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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
Study period:
02 October 2013 - 30 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (GLP)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997-07-21
Deviations:
no
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)
Version / remarks:
1998-08
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of the test substance: Hydroxypivalic acid neopentylglycolester
- Test substance No.: 03/0084 - 3
- Lot/batch No.: 95078568E0
- Purity: 99.0 corr.area-%
- Physical state: White, solid, flakes
- Stability under test conditions: Confirmed indirectly by dose formulation analytics
- Storage condition of test material: room temperature; protected against moisture

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: mice / Crl:NMRI from Charles River Laboratories Germany GmbH
- Age at study initiation: 9 - 10 weeks
- Weight at study initiation: 38.6 g
- Assigned to test groups randomly: yes
- Housing: single housing in Makrolon cages, type M II/III, single housing
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum, Harlan Laboratories
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
Fully air-conditioned rooms with central air conditioning
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 45-65%
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 1 % Carboxymethylcellulose;
- Justification for choice of solvent/vehicle: due to the relativ non-toxicity for the animal
- Concentration of test material in vehicle: 500, 1000 and 2000 mg/ml
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in 1% Carboxymethylcellulose (CMC), which was also used as vehicle control. The volume administered orally was 10 mL/kg b.w..
Duration of treatment / exposure:
once
Frequency of treatment:
once
Post exposure period:
24 (all groups) or 48 hours (for additional 5 animals in in the vehicle and positve control group)
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
7 (5 in the vehicle control group)
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide (CPA; dissolved in sterile water, 10 ml/kg)
- Route of administration: orally
- Doses / concentrations: 40 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow; polychromatic erythrocytes, normochromatic erythrocytes,
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items. The maximum tolerated dose level is determined to be the dose that causes toxic reactions
without having major effects on survival within 48 hours. The administered volume was 10 mL/kg b.w.. Three adequately spaced dose levels spaced by a factor of 2 were administered, and samples were collected at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Merck, 64293 Darmstadt, Germany)/Giemsa (Merck, 64293 Darmstadt, Germany). Cover slips were mounted with EUKITT (Kindler, 79110 Freiburg, Germany). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To investigate a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.
All animals were evaluated as described.

Evaluation criteria:
Acceptance criteria:
The study was considered valid as the following criteria are met:
- at least 5 animals per test group can be evaluated.
- PCE to erythrocyte ratio should not be less than 20 % of the vehicle control.
- the positive control shows a statistically significant and biological relevant increase of micronucleated PCEs compared to the vehicle control.

A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of thenon-parametric Mann-Whitney test.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
abdominal positon, eyelid closure and/or ruffled fur (2000mg/kg); ruffled fur (1000 mg/kg); Reduction of spontaneous activity, eyelid closure (500mg/kg)
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test item Hydroxypivalic acid neopentylglycolester was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was dissolved in 1% Carboxymethylcellulose (CMC), which was also used
as vehicle control. The volume administered orally was 10 mL/kg b.w.. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.
Seven males per test group (except the vehicle and positive control groups with 5 males only) were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei. To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per
2000 erythrocytes.
After treatment with the test item at 24h and 48h preparation interval the number of PCEs per 2000 erythrocytes was not substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control thus indicating that Hydroxypivalic acid neopentylglycolester did not induce cytotoxic effects in the bone marrow. In comparison to the corresponding vehicle controls there was no statistically significant or biologically
relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with Hydroxypivalic acid neopentylglycolester were below or near to the value of the vehicle control group and all values in all dose groups were very well within the historical vehicle control data range.

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the study described and under the experimental conditions reported, Hydroxypivalic acid neopentylglycolester did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.