Acetic acid, 2-chloro-, reaction products with 2-C11-13-alkyl-4,5-dihydro-1H-imidazole-1-ethanol and sodium hydroxide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

11 Sep 2017 - 19 Nov 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In vivo testing was triggered by the positive outcome of an in vitro study. The in vivo study was initiated after final approval by ECHA.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.11 (Mutagenicity - In Vivo Mammalian Bone-Marrow Chromosome Aberration Test)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian bone marrow chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C7-C17 odd-numbered, C17-unsatd. alkyl) derivs. and sodium hydroxide and chloroacetic acid

EC Number:

931-291-0

Molecular formula:

Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)

IUPAC Name:

Reaction products of 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C7-C17 odd-numbered, C17-unsatd. alkyl) derivs. and sodium hydroxide and chloroacetic acid

Test material form:

liquid

Details on test material:

- Physical appearance: colourless to light yellow liquid
- Storage conditions: At 20 ± 5 °C, in the dark

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

NMRI mice (SPF) were used as test system. These mice are recommended by international guidelines (e.g. OECD, EC).

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 weeks
- Weight at study initiation: 29 - 39 g
- Assigned to test groups randomly: yes
- Fasting period before study: yes, 3 - 4 hours prior to dosing until maximum 1 hour after administration
- Housing: animals were group housed (maximum 5 animals per sex per cage) in labelled polycarbonate cages containing sterilized sawdust as bedding material. Paper bedding was provided as cage enrichment.
- Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), except prior to dosing
- Water: free access to tap-water
Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 44-60
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 11 Sep 2017 To: 13 Oct 2017

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Solvent used: water for injection
A solubility test was performed based on visual assessment. Miranol Ultra C32 was dissolved in water for injection. The dosing volume was 10 mL/kg body weight.

Details on exposure:

Miranol Ultra C32 concentrations were dosed within 3 hours after preparation.

Duration of treatment / exposure:

12 - 18 hours (three doses) and 36 - 44 hours (high dose only)

Frequency of treatment:

Single exposure

Post exposure period:

12 - 18 hours (three doses) and 36 - 44 hours (high dose only)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

(based on dry weight)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

(based on dry weight)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

(based on dry weight)

No. of animals per sex per dose:

6 males (dose range finding study); 5 males (main study)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide, 40 mg/kg bw

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

Three to five hours prior to sacrifice, animals are treated with the metaphase-arresting chemical colchicine. 12 - 18 hours (1.5 x normal cell cycle length) and 36 - 44 hours (24 hours after the first sample time) after dosing the animals are sacrificed and bone marrow cells are collected. The cells are treated with a hypotonic solution and fixed. Finally, the cell material is spread on slides and stained. Metaphase cells are examined microscopically for the presence of structural chromosome aberrations such as breaks, gaps, minutes, dicentrics and exchange figures.
The mitotic index of bone marrow metaphase slides was determined by counting the number of metaphases from at least 1000 (with a maximum deviation of 5%) cells per animal. To prevent bias, all slides were randomly coded before examination of chromosome aberrations. One hundred twenty-five to two hundred metaphase chromosome spreads per animal were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 50 in 100 metaphases no more metaphases were examined. Only metaphases containing 40 ± 2 centromeres (chromosomes) were analyzed. Type and number of aberrations were given separately for each animal, the total number of aberrations and the number of cells with aberrations per group was calculated.

Evaluation criteria:

A bone marrow metaphase test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database and produce a statistically significant increase compared to the negative control. If (one of) the acceptability criteria are not met and the Study Director decides that this has a critical effect on the study, the test will be rejected and repeated.

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.
A test item is considered positive in the bone marrow metaphase test if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of cells with structural chromosome aberrations (excluding gaps) compared with the concurrent negative control
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the historical control data range.
A test item is considered negative in the bone marrow metaphase test if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05)
increase in the frequency of cells with structural chromosome aberrations (excluding
gaps) compared with the concurrent negative control.
b) There is no dose related increase when evaluated with a trend test.
c) All results are within the negative historical control data range.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- In the dose range finding test, three males and three females were dosed with 2000 mg Miranol Ultra C32/kg body weight. The animals showed no clinical signs after dosing. Since there were no differences between sexes in toxicity only male animals were used in the main study, five male animals were used in each treatment group.

RESULTS OF DEFINITIVE STUDY
- The animals of the groups treated with 2000, 1000 and 500 mg Miranol Ultra C32/kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs or mortality.
- No biologically relevant decrease in the mitotic index was observed in animals treated with Miranol Ultra C32. The inhibition of the mitotic index of the positive control was 10%.
- The scores for the number of aberrant cells (gaps included and excluded) and the number of the various types of chromosome aberrations at the various concentrations of Miranol Ultra C32 are attached.
- The number of cells with chromosome aberrations found in the animals dosed with the vehicle control was within the laboratory historical control data range. The positive control chemical produced a statistically significant increase in the frequency of aberrant cells. It was therefore concluded that the test conditions were appropriate for the detection of a clastogenic response. Miranol Ultra C32 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations, at both sampling times. No effects of Miranol Ultra C32 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed.

Applicant's summary and conclusion

Conclusions:

Based on the results of a mouse bone marrow cytogenetic assay was performed according to OECD/EC guidelines and GLP principles, it is concluded that Miranol Ultra C32 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations in vivo.

Executive summary:

A mouse bone marrow cytogenetic assay was performed with Miranol Ultra C32 according to OECD/EC guidelines and GLP principles. Male mice (5/group) were exposed to 500, 1000 or 2000 mg/kg bw/day and bone marrow was sampled 12-18 (all doses, vehicle control group and positive control group (treated with cyclophosphamide) or 36-44 (highest dose only) hours after dosing. No mortality occurred, no clinical signs were noted in any of the mice. The number of cells with chromosome aberrations found in the vehicle control animals was within the laboratory historical control data range. The positive control animals treated with cyclophosphamide induced a statistically significant increase in the number of cells with chromosome aberrations, indicating that the test conditions were adequate. Miranol Ultra C32 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations, at both sampling times.

Based on these results it is concluded that Miranol Ultra C32 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations in vivo.