Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Link to relevant study records
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
12 Februaury to 18 March 1991
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Study undertaken at GLP accredited laboratory to internationally accepted guidelines. The restriction is due to the use of the read across approach: the test was performed not with S-205 but with Black 400, a substance which has been demonstrated to be very similar in structure, physical/chemical properties and the toxicological profile .
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
not specified
according to guideline
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
not specified
according to guideline
other: US EPA (TSCA) (10 September 1987) 798, 5395. In vivo mammalian bone marrow cytogenetics test: Micronucleus assay. Chemical Regulation Reporter 11, 7691-7692.
not specified
according to guideline
other: JEPA/MOHW/MITI Joint Directive (31 March 1987) Kampo-gyo No. 237 (JEPA), Yakuhatsu No. 306 (MOHW) and 62 Kikyoku No. 303 (MITI), Mutagenicity test/micronucleus test in rodents.
GLP compliance:
Type of assay:
micronucleus assay
Details on test animals or test system and environmental conditions:
- Age at study initiation: 39 days old
- Weight at study initiation: 22 and 24 g
- Assigned to test groups randomly: yes
- Fasting period before study: the animals were deprived of diet overnight prior to and for two hours after oral dosing
- Housing: a plastic disposable cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: four days

- Temperature (°C): 22°C
- Humidity (%): no data
- Air changes (per hr): 30 changes of air per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

IN-LIFE DATES: From: 12 February To: 18 March 1991
Route of administration:
oral: gavage
- Vehicle(s)/solvent(s) used: aqueous 1% methylcellulose
- Concentration of test material in vehicle: 250 mg/ml
- Amount of vehicle (if gavage or dermal): 20 ml/kg bodyweight
Details on exposure:
Suspensions of ODB-2 were prepared in aqueous 1% methylcellulose on the morning of the test at 250 mg/ml.
Stability of the test compound in the vehicle was not assessed in this test.
Duration of treatment / exposure:
Single dose
Frequency of treatment:
single dose
Post exposure period:
24, 48 and 72 hours after dosing
Doses / Concentrations:
5000 mg/kg
actual ingested
No. of animals per sex per dose:
5000 mg/kg ODB-2 group: 15 male and 15 female mice (one male and 2 females were also dosed concurrently, to replace any animals that might die)
Vehicle control group: 15 male and 15 female mice
Positive control group: 5 male and 5 female mice
Positive control(s):
Mitomycin C was used as the positive control compound.
- Route of administration: oral gavage
- Doses / concentrations: It was prepared as a solution in sterile 0.9% saline at a concentration of 0.6 mg/ml.
Tissues and cell types examined:
Bone marrow and erythrocytes
Details of tissue and slide preparation:
Based on a preliminary toxicity study in which 2 male and 2 female mice dosed with 5000 mg/kg ODB-2 per kg bw showed only limited signs of toxicity

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
15 (+1) male and 15 (+2) female mice were dosed by oral gavage with 5000 mg/kg ODB-2 per kg bw. Following dosing the animals were examined regularly and any mortalities or clinical signs of reaction to the test compounds were recorded. Five males and five females from the negative control and test compound groups were sacrificed 24, 48 and 72 hours after dosing. The positive control group was sacrificed 24 hours after dosing.

A direct bone marrow smear was made from each femur onto a slide containing a drop of calf serum. The prepared smears were air-dried and fixed in methanol (>10 minutes). The smears were air-dried and stained for 10 minutes in 10% Giemsa (prepared by 1 : 9 dilution of Gurr's improved R66 Giemsa (BDH) with distilled water). Following rinsing in distilled water and differentiation in buffered distilled water (pH 6.8), the smears were air-dried and mounted with coverslips using DPX.

The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 1000 polychromatic erythrocytes per animal.

Micronuclei are identified by the following criteria:
(i) Large enough to discern morphological characteristics
(ii) Should possess a generally rounded shape with a clearly defined outline
(iii) Should be deeply stained and similar in colour to the nuclei of other cells - not black
(iv) Should lie in the same focal plane as the cell
(v) Lack internal structure i.e. they are pyknotic
(vi) There should be no micronucleus-like debris in the area surrounding the cell
Evaluation criteria:
A positive response is normally indicated by a substantial, dose-related and statistically significant increase in the incidence of micronucleated polychromatic erythrocytes compared to the incidence for the concurrent vehicle control group.
Bone marrow cell toxicity (or depression) is normally indicated by a substantial, dose-related and statistically significant decrease in ·the ratio of polychromatic to normochromatic erythrocytes. This decrease would normally be evident at both the 48 and 72 hour sampling points, a decrease at the 24 hour time point is not necessarily expected because of the relatively long transition time of erythroid cells.
Non-parametric statistical methods, based on rank, are chosen for analysis of results because:
(a) They are suited to analysis of data consisting of discrete/integer values such as the incidence of micronucleated polychromatic erythrocytes.
(b) The methods make few assumptions about the underlying distribution of data and therefore the values do not require transformation to fit a theoretical distribution (where data can be approximately fitted to a normal distribution, the results of non-parametric analysis and classical analysis of variance are very similar).
(c) 'Outliers' are frequently found in the polychromatic erythrocyte to normochromatic erythrocyte ratios for both control and treated animals; non-parametric analysis does not give such values an undue weighting.
For a comparison of an individual treated group with a concurrent control group, Wilcoxon's sum of ranks test is used.
no effects
Vehicle controls validity:
Negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
- Dose: 5000 mg ODB-2/kg bw
- Solubility:almost insoluble in water (approximately 0.8 ppm)
- Clinical signs of toxicity in test animals: no mortality, slight piloerection between 1 and 2 hours after dosing

- Clinical signs of toxicity in test animals: no mortality, all animals showed slight piloerection immediately after dosing, and one animal showed severe piloerection and lethargy, moderate hunched posture and ptosis 54 hour after dosing.
- Induction of micronuclei (for Micronucleus assay): ODB-2 did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes at any of the three kill times, while Mitomycin C caused large, highly significant increases in the frequency of micronucleated polychromatic erythrocytes. ODB-2 did not cause any substantial increases in the incidence of micronucleated normochromatic erythrocytes at any of the three kill times.
- Ratio of PCE/NCE (for Micronucleus assay): ODB-2 failed to cause any significant decreases in the ratio of polychromatic to normochromatic erythrocytes.
- Appropriateness of dose levels and route: this dose level is the limit dosage recommended by the US EPA and the JEPA/MOHW/MITI Joint Directive. The animals dosed with ODB-2 showed some signs of toxicity.
- Statistical evaluation: P>0.05 using Wilcoxon's sum of ranks test
Interpretation of results (migrated information): negative
It is concluded from the results obtained that ODB-2 shows no evidence of mutagenic potential or bone marrow cell toxicity when administered to male or female mice as a single oral dose of 5000 mg/kg bw in this in vivo test procedure.
Executive summary:

S-205 and ODB-2 (Black 400), which is tested for its genotoxicity in a mouse micronucleus test, are both colour precursor for heat sensitive record sheets. These substances have been demonstrated to be very similar in structure, physical/chemical properties and the toxicological profile. Due to the fact that S-205 and ODB-2 have nearly the same chemical structure. The same mode of interaction with bio-macromolecules, living cells and tissue and metabolic pathway is expected. Therefore, a read-across from S-205 to data obtained with ODB-2 is scientifically justified.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Justification for selection of genetic toxicity endpoint
This is the only in vivo study and is more meaningful that in vitro studies.

Justification for classification or non-classification

The three in vitro and the single in vivo studies all returned a negative result for genotoxicity. Therefore S-205 will not be classified as genotoxic.

Categories Display