4.20
3	30	female	50- Endoxan®	75	42	0.38	2.10

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The results lead to the conclusion that REAKTIV-ORANGE DYPR 934 did not cause a substantial increase of micronucleated polychromatic erythrocytes and is not mutagenic in the micronucleus test under the conditions described in this report.

Executive summary:

A micronucleus test was carried out with Reaktiv-Orange DYPR 934. The test compound was dissolved in deionized water and was given twice at an interval of 24 hours as an orally dose of 2000 mg per body weight to approx. 7 weeks old male and female mice, based on the results of a previous dose range finding assay. According to the procedure the animals were killed after 24 hours administration.

The main purpose of the study was to assess the potential of the test compound to cause chromosomal damage (clastogenicity) in a mouse bone micronucleus test.

Endoxan ® was used as positive control substance and was administrated once orally at dose of 50 mg per kg body weight.

Endoxan ® (cyclophosphamide) induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system.

The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

The number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes remained unaffected by the treatment with Reaktiv-Orange DYPR 934 and was not less 20 % on the control value.

Both biological and statistical significances were considered together for evaluation purposes.

Under the conditions of the present study the results in lead to the conclusion that REAKTIV-ORANGE DYPR 934 did not cause a substantial increase of micronucleated polychromatic erythrocytes and is not mutagenic in the micronucleus test under the conditions described in this report.