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Toxicological information

Genetic toxicity: in vivo

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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Fully GLP- and guideline compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Dimethylolpropanal, 3-hydroxy-2-(hydroxymethyl)-2-methylpropionaldehyde
- Physical state: pale solid (gel)
- Analytical purity: 100%
- Lot/batch No.: 182186/1
- Expiration date of the lot/batch: 30 June 2014
- Storage condition of test material: ambient temperature (15-25ºC)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 165.2 - 182.7 g
- Assigned to test groups randomly: yes, under following basis: proportionally to body weight
- Fasting period before study: no
- Housing: 5 animals per macrolon cages (type IV) with wood shavings (Lignocel, type ¾) as bedding material and a wooden block and strips of paper as environmental enrichment (Enviro-dri)
- Diet: cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3; pelleted) from a commercial supplier (SDS Special Diets Services, Witham, England), ad libitum
- Water: domestic mains tap water suitable for human consumption, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 10

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water for injection
- Amount of vehicle: 10 ml/kg bw
- Lot/batch no.: 11054402
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was freshly formulated in water for injection. The exact volume of the dose formulation was calculated for each animal individually based on body weight.
Duration of treatment / exposure:
2 days
Frequency of treatment:
once daily
Post exposure period:
24 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
2000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin C (MMC)
- Route of administration: intraperitoneal
- Doses / concentrations: 1.5 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow: polychromatic and normochromatic erythrocytes
Details of tissue and slide preparation:
BONE MARROW COLLECTION AND PROCESSING
Immediately following sacrifice, the bone marrow cells of one of the femurs was collected into foetal calf serum and processed into glassdrawn smears according to the method described by Schmid (1976). Two bone marrow smears per animal were made, air-dried and fixed in methanol. One fixed smear was stained with a May-Grünwald Giemsa solution. The other fixed smear was kept in reserve and discarded after analysis of the stained smear.
Evaluation criteria:
The following criteria were used for the scoring of cells:
- A polychromatic erythrocyte (PE) is an immature erythrocyte that still contains ribosomes and can be distinguished from mature, normochromatic erythrocytes by a faint blue stain.
- A normochromatic erythrocyte (NE) is a mature erythrocyte that lacks ribosomes and can be distinguished from immature, polychromatic erythrocytes by a yellow stain.
- A micronucleus is a small, normally round, nucleus with a diameter of circa 1/20 to 1/5 of an erythrocyte, distinguished from the cytoplasm by a dark blue stain.

The numbers of PE and NE were recorded in a total of at least 200 erythrocytes (E) per animal. If micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total of 200 E (PE + NE) were scored, an additional number of PE were scored for the presence of micronuclei until a total of 2000 PE were scored.

The study was considered valid if the positive controls showed a statistically significant increase in the mean number of MPE/2000 PE and the vehicle controls
where within the historical range.
A test substance was considered to cause chromosomal damage and/or damage to the mitotic apparatus if it showed a positive response, i.e. if the mean number of MPE/2000 PE was statistically significant higher compared to the vehicle control group.
The test substance or its metabolites were considered to be cytotoxic to the bone marrow via general circulation, if the test substance statistically significantly
reduced the mean number of PE.
Statistics:
Two ANOVA models were applied for both PE/200E and MPE/2000PE. The first ANOVA model tested if the positive control differed from the vehicle control (t test). The second ANOVA model tested if the vehicle control differed from the test substance. If the ANOVA assumptions were not valid (i.e. if variances were not
equal) non-parametric testing was performed and Mann Whitney p-values were reported. In all statistical tests a significance level of 5% was used (α = 0.05). All statistical tests were performed using GraphPad Prism®, Version 5.03, Copyright © 1992-2010 GraphPad Software, Inc., CA, USA.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: No abnormalities were observed in any of the animals either ca. 1 h, 4 h or 24 h after each dosing

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: no
- Ratio of PE/200E: 109 ± 11
- Appropriateness of dose levels and route: Indications of systemic availability of the test substance were obtained in the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (induction of liver enzymes was observed), demonstrating that the negative response observed in this bone marrow micronucleus test is not due to lack of systemic availability of the test substance or its metabolites.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test substance Dimethylopropanal did not show any indication of chromosomal damage and/or damage to the mitotic spindle apparatus of the bone marrow target cells of male rats, when orally dosed on two consecutive days with approximately 24 h interval, up to the limit dose of 2000 mg/kg bw.
Executive summary:

The test substance Dimethylolpropanal was examined for its potential to cause damage to the chromosomes and/or the mitotic apparatus of erythroblasts by analysis of erythrocytes as sampled in bone marrow of male rats.

Prior to the bone marrow micronucleus test, a dose confirmation study was performed in rats to determine tolerability of the test substance at the limit dose level of 2000 mg/kg-bw, in combination with the dosing regimen used in the current study. Two male rats were dosed twice orally (by gavage) on two consecutive days with approximately 24 h interval, with a dose level of 2000 mg/kg-bw Dimethylolpropanal (dosing volume 10 ml/kg-bw). No abnormalities were observed in either of the animals ca. 1 h, 4 h or 24 h after each dosing. Based on these observations a dose level of 2000 mg/kg-bw Dimethylolpropanal was considered suitable for the bone marrow micronucleus test.

In the bone marrow micronucleus test, rats were dosed twice orally (by gavage), on two consequentive days with approximately 24 h interval, with 2000 mg/kg-bw of the test substance or water for injection (vehicle control). Rats of the positive control group were injected once intraperitoneally with 1.5 mg/kg-bw mitomycin C. The dosing volume was 10 ml/kg-bw for all treatment groups. Prior to each dosing the rats were weighed and the dosing volume was adjusted to the body weight. Five rats per treatment group were used.

No abnormalities were observed in any of the groups and no mortality occurred during the study.

Approximately 24 h after the final treatment, rats were sacrificed by decapitation whilst under CO2/O2 anaesthesia, and bone marrow cells were collected from one of the femurs and processed into smears for microscopic examination. The number of polychromatic erythrocytes (PE) per 200 erythrocytes (E) and the number of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) were counted for each rat.

The incidence of MPE per 2000 PE, found in the positive control group, was statistically significantly higher compared to that of the vehicle control group (p<0.01). The incidence of MPE per 2000 PE in the vehicle control group was within the range of historical control data. Therefore, the study was considered valid.

The incidence of MPE per 2000 PE in the rats treated with 2000 mg/kg-bw Dimethylolpropanal showed no statistically significant difference from that observed in the vehicle control group.

The incidence of PE per 200 E in the rats treated with either 2000 mg/kg-bw Dimethylolpropanal or the positive control substance MMC showed no statistically significant difference from that found in the vehicle control group, which reflects a lack of toxic effects of these substances on erythropoiesis.

Under the conditions used in this study it is concluded that the test substance Dimethylolpropanal did not show any indication of chromosomal damage and/or damage to the mitotic spindle apparatus of the bone marrow target cells of male rats, when orally dosed on two consecutive days with approximately 24 h interval, up to the limit dose of 2000 mg/kg-bw.

Indications of systemic availability of the test substance were obtained in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (induction of liver enzymes was observed), demonstrating that the negative response observed in this bone marrow micronucleus test is not due to lack of systemic availability of the test substance or its metabolites.