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Toxicological information

Genetic toxicity: in vivo

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Administrative data

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Nov 2018 - 15 Jan 2019
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 2016
GLP compliance:
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River (UK) Ltd., Kent, England
- Age at study initiation: approximately 5 to 6 weeks old
- Weight at study initiation: 128-174 g (males), 116-150 g (females)
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing: The animals were housed in groups of up to 3, by sex, in solid floor cages with appropriate bedding.
- Diet: pelleted rodent diet (Teklad 2014C Rodent Maintenance Diet, Envigo RMS (UK) Limited) ad libitum
- Water: mains tap water (in bottles) ad libitum
- Acclimation period: at least 4 days

- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): room was air-conditioned
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 12 Nov 2018 To: 11 Jan 2019

Administration / exposure

Route of administration:
oral: gavage
- Vehicle/solvent used: methylcellulose with 0.1 % v/v Tween 80
- Justification for choice of solvent/vehicle: Based on a formulation trail, where the suitability of the vehicle was confirmed.
- Amount of vehicle: 10 mL/kg bw
Details on exposure:
The test item was formulated on the day of use as a suspension in the vehicle methylcellulose (medium viscosity) supplemented with 0.1 % v/v Tween 80. Separate formulations were prepared for each dose level, with the weighed quantity of test item being suspended in the appropriate quantity of vehicle.
Duration of treatment / exposure:
24 h
Frequency of treatment:
Rats were dosed twice, approximately 24 hours apart.
Post exposure period:
Animals were observed periodically for 48 hours after the last dosing.
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
200 mg/kg bw/day
No. of animals per sex per dose:
Range-finder: 3/sex/dose
Main test: 6 males/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CPA)
6 Samples from an existing positive control bank of blood obtained from rats dosed with CPA were considered.
- Doses / concentrations: 15 mg/kg bw


Tissues and cell types examined:
Details of tissue and slide preparation:
A range-finding study was carried out in order to select the highest dose of the test item that did not produce mortality or severe signs of clinical toxicity up to the maximum tolerated dose level (MTD). Groups of 3 male or 3 female rats were dosed with the test substance in a range of 200-500 mg/kg bw/day. The results of the range-finder study revealed no gender-related difference in toxicity and therefore, the main study was conducted with male rats only.

Groups of 6 male rats were dosed twice, approximately 24 h apart, with the vehicle alone (negative control) or the test item (50, 100 or 200 mg/kg bw/day). At the end of the observation period (approximately 48 h after the second test item administration), animals had a terminal blood sample taken for micronucleus scoring. At least 0.2 mL whole blood was taken from each main study animal into tubes containing K2EDTA. Samples were gently flicked to mix the blood and anticoagulant and then mixed, by inverting several times, before being placed on a roller and then held on water ice until storedat between 2 °C and 8 °C prior to fixing.

Blood samples were diluted, fixed, stored, labelled and analysed according to the biomarker method BMK034FC. In brief, blood samples were fixed in two separate methanol aliquots and stored at ≤ -70 ˚C for at least 3 days. One set of samples was then washed out of fixative and analysed. The remaining set of samples was transferred to long term storage solution for continued storage at ≤ -70 ˚C. The six samples from the positive control bank were washed out of fixative and analysed according to the biomarker method BMK034FC.

The analysis of micronuclei (MN) was performed by the biomarker method BMK034FC. It is a flow cytometric method designed by Litron Laboratories to detect micronuclei based on expression of CD71 (identifies reticulocytes, RET), CD61 (identifies platelets) and propidium iodide (PI, DNA stain). The machine (FACSVerse flow cytometer) was calibrated for the detection of MN at the beginning of each run using the kit supplied Malaria Biostandard. A minimum of 4000 and a maximum of approximately 20000 RET were scored for the presence of MN for each animal.
Evaluation criteria:
For the test to have been considered positive if the following criteria needed to be met:
1. A statistically significant increase in the frequency of MN-RET occurred at 1 or more
dose levels.
2. The incidence and distribution of MN-RET in individual animals at the dose level(s)
showing statistical significance exceeded the laboratory’s historical negative Control
data (e.g. exceeded control limits).
3. A dose-related increase in the frequency of MN-RET (where more than 2 dose levels
are analysed) was observed.
Results which only partially satisfy the above criteria were dealt with on a case-by-case basis.
Evidence of a dose-related effect was considered useful but not essential in the evaluation of a
positive result. Biological relevance was also taken into account.

For a test to be considered negative if the following criteria needed to be met:
1. None of the dose levels exhibited a statistically significant increase in the frequency
of MN RET, compared with the concurrent negative Control.
2. There was no dose-related increase when evaluated by an appropriate trend test.
3. All results were within Sequani’s historical negative Control data (e.g. within Control
4. Bone marrow exposure to the test item has occurred.
For the % RET data, the treated group(s) and the positive control were compared with the negative control using one-tailed exact Wilcoxon Rank Sum tests. A Jonckheere trend test was conducted for dose-response relationship investigation.

Micronucleated reticulocytes (MN-RET) data was analysed using a Poisson model with a log link function using GENMOD procedure in SAS. The effect of the test item was evaluated using two statistical approaches:
1. A linear trend test, built as a linear combination of the logs of the proportions of micronuclei, for dose-response relationship investigation.
2. Pairwise comparisons to the negative control, using the one-sided Likelihood Ratio test, to detect the doses significantly different from the control.

All the tests were one-sided, a decreasing trend being not of interest. So, in case of decrease and calculated p value < 0.5, the final p value was estimated as 1-[p value].
Overdispersion of the data was estimated by the deviance of the model divided by the associated degrees of freedom. Where overdispersion was detected (dispersion parameter >1), a correction was applied on the model.
The positive control group was not included in the model with the treated groups, but was calculated separately in a model that contained only the two control groups.

Results and discussion

Test results
Key result
at and above 100 mg/kg bw
Vehicle controls validity:
Negative controls validity:
not examined
Positive controls validity:
Additional information on results:
- Dose range: 200-500 mg/kg bw/day
- Clinical signs of toxicity in test animals: Minor body weight loss was observed in all animals. Clinical signs were observed at 320 and 500 mg/kg bw/day. For details, please refer to the attached background material 1. There were no clinical signs observed in males or females following administration at 200 mg/kg bw/day. Based on the results, the MTD was considered to be 200 mg/kg bw/day in males and females.

- Clinical signs: clinical signs were observed at 100 and 200 mg/kg bw/day. For details, please refer to the attached background material 2.
- Induction of micronuclei: no statistically significant increases in MN-RET frequency was noticed at any dose level (please also refer to the attached background material 3).
- % RET: statistically significant decrease in the % RET at 100 and 200 mg/kg bw/day, indicating toxicity to the bone marrow and proving exposure of the bone marrow to the test substance.
- Positive control: the samples from the positive control bank had statistically significant increases in the number of MN-RET compared with the concurrent control group which demonstrated that the test system was capable of detecting a known clastogen. There was a statistically significant decrease in the % RET in the positive control group, indicating toxicity to the bone marrow.
- Historical control data: the negative and positive control are within the historical controls from 2015 - 2018 (refer to the attached background material 4).

Applicant's summary and conclusion

The study was performed according to OECD guideline 474 and compliant with GLP. According to the bone marrow micronucleus assay, there was no evidence of clastogenicity or aneugenicity following oral (gavage) administration of the test substance up to the MTD of 200 mg/kg bw/day in male rats.