Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Mouse Bone Marrow Micronucleus Assay
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11-04-2017 to 16-08-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
We understand that REACH requires that new testing of a substance involving vertebrate animals is only carried out as a last resort. A registrant shall submit a testing proposal and ECHA publishes all test proposals involving vertebrate animals, for endpoints specified in Annexes IX and X under REACH before the testing is carried out (REACH Art 40(2)). The test according to OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test) is listed in Annex IX. Registrant made the study without a test proposal to ECHA because of the reasons stated below.

1. Uses in Section 3 and needs for global market
The substance in this dossier is an enzyme which should be used for uses stated in Section 3. The enzyme has been produced in Registrant’s site in EU, however the business decided to market the product first in Philippines. It is a new substance in accordance with the Philippine Inventory of Chemicals and Chemical Substances (PICCS). The enzyme was first notified for the low volume (less than 1 ton/year) with Small Quantity Importation (SQI) by Registrant’s business partner in Philippines. Along development of the market, there were needs for a Pre-Manufacture Pre-Importation Notification (PMPIN) for permit of higher tonnage import. Registrant conducted the in-vivo study for this purpose. Registrant’s business partner finally submitted PMPIN on January 24, 2018.

2. Other uses which are not in the scope of REACH
In addition, Registrant consider further development of uses of the enzyme within the food industry in EU and other countries. In EU, such an enzyme used as processing aid for food manufacture is in the scope of the EU regulatory framework, the Food Improvement Agents Package (FIAP) and a company must obtain approval from EFSA by submitting a dossier. According to the EFSA (CEF panel) guidance for FIAP dossiers, there are certain requirements for assessment of genotoxicity as follows.

“At least two in vitro assays should be performed. […] A positive result in genotoxicity testing would then require further assessment to determine whether it is genotoxic in vivo. […] One or more positive in vitro tests normally require follow-up by in vivo testing, unless it can be adequately demonstrated that the positive in vitro findings are not relevant for the in vivo situation. This is in line with the general strategy elaborated in the updated WHO/IPCS Harmonised Scheme on mutagenicity testing (Eastmond et al., 2009).” (page 16 - 17)

link to the FIAP guidance is http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2009.1305/epdf.

Thus the in vivo study was also preparation for approval according to the FIAP guidance. It in general takes years to develop a new enzyme and new markets. Registrant’s R&D and Market often seek various market opportunities in parallel. In addition many enzymes are already used for food manufacture as well as manufacture of non-food such as textile and paper. It was therefore a natural track for Registrant to follow FIAP guidance on this endpoint at early stages of R&D activities.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Reference substance name:
Nuclease, deoxyribo-
EC Number:
232-667-0
EC Name:
Nuclease, deoxyribo-
Cas Number:
9003-98-9
Molecular formula:
n.a.
IUPAC Name:
Nuclease, deoxyribo-
Test material form:
liquid
Details on test material:
- Lot/batch No.: U8AGS
- Expiration date of the lot/batch: Usually 10 years after production of the batch.
- Storage condition of test material: N/A
Specific details on test material used for the study:
The test material is a purified version of the batch PPW42035, as the test material was to be dosed intravenously to ensure maximum exposure of target tissue.

Test animals

Species:
mouse
Strain:
CD-1
Details on species / strain selection:
Out-bred CD-1 mice (Crl:CD1(ICR))
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Obtained from Charles River (UK) Ltd., Margate, UK.
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 28-35 g
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: Animals were housed in wire topped, solid bottomed cages, with (up to) three animals of the same sex per cage. Wood bedding (Aspen) along with wooden Aspen chew blocks and nesting material were provided.
- Diet: ad libitum access to 5LF2 EU Rodent Diet 14%
- Water: ad libitum via water bottles.
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 45-65%
- Air changes (per hr): 15-20 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light (0600 to 1800) and 12 hours dark

IN-LIFE DATES: From: 24 April 2017 To: 25 May 2017

Administration / exposure

Route of administration:
intravenous
Vehicle:
Phosphate buffered saline pH 7.4, with a composition of 10 mM KH2PO4/K2HPO4, 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2.
Details on exposure:
All treatments were given via intravenous bolus injection in order to maximise exposure of the target organ to the test article. Animals were dosed in ascending group order. The test article and vehicle control were given as two administrations, at 0 and
24 hours. This has been shown to be of sufficient duration for the expression of any genotoxic potential. The positive control was administered once only at 24 hours. All animals were sampled at 48 hours.
Duration of treatment / exposure:
24 hours
Frequency of treatment:
Two administrations, at 0 and 24 hours.
Post exposure period:
24 hours
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 male and 12 female animals were dosed in the Range-Finder Experiment.
27 male animals were dosed in the Main Experiment.
Control animals:
yes
Positive control(s):
Cyclophosphamide (CPA) 40 mg/kg, single oral administration at 5 mL/kg on Day 2 (T=24 hours).

Examinations

Tissues and cell types examined:
No tissues were retained from Range-Finder Experiment animals.
Both femurs were removed and bone marrow isolated from all Main Experiment animals at necropsy.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The maximum solubility of deoxyribonuclease, batch U8AGS in PBS is 53 mg/mL and the standard dose volume for bolus intravenous administration in mice is 5 to 10 mL/kg, therefore, the maximum feasible dose was 265 to 530 mg/kg/day. Based on this information an initial dose of 100 mg/kg/day was administered in a Range-Finder Experiment. Additional doses were tested until an estimate of the maximum tolerated dose (MTD) was determined. From the results of the Range-Finder Experiment dose levels of 100, 200 or 400 mg/kg/day 53 mg/mL Deoxyribonuclease, batch U8AGS, formulated in phosphate buffered saline (equivalent to 25% MTD, 50% MTD and the MTD respectively) were tested in the Main Experiment.

DETAILS OF SLIDE PREPARATION: Both femurs from each animal were exposed, removed, cleaned of adherent tissue and the ends removed from the shanks. Using a syringe and needle, bone marrows were flushed from the marrow cavity with 1 mL foetal bovine serum into appropriately labelled centrifuge tubes. A further 1 mL of foetal bovine serum was added to the tubes, and then centrifuged at 200 x 'g' for approximately five minutes; the serum was aspirated and a further 1 mL of foetal bovine serum was added and the centrifugation step was repeated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube by using a Pasteur pipette, and from each tube one drop of suspension was placed on the end of two uniquely labelled slides. A smear was made from the drop by drawing the end of a clean slide along the labelled slide. Slides were air-dried and then fixed for 5 minutes in absolute methanol and rinsed several times in distilled water before being stained. One slide from each set was taken (the remaining slides kept in reserve). After rinsing several times in distilled water, slides were stained for 10 minutes in filtered Giemsa stain diluted in distilled water (typically 1:4 (v/v), actual ratio recorded in raw data). Slides were rinsed and dried before clearing in xylene for 3 minutes. When dry, slides were mounted with coverslips and stored at room temperature prior to analysis.

METHOD OF ANALYSIS: Scoring was carried out using light microscopy at an appropriate magnification. Slides from the vehicle and positive control animals were checked for quality and/or response prior to analysis. All slides were allocated a random code and analysed by an individual not connected with the dosing phase of the study. All animals per group were analysed. Initially the relative proportions of PCE, seen as purple/blue enucleate cells, and normochromatic erythrocytes (NCE), seen as pink/orange stained enucleate cells, were determined until a total of at least 500 cells (PCE plus NCE) had been analysed. Counting continued until at least 4000 PCE per animal where possible had been examined. All PCE containing MN observed during these two phases of counting were recorded.
The following criteria were used for analysis of slides:
1. Cells were of normal cell morphology
2. Areas where erythrocytes overlap were to be ignored
3. A MN was to be round or oval in shape
4. A cell containing more than one MN was scored as a single micronucleated cell
5. MN that were refractive, improperly stained or not in the focal plane of the cell were judged to be artefacts and were not scored.
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic / aneugenic damage if:
1. A statistically significant increase in the frequency of MN PCE occurred at one or more dose levels.
2. The incidence and distribution of MN PCE exceeded the laboratory’s historical vehicle control data.
3. A dose-response trend in the proportion of MN PCE was observed.

The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met and bone marrow exposure was confirmed.
Results which only partially satisfied 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 taken into account, for example consistency of response within and between dose levels.
Statistics:
For each test article and vehicle control group, inter-individual variation in the numbers of MN PCE was estimated by means of a heterogeneity chi-square calculation (Lovell et al., 1989). The numbers of MN PCE in each treated group were compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine chi-square (Lovell et al., 1989). The tests were interpreted with one-sided risk for increased frequency with increasing dose. Probability values of p≤0.05 were accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 100, 265, 400, 530 mg/kg/day
- Solubility: Enzymes are water soluble
- Clinical signs of toxicity in test animals: 1/3 male animals dosed at 530 mg/kg/day died during the Day 3 observation period, confirming that 530 mg/kg/day exceeded the maximum tolerated dose for this study. A dose of 400 mg/kg/day produced moderate signs of toxicity and body weight loss but no morbidity or mortality in either male or female mice.
- Evidence of cytotoxicity in tissue analyzed: Not analysed
- Rationale for exposure: The maximum solubility of deoxyribonuclease, Batch U8AGS in PBS is 53 mg/mL and the standard dose volume for bolus intravenous administration in mice is 5 to 10 mL/kg, therefore, the maximum feasible dose was 265 to 530 mg/kg/day. The rodent bone marrow micronucleus test is recommended by various regulatory authorities as an appropriate test to determine the genotoxic potential of a compound in vivo.
- High dose with and without activation: 530 mg/kg/day

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Animals treated with all doses exhibited MN PCE frequencies that were similar to the concurrent vehicle control group.
- Ratio of PCE/NCE (for Micronucleus assay): Same as control
- Appropriateness of dose levels and route: 400 mg/kg/day identified as the MTD from the range-finding study. Intravenous dosing was applied to ensure maximum exposure of target tissue.
- Statistical evaluation: For each test article and vehicle control group, inter-individual variation in the numbers of MN PCE was estimated by means of a heterogeneity chi-square calculation (Lovell et al., 1989). The numbers of MN PCE in each treated group were compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine chi-square (Lovell et al., 1989). The tests were interpreted with one-sided risk for increased frequency with increasing dose. Probability values of p≤0.05 were accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.

Applicant's summary and conclusion

Conclusions:
It was concluded that deoxyribonuclease, batch U8AGS, formulated in phosphate buffered saline did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male mice when dosed intravenously with up to 400 mg/kg/day (an estimate of the maximum tolerated dose for this study), under the experimental conditions employed.
Executive summary:

Deoxyribonuclease, batch U8AGS, formulated in phosphate buffered saline was tested for its potential to induce micronuclei (MN) in the polychromatic erythrocytes (PCE) of the bone marrow of treated mice.

- Strain / Species: CD-1 mice.

- Vehicle: Phosphate buffered saline pH 7.4.

- Administration route: Intravenous bolus injection, to ensure systemic exposure.

- Dosing regimen: Two administrations at 0 hours (Day 1) and 24 hours (Day 2).

- Gender: Males only (no gender differences observed in the Range-Finder Experiment).

- Dose levels: 100, 200 or 400 mg/kg/day.

- Dose volume: 10 mL/kg.

- Maximum dose: Maximum tolerated dose based on Range-Finder data.

- Positive control: Cyclophosphamide (CPA) 40 mg/kg, single oral administration at 5 mL/kg on Day 2.

- Animals per group: Six (three for the positive control group).

- Clinical signs of toxicity: Following dosing on Day 2 and/or prior to necropsy on Day 3, observations of raised hair and/or hunched appearce were observed in the majority of animals dosed at 200 or 400 mg/kg/day. Body weight loss was observed in all animals dosed at 400 mg/kg/day (Day 1 to Day 3).

- Bone marrow sampled: 24 hours after the last dose administration (Day 3, equivalent to 48 hours).

- Assay validity: The vehicle control data (%PCE and MN PCE) were within the laboratory’s historical vehicle control data ranges. The positive control induced a significant increase in MN PCE that was comparable with the laboratory’s historical positive control data. The assay was therefore accepted as valid.

Animals treated with deoxyribonuclease, batch U8AGS, formulated in phosphate buffered saline at all doses exhibited group mean %PCE that were generally similar to the concurrent vehicle control group and which were within the laboratory’s historical vehicle control data, thus confirming there was no evidence of test article related bone marrow toxicity.

Animals treated with deoxyribonuclease, batch U8AGS, formulated in phosphate buffered saline at all doses exhibited MN PCE frequencies that were similar to the concurrent vehicle control group and that fell within laboratory's historical vehicle control data. There were no statistically significant increases in MN frequency for any of the groups receiving the test article, compared to the concurrent vehicle controls.

It was concluded that deoxyribonuclease, batch U8AGS, formulated in phosphate buffered saline did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male mice when dosed intravenously with up to 400 mg/kg/day (an estimate of the maximum tolerated dose for this study), under the experimental conditions employed.