Polar modified Rice Bran Wax

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20-Jul-2015 to 13-Aug-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1 (direct plate assay)
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 17, 52, 164, 512 and 1600 µg/plate
Experiment 2 (pre-incubation assay):
Without and with S9-mix: 17, 52, 164, 512 and 1600 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
The test item could not be dissolved or homogeneously suspended in water, dimethyl sulfoxide and ethanol at a concentration of 50 mg/ml. At lower concentrations a homogenous suspension was obtained in DMSO. DMSO is accepted and approved by authorities and international guidelines

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 Migrated to IUCLID6: 650 µg/plate in DMSO for TA100

Positive control substance:

2-nitrofluorene

Remarks:

without S Migrated to IUCLID6: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA

Positive control substance:

sodium azide

Remarks:

without S9 Migrated to IUCLID6: 5 µg/plate in saline for TA1535

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation and pre-incubation methods

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.


A test substance is considered positive if:
a) a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without S9 at 1600 µg/plate in second mutation experiment

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without S9 at 512 µg/plate in second mutation experiment

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without S9 at 1600 µg/plate in second mutation experiment

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without S9 at 1600 µg/plate in first mutation experiment and without S9 at 512 µg/plate as well as with S9 at 1600 µg/plate in second mutation experiment

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at dose levels of 512 (TA100 dose range finding only), 1600 and 5000 µg/plate, except in the tester strains TA1537 and TA100 (second experiment absence of S9-mix), where no precipitate on the plates was observed. Since Licocare RBW 106 precipitated heavily on the plates at the test item concentration of 5000 μg/plate, the number of revertant colonies of this dose level could not be determined.

RANGE-FINDING/FIRST MUTATION EXPERIMENT (direct plate assay):
- Cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain TA100 in the absence of S9-mix at 1600 µg/plate.
- In all other strains, no toxicity or mutagenicity was observed

SECOND MUTATION EXPERIMENT (pre-incubation assay):
- Toxicity was observed in all tester strains, except in the tester strains TA1535, TA1537 and TA98 in the presence of S9-mix and tester strain WP2uvrA in the absence and presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY (SECOND MUTATION EXPERIMENT):
TA1535: without S9: 1600µg/plate and with S9:no toxicity was observed
TA1537: without S9: 512 µg/plate and above and with S9:no toxicity was observed
TA98: without S9: 1600 µg/plate and with S9:no toxicity was observed
TA100: without S9: 512 µg/plate and above and with S9: 1600 µg/plate
WP2uvrA: no toxicity was observed

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that Licocare RBW 106 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

 

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 February 2020 to 08 May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted on 29th July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Clariant Plastics and Coatings (Deutschland) GmbH, BU Additive
Ludwig-Hermann-Straße
86368 Gersthofen/Germany
- batch No.of test material: DEF2105728
- Expiration date :16.01.2022
- Purity : 99.63%

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29 ºC)
- Solubility of the test substance in the solvent/vehicle: Acetone
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

Target gene:

Hprt locus of CHO AA8 cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO AA8 cells, Batch No. 5000062 procured from American Type Culture Collection (ATCC) was used for the test

Metabolic activation:

with and without

Metabolic activation system:

S9 homogenate rat liver

Test concentrations with justification for top dose:

0.50 mg/mL as there was no evidence of excessive cytotoxicity (˂10% RS) at and upto 0.5 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:Acetone
- Justification for choice of solvent/vehicle: Licocare RBW 106 TP formed a uniform suspension in acetone at 200 mg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 2×106 cells for gene mutation

DURATION
- Exposure duration:3 hours and 2 minutes for pre study and 3 hours and 51minutes for main study

SELECTION AGENT (mutation assays): 6-Thioguanine

STAIN (for colony counts): Giemsa

NUMBER OF REPLICATIONS: 03 for cytotoxicity study and 05 for gene mutation selection study

NUMBER OF CELLS EVALUATED: 20×106 cells per group

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative survival

Rationale for test conditions:

NA

Evaluation criteria:

•The concurrent vehicle control is considered acceptable for addition to the laboratory historical vehicle control database as described in OECD guidelines for testing of chemicals, No. 476.
•Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative/vehicle control.
• Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
• Adequate number of cells and concentrations are analysable (according to OECD guidelines for testing of chemicals, No. 476).
•The criteria for the selection of top concentration are consistent with those described in OECD guidelines for testing of chemicals, No. 476.
All above criteria was met and hence the test is considered valid

Statistics:

Data of mutant frequencies were analyzed for differences among vehicle control, treatment and positive control groups by performing power transformation procedure by Snee and Irr (1981) with which, the observed mutant frequency was transformed using the formula:
Y=〖(X+A)〗^B

Where,
Y = transformed mutant frequency, X = observed mutant frequency
[Where X=(No. of mutant colonies per replicate)/(ACE value)×100
and A, B = constants (viz. A = 1 and B = 0.15)]
Statistical analysis of the experimental data was carried out using SPSS Statistical package version 22.0.
The statistical significances are designated by the superscripts as given below:
* Statistically significant (p<0.05) change than the vehicle control group

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Water solubility: No
- Precipitation: No precipitation was observed at the tested concentrations of 0.0625, 0.125 and 0.25 mg/mL, mild precipitation was observed at 0.5 mg/mL, moderate precipitation was observed at 1.0 mg/mL and heavy precipitation was observed at 2 mg/mL.

RANGE-FINDING/SCREENING STUDIES: Yes

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Positive Control- Benzo(a)pyrene (3 µg/mL) and 4-Nitroquinoline N-oxide (1 µg/mL)
95% Confidence level
With Metabolic Activation
(3 to 6 hours)
[Benzo(a)pyrene] Without Metabolic Activation
(3 to 6 hours)
[4 Nitroquinoline N-oxide]
Mean Data of Mutant Frequency/2x106 Cells
Mean 264.63 264.08
Standard
Deviation 16.59 10.82
No. of Studies conducted 30 30
Margin of Error 5.94 3.87
Upper bound 270.57 267.95
Lower bound 258.70 260.21
95% Confidence level 1.96 1.96
Max 297.22 295.77
Min 208.97 240.51

- solvent historical control data:
Vehicle-DMSO
95% Confidence level
With Metabolic Activation
(3 to 6 hours) Without Metabolic Activation
(3 to 6 hours)
Mean Data of Mutant Frequency/2x106 Cells
Mean 24.58 25.33
Standard
Deviation 1.68 1.32
No. of Studies conducted 29 29
Margin of Error 0.61 0.48
Upper bound 25.19 25.81
Lower bound 23.97 24.85
95% Confidence level 1.96 1.96
Max 27.85 27.50
Min 19.05 21.95

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cyt

Conclusions:

Based on the results obtained, the test item, Licocare RBW 106 TP is considered as non-mutagenic at and up to the concentration of 0.5 mg/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions

Executive summary:

The test itemLicocare RBW 106 TPwas evaluated for gene mutation test in CHO AA8 cells.The test item formed a uniform suspension in acetone at 200 mg/mL. Precipitation test was conducted at 0.0625, 0.125, 0.25, 0.50, 1 and 2 mg/mL. Post 4 hours of incubation, no precipitation was observed at the tested concentrations 0.0625, 0.125 and 0.25 mg/mL, mild precipitation was observed at 0.5 mg/mL, moderate precipitation was observed at    1 mg/mL and heavy precipitation was observed at 2 mg/mL. On the basis of the results, 0.5 mg/mL was selected as the highest concentration for the initial cytotoxicity test. Initial cytotoxicity test was conducted at the concentrations of 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/mL using acetone as a vehicle in tetra plates/group in the presence and absence of metabolic activation (3 to 6 hours)The results of the initial cytotoxicity test indicated that the Relative Survival was greater  than 10% at 0.5 mg/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Based on these results, 0.5 mg/mL was selected as highest concentration for gene mutation test.The gene mutation test was conducted at the concentrations of 0.0625, 0.125, 0.25 and 0.50 mg/mLusing acetone as a vehicle in four plates/group in the presence and absence of metabolic activation (3 to 6 ho                                                                                                                                                                                                                                                                                                                             Benzo(a) pyrene and4 Nitroquinoline N-oxidewere used as Positive controlsfor the gene mutation test.Cytotoxicity was assessed by determining the Adjusted Cloning Efficiency and Relative Survival in the test. There was no evidence of cytotoxicity (˂10% RS) at any of the concentrations tested both in presence and absence of metabolic activation.There was no statistically significant increase in mutant frequencies at any of the concentrations tested when compared with the vehicle control. Moreover, treatment withLicocare RBW 106 TPresulted in mutant frequencies which fell within acceptable ranges with regard to historical controls.

The reference control (DMSO) cultures showed mutant frequencies well within the laboratory’s historical control ranges. The actual vehicle control showed responses which were comparable to the reference control responses and fell within acceptable ranges. The positive controls resulted in mutant frequencies,which were statistically significant when compared with the vehicle control and were well within the historical control ranges. Therefore, the study was considered to be valid

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 January 2020 to 28 April 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro Mammalian Chromosomal Aberration Test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:Clariant Plastics And Coatings (Deutschland) GmbH
- Expiration date : 16.01.2022

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Human blood
- Sex, age and number of blood donors if applicable:33 (female) and 26 (male)
- Whether whole blood or separated lymphocytes were used if applicable: separated lymphocytes
- Methods for maintenance in cell culture if applicable: Co2 incubator
- Modal number of chromosomes:46±2

MEDIA USED
- Type and identity of media including Co2 concentration if applicable:RPMI 1640 Culture media and 5% Co2.
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 Liver Homogenate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item formed suspension in acetone at 200 mg/mL.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

- Cell density at seeding (if applicable):

DURATION
- Exposure duration: 3 to 6 hours and 20 to 24 hours..

STAIN : Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:Pellets were mixed with 4 mL of freshly prepared warm 0.56% Potassium chloride. Cell suspension was incubated for 10 minutes at room temperature and later it was centrifuged at 1800 rpm for 10 minutes. Supernatant was discarded and cell pellet was mixed with 4 mL of freshly prepared cold acetic acid:methanol fixative (1:3). Cell suspension was incubated for 10 minutes at room temperature and later suspension was centrifuged at 2200 rpm for 10 minutes. The procedure was repeated twice by adding 4 mL of cold acetic acid: methanol fixative (1:3).

Clean slides were stored in a beaker with distilled water and kept in the refrigerator for at least 1 hour before use. The cell suspension was mixed using a pipette and few drops of the suspension were aspirated and dropped onto the chilled slide pre labeled with study number, with (+S9) or without metabolic activation (-S9), treatment/group and slide number. The slides were air dried.
Minimum of 3 slides were prepared for each treatment replicate. Slides were stained using 5% Giemsa stain for 15 minutes.

NUMBER OF CELLS EVALUATED: 500 per replicate

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 200 metaphase

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index


OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Methods, such as or fragmented chromosomes (if applicable): Microscopy

Evaluation criteria:

Considered acceptable for addition to the laboratory historical negative control database.
Adequate number of cells (at least 300 well spread metaphases per concentration) and concentration
s (at least three analyzable concentrations) should be analyzed.

Statistics:

Yes

Key result

Species / strain:

lymphocytes: Source of cells: Human blood

Remarks:

up to the highet dose tested (1 mg/mL)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

up to the highet dose tested (1 mg/mL)

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

up to 1mg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No change
- Water solubility: No
- Precipitation: Observed at 1 and 2 mg/mL


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
With S9 (3 to 6 hours) - Cyclophosphamide monohydrate Without S9 (3 to 6 hours) - Mitomycin C Without S9 (20 to 24 hours) - Mitomycin C
Average 10.08 10.08 10.56
Standard Deviation 1.31 1.51 1.79
Sample Size 41.00 41.00 41.00
Margin of Error 0.40 0.46 0.55
Upper Bound 10.48 10.54 11.11
Lower Bound 9.68 9.62 10.01
95% Confidence level 1.96 1.96 1.96
Max 12.70 14.70 16.70
Min 7.70 7.35 7.00

- Solvent/vehicle historical control data: (DMSO instead of Acetone as no sufficient inhouse historical control data)

With S9
(3 to 6 hours) Without S9 (3 to 6 hours) Without S9 (20 to 24 hours)
Average 0.74 0.87 0.79
Standard Deviation 0.34 0.35 0.41
Sample Size 34.00 34.00 34.00
Margin of Error 0.11 0.12 0.14
Upper Bound 0.86 0.99 0.93
Lower Bound 0.63 0.76 0.66
95% Confidence level 1.96 1.96 1.96
Max 1.67 2.00 1.33
Min 0.00 0.35 0.00

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Reduction in Mitotic index

Conclusions:

Based on the results obtained, the test item, Licocare RBW 106 TP is considered as non-clastogenic up to the concentration of 1 mg/mL, both in the presence and absence of metabolic activation under the presented test conditions.

Executive summary:

Summary :

The test item,Licocare RBW 106 TPwas evaluated for chromosomal aberrations in human lymphocytes according to OECD TG 473, “In vitroMammalian Chromosomal AberrationTest” adopted on 29^thJuly 2016 .The test item formed a suspension in acetone at 200 mg/mL. Precipitation and pH test was conducted at 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL. Post 23 hours and 30 minutes of incubation, mild and heavy precipitation was observed at 1 and 2 mg/mL respectively. No precipitation was observed at any other concentrations (0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/mL). No change in pH was observed in any of the concentrations tested upto 2 mg/mL. Hence, 1 mg/mL was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were 0.125, 0.25 and 0.5 mg/mL of test item.As the percentage reduction in Mitotic Index was not more than 45±5% at the concentration of 1 mg/mL, same was selected as the highest concentration for the chromosomal aberration test. Other concentrations tested were 0.25 and 0.5 mg/mL.In the chromosomal aberration test, the cells were treated withthe test itemat the concentrations of 0.25, 0.5 and 1 mg/mL using acetone as the vehicle. The treatment was carried out in duplicates for the short term period (3 to 6 hours) both in the presence and absence of metabolic activation and for the long term period (20 to 24 hours) in the absence of metabolic activation. Cyclophosphamide Monohydrate (+S9 for short term) at the concentration of 10 µg/mL and Mytomycin-C at the concentration of 0.05 µg/mL (-S9 both for short term and long term) were used as positive controls. Cells without any treatment were maintained as negative control.The treated cells were harvested at about 1.5 normal cell cycle length after treatment. During harvesting of cultures, thecells were treated with a metaphase-arresting substance (colchicine), harvested, stained and metaphase cells were analysed microscopically for the structural chromosomal aberrations.The percentage reduction in MI and percent aberrated cells of vehicle control were comparable with the negative control. There was no statistically significant increase in the number of aberrated cells when compared with vehicle control at any of the test item concentration levels tested.The cultures treated with positive controlsfor the short-term period(3 to 6 hours) both in the presence and absence of metabolic activation, and for the long-term period   (20 to 24 hours) in the absence of metabolic activation induced aberrated cells which were statistically significant compared with the respective vehicle controls. This demonstrated sensitivity of the test system towards positive controls and confirmed that the test conditions were adequate. 

The concurrent vehicle control values were within the 95% control limits of the distribution of the laboratory’s historical vehicle control database.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

genetic toxicity in vivo, other

Remarks:

Mammalian Erythrocyte Micronucleus Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 January 2020 to 28 April 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Adopted on 29th July 2016.

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Mammalian Erythrocyte Micronucleus Test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Clariant Plastics and Coatings (Deutschland) GmbH
- Expiration date :16.01.2022
- lot/batch: DEF2105728
- Purity test date:9 9.63%

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Solubility of the test substance in the solvent/vehicle: 0.5% carboxymethyl cellulose
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

Species:

mouse

Strain:

Swiss

Remarks:

Albino

Details on species / strain selection:

Mouse is one of the recommended species by regulatory agencies for conducting in vivo Micronucleus test among rodents

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: Male - 18.54 to 20.71 g, Female - 18.31 to 19.93 g (Pre study)
Male - 18.29 to 20.85 g, Female - 18.03 to 22.76 g (Main study)

- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: Maximum of five animals of same sex and group were housed together in a standard polypropylene cage (L 280 × B 220 × H 140 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Sterilized paddy husk was used as a bedding material.
- Diet (e.g. ad libitum): Altromin Maintenance Diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period
- Water (e.g. ad libitum): Water was provided ad libitum throughout the acclimatization and experimental period
- Acclimation period: Healthy adult young animals were acclimatized for six days in pre study and five days in limit study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.5 to 22.8°C
- Humidity (%): 47 to 69%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours

IN-LIFE DATES: From: 10 January 2020 To: 18 March 2020

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5 % w/v CMC (carboxymethyl cellulose);
- Justification for choice of solvent/vehicle: universally accepted vehicle for acute oral dosing
- Concentration of test material in vehicle: 2000 mg/kg body weight
- Lot/batch no. (if required): 210515

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Required quantity of test item was weighed as per the dose. The weighed test item was transferred to a clean mortar and ground using pestle by adding a small quantity of vehicle to get a uniform suspension. The content was transferred to a measuring cylinder. Again, a small quantity of 0.5% carboxymethyl cellulose was added to the mortar rinsed and transferred to the measuring cylinder. The rinsing procedure was repeated until the test item is transferred completely into the measuring cylinder. The final volume was made up with 0.5% carboxymethyl cellulose to get the desired concentration as per the dose requirement. Formulation of the test item was prepared freshly every day before dosing

Duration of treatment / exposure:

24 hours interval

Frequency of treatment:

Once in a day for two days

Post exposure period:

18 to 24 hours

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Positive control(s):

none; cyclophosphamide;
- Justification for choice of positive control(s): As per inhouse validation study and as per the OECD 474
- Route of administration: Oral
- Doses / concentrations: 100 mg/kg

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: As per the OECD Guideline No. 474, 2000 mg/kg body weight was selected as highest dose

DETAILS OF SLIDE PREPARATION: The femur was isolated from each animal for bone marrow collection. Bone marrow cells were obtained by cut opening the epiphyses of femur bone immediately following sacrifice. The marrow was flushed out in to a centrifuge tube using the Fetal Bovine Serum (FBS). The femur bone marrow cells were centrifuged at about 2700 rpm for 10 minutes. Prior to smear preparation, the supernatant was discarded and the cell pellet is then resuspended in approximately 50 µL of Fetal Bovine Serum (FBS).
Minimum of three slides were prepared for pre study and main study per animal. Smears before staining was fixed by immersing the slides in methanol for 5 minutes.The air dried slides were stained with May-Gruenwald and Giemsa stain for evaluation.

METHOD OF ANALYSIS: Slide evaluation

Evaluation criteria:

In the dose range finding study for each animal, minimum of 500 erythrocytes (which include mature and immature erythrocytes) were scored to determine the Polychromatic Erythrocytes (PCE) and Normochromatic Erythrocytes (NCE) ratio, in order to determine PCE: total RBC ratio. This result was used to determine the cytotoxicity of the test item.
In the limit study, all the slides including those of positive and vehicle controls were coded before microscopic evaluation to avoid group bias during evaluation.
For each animal, a minimum of 500 erythrocytes (which included mature and immature erythrocytes) were scored from first slide of the animal to determine PCE: total RBC ratio along with the incidence of micronucleus. The subsequent slides were scored only for the number of PCEs and incidence of micronucleated PCEs. For each animal, a minimum of 4000 Polychromatic Erythrocytes (PCEs) were scored for the incidence of micronucleated immature erythrocytes (MNPCEs).

Statistics:

Body weight of both pre study and limit study was analyzed by SPSS, version No. 22 at a 95% level (p≤0.05) of significance. Inter group comparison of Body weight of Day 1, 2 and 3 were done. Slides from limit study were decoded after analysis, the number of PCE (Polychromatic erythrocytes), RBC (Red blood corpuscles), MNPCE (Micronucleated Polychromatic erythrocytes) and PCE/ total erythrocytes ratio (Polychromatic erythrocytes/ total erythrocytes) and frequency of MNPCE was calculated. The data of positive control and the treatment groups were compared with that of the vehicle control for the incidence of MNPCEs and the proportion of PCEs among total RBCs by SPSS at a 95% level (p≤0.05) of significance. All analysis and comparisons were evaluated at the 95% level of confidence (p<0.05).
Statistically significant changes obtained were designated by the superscripts in the summary table throughout the report as stated below:
*: Statistically significant (p<0.05).

Key result

Sex:

male/female

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: Yes
- Solubility: Soluble in 0.5% W/V CMC
- Clinical signs of toxicity in test animals: No
- Evidence of cytotoxicity in tissue analyzed: No
- Rationale for exposure: No

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No
- Ratio of PCE/NCE (for Micronucleus assay): Yes
- Appropriateness of dose levels and route: Yes
- Statistical evaluation:Yes

Conclusions:

Based on the results obtained under the conditions employed during this experiment, it is concluded that the test item, Licocare RBW 106 TP is non-genotoxic at the limit dose of 2000 mg/kg.

Executive summary:

The test itemLicocare RBW 106 TPwas evaluated in the MammalianErythrocyte Micronucleus Test .

 This study was conducted to determine the genotoxic potential ofLicocare RBW 106 TP inthe micronucleus test using bone marrow cells of Swiss Albino mice. The pre study consisted of four groups, vehicle control, 500, 1000 and 2000 mg/kg.Licocare RBW 106 TPwasadministered at a dose volume of 10 mL/kg. In the pre study, each group of mice consisted of 3 males and 3 females. The limit study consisted of 3 groups of mice and each group consisted of 5 males and 5 females. G1 group animals were administered with vehicle, G2 group animals were administered with cyclophosphamide monohydrate at 100 mg/kg and animals of G3 group were administered with 2000 mg/kg for two consecutive days by oral route using gavage cannula. Post 18 to 24 hours of last dosing, all mice were sacrificed by cervical dislocation and bone marrow cells were collected. The slides of bone marrow cells were stained with May-Gruenwald and Giemsa stain and observed for incidences of micronucleated polychromatic erythrocytes (MNPCE).In the pre study, no clinical sign,no body weight changes, no gross pathological findingsand no mortality were observed in any of the animals dosed with  Licocare RBW 106 TPin any of the dose levels.

Percent reduction in P/E: total RBC ratio was1.92, 3.85 and 3.85 in males and 1.92, 1.92 and 3.85 in females dosed at 500, 1000 and 2000 mg/kg respectively when compared to respective vehicle control.There was no statistical change in theP/E: total RBC ratio at 500, 1000 and 2000 mg/kg in both the sexes.

In pre study, none of the doses produced observable toxic effects (no depression of bone marrow proliferation or other evidence of cytotoxicity) hence,2000 mg/kg was selected as the limit dose for both the sexes in limit study.

In the limit study, there was no clinical signs, no body weight changes, no gross pathological findings and no mortality were observed in any of the animals dosed with test item at 2000 mg/kg and also in positive control dosed animals. There was no statistical significant change in proportions of PCE: total RBC ratio in animals dosed at 2000 mg/kg of test item and there was statistically significant decrease in proportions of PCE: total RBC ratio in positive control group in both the sexes when compared to vehicle control groups respectively. The average percentage of MNPCEs was 0.05 in males and 0.04 in females dosed with vehicle. There was no statistically significant increase in thepercentageof micronucleated PCEs (per 4000 PCEs scored) at 2000 mg/kg in comparison with vehicle control.

The positive control,cyclophosphamide monohydrate at 100 mg/kgexhibited statistically significant increase in the percentage of MNPCE, when compared to vehicle control. Thisdemonstrated the sensitivity of the test system towards positive controls and confirmed that the test conditions were adequate.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)